CALCIUM A M EDICAL D ICTIONARY , B IBLIOGRAPHY , AND A NNOTATED R ESEARCH G UIDE TO I NTERNET R EFERENCES
J AMES N. P ARKER , M.D. AND P HILIP M. P ARKER , P H .D., E DITORS
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ICON Health Publications ICON Group International, Inc. 4370 La Jolla Village Drive, 4th Floor San Diego, CA 92122 USA Copyright ©2003 by ICON Group International, Inc. Copyright ©2003 by ICON Group International, Inc. All rights reserved. This book is protected by copyright. No part of it may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without written permission from the publisher. Printed in the United States of America. Last digit indicates print number: 10 9 8 7 6 4 5 3 2 1
Publisher, Health Care: Philip Parker, Ph.D. Editor(s): James Parker, M.D., Philip Parker, Ph.D. Publisher's note: The ideas, procedures, and suggestions contained in this book are not intended for the diagnosis or treatment of a health problem. As new medical or scientific information becomes available from academic and clinical research, recommended treatments and drug therapies may undergo changes. The authors, editors, and publisher have attempted to make the information in this book up to date and accurate in accord with accepted standards at the time of publication. The authors, editors, and publisher are not responsible for errors or omissions or for consequences from application of the book, and make no warranty, expressed or implied, in regard to the contents of this book. Any practice described in this book should be applied by the reader in accordance with professional standards of care used in regard to the unique circumstances that may apply in each situation. The reader is advised to always check product information (package inserts) for changes and new information regarding dosage and contraindications before prescribing any drug or pharmacological product. Caution is especially urged when using new or infrequently ordered drugs, herbal remedies, vitamins and supplements, alternative therapies, complementary therapies and medicines, and integrative medical treatments. Cataloging-in-Publication Data Parker, James N., 1961Parker, Philip M., 1960Calcium: A Medical Dictionary, Bibliography, and Annotated Research Guide to Internet References / James N. Parker and Philip M. Parker, editors p. cm. Includes bibliographical references, glossary, and index. ISBN: 0-597-83614-0 1. Calcium-Popular works. I. Title.
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Disclaimer This publication is not intended to be used for the diagnosis or treatment of a health problem. It is sold with the understanding that the publisher, editors, and authors are not engaging in the rendering of medical, psychological, financial, legal, or other professional services. References to any entity, product, service, or source of information that may be contained in this publication should not be considered an endorsement, either direct or implied, by the publisher, editors, or authors. ICON Group International, Inc., the editors, and the authors are not responsible for the content of any Web pages or publications referenced in this publication.
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Acknowledgements The collective knowledge generated from academic and applied research summarized in various references has been critical in the creation of this book which is best viewed as a comprehensive compilation and collection of information prepared by various official agencies which produce publications on calcium. Books in this series draw from various agencies and institutions associated with the United States Department of Health and Human Services, and in particular, the Office of the Secretary of Health and Human Services (OS), the Administration for Children and Families (ACF), the Administration on Aging (AOA), the Agency for Healthcare Research and Quality (AHRQ), the Agency for Toxic Substances and Disease Registry (ATSDR), the Centers for Disease Control and Prevention (CDC), the Food and Drug Administration (FDA), the Healthcare Financing Administration (HCFA), the Health Resources and Services Administration (HRSA), the Indian Health Service (IHS), the institutions of the National Institutes of Health (NIH), the Program Support Center (PSC), and the Substance Abuse and Mental Health Services Administration (SAMHSA). In addition to these sources, information gathered from the National Library of Medicine, the United States Patent Office, the European Union, and their related organizations has been invaluable in the creation of this book. Some of the work represented was financially supported by the Research and Development Committee at INSEAD. This support is gratefully acknowledged. Finally, special thanks are owed to Tiffany Freeman for her excellent editorial support.
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About the Editors James N. Parker, M.D. Dr. James N. Parker received his Bachelor of Science degree in Psychobiology from the University of California, Riverside and his M.D. from the University of California, San Diego. In addition to authoring numerous research publications, he has lectured at various academic institutions. Dr. Parker is the medical editor for health books by ICON Health Publications. Philip M. Parker, Ph.D. Philip M. Parker is the Eli Lilly Chair Professor of Innovation, Business and Society at INSEAD (Fontainebleau, France and Singapore). Dr. Parker has also been Professor at the University of California, San Diego and has taught courses at Harvard University, the Hong Kong University of Science and Technology, the Massachusetts Institute of Technology, Stanford University, and UCLA. Dr. Parker is the associate editor for ICON Health Publications.
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About ICON Health Publications To discover more about ICON Health Publications, simply check with your preferred online booksellers, including Barnes & Noble.com and Amazon.com which currently carry all of our titles. Or, feel free to contact us directly for bulk purchases or institutional discounts: ICON Group International, Inc. 4370 La Jolla Village Drive, Fourth Floor San Diego, CA 92122 USA Fax: 858-546-4341 Web site: www.icongrouponline.com/health
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Table of Contents FORWARD .......................................................................................................................................... 1 CHAPTER 1. STUDIES ON CALCIUM .................................................................................................. 3 Overview........................................................................................................................................ 3 The Combined Health Information Database................................................................................. 3 Federally Funded Research on Calcium ....................................................................................... 10 E-Journals: PubMed Central ....................................................................................................... 65 The National Library of Medicine: PubMed ................................................................................ 88 CHAPTER 2. NUTRITION AND CALCIUM ....................................................................................... 179 Overview.................................................................................................................................... 179 Finding Nutrition Studies on Calcium...................................................................................... 179 Federal Resources on Nutrition ................................................................................................. 187 Additional Web Resources ......................................................................................................... 188 CHAPTER 3. ALTERNATIVE MEDICINE AND CALCIUM ................................................................ 201 Overview.................................................................................................................................... 201 National Center for Complementary and Alternative Medicine................................................ 201 Additional Web Resources ......................................................................................................... 217 General References ..................................................................................................................... 238 CHAPTER 4. DISSERTATIONS ON CALCIUM .................................................................................. 239 Overview.................................................................................................................................... 239 Dissertations on Calcium........................................................................................................... 239 Keeping Current ........................................................................................................................ 261 CHAPTER 5. CLINICAL TRIALS AND CALCIUM ............................................................................. 263 Overview.................................................................................................................................... 263 Recent Trials on Calcium........................................................................................................... 263 Keeping Current on Clinical Trials ........................................................................................... 274 CHAPTER 6. PATENTS ON CALCIUM ............................................................................................. 277 Overview.................................................................................................................................... 277 Patents on Calcium.................................................................................................................... 277 Patent Applications on Calcium ................................................................................................ 363 Keeping Current ........................................................................................................................ 444 CHAPTER 7. BOOKS ON CALCIUM ................................................................................................. 445 Overview.................................................................................................................................... 445 Book Summaries: Federal Agencies............................................................................................ 445 Book Summaries: Online Booksellers......................................................................................... 446 The National Library of Medicine Book Index ........................................................................... 473 Chapters on Calcium.................................................................................................................. 474 CHAPTER 8. MULTIMEDIA ON CALCIUM ...................................................................................... 477 Overview.................................................................................................................................... 477 Video Recordings ....................................................................................................................... 477 Bibliography: Multimedia on Calcium....................................................................................... 480 CHAPTER 9. PERIODICALS AND NEWS ON CALCIUM ................................................................... 483 Overview.................................................................................................................................... 483 News Services and Press Releases.............................................................................................. 483 Newsletters on Calcium ............................................................................................................. 486 Newsletter Articles .................................................................................................................... 487 Academic Periodicals covering Calcium .................................................................................... 489 CHAPTER 10. RESEARCHING MEDICATIONS................................................................................. 491 Overview.................................................................................................................................... 491 U.S. Pharmacopeia..................................................................................................................... 491 Commercial Databases ............................................................................................................... 494 Researching Orphan Drugs ....................................................................................................... 495
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APPENDIX A. PHYSICIAN RESOURCES .......................................................................................... 499 Overview.................................................................................................................................... 499 NIH Guidelines.......................................................................................................................... 499 NIH Databases........................................................................................................................... 501 Other Commercial Databases..................................................................................................... 507 The Genome Project and Calcium.............................................................................................. 508 APPENDIX B. PATIENT RESOURCES ............................................................................................... 517 Overview.................................................................................................................................... 517 Patient Guideline Sources.......................................................................................................... 517 Associations and Calcium.......................................................................................................... 524 Finding Associations.................................................................................................................. 528 APPENDIX C. FINDING MEDICAL LIBRARIES ................................................................................ 531 Overview.................................................................................................................................... 531 Preparation................................................................................................................................. 531 Finding a Local Medical Library................................................................................................ 531 Medical Libraries in the U.S. and Canada ................................................................................. 531 ONLINE GLOSSARIES................................................................................................................ 537 Online Dictionary Directories ................................................................................................... 541 CALCIUM DICTIONARY ........................................................................................................... 543 INDEX .............................................................................................................................................. 665
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FORWARD In March 2001, the National Institutes of Health issued the following warning: "The number of Web sites offering health-related resources grows every day. Many sites provide valuable information, while others may have information that is unreliable or misleading."1 Furthermore, because of the rapid increase in Internet-based information, many hours can be wasted searching, selecting, and printing. Since only the smallest fraction of information dealing with calcium is indexed in search engines, such as www.google.com or others, a non-systematic approach to Internet research can be not only time consuming, but also incomplete. This book was created for medical professionals, students, and members of the general public who want to know as much as possible about calcium, using the most advanced research tools available and spending the least amount of time doing so. In addition to offering a structured and comprehensive bibliography, the pages that follow will tell you where and how to find reliable information covering virtually all topics related to calcium, from the essentials to the most advanced areas of research. Public, academic, government, and peer-reviewed research studies are emphasized. Various abstracts are reproduced to give you some of the latest official information available to date on calcium. Abundant guidance is given on how to obtain free-of-charge primary research results via the Internet. While this book focuses on the field of medicine, when some sources provide access to non-medical information relating to calcium, these are noted in the text. E-book and electronic versions of this book are fully interactive with each of the Internet sites mentioned (clicking on a hyperlink automatically opens your browser to the site indicated). If you are using the hard copy version of this book, you can access a cited Web site by typing the provided Web address directly into your Internet browser. You may find it useful to refer to synonyms or related terms when accessing these Internet databases. NOTE: At the time of publication, the Web addresses were functional. However, some links may fail due to URL address changes, which is a common occurrence on the Internet. For readers unfamiliar with the Internet, detailed instructions are offered on how to access electronic resources. For readers unfamiliar with medical terminology, a comprehensive glossary is provided. For readers without access to Internet resources, a directory of medical libraries, that have or can locate references cited here, is given. We hope these resources will prove useful to the widest possible audience seeking information on calcium. The Editors
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From the NIH, National Cancer Institute (NCI): http://www.cancer.gov/cancerinfo/ten-things-to-know.
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CHAPTER 1. STUDIES ON CALCIUM Overview In this chapter, we will show you how to locate peer-reviewed references and studies on calcium.
The Combined Health Information Database The Combined Health Information Database summarizes studies across numerous federal agencies. To limit your investigation to research studies and calcium, you will need to use the advanced search options. First, go to http://chid.nih.gov/index.html. From there, select the “Detailed Search” option (or go directly to that page with the following hyperlink: http://chid.nih.gov/detail/detail.html). The trick in extracting studies is found in the drop boxes at the bottom of the search page where “You may refine your search by.” Select the dates and language you prefer, and the format option “Journal Article.” At the top of the search form, select the number of records you would like to see (we recommend 100) and check the box to display “whole records.” We recommend that you type “calcium” (or synonyms) into the “For these words:” box. Consider using the option “anywhere in record” to make your search as broad as possible. If you want to limit the search to only a particular field, such as the title of the journal, then select this option in the “Search in these fields” drop box. The following is what you can expect from this type of search: •
Foscarnet - Induced Hypocalcemia and Effect of Foscarnet on Calcium Metabolism Source: Journal of Clinical Endocrinology and Metabolism; Vol. 72, No. 5, May 1991. p. 1130-1135. Contact: University of California Positive Health Program, San Francisco General Hospital, AIDS Program, Clinical Research Section, PO Box 0881, San Francisco, CA, 94110-0881, (415) 514-0550. Summary: Toscarnet (trisodium phosphonoformate), an investigational pyrophosphate analog increasingly used to treat refractory cytomegalovirus retinitis and mucocutaneous herpes simplex virus infections in immunocompromised patients, has been reported to cause abnormalities in serum calcium and phosphate, including cases of fatal hypocalcemia. To further elucidate the magnitude and mechanism of these
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abnormalities in humans treated with foscarnet for opportunistic herpes virus infections, we analyzed anaerobic serum specimens and 24-h urine samples before and after single and multiple doses of iv foscarnet and performed a series of in vitro experiments with normal human serum and plasma. Plasma ionized calcium concentrations acutely decreased by a mean 0.17 mmol/L in the 6 individuals who received a 90 mg/kg dose of foscarnet and by a mean 0.28 mmol/L in the 11 individuals who received a 120 mg/kg dose (P = 0.016, 90 vs. 120 mg/kg dose). Results of in vitro experiments showed a highly significant inverse linear relationship between foscarnet and ionized calcium concentrations, but no correlation between foscarnet and total calcium or phosphate concentration. Dialysis experiments suggested that the complexing of foscarnet with ionized calcium could be a cause of this ionized hypocalcemia. Physicians must be aware of this phenomenon and should measure serum ionized calcium during foscarnet therapy (preferably at the end of a foscarnet infusion) whenever neurological or cardiological abnormalities occur. •
Calcium Hypothesis of Alzheimer's Disease and Brain Aging Source: Annals of the New York Academy of Sciences. 15(747): 1-11. December 1994. Summary: This introductory article, in the volume that represents the proceedings of a workshop with the same title, reevaluates and defends the calcium hypothesis in light of new evidence. This evidence might support or refute the proposition that cellular mechanisms for maintaining homeostasis of cytosolic calcium concentration play a key role in aging, and that sustained changes in cytosolic calcium homeostasis could provide the common pathway for the neurological changes associated with Alzheimer's disease. The article discusses the rationale behind the calcium hypothesis, the parameters needed for it to become a unifying hypothesis, the essential features and challenges it must meet, and its key elements. 1 figure, 22 references.
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Calcium Antagonists May Help Slow Dementia Source: Medical World News. 32(11): 10. November 1991. Summary: This brief report in a medical news journal discusses accumulating clinical evidence suggesting that calcium antagonists can retard and perhaps halt the progression of dementia. Results of a number of trials indicate that nimodipine benefits patients with organic brain syndrome. The data are as yet inconclusive, and the possible effects on Alzheimer's disease cannot be predicted.
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Role of Calcium Regulation in Brain Aging: Reexamination of a Hypothesis Source: Aging (Raven Press). 1(1): 17-34. March 1989. Summary: Studies of the central nervous system have a long history; however, it is only recently that researchers have begun to understand brain function in health and disease states. And, the topic of the aging brain has become a subject of intense study for a short period. At present, the process of normal aging is relatively poorly understood. Although there are a number of theories of aging, no single theory appears to account for most age-dependent brain changes. This review article provides a re-evaluation of the 'Calcium Hypothesis of Brain Aging' in light of new evidence that suggests that cellular mechanisms involved in the maintenance of cytosol Ca2 homeostasis play a key role in brain aging, and that sustained changes in [Ca2] homeostasis provide a common pathway for age-as{sociated brain changes. Revision of this hypothesis suggests that there is a complex interaction between the amount of [Ca2] perturbation and the duration of deregulation of Ca2 homeostasis, and it proposes that a small disturbance in
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Ca2 homeostasis with a sustained increase in [Ca] over a long period has similar cell injuring consequences as that produced by a large increase in [Ca] over a shorter period. Although there are several alternative mechanisms through which the regulation of{ cytosol [Ca2] can be disrupted (such as changes in ion channels, extrusion pumps, and sequestration), this review focuses on disruptions in energy metabolism and changes in the structure and function of membranes as the most likely antecedent events which lead to disruption of Ca2 homeostasis. The principle purpose of this review is to identify scientific opportunities and stimulate further research into cellular mechanisms of brain aging. 84 references. •
Effect of Exercise Intensity on Bone Density, Strength, and Calcium Turnover in Older Women Source: Medicine and Science in Sports and Exercise. 32(6):1043-1050. June 2000. Summary: This journal article provides health professionals with information on a study that examined the effects of 24 weeks of high intensity strength training or low intensity walking on lumbar bone mineral density (BMD), muscular strength, and calcium turnover in Australian women either taking hormone replacement therapy (HRT) or not taking HRT. A subject pool of 64 women between 45 and 65 years of age and randomly allocated into weights, walking, weights and HRT, and walking and HRT groups completed the study. All subjects trained twice weekly in either a 50 minute walking or weight training program. Measurements included maximal isometric knee strength, 1RM bench press, 1RM squat, isokinetic back strength, lumbar BMD, serum osteocalcin, and urinary deoxypyridinoline crosslinks. The study found no significant group differences in BMD at the completion of training. However, a significant within group change was apparent for the walking group since BMD decreased 1.3 percent below baseline testing. Osteocalcin levels increased significantly in the walking group. Maximal bench press and squat strength improved significantly in the weight and weights and HRT groups. The weights group also increased significantly in isokinetic back strength. The article concludes that short term high intensity resistance training using free weight lifts provides an effective means for increasing muscular strength in women between 45 and 65 years old. 3 figures, 1 table, and 40 references. (AA-M).
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Bone Mineral Density in Mother-Daughter Pairs: Relations to Lifetime Exercise, Lifetime Milk Consumption, and Calcium Supplements Source: American Journal of Clinical Nutrition. 63:72-79; 1996. Summary: This journal article for health professionals describes a study that investigated associations between lifetime milk consumption, calcium intake from supplements, lifetime weight-bearing exercise, and bone mineral density (BMD) among 25 elderly women and their premenopausal daughters. The BMD of the total, axial, and peripheral skeleton was measured by dual-energy X-ray absorptiometry. Lifetime milk consumption, supplemental calcium intake, and weight-bearing exercise were estimated retrospectively by questionnaire and interview. Results from multiple-linear-regression analyses show that the total and peripheral BMD of mothers were positively associated with supplemental calcium intake after age 60 years, body weight, current estrogen replacement therapy (ERT), and past oral contraceptive (OC) use. Negative associations were found for age and height. The axial BMD of mothers was positively correlated with body weight and past OC use. Among daughters, lifetime weight-bearing exercise was a predictor of total and peripheral BMD, whereas total lean mass was a predictor of axial BMD. The lifetime milk consumption of mothers was positively associated with that of their daughters. The peripheral BMD values of both mothers and daughters were
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positively correlated after adjustment for daughters' exercise and mothers' age, body weight, and ERT. Results suggest that calcium supplementation and exogenous estrogen positively influence bone mass in postmenopausal years. Findings lend support to recommendations for physical activity as a means of osteoporosis prevention. In the age groups studied, the effects of behavioral and hormonal factors on BMD appeared to dominate over familial similarity, which suggests that women may successfully enhance their genetically determined bone mass through weight-bearing exercise, postmenopausal ERT, and adequate calcium intake. 60 references and 6 tables. (AA-M). •
Vitamin D and Calcium in the Prevention of Corticosteroid Induced Osteoporosis: A 3 Year Follow up Source: Journal of Rheumatology. 23(6):995-1000; 1996. Summary: This journal article for health professionals describes a study that determined the efficacy and safety of using 50,000 units per week of vitamin D and 500 milligrams per day of calcium in the prevention of corticosteroid-induced osteoporosis. A minimized double blind, placebo controlled trial was used to evaluate corticosteroidtreated patients in a tertiary care university affiliated hospital. Patients were eligible for the study if they had polymyalgia rheumatica, temporal arteritis, asthma, vasculitis, or systemic lupus erythematosus. There were 22 women and 9 men in the treatment group, and 20 women and 11 men in the control group. The primary outcome measure was the percentage change in bone mineral density (BMD) of the lumbar spine in the two treatment groups from baseline to 36 months followup. Results indicate that BMD of the lumbar spine in the vitamin D and calcium treated group decreased by a mean of 2.6 percent at 12 months, 3.7 percent at 24 months, and 2.2 percent at 36 months. In the placebo group, there was a decrease of 4.1 percent at 12 months, 3.8 percent at 24 months, and 1.5 percent at 36 months. The observed differences between groups were not statistically significant. Results suggest that vitamin D and calcium may help prevent the early loss of bone seen in the lumbar spine as measured by densitometry of the lumbar spine; however, long-term vitamin D and calcium in those undergoing extended therapy with corticosteroid does not appear to be beneficial. 14 references, 2 figures, and 7 tables. (AA-M).
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Calcium and Colorectal Adenoma Recurrence Source: Current Practice of Medicine. 2(6):115-117, June 1999. Summary: The author discusses the role of diet, particularly calcium, in the development and recurrence of colorectal adenoma. Diet is believed to be the main environmental factor in the pathogenesis of colorectal cancer. High-fat intake received the most initial attention as the responsible dietary component in the 1960's and 1970's. The sequence of changes in the colorectal mucosa during the progression from normal mucosa to the development of colorectal cancer and metastasis has received much attention over the past two decades. In patients at high risk for colonic cancer, cryptproliferating cells change. This change is thought to lead to adenoma formation, the precursor to colorectal cancer. A growing body of data shows the importance of calcium and/or vitamin D in the prevention of colorectal cancer. Epidemiological studies suggest that there is an inverse relationship between the incidence of colon cancer and the dietary intake of calcium or vitamin D. Research has shown that (1) supplementation with calcium carbonate to persons at risk for colon cancer reduces colonic crypt cell proliferative rates; (2) patients at risk for colon cancer who had recent adenomatous polyps removed and were fed a diet high in low-fat dairy foods have improved proliferative and differentiation markers of risk; and (3) calcium or calcium-containing
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foods reduce the recurrence rate of adenomatous polyps. Other possible chemopreventive agents include (1) nonsteroidal antiinflammatory drugs, (2) Cox-2 inhibitors, (3) sulphone, (4) fiber, and (5) folic acid. The author concludes that a combination of chemopreventive agents may be required to have a maximal effect on the development of colorectal cancer. 20 references. •
Is My Child Getting Enough Calcium? Source: Newsletter for People with Lactose Intolerance and Milk Allergy. Contact: Available from Jane Zukin. C/O Commercial Writing and Design, P.O. Box 3129, Ann Arbor, MI 48106-3129. Summary: This article, from a newsletter about lactose intolerance and milk allergy, reviews the role of recommended daily allowances (RDAs) of calcium and other vitamins and minerals, and focuses on ways to ensure that children are receiving adequate amounts of calcium. The author considers the use of calcium supplements and lists the RDA of calcium for kids, broken down by age, from infants through adolescents. The author also lists alternative calcium sources, with the typical serving size and milligrams of calcium in each noted.
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Cytoprotective Effects of Calcium Channel Blockers: Mechanisms and Potential Applications in Hepatocellular Injury Source: Journal of Hepatology. 12(2): 251-255. March 1991. Summary: This review article considers the cytoprotective effects of calcium channel blockers, focusing on their mechanisms and potential applications in hepatocellular injury. The authors discuss intracellular calcium homeostasis, calcium dependence of hepatocellular injury, targets of calcium-mediated liver cell injury, calcium entry and blockade, the potential cytoprotective mechanisms of calcium channel blockers, in vivo studies, and clinical applications of calcium channel blockers. The authors conclude that studies relating to the liver are few, but suggest that calcium channel blockers may have a role to play in limiting hepatocellular damage, especially that arising from exposure to a variety of toxic agents. 1 figure. 42 references.
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Counterpoint: Do People with Diabetes Benefit from Coronary Calcium Scans? Source: Diabetes Care. 26(2): 543-544. February 2003. Contact: Available from American Diabetes Association. 1701 North Beauregard Street, Alexandria, VA 22311. (800) 232-3472. Website: www.diabetes.org. Summary: Cardiovascular disease is responsible for most death and disability associated with diabetes. This editorial considers the role of coronary calcium scans as a noninvasive approach to screen and diagnose coronary syndromes in patients with diabetes. The author discusses calcium screening, the interplay of coronary calcium and future new coronary events in patients with diabetes, and diabetic atherosclerosis. The author contends that those who promote the unproven application of calcium screening for diabetes patients do them a disservice. Calcium screening has not been proven and the question of its therapeutic effectiveness must be addressed. The author concludes that it is far better to offer patients only those tests and therapies that are likely to help them. Elsewhere in the same publication is an article that offers the opposing viewpoint on coronary calcium testing. 20 references.
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Point: Diabetic Patients and Coronary Calcium Source: Diabetes Care. 26(2): 541-542. February 2003. Contact: Available from American Diabetes Association. 1701 North Beauregard Street, Alexandria, VA 22311. (800) 232-3472. Website: www.diabetes.org. Summary: Cardiovascular disease is responsible for most death and disability associated with diabetes. This editorial considers the role of coronary artery calcium (CAC) scans (as measured by electron beam tomography or EBT) as a noninvasive approach to screen and diagnose coronary syndromes in patients with diabetes. The author discusses risk stratification for individuals with diabetes, and then outlines the four possible times when EBT may be useful in the diabetes cohort: type 1 diabetes, younger patients with diabetes, improving compliance, and tracking effectiveness of therapy. The author concludes that while the vast majority of patients with diabetes would not need to undergo further noninvasive testing for CAC, there are certain cohorts that will benefit. The use of EBT to better risk stratify certain patients with diabetes, to improve compliance, and to allow for noninvasive tracking of atherosclerotic plaque could potentially reduce both mortality and costs and better direct therapy. Elsewhere in the same publication is an article that offers the opposing viewpoint on coronary calcium testing. 11 references.
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Calcium Antagonists and Cardiovascular Risk in Diabetes: A Review of the Evidence (commentary) Source: Diabetes Care. 22(7): 1206-1208. July 1999. Contact: Available from American Diabetes Association. 1701 North Beauregard Street, Alexandria, VA 22311. (800) 232-3472. Summary: This commentary highlights findings from large hypertension studies on the use of calcium antagonists (CAs) and the risk of cardiovascular disease in people who have diabetes. These studies provide evidence in support of CA based therapy for treating high blood pressure in people who have diabetes. In addition, the commentary examines fundamental assumptions used to support the hypothesis that there is an adverse change in dihydropyridine CA activity associated with a systemic decrease in the relative membrane cholesterol content of people who have diabetes. The first assumption is that the cholesterol to phospholipid mole ratio is lower in diabetes, leading to an increase in the accumulation of lipophilic agents such as CAs. The second assumption is that an increase in the membrane accumulation of CAs results in a change in their pharmacologic activity. A review of relevant evidence does not support either assumption and fails to predict an adverse pharmacologic effect for CAs in the hypertensive patient with diabetes. The article therefore concludes that there is an important role for CA based therapy in managing hypertension in people who have diabetes. 17 references.
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Effects of Aging on Serum Ionized and Cystosolic Free Calcium Source: Hypertension. 34(4 Part 2): 902-906. October 1999. Contact: Available from American Heart Association. 7272 Greenville Avenue, Dallas, TX 75231-4596. Summary: Calcium ions, both intracellular (within the cells) and extracellular (outside the cells) are critically important for a wide spectrum of cellular processes, including muscle contraction, nerve excitability, and stimulus to secretion coupling in all
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endocrine and exocrine tissues. Elevated cyctosolic free calcium (Cai) and reciprocally reduced, extracellular ionized calcium (Ca ion) levels are observed in both hypertension (high blood pressure) and type 2 diabetes. Because the changes of vascular (blood vessel) function and insulin sensitivity in these conditions resemble the changes associated with 'normal' aging, the authors of this article considered to what extent similar alterations in calcium metabolism occur with aging per se in the absence of overt hypertension or diabetes. This article reports on a study in which platelet Cai levels and serum C ion levels were measures in normotensive, nondiabetic, healthy, normal elderly (older than 65 years) subjects (n = 11); in 10 young adults with hypertension; and in 10 normotensive adults with type 2 diabetes. Platelet Cai levels were higher and Ca ion levels lower in normal elderly compared with young control subjects, but normal elderly Cai and Ca ion levels were indistinguishable from those in hypertensive and type 2 diabetes subjects. In normal subjects, significant correlations were found between platelet Cai levels and age, and between Cai levels and systolic blood pressure. The authors conclude that aging is associated with alterations of Cai and Ca ion levels resembling those changes present at any age in hypertension and type 2 diabetes. The authors hypothesize that these alterations of calcium metabolism underlie the predisposition to the alterations of blood pressure and insulin sensitivity characteristics of 'normal' aging. The data also suggest that studies of the aging process should be limited to subjects with normal blood pressure and glucose tolerance. 2 figures. 2 tables. 35 references. •
Bone Up On Calcium Source: Diabetes Forecast. 45(3): 38, 40-41. March 1992. Contact: Available from American Diabetes Association. 1701 North Beauregard Street, Alexandria, VA 22311. (800) 232-3472. Website: www.diabetes.org. Summary: This article reviews the role of calcium in the diet, particularly for people who have diabetes mellitus. Topics include specific circumstances that call for increased dietary calcium, the role of calcium in avoiding osteoporosis, guidelines for how much calcium should be ingested and the best sources for calcium, increasing calcium intake without increasing cholesterol intake, and calcium supplements. Four recipes from a menu designed to provide the U.S. Recommended Dietary Allowance of calcium are included: broccoli quiche, tomato soup, cucumber yogurt salad, and peachy rice pudding. Each recipe includes the full nutritional and exchange list information.
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Calcium Antagonists and Diabetic Nephropathy (editorial) Source: Archives of Internal Medicine. 151(12): 2361-2364. December 1992. Summary: This editorial considers some of the issues raised by the emerging body of evidence suggesting that calcium antagonists may also have beneficial effects in diabetic renal disease. The author also considers the controversy surrounding the theoretic framework that has been proposed to account for these beneficial therapeutic effects. Topics discussed include studies in experimental diabetes in the rat; the role of nonhemodynamic factors in affording protection; limitations in extrapolating from animal models; calcium antagonists; and how calcium antagonists differ in their ameliorative effects on proteinuria. 38 references.
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Federally Funded Research on Calcium The U.S. Government supports a variety of research studies relating to calcium. These studies are tracked by the Office of Extramural Research at the National Institutes of Health.2 CRISP (Computerized Retrieval of Information on Scientific Projects) is a searchable database of federally funded biomedical research projects conducted at universities, hospitals, and other institutions. Search the CRISP Web site at http://crisp.cit.nih.gov/crisp/crisp_query.generate_screen. You will have the option to perform targeted searches by various criteria, including geography, date, and topics related to calcium. For most of the studies, the agencies reporting into CRISP provide summaries or abstracts. As opposed to clinical trial research using patients, many federally funded studies use animals or simulated models to explore calcium. The following is typical of the type of information found when searching the CRISP database for calcium: •
Project Title: ACTIVITY DEPENDENT REGULATION OF BDNF EXPRESSION Principal Investigator & Institution: Ghosh, Anirvan; Associate Professor; Neuroscience; Johns Hopkins University 3400 N Charles St Baltimore, MD 21218 Timing: Fiscal Year 2001; Project Start 01-DEC-1999; Project End 30-NOV-2004 Summary: Synaptic activity has a profound effect on the formation of appropriate connections in the brain. Work during the last decade has indicated that such activitydependent plasticity requires calcium influx, which triggers a cascade of intracellular biochemical events that lead to long lasting physiological and anatomical changes. Many of these changes require calcium-dependent gene expression, and several lines of evidence suggest that the Brain-derived neurotophic factor (BDNF) gene is an important target of calcium signaling in neurons. The focus of the proposed experiments is to identify the mechanisms by which calcium influx leads to BDNF expression in cortical neurons. Our preliminary studies suggest that calcium- dependent expression of BDNF is regulated by two calcium-response elements. The first is a novel element (CRS-I) that is regulated by an as yet unidentified transcription factor. The second element is homologous to the cAMP response element (CRE) and is regulated by the transcription factor CREB. To gain further insight into the mechanisms by which calcium regulates BDNF expression, we propose to (i) identify and characterize the transcription factor that mediates transactivation via CRS-I, and (ii) determine the mechanism by which CRS-I and BIII-CRE cooperate to regulate BDNF expression. The mechanisms underlying activity-dependent BDNF expression are of scientific as well as clinical interest. It is clear that BDNF plays a critical role in brain development and plasticity, ans therefore perturbation of BDNF regulation might contribute to neurological and neuropsychiatric disorders. Our investigations should lead to the identification of molecules involved in regulating BDNF expression, and it is likely that this molecular understanding will eventually be useful in developing clinical strategies to correct disorders of the nervous system. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
Healthcare projects are funded by the National Institutes of Health (NIH), Substance Abuse and Mental Health Services (SAMHSA), Health Resources and Services Administration (HRSA), Food and Drug Administration (FDA), Centers for Disease Control and Prevention (CDCP), Agency for Healthcare Research and Quality (AHRQ), and Office of Assistant Secretary of Health (OASH).
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Project Title: ACTIVITY-DEPENDENT HOMEOSTASIS
DEVELOPMENT
OF
CALCIUM
Principal Investigator & Institution: Zirpel, Lance; Neurobiology and Anatomy; University of Utah 200 S University St Salt Lake City, UT 84112 Timing: Fiscal Year 2001; Project Start 01-JAN-2000; Project End 31-DEC-2002 Summary: The proposed research project is aimed at understanding how interactions between afferent activity and central auditory neurons affects development of those neurons and their abilities to cope with changing activity levels. The first set of experiments will address how neurons in the cochlear nucleus respond to the onset and increasing levels of afferent input from the cochlear nerve during development with the implementation of mechanisms to regulate intracellular ion homeostasis. Previous studies have indicated that these mechanisms become prominent at about the same time as onset of afferent activity. The hypothesis that activity determines that implementation of these mechanisms will be experimentally evaluated in chick embryos by surgically or pharmacologically eliminating afferent activity to the nucleus during development. The homeostatic mechanisms will then be evaluated in cochlear nucleus neurons and compared to neurons from normal embryos. Protein quantification and dynamic optical imaging will be used for these evaluations. The ability of a neuron to regulate intracellular calcium is critical for its survival. Many proteins are involved in this regulation and combined to restrict not only the amplitude of a calcium signal, but also the time course and area of that signal. During development of the auditory system, the input that generates these calcium signals changes dramatically. The second set of proposed experiments aims to understand how this input affects the ability of cochlear nucleus neurons to regulate these changing calcium signals. Similar to the first set of experiments, surgical and pharmacological techniques will be used to eliminate afferent activity during development of the brain stem auditory nuclei, and protein quantification and dynamic optical imaging will be used to evaluate specific components and functions of the cellular calcium regulatory machinery. In addition, a specialized imaging technique will be used that allows great precision in measuring the temporal and spatial aspects of the calcium signals within neurons. These studies will contribute to understanding the interactions between afferent activity and the development of specialized central auditory neurons. This understanding will allow for more accurate evaluations of the impact of early sensory deprivation on central neurons. In addition, they will contribute to an animal model of congenital hearing deficits that is widely used for studies on the development and regeneration of auditory function. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: ADAPTIVE MECHANISM TO LOW DIETARY CALCIUM INTAKES DURING PUBERTY Principal Investigator & Institution: Abrams, Steven A.; Baylor College of Medicine 1 Baylor Plaza Houston, TX 77030 Timing: Fiscal Year 2001 Summary: Although many lines of evidence document the importance of maintaining a relatively high calcium intake during pubertal development, the calcium intake of as many as 20% of pubertal American girls is less than one-third of the currently recommended level. Virtually no information is available regarding the physiological adaptation to these low intakes. For example, despite a lower average calcium intake, peak bone mass is greater and the risk of osteoporosis is lower in African-American compared with Caucasian girls. This proposal will evaluate the adaptation to low
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calcium intakes by girls during puberty and compare the differences in that adaptation between Caucasian and African-American girls. The specific aims of the project have not changed during the initial year since the project began. To summarize, these were to: 1) To assess the effects of decreasing calcium intake to 400 mg/day in girls on calcium absorption, excretion and bone formation/resorption; 2)To determine the changes in calcium retention and kinetics after supplementing the intake of girls with very low habitual intakes of calcium (Group 2) with 600 mg of calcium for 6 months; 3) To evaluate racial differences in adaptation to low calcium intakes which may partially account for the lower incidence of osteoporosis in African-Americans compared to Caucasians despite the lower calcium intake of African-Americans; 4) To determine the relationship between hormonal and pubertal status and adaptation to low calcium intake in healthy girls. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: ADP-RIBOSE DEPENDENT CALCIUM ENTRY IN THE IMMUNE SYSTEM Principal Investigator & Institution: Scharenberg, Andrew M.; Assistant Professor; Pediatrics; University of Washington Seattle, WA 98195 Timing: Fiscal Year 2001; Project Start 01-FEB-2001; Project End 31-JAN-2006 Summary: (Applicant's Abstract): The molecular mechanisms involved in the regulation of calcium entry into immune system cells are poorly understood. My preliminary data describe the molecular characterization of a novel calcium entry pathway controlled by ADP-ribose (ADPR). This characterization includes the identification of a novel highly specific vertebrate ADP-Ribose Hydrolase (ADPRH-1), and the functional demonstration that a previously identified gene encodes a protein product which is an ADPR gated calcium channel. Based on these data, I propose to explore ADPR metabolism and the mechanisms which regulate ADPR-mediated calcium entry within the immune system through three specific aims: Specific Aim 1: Characterization of free ADPR metabolism in the immune system. This aim will characterize the cell biology, structure/function relationships, and physiologic roles of ADPRH-1 in the regulation of ADPR metabolism. These analyses will include the production of an ADPRH- 1 deficient cell line and its complementation using various forms of ADPRH-1. Specific Aim 2: Immune system mechanisms for the regulation of free ADPR levels. This aim will focus on analyses of ADPR metabolism in relation to immune cell manipulations plausibly linked to the production of free ADPR. It will utilize both classic methods for measurement of total cellular free ADPR and a novel electrophysiologic assay for cytosolic free ADPR. Specific Aim 3: Characterization of the cell biology and structure/function relationships of the ADPR gated cation channel: a possible key effector of free ADPR. These analyses will include screening of cell lines and primary cell types for channel expression and ADPR-mediated currents, and a structure/function analysis of the role of an ADPRH- 1 homology region of the ADPR gated channel in the gating process. Together, the experiments in these aims will provide us with substantial new information regarding the metabolism of free ADPR and the role of ADPR dependent calcium entry in immune system function. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: ALCOHOL MODULATION OF CARDIAC CALCIUM CHANNELS Principal Investigator & Institution: Aistrup, Gary L.; Mol Pharm & Biol Chemistry; Northwestern University Office of Sponsored Programs Chicago, IL 60611
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Timing: Fiscal Year 2001; Project Start 01-APR-1999; Project End 31-MAR-2004 Summary: Alcohol exerts a variety of actions on the cardiovascular system, the nervous system, and other organs. Clinical studies, have linked alcohol consumption with a number of asymptomatic and overt cardiovascular abnormalities, including cardiomyopathy, hypertension, arrhythmias, heart failure, and stroke. The mechanisms responsible for these various problems are not well understood. In the nervous system, voltage activated calcium channels and certain ligand-gated channels arc particularly sensitive targets of alcohol. These channels are suspected of being instrumental in acute intoxication and withdrawal. In cardiac tissues, calcium channels play a key role in rhythmicity, conduction, and excitation-contraction coupling. These channels are a major site of control by endogenous hormones and transmitters, and by therapeutic drugs. Calcium channels have been directly linked to a number of the actions of ethanol on the heart. Ethanol interferes with contractility in a variety of models, and it reduces electrically-stimulated calcium transients in ventricular myocytes. Our preliminary data with rat myocytes, and results from other laboratories, have confirmed that ethanol blocks L-type calcium channels in isolated cardiac cells. Defining how alcohol affects the physiology and regulation of these channels is essential in explaining immediate consequences of alcohol ingestion, as well as events that occur during prolonged periods of alcohol ethanol abuse. The overall objective of the proposed studies is to use whole-cell patch clamp techniques to analyze ethanol modulation of cardiac calcium channels. Ventricular myocytes will be dissociated from cardiac tissues of adult rats, and subjected to acute alcohol exposure. Biophysical and pharmacological experiments will evaluate calcium channel function under these conditions, and impossible mechanisms of channel modulation. Certain second messenger systems are known to exert regulatory control over calcium channel function in heart cells. Among these, the betaadrenergic/cAMP/PKA pathway is a critical mechanism for enhancing L-type calcium channels and stimulating cardiac contractility. We will therefore test the hypothesis that ethanol alters regulation of channels through this signal transduction system. Our preliminary data have shown that ethanol not only blocks currents stimulated via the beta-adrenergic system, but it also inhibits desensitization of the coupling process. We have also just completed exciting new preliminary studies demonstrating that ethanol is capable of reversing or occluding nifedipine-induced channel block. This novel action may have major implications, given the widespread clinical use of dihydropyridines and other calcium channel antagonists. Drug interactions of this type will be an important focus of the project. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: ALTERED CALCIUM AND VITAMIN D METABOLISM IN PMDD Principal Investigator & Institution: Thys-Jacobs, Susan; St. Luke's-Roosevelt Inst for Hlth Scis Health Sciences New York, NY 10019 Timing: Fiscal Year 2001; Project Start 01-APR-2000; Project End 28-FEB-2004 Summary: (adapted from investigator's abstract): Premenstrual Dysphoric disorder (PMDD) is widely recognized as a recurrent disorder related to hormonal variations of the menstrual cycle. PMDD shares many of the features of depression anxiety and the dysphoric states. Whereas alterations in calcium homeostasis have long been associated with many affective disturbances, recent evidence has suggested that luteal phase symptomatology may be associated with a perturbation in calcium homeostasis. Recently reported abnormalities in calciotropic hormone regulation, as well as the beneficial response to calcium treatment, all support the hypothesis that disordered calciotropic hormone regulation is a major provocative factor in luteal phase
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symptomatology. The purpose of the investigation is to understand more completely the extent to which calcium regulation is disturbed in PMDD by utilizing new tools to assess calcium and bone turnover. This investigation seeks to confirm our preliminary data in a definitive study. The long-term objective is to elucidate the pathophysiology of PMDD as it relates to the calciotropic hormones and bone markers. The experimental design involves enrolling 70 with PMDD and 35 controls. Following 2 months of baseline symptom documentation, women with PMDD and controls will be enrolled in a 1-month observational period with frequent hormonal samplings, urinary collections and daily ratings. Clinical evaluation will involve a detailed history and physical examination, a structured psychiatric interview and a dietary assessment. Hormonal evaluation will include total and ionized calcium intact parathyroid hormone, 25 hydroxyvitamin D, 1,25 dihydroxyvitamin D, estradiol, progesterone, osteocalcin, urinary N-telopeptide, IGF-1 and TNF-A. Each subjects will be initially screened prospectively for 2 menstrual cycles with a daily, self assessment scale; symptoms will then be monitored and quantified for another 1 month while undergoing blood and urine sampling. The observation period will provide extensive characterization of the calciotropic hormones and biochemical markers of bone turnover across the menstrual cycle with simultaneous symptom ratings in women with PMDD and controls. Bone density evaluation will be performed as well on all subjects. Understanding the pathophysiology associated with PMDD may lead to effective therapeutic strategies to prevent the neuropsychiatric disturbances and abnormal calcium regulation that are characteristic of this disorder. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: AMACRINE CELL SIGNALING Principal Investigator & Institution: Gleason, Evanna L.; Biological Sciences; Louisiana State Univ A&M Col Baton Rouge Office of Sponsored Programs Baton Rouge, LA 70803 Timing: Fiscal Year 2003; Project Start 01-JUL-1998; Project End 30-JUN-2007 Summary: (provided by applicant): Retinal amacrine cells are interneurons that help to shape the visual signal that ganglion cells send to other visual centers in the brain. The ultimate goal of the research proposed here is to understand the factors that underlie the dynamics of calcium and calcium dependent signaling within and between amacrine cells. A better understanding of signal processing in the retina will be valuable in helping us to better understand how signals are shaped elsewhere in the brain. This proposal specifically addresses the role of mitochondria in shaping the temporal and spatial properties of calcium signals and calcium-dependent processes in amacrine cells. Mitochondria are especially interesting because they can be a spatially discrete sink and source of calcium. This is relevant to amacrine cell function in particular because it is thought that much of the synaptic signaling in amacrine cells is spatially localized rather than spread over broad regions of the cell. To understand the role of mitochondria in amacrine cell signaling, we propose the following three specific aims: Specific Aim 1: To elucidate the impact of mitochondrial calcium buffering on the spatial and temporal aspects of calcium elevations in amacrine cells. Specific Aim 2: To discover the role of mitochondrial calcium buffering during synaptic transmission between GABAergic amacrine cells. Specific Aim 3: To uncover the interactions between mitochondria and other mechanisms and organelles relevant to calcium signaling in amacrine cells. To achieve these three aims, amacrine cells cultured from the chick retina will be used as a model system for amacrine cells in the intact retina. This system will allow the examination of single amacrine cells or pairs of amacrine cells participating in functional GABAergic synapses, an extremely common type of synapse found in the retina. The
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proposed research will employ calcium imaging, whole cell voltage clamp recordings, immunocytochemistry and confocal microscopy to achieve the stated aims. These studies will lead to insights into the mechanisms that underlie the processing of the visual signal in the vertebrate retina. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: AMLODIPINE TREATMENT FOR COCAINE DEPENDENCE Principal Investigator & Institution: Malcolm, Robert J.; Professor; Psychiatry and Behavioral Scis; Medical University of South Carolina 171 Ashley Ave Charleston, SC 29425 Timing: Fiscal Year 2001; Project Start 05-JAN-1998; Project End 31-DEC-2002 Summary: Pharmacotherapies have not proven to be clinically useful in the treatment of cocaine dependence. Two lines of research indicate that calcium channel antagonists are promising in the treatment of cocaine dependence. 1) Clinical studies of chronic cocaine use have demonstrated multiple neurovascular changes on PET, SPECT and MRI scans of the brain. These studies demonstrate functional abnormalities in energy utilization and reductions in regional cerebral blood flow. Some improvement in these deficits have been noted with lengthening abstinence from cocaine; however, recent work indicates that some regional blood flow changes are persistent over many months. It is possible that these neurovascular abnormalities decrease cognitive abilities and thus reduce the benefits of rehabilitative treatment for cocaine dependence such as cognitive-behavioral therapy (CBT). Calcium channel antagonists antagonize cerebral vasoconstriction, enhance regional cerebral blood flow and possibly could lead to cognitive improvement. 2) Preclinical studies indicate that dihydropyridine type channel antagonists have specific binding sites in rat brains and play a role in modulating dopaminergic reinforcement. Calcium channel antagonists reduce cocaine- related behaviors. One human trial demonstrated that a calcium channel antagonist decreased the subjective rush of cocaine and reduced euphoric properties of cocaine. Thus, there is animal and human evidence that calcium channel antagonists may improve cerebral circulation, enhancing cognitive functioning, and alter the subjective rewarding experience of cocaine. This protocol proposes to evaluate amlodipine, a marketed dihydropyridine calcium channel antagonist which has a well-established safety profile and has known effects of enhancing regional cerebral blood flow. In conjunction with CBT, amlodipine versus placebo will be tested for efficacy in 160 males and females, Caucasians and African-Americans. After screening and informed consent, cocaine dependent individuals will enter a two week placebo period followed by randomization to 12 weeks of amlodipine plus CBT or placebo plus CBT. Follow-up will occur three months after treatment ends. Treatment outcome will be measured with twice weekly quantitative urine drug screens for cocaine and cocaine metabolites, retention in treatment,subjective cocaine craving, and psychometric testing of cognitive functions. Evaluation of this agent for effectiveness and safety provides the opportunity to examine the rationale that improvements in regional cerebral blood flow or modulation of dopamine mechanisms through calcium channel antagonism will enhance recovery from cocaine dependence. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: AN ADP-RIBOSE DEPENDENT CATION CHANNEL ENCODED BY LTRPC2 Principal Investigator & Institution: Penner, Reinhold; Queen's Medical Center 1301 Punchbowl St Honolulu, HI 96813
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Timing: Fiscal Year 2002; Project Start 01-JUN-2002; Project End 31-MAY-2006 Summary: (provided by applicant): Sustained calcium entry, in many cells, is fundamental to the initiation and maintenance of specific cellular responses. In addition to the widespread store-operated calcium influx mechanism, whose molecular nature remains elusive, other putative calcium influx pathways have emerged through identification of a growing number of genes coding for calcium-permeable cation channels. Our preliminary data describe the molecular characterization of a novel calcium-entry pathway controlled by ADP-ribose (ADPR). This characterization includes the identification of a novel highly specific ADP-ribose hydrolase, NUDT9, which shares high homology with the C-terminal region of a previously identified gene, LTRPC2, and the functional demonstration that LTRPC2 encodes a protein product that is an ADPR-gated calcium-permeable cation channel. We also identified natively expressed ADPR-dependent conductances in pancreatic beta cells and human monocytes. Based on these data, we propose to explore the mechanisms that regulate ADPRmediated calcium entry in recombinant and physiological systems. In Specific Aim 1, we will analyze the biophysical and molecular aspects of LTRPC2 ion channel function by using a combination of calcium imaging and electrophysiological analysis of cells that express recombinant LTRPC2. To identify the structural basis for ADPRdependent gating of LTRPC2, we will express and characterize truncated or chimeric LTRPC2 constructs directed towards the C-terminal nudix domain, the putative ADPR binding region. We also propose to systematically alter amino acids within this domain to assess structure-function relationships that confer selectivity for ADPR gating. In Specific Aim 2, we will address the functional and physiological role of ADPR-gated channels in the regulation of calcium homeostasis of beta cells. We will investigate the specific properties of native ADPR-gated channels and compare them with those of recombinant LTRPC2. We will assess their functional role in the cellular responses of the above cells by comparing the relative contributions of ADPR-gated Ca2+ signals to those of store-operated Ca2+ influx and voltage-dependent Ca2+ channels. Finally, we will seek to identify the mechanisms responsible for ADPR production by investigating the major enzymes and pathways involved in its metabolism. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: ANALYSIS OF NEAR-MEMBRANE CALCIUM USING TIRFM AND NSOM Principal Investigator & Institution: Clark, Heather A.; Physiology; University of Connecticut Sch of Med/Dnt Bb20, Mc 2806 Farmington, CT 060302806 Timing: Fiscal Year 2001; Project Start 01-SEP-2000 Summary: Many in vitro experiments demonstrate that the transient calcium concentrations needed to trigger cellular events such as signaling cascades and synaptic transmission are actually much higher than the average resting concentrations of calcium experimentally measured in the cytoplasm of the cell. These findings suggest that calcium reaches transient concentrations that are much higher than that measured in the bulk of the cell. We propose to utilize two optical imaging methods to obtain better resolution of near- membrane dynamics and hotspots: Total Internal Reflection Fluorescence Microscopy (TIRFM) and Near-field Scanning Optical Microscopy (NSOM). Both of these techniques effectively excite only 100 -200 nm into the bulk of the cell. TIRFM will provide a high resolution method of imaging near the membrane of a neuroblastoma cell, and NSOM will monitor a single channel with fast temporal resolution. The combination of the two techniques will elucidate the dynamics of
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calcium microdomains, including information on their size, concentration, location, and lifetime. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: ANALYTICAL ELECTRON MICROSCOPY AND IMAGING Principal Investigator & Institution: Somylo, Andrew P.; University of Virginia Charlottesville Box 400195 Charlottesville, VA 22904 Timing: Fiscal Year 2001 Summary: This project is dedicated to the interdisciplinary development of analytical electron microscopic and other structural approaches and their application to the determination of calcium-dependent and independent mechanisms of regulation in vascular smooth and cardiac muscle. A major goal is to perfect the sensitivity and spatial resolution of energy- filtered transmission electron microscopy (STEM-EELS) to a previously un- attained (2-4 nm) level suitable for sensitive compositional imaging of subcellular domains. STEM-EELS and X-ray mapping with an improved solid- state detector will be used to determine the functionally important, binding of calcium to membranes of cardiac and vascular smooth muscle, the sub-mitochondrial distribution of calcium and the composition of (putative) Ca- and Na buffering domains beneath the plasma membrane. The hypotheses, that the calcium content of mitochondria varies depending on their cellular location relative to the plasma membrane or the sarcoplasmic reticulum, will be quantitatively tested with electron probe microanalysis (EPMA). Manganese (Mn), used to quench the fluorescence of calcium-sensitive fluorophores used for measuring intracellular free calcium, will be quantitatively localize to assess the contribution of Mn accumulation by mitochondria and sarcoplasmic reticulum to fluorescent signals. The distribution of aluminum and fluoride, used to activate G- proteins will be determined in order to ascertain whether AIF/4 directly interacts and co translocates with monomeric GTP-binding proteins in cells or only interacts with trimeric G-proteins. Fluorescence confocal microscopy will be used to related to function the stimulus-dependent distribution of telokin, Rho and Rhoassociated proteins and smooth muscle myosin phosphatase in vascular smooth muscle. Health-related aspects of the research include the role of calcium binding to cardiac gap junctions in ventricular fibrillation, the most common cause of sudden cardiac death, and the importance of abnormalities of vascular and bronchial smooth muscle regulation in diseases such as high blood pressure and asthma. The STEM-EELS methodology developed for mapping,a t 2-4 nm resolution, biologically important elements, such as calcium, will be widely applicable to a broad range of problems related to normal and diseased cardiovascular function and to cellular physiology and pathology of nearly every biological system. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: ANESTHETICS AND CALCIUM CHANNELS Principal Investigator & Institution: Lingle, Christopher J.; Professor; Washington University Lindell and Skinker Blvd St. Louis, MO 63130 Timing: Fiscal Year 2001 Summary: Anesthetics exhibit considerable diversity in the nature of their anesthetic effects. The underlying mechanism of anesthetic action presumably involves alteration of one or more membrane conductances. Relative differences among anesthetics in their effects on these different targets may underlie the important clinical differences among anesthetics. Calcium channels are particularly attractive as potential targets of
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anesthetic action because of their central role in synaptic transmission. Some voltagedependent calcium channels are inhibited by anesthetics within clinical concentrations. Inhibition of particular components of Ca plus current may contribute to some of the diverse effects of different anesthetics including anticonvulsant activity and analgesic activity. Steroid anesthetics provide a particularly interesting group for evaluation of potential targets of anesthetic action, because the structurally defined nature of steroids make them more amenable to structure/function investigations and more useful for the ultimate identification of binding sites. Recent studies indicate that specific steroids exhibit remarkable selectivity in blocking particular components of calcium current. Based on these studies, this proposal addresses a series of aims concerned with the molecular mechanisms of action of steroids on calcium channels. First, we will examine the structure-activity relationships for blockade of particular calcium current components by steroids. Second, we will extend earlier work in which we are defining the pharmacological similarities and differences between different low voltage-activated calcium currents among different neurons. Third, using steroids which selectively block particular components of calcium current in native cells, we will examine the effects of those agents on cloned neuronal calcium channel variants. Finally, we will examine the contribution of different high-voltage-activated calcium currents in synaptic transmission in cultures of hippocampal neurons and the effects of steroids on that transmission. These experiments will provide a whole new series of agents for selective inhibition of different calcium current components which will help advance our understanding of the clinical and behavioral roles of different calcium channel subtypes. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: ANTECEDENTS OF PEAK BONE MASS IN BLACK & WHITE WOMEN Principal Investigator & Institution: Kimm, Sue Y.; Professor of Internal Medicine; Family Med/Clin Epidemiology; University of Pittsburgh at Pittsburgh 350 Thackeray Hall Pittsburgh, PA 15260 Timing: Fiscal Year 2001; Project Start 18-MAR-1999; Project End 28-FEB-2004 Summary: The aim of this study is to investigate environmental, biologic, and genetic determinants of bone mass in a large (n=650) biracial of young women enrolled in the NHLBI Growth and Health Study. Eleven-year longitudinal data are available data are available on dietary intake, physical activity, body composition, pubertal milestones, pregnancy, oral contraceptive use, smoking, and alcohol intake. This cohort is also concurrently enrolled in a second study which obtains annual total body DEXA scans until year 2000. Nested within this study, we will 1) determine what environmental (current and antecedent) and genetic factors determine bone mass (BMD) at the lumbar spine and proximal femur at ages 20-22 years; 2) determine racial difference in the effects of the significant environmental and genetic factors found in aim #1; and 3) determine what environmental and genetic factors influence biochemical markers of bone metabolism and calcium homeostasis. Bone mass will be assessed several different ways in part to address the biethnic nature of the population. Genotyping will be done for variation at genetic loci intimately involved in bone metabolism and calcium homeostasis. Biomarkers include serum osteocalcin and ionized calcium, intact parathyroid hormone, and urinary N-telopeptides and calcium. The main hypotheses involve the effects of dietary calcium, vitamin D, physical activity, and pubertal maturation on BMD, and their interaction with each other as well as with genotypes. Attention will be directed to understanding the extent to which selected environmental and genetic factors influence the well-recognized black-white differences in BMD. The
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relative importance of the timing of "exposure" to these environmental factors will be assessed by analyzing data in biologically meaningful time periods defined by pubertal milestones. This study will provide a comprehensive evaluation of the role of an exhaustive set of environmental measures, genotypes, and biomarkers of bone/calcium homeostasis in determining BMD as well as in determining the racial differences in BMD. Thus, by capitalizing on the efficiency and cost- saving afforded by the two ongoing studies at Cincinnati Children's Hospital, our study can "telescope" the 11 years of follow-up to uncover important childhood antecedents of peak bone mass in black and white women. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: ANTHRACYCLINE CARDIOTOXICITY AND CALSEQUESTRIN FUNCTION Principal Investigator & Institution: Shadle, Susan E.; Chemistry; Boise State University 1910 University Dr Boise, ID 83725 Timing: Fiscal Year 2002; Project Start 01-FEB-2002; Project End 31-JAN-2004 Summary: (provided by applicant): Recent work has shown pre-incubation with nanomolar concentrations of anthracyclines inhibits caffeine- 'induced calcium release from canine cardiac sarcoplasmic reticulum (SR) vesicles (Olson, et al., Tox. Appl. Pharm., 2000, 169, 168-176) This inhibition is important to understand because it may contribute to the clinical cardiotoxicity of anthracyclines. The inhibitory effects are hypothesized to be a result of a direct interaction between anthracyclines and the SR calcium binding protein, calsequestrin, which results in a drug-dependent disruption of proper calsequestrin function. This proposal is designed to probe the mechanism of inhibition, with particular attention to the roles of calsequestrin and the ryanodine receptor calcium release channel. Comparison of the anthracyclines daunorubicin, daunorubicinol and 5-iminodaunorubicin, which vary in their ability to inhibit calcium release, will allow the importance of various structural moieties for inhibition to be explored. The proposed research consists of four specific aims: 1) to study calciumdependent conformational changes in calsequestrin in the presence of anthracyclines using fluorescence spectroscopy; 2) to investigate whether anthracyclines alter the aggregation behavior of calsequestrin using flow-field flow fractionation and multiangle light scattering detection (flow-FFF/MALS); 3) to probe the ability of anthracyclines to inhibit calcium binding to calsequestrin using Ca-45 competitive binding assays; 4) to determine the conformation of the cardiac ryanodine receptor (RyR2) in isolated SR vesicles after preincubation with anthracyclines; ryanodine binding assays will be used. The proposed experiments will determine the ability of anthracyclines to interfere with normal calsequestrin function and will define the contribution of RyR2 channel gating to inhibition of SR calcium release. The results will provide insight into the details of molecular mechanisms involved in disruption of proper cardiac calcium handling by anthracychnes and will thus inform the design of improved anthracyclines with decreased cardiotoxicity. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: AORTIC CALCIUM: EPIDEMIOLOGY AND PROGRESSION Principal Investigator & Institution: Criqui, Michael H.; Professor; Family and Preventive Medicine; University of California San Diego 9500 Gilman Dr, Dept. 0934 La Jolla, CA 92093 Timing: Fiscal Year 2003; Project Start 10-MAR-2003; Project End 28-FEB-2007
20 Calcium
Summary: (provided by applicant): Aortic calcium, measured by computed tomography, occurs earlier in life than other subclinical (that is, asymptomatic) markers of cardiovascular disease (CVD), shows a wide range, and is common in women. The large size of the aorta and relative lack of image artifact from motion make it ideal for radiographic quantitative imaging. This application is coordinated with the Multi-Ethnic Study of Atherosclerosis (MESA), a large prospective epidemiologic study investigating multiple subclinical CVD measures and CVD risk factors. Subclinical measures in MESA include coronary calcium, carotid ultrasound, cardiac magnetic resonance imaging, and the ankle/brachial blood pressure index. The extensive CVD risk factor measurements include both traditional risk factors and newer measures such as inflammatory and genetic markers. The primary long-term objective of this application is to determine the epidemiology of aortic calcium. 2000 randomly selected (from 6500 total) MESA participants will be studied. Questions to be addressed include predictors of aortic calcium progression; associations of aortic calcium and aortic calcium progression with other subclinical CVD measures, CVD risk factors, and demographics; and the prognostic significance of aortic calcium. The project has three primary specific aims: 1) to predict the cross-sectional aortic calcium burden as a function of other subclinical CVD measures and CVD risk factors; 2) to predict aortic calcium progression as a function of other subclinical CVD measures and CVD risk factors, and 3) to predict aortic calcium progression as a function of progression of selected subclinical CVD measures and CVD risk factors. The two secondary specific aims are 1) to contrast the results of the three primary specific aims for men vs. women, and for four major ethnic groups (White, Hispanic, Black, and Asian); and 2) to provide a database for future evaluation of whether aortic calcium and/or aortic calcium progression independently predict subsequent myocardial infarction, stroke, and other CVD events. This application potentially has major significance for cardiovascular health. "Total body scanning" including measurement of aortic calcium, has become a popular diagnostic tool. This application, in conjunction with the extensive subclinical and risk factor measurements available in MESA, will uniquely allow determination of the independent and incremental importance of measuring aortic calcium in CVD risk assessment. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: ASSOCIATION BETWEEN KCA AND VOLTAGE-GATED CA2+ CHANNELS Principal Investigator & Institution: Cibulsky, Susan M.; Neuroscience; University of Pennsylvania 3451 Walnut Street Philadelphia, PA 19104 Timing: Fiscal Year 2002; Project Start 01-APR-2002 Summary: (provided by applicant): Calcium-activated potassium and voltage-gated calcium channels participate in an intimate interplay in excitable cells, by which each kind of channel regulates the function of the other. The calcium that enters a cell through voltage-gated calcium channels activates nearby calcium-activated potassium channels that in turn hyperpolarize the cell arid cause calcium channels to shut. This functional coupling plays important roles in determining electrical excitability and influencing signaling events in neurons and muscle cells. In order for the channels to interact functionally, they must be localized close together. Furthermore, in a cell expressing multiple types of each channel, functional coupling is often channel type specific. The work proposed here is designed to determine the mechanisms by which functional coupling is achieved, including the molecular components necessary for channel association and the determinants of channel type specificity. The results will
Studies 21
contribute to our understanding of the physiology of processes such as the regulation of neurotransmitter release and the determination of smooth muscle tone. Such information could lead to useful strategies for the treatment and/or prevention of neurological and vascular diseases of the central nervous system. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: BCL 2 FUNCTION IN ENDOPLASMIC RETICULUM Principal Investigator & Institution: Distelhorst, Clark W.; Professor; Medicine; Case Western Reserve University 10900 Euclid Ave Cleveland, OH 44106 Timing: Fiscal Year 2001; Project Start 03-FEB-2000; Project End 31-JAN-2005 Summary: (Adapted from the investigator's abstract) The Bcl-2 protein family regulates cell death induction by a wide range of apoptotic signals and thereby plays an important role in the pathogenesis of cancer. Although the antiapoptotic proteins Bcl-2 and Bcl-xL are localized to both mitochondria and the endoplasmic reticulum (ER) recent studies have focused almost exclusively on their role in mitochondria. Therefore, the purpose of this proposal is to investigate how Bcl-2/Bxl-xL function in the ER. Based on the central role the ER plays in intracellular calcium homeostasis and signaling, this proposal will test the overall hypothesis that Bcl-2/Bcl-xL work either as ion channels or regulators of ion channels to preserve calcium homeostasis within the ER lumen, thereby preventing organelle dysfunction that triggers apoptosis. Aim 1 will investigate the hypothesis that the transmembrane alpha 5, 6 helices of Bcl-2/Bcl-xL function as a calcium sensor that regulates the ion conductivity of Bcl-2/Bcl-xL in response to changes in calcium concentration within the ER lumen, thereby forming a feedback loop that maintains calcium homeostasis in the ER. Aim 2 will investigate the hypothesis that the BH4 domain of the Bcl-2/Bcl-xL regulates the ion conductivity of Bcl-2/Bcl-xL in response to changes in intraluminal calcium concentration. Aims 1 & 2 will employ planar lipid bilayer techniques to measure ion channel activity in ER-targeted fluorescent proteins, cameleons, to monitor effects of Bcl-2/Bcl-xL on intraluminal calcium concentration on a single cell basis. Aim 3 will investigate the hypothesis that Bcl-2/Bcl-xL regulate calcium efflux through the inositol 1.4.5-triphosphate receptor, a calcium channel located in the ER membrane. Aim 4 will test the hypothesis that apoptotic signals disrupt calcium-dependent ER function, as measured by the calciumdependent processing of the lysosomal aspartic protease cathepsin D, and that Bcl2/Bcl-xL preserve calcium-dependent protein processing in the ER, thereby inhibiting organelle dysfunction and preserving cell viability. The latter studies will employ both pulse-chase labeling techniques and microscopic imaging of green fluorescent proteincathepsin D fusion proteins to determine effects of apoptotic signals and Bcl-2/Bcl-xL on protein processing within the ER. Collectively, the aims of this proposal investigate novel concepts regarding the mechanism of Bcl-2/Bcl-xL function at the level of the ER. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: BRAIN INJURY AND INTERCELLUAR CALCIUM WAVES Principal Investigator & Institution: Floyd, Candace L.; Psychiatry; University of California Davis Sponsored Programs, 118 Everson Hall Davis, CA 95616 Timing: Fiscal Year 2002; Project Start 01-JUN-2002 Summary: (provided by applicant): The goal of the current proposal is to evaluate the hypothesis that traumatic brain injury alters intercellular communication between astrocytes, a potentially crucial and often overlooked aspect of brain processing. Astrocytes were once viewed as passive cells, but are now characterized as active
22 Calcium
contributors to signal processing in the brain. Yet, the effect of traumatic injury on calcium-mediated astrocyte intercellular signaling has not been evaluated. Therefore, the proposal evaluates the hypothesis that traumatic injury alters intercellular calcium signaling between astrocytes in the glial syncytium using the following specific aims: Specific Aim 1: Injury of cortical astrocytes alters origination and propagation of intercellular calcium waves. We will compare the velocity and distance of intercellular calcium waves in injured astrocytes to that of uninjured astrocytes. (Method: Use quantitative fluorescent microscopy to evaluate intercellular calcium waves). Specific Aim 2: Injury alters gap junction coupling. We will compare fluorescent dye transfer through gap junctions, expression of Connexin 43 protein, and phosphorylation of Connexin 43 protein between uninjured and injured astrocytes. (Method: Use lucifer yellow transfer to measure coupling of gap junctions; use Western Blots to measure expression and phosphorylation of Connexin 43 protein). Specific Aim 3: Intercellular calcium signaling will be maintained in astrocytes by pharmacological manipulation of IP3-mediated intracellular calcium signaling. (Method: Use agonists and antagonists of elements in the signaling pathway to determine if alterations in intercellular calcium waves can be attenuated). These proposed experiments evaluate, for the first time, the effect of traumatic brain injury on the intricate and extensive calcium signaling network among astrocytes. Aim #1 directly examines the effects of mechanical injury on intercellular calcium signaling in astrocytes. Aim #2 examines the potential mechanisms (i.e. gap junctions) involved in injury-induced alterations in intercellular calcium signaling. Aim #3 examines potential therapeutic interventions which could restore intercellular signaling. The results of the proposed experiments may provide not only new insights into the pathophysiology of TBI, but also potentially lead to the development of novel therapeutic approaches for the treatment of the traumatically injured brain. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: BUILDING BETTER BONES IN CHILDREN Principal Investigator & Institution: Zemel, Babette S.; Children's Hospital of Philadelphia 34Th St and Civic Ctr Blvd Philadelphia, PA 19104 Timing: Fiscal Year 2001; Project Start 01-JUN-1999; Project End 31-MAY-2004 Summary: Increased calcium intake has proven effective in increasing bone mineral density in children, but the effect disappears when calcium supplements are discontinued. Increased dietary calcium from daily and other food sources may have an even greater impact on bone density than that achieved by calcium supplements, but achieving sustained increased calcium from food sources has not been demonstrated. In addition, the effects of baseline characteristics of calcium intake and bone density and puberty status may influence the response to intervention. This study will develop and implement a Behavioral Modification-Nutrition Education (BM-NE) Intervention Program aimed at increasing dietary calcium Male and female subjects (n=154), ages 710 years (Tanner stage I or II), will be randomly assigned to participate in an intensive BM-NE intervention group to increase intake to 1500 mg/d or a group that will receive usual care (UC) as counseling on bone health. The BM-NE Program will consist of five separate group sessions for parents and children over a five to six week period, and use individualized plans to increase calcium intake. Participants will be recruited into two groups: a group of healthy children (i.e.,no known chronic disease or previous oral steroid exposure) with no known risk factors, and a group of healthy children with potential risk factors for low bone density (previous fracture from usual childhood activities, daily refusal, or lactose intolerance, family history of osteoporosis). These two
Studies 23
groups will be equally represented in their assignment to BM-NE and UC groups. This latter strategy will be used to determine whether the presence of risk factors influences participant compliance with the programs. We hypothesize that (a) at the end of 36 months the BM-NE group will have increased dietary calcium of at least 300 mg/d in the no-risk BM-NE group compared to those receiving UC, (b) baseline calcium intake and presence of risk factors will be associated with changes in calcium intake over the course of the study, and (c) after controlling for important co-variates such as increases in body size and sexual and skeletal maturation, changes in BMD will be associated with calcium intake and physical activity. These findings will help define important behavioral strategies for increasing peak bone mass and prevention of osteoporosis later in life that can be implemented in a short period of time with long-lasting effects. Furthermore, it will help quantify the impact of increased dietary calcium on bone density during growth and development with possible identification of the characteristics of children most in need of and responsive to this treatment. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CA HANDLING & ARRHYTHMIAS ASSOCIATED WITH LQT SYNDROME Principal Investigator & Institution: Laurita, Kenneth; Medicine; Case Western Reserve University 10900 Euclid Ave Cleveland, OH 44106 Timing: Fiscal Year 2002; Project Start 01-FEB-2002; Project End 30-NOV-2005 Summary: (provided by applicant): Long QT syndrome (LQTS) is a genetic disease characterized by prolonged QT interval and a high incidence of sudden cardiac death (SCD). Despite recent advances in our understanding of the genetic and molecular abnormalities underlying LQTS, the mechanistic relationship between such abnormalities and SCD is not well understood. In patients with LQTS, episodes of syncope and SCD are caused by torsade de pointes (TdP), where afterdepolarizations are believed to play a critically important role. Abnormal management (i.e. handling) of intracellular calcium has been implicated as an important mechanism of afterdepolarizations, including afterdepolarizations that are enhanced by Calcium/calmodulin-dependent protein kinase II (CaM kinase). We hypothesize that heterogeneities of calcium handling are present normally and are enhanced by electrophysiological changes that occur in LQTS. As a result, regional "hot spots" of abnormal calcium handling develop that are prone to the formation of early afterdepolarizations (EADs) and delayed afterdepolarizations (DADs). It is further hypothesized that the location and timing of afterdepolarizations and their interaction with transmural repolarization gradients can critically influence the initiation of TdP. The specific aims of his proposal are to: 1) Determine the regional differences (i.e. heterogeneities) of intracellular calcium handling and action potential duration across the ventricular transmural wall that occur normally and in models of LQTS (i.e. LOT1, LQT2, LQT3). 2): Determine the cellular/molecular mechanisms of calcium handling heterogeneities that occur normally by measuring the level of calcium regulatory protein expression under control conditions. 3) Determine the cellular/molecular mechanisms of enhanced heterogeneities of (i.e. abnormal) intracellular calcium handling and afterdepolarization in models of acquired LQTS, where calcium release from the sarcoplasmic reticulum and CaM klnase are important mechanisms. 4) Determine the mechanistic relationship between the regional occurrence of EADs, repolarization gradients, and episodes of TdP in all three models of acquired LQTS, with an emphasis on the mechanisms of initiation by pauses in cycle length. New optical mapping techniques developed and validated by the PI to measure transmembrane
24 Calcium
potential and intracellular calcium simultaneously from 256 sites across intact heart preparations will be used. A major advantage of this experimental system is that multiple cellular parameters can be measured during arrhythmia initiation, providing the unique ability to bridge cellular and molecular abnormalities with arrhythmias that are a consequence. The long term objectives of this study are to determine the mechanistic relationship between abnormal intracellular calcium handling and arrhythmias associated with LQTS (i.e. TdP). Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CA KINETICS IN PREADOLESCENT/ADOLESCENT GIRLS W /CROHN'S Principal Investigator & Institution: Caballero, Benjamin H.; Director and Professor; Johns Hopkins University 3400 N Charles St Baltimore, MD 21218 Timing: Fiscal Year 2001 Summary: Background & Aims: Children with Crohn's disease are at risk for osteoporosis because of undernutrition, prolonged corticosteroid therapy, inflammatory process itself, inadequate calcium intake or absorption and vitamin D deficiency. The aim of this study is to address the effects of Crohn's disease and cumulative corticosteroid dose on calcium metabolism, efficiency of calcium absorption, calcitropic hormone levels and bone mineral density in preadolescent and adolescent girls. Methods: Seven female subjects (12 to 1 yrs. of age) have been enrolled. Patients are undergoing anthropometric assessment, pubertal staging, bone age radiography, and BMD assessment by dual energy 2-ray absorptiometry (DEXA) of the lumbar spine and total body. Clinical disease activity of CD is assessing by te Pediartic Crohn's Disease Activity Index (PCDAI). The cumulative corticosteroid dose (i grams) is calculating. Laboratory evaluations include serum levels of albumin, prealbumin, ionized Ca, phosphated, alkaline phosphatase, 25-hydroxyvitamin D [25-OHD], 1,25dihydroxyvitamin D [1,25-(OH)xD], calcitonin, parathyroid hormone (PTH), estradiol, osteocalcin and interleukin-6, and urinary levels of N-telopeptide. We are measuring calcium absorption and urinary calcium excretion, using a dual-tracer, stable-isotope technique and thermal ionization mass spectrometry. Results: We have not completed this study yet. We hypothesize that the low bone mineral density reported in girls with Crohn's disease is related to disturbances in calcium metabolism. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: CA REGULATION IN INTACT VENTRICULAR MYOCYTES Principal Investigator & Institution: Bers, Donald M.; Professor and Chairman; Physiology; Loyola University Medical Center Lewis Towers, 13Th Fl Chicago, IL 60611 Timing: Fiscal Year 2001; Project Start 15-JAN-2000; Project End 31-DEC-2004 Summary: (adapted from the applicant's description): This proposal studies calcium regulation in intact ventricular cells with a focus on sarcoplasmic reticulum (SR) calcium release and the sodium/calcium (Na/Ca) exchanger. The proposal has three specific aims. Specific aim 1 determines the amount of calcium released during a calcium spark and the number of SR calcium release channels (ryanodine receptors, RYRs) contributing to a calcium spark (i.e. cluster size). Specific aim 2 measures the flux of SR calcium release during an action potential. Specific aim 3 explores the contribution of Na/Ca exchange current to the action potential and the trigger for SR calcium release. The majority of these studies will be performed on single cardiac ventricular cells isolated from adult rabbits, rats, mice and ferrets. Membrane currents (both whole cell
Studies 25
and single channel) will be measured with the patch clamp technique. Whole-cell calcium transients will be measured with indo-1 by standard techniques in epifluorescence microscopy. Local calcium transients (calcium sparks and sarcolemmal calcium transients) will be visualized as fluo-3 fluorescence with laser scanning confocal microscopy. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CA/CALMODULIN ACTIVATED PHOSPHOLIPASE A2 AND NA K ATPASE Principal Investigator & Institution: Yingst, Douglas R.; Wayne State University 656 W. Kirby Detroit, MI 48202 Timing: Fiscal Year 2002 Summary: (Applicant's abstract): A new form of phospholipase A2 (PLA2) associated with the cytoskeleton of human red cells inhibits Na,K-ATPase at the concentrations of intracellular free calcium that occur during cell activation. This form of PLA2 may also be present in neurons, where it could mediate the effects of intracellular calcium on Na,K-ATPase. Regulation of Na,K-ATPase in neurons by calcium has been linked to the control of neurotransmitter release and has been implicated in the development of bipolar disorders. The data in red cells suggest a single PLA2 that exists in two forms: phosphorylated and unphosphorylated. When phosphorylated, PLA2 is activated by micromolar (0.2-10 uM) free calcium by the binding of calmodulin (CaM). In the absence of phosphorylation, PLA2 is directly activated my millimolar (0.1-1 mM) calcium. This membrane-associated PLA2 has properties distinct from other PLA2s based on its activation by CaM, its sensitivity to both micromolar and millimolar calcium, and its response to inhibitors. The specific aims are to determine (1) the molecular basis for the Ca/CaM stimulated PLA2 and (2) how the Ca/CaM PLA2 affects the Na,K-ATPase. The results will define a new Ca/CaM-PLA2 capable of participating in cell signaling and of regulating Na,K-ATPase in response to changes intracellular free calcium. The studies will be carried out in red cells due to their advantages as a simple model system, but should be applicable to other cells, including neurons, where Na,K-ATPase is also inhibited by changes in intracellular free calcium. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: CA/NA EXCHANGERS--HORMONE & ETHANOL ACTION IN HEPATOCYTE Principal Investigator & Institution: Bernstein, Jaime; University of Puerto Rico Med Sciences Medical Sciences Campus San Juan, PR 00936 Timing: Fiscal Year 2001 Summary: The intention of the present proposal is to describe in isolated rat liver cells the operation of the Na/Ca exchanger and to determine whether the combined operation of this exchanger and that of the Na/H Exchanger System can contribute to the increased [Ca]i which is observed after the action of substances which promote calcium entry into the hepatocyte. Normally, the Na/Ca anti-porter seems to operate in the liver by exchanger intracellular sodium for extracellular calcium. It has been estimated that in rat liver slices about 60% of the resting calcium flux across the cell membrane would occur via the exchanger. This fact would make the Na/Ca anti-porter an important target for effectors which affect [Ca]i such as the calcium modeling hormones and hematotoxins like ethanol. In isolated hepatocytes liver slices and the perfused liver, these hormones and ethanol are known to increase [Ca]i and calcium
26 Calcium
uptake. However, the role of the Na/Ca Exchanger on the calcium entry across the cell membrane induced by these dependent calcium influx into the cell, and that in isolated rat hepatocytes the cytosolic calcium concentration markedly rises when intracellular sodium is increased by the use of ouabain, a well known inhibitor of the Sodium Pump (see preliminary data). It is also known that increases in intracellular calcium may constitute an important link in the development of cell pathologies and cell death. Using the isolated hepatocyte, it is specifically proposed to kinetically describe, in terms of Km and Vmax, the Na/Ca as well as to determine the kinetic characteristics of the exchanger function as a calcium extruder by studying the [Ca]i-dependent 22Na uptake at different [Ca]i and [Na]o. The stoichiometry of the anti-porter will be determined by analyzing the dependence of sodium-efflux on [Ca]o. In addition, the actions of ethanol on the activity and kinetics of the Na/Ca Exchanger will be analyzed and the effect of the calcium-mobilizing hormones, phenylephrine, vasopressin, angiotensin II, and glucogen on the activity and kinetic parameters of the Na/Ca anti-porter, will be determined by analyzing the dependence of sodium-efflux on [Ca]o. In addition, the actions of ethanol on the activity and kinetics of the Na/Ca Exchanger will be analyzed and the effect of the calcium-mobilizing hormones, phenylephrine, vasopressin, the effects of ethanol on the calcium-mobilizing activity of these hormones through the Na/Ca Exchanger will be studying 45Ca uptake and [Ca]i increases in cells at normal or at low pHi [proportionate- loaded) in the presence or absence of specific inhibitors of the Na/Ca, or Na/H exchangers, respectively. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: TRANSMISSION
CA++
&
PRESYNAPTIC
MODULATION--SYNAPTIC
Principal Investigator & Institution: Saggau, Peter; Neuroscience; Baylor College of Medicine 1 Baylor Plaza Houston, TX 77030 Timing: Fiscal Year 2001; Project Start 01-AUG-1995; Project End 31-MAR-2003 Summary: There is compelling evidence for the involvement of presynaptic inhibition in controlling normal synaptic transmission and preventing excessive neurotransmitter release at mammalian central synapses. The presynaptic site is an effective target for modulation of synaptic transmission and presynaptic voltage-dependent calcium channels play a significant role in controlling transmitter release. To address our longterm objective of understanding the basic presynaptic mechanisms underlying modulation of synaptic transmission in mammalian central synapses, we propose to investigate the direct and indirect role of presynaptic calcium during inhibition of synaptic transmission at hippocampal CA3/CA1, MF/CA3 and PP/GC excitatory synapses. This in vitro study will employ hippocampal brain slices and optical imaging techniques. We will selectively load presynaptic terminals with ion-sensitive indicators to investigate resting levels and transients of presynaptic calcium and presynaptic potassium. In addition, we will use voltage- sensitive dyes to measure presynaptic action potentials. Specific blockers will be utilized to identify and quantify the types of presynaptic voltage-dependent calcium channels involved in synaptic transmission. We will study presynaptic calcium during the application of neuromodulators with presumed inhibitory presynaptic action and identify the types of calcium channels involved and their quantitative inhibition. Through the use of advanced optical techniques, the proposed studies will provide new and important insight into the presynaptic modulation of mammalian synaptic transmission. This insight will contribute to the understanding of normal and pathological synaptic transmission. Excessive release of excitatory neurotransmitter has been observed during episodes of
Studies 27
epilepsy and after brain damage. Control of this release by presynaptically acting endogenous neuromodulators could be the basis of future therapeutic interventions. We will address the following specific aims: 1) To discriminate the types of presynaptic VDCCs at principal hippocampal excitatory synapses. 2) To investigate resting levels and influx of presynaptic Ca2+ during presynaptic inhibition of evoked synaptic transmission. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CA++ HOMEOSTASIS IN ISOLATED HYPERTROPHIED HEART CELLS Principal Investigator & Institution: Houser, Steven R.; Professor; Physiology; Temple University 406 Usb, 083-45 Philadelphia, PA 19122 Timing: Fiscal Year 2001; Project Start 01-JUN-1985; Project End 30-JUN-2005 Summary: (adapted from the applicant's description): The overall hypothesis of this proposal is that alterations in myocyte calcium handling are centrally involved in the abnormal contractility of failing myocytes and contribute to the progressive decline of cardiac function in CHF. Two specific hypotheses will be tested. The first hypothesis is that the "signature" defects in calcium handling in CHF are due to an increase in the relative transport rate and capacity of the Na/Ca exchanger (NCX) with respect to the sarcoplasmic reticulum (SR) rather than an increase or decrease in the absolute rate or capacity of either transport mechanism. This will be tested by (1) measuring the calcium transport capacities of the SR and NCX in single myocytes from normal, hypertrophied and failing feline hearts and from non-failing, failing and left ventricular assisted device-supported (LVAD) human hearts, and (2) manipulating the calcium transport capacity of the SR and/or NCX in feline and human myocytes transfected with SERCA2a, native or mutant phospholamban (PLB), and/or NCX. The second hypothesis is that calcium influx via reverse-mode NCX makes a greater than normal contribution to excitation-contraction (EC) coupling and SR calcium loading because NCX density increases and the density of the L-type calcium channels and t-tubules decreases in CHF. This will be tested by (1) determining if calcium influx via reverse mode NCX modulates calcium-induced calcium release (CICR), (2) determining if calcium influx via reverse mode NCX replaces calcium entry via L-type calcium channels as the primary source of trigger calcium in CICR, and (3) measuring the amount of L-type calcium current, NCX current and SR calcium loading that is lost when T-tubules are disrupted by osmotic shock from the surface membranes of non-failing and failing feline and human myocytes. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: CA2+ HYPERPARATHYROIDISM
SENSING
RECEPTOR
EXPRESSION
IN
Principal Investigator & Institution: Brown, Alex J.; Medicine; Washington University Lindell and Skinker Blvd St. Louis, MO 63130 Timing: Fiscal Year 2001; Project Start 01-FEB-2000; Project End 31-JAN-2003 Summary: Parathyroid glands (PTGs) express a calcium-sensing receptor (CaR) that detects the concentration of extracellular calcium and signals the glands to secrete the proper amount of parathyroid hormone (PTH) to maintain normal calcium levels. The calcium-PTH relationship is sigmoidal and can be defined by four parameters: maximum PTH, minimum PTH, the midpoint of the curve (set-point), and the slope at the midpoint. The CaR levels are decreased in the hyperplastic PTGs of patients with
28 Calcium
secondary HPT (2oHPT) due to chronic renal failure (CRF), but the factors responsible are not known. The impact of this down-regulation on calcium sensitivity remains controversial. In vitro studies indicate decreased calcium sensitivity (elevated set-point) in parathyroid cells from CRF patients, but in vivo assessment of the set-point in CRF patients have yielded mixed results. Furthermore, in patients it is not possible to relate CaR levels to the four parameters of the calcium-PTH relationship. We have now observed a similar reduced CaR expression in hyperplastic PTGs of CRF rats. This model can be used to determine the role of decreased CaR in PTG hyperplasia and the abnormal calcium-PTH relationship, and to identify the factors that regulate the CaR. Definitive identification of the factors that directly control CaR levels requires an in vitro model with stable expression of the CaR. Traditional monolayer cultures do not respond to calcium. We have developed a unique parathyroid cell culture system that preserves the normal cellular architecture, allowing stable CaR expression and calcium response for several weeks. These cultures, which we refer to as pseudoglands or psGs, provide the best available model for studying the regulation off parathyroid cell CaR expression and provide a unique in vitro model t9o examine the effects in CaR levels on the calcium-PTH relationship. This grant presents an experimental approach that utilizes these in vivo and in vitro models to examine both the causes and effects of the downregulation of the CaR in renal failure. The specific aims are: 1. To define the relationships, temporal and spatial, between parathyroid gland hyperplasia and downregulation of the CaR in vivo. 2. To examine the effects of established therapies for uremic secondary hyperparathyroidism (phosphate restriction and vitamin D compounds) on CaR expression in uremic rats. 3. To determine the direct effects of potential regulators of CaR expression in vitro using the psG model. 4. To determine the effects of down-regulation of CaR on the calcium- PTH relationship and PTH expression in vivo and in vitro. These studies will provide new information about the effect of decreased CaR in CRF on the calcium-PTH relationship, define the factors responsible for the decrease, and test therapeutic strategies to correct the abnormality. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CA2+/CAMP SIGNALING IN DEVELOPMENT OF FOREBRAIN CIRCUITS Principal Investigator & Institution: Pham, Tony A.; Psychiatry and Behavioral Scis; University of Washington Seattle, WA 98195 Timing: Fiscal Year 2001; Project Start 01-SEP-2000; Project End 31-AUG-2005 Summary: The precise synaptic connectivity of neurons in the mammalian brain is the underlying cellular substrate that governs perception and behavior. The major psychiatric illnesses, such as schizophrenia and depression, may be caused by abnormalities in the connectivity of forebrain neurons. However, the molecular mechanisms that instruct the precise development of forebrain connectivity are largely unknown. This application examines the hypothesis that cellular processes mediated by calcium and cyclic AMP are critical for the refinement and maturation of neural circuits in the thalamus and neocortex, key forebrain structures that mediate conscious perception. Using the visual system as the experimental model, the specific aims of the research are: (1) to examine the mechanisms that drive calcium/cAMP-regulated gene expression during the development of neocortical and thalamic circuitry, (2) to examine the processes that lead to the down-regulation of neocortical calcium/cAMP-regulated gene expression with maturation, (3) to determine the impact of disruption of calcium/cAMP signaling on neocortical plasticity and thalamic connectivity, (4) to construct a transgenic mouse line that has a thalamus specific disruption of the
Studies 29
calcium/cAMP-regulated transcription factor CREB. These experiments will help elucidate how calcium/cAMP signals translate sensory experience into patterns of gene expression, and determine the impact of disruption of calcium/cAMP signaling on neural connectivity. The investigator is a psychiatrist whose long-term interest is to understand how neuronal connectivity develops early in life and how it is modified by experience, with the hope that this knowledge will provide insight into the cellular and molecular abnormalities that underlie behavioral and perceptual disorders. He will have an appointment as assistant professor at the University of Washington School of Medicine. His transition towards research independence will be undertaken with mentoring from Dr. Daniel Storm and Dr. Mark Hamlin of the University of Washington. Also, several collaborations are planned, namely with Dr. Alcino Silva (UCLA), Dr. Barbara Gordon-Lickey (University of Oregon), and Dr. John Neumaier (University of Washington), that will result in a significant broadening of research experience. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CALCIUM ABSORPTION IN CACO-2 CELLS: MOLECULAR MECHANISM Principal Investigator & Institution: Fleet, James C.; Associate Professor; Foods and Nutrition; Purdue University West Lafayette West Lafayette, IN 479072040 Timing: Fiscal Year 2003; Project Start 15-SEP-1997; Project End 29-SEP-2004 Summary: (provided by applicant): 1,25 dihydroxyvitamin D (1,25(OH)2 D)-mediated intestinal calcium absorption is an important adaptive mechanism utilized by the body when calcium status is low. Low calcium absorption efficiency is a risk factor for osteoporosis and intestinal resistance to 1,25(OH)2 D action develops with aging and after menopause. Our long-term objective is to clarify the mechanisms used by 1,25(OH)2 D to promote calcium absorption and to determine how dysfunction in the regulatory control of intestinal calcium absorption during aging or due to menopause leads to reduced absorption efficiency and, ultimately, low bone density. Recent research demonstrates that 1,25(OH)2 D rapidly activates scr kinase, protein kinase C (PKC), and MAP kinases and that inhibition of these kinases blunts 1,25(OH)2 Dmediated activation of the CYP24 gene. The goal of the proposed research is to determine how the adaptive increase in intestinal calcium absorption due to 1,25(OH)2 D-dependent; vitamin D receptor (nVDR)-mediated gene activation is influenced by the basal or induced activity of these kinases. The specific aims of this project are to: (1) Identify the nVDR-mediated genomic pathways controlling intestinal calcium absorption that are modulated by 1,25(OH)2-induced activation of src kinase, PKC, and MAPK, (2) Establish the mechanisms by which 1,25(OH)2 D-induced kinase activation promotes 1,25(OH)2 D-mediated gene expression and intestinal calcium absorption, and (3) Identify new, 1,25(OH)2 D-regulated transcripts in differentiated enterocytes and determine whether their regulation is modulated by activation of protein kinases. We will accomplish these aims by studying the effect of 1,25(OH)2 D in a well-characterized cell culture model (Caco-2 cells) and in the small intestine of mice. Biological actions of 1,25(OH)2 D will be studied in the presence of activators and inhibitors of protein kinases (pharmacologic inhibitors, dominant negative kinases) and the rapid actions of vitamin D (vitamin D analogs), nVDR action and function will be studied with cellular imaging, RT-PCR, DNA microarrays, Western blotting and chromatin immunoprecipitation (CHIP) assays. Elucidating the mechanism for how signal transduction pathways influence the genomic actions of 1,25(OH)2 D will permit us to design prevention and pharmacologic intervention strategies to enhance intestinal
30 Calcium
calcium absorption, especially when vitamin D resistance associated with aging or estrogen deficiency is present. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CALCIUM AND CYTOTOXIC T LYMPHOCYTES Principal Investigator & Institution: Zweifach, Adam; Assistant Professor; Physiology; University of Colorado Hlth Sciences Ctr P.O. Box 6508, Grants and Contracts Aurora, CO 800450508 Timing: Fiscal Year 2003; Project Start 01-APR-2003; Project End 31-MAR-2008 Summary: (provided by applicant): The long-term goal of this proposal is to understand the role of intracellular calcium signals in the function of cytotoxic T lymphocytes (CTLs). These critical effectors of the immune system kill virus-infected cells and cancer cells and play a major role in the immune response to transplanted tissues; inappropriate killing can cause autoimmune diseases such as Lupus, certain forms of diabetes, and arthritis. Understanding CTL function is therefore important for preventing and treating naturally occurring viral diseases such as AIDS and influenza, and viral diseases such as smallpox used as biological weapons. It is also important for understanding and treating cancers and autoimmune diseases. Finally, the ability to suppress CTL function is vital for successful organ transplantation. One of the main mechanisms CTLs use to kill is the perforin pathway, which involves the exocytotic release of pore-forming peptides and hydrolytic enzymes contained in specialized lytic granules into an area of close apposition formed with the target. Granule exocytosis is known absolutely to require increased intracellular calcium caused by influx across the plasma membrane. However, the specific role(s) of calcium in granule exocytosis are unknown, the number of calcium-dependent steps is unclear, and molecules that confer calcium-dependence have not been identified. The specific aims of this proposal will use a battery of techniques, including novel fluorescence imaging methodologies we have developed, to: 1) determine whether bulk cytosolic calcium increases are sufficient to support granule exocytosis, or whether higher-than-cytosolic calcium increases in microdomains are required. 2) Investigate the calcium dependence of granule reorientation and of reorientation-independent exocytosis. 3) Determine whether immunological synapse formation is calcium dependent, and acts as a slow step in granule reorientation. 4) Investigate the role of the calcium-dependent phosphatase calcineurin in granule exocytosis. These studies will significantly further our understanding of the role of calcium influx in lytic granule exocytosis. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: CALCIUM AND ENDOCYTOTIC MEMBRANE RETRIEVAL Principal Investigator & Institution: Lambert, Nevin A.; Inst/Molecular Med & Genetics; Medical College of Georgia 1120 15Th St Augusta, GA 30912 Timing: Fiscal Year 2003; Project Start 24-MAY-2000; Project End 29-FEB-2004 Summary: (Applicant's Abstract) Endocytotic membrane retrieval compensates for excess surface membrane following exocytosis but the mechanism of exocytosisendocytosis coupling is not known. We have shown in sea urchin eggs that membrane retrieval requires calcium influx through agatoxin sensitive channels. Thus, in addition to their role in signaling for exocytosis at synapses, P-type calcium channels are required for endocytotic membrane retrieval in eggs. We hypothesize that exocytosis regulates P-type calcium channel gating to coordinate exocytosis and endocytotic membrane retrieval. We will use microscopy, electrophysiology, as well as cell and
Studies 31
molecular biological techniques to determine how exocytotic activity regulates calcium influx through P-type channels in sea urchin eggs, a model system for understanding calcium-triggered exocytosis and endocytosis. Specifically we will determine how exocytotic activity influences membrane depolarization and the cellular distribution of P-type calcium channels. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CALCIUM AND THIAZIDE PHENOTYPES AND GENETIC HYPERTENSION Principal Investigator & Institution: Fanestil, Darrell D.; University of California San Diego 9500 Gilman Dr, Dept. 0934 La Jolla, CA 92093 Timing: Fiscal Year 2001 Summary: Quantitative Trait Loci (QTLs) were found in a population of male F2 animals derived from an SHR(LJ)x WKY(LJ) cross for a number of phenotypic traits regulated by renal function. One QTL (LOD=4.45) is located on chromosome #7 and is associated with a lower plasma ionized calcium concentration in SHR. A second QTL (LOD greater than 3.40), located on chromosome #2, is associated with increased urinary excretion of calcium in the SHR. This QTL is the only one related to renal excretion that is present when the excretion data are normalized both per unit of excreted creatinine and as amount excreted per body weight. We consider these two calcium-related traits as traits most likely to yield information in studies of SHR-WKY congenics. Therefore, congenic strains of animals containing the relevant chromosome segments from SHR chromosomes #2 or #7 will be placed on a WKY background and the reciprocal congenics developed. These congenic strains will be raised after weaning on one of two diets, a low (0.3% Ca) or high (1.9% Ca) calcium diet traditionally used in this program. All animals will be studied for blood pressure, plasma ionized Ca&Mg, Na, K & Cl; for urinary excretion of creatinine and of Ca, Na, K and Cl; and for renal thiazide receptor density. These QTLs were identified by genomic scanning of a limited number of the male f2 animals on whom these renal traits have been determined. We will continue collaborative efforts with Core C, Molecular Genetics, in order to identify additional QTLs predicted to be present by the patterns of inheritance we found in this population. In addition, this application requests support for the initiation of genotyping of the DNA from a large population of female F2 animals on whom these renal traits have already determined. Since the difference between SHR and WKY for several of these traits are greater in females than males, there is enhanced likelihood that QTLs of major interest for these traits will be detected. Completion of these proposed studies will provide a most rigorous test of the postulates (a) that alterations in calcium homeostasis are of importance in the regulations (1) of blood pressure, in both "permissive" and "non- permissive" genetic backgrounds and (2) of the renal thiazide receptor, and (b) that genes modulating renal function can be identified by this genetic paradigm. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: CALCIUM AND VITAMIN D MALNUTRITION IN ELDERLY WOMEN Principal Investigator & Institution: Lappe, Joan; Medicine; Creighton University 2500 California St Omaha, NE 68178 Timing: Fiscal Year 2001; Project Start 30-SEP-1999; Project End 30-JUN-2004 Summary: (Adapted from the Applicant's Abstract): Postmenopausal osteoporosis is a condition of excessive skeletal fragility which results in low impact fractures. The best
32 Calcium
studied measurable determinant of skeletal fragility is low bone mass. One of the easily correctable, non-genetic determinants of bone loss is calcium nutritional deficiency, particularly for older women for whom secondary hyperparathyroidism is considered to be universal. Calcium nutritional deficiency may be due to low dietary calcium as well as low 25(OH)D which promotes calcium absorption. Numerous reports demonstrating a bone sparing effect, and a few reports demonstrating an anti-fracture effect of increasing calcium intake among vitamin D replete women strongly support the hypotheses that increasing calcium intake among vitamin D intake benefits bone health in older women. Small scale intervention studies have shown that older women who consume calcium intakes up to 2400mg/d have a reversal of secondary hyperparathyroidism and experience no adverse effects due to the "high", in terms of the American diet, calcium intake. Despite these data, the behavior of many in the scientific community indicates doubt about the therapeutic value of calcium. The proposed study is designed to test whether calcium supplementation alone (1500mg/d) or calcium 1500mg/d) plus vitamin D (800IU/d) reduces the incidence of fractures, eliminates secondary hyperparathyroidism, and halts bone loss in a population-based sample of women 60+ years of age. An Achilles heel for intervention trials is the problem of subject compliance. This study will use an electronic monitoring system to track the number of time the treatment bottle is opened. This compliance measure will be used, not only to determine the amount of medication taken, but as a method to counsel the study subjects regarding protocol problems. This is a valid method to determine compliance, far superior to the traditional pill counts, and thus a substantial aid to measure the anti-fracture efficacy of calcium alone or calcium plus vitamin D interventions. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CALCIUM CHANNEL CAT 1 IN PROSTATE CANCER PREVENTION Principal Investigator & Institution: Hediger, Matthias A.; Associate Professor of Medicine; Brigham and Women's Hospital 75 Francis Street Boston, MA 02115 Timing: Fiscal Year 2003; Project Start 01-JUN-2003; Project End 31-MAY-2008 Summary: (provided by applicant): Prostate cancer is among the leading causes of cancer-related death of men in Western countries. Strategies for its primary prevention through nutritional modifications have recently become a major focus. There is substantial epidemiologic evidence that an excess of dietary calcium, mainly from dietary sources, increases the risk of prostate cancer. The exact biological basis for this, however, is unknown. One possible explanation is that calcium can reduce the body's level of 1,25-vitamin D3 which has been shown to protect the prostate, but various scientists have questioned the significance of this concept. Understanding the molecular properties of calcium channels, including their regulation by 1,25-vitamin D3, may help explain the role of the nutrient calcium in prostate cancer development. Calcium channels play a important roles in the metabolism of prostate cancer epithelial cells but little is known about their implications in prostate cancer development. We propose to investigate the role of the epithelial calcium entry channel, CaT1 (TRPV6), and other calcium permeable channels in prostate cancer malignancy. Our laboratory has recently demonstrated that CaT1 is upregulated several fold in prostate malignancy, indicating that it might be involved in proliferation and tumor cell growth. To investigate the relationship of dietary calcium, calcium channels and prostate cancer risk, we propose to study the following: (1) The regulation of CaT1 in response to high and low calcium diets in various prostate cancer cell lines, the prostates of control mice and the prostate carcinomas of TRAMP (transgenic adenocarcinoma of mouse prostate) mice. (2) The
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effect of reduced expression of CaT1 on tumor progression by crossing the TRAMP mouse with the CaT1 (+/-) or CaT1 (-/-) mouse. (3) The implication of the level of CaT1 expression in regulating proliferation and apoptosis in prostate cancer cells. (4) The molecular identity, biophysical properties and roles in prostate tumor progression of other putative calcium channel proteins. Since the human body depends on dietary calcium intake to maintain the integrity of the skeleton, dietary calcium restriction would not be appropriate to reduce the risk of prostate cancer. The molecular characterization of CaT1 and related calcium channels, however, may lead to alternative strategies for the treatment of prostate cancer, involving inhibition of calcium influx into prostate cancer cells to impair tumor growth. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CALCIUM TRANSMISSION
CHANNELS
AND
INHIBITORY
SYNAPTIC
Principal Investigator & Institution: Mynlieff, Michelle; Biomedical Sciences; Marquette University P.O. Box 1881 Milwaukee, WI 532011881 Timing: Fiscal Year 2001; Project Start 01-AUG-1997; Project End 31-JUL-2003 Summary: (from applicant's abstract) Modulation of the voltage-dependent calcium channels regulating release by neurotransmitters is likely to be a common mechanism of regulating synaptic plasticity. There is currently no consensus on the identity of the calcium channel controlling neurotransmitter release, thus raising the possibility that different synapses may use different channel types. This project exploits the well characterized circuitry of the hippocampus to investigate the identity of calcium channels controlling release in specific inhibitory synapses, and also the modulation of these channels by neurotransmitter. The inhibitory synapses between the CA1 pyramidal cells and the vertical cells of the stratum oriens/alveus, the basket cells in the stratum pyramidale, and the stellate cells in the stratum laciunosum/moleculare will be studied. The vertical and basket cells mediate both feedforward and recurrent inhibition primarily by activation of GABAA receptors. The stellate mediate feedforward inhibition by activation of GABAB receptors. The first specific aim is to provide thorough characterization of the voltage-dependent calcium channels in the presynaptic cells of each of these synapses, using whole cell voltage-clamp recording in dissociated cells. The data obtained in these experiments will provide a foundation for the subsequent studies in neurotransmitter release and modulation of calcium channels. The second specific aim is to determine which calcium channel type controls release in each of the synapses in question, by application of specific calcium channel antagonists during whole cell voltage clamp recording in the postsynaptic cell in hippocampal slice preparation. The final specific aim is to determine if a decrease in calcium influx can account for the decrease of inhibitory synaptic transmission produced by activation of presynaptic GABAB and mu opioid receptors. The effect of GABA and opiates on calcium currents will be studied using whole cell voltage clamp recording in dissociated vertical, basket and stellate interneurons and the involvement of specific G proteins in this process will be explored. Understanding how neurotransmitter regulate specific synapses by modulation of distinct calcium channels types will provide insight into some of the mechanisms of synaptic plasticity. In addition, these studies will provide a better foundation for pharmaceutical therapies in diseases such as epilepsy and neurodegeneration where the use of neuromodulators is being actively pursued. Moreover, this information will also provide insights into the cellular pathology of various neuronal disorders in which synaptic function is altered. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: CALCIUM CITRATE AND PROPENSITY FOR CRYSTALLIZATION OF CALCIUM SALTS Principal Investigator & Institution: Sakhaee, Khashayar; University of Texas Sw Med Ctr/Dallas Dallas, TX 753909105 Timing: Fiscal Year 2001 Summary: Aims are: 1) to test the hypothesis that the rise in urinary calcium and in the propensity for the crystallization of calcium salts following calcium supplementation is transient, owing to the "intestinal adaptation process". and 2) that calcium citrate confers protection against calcium stone formation due to its persistent citraturic response. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: CALCIUM DEPENDENT GENE EXPRESSION IN MUSCLE Principal Investigator & Institution: Rosenberg, Paul B.; Medicine; Duke University Durham, NC 27706 Timing: Fiscal Year 2003; Project Start 15-DEC-2002; Project End 30-NOV-2006 Summary: (provided by applicant): This proposal describes a four-year training program for an academic career in molecular cardiology. The principal investigator has completed clinical training in internal medicine residency as well as clinical cardiology with additional training in heart failure/transplantation. He has also completed two additional years of post-doctoral work in the laboratory of the sponsor, R. Sanders Williams, M.D. During this time the principal investigator has developed fundamental skills in molecular biology and cellular physiology in order to establish a program focusing on calcium imaging in myocytes. The Mentored Clinical Scientist Development Award is an excellent opportunity to further his scientific development under Dr. Williams. The sponsor is the Dean of Duke Medical School in Durham, NC and an expert in cardiac and skeletal muscle biology. He has successfully mentored numerous molecular cardiologists, post-doctoral fellows and graduate students and junior faculty members over the years. This program will build on the extensive training initiated over the last two years and focus on calcium signaling and transcriptional activation in models of muscle hypertrophy and specialization. Work in the Williams' laboratory has established the link between neural activation of gene expression and the calcium regulated serine-threonine phosphatase, calcineurin. Loss of function and gain of function studies have established the calcineurin pathway as a critical nodal point for excitation transcription coupling. Research in this program will focus on the proximal aspects of the calcineurin pathway in order to understand calcium activation and identify upstream aspects of this pathway in muscle. Specific aims will include: 1) to determine the spatial and temporal aspects of calcium signaling that regulate calcineurin and NFAT signaling in isolated single fibers and cardiomyocytes, 2) to define the calcium signals responsible for NFAT activation in an intact animal, 3) to identify calcium signaling molecules that are differentially expressed among specialized myofiber subtypes and are important in gene regulation. The research proposed will establish a better understanding of calcium signaling and its influence on cardiac and skeletal myopathies. The training environment established in the Division of Cardiology at Duke University has demonstrated a continued commitment to the career development of young faculty, particularly in molecular cardiology. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: CALCIUM IN THE PREVENTION OF NEOPLASTIC POLYPS Principal Investigator & Institution: Baron, John A.; Professor of Medicine; Medicine; Dartmouth College 11 Rope Ferry Rd. #6210 Hanover, NH 03755 Timing: Fiscal Year 2001; Project Start 30-SEP-1988; Project End 31-DEC-2003 Summary: This application, a resubmission for the renewal of "Calcium Prevention of Neoplastic Polyps," is a proposal to collect further information on subjects in our randomized, placebo-controlled trial of calcium supplementation for the prevention of colorectal adenomas. The study began in response to animal and human experimental data and some epidemiological findings suggesting that calcium intake may exert a protective effect on large bowel neoplasia. The trial studied whether supplementation with calcium carbonate (1200 mg calcium per day) prevented the recurrence of colorectal adenomas in 930 patients with a recent history of these tumors. Treatment in the trial ended in December, 1996. Calcium lowered the risk of any recurrent adenoma by 18% (95% CI 0.66-0.98), and reduced the number of adenomas by 25% (95% CI 0.550.94). We now propose to investigate whether the effect of calcium persisted beyond active treatment, or if there was a subsequent rebound. We also plan to study some related biological mechanisms. Studies of calcium supplementation and mucosal proliferation in the bowel have been conflicting, but the relationship between calcium intake and apoptosis has been investigated and merits study. Further evidence that dietary calcium may preferentially affect the risk of adenomas harboring ras mutations needs confirmation. As well, animal models and some epidemiological data suggest that vitamin D may protect against bowel carcinogenesis. We propose to investigate this in the context of our trial, and to examine polymorphisms in the vitamin D receptor gene. With the funds requested here, we will use our established subject population and available biological specimens to study these mechanisms. We will follow the subjects in the study until the year 2003, monitor their medical history, and track adenoma recurrence after the end of treatment in the trial. All subjects provided blood specimens at study entry and at study exit. Using these samples, we will measure 25-OH vitamin D and 1,25-OH vitamin D to document how calcium supplementation affected these levels, and how the baseline levels and polymorphisms in the vitamin D receptor related to adenoma occurrence. We will also obtain slides from each adenoma detected during the risk period of the trial or during the additional follow-up period and investigate the association between calcium treatment and ras mutation. Finally, we propose to conduct a pilot study in preparation for a study of calcium supplementation and apoptosis both in normal rectal mucosa and in adenomas. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: CALCIUM INFLUX PATHWAYS IN ALLERGY Principal Investigator & Institution: Kinet, Jean-Pierre M.; Director, Laboratory of Allergy & Immuno; Beth Israel Deaconess Medical Center St 1005 Boston, MA 02215 Timing: Fiscal Year 2001; Project Start 01-SEP-2000; Project End 31-AUG-2003 Summary: (Adapted from the applicant's abstract) Sustained calcium entry in mast cells plays a critical role in the initiation and maintenance of allergic responses associated with ligand binding to Fc epsilon RI. It is believed that the sustained calcium entry associated with engagement of Fc epsilon RI is mediated by the opening of calcium channels in the plasma membrane in response to depletion of a subset of calcium stores by the second messenger inositol-1,4,5-trisphosphate (IP3). Although the functional relationship between calcium store depletion and calcium entry is a well-documented phenomenon, there is little or no definitive data concerning the nature of the relevant
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calcium channels involved (referred to as Store Operated Channels or SOC), or the molecular mechanisms by which these channels are gated in response to calcium store depletion. Moreover, although recent data suggest that calcium entry may be regulated by pathways associated with the production of the second messengers sphingosine-1phosphate, cyclic ADP-ribose or NAADP, the relationship between these putative pathways and calcium store depletion, and the generality of these pathways and the potential targets of these specific second messengers are either unknown or controversial. In summary, there is a significant lack of specific knowledge concerning the molecular mechanisms which regulate calcium entry into mast cells and other nonexcitable cells. Because of the fundamental importance of calcium entry to mast cell function, the investigator's laboratory has embarked upon a series of experimental approaches to identify calcium entry regulatory proteins. In the preliminary data provided, experiments describe the identification and initial characterization of a novel family of putative calcium channels (CeCH proteins) which are widely expressed in non-excitable cells including mast cells. In the current application, experiments are proposed to analyze the function of these proteins in both mast cell and non-mast cell lines. In specific aim 1, studies will be performed to analyze the assembly and transport of wild type CeCH proteins, to conduct structure/function analyses to identify structural features required for proper assembly and transport, and to isolate and characterize the role of CeCH-associated proteins in CeCH function. In specific aim 2, experiments will analyze CeCH function in regulating calcium homeostasis and signaling through a combination of calcium imaging and electrophysiologic analysis of cultured cells expressing defined combinations of CeCH proteins under various types of stimulus conditions. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CALCIUM INHIBITION OF CAMP IN ENDOTHELIAL CELL PERMEABILITY Principal Investigator & Institution: Stevens, Troy; Professor; University of South Alabama Mobile, AL 366880002 Timing: Fiscal Year 2001; Project Start 01-DEC-2001; Project End 30-NOV-2006 Summary: Calcium agonists disrupt macrovascular and not microvascular cell barrier function, suggesting distinct responses to similar inflammatory mediators may represent a mechanism for targeting lung inflammation to specific vascular segments. Our prior work indicated that activation of store operated calcium entry by inflammatory calcium agonists inhibits adenylyl cyclase activity in pulmonary artery endothelial cells resulting in decreased cyclic AMP (cAMP) sufficient to increase permeability. Although microvascular endothelial cells (PMVECs) express a calcium inhibited isoform of adenylyl cyclase (AC6), calcium agonists neither decrease cAMP content nor disrupt the PMVEC barrier. Preliminary data indicate this calcium insensitivity is accompanied by a high rate of cAMP synthesis and turnover; when cAMP hydrolysis is disrupted in PMVECs calcium inhibits cAMP formation and inflammatory mediators induce intercellular gaps, demonstration AC6 critically regulates both PAEC and PMVEC barrier function. One cAMP-sensitive target that regulates endothelial cell permeability is membrane associated, on-erythroid spectrin. Disruption of spectrin binding to F-actin promotes intercellular gap formation in both PAECs spectrin. Disruption of spectrin binding to F-actin promotes intercellular gap formation in both PAECs and PMVECs, demonstrating spectrin's important role in regulating cell shape. Taken together our data support the idea that calcium inhibition of cAMP reduces the spectrin-F-actin association. In PAECs, whereas preservation of
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cAMP content promotes the spectrin-F-actin association in PMVECs. Thus, this proposal test the overall Hypothesis that calcium inhibition of cAMP formation decreases spectrin binding to F-actin important to increase lung endothelial cell permeability. Specific Aims test the related Hypotheses that: [1] cAMP accumulation regulates calcium inhibition of AC6 in PMVECs; [2] non-erythroid spectrin from F-actin increases macro- and microvascular endothelial cell permeability. Completion of this work will be important to further our understanding of the key signaling events that link calcium agonists to endothelial barrier disruption, a cardinal feature of multiple inflammatory lung diseases including acute respiratory distress syndrome, asthma, and reperfusion pulmonary edema. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CALCIUM ADOLESCENTS
KINETICS
IN
PREGNANT
&
LACTATING
Principal Investigator & Institution: O'brien, Kimberly O.; Professor of International Health; Johns Hopkins University 3400 N Charles St Baltimore, MD 21218 Timing: Fiscal Year 2001; Project Start 01-DEC-2000; Project End 30-NOV-2001 Summary: Adolescent pregnancy is a significant problem in the United States. Special nutritional concerns are present in this group to insure that optimal nutritional needs can be met by both the fetus and the growing adolescent. The additional nutritional demands of pregnancy may be of primary concern when the mother has not yet completed her linear growth and has not fully achieved her peak bone mass. The aim of the proposed study is to address the impact of pregnancy and lactation on the efficiency of calcium absorption and rates of bone calcium deposition and resorption in adolescent non-Hispanic white and non-Hispanic black girls between the ages of 11-17 y. Fifteen girls from each group who intend to breastfeed their infants will participate in a longitudinal calcium kinetic study designed to address the efficiency of calcium absorption and rates of bone calcium turnover during both the third trimester of pregnancy (34-36 weeks of gestation) and during lactation (1-2 months post partum). All adolescents will be screened for positive predictors of lactation performance prior to the start of the study. In each kinetic study, girls will be admitted to the General Clinical Research Unit at Johns Hopkins Hospital. On the morning of each study, girls will consume a glass of milk containing a stable calcium isotope (46Ca), and following breakfast a second stable calcium isotope (42Ca) will be administered intravenously. Timed blood samples will be obtained for 8 hours following the isotope dosing, and a complete 24 hour urine demands of adolescence and the calcium demands of pregnancy and lactation. collection will be made. Additional spot urine samples will be collected for 5 days following each isotope study. To address the hormonal response to pregnancy and lactation, calcitropic hormones will be measured during each study using a fasting blood sample. Rates of bone calcium deposition and resorption in response to these physiological changes will be determined using multicompartmental modeling. These studies will provide novel information on the physiological alterations in calcium absorption and bone calcium turnover which occur to support both the increased calcium Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CALCIUM METABOLISM IN ADOLESCENT BOYS Principal Investigator & Institution: Weaver, Connie M.; Head and Distinguished Professor; Foods and Nutrition; Purdue University West Lafayette West Lafayette, IN 479072040
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Timing: Fiscal Year 2001; Project Start 01-JUL-1993; Project End 30-JUN-2003 Summary: (Adapted from the investigator's Abstract): From previous work on this project, the investigators determined calcium retention and kinetics in black and white adolescent girls. They developed a nonlinear regression model of the relationship between calcium intake and calcium retention in order to establish calcium requirements for maximal skeletal accretion. They now propose to study these same relationships in boys, hypothesizing that both calcium intake and stage of sexual maturity (associate with change in endocrine factors) will explain much of the variance in calcium accretion in boys as it did in girls. By controlling calcium intake in a metabolic study, the non-dietary factors, which drive calcium accretion, can be better understood. Boys are a better model than girls for studying endocrine factors which drive calcium accretion because of the fluctuation associated with menses in girls. The first aim establishes calcium requirements in boys. Peak velocity of calcium accretion determined by bone mineral densitometry in boys is 75 mg/d higher in boys than girls; thus calcium requirements may be higher for boys than girls. The second aim is to identify candidate hormones/growth factors which drive non-dietary changes in calcium. IGF-1 may be most predictive of initial gains in calcium accretion until attainment of peak velocity of calcium accretion in puberty and increases in sex steroid hormone levels may predict the subsequent decline in rate of calcium accretion. Calcium retention in boys will be determined over a range of calcium intakes between 700-2100 mg/d during three-week metabolic camps. Stable isotopes of calcium will be administered orally and by intravenous injection after the first week equilibrium period in a subset of boys. Analysis of complete urine and fecal collections and periodic blood samples will provide data for multicompartmental and stochastic analysis of calcium metabolism. Bone mass and total body and skeletal site calcium measured by dual energy X-ray absorptiometry, hormone levels (PTH, vitamin D metabolites, IGF-1, IGFBP3, estrogen, testosterone, and SHBG), and biochemical markers of bone turnover (serum osteocalcin and bone specific alkaline phosphatase and urinary collagen crosslinks) will be determined in all boys. The relationship of calcium retention and bone turnover to parameters of sexual maturity and size will be analyzed. Optimizing calcium accretion during this rapid period of growth should maximize peak bone mass within the genetic potential and reduce risk of osteoporosis later. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CALCIUM OXALATE IN BIOLOGICAL SYSTEMS Principal Investigator & Institution: Hatch, Marguerite; Associate Professor; Federation of Amer Soc for Exper Bio Bethesda, MD 208143998 Timing: Fiscal Year 2002; Project Start 01-AUG-2002; Project End 31-JUL-2003 Summary: (provided by applicant): This is an application for funds to provide partial support for a FASEB Summer Conference entitled: "Calcium Oxalate in Biological Systems" at the Vermont Academy in Saxtons River, Vermont, on August 3-8, 2002. This Conference is a sequel to the first FASEB Summer Conference on this topic which was very successful and subsequently propagated collaborations between newly found colleagues among the diverse scientists who attended. Perhaps the most intriguing and enigmatic aspect of the complexation of calcium and oxalate is the fact that it is crucial to the normal physiology of plant life, whereas in humans and animals, its primary importance is derived from its role in the pathophysiology of kidney stone disease. This contrast in function between plant and animal physiology highlights one of the important features of this Conference. In contrast to bacteria and fungi that can degrade oxalate, mammalian cells have no capacity in this regard. Clearly, there is tremendous
Studies 39
potential for both exchange and cross-fertilization of ideas when the topic is discussed by such a diverse group of investigators from a wide range of disciplines. This conference has been specifically planned to include young/new investigators since the most exciting and rapid growth in the area of calcium oxalate research is in the application of newer technology in addressing some of the nagging questions and issues that have remained elusive up until now. Approximately 150 scientists are expected to attend this second Conference. Presently, 50% of the Speakers/Session Chairs are young new researchers/new participants to this Summer Conference and the remainder are individuals who are highly respected and recognized in their respective areas of research. Nine scientific sessions will be held on the following topics: 1) Metabolic Pathways leading to Oxalate Formation; 2) Handling of Oxalate/Precursors by Cells; 3) Calcium Oxalate Crystallization Processes; 4) Calcium Oxalate Crystal Inhibition; 5) Crystal and Cell Interactions; 6) Cellular Responses to Oxalate and Calcium Oxalate; 7) Genetic Aspects; 8) Pathophysiology of Stone Disease; 9) Integration of Information from Diverse Systems. The overall goal of this Conference is to provide a forum where a diverse group of scientists in calcium oxalate research can gather and share the broad scope of knowledge among the completely different scientific disciplines. No where else can a botanist, a urologist, and a chemical engineer, (to name just three of the fifteen disciplines represented) gather at the same venue to discuss the same topic each from his/her own point of view. In our view the hallmark of this Conference is underscored by the final program session which acknowledges that collectively we can learn from each other by bringing a multidiscipinary approach to scientific discussion and experimentation. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CALCIUM PHOSPHATE BONE REPAIR MATERIALS Principal Investigator & Institution: Chow, Laurence C.; Chief Research Scientist; American Dental Assn Health Fdn 211 E Chicago Ave Chicago, IL 60611 Timing: Fiscal Year 2001; Project Start 01-MAY-1996; Project End 30-JUN-2004 Summary: (Adapted from the Investigator's Abstract): A calcium phosphate cement (CPC), developed under this research project, was approved by the FDA in 1996 for cranial defects repair applications in humans, thus becoming the first material of its kind to be available for clinical use. While CPCs were shown to be very useful in a number of dental and medical applications for which other materials do not work well, in vivo study results suggest that in order to achieve the best results, CPC should have handling and in vivo properties that are best suited for the particular clinical application. The objectives of the proposed research are to elucidate mechanisms of cement setting reactions and to understand the physicochemical factors that influence cements= handling and in vivo properties. Four specific aims are proposed. Aim 1 proposes to understand factors that control the hydrolysis reactions of tetracalcium phosphate (TTCP), alpha-tricalcium phosphate (alpha-TCP), dicalcium phosphate dihydrate (DCPD), dicalcium phosphate anhydrous (DCPA) and calcium hydroxide. These calcium phosphate salts are the major components of different CPCs. Hydrolysis of one or more of the salts that form hydroxyapatite (HA) is responsible for the hardening of the cement. A better understanding of the hydrolysis reaction of each of these salts will provide important insights into factors that influence some important cement properties, including the rate of conversion to HA, formation of Ca-deficient or stoichiometric HA, the crystallinity HA, etc. Defective or non-stoichiometric HAs are believed to be more bioresorbable. Aim 2 proposes to study the dissolution rate of cement products in demineralizing solutions having ionic compositions mimicking the
40 Calcium
acidic environment produced by osteoclasts. A dual constant-composition titration system was developed during the report period for measuring dissolution rates of calcium phosphate biomaterials under simulated acidified physiological solutions. The Principal Investigator proposes to use this technique as an in vitro model for predicting resorption rates of CPC and to understand factors that control the dissolution rate. Aim 3 proposes to study properties of non-rigid and resorbable calcium phosphate cements. Experiments are described to study composites of CPC and chitosan, a biocompatible polymer to form self-hardening, bioresorbable, and non-rigid bone graft materials. These materials should be useful in a number of applications in which the implant can remain stable and firmly attached to the bone defect surface despite micro-movements of the defect walls. Aim 4 will study properties of injectable premixed calcium phosphate cement pastes. Premixed CPC pastes have the advantages that they are stable in the package and harden only after delivery to the defect site where the non-aqueous liquid is replaced by water from the surrounding tissue. While premixed CPC is considerably easier to use and is injectable, its properties are quite different from the conventional CPCs. The hardening time, resistance to washout, and HA conversion of premixed pastes consisting of CPC powder and non-aqueous liquids, such as glycerin, will be studied. The Principal Investigator also proposes to determine mechanical properties of the hardened CPC pastes. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CALCIUM RECEPTOR SIGNALING OF P450 ARACHIDONIC ACID METABOLITES Principal Investigator & Institution: Hebert, Steven C.; Vanderbilt University 3319 West End Ave. Nashville, TN 372036917 Timing: Fiscal Year 2001 Summary: The hypothesis to be explored in Project #3 of this Program Project is that cytochrome P450 (P450) arachidonic acid (AA) metabolites provide a major signaling system for the extracellular calcium/polyvalent cation- sensing G protein-coupled receptor (CaSR) in the mammalian kidney. Genetic and physiological studies in man and laboratory animals have demonstrated that the CaSR is crucial not only for the extracellular calcium-dependent regulation of parathyroid hormone form parathyroid glands, but also for normal divalent mineral handling by the kidney. The CaSR is expressed in many epithelial segments of the mammalian kidney nephron, particularly at the urinary face of cells forming the initial proximal tubule and at the bloodinterstitial face of the ascending segment of the loop of Henle called the thick ascending limb (TAL). Since salt transport processes in both the proximal tubule and TAL are involved in regulated divalent mineral ion excretion, activation of the CaSR in these nephron segments is expected to modulate calcium and magnesium loss in the urine. Modulation of transport processes by the CaSR in these segments also alter the kidney excretion of salt and water, processes that help to ensure that calcium and magnesium can be excreted with a reduced likelihood for kidney stone formation or nephrocalcinosis. Specifically, calcium activation of the CaSR in the TAL functions as in endogenous "loop" diuretic, and this mechanism may account for the beneficial effect of dietary calcium intake on blood pressure seen in laboratory animals and man. Using a combination of molecular, biochemical and physiological approaches, we will: a) identify the specific P450 gene(s) involved in CaSR signaling in the kidney tubule epithelial cells; b) define the specific P450 omega/omega-1 AA metabolites generated by activation of the CaSR; c) identify the enzymatic components of the CaSR-P450 signaling pathway; and d) define the physiological effects of CaSR action on salt transport
Studies 41
process. Results of these studies should define roles for the CaSR in the renal regulation of divalent mineral homeostasis and salt and water balance. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CALCIUM REMOVAL AND REGULATION OF MUSCLE AND NEURONS Principal Investigator & Institution: Schneider, Martin F.; Professor; Biochem and Molecular Biology; University of Maryland Balt Prof School Baltimore, MD 21201 Timing: Fiscal Year 2001; Project Start 01-MAY-1994; Project End 30-NOV-2002 Summary: The overall objective of this project is to gain deeper understanding of fiber type-specific gene expression skeletal muscle and of calcium signalling in muscle and neurons. Activity dependent gene expression will be studied in skeletal muscle and role of cytosolic [Ca/2+] in such "excitation-transcription" (ET) coupling will be determined. Understand of the mechanisms of ET coupling is of central importance for maintenance of muscle tone and function in health, inactivity and after traumatic nerve section or during neuromuscular disease. The subcellular details of calcium signaling will be explored in isolated neurons to explain important local effects of [Ca/2+] relevant to wide range of neuronal functions. The contributions of Ca/2+ transport systems and Ca/2+ binding proteins to the decline of muscle [Ca/2+] after Ca/2+ release, which is crucial to the relaxation of normal and diseased muscle, will be determined for both fastand slow-twitch muscle. Chronic electrical stimulation will be used to study plasticity of gene expression in individual enzymatically isolated adult mouse skeletal muscle fibers in culture. The localization and functional properties of the calcium uptake, storage, and release organelles in neurons will be determined by high spatio-temporal resolution confocal microscopy of individual sympathetic ganglion neurons. Protein isoform composition will be determined in the same individual muscle fibers from which [Ca/2+] transients were recorded to define the molecular basis for the diversity of calcium removal mechanisms in fibers of the same and different types. These studies will provide a better understanding of the cellular and molecular mechanisms underlying fiber type conversion in skeletal muscle, the control of a variety of calcium dependent processes in both muscle and neurons, and relaxation of skeletal muscle fibers. They will provide deeper insight into calcium regulation and its adaptability in muscle and neurons under a variety of physiological states as well as during disuse, aging and under possible pathological conditions. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: CALCIUM SIGNALING AND OVARIAN CANCER Principal Investigator & Institution: Rodland, Karin D.; Senior Staff Scientist; Cell and Developmental Biology; Oregon Health & Science University Portland, OR 972393098 Timing: Fiscal Year 2001; Project Start 01-JUL-1998; Project End 30-SEP-2001 Summary: Ovarian carcinoma is one of the leading causes of cancer death among women. The low survival for victims of ovarian cancer (less than 13 percent for stage III disease) reflect, in large part, the highly aggressive and metastatic character of ovarian adenocarcinomas. Despite the obvious seriousness of ovarian cancer, very little is known about the normal biology of the ovarian surface epithelial cells which give rise to the most malignant forms of ovarian carcinoma. Data presented in this proposal indicate that increased extracellular calcium exerts a significant proliferative effect on ovarian surface epithelial cells, as measured by both thymidine incorporation and cell growth curves. Ovarian surface epithelial cells express mRNA and protein for the recently
42 Calcium
cloned Calcium-sensing Receptor (CaR). This G-protein coupled receptor has been shown to be responsible for triggering parathormone release from parathyroid cells in response to elevated extracellular calcium. We have shown that the CaR in ovarian surface epithelial cells displays the same functional responses to extracellular calcium and other agonists (including gadolinium) as does the parathyroid CaR. Activation of the CaR in ovarian surface epithelial cells is associated with increases in tyrosine phosphorylation, increased activity of the mitogen-activated kinase ERK2, and increased src kinase activity. Expressing a non-functional mutant of the CaR (R796W) inhibited the increases in tyrosine phosphorylation and ERK2 activity observed in response to agonists of the CaR, indicating that the presence of functional CaR is required for these responses to increased extracellular calcium. Furthermore, we have observed that two of four ovarian tumor cell lines examined appear to over-express CaR mRNA, as well as expressing a novel CaR transcript. These ovarian tumor cell lines are no longer growthinhibited in low calcium media. This proposal is based on the hypotheses that signal transduction downstream of the CaR leads directly to activation of ERK kinase and increased proliferation, and that disruption of normal CaR expression and/or function contributes to the loss of normal growth controls in ovarian carcinogenesis. These hypotheses will be tested by 1) disrupting known signaling mechanisms and observing the effect on CaR-dependent activation of src and/or ERK2, using transfection of dominant negative mutants or selective chemical inhibitors, 2) using a prospective study of CaR expression in patients with ovarian cancer to test clinical relevance. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CALCIUM SIGNALING AND TRANSPORT IN S CEREVISIAE Principal Investigator & Institution: Cunningham, Kyle W.; Biology; Johns Hopkins University 3400 N Charles St Baltimore, MD 21218 Timing: Fiscal Year 2001; Project Start 01-APR-1996; Project End 31-MAR-2004 Summary: Calcium signaling is a critical component of normal immune cell function and development of the heart and brain. The budding yeast Saccharomyces cerevisiae utilizes a calcium signaling mechanism that is closely related to those operating in human cells such as T lymphocytes in their response to antigens. In both of these systems, calcium signals can be sensed by calcineurin, a protein phosphatase that is activated by Ca2+/calmodulin and inhibited by the immunosuppressive drugs Cyclosporin A and FK506. In turn, activated calcineurin dephosphorylates specific transcription factors which then concentrate in the nucleus and induce expression of specific target genes. A new family of proteins conserved from yeast to humans that binds and regulates calcineurin function has been recently identified. One major goal of this proposal is to determine how the yeast protein Rcn1p exerts both positive and negative effects on calcineurin functions. Recent studies also show that yeast maintains a regulatory mechanism related to capacitative calcium entry (CCE) in lymphocytes that helps generate calcium signals. Depletion of Ca2+ from secretory organelles in yeast through inactivation of the Pmr1p Ca2+ pump strongly enhances the activity of a highaffinity Ca2+ channel involving Cch1p, a homolog of voltage-gated Ca2+ channels in animals. Additional goals of this proposal include the identification and characterization of factors involved in releasing Ca2+ from intracellular organelles, sensing Ca2+ concentration in the lumen of these compartments, regulating the Cch1p-dependent Ca2+ channel, and activating another unidentified Ca2+ influx channel in the plasma membrane. Calcineurin- dependent feedback regulation of these Ca2+ channels and of the Ca2+ transporters that dissipate calcium signals also will be evaluated. These goals will be accomplished by combining genetic, molecular, physiological, and biochemical
Studies 43
methods. The broad long-term objectives of this project are to elucidate the components of the calcium signaling mechanism operating in all cells and to develop a detailed working model for their interactions and functions. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CALCIUM CONTRACTION
SIGNALING
IN
CARDIAC
EXCITATION-
Principal Investigator & Institution: Gyorke, Sandor; Professor; Physiology; Texas Tech University Health Scis Center Health Sciences Center Lubbock, TX 79430 Timing: Fiscal Year 2001; Project Start 20-MAY-2001; Project End 30-APR-2004 Summary: This research will be done primarily in Slovak Republic as an extension of NIH grant # ROl HL52620. The overall goal of this proposal is to elucidate the mechanisms of calcium signaling in dyads of mammalian cardiac muscle cells. These structures, representing excitation-contraction coupling units, contain clusters of L-type calcium channels (the excitation unit) in apposition to clusters of ryanodine receptor calcium release channels (the release unit) functionally coupled by calcium ions. The feed-forward calcium signaling steps are important for activation of calcium release by excitation, the feedback calcium signaling steps are important for inactivation of calcium current and calcium release, and their relationship to the topology of channels in the excitation-contraction coupling unit will be determined. To this end, measurements of the kinetics of whole-cell calcium currents, whole-cell calcium transients, and dyadic calcium release events during stimulation and during recovery from inactivation will be carried out. The properties of the excitation-contraction coupling unit, such as calcium channel gating and permeation, the fraction of active calcium channels, and the rate of calcium binding to the ryanodine receptor, will be manipulated. Quantitative analysis of the kinetics of calcium current and calcium release in parallel will be used to estimate parameters of calcium signaling critical for control of gradation and efficiency of excitation-contraction coupling. Computer simulations of experiments on channel clusters that interact by way of calcium signaling will be used to ascertain possible mechanisms and to design experiments with high predictive power. Understanding the regulation of cardiac E-C coupling is important since it might become a strategic site for therapeutic intervention in cardiac muscle dysfunctions. Furthermore, defining mechanisms of calcium-based interaction between channels in co-localized clusters has a broader significance for understanding processes such as synaptic transmission, neuroendocrine secretion and regulation of vascular tone. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CALCIUM SIGNALING IN DENDRITIC CELL FUNCTION Principal Investigator & Institution: Ahern, Gerard P.; Pharmacology; Georgetown University Washington, DC 20057 Timing: Fiscal Year 2003; Project Start 15-FEB-2003; Project End 31-JAN-2007 Summary: (provided by applicant): Dendritic cells (DC) are a heterogeneous population of rare leukocytes highly specialized for immune-surveillance, and the induction and regulation of primary immune responses. This unique capacity reflects their ability to continuously sample the microenvironment and ingest foreign and self-antigens. After encountering a "danger" stimulus in the form of microbes, inflammatory molecules or allergens, DC transform into potent stimulatory cells and migrate to secondary lymphoid tissues, where they trigger the activation of antigen-specific effector T cells. The signaling pathways that underlie these processes are undoubtedly complex, but
44 Calcium
intracellular calcium appears to play a crucial role. We have recently characterized two novel calcium signaling pathways in DC. First, we have identified the skeletal muscletype ryanodine receptor (RyR1) in DC. RyR1 is a massive intracellular channel that can amplify small calcium transients within a cell to produce much larger, sustained calcium rises. Second, we have demonstrated that calcium fluxes trigger rapid secretion by DC. Such pathways enable DC to respond rapidly to external stimuli, and release autocrine and paracrine signaling factors including exosomes and leaderless secretory proteins. The goal of this proposal is to determine the RyRl-calcium regulated pathways in DC. We hypothesize that RyR1 integrates diverse cellular stimuli, and mediates the calcium pathways that drive DC function. An inter-disciplinary approach is outlined to investigate the properties of these calcium signaling mechanisms and understand how they participate in DC biology. We will accomplish the objectives of this proposal by pursuing the following specific aims: Aim 1 is designed to determine the role of RyR1 during DC development and function. Aim 2 tests the impact of endogenous and pharmacologic activators of RyR1 on DC. In Aim 3, we will elucidate the role of calcium-triggered secretion in DC. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CALCIUM SIGNALING IN THE TESTIS Principal Investigator & Institution: Beckingham, Kathleen M.; Biochemistry and Cell Biology; Rice University 6100 S Main Houston, TX 77005 Timing: Fiscal Year 2001; Project Start 28-MAR-2001; Project End 28-FEB-2005 Summary: (Adapted from the applicant's abstract) The long-term objectives of the proposed work are to dissect the roles of calcium regulation in sperm development and mature sperm function. The initial studies proposed here concern the role(s) of a newly identified calcium-binding protein implicated in calcium-regulatory events in the testis. The protein, termed Androcam, is specifically expressed in the testis and appears to be evolutionarily conserved from insects to mammals. The characteristics of Androcam indicate that it is a calcium sensor-a protein that converts transient changes in intracellular calcium into changes in cellular responses as a result of calcium-induced changes in its protein-protein interactions. Two major approaches to understanding Androcam function will be pursued: i) the disruptions in spermatogenesis and mature sperm function produced by mutations to the Androcam gene of Drosophila melanogaster will be analyzed; ii) the protein interaction partners of Androcam, both in the calcium-bound and calcium-free state will be identified in the same organism. Prior studies already allow the prediction that two types molecular motor proteins may be Androcam targets. The Androcam homologs in other species will also be isolated with a particular view to pursuing studies of Androcam-related proteins in mammalian species. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: CALCIUM SIGNALLING AMONG NON-NEURONAL BRAIN CELLS Principal Investigator & Institution: Nedergaard, Maiken; Professor; Cell Biology and Anatomy; New York Medical College Valhalla, NY 10595 Timing: Fiscal Year 2001; Project Start 01-AUG-2001; Project End 31-JUL-2005 Summary: In previous studies, we have reported that calcium waves among gap junction-coupled glia may form the cellular substrate for spreading depression in vivo. Recently, we have noted that calcium waves initiated in astrocytes in slices can propagate to brain endothelial and meningeal cells; all of these cell types express
Studies 45
connexin43, which may allow their mutual heterotypic syncytial interaction through homotypic gap junctions. On this basis, we propose to test the hypothesis that astrocytic calcium waves may thereby invade the brain by propagating along the capillary vasculature, as well as through the astrocytic syncytium. These experiments will test the possibility that endothelial calcium waves may follow the venular endothelium to invade the meningeal vasculature, thereby recruiting both meningeal cells and trigeminal sensory afferents. This proposed pathway, by bypassing and traversing the restrictive barrier of the pia limitans, would permit the recruitment of both the meningeal vasculature and its trigeminal sensory afferents into parenchymal waves of spreading depression. We propose here that this scenario might operationally model the initiation of migraine headache in adults. In parallel experiments, we will also follow-up our recent observation of a steroid-induced accentuation of astrocytic calcium signaling, by asking whether calcium signaling among non-neuronal brain cell types may be modulated by gonadal steroids. In particular, we seek to determine whether the cyclical female hormones estrogen and progesterone potentiate signaling from astrocytes to endothelial cells, and if so, whether the likelihood of meningovascular recruitment into a parenchymal calcium wave is thereby increased. This pathway might account for much of the symptomatology of migraine headache, while steroidal accentuation of calcium signaling might account for the cyclicity of migraine occurrence. The longdistance multicellular calcium signaling pathway that we propose, and its attendant hormonal regulation, suggests immediately testable strategies for its abrogation. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CALCIUM SPARKS IN HEART MUSCLE Principal Investigator & Institution: Lederer, W Jonathan.; Professor; Physiology; University of Maryland Balt Prof School Baltimore, MD 21201 Timing: Fiscal Year 2001; Project Start 01-DEC-1986; Project End 30-NOV-2001 Summary: The planned work will investigate a new phenomenon in heart muscle that we have recently discovered: "Calcium sparking". Calcium-sparking can be observed when imaging the distribution of calcium in normal heart cells with very high temporal and spatial resolution. The calcium spark is a highly localized (microns) elevation of intracellular calcium that arises spontaneously and lasts only briefly (msec). The "calcium spark" occurs largely randomly in time and space within a heart muscle cell. In normal, healthy cardiac myocyte the calcium spark event is rare. However, it produces a significant elevation of intracellular calcium (near-micromolar) that, surprisingly, does not activate additional calcium release from the sarcoplasmic reticulum (SR). Our preliminary investigation of calcium sparks in isolated rat heart muscle cells suggests that the calcium spark could result from the opening of a single SR calcium release channel, but this finding will be examined during the planned work. The calcium spark may activate propagated waves of elevated calcium when the heart cells are "overloaded" with calcium. Over the next five years we plan to examine the cellular, biophysical and molecular basis of [Ca2+]i- sparking and explore its cellular consequences. Four experimental series are planned that will address the following broad questions: (1) What controls the [Ca2+]i sparking process? (2) What is the relationship between [Ca2+]i sparks and the normal [Ca2+]i transient? (3) What is the relationship between [Ca2+]i sparks and propagated waves of elevated [Ca2+]i? (4) Are the kinetic and functional properties of the SR Ca-release channel appropriate to explain calcium sparking? We expect this planned investigation to resolve issues that have been raised by our preliminary results: (a) What activates the calcium-spark? (b). How many SR Ca-release channels are involved in contributing calcium to a spark? (c) Why doesn't
46 Calcium
a spark usually give rise to a propagated wave of elevated calcium? (d) Is the frequency of calcium sparking a direct function of the open probability (P) of the SR- Ca-release channel? We will use isolated guinea pig and rat heart cells examined by high-speed confocal fluorescence microscopy in combination with a patch-clamp method in wholecell mode. Additionally, the novel planar lipid bilayer method of Gyorke & Fill (1993) will be used to examine the SR Ca-release channels from rats and guinea-pig hearts. Both bilayer and imaging methods will exploit flash photolysis of caged calcium in the planned work. Fundamental new information on the cellular processes that control SR Ca- release channels and EC coupling in the heart muscle should result from the propose experiments. The work should thus broaden our understanding of calciumdependent arrhythmias and diverse forms of heart failure. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CALCIUM, DIFFERENTIATION
S100
PROTEINS
AND
KERATINOCYTE
Principal Investigator & Institution: Eckert, Richard L.; Professor; Physiology and Biophysics; Case Western Reserve University 10900 Euclid Ave Cleveland, OH 44106 Timing: Fiscal Year 2001; Project Start 03-MAY-2000; Project End 30-APR-2005 Summary: (Adapted from the applicant's abstract) - Calcium is the single most important regulator of keratinocyte differentiation. A major goal in keratinocyte biology is to understand how the calcium signal is transduced to drive keratinocyte differentiation. S100 proteins are an important class of protein that are required for calcium signal transduction. They are activated by a calcium binding-dependent conformation change, and then bind to and regulate the function of target proteins. The target proteins include regulators of cell proliferation and differentiation. Although keratinocytes express eight S100 proteins, little information is available regarding S100 protein function in keratinocytes. S100 proteins are thought to function as noncovalently associated homodimers. However, their recent study shows that the calcium-dependent enzyme, transglutaminase (TG), can modify S100 proteins by adding two interprotein covalent links, one linking each end of the antiparallel homodimer. They hypothesize that this regulates S100 function by altering the calcium-dependent conformation change. This hypothesis links transglutaminase, S 100 proteins, and calcium as components of a common regulatory unit. In this model, TG modification of S100 protein structure could provide a trigger to promote differentiation. The overall goals of this study are to characterize the S100 proteins expressed in keratinocytes with respect to (i) ability to act as a TG substrates and the effects of TG modification on function, (ii) subcellular and tissue distribution and the effects of physiological agents in this distribution, (iii) interaction with target proteins, and (iv) alteration in psoriasis. In preparation for these studies they have cloned cDNAs, expressed protein, and produced antibodies against S100A11, S100A10, and S100A7. They show that each of these proteins are TG substrates and in one case, S100A11; they identified the TG reactive sites by amino acid microsequencing. Their study is designed to provide new information on S100 protein function in keratinocytes. In Specific Aim 1, they clone, express, and generate antibodies to the remaining keratinocyte S100 proteins, S100A2, S100A4, S100A6, S100A8, and S100A9. These reagents are used (i) to determine whether these S100 proteins form TG-dependent covalent multimers in vivo and in cultured keratinocytes, (ii) to determine the structure of crosslinked products, (iii) to identify the amino acids involved in crosslink formation, and (iv) to study S100 protein function in psoriasis. The studies described in Specific Aim 2, are designed to determine whether TG-dependent modification of S100A11, their prototype S100 protein, alters its ability to
Studies 47
interact with its target substrate, annexin I. This, they expect, will provide a direct test of their hypothesis that TG modification is a regulatory mechanism designed to alter S100 protein function. Localization can profoundly influence function. Their preliminary studies show that S100A11 is mobilized to the plasma membrane in response to calcium. In Specific Aim 3 they (i) use anti-S100 protein antibodies to localize the proteins in epidermis (normal and psoriatic), and in cultured cells, (ii) and examine the effects of physiological agents (calcium, etc.) on their subcellular distribution. S100 proteins transduce calcium signals by binding to target proteins; however, the targets are not known in keratinocytes. The goal of Specific Aim 4 is to identify these target proteins using ligand blots, co-immunoprecipitation, affinity chromatography, and protein microsequencing. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: TOLERANCE
CALCIUM-DEPENDENT
MECHANISMS
IN
NICOTINE'S
Principal Investigator & Institution: Damaj, M. Imad.; Associate Professor; Pharmacology and Toxicology; Virginia Commonwealth University Richmond, VA 232980568 Timing: Fiscal Year 2001; Project Start 01-FEB-2001; Project End 31-JAN-2004 Summary: The broad objective of this proposal is to elucidate the calcium- dependent mechanisms of nicotinic receptor function. It was recently shown that neuronal nicotinic receptors (nAChRs) have a high Ca2+ permeability and our initial studies indicate that changes in calcium homeostasis and Ca2+/calmodulin-dependent protein kinase (CaMKinase II) can modulate nicOtine's pharmacological effects and are involved in neuroadaptative processes after chronic exposure to nicotine in the animal. In this project we plan to study calcium-dependent intracellular mechanisms and their modulation after short and long-term activation of nAChRs. We will assess the potential role of CaM-kinase II activatiOn in the behavioral effects of nicOtine using pharmacological, biochemical and genetic approaches. Tolerance to nicotine's effects could involve an adaptation in the calcium signaling system activated by nicotine leading to a decrease in in CaM-kinase II activity. Such a hypothesis will be investigated by measuring quantitative and qualitative changes in Ca2+-calmodulin-dependent signaling in in vitro and ex-vivo preparations during long-term exposure to nicotine. Finally, we will attempt to modulate tolerance to nicotine's effects by administering drugs that alter calcium entry. In vivo studies with calcium channel modulators and CaM-Kinase II inhibitors as well as NMDA receptor antagonists will be conducted in animals chronically infused with nicotine. The proposed studies will elucidate the calcium-dependent processes involved in nicotine's actions in the brain as well as the neuroadaptation that occurs after short-and long-term exposure to this drug of abuse. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CALCIUM-DEPENDENT SURVIVAL OF DEVELOPING NEURONS Principal Investigator & Institution: Mao, Zixu; Rhode Island Hospital (Providence, Ri) Providence, RI 02903 Timing: Fiscal Year 2001; Project Start 01-JUL-2000; Project End 30-JUN-2005 Summary: (From the applicant's abstract): Afferent input supports many neuronal functions in vivo including neuronal survival in the developing brain. Imbalance of the process of neuronal survival and apoptosis is linked to a growing list of developmentand aging related neurological abnormalities characterized by neuronal death including
48 Calcium
Alzheimer's and Huntington's diseases. Neuronal activity-dependent cell survival is mediated by calcium influx. But the process by which calcium influx promotes neuronal survival remains largely unknown. The long-term objective of our research is to understand the mechanisms by which neuronal activity regulates gene expression. This present application is specifically designed to study how membrane depolarizationinduced calcium influx affects the regulation and function of the transcription factor, myocyte enhancer factor 2 (MEF2), in the context of a cell survival model of cultured primary neonatal cerebellar granule neurons. The specific aims of this application include: 1. Identify the kinases and phosphatases that regulate MEF2 activity in calciumdependent neuronal survival; 2. determine calciium-regulated sites of phosphorylation and dephosphorylation on MEF2A; 3. characterize the mechanisms by which MEF2 mediates calcium-dependent neuronal survival. Membrane depolarizing concentrations of extracellular potassium chloride mimics neuronal activity in vivo to promote survival of neonatal cerebellar granule neurons. This model will be used to determine by kinase assay or phospho Westernblot analysis the activities of specific isoforms of p38 MAPK, PKCs as well as calcineurin in response to calcium influx. The critical sites of (de)phosphorylation on MEF2A by these kinases and phosphatase will be determined by a combined approach of phosphopeptide mapping and phosphoamino acid analysis. Dimerization, subcellular localization, DNA binding, and transcription activation assays, combined with mutagenesis and phospho-antibody analysis, will be used to determine the effects of (de)phosphorylation of these sites on MEF2 function following calcium signals. Finally, the mechanisms by which MEF2 mediates survival will be studied by establishing whether KC1 and MEF2 regulate the expression of pro-survival gene bc1-2 in bc1-2 promoter-driven luciferase reporter assay and whether Bc1-2 can rescue neurons from apoptosis when calcium-dependent activation of MEF2 is blocked. These experiments will define the mechanisms of calcium-dependent, MEF2-mediated neuronal survival which are essential to our understanding of both physiological and pathological changes in developing neurons. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: DEVELOPMENT
CALCIUM-MEDIATED
MODULATION
OF
CARDIAC
Principal Investigator & Institution: Porter, George A.; Pediatrics; Yale University 47 College Street, Suite 203 New Haven, CT 065208047 Timing: Fiscal Year 2002; Project Start 08-JUL-2002; Project End 30-JUN-2007 Summary: (provided by applicant): This Mentored Clinical Scientist Developmental Award application describes a program of career development and training for the applicant, who has completed a residency in pediatrics and a fellowship in pediatric cardiology. This program will be under the mentorship of Dr. Scott Rivkees, who has experience grooming young M.D. and Ph.D. trained scientists for independent research and who is a recognized leader in the field of receptor biology. The applicant intends to study the origins of abnormal heart development by using his expertise in muscle cell biology and in the physiology of congenital heart lesions and by continuing his training in developmental molecular biology. The research plan presented herein will focus on the role of intracellular calcium signals in controlling cardiac development. Calcium's role as an intracellular messenger is well-recognized. Preliminary data demonstrate that alterations in intracellular calcium levels and the signal cascades that lie downstream of calcium can dramatically alter cardiac development. In addition, previous studies have demonstrated the presence of calcium regulatory mechanisms in the early mammalian heart. To further understand these phenomena, following Specific Aims are proposed.
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(1) I will examine whether a reduction of intracellular calcium levels affect cardiac myocyte differentiation and cardiac morphogenesis. (2) I will examine the role of CaMK II in the regulation of cardiac morphogenesis and myocyte differentiation. Finally, (3) I will determine whether calcium signals modulate TAF expression and function in the developing heart. Yale University is a world leader in biomedical research, and the Department of Pediatrics has proven itself to be an ideal setting to train pediatric physician-scientists. A prestigious advisory committee drawn from the faculty at Yale will monitor the applicant's progress during this project. Combined with Dr. Rivkees mentorship, these resources should allow the applicant to develop into a successful independent investigator and make great contributions to the field of developmental cardiology. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CALMODULIN AND CGMP CONTROL OF OSTEOCLASTIC H+ SECRETION Principal Investigator & Institution: Blair, Harry C.; Professor of Pathology and of Cell Biolo; Pathology; University of Pittsburgh at Pittsburgh 350 Thackeray Hall Pittsburgh, PA 15260 Timing: Fiscal Year 2001; Project Start 01-SEP-1994; Project End 30-JUN-2004 Summary: Management of osteoporosis is limited by poor understanding of how bone mass is regulated. Osteoclasts are the acid-secreting cells that degrade bone. We found that Ca+2/calmodulin at the acid-secreting membrane of osteoclasts is required for HCI production. In contrast, inactivation of acid secretion is calmodulin independent, but is mediated by the cGMP- dependent enzymes function in a coordinated counterregulatory processes that mediates the reversible phosphorylation of proteins in the acid secreting membrane to regulate osteoclastic activity. We will study this hypothesis using human osteoclasts in vitro. Aim 1 will determine the role of local intracellular Ca+2 increases (calcium puffs or sparks) that occur near the acid-secreting cell membrane. H+- ATPase or acid secretion will be modified, and effects on intracellular calcium puffs will be measured using fluorescent indicators. The effects of calcium transport antagonists will be studied to identify the mechanism(s) that cause the calcium puffs, and the effects of calcium puffs on membrane protein phosphorylation will be determined. Aim 2 will use adenovirus infection to modify osteoclastic expression of the cGMP- dependent protein kinase and calcineurin (the calmodulin-dependent phosphatase). Eliminating the kinase should promote acid transport and abolish cGMP effects in osteoclasts; constitutive kinase should inactivate acid secretion. In cells without calcineurin, phosphorylation of membrane regulatory proteins should increase, decreasing acid secretion. Aim 3 will characterize the interaction of cGMP and Ca+2 signals in osteoclasts. How calmodulin-dependent cGMP phosphodiesterase activity varies with cGMP, acid secretion, and calcium puff activity will be assessed, using Western analysis and immune localization, to establish the role of this enzyme in balancing cGMP and Ca+2/calmodulin effects. Interactions of cGMP- and calmodulindependent kinases/phosphatases in the acid-secreting cell membrane will also be studied, using kinase, membrane activity, and immune localization assays. This will show whether Ca+2/calmodulin and cGMP counter- regulation occurs via common target proteins, and how distribution of regulatory proteins varies with cGMP and calcium signals. These studies will characterize an intracellular regulatory system that controls osteoclastic activity. This knowledge will be useful in understanding how osteoporosis develops, and may assist in the design of treatments to reduce bone resorption and prevent osteoporosis.
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Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CALMODULIN, AGING AND CALCIUM HOMEOSTASIS Principal Investigator & Institution: Squier, Thomas C.; Associate Professor; University of Kansas Lawrence Lawrence, KS 66045 Timing: Fiscal Year 2001; Project Start 01-SEP-1995; Project End 31-MAR-2006 Summary: Our long term goal is to identify the molecular mechanisms underlying the proposed linkage between oxidative stress and the age-dependent loss of calcium regulation. Based on our previous findings that during aging multiple methionines in the calcium regulatory protein calmodulin (CaM) are oxidatively modified to their corresponding sulfoxides and the key role that CaM plays in intracellular signaling, we hypothesize that age- related decreases in CaM function play a major role in the loss of calcium homeostasis observed in senescent cells. The accumulation of oxidatively modified and functionally inactive CaM during aging is consistent with a decreased function of cellular repair and degradative enzymes in senescent animals. Thus the specific activity of methionine sulfoxide reductase (MsrA), which is able to repair oxidized CaM in vitro and restore the ability of oxidized CaM to activate the plasma membrane Ca- ATPase (PMCA), may be compromised during aging. Likewise, agerelated decreases in the function of the proteasome, which normally degrades oxidized proteins, may result in the accumulation of inactive CaM. Therefore, to identify the cellular effects of CaM oxidation, we propose to test whether changes in CaM function observed in vitro using purified CaM are important in vivo for modulating cell function. These measurements will combine biochemical measurements of protein function using genetically engineered CaM mutants with altered sensitivities to oxidative stress with well defined measurements of cellular function and calcium regulation of neurons in cell culture. It is our expectation that the oxidative modification of CaM correlates with the loss of calcium regulation and enhanced sensitivity of neurons to oxidative stress. We therefore propose the following specific aims: (1) Identify how methionine oxidation in CaM alters target protein activation, (2) Investigate the cellular consequences of CaM oxidation on calcium homeostasis and neuronal visibility, (3) Determine the physiological role of MsrA in the maintenance of cell function, and (4) Investigate functional modifications to cellular repair and degradative systems during aging. An understanding of cellular mechanisms that modify calcium homeostasis under conditions of oxidative stress in the role of CaM oxidation in modifying cellular survival will be important to the development of possible therapies that could alleviate the decline in cellular functions associated with aging. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: CALPAINS AND AGING AND AD PATHOGENESIS Principal Investigator & Institution: Adamec, Emil; Mc Lean Hospital (Belmont, Ma) Belmont, MA 02478 Timing: Fiscal Year 2001; Project Start 01-JUN-1997; Project End 30-APR-2003 Summary: (Adapted from the application) Changes in the rate or efficiency of proteolysis have been implicated as potentially important factors in the mechanism of aging. One of the pathophysiological abnormalities associated with aging and agerelated neurodegenerative disorders might be abnormal regulation of intracellular calcium homeostasis, which can lead to excessive activation of calcium-dependent enzymes, such as calpains, as seen in Alzheimer s disease. Calcium-mediated injury is considered as one potentially important contributing mechanism in neurodegeneration.
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This application is based on the hypothesis that abnormalities in the regulation of calcium-activated neutral proteinase (calpain) represent important pathophysiological mechanisms in etiopathogenesis of neurodegeneration and aging. The broad, long-term objectives of this proposal are to provide the educational, environmental, and technical resources necessary for the candidate to become an independent researcher. In the educational part of the proposal, the candidate will gain further knowledge of molecular biology, advanced neurobiology, mathematical modeling and statistical analysis. In the scientific part of the proposal, the candidate will apply the knowledge to answering specific questions of the pathogenesis of neurodegeneration and aging. Specific aim 1 will determine which factors regulate the recovery of neurons from calcium load, using single cell digital imaging of cell calcium. Specific aim 2 will determine if chronic decrease in the cellular concentration of calpastation will affect the vulnerability of cells to calcium-mediated injury, using antisense oligodeoxynucleotides to generate gradual and prolonged depletion of calpastatin. In specific aim 3 the candidate will establish neuronal cultures with downregulated or upregulated calpain/calpastatin system using genetic engineering and determine the pathophysiological consequences of the manipulation. Specific aim 4 will test if combinational therapy with calpain inhibitors together with other putative neuroprotective agents provides higher level of neuroprotection as compared to calpain inhibitors alone. Successful completion of the proposed experiments will provide new insight into fundamental mechanisms of neurodegeneration. The results might suggest new avenues and approaches for the prevention and treatment of neuronal death in age-related neurodegenerative disorders and stroke. Successful completion of the proposal will give the candidate firm foundation in neuropharmacology, molecular biology, cellular physiology, and computational neuroscience to become a successful and independent researcher in cellular biology of neuronal degeneration. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CARBONATED DAIRY CALCIUM SOFT DRINK Principal Investigator & Institution: Schroder, Bruce G.; Dairilean, Inc. Box 88647 Sioux Falls, SD 57105 Timing: Fiscal Year 2001; Project Start 30-SEP-1999; Project End 31-JAN-2003 Summary: (Scanned from the Applicant's Abstract): Americans, especially teenage girls and young women, do not consume the recommended amount of calcium per day. This inhibits proper bone development and contributes to other health problems including, osteoporosis. In recent decades consumption of milk has declined significantly and the consumption of soft drinks has increased. Teenagers consume on average more than 64 gallons of soft drinks annually. During Phase I research a good tasting carbonated soft drink, containing 300 mg of dairy calcium and minerals per 10 ounce serving was developed. The Phase II research will refine and test a dairy calcium soft drink product and an orange juice product supplemented with dairy calcium. The bioavailability of the calcium in the two products will be tested against milk and Tropicana calcium supplemented orange juice. Introduction of the product along with a calcium education program into schools and retail outlets will also be tested. At the completion of Phase II research Dairilean will market the product either through a regional marketing plan that has been developed or through a licensing agreement with a large soft drink manufacturer. PROPOSED COMMERCIAL APPLICATION: Not available Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: CAROTID BODY EXCITATION--NEW CONCEPT Principal Investigator & Institution: Shirahata, Machiko; Associate Scientist; Environmental Health Sciences; Johns Hopkins University 3400 N Charles St Baltimore, MD 21218 Timing: Fiscal Year 2001; Project Start 05-JUL-1999; Project End 30-JUN-2003 Summary: (Applicant's abstract): The carotid body is a major chemoreceptor organ whose excitation causes reflex responses in cardiopulmonary, renal and endocrine systems. Although the mechanisms of carotid body excitation are not yet clear, essential steps include the depolarization of chemosensitive glomus cells, the increase in glomus cell intracellular calcium and the release of neurotransmitters. Many studies point to the involvement of oxygen-sensitive potassium channels, but a causal relationship between the inhibition of these channels and the depolarization of glomus cells during hypoxia has not yet been established. Since cat glomus cells release acetylcholine even under normoxic/normocapnic conditions, we hypothesize that hypoxia augments the activity of neuronal nicotinic acetylcholine receptors and/or enhances the sensitivity of acetylcholine receptors for acetylcholine. This initiates the depolarization of glomus cells and the increase in intracellular calcium. Oxygen-sensitive potassium channels and voltage-gated calcium channels participate in the later phase of the changes. Preliminary data have shown that: 1) cat glomus cells expressed alpha-4 subunit containing nicotinic acetylcholine receptors, 2) acetylcholine-induced inward current and carotid body neural output were enhanced by a mild decrease in oxygen tension from normoxic levels, 3) acetylcholine increased calcium of carotid body cells, 4) oxygen-sensitive potassium current was linearly inhibited by decreasing oxygen, and 5) increased carotid body neural output in hypoxia was inhibited by L-type voltage gated calcium channels. Specific aims are to investigate: 1) the role of acetylcholine and nicotinic acetylcholine receptors for initiating the depolarization of glomus cells and the increase in calcium, 2) the contribution of oxygen sensitive potassium channels in the late phase of glomus cell depolarization during hypoxia, 3) the contribution of voltage gated calcium channels to the late phase of the calcium increase in glomus cells during hypoxia. Patch clamp, microfluorometric, and immunocytochemical techniques are to be used. This innovative proposal will advance the understanding of the excitation mechanisms of glomus cells. Once the chemotransductive mechanisms are understood, pharmacological or genetic tools can be developed to alter the carotid body function to levels desirable for treating carotid body related pathological conditions such as sudden infant death syndrome and hemodynamic changes in sleep apnea patients. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: CELLULAR CALCIUM TRANSPORT IN URINARY EPITHELIA Principal Investigator & Institution: Friedman, Peter A.; Professor; Pharmacology; University of Pittsburgh at Pittsburgh 350 Thackeray Hall Pittsburgh, PA 15260 Timing: Fiscal Year 2001; Project Start 01-SEP-1998; Project End 31-MAR-2003 Summary: The long-term objective of our work is directed toward a complete understanding of the cellular mechanisms and regulation of calcium transport by renal tubular epithelial cells. Although most calcium absorption proceeds in proximal tubules, distal tubules are the site of the physiological regulation of calcium transport by parathyroid hormone (PTH), calcitonin and vitamin D3. The specific aims of the present proposal are to: 1) characterize the mechanism of calcium entry across apical plasma membranes of distal convoluted tubule (DCT) cells; 2) evaluate the participation and regulation of Na+/Ca2+ exchange in mediating calcium efflux; and, 3) define the
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signaling pathways activated by PTH and calcitonin in DCT cells, and to examine the coordinate regulation of PTH-dependent calcium transport by 1,25(OH)2 vitamin D3 and estradiol. We identified and partially characterized a novel calcium channel that mediates calcium entry. To acquire additional information about these channels we will: 1) characterize the properties of these channels with regard to their ion selectivity, pharmacologic sensitivity and voltage- dependence; 2) determine the regulation of these calcium channels by protein kinases; 3) define the participation of G-proteins in regulating calcium entry channels; and, 4) identify and clone calcium channel transcripts. Extrusion of calcium across basolateral plasma membranes of distal tubules is mediated by Na+/Ca2+ exchange and by /ca2+-ATPase. Pilot studies suggest that Na+/Ca2+ exchange is the dominant efflux mechanism in DCT cells. We will: 1) Test the hypothesis that thiazide diuretics can inhibit Na+/Ca2+ exchange; and 2) Characterize the stimulatory effects of PTH on Na+/Ca2+ exchange. Calcium transport in distal nephrons is regulated by PTH, calcitonin and 1,25(OH)2 vitamin D3. The following studies will evaluate the mechanism mediating the hormonal regulation of calcium transport in DCT cells. The goals of these experiments are to: 1) Characterize the signaling pathways with particular regard to the phospholipases responsible for activating PKA and PKC and characterize the temporal sequence in which PKA and PKC are activated. We will test the hypothesis that PTH and calcitonin activate PKC via phospholipase D. 2) Identify the mechanism by which PTH activates C1- channels, a primary event in membrane hyperpolarization and stimulation of calcium transport in DCT cells. We will test the hypothesis that these chloride channels are regulated by the PKA limb of the PTH-activated signaling pathway. 3) Examine the regulation of vitamin D3 and estradiol accelerate PTH dependent calcium transport by up-regulating PTH receptor expression. The specific aims will be achieved by applying single cell fluorescence, patch clamp, tracer flux measurements, biochemical and molecular techniques to a DCT cell line that we developed, which expresses an appropriate phenotype. In selected studies, primary cell cultures of proximal or distal tubule cells will be used to verify results in transformed cells or as controls. All procedures are established in our lab. Results from the proposed experiments will provide new information on the mechanism and regulation of calcium transport in the kidney under normal conditions, but also in calcium-wasting syndromes including hyperparathyroidism, renal failure, osteoporosis and malignancy- associated hypercalcemia. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CGMP DIFFERENTIATIONS
CHANNEL
ISOFORMS
AND
KERATINOCYTE
Principal Investigator & Institution: Mauro, Theodora M.; Dermatology; University of California San Francisco 500 Parnassus Ave San Francisco, CA 94122 Timing: Fiscal Year 2001; Project Start 01-AUG-1997; Project End 30-JUN-2003 Summary: (Adapted from the applicant's abstract) - Calcium influx, required for keratinocyte differentiation, is enhanced by treatment with permeant cGMP. Moreover, the investigators have found a keratinocyte channel that is activated by cGMP. Finally, they have cloned complete and alternatively spliced isoforms of a cGMP channel that is similar to the calcium-permeable channel found in photoreceptor rod cells. The isoforms differ in their cGMP sensitivity, and their expression is unique in that it is developmentally regulated. The investigators hypothesize that this channel regulates keratinocyte differentiation through its role as cGMP-gated calcium influx pathway. The broad, long-term objective of these studies is to develop methods that modify
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keratinocyte differentiation by manipulating calcium influx into these cells. The healthrelatedness of the project stems from the fact that abnormal keratinocyte differentiation underlies common skin diseases, both benign; psoriasis, abnormal wound healing; and malignant: squamous cell carcinoma. Thus, new approaches to modifying cell differentiation would have wide clinical applications. The specific aims of the project are: 1) To correlate the exact proportion of each isoform with the degree of keratinocyte differentiation; 2) to compare channel characteristics in differentiating and undifferentiated keratinocytes; 3) to examine the channel's role in keratinocyte differentiation by overexpressing the native, alternatively spliced, or mutant isoforms in undifferentiated and differentiating keratinocytes. To accomplish these aims, the research design will first quantify the proportion of each isoform using the RNAse protection assay. Next, the functional difference conferred by the different isoforms will be examined by comparing 1) ion channel and whole cell current characteristics, using the patch clamp method; 2) calcium influx, using calcium-45 studies; and 3) intracellular calcium concentration, using Fluo-3 measurement. Finally, alteration of calcium influx and keratinocyte differentiation by overexpression of channel isoforms in keratinocytes will be tested. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: CHEMORESPONSE IN PARAMECIA: ROLE OF THE CA2+ PUMP Principal Investigator & Institution: Gannon-Murakami, Laura J.; Biology; University of Vermont & St Agric College 340 Waterman Building Burlington, VT 05405 Timing: Fiscal Year 2002; Project Start 01-FEB-2002 Summary: (provided by applicant): Calcium levels in cells are controlled in part by the plasma membrane calcium ATPases. The calcium pump is highly conserved from paramecia to humans and has many sites of control including phosphorlation by protein kinases and binding calmodulin. Mutations in this plasma membrane pump have been shown to cause such diseases as deafness and balance deficits. We are interested in its role in chemoresponse. We have found indirect evidence that activation of the calcium pump figures into the transduction pathways for chemoreception in Paramecium. (See evidence described in the proposal.) I propose to examine the regulation of the Paramecium calcium pumps and their potential roles in chemosensory signal transduction. Specifically, I plan to test the hypotheses that the calcium pump is activated by phosphorylation of and by calmodulin binding to the autoinhibitory domain (also called calmodulin binding domain, or CBD) in two different chemosensory pathways. In preparation, I have created CBDs in which two conserved serines are replaced by alanines (AA) or glutamates (EE). As expected, the wild type CBD is phosphorylated by protein kinases A and C in vitro, but the mutant AA and EE CBDs were not. Calmodulin binds to the wild type and AA CBD, but not to the EE mutant. I will use the CBDs as specific inhibitory reagents to probe for the role of phosphorylation and calmodulin binding in chemosensory signal transduction and to test my hypotheses. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen
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Project Title: CORONARY ARTERY REGULATION BY CA2+SIGNALING & ESTROGEN Principal Investigator & Institution: Nelson, Mark T.; Professor and Chair; Pharmacology; University of Vermont & St Agric College 340 Waterman Building Burlington, VT 05405
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Timing: Fiscal Year 2001; Project Start 15-SEP-2000; Project End 31-JUL-2004 Summary: (Verbatim from the application): The cardioprotective effect of estrogen is complex, and incompletely understood. We have identified one potentially important action of physiological circulating estrogen on coronary arteries, which involved an elevation of basal nitric oxide release from the endothelium and an ensuing smooth muscle relaxation through activation of one type of calcium-sensitive (large conductance; BK) potassium channel in the smooth muscle. Our results indicate that estrogen causes fundamental alterations in Ca2+ signaling in the coronary endothelium, which leads to an elevation of nitric oxide production, which in turns alters Ca2+ signaling in the smooth muscle. This proposal focuses on novel, interlinked Ca2+ signaling mechanisms to explain the effects of estrogen and nitric oxide on coronary artery diameter. Specifically, Aim 1 will determine the mechanisms by which estrogen leads to an elevation of endothelial [Ca2+]i; Specific Aim 2 will explore positive feedback regulation of endothelial Ca2+ by local and global Ca2+ signaling from ryanodine receptors (RyRs) in the endoplasmic reticulum to small conductance calciumsensitive (SK) and BK channels; Specific Aim 3 will determine the negative feedback mechanisms by which endogenously produced nitric oxide activates RyRs and BK channels in coronary artery smooth muscle, with a focus on the key roles of phospholamban, which regulates SR Ca2+-ATPase activity, and on the beta-subunit of the BK channel. To address these issues, we have developed techniques to measure global and local calcium in the endothelium and smooth muscle of intact pressurized coronary arteries, including coronary arteries from phospholamban and beta-subunit gene-ablated mice, and developed novel, selective peptide inhibitors of cGMPdependent protein kinase. The proposed study should significantly deepen our understanding of the regulation of Ca2+ signaling in coronary arteries, and the important influences of physiological estrogen and nitric oxide on coronary artery function. This work should also suggest novel mechanisms for therapeutic interventions to mimic the beneficial effects of estrogen and nitric oxide on calcium signaling in coronary arteries. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •
Project Title: DAIRY PRODUCTS, CALCIUM, AND RISK OF PROSTATE CANCER Principal Investigator & Institution: Lee, I-Min M.; Assistant Professor of Medicine; Epidemiology; Harvard University (Sch of Public Hlth) Public Health Campus Boston, MA 02460 Timing: Fiscal Year 2003; Project Start 01-JUN-2003; Project End 31-MAY-2005 Summary: (provided by applicant): Prostate cancer is the most common cancer among men in the U.S. (apart from non-melanoma skin cancer), and is a leading cause of death. Unfortunately, well-established risk factors for this disease (e.g., age) are not modifiable. In the search for modifiable predictors, calcium intake has been proposed to be a risk factor. A high intake of calcium may increase risk by down-regulating 1,25 hydroxyvitamin D3, a hormone that inhibits prostate cancer cell growth and development. It is important to clarify the relation between calcium intake and risk of prostate cancer because calcium is important in the prevention and treatment of osteoporosis. Epidemiologic data on the association of calcium intake and prostate cancer risk have been inconsistent. Few studies have specifically investigated intake of calcium from dairy products and risk of prostate cancer. The data on calcium supplement use and prostate cancer risk are even sparser. Additionally, little information is available on whether the association of calcium intake and prostate cancer risk is modified by age. This is an important question since older men are at
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higher risk of osteoporosis. Finally, there is a suggestion that calcium intake may be more strongly related to advanced prostate cancer, but few data are available. We propose to analyze the associations of calcium intake from dairy products and supplements among 11,122 men (mean age, 67 years) who were free of cancer in 1988. Men reported on their diet, including intake of dairy products and calcium supplements, in 1988. Information on the development of nonfatal prostate cancer is available through 1998; fatal prostate cancer, 1995. We propose to collect another 3 years of mortality data through 1998. We anticipate a total of 800 prostate cancers (including 116 fatal cancers) to occur among subjects, providing sufficient statistical power to address the questions of interest. We will examine the associations of calcium intake from dairy products and supplements with risk of all and fatal prostate cancer. A consistent increased risk seen with both calcium from dairy products and calcium supplements lends weight to the hypothesis that it is calcium, rather than other nutrients in the dairy products, that is responsible for the increased risk. We also will examine men aged
