Oxygen sensing: molecule to man

OXYGEN SENSING MOLECULE TO MAN

ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY Editorial Board: NATHAN BACK, State University of New York at Buffalo IRUN R. COHEN, The Weizmann Institute of Science DAVID KRITCHEVSKY, Wistar Institute ABEL LAJTHA, N. S. Kline Institute for Psychiatric Research RODOLFO PAOLETTI, University of Milan Recent Volumes in this Series Volume 467 TRYPTOPHAN, SEROTONIN, AND MELATONIN: Basic Aspects and Applications Edited by Gerald Huether, Walter Kochen, Thomas J. Simat, and Hans Steinhart Volume 468 THE FUNCTIONAL ROLES OF GLIAL CELLS IN HEALTH AND DISEASE: Dialogue between Glia and Neurons Edited by Rebecca Matsas and Marco Tsacopoulos Volume 469 EICOSANOIDS AND OTHER BIOACTIVE LIPIDS IN CANCER, INFLAMMATION, AND RADIATION INJURY, 4 Edited by Kenneth V. Honn, Lawrence J. Marnett, and Santosh Nigam Volume 470 COLON CANCER PREVENTION: Dietary Modulation of Cellular and Molecular Mechanisms Edited under the auspices of the American Institute for Cancer Research Volume 471 OXYGEN TRANSPORT TO TISSUE XXI Edited by Andras Eke and David T. Delpy Volume 472 ADVANCES IN NUTRITION AND CANCER 2 Edited by Vincenzo Zappia, Fulvio Delia Ragione, Alfonso Barbarisi, Gian Luigi Russo, and Rossano Dello Iacovo Volume 473 MECHANISMS IN THE PATHOGENESIS OF ENTERIC DISEASES 2 Edited by Prem S. Paul and David H. Francis Volume 474 HYPOXIA: Into the Next Millennium Edited by Robert C. Roach, Peter D. Wagner, and Peter H. Hackett Volume 475 OXYGEN SENSING: Molecule to Man Edited by Sukhamay Lahiri, Naduri R. Prabhakar, and Robert E. Forster, II

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OXYGEN SENSING MOLECULE TO MAN Edited by

Sukhamay Lahiri University of Pennsylvania Medical Center Philadelphia, Pennsylvania

Nanduri R. Prabhakar Case Western Reserve University Cleveland, Ohio

and

Robert E. Forster, II University of Pennsylvania Medical Center Philadelphia, Pennsylvania

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PREFACE By a close vote in Chile, 1996, the membership decided to hold the XIV meeting of the International Society of Arterial Chemoreception (ISAC) in Philadelphia,, USA, with Sukhamay Lahiri as its president. The XIV meeting of the ISAC was held on June 24-28, 1999, in Philadelphia. Since its inception, these meetings have been focused on arterial chemoreceptors and their functions. This time, it was expanded to include oxygen sensing in other tissues and cells in the body, and the genes involved. This genetic flavour made the meeting more exciting, and it was attended by more than two hundred participants at a time. The idea was to bring together scientists from cellular and systemic boundaries of physiology, working at the interface of cellular and molecular biology. As a result, a new conference was born and the title of the conference was Oxygen Sensing: Molecule to Man. The organizers of the conference were Sukhamay Lahiri and Nanduri Prabhakar, Dr. Robert W. Torrance died on January 8, 1999. C.C. Michel who was a student of his and was close to him presented a Tribute. Two other students of his, Mark Hanson and Prem Kumar, also wrote reminiscences. Bob also collaborated with Patricio Zapata in Chile occasionally during the last few years. Patricio and Carolina also wrote a reminiscence. Bob was a founder member of the ISAC, and as the president of the Oxford meeting, 1966, when he wrote a seminal article, prolegomena, reviewing the state of knowledge of arterial chemoreception at that time. This article was a landmark in the history of chemoreception. One special feature of the symposium was that twelve experts had been asked to write articles for a volume of Respiration Physiology pertaining to the symposium, which was precirculated to the participants. This volume gave a preview of the symposium, thanks to the Editor of Respiration Physiology, Dr. Peter Scheid. It was to provide a focus for the participants and background reading for the forthcoming

meeting.

The first two days of the symposium were devoted to the genes and genetic expression of oxygen sensing, beginning with systemic phenomena in man and animals. It quickly went onto bacteria and yeast, bringing out how similar their oxygen sensing was to the mammals at the molecular level. As the meeting proceeded, it became clear that oxygen sensing in mammals can be divided primarily into two distinct categories: one is the membrane based NAD(P)H oxidase systems and another is mitochondrial. These were present in various cells and systems. A controversy arose as to how more oxygen radicals are generated during hypoxia. It was felt that the differences probably pertained to demanding methods using fluorescent dye. Potassium ion channels figured most prominently in the glomus cell membrane depolarization. Inhibition of the classical oxygen sensitive potassium currents did not inhibit oxygen sensing, as a result the role of voltage insensitive leak potassium current was v

resurrected, in addition to the of voltage sensitive HERG–like potassium current in all the glomus cells. Several features of glomus cells fit the criteria of oxygen chemoreceptors but their sensory discharge did not always fit these criteria. It was therefore proposed that sensory nerves may be the ultimate sensors whereas glomus cells play only a secretory role, but recoding from the petrosal ganglion cells did not support this notion. There were fourteen papers from the young investigators competing for the awards, four of whom were judged winners by the audience: two for the Heymans-de Castro-Neil Awards, (Beth Ann Summers and Roger J. Thompson) and two for Comroe, Forster & Lambertsen Awards (Ricardo Pardal and Nicholas A. Ritucci). There was a roundtable conference at the end of the second day, discussing the genomic aspects. A similar roundtable conference was originally planned to take place at the end of fourth day but by then there had been so much discussion that it was felt unnecessary. The original gramophone recordings of the carotid body sensory discharged, which were made in the 1930s by Drs. Zotterman, von Euler,and Liljestrand, were played to the audience at the banquet. We are grateful to Professor Curt von Euler, Karolinska Institut, Stockholm, Sweden, for the gift. The council of the ISAC met and at a business meeting the membership decided on the venue of the next meeting which is to be Lyon, France, in 2002, with Jean-Marc Pequignot as its president. Tentatively the meeting after that is to be held

in Kita-kyushu, Japan,in 2005. There will be a section on Arterial Chemoreception at IUPS Meeting in New Zealand,in 2001. The Symposium was only possible because of the funds made available to us by generous gifts, particularly from the Barra Foundation, US Army Research Administration, Ecosystems Tech Transfer, Inc.,and by Merck & Company, and for the contribution of David E. Millhorn. The Division of Lung Diseases National Heart, Lung,and Blood Institute provided a conference grant (R13-III–60955). We are also fortunate to have received additional anonymous donations. We are grateful to them all. Finally, we are grateful to the participants who came and contributed to the success of the Symposium. Special thanks are due to Mrs. Mary Pili (University of

Pennsylvania, Philadelphia, PA., USA) and Mrs. Marianne Sperk (Case Western Reserve University, Cleveland, Ohio, USA) and Mrs. Michele Deparc, Ruhr-University at Bochum, Germany, for their secretarial managements.

The Editors Sukhamay Lahiri (Philadelphia, PA, USA) Nanduri R. Prabhakar (Cleveland, OH, USA) Robert E. Forster, II (Philadelphia, PA, USA) August 12, 1999

vi

CONTENTS

A Tribute to Robert W. Torrance................................................ Charles C. Michel

1

Reminiscence of Bob Torrance (1) ............................................. Patricio and Carolina Zapata

7

Reminiscence of Bob Torrance (2) ........................................... Mark Hanson and Prem Kumar

9

Genomics of Oxygen Sensing Placticity and Multiplicity in the Mechanisms of Oxygen Sensing ....... Sukhamay Lahiri

13

Evolution of Human Hypoxia Tolerance Physiology ........................

25

Comparative Aspects of High-Altitude Adaptation in Human Populations..........................................................................................

45

Peter W. Hochachka, and C. Carlos Monge

Lorna G. Moore, V. Fernando Armaza, Mercedes Villena, and Enrique Vargas

Tibetan and Andean Contrasts in Adaptation to High-Altitude Hypoxia............................................................................................

63

A Genomic Model for Differential Hypoxic Ventilatory Responses .... Clarke G. Tankersley

75

Cynthia M. Beall

Regulation of the Hypoxia-Inducible necessary for hypoxic induction of

: ARNT is not in the nucleus ..................

Max Gassman, Dmitri Chilov, and Roland H. Wenger

Intracellular Pathways Linking Hypoxia to Activation of c-fos and

AP-1..................................................................................

Daniel R. Premkumar, Gautam Adhikary, Jeffery L. Overholt, Michael S. Simonson, Neil S. Cherniack, and Nanduri R. Prabhakar

87

101

vii

Hypoxia-Induced Regulation of mRNA Stability ...................................... Waltke R. Paulding and Maria Czyzyk-Krzeska

111

Hypoxia, HIF-1, and the Pathophysiology of Common Human Diseases..... Gregg L. Semenza, Faton Agani, David Feldser, Narayan Iyer, Lori Kotch, Erik Laughner, and Aimee Yu

123

Gene Regulation during Hypoxia in Excitable Oxygen-Sensing Cells: Depolarization-Transcription Coupling ............................................................. David E. Millhorn, Dana Beitner-Johnson, Laura Conforti, P. William Conrad, Suichi Kobayashi, Yong Yuan, and Randy Rust Regulation of CREB by Moderate Hypoxia in PC 12 Cells............................ Dana Beitner-Johnson, Randy T. Rust, Tyken Hsieh, and David E. Millhorn Reactive Oxygen Species as Regulators of Oxygen Dependent Gene Expression................................................................................................. Jochim Fandrey and Just Genius A Glycolytic Pathway to Apoptosis of Hypoxic Cardiac Myocytes:

Molecular Pathways of Increased Acid Production .................................. Keith A. Webster, Daryl J. Discher, Olga M. Hernandez, Kazuhito Yamashita, and Nanette H. Bishorpric Mitochondrial-Nuclear Crosstalk Is Involved in Oxygen-Regulated Gene Expression in Yeast................................................................................ Robert O. Poyton and Christopher J. Dagsgaard Rox1 Mediated Repression: Oxygen dependent repression in yeast............ Alexander J. Kastaniotis, and Richard S. Zitomer Oxygen Dependence of Expression of Cytochrome c and Cytochrome c Oxidase Genes in S. cerevisiae ................................................................... Pastricia V. Burke, and Kurt E. Kwast Hypoxic and Redox Inhibition of the Human Cardiac L-Type Channel........................................................................................... I.M. Fearon, A.C.V. Palmer, A.J. Balmforth, S.G. Ball, G. Varadi, and C. Peers Molecular Identification of and Poassium Channels in the Pulmonary Circulation ........................................................................ Stephen L. Archer, E. Kenneth Weir, Helen L. Reeve, and Evangelos Michelakis

viii

131

143

153

161

177

185

197

209

219

Chemosensing at the Carotid Body: Involvement of a HERG-like potassium current in glomus cells ...................................................... Jeffrey L. Overholt, Eckhard Ficker, Tianen Yang, Hashim Shams, Gary R. Bright, and Nanduri R. Prabhakar

Oxidant Signalling and Vascular Oxygen Sensing: Role of Responses of the Bovine Pulmonary Artery to Changes in

in ...............

241

249

Kamal M. Mohazzab-H. and Michael C. Wolin

Tissue and Mitochondrial Enzymes: Cytochrome c Oxidase as as Sensor.............................................................................................. D.F. Wilson, A. Mokashi, S. Lahiri, and S.A. Vinogradov

259

Regulation of Shaker-Type Potassium Channels by Hypoxia: Oxygen-sensitive channels in PC12 cells ...................................... Laura Conforti and David E. Millhorn

265

HIF-1 Is Essential for Multilineage Hematopoiesis in the Embryo ......... David M. Adelman, Emin Maltepe, and M. Celeste Simon

275

Dual Influence of Nitric Oxide on Gene Regulation during Hypoxia ...... Gautam Adhikary, Daniel R.D. Premkumar, and Nanduri R. Prabhakar

285

Hypoxia Differentially Regulates the Mitogen- and Stress-Activated Protein Kinases: Role of in the activation of MAPK and p38y .............................................................................. P. William Conrad, David E. Millhorn, and Dana Beitner-Johnson Chairman’s Summary: Mechanisms of Oxygen Homeostasis, Circa 1999 ......................................................................................... Gregg L. Semenza

293

303

Arterial Chemoreceptors

Oxygen, Homeostasis, and Metabolic Regulation ................................... Peter W. Hochachka

311

Evidence that Nitric Oxide Plays a Role in Sensing from Tissue NO and Measurements in Cat Carotid Body .................................. Donald G. Buerk and Sukhamay Lahiri

337

Carotid Body Gap Junctions: Secretion of Transmitters and Possible Electric Coupling between Glomus Cells and Nerve Terminals .............. Carlos Eyzaguirre

349

ix

Short- and Long-Term Regulation of Rat Carotid Body Gap Junctions by cAMP Identification of Connexin43, a Gap Junction Subunit............... Verónica Abudara, Carloa Eyzaguirre, and Juan C. Sáez

Subcellular Localization and Function of B-Type Cytochromes in Carotid Body and Other Paraganglionic Cells ..................................... Wolfgang Kummer, Brigette Höhler, Anna Goldenberg,and Bettina Lange Acetylcholine Sensitivity of Cat Petrosal Ganglion Neurons .................

Machiko Shirahata, Yumiko Ishizawa, Maria Rudisill, James S.K. Sham, Brian Schofield, and Robert S. Fitzgerald

Responses of Petrosal Ganglion Neurons in vitro to Hypoxic Stimuli

and Putative Transmitters ...............................................................

359

371

377

389

J. Alcayaga, R. Varas, J. Arroyo, R. Iturriaga, and P. Zapata The Metabolic Hypothesis Revisited .................................................. Charmaine Rozanov, Arijit Roy, Anil Mokashi, Shinobu Osanai, Peter Daudu, Bayard Storey, and

397

Sukhamay Lahiri

Effect of Adenosine on Chemosenstivity, Functional, Cellular and Molecular Studies ........................................................................ P. Kumar, A.F. Conway, C. Vandier, N.J. Marshall,

405

J. Bruynseels, and G.M. Matthews

The Present Status of the Mechanical Hypothesis for Chemoreceptor Stimulation............................................................................................ Ashima Anand and A.S. Paintal

411

Identification of An Oxygen-Sensitive Potassium Channel in Neonatal Rat Carotid Body Type I Cells .......................................................... Betrice A. Williams and Keith J. Buckler

419

Significancy of ROS in Oxygen Chemoreception in the Carotid Body Chemoreception: Apparent Lack of a Role of NADPH Oxidase ........... A. Obeso, G. Sanz-Flfayate, M.T. Agapito , and C. Gonzalez

425

ATP-Dependent and Voltage-Gated Channels in Endothelial Cells of Brain Capillaries: Effect of Hypoxia ..................................... Marco A. Delpiano

435

Different

441

x

Mechanisms by Different Gabriel C. Haddad and Huajun Liu

Channels................

Response of Intracellular pH to Acute Anoxia in Individual Neurons from Chemosensitive and Nonchemosensitive Regions of the Medulla..... Laura Chambers-Kersh, Nick A. Ritucci, Jay B. Dean, and Robert W. Putnam

453

Hyperbaric Oxygen Depolarized Solitary Complex Neurons in Tissue Slices of Rat Medulla Oblongata ............................................. Daniel K. Mulkey, Richard A. Henderson III, and Jay B. Dean

465

Chronic Hypoxia Induces Changes in the Central Nervous System Processing of Arterial Chemoreceptor Input......................................... M.R. Dwinell, K.A. Huey, and F.L. Powell

477

Acetylcholine Is Released from in vitro Cat Carotid Bodies during

Hypoxic Stimulation.............................................................................. R.S. Fitzgerald, M. Shirahata, and H-Y. Wang

485

Interactions between Acetylcholine and Dopamine in Chemoreception..... P. Zapata, C. Larraín, R. Iturriaga, J. Alcayaga, and C. Eyzauirre

495

Interactions between Catecholamines and Neuropeptides in the Carotid Body: Evidence for Dopamine Modulation of Neutral Endopeptidase Activity..................................................................................................... Ganesh K. Kumar, Eui K. Oh, and Myeong-Seon Lee

507

Pharmacological Effecs of Endothelin in Rat Carotid Body: Activation of Second Messenger Pathways and Potentiation of Chemoreceptor Activity.................................................................................................... J. Chen, L. He, B. Dinger, and S. Fidone

517

Oxygen and Acid Chemoreception by Pheochromocytoma (PC 12) Cells.... S.C. Taylor and C. Peers

527

Postnatal Changes in Cardiovascular Regulation during Hypoxia .............. Phyllis M. Gootman and Norman Gootman

539

Expression and Localization of A2a and Al-Adenosine Receptor Genes in the Rat Carotid Body and Petrosala Ganglia: A2a and A1-adenosine receptor mRNAs in the rat carotid body ................................................. E.B. Gauda Serotonin and the Hypoxic Ventilatory Response in Awake Goats .......... J.K. Herman, K.D. O’Halloran, and G.E. Bisgard

549 559

xi

Peripheral Chemosensitivity in Mutant Mice Deficient in Nitric Oxide Synthase.................................................................................................. David D. Kline and Nanduri R. Prabhakar

571

Dopaminergix Excitation in Goat Carotid Body May be Mediated by Serotonin Receptors ...... ........................................................................ K.D. O’Halloran, J.K. Herman, P.L. Janssen, and G.E. Bisgard

581

Augmentation of Calcium Current by Hypoxia in Carotid Body Glomus Cells........................................................................................ B.A. Summers, J.L. Overholt, and N.R. Prabhakar

589

in Developing Rat Adrenal Chromaffin Cells ......... Roger J. Thompson and Colin A. Nurse by Model Airway Chemoreceptors: Hypoxic inhibition of channels in H146 cells .................................................................... Ita O’Kelly, Chris Peers, and Paul J. Kemp Morphological Adaptation of the Peptidergic Innervation to Chronic Hypoxia in the Rat Carotid Body ........................................................ H. Matsuda, T. Kusakabe, Y. Hayashida, F.L. Powell, M.H. Ellisman, T. Kawakami, and T. Takenaka Continuous But Not Episodic Hypoxia Induces CREB Phosphorylation in Rat Carotid Body Type I Cells .......................................................... Z.-Y. Wang, T.L. Baker, I.M. Keith, G.S. Mitchell, and G.E. Bisgard Intracellular of the Carotid Body .................................................... D.F. Wilson, S.M. Evans, C. Rozanov, A. Roy, C.J. Koch, K.M. Laughlin, and S. Lahiri Redox-Based Inhibition of Channel/Current Is Not Related to Hypoxic Chemosensory Responses in Rat Carotid Body ........................................ Arijit Roy, Charmaine Rozanov, Anil Mokashi, and Sukhamay Lahiri

Effects of 2, 4-Dinitrophenol (DNP) on the Relationship between Intracellular Calcium of Glomus Cells and Chemosensory Activities of the Rat Carotid Body ........................................................................... Peter A. Dauda, Charmaine Rosanov, Arijit Roy, Anil Mokashi, and Sukhamay Lahiri

xii

601

611

623

631

637

645

655

Estimation of Chemosensitivity from the Carotid Body in Humans .... M. Tanaka, A. Masuda, T. Kobayashi, and Y. Honda Adenosine-Dopamine Interactions and Ventilation Mediated through Carotid Body Chemoreceptors .............................................................. Emilia C. Monteiro and J. Alexandre Ribeiro Carotid Body NO-CO Interaction and Chronic Hypoxia .......................... C. Di Giulio, A. Grillo, I. Ciocca, M.A. Macrì, F. Daniele, G. Sabatino, M. Cacchio, M.A. De Lutiis, R. Da Porto, F. Di Natale, and M. Felaco

663

671 685

Interplay between the Cytosolic Increase and Potential Changes in Glomus Cells in Response to Chemical Stimuli..................................... Yoshiaki Hayashida, Katsuaki Yoshizaki, and Tatsumi Kusakabe

691

Characteristics of Carotid Body Chemosensitivity in the Mouse: Baseline Studies for Future Experiments with Knockout Animals ........................ L. He, J. Chen, B. Dinger, and S. Fidone

697

Role of Substance P in Neutral Endopeptidase Modulation of Hypoxic Response of the Carotid Body ............................................................ Ganesh K. Kumar, Yu Ru-Kou, Jeffrey F. Overholt, and Nanduri R. Prabhakar Effect of Barium on Rat Carotid Body Glomus Cell and Carotid Chemosensory Response........................................................................ A. Mokashi, A. Roy, C. Rozanov, P. Daudu, and S. Lahiri

705

715

A Dual-Acid Influx Transport System in the Carotid Body Type I Cell: Acid influx in carotid body type I cells .......................................... Ke-Li Tsai, Richard D. Vaughan-Jones, and Keith J. Buckler

723

L-Dopa and High Oxygen Influence Release of Catecholamines from the Cat Carotid Body ........................................................................... Hay-Yan J. Wang, Machiko Shirahata, and Robert S. Fitzgerald

733

Effects of a Dopamine Agonist on Cytosolic Changes Induced by Hypoxia in Rat Glomus Cells ................................................................ Katsuaki Yoshizaki, Hideki Momiyama, and Yoshiaki Hayashida

743

Carotid Chemoreceptors Participation in Brain Glucose Regulation: Role of arginine-vasopressin .................................................................. Sergio A. Montero, Alexander Yarkov, and Ramon Alvarez-Buylla

749

xiii

Nitric Oxide Modulation of Carotid Chemoreception ............................ Rodrigo Iturriaga, Sandra Villanueva, and Julio Alcayaga and Respiration in Exercising Human Muscle: The Regulation of

761

Oxidative Phosphorylation in vivo .............................................................. Youngran Chung, Paul Mole, Tuan K. Tran, Ulrike Kreutzer, Napapon Sailasuta, Ralph Hurd, and Thomas Jue

769

pH Sensitivity in the Isolated CNS of Newborn Mouse ............................. Claudia D. Infante and Jaime Eugenin

785

Aortic Body Chemoreflex of the Anesthetized Rat: Electrophysiological

morphological and reflex studies ........................................................... James F.X. Jones

Changes in the Peptidergic Innervation of the Rat Carotid Body a Month after the Termination of Chonic Hypoxia ................................................ T. Kusakabe, Y. Hayashida, H. Matsuda, T. Kawakami, and T. Takenaka

789

793

Carotid Bodies and the Sigh Reflex in the Conscious and Anesthetised Guine-Pig....................................................................................................

801

Immunohistochemical Study of the Carotid Body during Hibernation.......

815

Daryl O. Schwenke and Patricia A. Cragg

Kazuo Ohtomo, Kohko Fukurara, and Katsuaki Yoshizaki

Neurochemical Reorganization of Chemoreflex Pathway after Carotid Body Denervation in Rats....................................................................... J.C. Roux, J. Peyronnet, O. Pascual, Y. Dalmaz, and

823

J.M. Pequignot

Index..........................................................................................................

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A TRIBUTE TO ROBERT W. TORRANCE

C. C. Michel Section of Cellular and Integrative Biology, Division of Biomedical Sciences, Imperial College School of Medicine, Exhibition Road, London SW7 2AZ, UK.

Bob Torrance, who organised the first international meeting entirely devoted to arterial chemoreceptors, died in Oxford on 8m January 1999. Bob will be remembered not only for that meeting, but for his own contributions to the subject both as a leader of a research group, as the author of reviews of great insight, a stimulating contributor to discussion and above all for his warm and generous personality. Bob Torrance spent nearly all his working life in Oxford, as a Fellow and Tutor of St John’s College and as Lecturer in the University Laboratory of Physiology. He did not start working on arterial chemoreceptors until the early 1960’s when he was in his late thirties. Appreciating the great stimulus which the Haldane Centenary Symposium had given to respiratory physiology in 1961, he toyed with idea of arranging a small meeting on chemoreceptors alone, to coincide with a visit which Carlos Eyzaguirre was planning to make to the UK in 1963. He discussed this with Sir Lindor Brown, who was head of the University Laboratory at the time and with Dan Cunningham, who with Brian Lloyd, had organised the Haldane Meeting. Lack of time in which to raise the necessary funds meant that the idea had to put aside but fortunately it was not forgotten. One Wednesday in the summer of 1965, Sir Lindor Brown happened to share a taxi with Sir Ifor Evans of University College, London. To pass the time of day, Evans told Brown that he was about to become a trustee of a small foundation which was being set up to support medical science. “What do you suggest that would be both different and worthwhile for us to encourage?” he asked. “Small scientific meetings” replied Brown and proceeded to expound enthusiastically on the benefits of face to face discussions and personal interactions in science particularly when people with slightly different expertise are brought together. “ There’s someone in my

Oxygen Sensing: Molecule to Man, edited by S. Lahiri et al. Kluwer Academic/Plenum Publishers, 2000

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department who has an interesting idea for a meeting of this kind,” he

concluded. “Let me have the proposal in three days” said Evans. So Brown returned to Oxford and told Bob Torrance. By Friday, the plans for a meeting on aortic and carotid chemoreceptors were in the post. On the following Wednesday, Bob heard that £2500 had been allocated for him set up the first Wales Foundation Symposium. Several factors made the meeting a remarkable success. First it was very much a family affair. While there was an organising committee (Sir Lindor

Brown, Eric Neil, Dan Cunningham and Bob), this met formally only once, though (according to the Preface of the Symposium Proceedings, 3) its members, encouraged by their secretary, communicated constantly. The secretary to the committee was Margaret Torrance (Bob’s wife) who, in

addition, acted as Bob’s secretary while also taking care of the general administration of the meeting including accommodation and social functions. The scientific programme was developed by Bob in consultation with Dan

Cunningham and Eric Neil. At that time the carotid and aortic chemoreceptors were usually described in the textbooks as “peripheral chemoreceptors.” By 1965, this term had lost its specificity for in the late 1950’s and early 1960’s there was speculation about the importance of chemoreceptors which detected changes in and in the mixed venous blood. To clarify that it was to be about the carotid and aortic receptors, the meeting was given the title “arterial chemoreceptors”, a term that has continued. Speakers were asked to address a particular topic so that a comprehensive view of the arterial

chemoreceptors could emerge. To provide participants with an up to date account of the subject, before the meeting they all received a scholarly review which Bob had written for the occasion. This review, which was circulated and eventually published in the proceedings of the symposium (3) under the title of “Prolegomena,” was the first of several important reviews on chemoreceptors that Bob was to write over the next two decades. It arose from a series of four advanced lectures which Bob had given in November and December 1965. They had been delivered on successive Saturday mornings and Bob wrote them up in a rough form for the benefit of one of his research students who had been unable to attend the lectures as he was captain of the Oxford University rugby

team. The rough copy began to circulate among other students and interested colleagues in the department, so Bob started to polish the text. At Eric Neil’s

suggestion, it was circulated to all participants “...for them to read or ignore as they think fit.” It was wonderful background reading for the meeting, and even those who were working on chemoreceptors at that time benefited from following Bob’s account of how an understanding of the chemoreceptors must be compatible with the broader picture of the regulation of breathing. Its main aim was to provide a focus for the speakers; to make it easier for them to address the specific questions which they had been asked to consider in their presentations. It was successful in this respect and perhaps even more so in

2

raising the standard of the general discussions. So successful were these, that instead of the meeting finishing on Friday lunchtime as planned, at the request of the participants, themselves, it continued until the end of the afternoon so that a final concluding discussion could be held. Over the following 20 years Bob Torrance and his students made a series of important contributions to our understanding of the properties of the chemoreceptors. Perhaps the two most important of these were first, the demonstration of their different transient responses to step changes in oxygen and carbon dioxide tensions (2) and then that impulses from the chemoreceptors (and other peripheral sense organs) only have reflex effects on breathing if they arrive in the central nervous system during inspiration (1). In addition, there were Bob’s reviews of the subject. These were always scholarly, intellectually stimulating and well written. Bob Torrance was born in Wolverhampton, Staffordshire on the 4th September, 1923 .His father taught Physics in the local grammar school. When the family moved to Yorkshire in the 1930’s, Bob attended Bradford Grammar School, going through the middle school on the classical side. Attracted to the idea of becoming a doctor, he switched to sciences in the sixth form. After some tutorials at home from his father, he developed a real enthusiasm for mathematics and physics. By contrast, he found the learning involved for Chemistry “rather dull,” so he concentrated on maths and physics, winning a Hastings Scholarship to The Queen’s College, Oxford, in November 1941. At Queen’s, he read Physiology and Medicine and was awarded First Class Honours in Physiology in the summer of 1945. This success led Professor J.H.Burn to invite him to interrupt his medical studies to spend a year doing research in the department of Pharmacology. Bob accepted the invitation but made little progress over the first few months with the project which he, himself, had chosen to work on. In December of that year, Burn agreed for Bob to move to the department of Physiology. Here under the supervision of David Whitteridge, his work flourished and by the summer of 1946 he had been elected to a research fellowship at St John’s College. Although Bob had plans to use the fellowship to follow up Leskell’s then very recent report of the motor innervation of muscle spindles, the College saw Bob as a future medical tutor and persuaded him to use the fellowship to qualify in medicine with a view to succeeding Professor C.G.Douglas who had held the position since 1908. Such opportunities were rare at the time and Bob agreed, going on to University College Hospital in London to pursue his clinical studies. After qualifying in medicine, he spent six months as a house physician and two years in the Royal Army Medical Corps before returning to St John’s College in 1952. In addition to his College duties, Bob became departmental demonstrator with teaching commitments and a small research room in the University Laboratory of Physiology. In the six years since his election to the Fereday

3

fellowship, several groups had started to investigate the motor innervation of the muscle spindles so Bob decided that instead he would investigate the

properties of receptors in the cardiovascular and respiratory systems. His first substantial piece of research, however, was with Jean Banister on the hemodynamics of the pulmonary circulation. In a series of well planned experiments they showed unambiguously how blood flow through the lungs is influenced by the pressure in the airways. Their single paper was followed by a flurry of publications from elsewhere, some of which acknowledged their precedence. With the paper in press, Bob departed for a year’s sabbatical leave, where he worked in Stanley Sarnoff’s laboratory at the National Institutes of Health in Bethesda. This proved to be the most important period of his personal life, for during this year he met Margaret Aspinwall, who was English but working in Washington, and before he returned to Oxford they were married. This was a most happy and successful partnership. Bob Torrance was a large man. Six feet five inches tall and at times

weighing over 280 pounds, he was always easy to find in a crowd and for over forty years, his huge figure on a not so large bicycle was a familiar sight in North Oxford. But after only a brief conversation with Bob, most people forgot his size and were impressed by the sharpness of his mind and the warmth and

generosity of his personality. He was particularly good in talking to young

people, quickly finding common experiences and allegiances he could share with them and use to introduce them to others. He delighted in being a fellow of St John’s College. He was a tutor for nearly forty years and his ability to review areas in a broad yet analytical way made him an inspiring teacher, particularly of more able students. He also served as Junior Dean and Tutor for Admissions at various times. His pleasure in the College increased when in the late 1960’s it repossessed St Giles House, which had formerly been the Judges’

Lodgings in Oxford. It was adjacent to the College and Bob moved his teaching rooms there, appreciating that with its elegant reception rooms and well planned garden, it would be a wonderful place to entertain pupils colleagues

and friends. With Margaret, he enjoyed arranging receptions and dinners in these beautiful surroundings. On such occasions he was a wonderful host, welcoming and particularly thoughtful with newcomers and visitors, always ready to laugh with old friends and tell stories against himself.

For someone who enjoyed conversation so much, it must have been particularly hard for Bob to discover in early middle age that he was becoming progressively deaf. He faced this stoically and sensibly, producing the microphone of his hearing aid to be spoken into if he had difficulty in following a conversation. It seemed as if he treated his hearing aid in the same way as he would treat a piece of laboratory equipment, continuously adjusting it to obtain the optimal signal. Although his deafness was a major handicap in following the discussions at scientific meetings, his hearing aid could occasionally help in a way of which he, himself, was unaware. When he felt

4

that he had a point to make, his intervention would be preceded by a squeaking and a whining which would invariably catch the chairman’s attention. On such occasions he could be most impressive. After detecting an error in a presentation at a Physiological Society meeting some years ago, Bob rose to his feet as soon as the discussion opened, took one massive stride over the front

row of seats and producing a piece of chalk from his pocket, proceeded to draw a diagram on the blackboard to give the correct explanation of the point in question. The paper had been given by a small man and Bob towered over him, not an aggressive, dominating figure but rather that of a patient schoolmaster concerned by the lack of understanding of a wayward pupil. Ill health blighted the last two and a half years of his life but he continued to think and write about physiology in spite of it. He published a biographical paper on Mabel Fitzgerald, one the early women physiologists who is best known for her collaboration with J.S.Haldane in his studies (with Douglas, Henderson and Schneider) on acclimatisation to high altitude; a paper on carbon monoxide transport and oxygen secretion and a paper on the scaling of blood flow in the aorta. He presented his last communication to the Physiological Society in September 1998, less than three and a half months before he died. To the end he retained his mental sharpness and the warmth of his personality. He leaves his wife, Margaret and their two sons.

REFERENCES 1 . Black, A..M. S., McCloskey, D. I. and Torrance, R.W. The responses of carotid body

chemoreceptors in the cat to sudden changes in hypercapnic and hypoxic stimuli. Respiration Physiology, 1971, 13, 3 6 - 4 9 . 2. Black, A.M.S. and Torrance, R.W. Respiratory oscillations in chemoreceptor discharge and the control of breathing. Respiration Physiology, 1971, 13, 221 - 237. 3. Torrance, R.W. (Editor) The Proceedings of the Wates Foundation Symposium on Arterial Chemoreceptors. Blackwell Scientific Publications, Oxford, 1968, pp 1 - 40.

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REMINISCENCE OF BOB TORRANCE

Patricio and Carolina Zapata Laboratory of Neurobiology, Catholic University of Chile, Santiago, Chile

It is almost impossible to realise that Bob is no longer with us. He was full of life, and plans, which he carried into practice with rare, characteristic

enthusiasm and consistency. He continued to publish scientific papers until quite recently. He kept his enthusiasm to the last, in the vigour of his

conversation, and in his warm and generous friendship. He was an exacting and outstanding scholar. His creative mind was always bursting with enthusiasm for work and advice. He had strong

convictions and defended them, but he left nothing to interfere with his enjoyment of life. He firmly believed that life is for involvement and

enjoyment. He lived every day with great eagerness. In addition, he was gentle and understanding with his friends. His broad range of interests was truly exceptional. He was refined in his tastes and a wine “connoisseur”. We had the opportunity to see his wine cellar at his home in Oxford, where he offered us one of his best, aged, and “dusty” red wines. He insisted on being called Bob, not Dr. Torrance. That was too formal to his taste. It was not difficult to do so, because we know of no person who would not call him Bob after a first conversation. He was always ready for help and counsel, rarely lost his trustworthy smile. He was a charming, intelligent and pleasant companion, with a wonderful sense of humour, whose friendship was greatly valued by the many people who knew him. From the period in which he lived in Chile in the early sixties, Bob turned

very fond of this country. In fact, he returned to Chile with Margaret on three occasions, where we had the opportunity to become good friends. Working together in the laboratory was always exciting because he kept his boyish fascination with discovery. It was not uncommon to see him going to

the library to search for an article or specific data right in the middle of an

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experiment. We wondered how he managed to make himself understood, since the librarians did not speak a word of English. But, apparently, he was quite successful in using signs and gestures. The last letter we sent to Bob was dated January 8th, the very same day he died. In that moment we had the strange feeling that something was wrong with him because he had not answered our Christmas card. He could not have done it: he was already in the hospital. Because of his fine human qualities, Bob’s passing has also left a great void. We will remember him with admiration as a forceful, dynamic yet unpretentious and warm personality. He had integrity, youthful enthusiasm, and great personal charm. Finally, we would like to transcribe part of a poem by Nezahual Coyotl, Prince of Tezcoco, Mexico (1402–1472), which was published in Biological Research 26:405 (1993). Bob Torrance and one of us (CL) translated it from Spanish into English. Bob worked very hard on this translation. He intended to grasp not only the right wording of the poem, but the poetic intonation and rhythm as well. It stands as follows: “Listen to me, my Lords! I have solved the mystery, I know the secret. I know what we are: We are all of us mortals! “All men who were born like us on this earth Have to move on from stage to stage of our lives: All of us have to die, here on this earth...” We shall forever keep memories of this remarkable man.

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REMINISCENCE OF BOB TORRANCE Mark Hanson* and Prem Kumar** *UCL Medical School. London, U.K, **University of Birmingham, Birmingham, U.K.

We have the same story to tell. It is a bright crisp morning in Oxford and a large man is laconically riding a bicycle, of a size more suited to a young teenager, round to the rear of the University Laboratory of Physiology. Bob Torrance leans the bicycle casually against the wall and strolls through the back door and along a short corridor, kicking his heels and singing to himself. One side of his raincoat flaps; the other holds a heavy volume in a capacious inside pocket. As he pushes through the door of his laboratory (Room 3) past boxes of assorted equipment and a blackboard, he extracts this volume, the Journal of Physiology, and lifts his glasses on to his forehead to focus on the

Table of Contents on the rear cover. His eyebrows rise in mild astonishment. After ten seconds he pulls his glasses down and in one movement swings his coat and the journal on to a side bench. The laboratory has a timeless atmosphere: a radio plays softly; in a corner a bunsen burner flickers under a large beaker of coffee. The walls are hung with stuffed animals and birds. Ignoring all this, he’s excited to find out what is happening in an inner room, metal-clad and lit only by a dissecting lamp. Someone looks up from the microscope. “Any joy, maestro?” Bob hails through the doorway, not going in. An answer is shouted, but imperfectly heard. Bob makes Caesar’s gesture questioningly: thumbs up? thumbs down? Thumbs up, the reply is grinned. “I’ll keep out of the way then” and he goes off to attend to other matters in the Department or in College. We don’t know how many times this little drama, or a version of it, was played out: maybe once for every chemoreceptor recording that was attempted by every one of a long line of D.Phil students and post-docs who worked with Bob Torrance over many years. For us, it encapsulates what was so special about him. Research was always a priority and his support for those engaged

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in it was boundless: to relish the excitement of it when it was going well, to

commiserate when it wasn’t. He could cap any account by a sad student, who had messed up the preparation or broken something, by a tale of some disaster that had befallen him as a student of David Whitteridge. Bob was a dedicated and inspiring teacher, not only to his postgraduate students, but also to undergraduates. For many years at St. John’s College, he transmitted his insight in physiological mechanisms to generations of medical and science students, sitting on the sofa in his room in St. Giles’ House, with the portrait of his predecessor, C.G. Douglas presiding. Bob would occasionally refer to a paper, or bring down a book from his impressive library: but usually he drew diagrams or graphs on paper with a soft pencil. Understanding science for him was intensely pictorial and depended on developing simple diagrams. If it couldn’t be shown in this way, it seemed that it wasn’t worth discussing. He also believed in working things out from first principles, an approach that, if mastered, could stand the student in good stead as it permitted a dramatic reduction in the rote learning which seemed essential for other subjects such as anatomy and histology.

Bob’s empirical approach seemed to extend to everything, for example when he remarked that the Chinese invention of the hemispherical wok

permitted the rapid cooking of large quantities of food with the minimum of expensive ingredients - oil and fuel. Clearly he thought about everything in

detail and time spent in his cottage in France would often result in letters back to the lab, containing not only ideas for experiments inspired by the French countryside but also anecdotes on home improvements - a long black pipe to warm sufficient water in the day’s sunshine for a shower (“one can get perfectly clean in a shower, but only asymptotically clean in a bath”). Even the

ailments which beset him, whether his deafness, migraine or later the sequelae of a coronary thrombosis, provided the focus for experimental approaches. He would breathe various concentrations of and oxygen to improve (or not as the case may be) his migraine and discuss the relative merits of diuretics acting, on him as a whole as opposed to the single carotid body chemoreceptor.

Equal importance seemed to be given to whether variable x had a ‘relation’ or a ‘relationship’ to variable y as to whether breath-by-breath oscillations in chemoreceptor discharge played any role in respiratory control. Fowler’s modern usage and D.Phil theses of previous students seemed, at times, to be the only universal truths on which we could base my findings. Statistics seemed to Bob only necessary when experiments hadn’t been designed properly.

Bob’s intelligence was prodigious: he could get to the root of a problem

long before others and because he excelled at thinking on his feet, he could often come to a conclusion of devastating clarity. But he had no arrogance: he could be as gentle and patient with his students as when he was dissecting

chemoreceptor fibers. He had a great sense of humor and loved an anecdote.

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He was incredibly kind and generous. But above all, he had an enthusiasm which was utterly infectious. When he said, delighted at a Michelangelo drawing of the head of a youth, which his technician Chris Hirst had framed for him “Isn’t it a beautiful thing?”, he might have been talking about the result of the experiment, later in the day when he came back to the lab to see how things were going. Countless students owe a love and a respect for nature to Bob Torrance.

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PLASTICITY AND MULTIPLICITY IN THE MECHANISMS OF OXYGEN SENSING

Sukhamay Lahin Department of Physiology, University of Pennsylvania Medical Center, Philadelphia PA. 19104, USA

1.

INTRODUCTION

This brief review about the plasticity and mechanisms of oxygen sensing in vertebrates is divided into the following sections. It describes the time-dependent appearance and disappearance of oxygen sensing after birth. The principle of plasticity is that the time dependent phenomena and resetting are related primarily to the availability of oxygen and, therefore, to oxygen related changes in gene expression. The mechanisms do not change with time but their multiplicity can be expressed differentially.

2.

RESULTS

2.1

PLASTICITY

2.1.1

Developmental

It is abundantly clear that plasticity is the rule rather than an exception in mammalian oxygen sensors. This is a time-dependent phenomenon involving gene expression. Until that occurs, the responses can be

Oxygen Sensing: Molecule to Man, edited by S. Lahiri et al. Kluwer Academic/Plenum Publishers, 2000

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considered as acute. From fetal life, sensing begins to develop in the carotid body at different rates in different species to reach a stable state in two to three weeks after birth (e.g., Wasicko, et al., 1999). Hypoxia stimulates increase in in glomus cells at about the same rate, and glomus cell currents are barely developed at birth (Hatton et al., 1997). Taken together, these results show that the hypoxic effect develops slowly with time. These oxygen sensing functions seem to suggest that there is a connection between these events. But there need not be. For example, adreno - medullary cells which are of the same neural crest origin, seem to undergo a loss of oxygen sensitivity as animals mature ( Mojet et al., 1997; Thomson and Nurse, 1998). It has been argued that fetal and neonatal animals need a surge of catecholamine secretion as a result of asphyxia by a non-neurogenic mechanism in adrenomedullary cells not innervated at that time. As animals mature, sympathetic innervations develop, and the hypoxic response is no longer needed and therefore is dispensed with (Seidler and Slotkin, 1985). The Ductus arteriosus is located as a bridge between the pulmonary artery and the descending aorta. At birth, the ductus arteriosus switches from the hypoxic environment in utero to an air-filled oxygen rich environment. At this time, the lumen of the ductus contracts to the point of self-obliteration whereas the pulmonary circulation vasodilates. Ductus constriction is supposed to be inhibited by inhibition of sensitive channels (Abman, 1996). Thus, the following are examples of plasticity. a) Increased hypoxic response of chemosensory discharge with time in animals grown in normoxia (Wasicko et al., 1999). b) Increased hypoxic response of glomus cell with time grown in normoxia (Wasicko et al., 1999). c) Increased responses of glomus cell to hypoxia with time grown in normoxia (Hatton el al., 1997). d) Loss of hypoxic response of adreno-medullary cells as the animal matures in normoxia (Mojet et al., 1997; Thompson and Nurse, 1998). e) Reversed sensitivity of ductus arteriosus (Abman, 1996). 2.1.2

Environmental

If the environment is kept low from birth, development of hypoxic sensitivity is delayed (Hanson et al., 1989). However, with time, full chemosensitivity develops. For example, children born at any altitude have a low ventilatory sensitivity initially but they do develop normal oxygen sensitivity with time (Lahiri et al., 1976). However, as they grow older their chemosensitivity may become blunted (Byrn-quinn et al., 1972; Lahiri and Milledge, 1965; Severinghaus et al., 1966). Soon after birth the sensitivity at

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any altitude is nearly zero (Lahiri et al., 1976). Then it develops with age. Often the sensitivity to hypoxia decreases, that is, the greater the altitude of residence, people become less sensitive to hypoxia (Lahiri et al., 1976). Hypoxia a) Ventilatory acclimatization to chronic hypoxia in adults (Lahiri and Milledge, 1965; Lahiri etal., 1976). b) Increased chemosensory response to chronic hypoxia in adult cats (Barnard et al., 1987). c) Diminished chemosensory response to acute hypoxia in kittens grown in chronic hypoxia (Hanson et al., 1989). d) Blunted ventilatory response to hypoxia in the natives of high altitude (Milledge and Lahiri, 1967). e) Loss of hypoxic ventilatory response without loss of erythropoietic response (Winslow and Monge, 1987). f) Hypertrophy of glomus cells and associated cellular changes in chronic hypoxia (McGregor et al., 1984). g) Increased tyrosine hydroxylase and catecholamine levels with chronic

hypoxia (Czyzyk-Kreska et al., 1992; Hanbauer et al., 1981). Hyperoxia a) Loss of chemosensory response to hypoxia and not to hypercapnia (Lahiri et al., 1987). b) Graded effects of hyperoxia (Lahiri et al., 1989). Reversal of blunted hypoxic response There is evidence that a blunted ventilatory response to hypoxia in adults

at high altitude is reversed if they come down to sea level, the reversal being more prompt if their return to sea level is earlier in life. This reversal is also seen in the patients with congenital heart disease who manifest a blunted hypoxic ventilatory response (Edelman et al., 1970) and show a normal response after surgical correction with return to normal arterial (Blesa et al., 1977). Carotid body Unlike many other organs, chronic hypoxia makes the carotid body grow. Much of this growth involves the type I cell (e.g, McGregor et al., 1984).These cells contain tyrosine hydroxylase, which also increases. As a consequence, catecholamine levels which include tyrosine hydroxylase increase significantly (Hanbauer et al., 1981; Pequignot et al., 1986). Obviously many other enzymes and proteins are modified or formed with altered gene expression (Guillemin and Krasnow, 1997).

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Hyperoxia and carotid body After exposure of cats to hyperoxia for approximately 60 hours carotid bodies lose their oxygen sensitivity (Lahiri et al., 1987). However, the response to hypercapnia remains augmented. These responses resemble those of oligomycin treatment of carotid body (Mulligan et al., 1981). This means that a blockade of oxidative phosphorylation is lost in hyperoxia. Nothing much is known about other aspects of carotid body except that its cells show signs of degeneration with hyperoxia.

2.2

Lessons from plasticity

It is safe to assume that the initial reaction of oxygen sensing is so fundamental that it isn’t going to change with time. It is the manifestation of the response that normally involves altered gene expression, which constitutes plasticity.

3.

CONCLUSIONS

3.1

MULTIPLICITY OF MECHANISMS

The mechanisms involve a heme-based oxygen sensor: cytochrome oxidase, NAD(P)H oxidase and ion channels. The focus of this section will be on the heme ligand carbon monoxide. It has been used in the mammalian carotid body and in yeast cells to show that oxygen sensing properties are shared by both organisms (Bunn and Poyton, 1996).

3.1.1

Aerobic CO effects on carotid body and yeast in the dark

a) High CO in the absence of hypoxia excites chemosensory discharge which is inhibited by light (Fig. 1) (Lahiri, 1994). b) This CO excitation is divisible into different wave-lengths (Fig. 2A) which is matched by a similar absorption spectra of respiratory enzyme in yeast

(Fig. 2B) as is shown in K. (1963). c) The effect of C O excitation in the carotid body is associated in the dark with the decrease in uptake which is reversed by light (Fig. 3). A similar effect of absence and presence of light on cytochrome oxidase in yeast (Keilin, 1966). d) The effect of CO on chemosensory excitation is associated with dopamine release from carotid body (Buerk et al., 1997). But the light effect on chemosensory discharge is not shared by dopamine release, suggesting different mechanisms for chemosensory discharge and dopamine secretion (Lahiri and Acker, 1999).

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3.1.2

CO effect during hypoxia on carotid body and yeast

a) Hypoxic chemosensory discharge is inhibited by CO (Lahiri et al 1993) (Fig. 4). b) CO (Lopez-Lopez and Gonzalez, 1992) (Fig. 4) relieves hypoxic suppression of current. But no light effect has been demonstrated in this paper. c) Induction of HIF-1 by hypoxia is blocked by CO, an intracellular event (Huang et al., 1999; Kwast et al., 1999). Light effect on suppression of HIF-1 by CO is not known.

3.1.3

CO induced excitation and dopamine release is dissociated with light effect

High Pco induced excitation is associated with dopamine release but light while suppressing excitation does not inhibit dopamine release (Fig. 5).

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4.

DISCUSSION

Plasticity is ingrained into the developmental aspects which are dependent on genes. Environment can modify these changes which can work through multiple mechanisms. Many of the mechanisms are heme-based

which can react with carbon monoxide. The effect of carbon monoxide provides the key evidence for such a role of heme proteins (Lahiri and Acker, 1999). In the absence of hypoxia high Pco excites carotid chemoreceptors. This excitation is inhibited by light. The effect of light is divisible into wavelengths (Wilson et al., 1994). This CO mediated

activity matches the action spectra for cytochrome c oxidase in yeast and

cardiac muscles. Also, CO inhibits oxygen uptake, which is relieved by light as shown in Fig. 3 (Lahiri et al., 1997). It is expected that oxygen uptake will be

dependent on the wavelength of light.

CO has been shown to offset the effect of hypoxia on chemosensory

discharge (Lahiri et al., 1997). This corresponds to reversal of

current

suppression of the glomus cell membrane by hypoxia (Lopez-Lopez and

Gonzalez, 1992). But the mechanisms need not be the same (Huang et al., 1999). CO also suppresses the hypoxic activation of HIF-1, and consequently

the subsequent series of gene expression (Guillemin and Krasnow, 1997). But this CO suppression resembles the effect of

currents of the glomus

cell membrane (Lopez-Lopez and Gonzalez, 1992). How these intracellular events are transferred to a membrane is not clear. CO also suppresses the

hypoxia excitation of chemoreception (Lahiri et al., 1997). It could be that

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release from stores up-regulates the current that accounts for the membrane phenomena and suppression of excitation. All the effects of CO related to cytochrome oxidase are expected to be inhibited by light. Thus, the suppression effect of CO on hypoxia activation should also be effected by light. However, the effect of light on this phenomenon has not been demonstrated. Also, in severe hypoxia, light excites the chemoreceptors. One explanation is that this phenomenon occurs due to a conformational change of hemoprotein (Lahiri and Acker, 1999). The light effect on chemosensory excitation should be reflected in changes which needs to be measured. However, light inhibition of chemosensory discharge is not shared by dopamine release (Buerk et al., 1997) which suggest two different sites for chemosensory excitation and dopamine secretion (Lahiri and Acker, 1999). The source of dopamine is the glomus cells. But neural excitation originates from nerve ending possibility without involving secretion from the glomus cells. This idea is reminiscent from the work of for examole, Mitchell et al.(1972). The transition metals, and , mimic the hypoxic effects on gene expression. But and also block channels and subsequently block the hypoxic response. However, the relationship between channel blockade and gene expression is not fully elucidated.

ACKNOWLEDGEMENTS Supported in part by the NIH grant R 37 HL-43413

REFERENCES Abman, S.H. (1996). Oxygen sensing, potassium channels, and the ductus arteriosus. J. Clin. Invest. 98: 1945–1946. Barnard, P.S., Andronikou, S., Pokorski, M., Smatrsk, NJ., Mokashi, A. and Lahiri, S.(1987). Time-dependent effect of hypoxia on carotid body chemosensory function. J. Appl. Physiol. 63: 851–691. Blesa, M., Lahiri, S., Rankind. W. and Fishman, A.P. (1977). Normalization of the blunted ventilatory response to acute hypoxia in congenital cyanotic heart disease. New. Engl. J. Med. 296: 237–241. Buerk, D.G., Chugh, O.K., Osanai, S., Mokashi, A. and Lahiri, S. (1997). Dopamine increases in cat carotid body during excitation by carbon monoxide: implications for a chromophore theory of chemoreception. J. Autonom. Nerv. System. 67: 130–136. Bunn, H.F. and Poyton, R.O. (1996). Oxygen-sensing and molecular adaptation to hypoxia. Physiol. Rev. 76: 839–855. Byrn-quinn, E., Sodal, I.E. and Weil, J.V. Hypoxia and hypercapnic ventilatory drive in children native to high altitude. J. Appl. Physiol. 32: 44–46, 1972

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Czyzyk-Kreska, M.F., Baliss, D.A., Luwson, E.E. and Milhorn, D.E. (1992). Regulation of tyrosine hydroxylase gene expression in carotid body by hypoxia. J. Neurochem. 58: 1538– 1546. Edelman, N.H., Lahiri, S., Cherniack, NS. and Fishman, A.P. The ventilatory response to hypoxia in cyanotic congenital heart disease. New Eng. J. Med. 282:405Guillemin, K. and Krasnow, M.H. (1997). The hypoxic response: Huffing and Miffing. Cell 89: 9-12. Hanbauer, I ., Karoum, F. Hellstorm, S. and Lahiri, S. (1981). Effect of long term hypoxia on the catocholamine content in rat carotid body. Nuerosciencc 6: 81-86. Hanson, M.A., Eden, G.J., Niglnis, JG. and Moore, P.J. (1989) Peripheral chemoreceptors and other oxygen sensors in the fetus and newborn. I n : Chemoreceptors and Reflexes in Breathing: Cellular and molecular aspects. Eds, S. Lahiri, R.E. Foster, I I . R.O. Davies and A.I Pack. pp. 113-120. Hatton, C.J., Carpenter, E., Pepper, D.R., Kumar, P. and Peers, C. (1997). Developmental changes in isolated rat type I carotid body cell K+ current and their modulation by hypoxia. J. Physiol. 501: 49-58 Huang, L.E., Willmore W., Gu J., Goldberg, M.A. and Bunn, H.F. (1999). Inhibition of H1F–1 activation by carbon monoxide and nitric oxide: implications for oxygen sensing and signaling. J. Biol. Chem. 274: 9038-9044. Kwast, K.E., Burke, P.V., Staahl, B.T. and Poyton, R.O. (1999). Oxygen sensing in yeast: evidence for the involvement of the respiratory chain in regulating the transcription of a subset of hypoxic genes. P.N.A.S. 96:5446-5451. Keilin, D. (1966). The history of cell respiration and cytochrome. Cambridge, UK. Cambridge Univ. Press. Lahiri, S. (1994). Chromophores in chemoreception: The carotid body model. NIPS 9: 161 –164 Lahiri, S. and Acker, H. (1999). Redox-dependent binding of to heme protein controls chemoreceptor discharge of the rat carotid body. Respir. Physiol. 169-177. Lahiri, S., Iturriaga, R., Mokashi, A., Ray, D.K. and Chugh, D. (1993). reveals dual mechanisms of chemoreception in the cat carotid body. Respir. Physiol. 94: 227-240. Lahiri, S., Buerk, D.G., Chugh, D., Osanai, S and Mokashi, A. (1997). Reciprocal photolabile consumption and chemoreceptor excitation by carbon monoxide in the cat carotid body: evidence for cytochrome as the primary sensor. Brain Res. 684: 194-200 Lahiri, S., Delaney, R.G., Brody, J.S., Simpser, M., Velasquez, T., Motoyama, E.K. and Polger, G. (1976). Relative role of environmental and genetic factors in respiratory adaption to high altitude. Nature 261: 133-135. Lahiri, S. and Milledge J.S (1965). Sherpa Physiology, Nature 207:610-612. Lahiri, S., Mulligan, E., Indronikou, S., Shirahata, M. and Mokashi, A. (1987). Carotid body chemosensory function in prolonged normobaric hyperoxia in the cat. J. Appl. Physiol 62: 1924–1931. Lahiri, S. M u l l i g a n , E., Smatresk, N.J., Barnard, P., Mokashi, A., Torbati, D., Pokorski, M., Zhang, R., P.G. Data and Albertine, K. (1989). Neurotransmission of carotid body responses to chronic low and high oxygen pressures. In: Chemoreceptors and Chemoreflexes in Breathing: Cellular and Molecular Aspects. Eds. S. Lahiri, R.E. Foster I I , R.O. Davies and A.I. Pack. Oxford Univ. Press, New York. pp. 215-227. Lopez-Lopez, J.R. and Gonzalez., C. (1992). Time course of current inhibition by tow oxygen in chemoreceptor cells of adult rabbit carotid body. Effects of carbon monoxide. FEBS Lett., 299: 251-254. McGregor, K.H., Gil, J. and Lahiri, S. (1984). A morphometric study of the carotid body in chronically hypoxic rats. J. Appl. Physiol 57: 1430-1438.

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Milledge, J.S. and Lahiri, S. (1967). Respiration control in lowlanders and sherpa highlanders at altitude. Respir. Physiol. 2: 323Mitchell, R.A., Shuiha, A.K. and McDonald, D.M. (1972). Chemoreceptive properties of regenerated endings of the carotid sinus nerve. Brain Res. 43: 1077-1088. Mojet, M.H., M i l l s , E.,and Duchen, M.R. (1997). Hypoxia-induced catecholamine secretion in isolated newborn rat adrenal chromaffin cells is mimicked by inhibition of mitochondrial respiration. J. Physiol. 504: 175-189. M u l l i g a n , E., Lahiri, S. and Storey, B. (1981). Carotid body chemoreception and mitochondrial oxidative phosphorylation. J. Appl. Physiol. 51:438-446. Pequignot, J.H., Cottet-Emrad, J.M., Dalmaz, Y., Dehaut D. Sigy, M. and Pey (1986). Biochemical evidence for dopamine and norepinephrine stores outside the sympathetic nerves in rat carotid body. Brain Res. 367:238-243. Seidler, F.J. and Slotkin, T.A. (1985). Adrenomedullary function in the neonatal rat: responses to acute hypoxia. Physiol. 385: 1 - 1 6 . Severihaus, J.W, Bainton, C.R. and Carcelen. (1966) Respiratory insensitativity to hypoxia in chronically hypoxic man. Respir. Physiol. 1: 308-334. Thompson, R.J. and Nurse, C.A. (1998). Anoxia differentially modulates multiple currents and depolarizes neonatal rat drenal chromaffin cells. J. Physiol. 512: 421-434. Wasicko, M.J., Sterni, L.M., Bramford, O.S., Montrose M.H. and Carrol I, J.L. (1999). Resetting and postnatal maturation pf oxygen chemosensitivity in rat carotid chemoreceptor cells. J. Physiol. 514: 493-503. Wilson, D.F., Mokashi, A., Chugh, D., Vinogradov S., Osanai, S. and Lahiri, S. (1994). The primary oxygen sensor of the cat carotid body is cytochrome of the mitochondrial respiratory chain. FEBS Lett.351: 370-374 Winslow R.M. and Monge, C. (1987). Hypoxia, polycythemia and chronic mountain sickness. The John Hopkins University Press, Baltimore, MD, USA.

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EVOLUTION OF HUMAN HYPOXIA TOLERANCE PHYSIOLOGY

Peter W. Hochachka* and Carlos Monge, C.** *Depts. of Zoology and Radiology and the Sports Medicine Division, University of British Columbia, Vancouver, B.C., Canada V6T 1Z4** Dept. of Physiological Sciences, Universidad Peruana Cayetano Heredia, Lima 100, Peru

Abstract

Analysis of human responses to hypobaric hypoxia in different lineages (lowlanders, Andean natives, Himalayan natives, and East Africans) indicates 'conservative' and 'adaptable' physiological characters involved in human

responses to hypoxia. Conservative characters, arising by common descent, dominant and indeed define human physiology, but in five hypoxia response systems analyzed, we also found evidence for 'adaptable' characters at all levels of organization in all three high altitude lineages. Since Andeans and Himalayans have not shared common ancestry with East Africans for most of our

species history, we suggest that their similar hypoxia physiology may represent the 'ancestral' condition for humans – an interpretation consistent with recent evidence indicating that our species evolved under 'colder, drier, and higher' conditions in East Africa where the phenotype would be simultaneously advantageous for endurance performance and for high altitude hypoxia. It is presumed that the phenotype was retained in low capacity form in highlanders and in higher capacity form in most lowland lineages (where it would be recognized by most physiologists as an endurance performance phenotype). Interestingly, it is easier for modern molecular evolution theory to account for the origin of ‘adaptable’ characters through positive selection than for conserved traits. Many conserved physiological systems are composed of so many gene products that it seems difficult to account for their unchanging state (for unchanging structure and function of hundreds of proteins linked in sequence to form the physiological system) by simple models of stabilizing selection.

Oxygen Sensing: Molecule to Man, edited by S. Lahiri et al. Kluwer Academic/Plenum Publishers, 2000

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1.

HOW TIME INTERPLAYS WITH HYPOXIA ADAPTATION OPTIONS It is widely accepted in comparative biology that how a species deals with

hypoxia, or any other selectively relevant factor, depends upon the time

available for orchestrating the response. Usually the timeline for response is divided into three categories: acute, acclimatory, and genetic or phylogenetic. The formal relationship [27] between these three timelines of responses begins first with sensing mechanisms which inform the organism when an limitation problem arises. Second, this information must be transduced at various levels of organization into appropriate acute responses. Third, either the same or different sets of sensing and signal transduction pathways may be utilized to achieve more complex acclimatory responses. Finally fourth, any of the above the sensing step, the signal transduction pathways, the acute response, and the acclimatory responses - during generational time (i) may change randomly due to genetic drift (characters arising by this means in extant lineages are not adaptations), (ii) may change due to positive natural selection at rates proportional to selection pressures (characters arising by this process are termed adaptations), or (iii) may be conserved or stabilized essentially

unchanged by negative or stabilizing selection pressures (these kinds of characters are expressed in extant lineages as a result of common descent and are designed for function in many settings; they may be used along with (ii) above in physiologically adaptive responses to hypoxia but technically they are not hypoxia adaptations per se). Except for the frequent casual use (or misuse) of the term ‘adaptation’ by mammalian physiologists, the acute and acclimatory responses to hypobaric hypoxia are fairly well known [3-8, 14,17,27,30,31 ]; however, much less insight is available on how these response systems change in humans through evolutionary time – the fourth process above, which we wish to focus upon in this overview.

2.

STUDIES OF DIVING ANIMALS SUPPLY GUIDELINES FOR HUMAN EVOLUTIONARY PHYSIOLOGY

Probably the main reason why the evolution of human hypoxia tolerance was not investigated earlier was because there were few if any guidelines for the study of the evolution of complex physiological systems in humans or in animals. In our case, initial guidelines [23,46] arose from recent quantitative analyses of the variability of the diving response in pinnipeds (seals and sea lions). Some 50 years of research established the main physiological features of diving to include apnea, bradycardia, and peripheral vasoconstriction, with

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hypometabolism of hypoperfused tissues. Together these features are referred to as the diving response which serves to conserve for the heart and brain; peripheral tissues in contrast may become more and more limited as diving is extended. Over the years significant amounts of information gradually accumulated for almost all of the 30 or so species of pinnpeds. This allowed a comparative analysis [23,46] and led to three principles of evolution of the diving response which we found useful as framework for probing the evolution of complex physiological systems: (i) some physiological / biochemical characters considered necessary in diving animals are conserved in all pinnipeds; these traits (including diving apnea, bradycardia, tissue hypoperfusion, and hypometabolism of hypoperfused tissues) probably arose in response to factors other than - or in addition to - diving requirements and presumably were and are maintained largely by negative or stabilizing selection (any mutations affecting them not surviving). At this stage in our understanding of diving physiology and biochemistry, we are unable to detect any correlation between these characters and diving capacity, even though they are clearly used during diving and are so important that (the cardiovascular control systems for) diving bradycardia plus peripheral vasoconstriction are often referred to in this literature as the ‘master switch of life’. It is reasonable to assume that the cardiovascular control systems in seals and sea lions in fact are ancient characters and are expressed in extant species as a result of common descent, (ii) A few other diving characters are more malleable and are clearly correlated with long duration diving and prolonged foraging at sea. These characters are more lineage specific, and include spleen weight, blood volume, and red blood cell (RBC) mass. The larger these are, the greater the diving capacity (defined as diving duration). Since the relationships between

diving capacity and any of these traits are evident even when corrected for body weight, it is reasonable to conclude that these three traits - large spleens, large blood volume and large RBC mass – are true adaptations, selected because they extend diving duration, probably through effects on storage and management during diving. That is, in contrast to conserved traits such as bradycardia, these kinds of characters have evolved presumably by positive selection to enable prolonged dive times, (iii) The evolutionary physiology of the diving response thus can be interpreted in terms of the degree of development of adaptable vs conservative categories of diving characters; i.e., in terms of how these patterns change through time and how the patterns are lineage specific. Using these studies as guidelines, we then turned our attention to the evolution of human responses to hypobaric hypoxia. Working with several low and high altitude lineages, our own studies [2,20-31, 41] focussed on four systems: integrated whole body physiology and metabolism, muscle metabolism at rest and during work, heart metabolism in normoxia vs hypoxia, and brain metabolic organization. In addition, we also relied on data already in

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the literature (see ref [22,27]). Guided by the diving studies above, our detailed earlier analysis [27] looked for and found strong evidence that the physiology of hypoxia tolerance in humans displays both conservative and adaptable characters. Brain metabolism can serve as an example of the former and heart metabolism as an example of the latter.

3.

BRAIN METABOLIC ORGANIZATION AS AN EXAMPLE OF A CONSERVATIVE CHARACTER

To evaluate the nature of brain metabolism through generational time, we compared mass-specific glucose metabolic rates (using Positron Emission Tomography, PET) in over 20 anatomically distinguishable regions of the brain in lowlanders and compared these patterns to those found in indigenous highlanders [24,25 ]. In all three lineages, (i) glucose is the preferred fuel of the brain, ( i i ) brain metabolic rates are qualitatively similar, and (iii) region by region comparisons indicate qualitatively similar metabolic organization. As these are measurements of the brain in physiological steady state, the results indicate that pathways of adenosine triphosphate (ATP) demand and ATP supply (and presumably associated regulatory mechanisms in brain ATP turnover) are all conserved in our phylogeny. In fact, studies from other laboratories indicate the above features apply for the brain in other (e.g., Japanese) lineages [42] also. As these different human groups have been evolving separately for a significant part of our species history, the above stability of expression implies that the genes specifying structure and function of the central nervous system (CNS) involved in regulated ATP demand and ATP supply pathways have been stabilized by negative selection through our phylogeny (any mutations causing change either being prevented or being deleted). Human brain metabolic organization thus well illustrates a physiological character that is strikingly conserved in our evolution and whose functions appear both in normoxia and in hypobaric hypoxia.

4.

MOST PHYSIOLOGICAL CHARACTERS IN OUR SPECIES ARE CONSERVATIVE

A key point is that the conservative nature of human brain metabolism is by no means exceptional. Since we here are assessing traits within a single species, conservative characters are probably the rule and are too numerous to outline in detail. Three additional, well worked out examples are hemoglobin (Hb) affinity and regulation [60], muscle organization into different fiber types [34,50], and, the cardiovascular control system (used in regulation of

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heart and perfusion in many physiological settings including diving, as mentioned above [46]). Such categories of physiological traits - in sum they make up most of our physiology - and the way they are used on hypoxia exposure appear common in humans no matter what the lineage or the content of the inspired air in the normal environment.

5.

HEART METABOLISM AS AN ADAPTABLE PHYSIOLOGICAL CHARACTER

In contrast to the brain, the heart displays a prominently adaptable metabolic organization. In lowlanders, heart metabolism is opportunistic and w i l l utilize free fatty acids (FFAs), glucose, or lactate on an availability basis. To evaluate the nature of heart metabolism through generational time, we used Magnetic Resonance Spectroscopy and compared the spectra in lowlanders to those found in indigenous highlanders in normoxia vs hypoxia. We found [26] that the concentration ratios of phosphocreatme (PCr)/ATP were maintained at steady state normoxic values (0.9 -1) that were unusually low, about 1/2 those found in normoxic lowlanders (1.8) monitored the same way at the same time. Because the creatine phosphokinase reaction functions close to equilibrium, these steady state PCr/ATP ratios presumably coincide with about 3-fold higher free adenosine diphosphate (ADP) concentrations. Higher ADP concentrations (i.e., lower [PCr]/[ATP] ratios) correlate with the Km values for ADP-requiring kinases of glycolysis and reflect elevated carbohydrate contributions to heart energy needs. This metabolic organization was presumably selected in highlanders because the ATP yield/02 is 25–60% higher

with glucose than with free fatty acids (the usual fuels utilized in the human heart in postfasting conditions). In addition, the effects of hypoxia acclimation on heart PCr/ATP signatures also differ in the two groups. In highlanders, the PCr/ATP signature of glucose fuel preference remains stable even after four weeks of deacclimation at low altitudes [26], while a similar period of acclimation in lowlanders leads to a modest shift towards the highlander pattern (P.W.Hochachka and R.V.Menon, unpublished data). Recent studies of rats also found that hypobaric hypoxia acclimation for 3 – 4 weeks leads to a decrease in fatty acid oxidation capacity, with a relative increase in glucose preference as fuel for the heart [51]. Taken together, these data indicate that heart adaptations seem to rely upon stoichiometric efficiency adjustments [26,31], improving the yield of ATP per mole of consumed, as in muscle [6-8], by increased preference for carbohydrate as a carbon and energy source. Together with increased blood volume and RBC mass (i.e. increased whole body carrying capacity), these adaptations imply dampened heart work requirements at any given altitude for

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a similar submaximal level of whole body exercise. The key point is that in contrast to the brain, in the heart, some component or components of the ATP supply pathways appear to be under positive selective pressure in high altitude natives, leading to two adaptations: elevated preference for glucose as fuel for the heart and a modified hypoxia acclimation response.

6.

ADAPTABLE CHARACTERS AND A HIGH ALTITUDE PHYSIOLOGICAL PHENOTYPE

In spite of the overwhelmingly conservative nature of human physiology, we also found evidence for other metabolic and physiological responses to hypobaric hypoxia that, like heart metabolism, appear to be true adaptations in Quechuas and Sherpas. Such adaptable characters seemingly occur at all levels of organization examined and can be summarized as five adjustable hypoxia response systems (AHRS) which seem to form a key and common basis for the complex physiology of hypoxia tolerance: (i) blunted hypoxic ventilatory response (HVR) mediated by the carotid body sensor [1,37], serving to counteract acid-base problems [54] arising from hyperventilation; (ii) blunted hypoxic pulmonary vasoconstrictor response (HPVR) mediated by pulmonary vasculature sensors [58], serving to minimize risks of pulmonary hypertension [18]; (iii) up regulated expression of vascular endothelial growth factor 1 (mediated by vascular sensors [15]), angiogenesis and hence increased blood volume [17,36,47]; (iv) maintained erythropoietin regulation of erythropoiesis (mediated by kidney sensors), and hence increased RBC mass and carrying capacity [9,43,57,59]; and (v) regulatory adjustments of metabolic pathways to alter fuel/pathway preferences (including the ratio of aerobic/glycolytic metabolism) and, in striated muscle, to attenuate concentrations of enzymes in energy metabolism (for representative data, see [20,30,33,34,39]). The AHRS in turn set the stage for additional ‘downstream’ effects. For example, we find that in Andean and Himalayan natives maximum aerobic and anaerobic exercise capacities are down regulated. The acute effects of hypoxia (making up the energy deficit due to lack) expected from lowlanders [7] are blunted, and metabolic acclimation effects [29,41] are also attenuated. The in vivo biochemical properties of skeletal muscles, in Quechuas formed predominantly of slow twitch fibers [34,50], are consistent with regulatory adjustments of glycolytic vs oxidative contributions to energy supply to improve the yield of ATP per mole of carbon fuel utilized. These fiber type distributions in indigenous highlanders are unchanged by acclimation [34] and correlate with better coupling between ATP demand and ATP supply pathways (lesser perturbation of phosphate metabolite pools during rest-exercise transitions [2,41]), with lower lactate accumulation [29]

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and with improved endurance [41]. Indeed, low lactate accumulation during exercise is one of the most characteristic metabolic features of indigenous highlanders [21,22,29]. Kenyans native to medium altitude environments, even

if not as much studied [52,53], show similar, if higher capacity, biochemical and physiological properties (adjustments at least in part based on preponderance of slow twitch fibers in skeletal muscles); this is not evident in Africans originating from lowland regions of West Africa, showing a much higher preponderance of fast twitch fibers in their muscles [3]. Finally, a blunted catecholamine response to hypoxia in indigenous highlanders indicates reduced hypoxic sensitivity of sympathadrenergic control [4,5,11,14,44], below the normally expected desensitization on exposure of human cells to hypoxia [48]. Compared to acute or acclimatory adjustments, these longer term (phylogenetic) adaptations appear to compensate pretty well for deficits caused by hypoxia, but the advantage appears to be gained at the cost of some attenuation of maximum aerobic and anaerobic metabolic capacities. On balance the picture emerging so far is that of a high altitude physiological phenotype based on numerous similar physiological traits (data mainly from Andean and Himalayan natives). Parenthetically, we might add that while overall hypoxia responses appear to involve fine tuning each of the above sensing and signal transduction pathway cascades, the hypoxia defense adjustments of Andean and Himalayan natives are not always exactly the same. For example, the HVR is more robust in Tibetans than in Quechuas [55]or Sherpas [38], altitude associated birth weight perturbations are less in Tibetan than in Quechau newborns [61], and hypoxia mediated increases in RBC mass in Andean natives [24,29] are more robust than in Himalayans [24,25]. Similarily, Hb affinities need special explanation. Most other vertebrates that tolerate extremes of high altitude display Hb homologues with increased affinities; as a result full saturation can be achieved at quite high altitudes (see ref [45,60]). In humans, as one of us (CM) has been well aware for nearly half a century, Hb seems to be designed for ‘low altitude’ function because in all lineages it displays a monotonic decline in saturation with altitude. Why this should be so has been perplexing to workers in this field, since intuitively it seemed reasonable that high altitude humans would be selected for higher affinity Hb to allow saturation at altitude. A solution to this paradox may be proposed which arises

from recent work [ 19] showing that the benefit of a left shifted saturation curve is achieved only at unusually high altitudes and high work rates. Since humans typically do not live above about 4500 m, there may have been little selective pressure for high affinity Hb. At sea level or low altitudes, a left shifted saturation curve is detrimental to unloading and leads to a decline in maximum whole body consumption rates. At intermediate altitudes, left shifting the saturation curve does not seem to have much effect on maximum aerobic metabolism [19]. These results, along with the finding that

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high altitude humans display low maximum consumption rates, help to explain why they have managed hypobaric hypoxia with essentially normal or lowlander types of Hb binding properties. However, as with HVR and developmental adaptations [61], there may be modest differences in Hb affinity regulation between Sherpas, Quechuas, and other high altitude natives [60). Given the length of time these lineages have been evolving separately (see below), some differences in a few physiological characters are not

unexpected and do not alter our impression of a high altitude physiological phenotype based on numerous similar traits in Andean and Himalayan natives.

7.

PHYSIOLOGICAL PHENOTYPES FOR HYPOXIA TOLERANCE AND FOR ENDURANCE PERFORMANCE

Because of well known effects of exercise and altitude [7,21,29,41,45], it is perhaps not surprising that most of the above adjustable hypoxia response systems (AHRS) are also found in humans adapted for endurance performance. The common traits often include a blunted HVR and HPVR, expanded blood

volume, altered expression of metabolic enzymes and metabolite transporters, fuel preference adjustments, enhanced ratio of aerobic/anaerobic contributions to exercise, high ratios of slow twitch/fast twitch fibers in skeletal muscle, and enhanced endurance [7,20,28,29,39,41,52,53]. In endurance athletes, who display much higher maximum aerobic capacities than do altitude natives, many of these series of traits appear as high performance versions of those found in high altitude natives, with up regulation of muscle mitochondrial volume density (of flux capacities at the working tissues) being perhaps the only serious modification to the physiological phenotype described above [34,52,53], The comparisons of lowlanders and highlanders under normoxia are qualitatively good descriptions of the difference between individuals who

are well adapted for endurance vs those who are not. For example, low plasma [ lactate] in exercise eliciting maximum aerobic metabolism is as characteristic of endurance performers as it is of highlanders [7,21]. Or, to put it another way, the biochemical and physiological organization of both indigenous highlanders and individuals adapted for endurance performance are similar to each other, but both differ strikingly from the homologous organization in 'burst performance' individuals [7,21]. Similar contrasts emerge when East Africans (medium altitude origins [52]) are compared to West Africans (lowland origins), where fast twitch fibers form a much larger percentage of skeletal muscle [3]. In the latter, exercise-induced lactate concentrations can

reach very high levels, and cardiovascular adjustments play as important a role in recovery from performance as they do during performance per se [7,21].

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Because the differences between acclimated and unacclimated (and trained vs untrained) individuals may be as great or greater than the difference between genetically distinct individuals (and because acclimation responses themselves are genotype dependent), genetic vs environmental contributions to these character traits are hard to quantify. Actually, most physiological studies are not properly designed to evaluate this issue, and in fact much of the physiological literature ignores genotype and usually reports no information at all on the genetics of the subjects under study. However, studies which have examined this issue usually find that genetic factors account for about 50% or more of the variance of these kinds of physiological systems [3,13]. It is important to remind physiologists that this estimate refers to genes affecting 50% or more of the variance, not the structural components, of physiological systems per se. Structural components - usually being gene products - could be nearly 100% ‘genetic’, which is a small but fundamental point often overlooked by physiologists (but not by pathophysiologists who well know the connection between genes and physiology). What is more, the % genetic contribution to variation in trait expression, such as HVR, may vary in different lineages (higher in Tibetans than in Andean natives [55] or Sherpas [38], for example). For any given system of course, natural selection can act only upon components that arc under genetic influence.

8.

PHYLOGENETIC ORIGINS OF HUMAN HYPOXIA TOLERANCE

If the AHRS compromises the primary 'solution' of our species to 'problems/requirements' of hypobaric hypoxia and/or endurance performance, the problem remains of when it arose in our species history. To explore this issue requires insight into the evolutionary pathways of our species. To this end, we relied upon a simplified 'phylogenetic tree' for the human species (Figure 1) from a recent summary of human genetics and evolution [10]. The main groups whose physiological responses to hypobaric hypoxia to date have been extensively studied [22,45,60] are shown on the Figure. The age of our species is not known exactly, but we can assume approximately 100,000 years (this is controversial, but if our species is older, the arguments below will be stronger). Several insights arise [22,27]. First, Figure 1 suggests that the last lime Caucasians, Sherpas and Quechuas shared common ancestors was over half the age of our species. Second, the last time the Himalayan highlanders (Sherpas and Tibetans) and the Andean highlanders (Quechuas and Aymaras) shared common ancestors was equivalent to about 1/3 of our species history. Third, divergence times between these groups and East Africans from medium altitude environments are even greater. Even if many details of human

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phylogeny remain unsettled and Figure 1 is overly simplified, accepting other possibly phylogenies would not alter our main conclusion that the lengths of

separation of the Andean, the Himalayan, and the East African lineages represent large fractions of our species’ history. Despite such phylogenetically deep divergences, the latter three groups express many similar metabolic and

physiological responses to hypobaric hypoxia. Fourth, in numerous other lineages (intermediate branches in our phylogeny) the AHRS features when present often are ‘high performance’ versions of the high altitude phenotype. These provocative phylogenetic data are consistent with two possible interpretations.

One plausible model is that, with only modest differences, similar metabolic and physiological 'solution' arose independently by positive natural

selection in the two high altitude (Andean and Himalayan) lineages for which we have the most data and possibly in a third east African lineage for which the data are not as extensive. If so, such convergence (same characters arising independently in different lineages) would satisfy one of the criteria of

evolutionary biology and would indicate that the above suite of physiological characters are defense adaptations against hypobaric hypoxia and arose by positive selection. Whereas this was our thinking initially, the phylogenetic

observations are not easily incorporated into this interpretation. An alternative view – hypothesis (ii) – is that the above suite of physiological and metabolic traits, the AHRS, while arising by positive natural selection, represents the 'ancestral’ condition, which would be consistent with

evidence suggesting that the origin of our species occurred under conditions that were getting colder, drier, and higher. As emphasized elsewhere [22,27], key environmental influences on the root of our phylogeny, on the origins of our species, go back a long way. During early phases, hommid evolution in the East African Rift was occurring under conditions of mild altitude hypoxia – ideal from a training point of view [39] – aggravated by drier and colder climates. These conditions, where the AHRS would be advantageous, prevailed at the origins of our species (indeed, they prevail in East Africa today) and may have been particular important during the so-called

‘bottleneck’ period, thought perhaps to have been caused by unusually harsh ice-age conditions beginning at around 75,000 years ago. At this time, human populations may have been driven to remarkably low levels (possibly close to extinction). In any event, according to this model, over some 5000 or more generations of our species history, the ancestral condition was 'retained' in a

down regulated form in high altitude groups and was 'retained' in an up regulated high capacity form in groups selected for endurance performance (including Kenyan highlanders, who continue to thrive on the same plateau that served as the colder, drier, and higher place of our species beginnings). The fact that most lineages have a significant proportion of endurance type

phenotypes indicates that the ancestral condition was ‘retained’ in many

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intermediate lineages in our species. In situations such as the moderate hypobaric hypoxia of East Africa, selection pressures for both hypoxia tolerance and endurance performance may well have been applied simultaneously (see ref [39] for a recent detailed analysis of the interaction between endurance performance and hypobaric hypoxia). In any event, our hypothesis (ii) predicts that the ancestral organization of our physiology was inherently very dependent upon efficient physiological delivery systems and upon 'aerobic' metabolic pathways and fiber types, with relatively minor development of, or reliance on, anaerobic metabolic systems to sustain short, intense bursts of whole body exercise. If this hypothesis is correct, then (in terms of our original framework for evaluating the evolution of complex physiological systems) it appears that much is determined by so called initial or ancestral conditions. Much of the evolution of physiological systems seems to involve stabilizing or negative selection (pruning out genotypes in which the ancestral 'models' are altered). Only a part of the evolution of our physiology seems to be the result of positive selection for new functional capacities and fundamentally new physiological characters - the AHRS described above and some parallel developmental adaptations [61], and even these traits appear be ancestral (to have arisen very early in our evolution). Finally, it is satisfying to note the similarities between these interpretations of the evolution of a complex physiological system within the hominids to the picture obtained in an analysis of the diving response in pinnipeds; in one case, we are dealing mainly with one species, or at most a few, evolving for about a million years, while in the latter case, we are dealing with 30 species whose phylogeny extends back about 20 millions years [46]. Nevertheless, these conclusions raise two problems for modern molecular evolution theory - how to account for conservative and for adaptable traits in the evolution of complex physiological systems.

9.

CAN MOLECULAR EVOLUTION MODELS EXPLAIN ADAPTABLE TRAITS IN HYPOXIA TOLERANCE?

Most evolutionary biologists today assume that selection can be an overwhelming force. Population genetics theory tells us that the response to selection depends on the amount of phenotypic variation upon which selection can operate (the % variation that is heritable) and on the intensity of selection. As mentioned above, most physiological studies are not designed for teasing out the values of these parameters. However, for physiological and morphological systems which have been studied quantitatively, the coefficient of variation is often about 10% or even higher. Heritabilities vary a lot, but a

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range of between about 0.3 – 0.7 is reported by physiological studies using family inheritance patterns or monozygotic vs dizygotic twins to quantity genetic contributions to variance in physiologcal traits. Selection coefficients also vary greatly; at the upper extreme values as high as 0.43 are known. A 43% selective advantage means a selection intensity so extreme that individuals one phenotypic standard deviation above the mean are more than twice as fit as individuals one deviation below the mean [35]. Armed with these values, evolutionary biologists calculate that positive selection can produce evolutionary rates in excess of 1% change in the mean value for a trait per generation. As our species history goes back at least 100,000 years (early hominid phylogeny, when much of our basic physiological phenotype was being formed, goes back even further, to 3–4 million years ago), it is not hard to conclude that characters arising under positive selection and observed within extant lineages of our human family can be easily accommodated by current evolutionary theory. Of course there remains a caveat; namely, that these kinds of calculations are almost always focussed onto a single trait. For situations where the evolution of two or more systems must be coordinated (as in AHRS above) the probability and mechanisms of such coevolution remain obscure. Nonetheless, at least in a general sense, the answer to the question posed appears affirmative.

10.

CAN MOLECULAR EVOLUTION MODELS EXPLAIN TRAITS CONSERVED THROUGH HUMAN PHYLOGENY?

Interestingly, when considering this second issue - can conserved traits such as brain metabolic organization or such as the pathways of cardiovascular or ventilatory control be explained by modern molecular evolution theory - the answer is not so obvious. To put it in perspective, we first must remind the reader of the enormous complexity of the physiological systems involved. A hint of this arose in our earlier analysis of a number of major sensing, signal transduction, and effector pathways involved in cardiovascular control in diving animals [23,46]; this control system is equally well conserved in human evolution. At the molecular level each arm of this enormously complex regulatory system is composed of up to 100 already identified gene products; from each protein being linked in a highly precise manner with others in the pathway emerges the coordinated (and evolutionanly conserved) physiological function of cardiovascular control [46]. In the case of the CNS, well over 200 currently known gene products are involved in pathways of ATP demand and ATP supply, in sensing, signal transduction, and regulation, and in structural integration. Conserving the emergent physiologies formed from the expression

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of linked function of many gene products means conserving the structure and function of at least key parts of gene products (those specifying ‘active’ or otherwise functionally important sequences). That is, it means deleting most mutations which arise - this is negative or stabilizing selection. To estimate how much negative or stabilizing selection is required to keep such systems from randomly changing through phylogenetic time we need to have an estimate of the selective cost of substitutions in each of the gene products in such integrated sequences. Of course this information is not available for all components of complex physiological systems such as the CNS. However, these kinds of values are known for other presumably comparable macromolecular systems (see ref [12] for a comparable such study of 46 genes in humans and other homimds). A particular clear example is given for 16/18S rRNAbydolding[16]. Ribosomal RNAs have been used to peer into the deepest recesses of the phylogeny of living organisms on earth because these macromolecules are ubiquitous and because their primary sequences change slowly due to powerful negative selection. To figure out how powerful, Golding [16] examined the primary sequences for 16/18S rRNAs from 51 species, including representatives from all major branches of life. The secondary structures of rRNAs are stabilized by many hydrogen bonded pairs of nucleotides, with stability of bonding being dependent upon the actual nucleotide pairs utilized and on their neighbors. In the total absence of selection, one would expect to see any base pair at any one site in the secondary structure among the 51 species, but this is not observed. Instead, most species are restricted to pairs that form strong hydrogen bonds and some sites are more conserved (are selected more strongly) than others. For estimating the selection coefficient for each hydrogen bonded pair in the secondary structure of the rRNA molecule, what was actually calculated was a composite parameter, 4Ns, where N is the effective population size and s is the selective advantage of hydrogen bonding at a site. Golding [16] found that was sufficiently large to account for complete conservation of hydrogen bonding at a site; i.e. for all 51 taxa to have a hydrogen bonded base pair at a site. Assuming a reasonable value for N (say 16,000), then a value of is large enough to maintain a specific site in a sequence over long phylogenetic time periods. This means that on average a 0.01% advantage is adequate to assure 100% conservation of a given site in rRNA; a 1% advantage would be consistent with the conservation of about 100 such sites, which in fact is close to what is observed, with the most conservative sites tending to be located in the middle of the molecule. (During the ‘bottleneck’ in human history, N may have been dangerously low. If in the above equation N is taken to be only 4000, then s rises drastically to about 0.0012. Now on average a 0.12% advantage is required to assure conservation at a site and a 12% advantage presumably would be required to assure conservation of 100 such

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sites. Such selective pressures may be inordinately high under most conditions; however, as mentioned above, even higher values of s have been previously reported [35]). While these calculations for gene products whose structures are necessarily extremely conservative may overestimate the selective disadvantage of ammo acid altering mutations for proteins generally, they should be fairly appropriate for ‘active’ or ‘functionally important’ regions of proteins generally. This is because similar structural constraints (requiring the conservation of weak bonding interactions at specific sites) are commonly observed and in fact are considered a ‘rule of thumb’ for the ‘active sites’ of macromolecules such as enzymes, channels, exchangers, pumps, metabolite transporters, and ligand (signal) specific receptors. In fact, the ‘rule of thumb’ applies pretty well across the board for functionally critical regions of all gene products since (from mRNA onwards) their functions almost ubiquitously depend upon weak bonding interactions. Such natural selection based conservation of structure (i.e., of specific sites in specific sequences) of course is the basis of maintained functional specificity of macromolecules through evolutionary time [28] and in principle should be applicable to the conservation of specific physiological systems in human hypoxia tolerance. To indicate the flavor of the problems involved, we here will stick to the example of ATP turnover in the CNS because it is relatively well described and understood. Assuming that similar selection pressures operate on the proteins in these

pathways as in conserving rRNA sequences [16], we can estimate s for simple multiple-component pathways (such as glucose transporter function coupled to glucose-glycogen conversion to pyruvate). Assuming modest sized active sites, we arrive at the conclusion that a small (about a 1.5%) selective advantage is adequate to assure that 10 sites in each of the 15 gene products in such a pathway in brain metabolism will be conserved (many metabolic enzymes are formed of more than one subunit and are highly conserved; hence, lor this example the actual numbers of genes involved and the numbers of conserved sites per gene are substantially higher, in which event the overall selective advantage would have to be proportionately higher than 1.5%). Still this seems helpful for it suggests that modern molecular evolutionary concepts should be able to readily accommodate the conservation of complex (multigene dependent) physiological systems. However, to experimental biologists, the implications are disturbing for two reasons. First, there is the practical problem of this being experimentally intractable - signal to noise ratios in physiological studies almost always are in the 5–10% range. In most physiological studies, a 1.5% advantage would be undetectable, which is one reason why physiologists should turn to evolutionary studies with caution [40]. Secondly, a serious theoretical problem seems to arise because the above illustrative estimates apply to only one pathway. In contrast, human hypoxia defense mechanisms involve many such highly conserved pathways. As

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mentioned above, in CNS energy supply + energy demand pathways alone, it is easy to demonstrate over 200 genes, not the 15 or so selected for illustrative purposes for a simple metabolic pathway. We are not certain about how far we can advance such analyses, but it seems clear that as the numbers of gene products required for given physiological systems increase towards 100s or even 1000s, the strength of selection required to conserve them unchanged (to conserve the complex physiological system unchanged) through evolutionary time may rise without limit. To molecular physiologists, his may not be very satisfying. Nevertheless, the relationship does suggest that the problem of conserving complex physiological systems through human phylogeny may be more difficult to explain than the appearance of so called ‘adaptable’ physiological traits, which in the context of this paper traditionally would be considered pivotal for extending tolerance of, and performance in, hypobaric hypoxia. Whereas evolutionary literature has addressed this problem in general terms [49J, to our knowledge the selective forces required to stabilize complex physiological systems composed of 100s or even 1000s of well defined gene products so far have never been quantified, or for that matter, even addressed.

Interestingly, a recent study of a random collection of 46 genes in humans and other hominids (12) unexpectedly found such high deleterious mutation rates in humans and other hominids that they doubted such species could survive if mutational effects on fitness were to combine in simple multiplicative way. The authors thus took their data to indicate that the effects of deleterious mutations may combine synergistically and such synergistic epistasis might help to explain the lower amount of ‘genetic death’ required to delete undesirable mutations and thus conserve ancestral physiological function. Be that as it may, this analysis appears to indicate that keeping complex physiological systems the same through long evolutionary time, keeping them the same as in our ancestors, may be more difficult than adjusting them to suite specific environmental challenges.

ACKNOWLEDGEMENTS This work was supported by NSERC (Canada). Especial thanks to Dr. Hans Christian Gunga whose questions and insights initiated this project in the first place.

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COMPARATIVE ASPECTS OF HIGHALTITUDE ADAPTATION IN HUMAN POPULATIONS

Lorna G. Moore 1, 2, Fernando Armaza V .3, 4 Mercedes Villena 3 , Enrique Vargas 3 1

Department Of Anthropology, University Of Colorado At Denver, Denver, CO 80217-3364 Center For Women ’s Health Research, University of Colorado Health Center, 4200 East Ninth Avenue, Denver, CO 80262; 3Caja Nacional de Salud, La Paz, Bolivia; 4Instituto Boliviano de Biolog’a de la Altura, La Paz, Bolivia 2

1.

POPULATION DIFFERENCES IN DURATION OF

HIGH-ALTITUDE RESIDENCE

Nearly 140 million people live permanently above 2500 m (8,000 ft) throughout the world, with about 17% of these persons residing in Africa, 56% in Asia, 26% in Central and South, and