International Review of Cytology: A Survey of Cell Biology, Volume 172

VOLUME 172 SERIES EDITORS Geoffrey H. Bourne James F. Danielli Kwang W. Jeon Martin Friedlander Jonathan Jarvik 1949...

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VOLUME 172

SERIES EDITORS Geoffrey H. Bourne James F. Danielli Kwang W. Jeon Martin Friedlander Jonathan Jarvik

1949-1 988 1949-1 984 19671984-1 992 1993-1 995

EDITORIAL ADVISORY BOARD Aimee Bakken Eve Ida Barak Howard A. Bern Robert A. Bloodgood Dean Bok Stanley Cohen Rene Couteaux Marie A. DiBerardino Charles J. Flickinger Nicholas Gillham Elizabeth D. Hay P. Mark Hogarth Anthony P. Mahowald M. Melkonian Keith E. Mostov Audrey L. Muggleton-Harris

Andreas Oksche Muriel J. Ord Vladimir R. Pantic Thomas D. Pollard L. Evans Roth Jozef St. Schell Manfred Schliwa Hiroh Shibaoka Wilfred D. Stein Ralph M. Steinman M. Tazawa Yoshio Watanabe Donald P. Weeks Robin Wright Alexander L. Yudin

Edited by

Kwang W. Jeon Department of Zoology University of Tennessee Knoxville, Tennessee

VOLUME 172

ACADEMIC PRESS San Diego London Boston New York Sydney Tokyo Toronto

Front cover photograph: SEM of coralline HA (Interpore) showing the interconnecting pore. (See Chapter 4, Fig. 19 for more details.)

This book is printed on acid-free paper.

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Copyright 0 1997 by ACADEMIC PRESS All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any mcans, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the Puhlishcr. The appearance of the code at the bottom of the first page of a chapter in this book indicates the Publisher’s consent that copies of the chapter may he made for personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. (222 Rosewood Drive, Danvers, Massachusetts 01 923), for copying heyond that permitted by Sections 107 or 108 ofthc US . Copyright Law. This consent does not extend lo other kinds ofcopying, such as copying for genera! distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Copy fccs for pre-1997 chaptcrs arc as shown on the title pages, if no fee code appears on the title page. the copy fee is the same as for current chapters. 0074-7696197 $25.00

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3 2 1

CONTENTS

Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ix

Transport of Glucose across the Blood-Tissue Barriers Kuniaki Takata, Hiroshi Hirano, and Michihiro Kasahara I. 11. 111. IV. V. VI.

Introduction . . . . . ............................ ............ Glucose Transporte .............. .................... Blood-Tissue Barriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glucose Transport in the Blood-Tissue Barriers . . . . . . . . . . . . . . . Regulation of Glucose Transporter Expression in Blood-Tissue Barriers . . . . . . . . . . . . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. ...... References

1 2 5 9 33 37 38

The Role of Suppressors in Determining Host-Parasite Specificities in Plant Cells Tomonori Shiraishi, Tetsuji Yamada, Yuki Ichinose, Akinori Kiba, and Kazuhiro Toyoda I. 11. II. IV. V. VI. VII.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Suppressors of Defense Response Produced by Phytopathogens .. Specific Production and Accessibility-Inducing Activity of Suppressors . . . . . . . . . . . . . Specific Suppression of the Establishment of Chemical Barriers . . . . . . . . . . . . . . . . . . Mode of Action of the Suppressors ........ Species-Specific Suppression of Cell Wall Function by the Suppressor . . . . . . . . . . . . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

............. V

55 59 62 65 67 76 81 85

vi

CONTENTS

Distribution and Functions of Platelet-Derived Growth factors and Their Receptors during Embryogenesis Paris Ataliotis and Mark Mercola I. II. 111, IV.

Introduction Biochemistry of PDGFs and Their Receptors Distributionof PDGFs during Embryogenesis Functional Studies on the Effects of PDGF V. Concluding Remarks References

95 96 104 110 117 119

Adaptive Crystal Formation in Normal and Pathological Calcifications in Synthetic Calcium Phosphate and Related Biomaterials G. Daculsi, J.-M. Bouler, and R. Z. LeGeros I. 11. 111. IV. V.

VI. VH. VIII. IX.

Introduction Analytical Techniques Crystal Formation, Composition, and Properties in Normal Calcifications Crystal Formation, Composition, and Properties in Pathological Calcifications Factors Affecting Crystal Formation and Properties of Biologically Relevant Calcium Phosphates Calcium Phosphates and Related Bone Graft Biomaterials Comparative Properties of Bone and Calcium Phosphate Materials Bone/BiomaterialInterface Summary and Conclusion References

129 135 139 145 154 159 165 167 175 177

The Biogenesis, Traffic, and Function of Cystic Fibrosis Transmembrane Conductance Regulator Tamas Jilling and Kevin L. Kirk I. Introduction II. Cystic Fibrosis Transmembrane Conductance Regulator Ill. CFTR Mutations That Cause Disease IV. Physiological Role of CFTR as an Apical CI' Channel in Epithelial Tissues V. Regulation of CFTR Function by the Cytoskeleton VI. CFTR as a Regulator of Other Channels VII. Itinerary of CFTR Traffic within Epithelial Cells

193 195 200 208 212 213 215

vii

CONTENTS

VIII. Regulation of Membrane Traffic by CFTR? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. Summary and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

220 231 232

Regulation of Phenylpropanoid Metabolism in Relation to Lignin Biosynthesis in Plants Mark S. Barber and Heidi J. Mitchell I. II. 111. IV. V. VI.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General PhenylpropanoidPathway . . . . . . . . . . . . . . . . . . . . . . . Lignin Branch Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Storage and Transport of Monolignols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PolymerizationProcess . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding Remarks,. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

....................................................

243 247 262 269 271 273 273

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

295

This Page Intentionally Left Blank

Numbers in parentheses indicate the pages on which the authors' contributions begin.

Paris Ataliotis (95), Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02 1 15 Mark S. Barber (243),School of Biological Sciences, University of Southampton, Southampton SO 16 7PX, United Kingdom J,-M. Bouler (129), Laboratoire Recherche surles Tissus Calcifies et les Biomateriaux, Faculte de Chirurgie Dentaire, 44042 Nantes Cedex 01, France G. Daculsi (129), Laboratoire Recherche sur les Tissus Calcifies et les Biomateriaux, Faculte de Chirurgie Dentaire, 44042 Nanfes Cedex 01, France Hiroshi Hirano (1 ), Depaltment of Anatomy, Kyorin University School of Medicine, Mitaka, Tokyo 181, Japan Yuki lchinose (55), Laboratory of Plant Pathology and Genetic Engineering, College of Agriculture, Okayama University, Okayarna 700, Japan Tamas Jilling (193), Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama Michihiro Kasahara ( 1), Laboratory of Biophysics, School of Medicine, Teikyo University, Hachioji, Tokyo 192-03,Japan Akinori Kiba (55),Laboratory of Plant Pathology and Genetic Engineering, College of Agriculture, Okayama University, Okayama 700, Japan Kevin L. Kirk (193), Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294

R. 2. LeGeros (129), Laboratoire Recherche sur les Tissus Calcifies et les Biomateriaux, Faculte de Chirurgie Dentaire, 44042 Nantes Cedex 01, France ix

X

CONTRIBUTORS

Mark Mercola (93, Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 021 15 Heidi J. Mitchell (243), Research School of Biological Sciences, Australian National University, Canberra ACT 0200, Australia Tomonori Shiraishi (55), Laboratory of Plant Pathology and Genetic ,Engineering, College of Agriculture, Okayama University, Okayama 700, Japan Kuniaki Takata (i), Laboratory of Molecular and Cellular Morphology, lnstifute for Molecular and Cellular Regulafion, Gunma University, Maebashi, Gunma 371, Japan KazuhiroToyoda (55),Laboratory of Plant Pathologyand Genetic€ngineering,College of Agriculture, Okayama University, Okayama 700, Japan Tetsuji Yamada (55),Laboratory of Plant Pathology and Genetic €ngineering, College of Agriculture, Okayama University, Okayama 700, Japan

Transport of Glucose across the Blood-Tissue Barriers Kuniaki Takata,* Hiroshi Hirano,t and Michihiro Kasaharat “Laboratory of Molecular and Cellular Morphology, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma 371, Japan; ?Department of Anatomy, Kyorin University School of Medicine, Mitaka, Tokyo 181, Japan; and $Laboratory of Biophysics, School of Medicine, Teikyo University, Hachioji, Tokyo 192-03, Japan

In specialized parts of the body, free exchange of substances between blood and tissue cells is hindered by the presence of a barrier cell layer@).Specialized milieu of the compartments provided by these “blood-tissue barriers” seems to be important for specific functions of the tissue cells guarded by the barriers. In blood-tissue barriers, such as the blood-brain barrier, blood-cerebrospinal fluid barrier, blood-nerve barrier, blood-retinal barrier, blood-aqueous barrier, blood-perilymph barrier, and placental barrier, endothelial or epithelial cells sealed by tight junctions, or a syncytial cell layer@), serve as a structural basis of the barrier. A selective transport system localized in the cells of the barrier provides substances needed by the cells inside the barrier. GLUTl , an isoform of facilitated-diffusionglucose transporters, is abundant in cells of the barrier. GLUT1 is concentrated at the critical plasma membranes of cells of the barriers and thereby constitutes the major machinery for the transport of glucose across these barriers where transport occurs by a transcellular mechanism. In the barrier composed of doubleepithelial layers, such as the epithelium of the ciliary body in the case of the blood-aqueous barrier, gap junctions appear to play an important role in addition to GLUT1 for the transfer of glucose across the barrier. KEY WORDS: Glucose transporter, GLUTl , Gap junction, Blood-tissue barriers, Placental barrier, Epithelial cells, Endothelial cells.

1. Introduction The mammalian body is a conglomerate of highly differentiated organs. The organs are made of specialized tissue-specific cells, blood vessels, nerves, etc. lnrernarional Review of cyfO/Ogy, V d I72 (H174-769hiY7 $25.00

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Copyright 0 1997 hy Academic Press All rights of reproduction i n any form reserved.

2

KUNlAKl TAKATA ET AL.

The normal function of these tissue cells is maintained by an uninterrupted supply of nutrients and oxygen and concomitant removal of metabolites and carbon dioxide via the circulating blood. In some of the tissues and organs, free exchange of substances between blood and tissue cells is hindered by the presence of barrier cell layers. A specialized environment provided by these “blood-tissue barriers” seems to be important for specific functions of the tissue cells enclosed by the barrier. Tracer experiments have demonstrated that either cndothelial or epithelial cells are the anatomical basis of the barrier. Specific transport mechanism across such bloodtissuc barriers must exist for a number of substances. In this chapter, we focus on the cellular and molecular basis for the transport of glucose across the blood-tissue barriers. II. Glucose Transporters Glucose is onc of the most important sources of energy as well as a substrate for a variety of cellular molecules. Cellular membranes, whose basic structure is made of phospholipid bilayers, are practically impermeable to small polar molecules such as glucose. Glucose transporters are integral membrane proteins that mediate the transport of glucose and related substances across the cellular membranes (Wheeler and Hinkle, 1985; Baly and Horuk, 1988; Carruthers, 1990; Silverman, 1991; Baldwin, 1993). Two types of glucose transporters have been discovered in animal cells: facilitateddiffusion glucose transporters and Na+-dependentactive glucose transporters (cotransporters) (Kasahara et al., 1985; Baly and Horuk, 1988; Nikaido and Saier, 1992). Facilitatcd-diffusion glucose transporter was first identiticd by Kasahara and Hinkle (1977) in human erythrocyte ghosts as a zone 4.5 protein. The gene was cloned and sequenced from a HepG2 human hepatoma cell cDNA library (Mueckler et al., 1985) and from a rat brain library (Birnbaum rt a/., 1986), and later termed GLUT1 (Bell et al., 1990). The N a ’ -dependent glucosc transporter is a cotransporter that mediates the active uptake of glucose across the membrane driven by the influx of Nat according to its chemical gradient. The gene was first cloned from a rabbit intestinal cDNA library by the expression cloning method using Xenopus oocytes (Hedigcr et al., 1987) and termed SGLTl (Hediger et al., 1989). Each transporter constitutes a family, and several isoforms have been identitied and characterized.

A. Facilitated-Diffusion Glucose Transporter Family Six isoforms of facilitated-diffusion glucose transporter GLUT family have been cloned in mammalian cells (Table 1) (Bell etal., 1990,1993; Silvcrman,

TABLE I GLUT Family Transporters in Animal Cells

Gene

Species

Major site of expression

GLUT1

Human

Blood-tissue barriers. erythrocyte. fetal tissues

Transport substrate

K,,, (mM)"

Number of amino acids

17

492

Glucose

492 492 492 492 Fragment 490

Rat Mouse Bovine Rabbit Pig Chicken

GLUT2

Human

Liver. pancreatic 6-cell. small intestine, kidney

42

Glucose, fructose

Rat Mouse Chicken

GLUT3

503145 X16986. XI5684 222932

Dog Chicken Sheep

495 496 494

Rat

GLUT4

522 523

Mouse

11

Glucose

Human Rat

Adipocyte, muscle

4

Glucose

510

Mouse

GLUT5

Human Rat

GLUT7

Rat

509 509

Small intestine

Fructose

Liver microsome

Glucose

6

Rabbit ~

501 502

Human chromosomal location

JCqofor cytochalasin B (PM) 0.1

1

0.4

M13979, M22063 M22998. M23384 M60448 M21747 X17058 LO7300

~03810

533

Brain

K0319S

524

496 493 493

Human

GenBank accession number

M20681

3

7

12

0.1-2

D13962, U17978 X61093, U11853, M75135 L35267 M37785 L39214 M20747, M91463 D28561.504524, X14771. M25482 M23383

486

M55531 D13871, D28562, LO5195 D26482

528

X66031

17 0.3

1

No inhibition

~

* K,, for 3-0-methyl glucose transport under equilibrium exchange conditions, except for GLUT5 in which fructose is the substrate.

4


1991; Gould and Bell, 1990; Lienhard et al., 1992; Pessin and Bell, 1992; Thomas el al., 1992; Baldwin, 1993; Gould and Holman, 1993; Marger and Saier, 1993; Takata et al., 1993a; Mueckler, 1994). Analyses of the amino acid sequences of the transporters indicate that GLUT glucose transporters are transmembrane proteins that span the lipid bilayer 12 times, with both the amino and carboxyl termini facing the cytoplasmic side (Mueckler rt al., 1985). GLUT transporters are a member of thc major facilitator superfamily which possesses a common structural motif of 12 transmembrane-spanning a-helical segments (Marger and Saier, 1993). The distribution of GLUT transporters in organs, tissues, and cclls has bccn determined by Northern blotting, immunoblotting, in situ hybridization, and immunohistochemical staining. GLUT transporters are produced in a tissue- and cell-specific manner and seem to be closely related to a variety of cellular activities, ubiquitous and specific (Gould and Bell, 1990; Takata et al., 19931; Gould and Holman, 1993; Mucckler, 1994). GLUTl is a widely distributed transporter isoform whose synthesis begins at a very early stage during development and is responsible for the uptake of glucose for the basal cellular activities. The most characteristic feature of GLUTl is its abundance in the blood-tissue barriers. GLUT2 is a low-affinity, highcapacity transporter produced in liver, intestine, kidney, and pancreatic /3 cells (Thorens et al., 1988, 19YOa,b; Fukumoto et al., 1988a; Johnson et af., 1990; Orci et al., 1989; Thorens, 1992). This transporter, in combination with other molecules such as glucokinase, is considered to play an important role in the sensing of blood glucose level in pancreatic /3 cclls (Orci et u1.,1989; Thorens et a/., 1990c; Matschinsky, 1990; Unger, 1991; Thorens, 1992; Vionnct et al., 1992). GLUT3 is predominantly produced in the brain and is considered to be a glucose transporter of neurons (Kayano et af., 1988; Nagamatsu et d., 1992). GLUT4 is an isoform produced in insulinsensitive cells such as adipocytes and skeletal and cardiac muscle cells (Birnbaum, 1989, 1992; James et al., 1989; Kaestner et al., 1989; Charron et al., 1989; Fukumoto et a/., 1989). This transporter is prefercntially localized in the intracellular compartments such as endosomes and the transGolgi reticulum (Slot et al., 1990, 1991a,b; Friedman et al., 1991; Smith et al., 1991; Takata et al., 1992a). Upon insulin stimulation, cytoplasmic vesicles containing GLUT4 fuse with the plasma membrane (Suzuki and Kono, 1980; Cushman and Wardzala, 1980; Kono et al., 1982; Ezaki et al., 1986) and thereby the number of surface GLUT4 molecules increases. Such translocation of GLUT4 molecules from the intracellular pool to the cell surface mainly accounts for the insulin-stimulated increase in glucose transport activity and serves as a mechanism for the homeostasis of blood glucose level (Birnbaum, 1992). GLUT5 is a fructose transporter expressed in the small intestine and is responsiblc for the dietary absorption of fructose (Kayano et al., 1990; Burant et al., 1992; Davidson et al., 1992). GLUT6 is

TRANSPORT OF GLUCOSE ACROSS BLOOD-TISSUE BARRIERS

5

a pseudogene (Kayano etal., 1990). GLUT7 was isolated from a liver cDNA library and has a homology to GLUT2 (Waddell et af.,1992). It is a glucose transporter of the microsomal membrane and seems to play an important role in the exit from the endoplasmic reticulum of glucose formed by the action of glucose-6-phosphatase (Burchell et af., 1994).

B. Nat-Dependent Glucose Transporter Family Transport of glucose by Na+-dependent glucose transporter SGLTl is dependent on extracellular Na', and therefore it can mediate the uphill transport of glucose. SGLTl does not have sequence homology to transporters of the GLUT family (Hediger etaf., 1989). Rather, it is a member of a distinct Na+-dependent cotransporter SGLTl family (Wright, 1993; Hediger and Rhoads, 1994; Hediger et uf., 199.5). Four different models have been proposed so far for the configuration of the molecule in the lipid bilayer (Hediger et al., 1987; Wright et al., 1991; Lee et af., 1994; Turk et al., 1996). SGLTl is a high-affinity glucose cotransporter with a Na+/glucose coupling ratio of 2: 1 (Lee et af., 1994), and it is present in both the kidney and the small intestine (Takata et af., 1991a,b, 1992b; Hwang et al., 1991; Yoshida et af., 199.5). SGLTl plays a pivotal role in the absorption of glucose in the small intestine because mutation of SGLTl resulted in a severe dysfunction of glucose absorption in the small intestine (Turk et al., 1991). SGLTl in combination with GLUT2 and GLUT1 serves in the active transepithelial transport of glucose (Thorens, 1993; Takata, 1996). Recently, two low-affinity, Na+-dependent glucose transporters, SGLT2 (Kanai et af., 1994) and pSGLT2 (SAAT1) (Kong et al., 1993; Hediger et af., 199.5), were identified. SGLT2 exhibited a Na+/glucose coupling ratio of 1: 1 and seems to be responsible for the reabsorption of glucose from the urinary filtrate in the kidney S1 proximal tubules. Na+-dependent rnyoinositol cotransporter SMIT (Kwon et al., 1992) and nucleoside cotransporter SNSTl (Pajor and Wright, 1992) are also members of this family.

111. Blood-Tissue Barriers A. Dietary Absorption of Glucose Dietary carbohydrates are hydrolyzed to monosaccharides and absorbed in the alimentary tract. In the small intestine, glucose is absorbed through the absorptive epithelial cells lining the surface of the villi. Nat-dependent glucose cotransporter SGLTl is localized at the microvillous apical plasma

ti


membrane of the absorptive epithelial cells (Takata et al., 1992b; Yoshida et nl., 1995). Glucose in the intestinal lumen is actively transported into the cytoplasm of the absvrptive epithelial cells by SGLTl driven by the chemical gradient of Na+ across the plasma membrane, which is maintained by the action of Na'/K+-ATPase at the expense of ATP (Wright 1993; Wright rt al., 1991, 1994; Hediger and Rhoads, 1994). Glucose leaves the absorptive epithelial cells by the action of facilitated-diffusion glucose transporter GLUT2 localized at the basolateral plasma membrane (Thorens et of., 1990a; Thorens, 1992, 1993). It then enters the capillaries in the core of the villi and, after passing through the liver via the portal vein, it is distributed throughout the body via the vast network of blood vessels (Unger, 1991; Takata et al., 1993a).

6. Blood-Tissue Barriers: Endothelium Type and Epithelium Type In most parts of the body, relatively free exchange of substances including glucose occurs between blood and tissue cells (parenchymal cells). In the specialized tissues and organs, in which a specific microenvironment is of great importance for their specific functions, such free exchange is hindered by the presence of barrier cell layers (Fig. 1). The barrier property of the tissues was well demonstrated by tracer experiments: Intravenously administered cytochemical tracers, such as dyes or horseradish peroxidase, b

a tissue cells

C

tissue cells

tissue cells

FIG. 1 Blood-tissue barriers. (a) The absence of blood-tissue barricrs. In most lissues, eudolhelial walls of blood vessels are highly permeable, and free exchange of a variety of substances occurs bctwcen blood and tissue cells. (b) Blood-tissue barrier of the cndothelium type. The endothelium is impermeable and constitutcs a barrier layer. (c) Blood-tissue barrier of the epithelium type. Although the blood vessels are permeable, the epithelial barricr layer prcvcnts the exchange of substances between blood constitucnts and tissue cclls.

7


failed to penetrate into these tissues. Such impermeable barriers, as summarized in Table 11, are found in the brain (blood-brain barrier and bloodcerebrospinal fluid barrier), the eye (blood-ocular barrier: i.e., bloodretinal barrier and blood-aqueous barrier), the inner ear (blood-perilymph barrier and blood-endolymph barrier), the peripheral nerves (blood-nerve

TABLE II Blood-Tissue Barriers and Glucose Transporters"

Barrier ~

Site of the barrier

~

~

Major glucose transporter in the barrier ~~

Blood-brain barrier

Endothelial cells (brain microvessels)

GLUT^ *

Blood-cerebrospinal fluid barrier

Endothelial cells (brain rnicrovessels)

GLUTl

Epithelial cells (choroid plexus)

GLUT1'

Blood-retinal barrier

Endothelial cells Pigment epithelial cell

GLUTl GLUTl

Blood-aqueous barrier

Epithelial cells (ciliary body) Endothelial cells (iris)

GLUTI"

Blood-perilymph barrier

Endothelial cells (inner ear)

GLUTl

Blood-endolymph barrier

Basal cells (stria vascularis)

GLUTl'

Blood-nerve barrier

Perineurial cells (perineurium) Endothelial cells (endoneurial blood vessels)

GLUTl GLUTl

Placental barrier (human)

Syncytiotrophoblast layer

Placental barrier (rat)

Syncytiotrophoblast layers

GLUTIK

Blood-testis barrier

Sertoli cells

NDhJ

Blood-thymus barrier (cortex)

Endothelial cells'

N D~

CLUTlf

See text for references. In addition to GLUTl, the presence of GLUT3 or GLUT4 was suggested. GLUTl is localized at the basolateral membrane. 'IGap junctions (connexin 43), in combination with GLUTl, serve as a transport machinery. GLUTl in the basal cells may also serve in the transport of glucose across the perilymphendolymph barrier. 'In addition to GLUTl, the presence of GLUT3 was suggested. Gap junctions (connexin 26), in combination with GLUTl, serve as a transport machinery of glucose. GLUT3 is also present. Not detected. ' GLUTl is localized in the endothelial cells of the blood vessels surrounding seminiferous tubules. 1 Contribution of macrophages is also suggested.

''

8

KUNIAKI TAKATA E r AL.

barrier) , the placenta (placental barrier), the testis (blood-testis barrier), and the thymus (blood-thymus barrier). These barriers are collectively called the blood-tissue barriers (Takata et al., 1990a,b, 1993a). Histological and ultrastruct ural examinations have revealed that endothelial cells sealed by tight junctions serve as an anatomical basis for the barrier when blood vessels are the barrier (Fig. l b ) (Takata et al., 1990b). In another case, the blood vessels are highly permeable, but the adjacent epithelial cells sealed by tight junctions function as an impermeable barrier (Fig. lc). In special cases, a syncytial cell layer works as a barrier layer instead of cells sealed by tight junctions. Specific transport machinery located in the barrier layer may ensure the supply of substances needed by the cells enclosed by the barrier layer. Glucose is a ubiquitous source of cellular metabolism in the mammalian body. Glucose transporters located in the cells of the barrier play a pivotal role in the passage of glucose across the barrier for the nourishment of the cells inside the barrier. Among the isoforms of glucose transporters, GLUTl is abundant in the cells of blood-tissue barriers and serves as the major glucose transporter isoform in these barriers (Figs. 2 and 3) (Takata et al., 1990a,b, 1993a; Harik et al., 1990a,b). In some cases, connexins of gap junctions in combination with GLUTl seem to be involved in the passage of glucose through the barrier (Takata et al., 1991c, 1994; Shin et ul., 1996a,b).

continuous capillary

fenestrated epithelial barrier

FIG. 2 GLUTI, a glucose transporter isoform, in blood-tissue barriers. The two basic types of blood-tissue barriers are illustrated. TJ, tight junction. (a) Endothelium type. A continuous capillary sealed by tight junctions serves as the structural basis for the barrier. Glucose transfer across the barrier is carried out by the transendothelial transport of glucose via plasma membrane GLUTI. Note that GLUTl is present at both the luminal and contraluminal domains of the plasma membrane. (b) Epithelium type. Fenestrated capillarics are highly permeable, An epithelium sealed by tight junctions, or a syncytial epithclial cell (not shown), serves as the structural basis for this barrier. Glucose transfer across the barrier is carried o u t by the transepithelial transport of glucose via plasma membrane GLUT1. Note that GLU'TI is present at both the apical and basolateral domains of the plasma membrane.

9


-200K -116K - 97K

-

66K

45K

FIG. 3 Immunoblotting with anti-GLUT1 antibody of rat tissues having blood-tissue barriers. Cell homogenates (10pgprotein each) were analyzed. GLUTl is detected in brain microvessels (brain vessel, 50 kDa), inner portion of the retina (retina, 43 kDa), pigment epithelium of the retina (pig. epithelium, 46 kDa), ciliary body and iris of the eye (ciliary B. & Iris, 46 kDa), and in the placenta (44 kDa). No comparable amount of GLUTl was found in testis and thymus. Reproduced from Takata et al. (1990b) with permission from Academic Press.

IV. Glucose Transport in the Blood-Tissue Barriers A. Blood-Brain Barrier 1. Structure Among the blood-tissue barriers, the blood-brain barrier has been extensively studied (Fishman, 1980; Betz and Goldstein, 1986; Cserr, 1986; Strand, 1988; Dermietzel and Krause, 1991). In the blood-brain barrier, tight junctions between endothelial cells are responsible for preventing the free passage of substances across the barrier (Reese and Karnovsky, 1967; Stewart et al., 1994). Intravenous injection of tracers such as horseradish peroxidase demonstrated that the tracers remained in the lumen of the blood vessels in the brain and were not found beyond the vascular endothelium (Reese and Karnovsky, 1967). A similar tracer experiment also established the blood-cerebrospinal fluid barrier, in which the epithelium of the choroid plexus, in addition to the cerebral blood vessels, functions as the structural basis for this barrier (Brightman and Reese, 1969).

10


2. GLUTl in the Blood-Brain Barrier Crone (1965) measured the transfer of glucose from the blood into brain tissues by the intracarotid injection of labeled glucose in dogs. A carriermediated. facilitated passage of glucose across the blood-brain barrier was observed. Based on these findings, Crone (1965) proposed a possible mechanism involving endolhelial cells in the brain capillaries for the transport of glucose across the blood-brain barrier. Dick et al. (1984) demonstrated that cerebral microvessels were rich in a 53-kDa glucose transporter by the binding of cytochalasin B, a specific ligand for glucose transporter, and by anti-human erythrocyte glucose transporter (GLUTl) antibody labeling. The abundance of GLUTl in the blood-brain barrier was confirmed in brain capillary specimens by immunoblotting (Fig. 3 ) (Kalaria et al., 1988; Gerhart et al., 1989; Pardridge et al., 1990; Takata et al., 1990b), Northern blotting (Flier et al., 1987; Boado and Pardridge, 1990), and binding of cytochalasin B (Kalaria et al., 1988). Comparison of quantitative immunoblotting for GLUT1 and cytochalasin B binding indicated that GLlJTl is the principal glucose transporter isoforni mediating glucose transport across the blood-brain barrier (Pardridge et al., 1990; Dwyer and Pardridge, 1993). GLUTl was shown to be localized in the endothelial cells of the brain microvessels by immunohistochemical staining (Fig. 4) (Kalaria et al., 1988; Gerhart et al., 1989; Kasanicki et al., 1989; Harik et al., 1990a; Pardridge et al., 1990; Takata et al., 1990b) and by in situ hybridization (Pardridge et aL, 1990). GLUTl localized at both the luminal and contraluminal plasma membranes (Gerhart et al., 1989;Takata et al., 1990b;Farrell and

FIG. 4 GLCJTI in the blood-brain barrier. GLUTl is localized at hoth the luminal (arrowheads) and contraluniinal (arrows) plasma membranes of microvessel cndothclial cells. Immunofluorescence (a) and corresponding Nomarski differential interference contrast (b) images arc shown. Bar = 10 Fm.


11

Pardridge, 1991) is thought to be the molecular basis of the transendothelial transfer of glucose in the blood-brain barrier. In an immunogold electron microscopic study, Gerhart et al. (1989) showed that GLUTl was equally abundant on the luminal and contraluminal membranes of endothelial cells of canine brain microvessels. Most of the label was restricted to the plasma membrane, and less than 10%of the GLUTl was found in the cytoplasm. Farrell and Pardridge (1991), on the other hand, showed GLUTl to be asymmetrically distributed on endothelial cells of the rat brain capillaries: There was a fourfold greater abundance on the contraluminal membrane compared with the density on the luminal membrane. Similar results were obtained for rabbit brain endothelial cells (Cornford f’t al., 1993). The difference observed between canine and rat/ rabbit blood vessels with respect to the distribution of GLUT1 is not clear but may be attributed to species differences or the method used. The asymmetrical distribution of GLUTl might create a faster rate of glucose transport across the contraluminal membrane compared with that across the luminal membrane, thereby minimizing the phosphorylation of glucose in the cytoplasm and maximizing the transfer of glucose across the barrier. In addition. in this case more than 40% of the GLUTl was observed within the cytoplasm of the endothelial cells (Farrell and Pardridge, 1991). This cytoplasmic pool of GLUT1 might serve as a reservoir and could be in a possible translocation machinery as seen for GLUT4 in the insulin-sensitive cells (Birnbaum, 1992). GLUTl is also expressed in astrocytes (Devaskar et al., 1991; Lee and Bondy, 1993; Morgello et al., 1995), epithelial cells of the choroid plexus (Kalaria et al., 1988; Bagley et al., 1989; Takata et al., 1990b; Farrell et al., 1992a), ependymal epithelial cells lining the ventricular wall (Farrell et al., 1992a), and in tanycytes (Young and Wang, 1990) in the brain. Differences in apparent molecular mass of GLUTl were observed following SDSpolyacrylamide gel electrophoresis: Whereas GLUTl of microvessels showed a molecular mass of 54 or 55 kDa, that of astrocytes and choroid plexus exhibited a lower one of 42-45 kDa (Maher et al., 1993, 1994; Vannucci, 1994; Kumagai et al., 1994a; Morgello et al., 1995). The apparent difference is caused by differential N-linked glycosylation (Kumagai et al., 1994a), the functional significance of which remains to be clarified. GLUT1 was not expressed in the endothelial cells devoid of barrier properties in the brain, nor was it detected in the blood vessels of the median eminence and area postrema, both of which lack barrier characteristics (Kalaria rt al., 1988; Young and Wang, 1990). The adenohypophysis was devoid of GLUT1, whereas some blood vessels were GLUTl positive in the neurohypohysis (Gerhart et al., 1989). Endothelial cells in the choroid plexus have numerous fenestrae and are thus highly permeable. GLUTl was not found in these cells. Instead, epithelial cells of the choroid plexus

12


were strongly positive for GLUTl (Kalaria et al., 1988; Bagley et al., 1989; Takata et al., 1990b). 3. Glucose Transporters Other Than GLUTl

GLUT3 is expressed in the brain (Kayano et al., 1988; Bell et al., 1990; Nagamatsu et al., 1992; Maher et af., 1994). Northern blotting and in situ hybridization as well as immunoblotting and immunohistochemical staining showed that GLUT3 is specifically localized in neurons in the rodent brain (Nagamatsu el al., 1992,1993a). Although GLUTl is the prime isoform of glucose transporters in the cerebral microvessels and astrocytes, and GLUT3 in the neurons, the contribution of transporter isoforms other than GLUT1 in the blood-brain barrier has been suggested. Using anti-peptide antibody raised against the carboxyl terminal end of human GLUT3 protein, Gerhart et al. (1992) detected GLUT3 in the blood-brain barrier in frozen sections from dogs and rats. Immunohistochemical staining demonstrated that neurons and microvessels were positively stained. Becausc the amino acid sequencc of the carboxyl terminus of GLUT3 differs considerably between human and rodent GLUT3 (Kayano et al., 1985; Nagamatsu et al., l992), some of these positive reactions may not represent GLUT3. In fact, antibody specific for rodent GLUT3 failed to result in positive staining in rat brain microvessels (Gerhart et nl., 1995). In the dog brain, however, endothelial cells in the blood brain barrier were positively stained with an antibody against canine GLUT3 (Gerhart et al., 1995). In the human brain, although GLUT3 is primarily expressed in neurons, the presence of GLIJT3 in the microvascular endothelial cells was reported (Mantych et nl., 1992). However, GLUT3 was not detected in isolated human or rat microvessels (Maher et al., 1993). The previously mentioned inconsistency in results with regard to the presence of GLUT3 in brain microvessels may be due to spccies differences andlor to the variation in carboxyl terminal sequences used to generate the antibodies, which would result in differences in the cross-reactivity of the antibodies as well as in nonspecific binding of the antibody to other molecules (Shepherd et al., 1992a). Although further studies are needed, GLUTl is clearly the major transporter in the bloodbrain barrier, and the contribution of GLUT3 to the transfer of glucose across the barrier is probably minimal, if any (Maher et al., 1993, 1994; Vannucci 1994). Fructose transporter GLUTS was detected in the human brain by immunoblotting (Shepherd et aL, 1992b; Mantych et al., 1993a). Immunohistochemical analysis demonstrated that only some of the brain microvascular endothelial cells were positive for GLUTS, although all the vessels were positive for GLUT1 and factor VlIl staining (Mantych et al., 1993a). Because fructose is not used as a source of nutrient in the brain, it is unlikely


13

that GLUTS carries fructose through the blood-brain barrier. The possible role of GLUT5 remains obscure. In addition to the facilitated-diffusion transfer mechanism, immunohistochemical and immunoblotting studies on bovine cortical vessels suggested the presence of a Na+-dependentglucose cotransporter of the SGLT family in these vessels (Nishizaki et af.,1995). Further examination will be needed to determine the possible involvement of a Na'-dependent system in the blood-brain barrier.

6 . Blood-Cerebrospinal Fluid Barrier

1. Structure Cerebrospinal fluid, which fills the cranial cavity, is an important determinant of the extracellular fluid that bathes neurons and glia in the central nervous system (Fishman, 1980; Wood, 1980; Cutler, 1980; Rowland et af., 1991). It is secreted mainly by the choroid plexus in the lateral ventricle and is absorbed through the arachnoid villi. Another source of the cerebrospinal fluid is parenchymal blood vessels. The fluid from the blood vessels enters the ventricular system through the ependymal cell layer. The permeability barrier between blood and cerebrospinal fluid is called the bloodcerebrospinal fluid barrier. The choroid plexus is made of cuboidal to columnar epithelial cells connected by tight junctions. Capillaries located underneath the choroid plexus epithelium are of the fenestrated type. Tracer experiments using horseradish peroxidase and lanthanum demonstrated that endothelial cells in the brain microvessels and epithelial cells of the choroid plexus, both of which are sealed by tight junctions, serve as the anatomical basis for the blood-cerebrospinal fluid barrier (Brightman and Reese, 1969).

2. Glucose Transport A carrier-mediated glucose transport system across the barrier was shown by the intravenous or intracisternal administration of sugars (Fishman, 1964) and by the ventriculocisternal perfusion method (Bradbury and Davson, 1964). The epithelial cells of the choroid plexus are rich in GLUT1, which is localized at the basolateral plasma membranes (Kalaria et al., 1988; Bagley et al., 1989; Takata et nf., 1990b; Farrell et al., 1992a). A 45- to 47-kDa form of GLUT1 was detected in the choroid plexus (Kumagai et al., 1994a; Vannucci, 1994). The apparent difference in M , of GLUT1 from that of brain microvessels is due to the differential glycosylation of GLUT1 in the choroid plexus (Kumagai et al., 1994a). Such differential glycosylation

14


might contribute to the basolateral targeting of GLUTl molecules in the choroid plexus. Glucose transporters in the apical plasma membrane of the epithelial cells in the choroid plexus have not been demonstrated so far. Because the disposition of glucose transporters at both the apical and basolateral plasma membranes is a prerequisite for the successful transepithelial transport of glucose (Thorens, 1993; Takata, 1996), the choroid plexus is not likely to be responsible for the secretion of glucose into the cerebrospinal fluid. Rather, GLUT1 at the basolateral membrane may contribute to the uptake of glucose to fuel the epithelial cells, which are very active in the transport of various substances in regulating the composition of the cerebrospinal fluid (Spector and Johanson, 1989). A similar basolateral localization of GLUTl was observed in the S3 segment of the kidney proximal tubules (Thorens et ul., 1990b; Takata et d.,1991b), where it may serve to supply glucose to these metabolically active cells.

C. Blood-Ocular Barrier The eyes develop from the neural tube and are often considered to be an extension of the central nervous system. The blood-ocular barrier (bloodeye barrier) therefore has similarities to the blood-brain barrier but has various unique aspects of its own as well. It consists of the blood-retinal barrier and blood-aqueous barrier (Raviola, 1977).

D. Blood-Retinal Barrier 1. Structure

The retina originates from the neural tube and develops a unique photoreception system. It is nourished from both inside and outside. In the former case, nutrients and oxygen are supplied directly from the blood vcsscls distributed inside the retina, which are branches of the central retinal blood vessels in the optic nerve. The blood vessels of the retina have a barrier property as is seen in the brain (Raviola, 1977; Bill et al,, 1980). When a tracer, such as thorium dioxide (Shakib and Cunha-Vaz, 1966), horseradish peroxidase (Shiose, 1970; Raviola, 1977), or microperoxidase (Smith and Rudt, 1975), was injected into the bloodstream, it failed to penetrate the endothelial cell layer of the capillaries in the retina. Similar results were observed in the permeability of the capillaries to endogenous serum proteins such as albumin and immunoglobulin (Pino and Thouron, 1983).Ultrastructural examination showed that retinal capillaries are of the continuous type,

TRANSPORT

OF GLUCOSE ACROSS BLOOD-TISSUE BARRIERS

15

i.e., the endothelial cells are devoid of fenestrae and are connected by tight junctions (Raviola, 1977). The retina is surrounded by the highly vascularized choroid layer. Blood vessels in the choroid adjacent to the retinal pigment epithelium have numerous fenestrae in their endothelial cells (Bernstein and Hollenberg, 1965; Raviola, 1977) and are highly permeable (Bill et al., 1980). Intravenously injected tracers easily escape from the blood vessels (Shiose, 1970; Raviola, 1977). Retinal pigment epithelial cells, which constitute the outermost layer of the retina, are connected by well-developed tight junctions and gap junctions (Hudspeth and Yee, 1973; Raviola, 1977). Observation of freeze-fracture replicas demonstrated elaborate arrays of intramembranous particles of tight junctions (Hudspeth and Yee, 1973). Intravenous administration of horseradish peroxidase demonstrated that the retinal pigment epithelium constitutes a permeability barrier, i.e., horseradish peroxidase was effectively blocked by the tight junctions connecting the retinal pigment epithelial cells (Shiose, 1970; Smith and Rudt, 1975; Raviola, 1977; Caldwell and McLaughlin, 1983). 2. Glucose Transport

The existence of the barriers in the inner and outer parts of the retina suggests the presence of selective machineries for the transport of nutrients and metabolites across the barrier, which would be crucial for the maintenance of retinal function. In fact, transport of glucose (Zadunaisky and Degnan, 1976; Pascuzzo et al., 1980; Masterson and Chader, 1981; Crosson and Pautler, 1982; Stramm and Pautler, 1982; DiMattio and Streitman, 1986; Miceli et al., 1990) as well as of ions (DiMattio et al., 1983; Kennedy, 1990), retinoids (Ottonello et al., 1987; Bok, 1990), myo-inositol (Khatami, 1988), amino acids (Pautler and Tengerdy, 1986; Sellner, 1986), and ascorbate (Khatami et al., 1986) was observed to occur in the retinal pigment epithelial cells. In the inner blood-retinal barrier, GLUTl is abundant in the endothelial cells of the blood vessels, the site of the barrier (Takata et al., 1990b, 1992c; Harik et al., 1990b; Mantych et al., 1993b). Electron microscopic immunohistochemistry revealed that both the luminal and contraluminal plasma membranes were positive for GLUTl together with cytoplasmic staining (Takata et al., 1992c; Kumagai et al., 1996). These observations indicate that glucose passes through the continuous capillaries via GLUTl localized at both the luminal and contraluminal plasma membranes of the endothelial cells (Fig. 5). Immunoblotting of the homogenate of rat retinal pigment epithelium demonstrated abundant GLUTl protein of 46 kDa, which was also in abundance in the ciliary body (Fig. 3) (Takata et al., 1990b, 1992~).A

16


FIG.5 Blood-retinal barrier and GLUTl. Both the cndothelium- and epithelium-type barriers are present in the retina. GLUTl plays a pivotal role in the transfer of glucose across the barrier in both cascs. RPE, rctinaf pigment epithelium; TJ, tight junction.

50-kDa protein was detected in the human retina with anti-GLUT1 antibody (Mantych et al., 1993b). Immunohistochemical staining showed that the retinal pigment epithelium is rich in GLUTl (Takata et al., 199Ob, 1992~; Harik et al., 199Ob; Mantych et al., 1993b). GLUTl was present along all aspects of the plasma membrane of the cell, i.e., it was found in the basolateral domain with well-developed infoldings as well as in the microvillous apical membrane facing the outer segments of the photoreceptor cells. The endothelial cells of the adjacent choriocapillaries were negative for GLUTl (Takata et al., 1992~).Taking into account the ultrastructure of the choriocapillaries and of the retinal pigment epithelium as well as the localization of GLUT1, we have proposed that glucose passes the outer blood-retinal barrier in a transepithelial manner as follows (Takata et a/., 1992~):Glucose (i) leaves the choriocapillary through the fenestrae into the extracellular matrix, (ii) is transported into the cytoplasm of the retinal pigment epithelial cell via GLUTl at the infolded basal plasma membrane, (iii) leaves the pigment epithelial cell via GLUTl at the microvillous apical plasma membrane (Fig. 5). The reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblotting demonstrated the expression and thc presence of the lowaffinity glucose transporter GLUT2 in the retina (Watanabe et af., 1994). Immunofluorescence staining showed that GLUT2 was localized in the boundary between the outer nuclear layer and the photoreceptor layer, corresponding to the location of the external limiting membrane. Immunoelectron microscopic examination showed that the microvilli extending from the apical ends of the Miiller cells were densely stained €or GLUT2 with less intense staining in their nonprojecting apical ends. Other structures including photoreceptor cells were negative for GLUTZ. Polarized distribution of GLUT2 was observed in pancreatic /3 cells (Orci er ul., 1989) and enterocytes (Thorens et al., 1990a; Thorens, 1992). Miiller cclls are located between two independent blood-retinal barrier systems: the inner system, composed of blood vessels inside the retina, and the outer system, conipris-


17

ing the retinal pigment epithelium and the choriocapillaries. Whereas GLUT1 is instrumental in transporting glucose across these barriers (Takata et al., 1990b, 1992c; Harik et al., 1990b), GLUT2 in the apices of Miiller cells may be involved in the transport of glucose into the anterior part of the retina. Because GLIJT2 is a low-affinity glucose transporter and is considered to be a part of the sensing machinery in pancreatic fl cells for the regulation of the blood glucose level (Unger, 1991; Thorens, 1992), GLUT2 in the Miiller cells might play a regulatory role in the intraretinal glucose homeostasis (Watanabe et al., 1994). In addition, GLUT3 was detected, but neither GLUT4 or GLUT5 was expressed, in the human retina (Mantych et al., 1993b). GLUT3 was restricted to the inner synaptic layer and therefore does not participate in the transport of glucose across the barrier.

E. Blood-Aqueous Barrier 1. Structure The aqueous humor is a transparent fluid filling the anterior and posterior chambers of the eye. It nourishes the lens and cornea, both of which are devoid of vascularization. This fluid is continuously produced by the ciliary body, flows from the posterior chamber into the anterior chamber, and drains into the venules through the canal of Schlemm (Stamper, 1979; Sears, 1981). The ciliary body epithelium is the site of aqueous humor production and is mainly responsible for the determination of the constituents of the aqueous humor. When compared with the composition of plasma, that of the aqueous humor is characteristic in having a low concentration of serum protein and a high concentration of ascorbic acid (Bito, 1977; Cole, 1984). The barrier between the blood and the aqueous humor is called the bloodaqueous barrier. The glucose level of the aqueous humor is comparable to that of blood, suggesting a specific transport machinery in the epithelium of the ciliary body (Bito, 1977; Cole, 1984). The ciliary body epithelium is made of two cell layers: the nonpigmented epithelial cell layer and the pigmented epithelial cell layer (Fig. 6a). These two layers originate from the two layers of the invaginated optic cup in the embryo. The bases of nonpigmented epithelial cells face the posterior chamber, whereas the bases of pigmented epithelial cells rest on the ciliary body stroma (Raviola, 1977). Thus, the nonpigmented and pigmented epithelial cells oppose with each other at their apical surfaces. The pigmented epithelial cell layer is a continuation of the pigmented epithelium of the retina posteriorly and of the anterior layer of the iridial epithelium. The nonpigmented epithelial cell layer is the continuation of the neural retina

18


b

a aqueous humor

C C Y t maternal blood glucose

FIG. 6 Glucose transfer across the blood-tissue barrier of a double-layered epithelium. (a) Ciliary body of the eye. Glucose leaves the capillary through fenestrae and enters the pigmented epithelial cells via GLUTl. Then glucose enters the nonpigmentcd epithelial cells through gap junctions made o€connexin 43 (Cx43) connecting pigmented and nonpigmented epithelial cells and is finally transferred to the aqueous humor via GLUTl in the pigmented epithelial cells. (b) Rat placenta. Glucose in the maternal blood easily crosses the cytotrophoblast layer through pores; glucose then enters the syncytiotrophoblast I layer via GLUTl. Next, the sugar mows into the syncytiotrophoblast 11 layer through gap junctions made ofconnexin 26 (Cx26). Glucose leaves the syncytiotrophoblast 11via GLUTl and finally enters the fetal blood through the fenestrae of thc endothelial cells. Note that in both cases, GLUTI, connexin of gap junctions, and GLIJTI located in series serve as the glucose transfer machinery across the barrier. The specificity of the transport of the system is determined by the specific transporter molccules assigned for the entry into and exit from each end of the double-layered epithelium. TJ, tight junction; PE, pigmented epithelial cells; NPE, nonpigmented epithelial cells; Cyt, cytotrophoblast; Syn 1, syncytiotrophoblast I; Syn 11, syncytiotrophoblast 11.

posteriorly and of the posterior layer of the iridial epithelium (Raviola, 1977). Ultrastructural examination revealed that tight junctions are formed between nonpigmented epithelial cells (Smith and Rudt, 1973; Raviola, 1977; Raviola and Raviola, 1978; Freddo, 1987). Endothelial cells lining the capillary wall have many fenestrations and are highly permeable (Bill et al., 1980). The barrier property of the ciliary body was analyzed by the intravascular administration of tracers (Shiose. 1970; Vegge, 1971; Smith and Rudt, 1973, 1975; Raviola, 1974, 1977). The injected horseradish peroxidase easily escaped from the fenestrated capillaries and filled the intercellular space between pigmented epithelial cells and that between pigmented and nonpigmented epithelial cells including the ciliary channels. The further penetration of the tracer was blocked by the tight junctions between nonpigmented epithelial cells, and the aqueous humor was free of the tracer. Similar results were obtained when the fluorescent dye acriflavine neutral was used as a tracer (Rodriguez-Peralta, 1975). The barrier function of the tight


19

junctions between nonpigmented epithelial cells was also demonstrated by the administration of colloidal lanthanum from the aqueous side (Raviola, 1977). These observations show that capillary walls in the ciliary body stroma are highly permeable and that nonpigmented epithelial cells connected by tight junctions are the principal site of the blood-aqueous barrier (Fig. 6a). Capillaries in the iris stroma are of the nonfenestrated continuous type connected by tight junctions (Freddo and Raviola, 1982a,b) and constitute another site of blood-aqueous barrier (Raviola, 1974, 1977; Smith and Rudt, 1975; Freddo and Raviola, 1982a). Intravascularly administered tracers failed to pass through the endothelial cells of these capillaries (Vegge, 1971; Raviola, 1974; Smith and Rudt, 1975; Freddo and Raviola, 1982a).

2. Glucose Transport in the Ciliary Body The concentration of glucose in the aqueous humor is maintained at a level simiIar to that found in the plasma (Bito, 1977; Cole, 1984). Thus, the existence of a glucose transporter system in the blood-aqueous barrier, i.e., in the ciliary body epithelium, was suggested (Bito, 1977; Cole, 1984). A high level of 46- or 47-kDa GLUTl protein was detected in the rat ciliary body specimens (Fig. 3) (Takata et al., 1990b, 1991~).Light microscopic immunohistochemistry revealed that GLUTl is abundant in the epithelial cells of the ciliary body and the iris (Figs. 7a and 7b) (Takata et al., 1990b, , Mantych et al., 1993b). Blood vessels in the ciliary 1991~; Harik et ~ l . 1990b; body stroma were negative for GLUT1, whereas those in the iris stroma, another site of blood-aqueous barrier, were positive for GLUTl (Takata et al., 1990b, 1991c; Harik et al., 1990b). Ultrastructural examination revealed that GLUTl is abundant in both the pigmented and nonpigmented epithelial cells (Figs. 7c and 7d) (Takata et al., 1990b, 1991~).Fenestrated endothelial cells beneath the pigmented epithelial cells were negative for GLUTl. In pigmented epithelial cells, GLUTl is present along the entire surface except at gap junctions and desmosomes. Well-developed basal infoldings are present, which drastically increase the surface area of the cells. Most GLUTl transporters, therefore, are localized along these basal infoldings. In the nonpigmented epithelial cells, GLUTl is also present along the entire surface except at junctional regions. Because basal infoldings facing the posterior chamber constitute the majority of the plasma membrane, most of the GLUTl molecules are present in these basal infoldings. Semiquantitative analysis of the colloidal gold label for GLUTl in ultrathin sections revealed an approximately twofold higher labeling density for GLUTl in the basal infoldings of the pigmented epithelial cells than in the basal infoldings of the nonpigmented epithelial cells (Takata et af., 1991~).

20


FIG. 7 GLUTl in the blood-aqueous barrier ofthe rat. (a, b) ImmunoHuorescencc localization of GLUTl in the ciliary body. GLUTl is present in the pigmented (PE) and nonpigmented

(NPE) epithelial cells (a). Corresponding Nomarski diffcrcntial interference contrast image is also shown (b). In NPE cells, QLIJTI is abundant along the posterior chamber (P). In PE cclls, strong positive labcling for GLUTl is seen along the connective tissue stroma. Only a


21

Because the pigmented and nonpigmented epithelial cells have a similar surface area of their respective basal infoldings (Okami er aL, 1989), these observations indicate that pigmented epithelial cells have approximately twofold more GLUTl than d o nonpigmented epithelial cells. Such asymmetrical distribution of GLUTl may be attributed to the consumption of glucose by the nonpigmented epithelial cells themselves rather than simply to the transfer of glucose. Another explanation is a possible kinetic asymmetry of the GLUTl molecule for the transport of glucose (Lowe and Walmsley, 1986; Carruthers, 1990). Asymmetric distribution of GLUT1 in the blood tissue barriers was observed in the endothelial cells of the blood vessels in the rat brain when immunogold labeling of ultrathin sections was carried out (Farrell and Pardridge, 1991). The luminal plasma membrane, which is the site of entry into the barrier, exhibited less labeling compared with the contraluminal membrane, the site of exit from the barrier cell. When considering the direction of the transport of glucose, this observation is in contrast to that in the ciliary body, where the site of entry into the barrier layer has more GLUTl.

3. Gap Junctions Gap junctions develop between pigmented epithelial cells, between pigmented and nonpigmented epithelial cells, and between nonpigmented epithelial cells (Bairati and Orzalesi, 1966; Smith and Rudt, 1973; Kogon and Pappas, 1975; Raviola and Raviola, 1978; Freddo, 1987), among which rows of gap junctions between pigmented and nonpigmented epithelial cells are prominent. Such junctions are hydrophilic channels connecting the cytoplasm of adjacent cells. They are made of transmembrane proteins named connexins (Beyer, 1993; Dermietzel and Spray, 1993). Spherical molecules as large as 900-1000 Da are allowed to pass the gap junction channels (Spray and Bennett, 1985;Pitts and Finbow, 1986).Fluorescencelabeled glucose was demonstrated to pass through gap junctions (Loewenstein, 1979), showing that cells connected by gap junctions are metabolically coupled. Because the pigmented and nonpigmented epithelial cells are connected by well-developed gap junctions, the cytoplasms of these

small amount of GLUTl is seen between PE and NPE cells. Bar = 10 pm. (c, d) Immunogold labeling of ultrathin frozen sections for GLUT1 . In NPE cells of the ciliary body (c), GLUTl is localized along the infolded plasma membrane (arrowheads) facing the posterior chamber (P). In PE cell (d), GLUTl is localized along the infolded plasma membrane (arrowheads). Endothelium (E) of the adjacent capillary is not labeled for GLUTl. Bars = 0.5 pm. Figure 7c was reproduced from Takata et al. (1990b) with permission from Academic Press.

22


cells are equivalent to a single cell cytoplasm as far as the cytoplasmic diffusion of glucose is concerned. How does glucose pass the blood-aqueous barrier during the course of the production of the aqueous humor in the ciliary body epithelium? Taking into account the presence of tight and gap junctions, and the localization of GLUT1, we suggested the following major pathway for the transport of glucose across the ciliary body epithelium (Takata et al., 1991c)(Fig. 6a): Glucose freely passes through the fenestrae of the endothelial cells and is then transported into the cytoplasm of the pigmented epithelial cells via GLUTl localized in the basal infoldings. Next, glucose enters the cytoplasm of nonpigmented epithelial cells by passing through arrays of gap junctions connecting the apposing apical plasma membranes of pigmented and nonpigmented epithelial cells. Finally, glucose leaves the cytoplasm of nonpigmented epithelial cells via GLUTl located in their infolded basal plasma membrane and thus passes into the aqueous humor. The existence of gap junctions in the epithelium of the ciliary body makes the double-layered epithelium functionally a single-layered epithelium. In this model, the specificity of the transport is determined by the specific transporters located at both ends of the system: Transport through the barrier is mediated by a combination of a specific transporter for the entry into the barrier, nonspecific gap junction channel for transfer between two cells, and a specific transporter for the exit from the barrier. Immunoblotting and immunohistochemistry revealed that the gap junction protein connexin 43 is concentrated in the gap junctions connecting the pigmented and nonpigmented epithelial cells (Coca-Prados etal., 1992; Shin et al., 1996a). In summary, the combined sequential action of GLUTl ,connexin 43, and GLUTl could be key in the transport of glucose across the blood-aqueous barrier and play a pivotal role in supplying glucose to the anterior and posterior chambers, thereby nourishing the lens and cornea. 4. Glucose Transport in the Iris

Another part of the blood-aqueous barrier is the endothelial cells of the blood vessels in the iridial stroma. Heavy labeling for GLUTl was observed in both the luminal and contraluminal plasma membranes of these endothel i d cells (Takata et al., 1991~).As occurs in the capillaries in other bloodtissue barriers, glucose may pass the endothelial cell layer by using GLUTl for entry into and exit from the cytoplasm of the endothelial cells. These capillaries may be substantial in nourishing the cells of the iris. The iridial epithelium, which is a continuation of the ciliary body epithelium (Raviola, 1977), is also rich in GLUT1. This epithelium is similar to that of the ciliary body, connected by tight and gap junctions, and a tracer experiment showed the barrier characteristics (Freddo, 1984). Ultrastruc-


23

turd examination revealed that GLUTl is abundant along the plasma membranes of these cells (Takata et al., 1991~).GLUT1 in the iridial epithelium may serve to facilitate the transfer of glucose between the anterior and posterior chambers.

F. Blood-Perilymph and Blood-Endolymph Barriers in the Inner Ear The mammalian inner ear is an enclosed compartment containing transparent Auids called perilymph and endolymph, both of which have a composition distinct from that of blood. Tracers administered via the bloodstream failed to enter these fluids (Duvall et al., 1971; Winther, 1971a,b; Gorgas and Jahnke. 1974; Santos-Sacchi and Marovitz, 1980). The barrier in the inner ear is composed of the blood-perilymph and blood-endolymph (blood-strial) barriers. Ferrary et ul. (1987) showed the transport of glucose by facilitated diffusion across the blood-perilymph barrier. Immunohistochemical staining revealed that GLUT1 is present in the microvascular endothelial cells in the soft tissues of the labyrinth (It0 et al., 1993). In the vestibular system, strong staining for GLUT1 was found in the capillaries in the crista ampullaris. The staining of the vascular network is similar to that seen in the blood-brain barrier. GLUT1 in these blood vessels serves to nourish cells in the inner ear while the barrier effectively prevents the nonspecific entry of other blood constituents including blood cells. In the cochlea, the stria vascularis is responsible for the production of the endolymph. Blood vessels are present inside the stria. The strial basal cells are connected by tight junctions, forming a barrier toward the perilymph (Winther, 1971b; Reale et al., 1975). The strial marginal cells are also sealed by tight junctions, forming a barrier toward the endolymph of the cochlear duct (Winther, 1971b; Reale et ul., 1975). In guinea pig ears, intravenously administered horseradish peroxidase easily penetrated the strial blood vessels but was blocked by these cells (Duvall et al., 1971; Winther, 1971b; Gorgas and Jahnke, 1974). Therefore, the basal and marginal cells of the stria vascularis are the anatomical basis for the bloodperilymph and blood-endolymph barriers, respectively. In the mouse, intravenously administered ferritin and iron dextran were blocked by the endothelial cells of the stria vascularis, demonstrating a blood-strial barrier (Santos-Sacchi and Marovitz, 1980). Strong staining for GLUT1 was observed in these endothelial cells, indicating that GLUTl is responsible for the transport of glucose across the blood-stria1 barrier (It0 et aZ., 1993). The apparent differential permeability of the intrastrial blood vessel wall between guinea pig and mouse may be attributed to the species difference or to the tracers used. In any event, at least strial cells (basal and marginal

24


cells) connected by tight junctions function as a barrier layer. GLUTl is present in the strial basal cells. GLUTl in these cells may serve in the selective uptake of glucose from the interstitial fluid and its transport across the basal cell layer, thus transferring glucose across the perilymphendolymph barrier. In summary, GLUT1 in the inner ear, whether it is in the endothelial cells or strial epithelial cells, may play an important role in the transfer of glucose across the barriers including blood-perilymph, blood-endolymph, blood-strial, and perilymph-endolymph barriers.

G. Blood-Nerve Barrier 1. Structure Peripheral nerves are a continuation of the central nervous system, which is protected from the free access of blood constituents by the blood-brain barrier. The outermost part of the nerve fibers are surrounded by an epithelial cell-likecell layer called the perineurium (Shanthaveerappa and Bourne, 1962). Ultrastructural examination revealed that cells of the perineurium are connected by tight junctions (Thomas, 1963). When horseradish peroxidase was injected as a tracer into the endoneural space of peripheral nerves, it was blocked by this perineurial sheath (BGck and Hanak, 1971; Olsson and Reese, 1971). Peroxidase injected locally around the sciatic nerve was prevented from diffusing into the nerve by the perineurium as well (Olsson and Reese, 1971).These observation show that perineurium sealed by tight junctions serves as a permeability barrier. Thick nerves have blood vessels inside them. These microvessels show barrier characteristics similar to those found in the brain microvessels. Intravascularly injected tracers, such as Ruorescence-labeled albumin and horseradish peroxidase, failed to pass through the endothelium of the blood vessels within the nerve (Olsson, 1966; Bock and Hanak, 1971; Olsson and Reese, 1971). These results clearly show that nerve fibers are separated from the bloodstream from both inside and outside. The blood-nerve barrier was also demonstrated for inorganic ions (Welch and Davson, 1972; Weerasuriya et ul., 1980). 2. Glucose Transport An in situ perfusion experiment with labeled glucose showed that saturable and stereospecific glucose transport machinery is present in the rat peripheral nerve (Rechthand et ul., 1985). Abundant GLUTl was demonstrated in the perineurial sheaths (Froehner et al., 1988;Takata et af.,1990b;Gerhart and Drewes, 1990; Harik et ul., 1990a; Handberg et al., 1992). lmmunogold


25

electron microscopy also demonstrated that GLUTl is localized at the plasma membrane of the perineurial cells (Gerhart and Drewes, 1990). GLUTl was also found to be localized in the endoneurial blood vessels (Froehner et af., 1988; Takata et al., 1990b; Gerhart and Drewes, 1990; Handberg el al., 1992). These results, obtained from both rats and dogs, show that peripheral nerve fibers are nourished from both inside and outside by the action of GLUTl located at the plasma membrane of the cells of the blood-nerve barrier. In the adult human sciatic nerve, the perineurium is rich in GLUTl, whereas only a few endoneurial capillaries stained positively for it (Muona et af., 1993). The expression of GLUTl in the human sciatic nerve during development was investigated (Muona et af.,1993). At 14 weeks of gestation the perineurial cells were negative for GLUTl , whereas endoneurial and epineurial blood vessels were intensely positive for it. During the course of development, positive staining for GLUTl in the endoneurial capillaries became reduced, and in the adult only a few of them were positive. In marked contrast, the intensity of positive staining for GLUTl in the perineurium increased with the maturation of the barrier properties of the perineurium. In the adult human nerves, GLUTl in the perineurium seems to play a major role in nourishing the nerve fibers. GLUT3, which is abundant in the brain, was not detected in the peripheral nerves (Haber et al., 1993; Muona et af., 1992). Other transporter isoforms, such as GLUT2 or GLUT4, were not detected in the rat peripheral nerve (Muona et al., 1992). In summary, GLUTl is concentrated at the sites of the blood-nerve barrier and plays an important role in supplying glucose to the nerve fibers and associated cells.

H. Placental Barrier The placenta is an organ in which exchange of gases, nutrients, and metabolites occurs between maternal blood and fetal blood, in the absence of any mixing of the two. The structure of the placenta differs considerably from species to species, and hence the structure of the barrier differs accordingly (Wimsatt, 1962; Enders, 1965a,b; Faber and Thornburg, 1983; Benirschke and Kaufmann, 1995a). The glucose transport mechanism across the placental barrier in humans and rats is reviewed. I. Human Placental Barrier

1. Structure In the human placenta, villous trees are directly surrounded by the maternal blood (Benirschke and Kaufmann, 1995b; Castellucci and Kaufmann, 1995).

26


In the first trimester, the human placenta is hemodichorial, i. e., the maternal blood faces two layers of trophoblasts. The superficial layer facing the maternal blood is syncytial and called the syncytiotrophoblast, underneath which are cytotrophoblasts (Langhans cells). Cytotrophoblasts, by fusing with the adjacent syncytiotrophoblast layer, serve as a source for the syncytium (Enders, 196Sb). At term, only a small number of the cytotrophoblasts remains, and a single syncytiotrophoblast layer lines the surface of the villous tree, thus forming the hemomonochorial placenta (Rhodin and Terzakis, 1962; Enders, 1965b; Benirschke and Kaufmann, 1995b). The structural basis of the human placental barrier is attributed to the syncytiotrophoblast layer (Benirschke and Kaufmann, 199%). The syncytiotrophoblast is a single continuous cell layer separating the maternal and fetal circulations. In the term placenta, from the maternal blood side to the fetal blood side, the syncytiotrophoblast directly faces the maternal blood. Next comes the cytotrophoblast sporadically found underneath the syncytiotrophoblast. The fetal blood is enclosed by the endothelial cells of the fetal capillaries, which are of the continuous type and develop tight junctions (Heinrich et al., 1976). The permeability of the fetal capillaries in the term human placenta resembles that of skeletal muscle (Eaton et al., 1993). Detailed examination revealed that tight junctions are not continuous, suggesting the contribution of the paracellular pathway across the capillary wall in the human placenta (Leach and Firth, 1992). 2. Glucose Transport

The continuous syncytiotrophoblast layer is the prime barrier layer in the human placenta and therefore must have the machinery for the exchange of various substances between mother and fetus. Glucose is a major nutrient for the fetal development and is supplied from the maternal blood through the placenta (Dancis, 1962; Smith et al., 1992). Glucose somehow must pass this syncytial layer. Facilitated diffusion is the main process for the transplacental transfer of glucose (Carstensen et al., 1977: Morris and Boyd, 1988). Carrier-mediated glucose uptake was observed in vesicles prepared from the apical microvillous (Johnson and Smith, 1980; Bissonnette et al., 1981, 1982) and basal (Johnson and Smith, 1985) plasma membranes of the syncytiotrophoblast of the human placenta. The uptake was inhibited by cytochalasin B and phloretin. Photoaffinity labeling of microvillous membrane with cytochalasin B revealed D-glucose-sensitive cytochalasin Bbinding proteins of 52 kDa (Johnson and Smith, 1982) and 60 kDa (Ingermann et al., 1983). A protein of 42-68 kDa was also detected by the photoaffinity labeling of the human placental microsomes with cytochalasin B (Wessling and Pilch, 1984).


27

Northern blot analysis showed that GLUT1 mRNA is abundant in the human placenta (Fukumoto et al., 1988b; Bell et al., 1990). GLUTl mRNA is detectable at an early stage of placental development and is developmentally regulated with mRNA levels fivefold higher in the first trimester than at term (Hauguel-De Mouzon et al., 1994). In situ hybridization demonstrated that GLUT1 is highly expressed in the syncytiotrophoblast, suggesting its involvement in transplacental glucose transport ( Jansson et al., 1994). GLUT1 was detected in the human term placenta by immunoblotting (Takata et al., 1992d), and the results agreed with those of photoaffinity labeling with cytochalasin B (Johnson and Smith, 1982; Ingermann et aL., 1983; Wessling and Pilch, 1984). Immunohistochemical staining revealed that GLUT1 is localized in the syncytiotrophoblast layer (Takata et al., 1992d). Immunofluorescence (Takata et al., 1992d) and immunoperoxidase (Jansson et al., 1993; Hahn et al., 1995) as well as immunogold electron (Takata et al., 1992d) microscopy demonstrated that GLUTl is localized at both the microvillous apical and infolded basal plasma membranes of the syncytiotrophoblast. Immunohistochemical staining of sections of the villi showed that the apical microvillous membrane is more intensely labeled for GLUTl (Jansson et al., 1993), although GLUTl was reported to be associated with the basal membrane by electron microscopic analysis (Arnott et al., 1994). lmmunoblotting of microvillous and basal membranes confirmed that GLUTl is present in both membranes but in different amounts: the amount of GLUTl in the microvillous membrane is about 20-fold larger than that in the basal membrane (Jansson et al., 1993). Such semipolarized distribution of GLUTl, together with its abundance, in the syncytiotrophoblast may be important in the efficient transfer of glucose across the barrier as well as in nourishing the placental cells. GLUTl is present at the plasma membrane of the cytotrophoblasts as well (Takata et al., 1992d; Hahn et al., 1995). This observation shows that a high level of GLUTl expression begins prior to the fusion to form the syncytiotrophoblast layer. Positive staining for GLUTl was also observed in the endothelial cells of the capillaries in the core of the villi (Takata et al., 1992d; Hahn et al., 1995). Because capillaries are relatively permeable as noted, GLUTl may serve for the uptake of glucose for the endothelial cells as well as contribute to the transendothelial transfer of glucose into the fetal circulation. In addition to the villi, GLUTl was detected in the fetal membranes, which also constitute the barrier in the term placenta (Wolf and Desoye, 1993). In the amnion epithelial cells, GLUTl is predominantly localized at their apical membrane and may cover their basal glucose requirement from the amniotic fluid. Aside from GLUTl, Northern blot analysis also revealed that a high level of GLUT3 is also expressed in the human placenta (Kayano et al.,

28


1988; Bell ef al., 1990). I n situ hybridization showed that GLUT3 mRNA is evenly distributed between syncytial and other placental cells at a low level ( Jansson et ul., 1994). Immunofluorescence staining showed that GLUT3 is localized at the apical membrane of the syncytiotrophoblast (Arnott et al., 1994). However, immunoblot analyses detected only a small amount of GLUT3 protein (Shepherd et al., 1992a; Haber et al., 1993) or failed to detect any significant amount of GLUT3 protein in the human placenta (Maher etaf., 1992;Jansson et al., 1993). These observations suggest that GLUT3 protein is not present, or at least is not as abundant as in the brain, in the human placenta. The inconsistent results may be attributed to the specificity of the anti-GLUT3 antibodies used (Shepherd rt al., 1992a; Jansson et al., 1993) as well as to the methods employed. The discrepancy between the abundance of GLUT3 mRNA and paucity of GLUT3 protein may be due to the possible blocking of the translation of GLUT3 mRNA in the placenta, or alternatively, to cross hybridization of the cDNA probe with other mRNA species (Haber ~t al., 3993). The human placenta is rich in insulin receptors (Siege1 et al., 1081; FujitaYamaguchi et al., 1983). Glucose transport in the human placenta, however, is insensitive to insulin (Johnson and Smith, 1980 Challier et d., 1986). GLUT4, an isoform of insulin-regulatable glucose transporter, is mainly rcsponsiblc for the insulin action in adipocytes and skeletal muscle cells. Northern blot analysis showed that only a very low level of GLUT4, if any, is expressed in the human placenta throughout pregnancy (Fukumoto et ul., 1989; Hauguel-De Mouzon et al., 1994). Immunohistochemical staining of human term placental tissues failed t o detect GLUT4 (Takata et al., 1992d). These results show that the GLUT4 insulin-regulatable glucose transporter is unlikely to participate in the transfer of glucose across the human placental barrier. A possible major route for transplacental glucose transfer across the human placental barrier may be envisioned as follows: Glucose in the maternal blood enters the cytoplasm of the syncytiotrophoblast via GLUTl localized at its microvillous apical membrane and the sugar leaves the syncytiotrophoblast via GLUTl localized at its basal membranc (Takata, 1994, 1996; Takata and Hirano, 1996). Contribution of GLUT1 to the entry into and exit from the barrier cell layer is basically the same as that seen in other blood-tissue barriers. The glucose concentration in the umbilical artery is about 80% of that in the maternal vein in the human placenta (Economides and Nicolaides, 1989). This concentration gradient serves as a driving force for the glucose transfer by facilitated diffusion across the placental barrier. In mid-gestation, there were fetuses whose glucose concentration in the umbilical vein exceeded the maternal concentration (Bozzetti et af., 1988). A similar reversal of glucose concentration gradient was observed in other species (Anand


29

et al., 1979; Thomas et al., 1990). These observations suggest a possible backtransfer of glucose to the trophoblast or to the maternal blood. In fact, the dually perfused isolated lobule of the human placenta indicates the bidirectional glucose transfer across the placental barrier (Reiber et al., 1991). However, the high efficiency of the transfer of glucose from the maternal to fetal circulation compared with the transfer in the reverse direction suggests a possible protective measure against glucose loss of the fetus under maternal hypoglycemia (Reiber et al., 1991). J. Rat Placental Barrier

1. Structure The rat develops hemochorial placentae where maternal and fetal blood flows are separated by the trophoblastic layers and endothelial cells. Instead of the villi in the human placenta, a complex of maternal and fetal circulation routes, the labyrinth, is formed in the rat placenta (Wimsatt, 1962; Enders, 1965a; Faber and Thornburg, 1983; Benirschke and Kaufmann, 1995a). Between maternal and fetal bloodstreams lie a single cytotrophoblast, two syncytiotrophoblastic layers (hereafter termed from the maternal blood side as syncytiotrophoblasts I and II), basal lamina, and the endothelial cells of the fetal capillary (Jollie, 1964, 1976; Enders, 1965a; Metz et al., 1976a,b;Metz, 1980 Faber and Thornburg, 1983). Ultrastructural examination showed that the cytotrophoblast, which lies next to the maternal blood, has numerous fenestrations in its thin cytoplasm. Tracers, such as horseradish peroxidase and lanthanum chloride, administered via the maternal circulation easily penetrated the cytotrophoblast layer through the fenestrations (Metz et a[., 1978). They failed to penetrate the syncytiotrophoblast I, demonstrating that syncytiotrophoblast I is the structural basis for the placental barrier from the maternal side. When tracers were injected into the umbilical artery, they rapidly traversed the capillary endothelium, where fenestration and pinocytotic vesicles and probably leaky junctions provided the pathway for the tracers (Aoki et al., 1978).The penetration of the tracers was blocked by the syncytiotrophoblast 11, showing that syncytiotrophoblast I1 is the structural basis for the placental barrier from the fetal side. These tracer experiments showed that two syncytial layers, syncytiotrophoblast layers 1 and 11, serve as a barrier between maternal and fetal circulations (Fig. 6b).

2. Glucose Transporters In situ hybridization showed that both GLUT1 and GLUT3 are expressed in the rat placental labyrinth (Zhou and Bondy, 1993). The level of GLUT3

30

KUNlAKl TAKATA ET AL

mRNA remained constant from mid-gestation through term, whereas a reduction in that of GLUTl mRNA was observed during this period. In addition, GLUTl expression was found in the junctional zone, where the highest uptake of 2-deoxyglucose was observed. Zhou and Bondy (1993) suggested that GLUT3 is important for glucose transfer to the embryo, whcreas GLUT1 is responsiblc for supplying glucose for use as a placental fuel. By the immunoblotting of the labyrinthine spccimens, abundant GLUTl (Fig. 3) (Takata et al., 1990b, 1994) and GLUT3 (Boileau et d, 1995) were detected. Two syncytial layers serve as the barrier, in which four layers of plasma membranes are the principal barrier. How do hydrophilic molecules such as glucose pass through these lipid bilayers? Immunohistochemical labeling showed that the interhemal membrane or the labyrinthine wall is rich in GLUTl (Fig. 8) (Takata et al., 1990b, 1993b, 1994; Takata, 1994; Hahn et al., 1995; Boileau et al., 1995). GLUT1 is abundant in the syncytiotrophoblast layers I and 11, whereas it is not detected in the cytotrophoblasts or endothelial cells of the blood vessels (Takata et al., 1990b, 1993b, 1994; Takata, 1994). In the syncytiotrophoblast layer I, GLUT1 is abundant along the highly infolded plasma membrane facing the cytotrophoblasts. The basal plasma membrane of syncytiotrophoblast layer I1 is also rich in GLUTl. The apposing plasma membranes of syncytiotrophoblasts I and 11, which have straight contour, exhibited poor labeling for GLUT1 (Takata et al., 1994).

FIG. 8 GLUT1 and gap junction protein connexin 26 in the rat placenta. Double irnrnunofluorcscence staining. (a) GLUT1 . (b) Connexin 26. (c) Noniarski differential interference contrast image. Note that GLlJTl (arrowheads), connexin 26 (arrows), and GLUT1 (double arrowheads) arc present in this order in the placcntal barricr from the maternal blood (M) sidc to the fctal blood (F) side. Bar = 10 pni.


31

Immunofluorescence staining showed that the expression of GLUT3 protein is restricted to the labyrinthine zone, whereas GLUTl protein is ubiquitously distributed in both the junctional and labyrinthine zones (Boileau et af., 1995). The expression of GLUT3 was stimulated under hyperglycemic conditions and seems to play an important role in the case of diabetic pregnancies (Boileau et al., 1995). The precise determination of the location of GLUT3 at the cellular level should shed light on the role of GLUT3 and GLUTl in rat placental function.

3. Gap Junctions Ultrastructural examination revealed the presence of numerous gap junctions between syncytiotrophoblast layers I and I1 (Forssmann et al., 1975; Heinrich et al., 1976; Metz et al., 1976a; Metz, 1980). These gap junctions can thus function as a channel for the glucose transfer between these two syncytial layers (Takata et af.,1993b, 1994; Takata, 1994) in a way similar to that proposed in the double epithelial cell layers in the ciliary body of the eye (Takata et al., 1991c, 1993a; Shin et al., 1996a). A high expression level of connexin 26, an isoform of gap junction proteins, was observed in the rat placenta, suggesting that connexin 26 may constitute a major fetomaternal exchange route (Risek and Gilula, 1991). Double immunofluorescence microscopy for GLUTl and connexin 26 demonstrated that connexin 26 is abundant and localized in between syncytiotrophoblasts I and I1 (Fig. 8) (Shin et al., 1996b). Such spatial distribution of GLUTl and connexin 26 indicates the transfer of glucose across the rat placental barrier as follows (Fig. 6b) (Takata, 1994; Takata and Hirano, 1996; Takata et al., 1994; Shin et al., 1996b): Glucose in the maternal blood passes through the cytotrophoblast via numerous pores penetrating the cytoplasm and is then transported into the cytoplasm of the syncytiotrophoblast I via GLUTl localized at the plasma membrane of the cytotrophoblastic side. Next, glucose passes the gap junction channels of connexin 26 connecting syncytiotrophoblasts I and I1 and enters the cytoplasm of the syncytiotrophoblast 11.The sugar leaves the cytoplasm of the syncytiotrophoblast I1 via GLUTl localized at the basal plasma membrane and finally enters the fetal circulation by passing through the fenestration of the endothelial cells of the capillary wall. A similar mechanism may possibly be at work for the transport of hydrophilic small molecules other than glucose.

K. Blood-Testis Barrier Germinal cells differentiate to become sperm inside the seminiferous tubules of the testis. The interior of the tubule is separated from the exterior

32


by well-developed arrays of tight junctions between Sertoli cells (Nicander, 1967; Dym, 1973; Ross, 1977; Russell, 1978; Nagano and Suzuki, 1983), thereby preventing the exposure of sperm antigen to the immune system as well as providing a favorable environment for sperm differentiation. The blood-testis barrier was demonstrated by tracer experiments: Lanthanum nitrate and intravascularly injected horseradish peroxidase were blocked by these tight junctions from gaining cntrance to the interior of the seminiferous tubules (Dym, 1973; Ross, 1977; Russell, 1978). Although cultured Sertoli cells express GLUTl (Ulisse et al., 1992), immunohistochemical staining revealed that Sertoli cells, the critical barrier cell layer, was not positive for GLUT1. Instead, GLUTl was concentrated in the endothelial cells of the blood vessels surrounding the seminiferous tubules (Takata et al., 1990b; Harik et al., 1990a; Holash et al., 1993). The capillaries surrounding the tubules are of the nonfenestrated continuous type. Holash et al. (1993) compared the blood-testis barrier with the bloodbrain barrier and found that P-glycoprotein (Cordon-Cardo et al., 1989; 1990) and y-glutamyl transpeptidase (Orlowski et al., 1974), both of which are markers of barrier properties of brain microvessels, are present in the testis rnicrovessels. Transferrin receptor, another marker of brain microvessels ( Jefferies etal., 1984), however, was absent in the testis vessels. Interestingly, intertubular Leydig cells, adjacent to the blood vessels, expressed the astrocyte marker proteins such as the glial fibrillary acidic protein, glutamine synthetase, and S-100 protein (Michetti et al., 1985; Holash ef al., 1YY3), suggesting a similarity between the blood-testis and blood-brain barriers (Holash et al., 1993). These observations lead to the idea that, in the testis, Sertoli cells are equivalent to astrocytes in the blood-brain barrier and possibly induce and maintain the brain microvessel-like characteristics of the endothelial cells in the testicular microvessels. Endothelial cells and Sertoli cells may constitute “in series” the blood-testis barrier to achieve a favorable environment for spermatogenesis inside the seminiferous tubule (Holash et al., 1993).The transport mechanism of nutrients including sugars across the Sertoli cell layer, however, remains to be clarified.

L. Blood-Thymus Barrier Tracer experiments showed that horseradish peroxidase, cytochrome c, catalase, ferritin, and lanthanum are retained in the lumen of capillaries in the cortex of the mouse thymus (Raviola and Karnovsky, 1972). These tracers failed to penetrate the endothelial cells connected by tight junctions, indicating the presence of the blood-thymus barrier, which would provide a favorable environment for differentiating lymphocytes. The barrier does not seem to be maintained solely by endothelial


33

cells, however. Tracers penetrating the endothelial cells were promptly sequestered by macrophages stretched out in a continuous row along the cortical capillaries (Raviola and Karnovsky, 1972). In the medulla of the thymus, on the other hand, blood vessels including postcapillary venules are leaky, and the tracers easily escaped from the lumen of the blood vessels. These results show that blood-thymus barrier is present but is limited to the cortex. Glucose, however, does not seem to be prevented from entering the cortex by the barrier maintained by the phagocytic macrophages, and hence a sufficient amount of glucose may pass the blood-thymus barrier. Immunoblotting and immunohistochemical staining so far have failed to demonstrate an abundance of GLUTl or of other glucose transporters in the blood-thymus barrier (Takata, 1990b). The apparent lack of glucose transporters in the barrier may also be due to the relatively low metabolic activity of the thymic cortex. Another possibility is that the cortex is nourished by glucose that diffuses from the medulla.

V. Regulation of Glucose Transporter Expression in Blood-Tissue Barriers

A. Developmental Regulation During mouse development, GLUT1 is detected as early as in the oocyte (Aghayan et al., 1992). GLUTl remained expressed in both the inner cell mass and the trophoectoderm throughout the preimplantation development (Hogan et al., 1991; Aghayan et al., 1992), suggesting that GLUTl is a basic glucose transporter in mammalian cells. The expression of GLUTl and GLUT3 is developmentally regulated (Devaskar et al., 1991, 1992; Bondy et al., 1992; Cornford et al., 1993, 3994; Dwyer and Pardridge, 1993; Harik et al., 1993; Vannucci, 1994; Vannucci et al., 1994; Nagamatsu et al., 1994; Bauer et al., 1995). In the course of the brain development, GLUTl is present in both the capillary and the neuroectoderm at first, and later GLUT1 expression is upregulated and mainly restricted to the endothelium of the blood vessels. During the development of the mouse telencephalon, GLUTl immunoreactivity in the intramural blood vessels is associated with the formation of the blood-brain barrier, as measured by the impermeability of the intravenously administered tracers such as trypan blue and horseradish peroxidase, and with the concomitant loss of GLUTl in the neuroectoderm cells (Bauer et al., 1995). In the human newborn brain, GLUTl is associated with the microvascular endothelium (Mantych et al., 1993~).The tightness of the cerebral endothelium, as demonstrated by

34


the exclusion of the intravascularly applied horseradish peroxidase and fluorescence-labeled dextrans, is accompanied by a reduction in the amount of GLUTl in neuroepithelial cells and confinement of the transporter to the endothelium during rat brain development (Dermietzel et al., 1992). Immunogold electron microscopy also revealed that GLUTl density in the capillary walls increases during the postnatal period (Cornford et al., 1993, 1994). These observations suggest that the development of the blood-brain barrier and the GLUTl expression in the brain capillaries seems to be closely related. During the postnatal development of rabbit brain, GLUT1 protein undergoes marked upregulation, whereas mRNA level remains unchanged, suggesting a posttranscriptional mechanism of regulation for GLUT1 gene expression (Dwyer and Pardridge, 1993).

6 . Induction of GLUT1 in Blood Vessels Stewart and Wiley (1981) demonstrated with a transplantation experiment that the blood-brain barrier is induced by the environment of the central nervous systcm. When astrocytes were injected into the anterior chamber of the eye, nonleaky endothelial cells were induced, whereas meningeal cells failed to induce nonleaky properties (Janzer and Raff, 1987). This result might suggest that astrocytes, which surround the microvessels in the brain, could be responsible for the induction of the barrier properties of the microvessels (Janzer and Raff, 1987; Maxwell et aE., 1987; Goldstein, 1988). The brain capillary is characteristic in its nonfenestrated continuous wall and high expression level of GLUTl. In cultured bovine brain capillary endothelial cells, GLUT1 expression is markedly downregulated ( Farrell eta/., 1992b). A bovine brain homogenate induced GLUTl at the transcriptional level, suggesting the aclion of a brain-derived trophic factor(s) for the expression of GLUTl (Boado et al., 1994b). Tumor necrosis factor a: partially mimicked this effect. Because astrocytes are closely situated around brain microvessels, they have been thought to be responsible for the induction of the characteristics of the blood-brain barrier. An increase in the mRNA level of GLUTl was observed by treatment with phorbol estcrs and serum (Farrell et al., 1992b).Hurwitz et al. (1993) cultured human astrocytes and brain endothelial cells on the opposite sides of a permeable membrane. The endothelial cells expressed GLUTl and y-glutamyl transpeptidase, markcrs of the blood-brain barrier. Such expression was dependent on the endothelial cells being in close apposition to or in direct contact with the astrocytes, suggesting that characteristics of brain microvessels including GLUTl expression are regulated by astrocytes.


35

C. Effect of Glucose and Diabetes

Expression of glucose transporters is regulated by glucose (Klip et al., 1994). It was proposed that GLUT1 belongs to the glucose-regulated protein family of stress-inducible proteins (Wertheimer et al., 1991). Low glucose treatment induced an increase in hexose uptake in primary cultures of bovine brain microvessel endothelial cells (Takakura et al., 1991). Deprivation of glucose in cultured bovine brain capillary endothelial cells resulted in an increase in GLUTl mRNA via its stabilization (Boado and Pardridge, 1993). In experimental chronic hypoglycemia in the rat, an approximately 50% increase in both mRNA and protein of GLUTl in the brain microvessels was observed (Kumagai et a!., 1995). The elevation of the GLUTl level in the endothelial cells in the blood-brain barrier upon hypoglycemia suggests that the increase occurs to compensate for the low blood glucose level so that proper nourishment of the central nervous system can be maintained. High glucose treatment, on the other hand, had no significant effect on hexose uptake. Moreover, in experiments using the intracarotid injection method, the blood-brain glucose transport was downregulated in the hyperglycemic mouse (Cornford et al., 1995). In the human diabetic retina, neovascular endothelium of proliferative retinopathy did not stain for GLUT1, showing that the loss of barrier function is associated with an absence of GLUTl (Kumagai et al., 1994b). In the diabetic retina with minimal or no clinical retinopathy, drastic localized upregulation of GLUTl in the retinal blood vessels was observed by quantitative immunoelectron microscopy (Kumagai et al., 1996). Such focal increase in the amount of GLUTl in the blood vessels may amplify the toxic effects of hyperglycemia, thus leading to the focal retinopathy encountered in diabetes (Kumagai et al., 1996).

D. Effect of Ischemia and Hypoxia In the ischemic hippocampus of the rat, the amount of GLUT3 decreased, possibly related to the loss of ischemically damaged neurons (McCall et af., 1995). Kinetic analysis suggested that ischemia downregulates the glucose transporter in the blood-brain barrier in the rat brain (Suzuki et al., 1994). In the gerbil brain, however, ischemia upregulated GLUTl in brain microvessels as well as GLUT3 in neurons (Gerhard et a/., 1994). In situ hybridization studies showed that expression of GLUTl increased in response to an ischemic insult in microvessels, astrocytes, and some neurons in the rat brain (Lee and Bondy, 1993). In cultured bovine aortic and human umbilical vein endothelial cells, hypoxia induced the expression of GLUTl

36


(Loike et ul., 1992). In the brain under chronic hypoxia, an increase in the amount of GLUTl mRNA was seen in fetal and developing rats, whereas a decrease was observed in the adult animals (Xia et uZ., 1995).

E. Degenerative Disease

In Alzheimer’s disease, GLUT1 in the brain microvessel endothelium was significantly reduced compared with its amount in age-matched normal brains, suggesting decreased glucose availability to the brain (Kalaria and Harik, 1989; Harik, 1992; Horwood and Davies, 1994; Simpson ef al., 1994). However, these was no change in the density of GLUT1 in erythrocytes, suggesting that the decrease is the result rather than the cause of the disease (Harik, 1992). In Huntington’s disease, a drastic decrease in GLUTl and GLUT3 proteins was observed (Gamberino and Brennan, 1994). These observations indicate that the amount of GLUTl in the blood-brain barrier is regulated by the existence and/or activity of neurons and the subsequent consumption of glucose.

F. Tumors Altered glucose transporter expression, especially in the induction of GLUT3, was observed in human brain tumors (Nishioka et aZ., 1992; Nagamatsu et ul., 1993b; Boado et al., 1994a). The blood vessels inside a brain tumor, whether the tumor was primary or the result of metastasis, usually lost GLUTl immunoreactivity (Harik and Roessmann, 1991). Expression of GLUTl depends on the type of tumors and not on the permeability of the vessels (Guerin et al., 1992a). In the rat intracerebral9L glioma model, dexamethasone treatment reduced the vascular permeability of tumor vessels as measured by Evans blue, with a concomitant increase in the number of GLUT1-positive blood vessels (Guerin et al., 1992b). This observation suggests that GLUTl in the brain tumor may be used to identify the aggressiveness of the tumor. The expression of GLUT1 in tumor blood vessels is also influenced by the location of the tumor, because positive GLUTl staining seen in the intracerebral rat 9L and F98 glioma was virtually lost in the subcutaneous implants of the same tumor (Arosarena et al., 1994). The glioma cells by themselves are not sufficient to induce the expression of GLUTl in the blood vessels. The induction of GLUTl in the microvascular endothelial cells inside the brain tumor may be closely related to the property of the tumor cells, which usually have a glial origin. Because astrocytes are in close apposition to the endothelial cells and


37

possibly affect the expression of GLUT1 in the blood-brain barrier, the ability of tumor cells to influence the endothelial cells to express GLUTl may be closely related to the loss of normal cell characteristics in the tumor cells. In addition, expression of GLUT3, whose expression is normally restricted to nerve cells, was observed in the tumor vessels in glioblastomas (Nishioka et al., 1992).

G. Defective Glucose Transporters Low cytochalasin B binding and decreased hexose uptake together with the loss of immunoreactivity to anti-GLUT1 antibody was observed in the erythrocytes of patients with persistent hypoglycorrhachia (low concentration of glucose in cerebrospinal fluid), seizures, and developmental delay (De Vivo et al., 1991; Harik, 1992). Because GLUT1 is responsible for the transport of glucose across the blood-brain barrier, reduced glucose transport activity across the barrier by the defective GLUTl may be responsible for these phenomena.

VI. Concluding Remarks Glucose transporters are one of the most extensively studied transporter molecules in mammalian cellular membranes. We proposed that GLUTl is the glucose transporter isoform of blood-tissue barriers (Takata et al., 1990a,b). Accumulating evidence has confirmed that GLUT1 is highly produced in the cells of blood-tissue barriers. GLUTl is present at the sites of both entry into and exit from the cells of the barrier, although semipolarized distribution is sometimes encountered. An abundance of GLUT1 at the critical plasma membranes of the cells of the blood-tissue barrier ensures a sufficient supply of glucose to cells isolated from the general circulation. Among the six isoforms, GLUT1 appears to serve as the main glucose transporter for the blood-tissue barriers. Transport of glucose via GLUTl is little affected by the regulatory mechanism under physiological conditions, which makes a marked contrast to the transport via GLUT2 or GLUT4. Such steady characteristics of GLUTl, together with its high affinity to glucose, may be ideal as a glucose transport machinery in the blood-tissue barriers in which a constant and stable supply of glucose is crucial. Further analysis of the glucose transport system across the blood-tissue barriers, along with comparative and developmental studies, will lead to a more detailed characterization of these barriers.

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Acknowledgments We thank S. Tsukui and M. Kanai for secretarial assistance. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan. and Japan Private School Promotion Foundation.

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The Role of Suppressors in Determining Host-Parasite Specificities in Plant Cells Tomonori Shiraishi, Tetsuji Yamada, Yuki Ichinose, Akinori Kiba, and Kazuhiro Toyoda Laboratory of Plant Pathology and Genetic Engineering, College of Agriculture, Okayama University, Okayama 700, Japan

Plant pathogens secrete the compounds that delay or prevent defense responses only of the host plants, with resultant conditioning of host cells such that they become susceptible even to avirulent microorganisms. The principles, which are called suppressors, have been characterized as glycoproteins, glycopeptides, peptides, or anionic and nonanionic glucans. Suppressors do not evoke drastic and visible damages of plant cells and, thus, they can be distinguished from host-specific toxins produced by several fungal species almost belonging to genera Helminfhosporiumand Alfernaria. The mode of action of these suppressors has been found to disturb fundamental functions of host cells. The suppressor from a pea pathogen, Mycosphaerelh pinodes, inhibits both the ATPase activity and the polyphosphoinositidemetabolism in pea plasma membranes, causing the temporary suppression of the signal transduction pathway leading to the expression of defense genes encoding key enzymes in the biosynthetic pathway to phytoalexin. Moreover, it affects the function of cell wall in a strict species-specific manner even in vitro. In this chapter, evidence for the role of suppressors in the determination of plant host-parasite specificity is summarized. KEY WORDS: Defense responses, Determinants of specificity, Suppressor, Susceptibility induction, Transmembrane signaling, Cell wall.

1. Introduction In nature, plants as well as other organisms are resistant or immune to the vast majority of pathogens. In other words, the number of pathogens that Inrernarif~n,nnlH e b ~ i c wof’ L:vrolqy, V n l 172 n 0 7 4 - 7 m i ~ 7$ZS.MI

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Copyright (0 1YY7 by Aczidemic Press All rights of reproduction in any form rrservcd.

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have the potential to attack a given plant species is extremely limited. For example, rice plants are severely diseased by only a few species of 50 fungal pathogens of rice plants among 8000 phytopathogenic fungi in a few hundred thousands species of mycota (Agrios, 1978). This phenomenon is universally observed and is called “host-parasite specificity,” of which the determining mechanism is one of the most intriguing issues. Thus, even in plant-parasite interactions, resistance is the rule and susceptibility is the exception (Oku, 1994). Plant resistance is physiologically classified as static and active. Both resistances are thought to protect plants phasedly and synergistically against the attack by pathogens. Static resistance includes preformed properties such as the strength of cell surfaces and the presence of constitutive antimicrobial compounds. On the other hand, active (induced) resistance involves the formation of chemical and physical barriers, such as phytoalexins, infection inhibitors, active oxygen species, pathogenesis-related (PR) proteins, lignin. callose, and hydroxyproline-rich glycoprotein (Lamb et al., 1989; Ouchi, 1991). The latter is thought to be more essential for the resistance mechanism because suppression of active resistance by treatment with certain metabolic inhibitors, by high temperature, or by inoculation with virulent (compatible) fungi allows avirulent pathogens to infect even nonhost plants. The idea for the molecular mechanism in the active resistance was first presented as “phytoalexin theory” by Miiller and Borger (1940) who hypothesi~edthat certain antimicrobial substances may be accumulated in the potato tissues based on the double inoculation with an incompatible and a compatible race of the late blight fungus Phytophthora in,festans.The following are their conclusions: 1. A principle, designated as “phytoalexin,” which inhibits the development of the fungus in a hypersensitive tissue, is formed or activated only when the host cells come into contact with the parasite. 2. The defensive reaction occurs only in living cells. 3. The inhibitory material is a chemical substance and may be regarded as the product of necrobiosis of the host cell. 4. This phytoalexin is nonspecific in its toxicity toward fungi; however, fungal species may be differentially sensitive to it. 5. The basic response that occurs in resistant and susceptible hosts is similar. The basis of differentiation between resistant and susceptible hosts is the speed of formation of the phytoalexin. 6. The defense reaction is confined to the tissue colonized by the fungus and its immediate neighborhood. 7. The resistant state is not inherited. It is developed after the fungus has attempted to infect. The sensitivity of the host cell that determines the speed of the host reaction is specific and genotypically determined.

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If the term “phytoalexin” is put in different words such as “newly and inductively formed chemical and physical barriers,” it seems to be more suitable to explain the mechanism in active resistance. A factor inducing active resistance was at first isolated from the mycelia of Monilinia fructicola and it was partly characterized as a peptide named monilicolin A, which induced a bean phytoalexin, phaseollin (Cruickshank and Perrin, 1968). Substances that induced phytoalexin production were named “elicitors” by Keen (1975). Later, the term elicitor has been used in a more wide sense for the substances that are able to induce active resistance in plants (DeWit, 1986). The term “inducers” has been also used for these resistance-inducing substances (Hayami el al., 1982). The elicitors were prepared from the culture filtrate, hyphal cell walls, or spore germination fluid of pathogenic fungi and they have been characterized as polysaccharide, glucan, chitin, chitosan, glycoprotein, peptide, lipid, and so on (Darvill and Albersheim, 1984; DeWit, 1986; Ralton et al., 1986). The significance of elicitors in plant-parasite interactions and their mode of action on induction of active resistance were reviewed by Lamb et al. (1989) and Yoshikawa et al. (1993). Compared to the amount of information on the mechanism of plant resistance, little is known about those of susceptibility or accessibility (local susceptibility; Ouchi et al., 1974a). However, an important phenomenon was reported that plant tissues, which had been preliminary infected by virulent or compatible pathogens, became accessible even to avirulent or hypovirulent pathogens (Yarwood, 19.59; Ouchi and Oku, 1981). Preliminary inoculation with a compatible pathogen (or race) was reported to predispose potato to an incompatible race of the late blight fungus (Tomiyama, 1966), barley to powdery mildew fungi (Moseman and Greeley, 1964; Tsuchiya and Hirata, 1973; Ouchi et al., 1974a,b; Kunoh et al., 1985), and oat to an incompatible race of crown rust (Tani et al., 1975). Tsuchiya and Hirata (1973) found that 45 of 51 powdery mildew fungi were able to infect mildewed barley leaves and 30 of the 45 species formed conidia. Ouchi et af. (1974a,b) demonstrated that, within 18 h after inoculation, a compatible race of Erysiphe graminis hordei conditioned barley leaves to be accessible not only to incompatible races of the fungus but also to the wheat and melon powdery mildew fungi. On the other hand, barley leaves that had been inoculated with an incompatible race became inaccessible even to a compatible race of barley fungus within 12 h. Thus, apparently such a phenomenon is inducible. They also clarified that both accessibility- and inaccessibility-induced areas in barley leaves were found to localize near the sites of the primary inoculation with fungi (Ouchi eta!., 1979) and that both cellular conditionings were indeed irreversible. Kunoh et al. (1985, 1988) clarified the timing of establishments of accessibility and inaccessibility by inoculating barley coleoptiles with a compatible race of E. graminis

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TOMONORI SHlRAlSHl ET AL

and an avirulent pathogen, E. pisi, respectively. That is, E. pisi alone never infected the barley coleoptile, but 30% of conidia established their infection on the cell whcre E. graminis penetrated 1 hr earlier than did E. pisi. The molecular mechanism in accessibility induction is also partly included in the phytoalexin theory. Their conclusions clearly indicate that active defense is delayed in the compatible combination. In fact, the delaying of defense responses was caused by inoculation with compatible races or virulent fungi (Bell et al., 1986; Cuypers et al., 1988; Oku et a!., 1975a,b; Yoshikawa et al., 1978; Yoshioka et al., 1995). This indicates two possible mechanisms: (i) compatible races or virulent pathogens do not produce elicitors that are effective only on the host plants at least during an early stage of infection, and (ii) the pathogens have an aggressive ability to suppress the active resistance in the hosts. If the former case was true, the challenging incompatible pathogens could not infect the plants preinoculated (predisposed) with compatible pathogens because the active resistance must be induced by the effective elicitors that are produced by the challenging pathogens themselves. However, even the incompatible challenger is able to establish itself on the predisposed plants as described. Furthermore, as far as we know, there is no pathogen that does not produce an elicitor. For example, the fragments of common compounds, such as P-glucans and chitin, constitutively present in the hyphal cell walls of many pathogenic fungi can act as nonspecific elicitors (Darvill and Albersheim, 1984). It was also reported that some polysaccharide or glycopeptide elicitors, which were secreted in spore germination fluid of pathogens at the infection sites, induced active resistance even in their own hosts (Hayami et ul., 1982; Shiraishi et al., 1978b; Toyoda et al., 1993b: Yamamoto et al., 1984, 1986; Yoshioka et a[., 1992b). Once the chemical and physical barriers have been established in plant tissues, the penetration, growth, and/or reproduction of the pathogens is crucially inhibited (Oku et al., 1976;Shiraishi et al., 1978a; Yamamoto et al., 1986). Thus, the rejection reaction is indeed irreversible. Therefore, the ability to overcome the host’s resistance is essential for the establishment of infection and colonization by pathogens (Oku, 1980). In other words, the specificity cannot be explained solely by the production of elicitors but is rather determined by the substances that are able to circumvent or negate the active resistance of host plants (Heath, 1981; Oku, 1980; Ouchi and Oku, 1981; Shiraishi et al., 1994). In medical science, nontoxic bacterial factors that inhibit the defense mechanism of multicellular organisms are called “aggressin” or “impedin.” The latter concept was originally presented by Torikata in 1917 (see Ouchi and Oku, 1981) who found that certain bacteria did not produce toxic compounds but produced nontoxic substances, disturbing the host’s immunoreaction. In immunological literature, Glynn (1972) recommended usage of thc term impedin because the term aggressin literally gave an impression


59

that it damaged host cells (Ouchi and Oku, 1981). Furthermore, the term aggressin was already used by M. C. Chou to define the factor from pollen that induced expanding or aggressive lesions with Botrytis cinerea on Vicia faba (Warren, 1972). A few plant pathologists proposed to extend “the impedin concept” to plant host-parasite interactions (Mahadevan, 1979; Ouchi and Oku, 1981). Ouchi and Oku (1981) further defined the term impedin as “the factor that is produced by a pathogen and actively or passively suppresses the defense reaction of host cells or tissues, enabling the producer or other potential pathogens to establish infection.” At that time, however, the term “suppressor” had been already used for such a substance that had no visible toxicity but suppressed phytoalexin production induced by elicitors or inoculation with avirulent pathogens and conditioned plant tissues to be accessible to avirulent fungi (Oku et al., 1977; Shiraishi et al., 1978b). In this chapter, therefore, available physiological information on the production, the mode of action, and the significant role in determination of specificity of these substances from plant pathogenic fungi, in particular those from M . pinodes, will be introduced by using the term suppressor.

II. Suppressors of Defense Response Produced by Phytopathogens Several phytopathogens were found to produce the metabolites to suppress plant active resistance that is induced by elicitors or avirulent pathogens in a strict species-specific or a race cultivar-specific manner as shown in Table I, in which toxins, endogenous suppressors, and suppressors in infected plants were excluded. Doke (1975) reported that zoospore constituents of a compatible race of Phytophthora infestuns blocked or delayed the hypersensitive cell death of potato tissues induced by inoculation with an incompatible race, but those of an incompatible race showed less activity. The substance was reported to be composed of 17-23 glucose units and contained /3-1,3- and /3-1,6-glycosidic linkages (Doke et al., 1979). Similar activity, which was released from germinating sporangia of P. infestans but not from mycelia, suppressed the hypersensitive reaction of tomato (Storti et al., 1988). The cause of pea Mycosphaerella blight, M . pinodes, secreted a high-molecular-weight elicitor and a low-rnolecular-weight peptidecontaining suppressor for biosynthesis of pea phytoalexin, pisatin, into the spore germination fluid (Oku et al., 1977; Shiraishi et al., 1978b). Later, similar substances in culture filtrate mycelia, or spore germination fluid of several pathogenic fungi were found to suppress NADPH-dependent generation of superoxides (Doke, 1983a), the accumulation of phytoalexins (Doke et al., 1979; Kessmann and Barz, 1986; Ziegler and Pontzen, 1982)

TABLE I Suppressors from Phytopathogenic Fungi

Fungus

Origin

Chemical nature

Host plant

Defense suppressed

Specificity

Accessibility

Site of action

Reference

?

?

Kessmann and Bar7 (1986)

Ascochyfa rahiei

Culture filtrate

Glycoprotein

Chick pea

Race cultivar

PA

Borrytis sp

Germination Huid

Peptide +l

Alliiini spp.

Genus (species)

General'?

Induced

MycoAphaerella ligulicola

Germination Huid

Glycopeptide?

Chrysanthemum

Species

General?

Induced

?

O k u er al. (1987)

M . melonif

Germination fluid

Genus (species)

General'?

Induced

?

Oku er ul. (1987)

Induced

CW, PM (ATPase, PI metabolism)

Oku r f nl. (1 977)

Kodama rt ol. (198Y)

PM?

Germination Huid

Glycopeptide

Pea

Species

General'? 1.1. PA. PR proteins

Phytophthora cupsici

Mycelia

Glucan

Sweet pepper. tomato

Species

HR, AOS

1

PM"

Sanchez era/. (1995)

P. riicotiana

Mycelia

Glucan

Tobacco. tomato

Species

HR, AOS

?

PMI

Sanchez e f nl. (1995)

P. infestans

Zoospore Mycelia

Glucan Phosphoglucan

Potato Tomato

Specie\ Race cultivnr

AOS. HR. PA

?

PM(Ca*+. NADPHoxidase)

Doke (1975)

P. infestans

Zoospore

Glucan?

Tomato

Race cultivar?

HR

?

?

Start1 e r a / . (1988)

P. glycinra

Culture filtrate

Mannan glycoprotein (invertase)

Soybean

Race cultivar

PA

f

?

Ziegler and Pontzcn (1982)

Uromyces pliaseoli

Infection structure

Kidney bean

Species

General? silicon deposits

'?

Heath (1981)

'?

Induced?

Note. AOS, active oxygen species; CW, cell wall; HR, hypersensitive reaction; I.I., infection-inhibitor; PA, phytoalexin; PI metabolism, polyphosphoinositide metabolism; PM, plasma membrane; PR proteins. pathogenesis-related proteins such as endochitinase and P-13gIucanase.


61

and of infection inhibitors (Yamamoto et al., 1986), and the deposition of silicon-containing compounds (Heath, 1980). A part of these metabolites was clarified to cause plant tissues to become accessible even to avirulent pathogens (Kodama et al., 1989; Oku et al., 1980, 1987; Shiraishi et al., 1978b). Furthermore, it was also demonstrated that M . pinodes substances affect both the ATPase and transmembrane signaling of host cells (Kato et al., 1993; Shiraishi et al., 1991a; Toyoda et al., 1992, 1993a; Yoshioka et al., 1990, 1992a,b,). In plant-bacterial pathogen interactions, the artificial administration of an exotoxin, phaseolotoxin, which was specifically produced by the cause of halo blight of bean-Pseudomonas syringae pathoverphaseolicola-only in the susceptible cultivar, was reported to result in suppression of both the hypersensitive reaction and the accumulation of bean phytoalexins and in stimulation of bacterial multiplication even in the resistant cultivar (Patil and Gnanamanickam, 1976). On the other hand, the substance(s) that was able to suppress or delay the production of pea phytoalexin in a race cultiver-specific manner was found in the culture filtrate and the water exudate of the cause of bacterial blight of pea-P. syringae pathovar pisi (Yamada et al., 1994). According to previous reports (Oku, 1980; Oku et al., 1980, 1987), suppressors were defined as “determinants for pathogenicity (specificity) without apparent phytotoxicity.” In detail, (i) they are produced by pathogens at the site of infection; (ii) they participate in suppression of general resistance and in induction of local susceptibility (accessibility) in host plants; (iii) they are host specific; and (iv) they are not toxic to plants. Although the host-specific or selective toxins (HSTs) were first discovered to be the substances that cause necrosis of host cells or tissues, suppressors did not cause any visible damage of the host tissues or protoplasts as far as has been examined. Thus, the suppressors should be distinguished from HSTs. However, the significant role of HSTs in determining specificity has been determined to be the function of suppressing the defense responses of their own hosts (Kohmoto et al., 1987; Hayami et al., 1982; Yamamoto et al., 1984) and conditioning host cells to be accessible to pathogens (Comstock and Scheffer, 1973; Otani et al., 1975; Yoder and Scheffer, 1969), as do the suppressors. The chemical nature of suppressors was determined to be a water-soluble glucan, phosphoglucan, glycopeptide, glycoprotein (such as invertase), or peptide, but unlike HSTs the structure of suppressors was unknown for a long time. However, the structures of two mucin-type suppressors, supprescins A and B, isolated from a pea pathogen. M. pinodes, were determined as GalNAc-0-Ser-Ser-Gly and Gal(P-1,4)GalNAc-O-Ser-Ser-Gly-AspGlu-Thr, respectively (Shiraishi et al., 1992).

62

TOMONORI SHIRAISHI E r AL.

111. Specific Production and Accessibility-Inducing Activity of Suppressors If the fungal metabolites, which show highly biological activities, do not exist at the site ol infection, their significance in host-parasite interactions or in the determination of specificity is little. The initial interaction between plants and pathogens is considered to be mediated by substances that are secreted into spore germination fluids because the majority of phytopathogenic fungi commonly infest and infect through their conidiospores. As mentioned previously, cystospores of P. infestans secrete anionic and nonanionic water-soluble glucans into the germination fluid, and the amounts of both types of glucans increase during incubation. These glucan suppressed, in a race cultivar-specific manner, hypersensitive cell death in and the production of phytoalexin by potato tubers that were induced by an incompatible race of the fungus or by treatment with hyphal cell wall components (elicitors) of the fungus (Doke et al., 1979, 1980). Two infection-inducing factors were isolated from spore germination fluid of Botrytis sp., the cause of scallion bulb rot (Kodama et al., 1989). The infection hyphae from conidiospores of a saprophytic or nonpathogenic strain of Alfernaria afternufawere formed at significant levels on plants in t h e genus Alliurn, such as scallion, onion, wakegi (Alliurn ~ ~ ~ t ~ L.), ~ l ~ ~ . s u Chinese chive, and garlic, that had been treated with the spore germination fluid or the factors from Botrytis sp., whereas the fluid was unable to inducc susccptibility on nonhosts such as strawberry, tomato, and Japanese pear (Kodama et al., 1989). It was also reported that these active substances ( M , 70,000) and glycopeptide suppressors ( M , UTP

IJTP

=

> PPi

CTP > GI'P > ATP = pNPP

Demand ol divalcnt cation

Mn2+,Mg2' (none; YO% loss)

Ca2+,Mn", Mg" (none; 20-4074) IOSS)

Inhibitor

Orthovanadate Ncomycin

Orthovanadate

In vitro action ofsupprcssor from Mycnsphu~wNu pinodes

Not specics-specific Inhi hit ion

Species-spccific Pea: inhibition Nonhosts: activation

79

ROLE OF SUPPRESSORS IN HOST-PARASITE SPECIFICITIES 7

8

6 3

5

6

3

k

7

.-

4

4

3

Y u

g

2

2

1

if

;-0

0

wc

S

E

wc

S

E

FIG. 4 Effects of the suppressor and elicitor from Mycosphaerella pinodes on ATPase activities in cell wall fractions from pea, cowpea, kidney bean, and soybean. The ATPase assay was carried out at 25°C for 20 min in 30 mM Tris-MES (pH 6 . 5 ) containing 3 mM Mg-ATP in the absence (water control; WC) or presence of 100 kg/ml elicitor alone (E) or 100 pg/ml suppressor alone (S) as described by Kiba et al. (1995). The protein contents of cell wall fractions from pea, cowpea, kidney bean, and soybean were 4.0, 14.6, 15.0, and 12.3 mgig dry wt respectively. Each value represents the mean with standard deviation (SD) of results from triplicate experiments. Note that the elicitor activated the ATPase activities in cell wall fractions of all species but that the suppressor inhibited the activity only in pea fraction and rather activated those of nonhosts of M. pinodes compared lo the water control.

ATPase activities of nonhost cells were never inhibited in vivo by the suppressor, and those of cell walls isolated from nonhosts in vitro were also not inhibited. Taken together with our previous reports, it is likely that the cell wall (or cell wall-bound ATPases) might affect or regulate the ATPases of other organella such as the plasma membrane and vacuole. In other words, inhibition of cell wall ATPases might result in a decrease in the activity of plasma membranes including ATPases with subsequent suppression of defense responses as described (Shiraishi et al., 1991b; Yoshioka et al., 1990, 1992a,b). If so, the cell walls might also participate in acceptance of a virulent pathogen as well as in rejection of an avirulent pathogen. Because tight connections between cell walls and cytosolic microtubles via plasma membranes were reported to exist (Akashi and Shibaoka,

80

TOMONORI SHlRAlSHl ET AL

1991; Shibaoka, 1993), the above concept may not bc distant from the facts. The finding that cell wall-bound ATPases are stimulated nonspecifically by the elicitor but are inhibited by the suppressor in a species-specific manner also indicates that the putative receptor for the fungal signals might bind tightly to and affect the cell wall-bound ATPases or that the cell wallbound ATPases might act as a receptor and/or a modifier to recognize and change these fungal signals. Alternatively, there remains the possibility that the cell wall-bound ATPasc itself might be the receptor for both signals from M. pinodrs.

B. Specific Suppression of 02-Generation It was reported that generation systems of H 2 0 2and 02-were contained in the cell walls of horseradish and tobacco, which may play an important role in lignin synthesis (Gross et af., 1977; Halliwell, 1978; Mader and Fussl, 1982). However, the relationship between such a gcncration system in cell wall and plant-microbe interactions has been obscure for a long time. We found that O2 was generated on the surface of uninjured lcavcs of pea and cowpea and in the fractions solubilized from cell walls of both plants (Kiba et al., 1996b). The elicitor from M. pinodes, which was placed on both leaves, induced the nitroblue tetrazolium-reducing activity sensitive to superoxide dismutase, whereas the concomitant presence of the M. pinodes suppressor markedly inhibited such a blue formazan formation on pea leaves but not on cowpea leaves. Moreover, thc formation of blue formazan on cowpca leaves was rather enhanced by the suppressor alone. A superoxide dismutase-sensitive NBT-reducing activity was also tound in the fraction of NaCl solubilized from cell walls, which were isolated from etiolated seedlings of pea and cowpea (Kiba et af., 1996a). The activity was NAD(P)H dependent and required manganese ion and p-CA as cofactors but was markedly reduced in the presence of a scavenger of H202,catalase, in a dose-dependent manner. The requirement of these cofactors and the inhibition by catalase intensively indicate that such an 02-generation system is sustained by a certain cell wall-bound peroxidase(s) as described by Halliwell (1 97X) with horseradish peroxidase. Inhibitors of NADPH oxidase, quinacrine and imidazole, which are bound to flavoprotein (Cross and Jones, 1991) and b-type cytochrome (Iizuka et al., 19X5), respectively, generation in the fraction solubilixed did not inhibit or scarcely affected 02from cell walls of both plants. On the other hand, SHAM, an inhibitor of pcroxidase, remarkably inhibited the 02-generation in both fractions. This result also supports the idea that cell wall-bound peroxidase(s) may mainly participate in the NADH-dependent O2 generation in cell wall fractions, whereas a NAD(P)H oxidase(s) might not. Such an hypothesis that cell


81

wall peroxidase(s) catalyzed the 02-production by a complex pathway involving NADH, NAD-, and NADt has been presented by several groups (Gross et al., 1977; Halliwell, 1978; Mader and Amberg-Ficher, 1982). Neomycin did not affect 02-generation in both fractions. On the other hand, orthovanadate markedly inhibited the activity of the fractions from both plants. Effects of both inhibitors on the formazan formation seem to coincide with those on cell wall-bound ATPases. That is, the cell wallbound ATPase was inhibited by orthovanadate but not by neomycin (Kiba et al., 1996a; Table IV), whereas the plasma membrane ATPases were inhibited by both inhibitors (Yoshioka et af., 1990; T. Shiraishi, T. Yamada, Y. Ichinose, A. Kiba, and K. Toyoda, unpublished results). These results suggest the possibility that the 02-generation in the cell wall fractions of pea and cowpea might be regulated together with cell wall-bound ATPases. However, because it was reported that vanadate acted as an inhibitor of peroxidase (Serra et al., 1990), it is yet unknown whether the inhibition of 02-generation by orthovanadate is a result of the inhibition of.cel1 wallbound peroxidase or ATPase. As shown in Fig. 5 , the elicitor from M . pinodes significantly enhanced the formazan formation in the fractions solubilized from cell walls of pea and cowpea in a nonspecific manner as well as the cell wall-bound ATPase. On the other hand, the suppressor inhibited the formazan formation only in pea fraction. Even the concomitant presence of the suppressor with the elicitor decreased the formazan formation in pea fraction to the level of water control. However, in cowpea fraction, the formation was not inhibited by the concomitant presence of the suppressor and was inversely enhanced even by the suppressor alone. These results showed that the activity of 02generation in the cell wall fraction is also regulated by both fungal signals and that the suppressor acts on the activity in a species-specific manner. Thus, the NADH-dependent O f generating system in cell wall seems to be tightly correlated with cell wall-bound ATPase. Our recent experiments showed that the cell wall-bound ATPases of pea and cowpea were copurified with the activity of peroxidase(s) by an affinity chromatography (T. Shiraishi, T. Yamada, Y. Ichinose, A. Kiba, and K. Toyoda, unpublished results). These results support the idea that the plant cell walls may recognize fungal signals and play a key role in determination of plant-microbe specificity. Further investigations are needed to elucidate not only the relationship between the receptors for these fungal signals and the regulatory systems of 02-generation in cell walls but also the role of 02-in signal transduction cascade leading to the early defense responses of plants.

VII. Concluding Remarks A pathogen must possess the capacities (i) to penetrate plant tissues, (ii) to overcome the host’s resistance, and (iii) to evoke disease (Gaumann

82

TOMONORI SHlRAlSHl ET AL.

c

30

. I

3)

g 25

1 Pea

I

P

40

35 30

? 20

25 20 15

10

5 3)

2

e9

0

0 0

60

120

0

60

120

Time [sl FIG. 5 Effect of the elicitor and suppressor from Mycosphaerella pinodes on superoxidc generation in the fraction solubilized from cell walls that were isolated from pea and cowpea seedlings. The assay was carricd out i n 30 mM Tris-MES (PIT. 6.5) containing 20 m M MnCI2, 2.5 pLg/ml of nitroblue tetrazolium, 0.5 rnM p-coumaric acid in the absence ( X ) or presence of 100 units of superoxide dismutasc (A), I00 p g h l of the elicitor alone (0),100 pglml of the suppressor alone (A), and the elicitor plus suppressor (B) by the method of Nathan et 01. (J. Clin. Invest. 48, 1895-1904, 1969). Each value represents the mean with standard deviation from triplicate experiments. Notc that the elicitor enhanced superoxide generation in both fractions and that the suppressor inhibited the generation in pea fraction but enhanced that in cowpea fraction.

1951; Oku, 1980). From the accumulated evidence (Oku cf al., 1977, 1980; Shiraishi et a/., 197%;Yamamoto et al., 1986; Yamada et af.,1989; Yoshioka ct al., 1990, 1992b), the suppressor of M . pinodes is thought to be a key factor in overcoming the general resistance of its host and in determining host specificity. Currently, we are most interested in how the suppressor overcomes the host defense responses induced by the nonspecific elicitor and establishes the specific accessibility. A model for the fungal signal transduction cascade in pca plants is summarized in Fig. 6. After the elicitor of M. pinodes has been recognized by a putative receptor on cell walls (the elicitor-binding molecule on pea plasma membranes is presented as RE2 in Fig. 6), it activates the ATPase and 0 -generation system in cell wall and plasma membranc PI metabolism, which involves Ptdlns kinase, PtdInsP kinasc, PLC, and PLA, with resultant production of second messengers such as DAG, IP3, and fatty acids. It is possible that certain protein kinases, present in plasma membranes, cytosol, and nuclei, also participate in such a process, but thcse have been omitted from Fig. 6. Although the second messengers

ROLE OF SUPPRESSORS IN HOST-PARASITE SPECIFICITIES 0 2

a3

02-

FIG. 6 Proposed rnodcl o f pea signal transduction cascades for the elicitor and suppressor from a pathogenic fungus. The enzymes and cascade that are presented by solid lincs a r e regulated by tlic suppressor and elicitor from Mycovphaerella pinodes. The broken lincs indicate putatiLc enzyme\ or cascades that might be affected by both fungal signals. CIIS. chalcone synthase; CW. cell wall; DAG. diacylgl~czrol:ER. endoplasmic reticulum; IP3, inositol 1,3,S~trisphosphate:LK. phospholipid kinases such as PtdIns kinase and 1'tdlnrP kinase: I.OX, lipoxygcnase; PA. phosphatidic acid: PAL. phenylalanine ammonia-lyase; PI. phospliatidylinositol: PIPz, phosphatidylinositol 1.5-bisphosphate: PK. protein kinasc: PKC, protein kinase C ; PLA,, phospholipase Xz: PLC, phospholipase C; PM, plasma membrane; POX. peroxidase; PK protein, pathogenesis-related protein; RE. receptor for elicitor; KS. receptor (or suppressor.

that act to induce the transcriptional activation of defense genes have not been elucidated yet, it is hypothesized that IP7 evokes the release of CaZ+ from the endoplasmic reticulum or tonoplast into cytosol and that the released a-TCP >> P-TCP > CdA >> HA For BCP, extent of dissolution depends on the P-TCPIHA ratio, the higher the ratio, the higher the extent of dissolution (LeGeros et id., 1988d; LeGeros and Daculsi, 1990; Daculsi et al., 1989a).

ADAPTIVE CRYSTAL FORMATION

165

D. Calcium Phosphate Coatings on Dental and Orthopedic implants The plasma-spraying process causes changes in the composition of the source material (HA ceramic) resulting in the formation of other calcium CaP phosphate phases besides HA-principally ACP, small amounts of a- and P-TCP, tetracalcium phosphate, and, sometimes, calcium oxide (LeGeros and LeGeros, 1991; LeGeros et al,, 1993, 199.5~;Klein et al., 1993). Variations in composition, principally in the ACP/HA ratios, were observed between the outer and inner layers of the coatings and among the coatings on implants from different manufacturers (LeGeros, 1991b; LeGeros and LeGeros, 1991; J. P. LeGeros et al., 1994; LeGeros et al., 1995d). Such differences in the composition of the coatings on dental and orthopedic implants are expected to affect the biodegradation of these coatings (LeGeros, 1993; LeGeros et al., 1993, 1995a).

VII. Comparative Properties of Bone and Calcium Phosphate Materials

Bone is an integrated composite of an organic phase (principally collagen) and an inorganic or mineral phase, with an inorganiciorganic ratio of approximately 75/25 by weight and 65/35 by volume. The inorganic or mineral phases of enamel, dentin, and bone were initially identified as an apatite, idealized as HA, with the structural formula, Calo(P04)6(OH)2(De Jong, 1926; Beevers and Mclntyre, 1942). Actually, the biological apatites (i.e., mineral phases of enamel, dentine, cementum, and bone) are associated with minor (e.g., carbonate, magnesium, sodium, potassium, chloride, fluoride, and HP04) and trace elements (e.g., strontium). Although carbonate substitution in biological apatites has been proposed by McConnel (1952), experimental evidence for such substitution was originally presented by LeGeros (1967; LeGeros et al., 1967, 1971) and later confirmed by other studies (LeGeros, 1981; Elliott, 1974; Monte1 et al., 1981; Nelson and Featherstone, 1982; Rey et al., 1991). Biological apatites are more accurately described as CHA, approximated by the formula, (Ca,Mg,Na,X)lo. (P04,C03,HP0&(OH,CI,F);?,where X may be other substituents for calcium (e.g., strontium, etc.). Magnesium is incorporated in synthetic and biological apatites to a very limited extent (LeGeros, 1984,1991b; Okazaki and LeGeros, 1992). The concentrations of carbonate and magnesium are higher in both dentin and bone apatite compared to that in enamel apatite (LeGeros, 1967, 1981, 1991b; LeGeros et al., 1995d). Biological apatites,

166

G. DACULSl ET AL.

unlike HA (Table V), are nonstoichiometric with Ca/P molar ratio 1.67< for bone apatite, depending on age and specie of the animal (LeGeros, 1981a, 1991b, 1994; Rey et al., 1991). Biological apatites can more appropriately be represented as (Ca,X)lo(P04,Y)6(OH,Z)2where X is possible substituents for calcium (e.g., Mg,Na,K,Sr, etc.), Y is possible substituents for PO4 (e.g., C 0 3 ,HP04, and SO4), and Z is possible substituents for OH (e.g., C1 or F) (LeGeros, 1967,1981a, 1991b; Monte1 et al., 1981; Young and Elliott, 1966). The bone apatite microcrystals have rod-like or plate-like morphology, with average dimensions of 25 X 3 nm, compared with about 200 nm for HA ceramic.

TABLE V Comparative Composition and Crystallographic and Mechanical Properties of Human Bone and Synthetic Hydroxyapatite Ceramic"

Weight %

Human bone

HA

24.5 11.5 1.65 0.1 0.03 0.55 5.8 0.02 0.1 65.0 25.0 9.7

39.6 18.5 1.67 Trace Trace Trace

Crystallographic propertics Lattice parameters (t0.003 11 axis b axis Crystallinity indcy" Crystallite sizc (A)

9.419 6.880 33-31 250 X 25-SO

9.422 6.880 100 2000

Products after sintering (950°C)

HA

Mechanical properties Elastic modulus (10' MPa) Tensile strcngth (MPa)

Cortical bone 0.020 150

Constituents Calcium (Ca2') Phosphorus (P) (CalP) molar ratio Sodium (Na') Potassium ( K ' ) Magnesium (Mg") Carbonate (CO? ) Fluoride (F ) Chloride (CI-) Ash (total inorganic) Total organic Adsorbed H20" Trace elcments: Sr2', Pb", Ba2+,Fe", ZnZ', Cu" etc.

-

I00

A)

" From LeGeros (1967, 1981a, 1991a).

+ CaO

HA HA 0.01 100

* Ratio of coherenthcoherent scattering in mineral HA crystal index value is 100.


167

Sintered bone-derived apatite is less soluble than unsintered bone apatite but more soluble than synthetic ceramic HA (LeGeros et al., 1995b,c,). Another unique property of bone apatite crystals is their association with an organic matrix that plays an important role in the formation, growth, orientation, and size of the biological apatite crystals (Boskey, 1992;Daculsi and Kerebel, 1978; Daculsi et al., 1989a;Wright et al., 1992). Collagen fibrils, for example, give the direction of epitaxies (Boskey, 1992). Moreover, biological crystals are covered by a proteic sheath. This sheath may influence their reactivity (Daculsi et al., 1989a) and their dissolution properties (LeGeros et al., 1995d). The protective effect of the organic phase is suggested from the observation that bone or dentin is less soluble before the removal of the organic phase (LeGeros et al., 1995d). Cellular activity causes biodegradation and dissolution of calcium phosphate materials, eventually contributing to the formation of carbonate apatite on the surfaces of these materials as described in Section VIII. Moreover, numerous lattice defects appear in biological apatite crystals, such as dislocations that disturb the normal arrangement of the lattice planes (Jongbloed et al., 1974; Daculsi and Kerebel, 1977, 1978; Kerebel et al., 1976; Lee and LeGeros, 1981; Voegel and Frank, 1975). Crystal defects have also been observed in HA ceramics that appeared to depend on sintering temperature (Daculsi and LeGeros, 1996; Daculsi et al., 1989b). These ultrastructural differences may explain the difference in mode of dissolution between the biological and ceramic apatite crystals, i.e, preferential dissolution of the crystal core in biological apatites compared to nonspecific dissolution in ceramic HA crystals, and the difference in bioactivity related to the sintering temperature of the implanted HA (Nagai and LeGeros, 1993).

VIII. Bone/Biomaterial Interface The main attractive feature of bioactive bone graft materials, such as CaP ceramic and special glass ceramics (bioactive glass), is their ability to form a strong direct bond with the host bone resulting in a uniquely strong interface compared to bioinert or biotolerant materials that form a fibrous interface (Daculsi et al., 1990b; De Groot, 1983; Hench et al., 1971; Hench, 1994; Osborn and Newesely, 1980). The strong bond associated with HA ceramics has been attributed to a “bonding zone,” also described as “electron dense” or “amorphous” zone (Jarcho et aL, 1976; Ganeles et al., 1986; Gross et al., 1983). The formation of this dynamic interface is believed to result from a sequence of events involving interaction with cells; formation of carbonate hydroxyapatite CHA (similar to bone mineral) by dissolution/

168

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precipitation processes, and mineralization (Hench, 1991; LeGeros et af., 1991, 1992).

A. Cellular Events The CaP materials elicit responses from bone cells and related cells in vitro and in vivo that are similar to those elicited by bone. These materials allow cell attachment, proliferation, and expression (Bagambisa et al., 1990; Blottier et al., 1995; Cheung and Haak, 1988; Davies, 1990; Frayssinet et al., 1991;Galgut etal., 1990; Gregoire et al., 1992; Gross et al., 1991;LeGeros et al., 1991;Nagai and LeGeros 1993; Niwa et al., 1980; Ohgushi et al., 1993; Soueidan et al., 1995). The first biological events after CaP ceramics implantation are biological fluid diffusion, followed by cells colonization. These cells include monocytes macrophages (Fig. 25), giant cells (Fig. 26), osteoclasts for resorption, and fibroblast and osteogenic cells for tissue repair (Fig. 27). The nature of the multinucleated cells involved in the resorption processes occurring inside macroporous calcium phosphate biomaterials grafted into rabbit bone was demonstrated using light microscopy, histomorphometric analysis, enzymatic detection of tartrate-resistant acid phosphatase (TRAP) activity, and SEM and TEM microscopy (Bade et al., 1993). The combination of these analytical techniques allowed the observation that osteogenesis and resorption occur at the surface of the biomaterials and inside the macropores as early as Day 7. Resorption of both newly formed bone and CaP materials was associated with two types of multinucleated cells. Giant cells were found only at the surface of biomaterials, showed a large number of nuclei, were TRAP negative, developed no ruffled border, and contained numerous vacuoles with large accumulation of ceramic crystals from the biomaterials. The number of these cells decreased with time. The other multinucleated cells observed were osteoclasts. These cells exhibited well-defined ruffed border and were TRAP positive. They were observed at the surface of the newly formed bone and on the surface and inside the macropores of the CaP biomaterials. The increase in microporosity of the CaP ceramics underneath this type of cell were greater than that observed underneath giant cells or in the depth of the biomaterials. Calcium phosphate ceramics elicit osteogenesis (bone ingrowth) and the recruitment of a double multinucleated cell population having resorbing activity, macrophages with monocytes (Blottiere et al., 1995; Benhamed et af., 1994, 1996; Soueidan et af., 1995), and multinucleated giant cells that resorb biomaterials and osteoclasts that resorb newly formed bone and biomaterials. These observations suggest that resorptionidissolution must occur before osteoblastic adhesion and

FIG. 25 TEM of monocytes macrophages in contact with calcium phosphate ceramics.

FIG.26 TEM of giant cells invading macropores of calcium phosphate ceramic. FIG. 27 Light microscopy of the interface of BCP ceramic (Triosite) ( C )and bone (B) showing osteoclasts and osteoblasts. FIG. 28 TEM of extracellular dissolution of HA single crystal (arrows) (a) and crystal core dissolution (b).

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phenotypic expression [similar to that described for bone remodeling to takc place (Daculsi and Dard, 1994)l.

B. Biodegradation and Biodissolution The degradation of the CaP ceramics after implantation has been described using light microscopy of decalcified sections (Drobeck et ul., 1984; Froum etal., 1982;Moskow and Lubarr, 1983;Osborn, 198.5) or TEM of decalcified and undecalcified sections (Daculsi et al., 1986, 1989a; Ganeles et al., 1986; Tracy and Dorcmus, 1984). The biodegradation of these ceramics included the dissolution of the individual HA or P-TCP crystals (Daculsi et al., 1989a; Daculsi and Delecrin, 1994; LeGeros and Daculsi, 1990; LeCeros et al., 1988a, 1991, 1992) as shown in Figs. 28a and 2%. The proportion of HA to 0-TCP crystals in BCP appeared greater after implantation (Richard et al., 1996), in agreement with previous reports (LeCeros et al., 1988a) and the known higher reactivity or solubility of P-TCP compared to HA (LeGeros, 1991b, 1993; Osborn, 1985). Dissolution of biological apatites occurs during natural processes such as dental caries and bone remodeling. Dissolution of human enamel apatites crystals was shown to be site specific, occurring preferentially at the crystal core (Bres et al., 1986; Daculsi et al., 1979; Daculsi and Kerebel, 1977; Daculsi and LeCeros, 1996; Jongbloed et al., 1974; Lee and LeGeros, 198.5; Scott et af., 1974; Voegel and Frank, 1975). The dissolution of some synthetic ceramic HA crystals, unlike biological apatite crystals, did not show site specificity (Daculsi et al., 1989b; Daculsi and Delccrin, 1994). Dislocations are crystal lattice defects, and it has been established that acid dissolution starts at the dislocation sites (Lee and LeGeros, 1985). Such phenomena were observed in the dissolution of both the ceramic HA and biological apatite crystals: dissolution on grain boundaries and crystal cores in ceramic HA and preferential dissolution in crystal cores in biological apatites (Daculsi et al., 198%). However, significant differences were also observed: (i) preferential core dissolution of biological apatites compared to the nonspecific crystal dissolution of ceramic apatite crystals and (ii) correlation with crystal c axis in biological apatite dissolution and noncorrelation observed with ceramic apatite. The observed differences in dissolution features, i,e., preferential dissolution at the crystal core (site specific) for biological apatite and nonspecific dissolution for ceramic HA (Fig. 28), may be attributed to the differences in the chemical and physical properties of the biological and ceramic apatite crystals resulting from the differences in the conditions of their formation: for example, (i) the presence of screw dislocation at the crystal base and along thc c axis in biological apatites (Jongbloed ef al., 1974): Thcse screw


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dislocations, responsible for the helicoidal crystal growth, could explain the elongated tunnel observed along the c axis of the biological apatite crystals subjected to in vivo and in vitro acid dissolution. This morphological aspect was not observed in ceramic HA crystals because, during the sintering process (at temperatures above 90OOC), the individual elongated crystals fuse into large equiaxed crystals; (ii) difference in the origin of crystal defects in biological and ceramic apatite crystals: Biological apatites form by nucleation and growth in aqueous systems of different chemical composition that may contribute to the observed preferential core dissolution. HA ceramic, on the other hand, is prepared by compacting and sintering apatites. These processes may induce different types of crystal defects causing the nonsite-specificdissolution process; (iii) intimate crystal/protein interaction associated with individual biological apatites (Bonucci, 1987; Daculsi and Kerebel, 1978; Daculsi et al., 1995) but not with ceramic apatites: Although serum-derived proteins can adsorb on ceramic HA implant, the interaction with individual HA crystals of the ceramic implant is purely physical. This crystal/protein interaction unique to biological apatites (Bonucci, 1987; Daculsi et al., 1984; Daculsi, 1995) appears to make the crystal surfaces less susceptible to acid dissolution (LeGeros et al., 1994~). The observed decrease in average size of crystals in BCP ceramics after implantation is associated with an increase in the size of microporosities in the surface and at the core of the ceramic indicating that in vivo dissolution has taken place (Daculsi, 1988; Daculsi and Passuti, 1990; Daculsi et al., 1990a; LeGeros and Daculsi, 1990; Passuti et al., 1989, 1991). The resorbability (reflecting in vivo dissolution) of BCP ceramics depends on their P-TCPIHA ratios; the higher the ratio, the greater the resorbability (Daculsi et al., 1989a, LeGeros et al., 1988a; LeGeros and Daculsi, 1990). Formation of microcrystals (which are able to diffract X rays) with Ca/P ratios similar to those of bone apatite crystals were also observed after implantation (Daculsi et al., 1988,1990a). The abundance of these crystals was directly related to the initial P-TCPIHA ratio in the BCP; the higher the ratio, the greater the abundance of the microcrystals associated with the BCP crystals (Daculsi et al., 1989a; LeGeros et al., 1988b; LeGeros and Daculsi, 1990). It was proposed that the formation of the bone apatite-like crystals may be due to the precipitation of calcium and phosphate ions released from the dissolving BCP crystals with the P-TCP component dissolving preferentially to the H A component (LeGeros et al., 1988b; LeGeros and Daculsi, 1990).

C. Bonding Zone

TEM of undecalcified sections from the bone/HA interface showed the presence of microcrystals, described as biological apatite, deposited perpen-

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dicular to the ceramic HA crystal surface and associatcd with collagen fibers (Heughebaert ef al., 1988; Jarcho, 1981; Tracy and Doremus, 1984) (Fig. 29). Using high-resolution TEM, Daculsi et a!. (1990a) demonstrated

FIG. 29 TEM of coalescing zone between CaP residual crystals (C) arid newly formed bone. FIG. 30 Biological apatite precipitation in microspores and at the surface of the implant. FIG. 31 Biological apatite precipitation in microspores and in close contact with CaP ceramic crystals. FIG. 32 High resolution TEM of biological apatite crystals growing heteroepitaxially on the surface of p-TCP ceramic crystal after implantation.


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for the first time that the formation of these microcrystals after implantation (Fig. 30) was nonspecific, i.e., not related to implantation site, subjects of implantation, and types of CaP ceramics. It was further observed that the new crystals were not necessarily deposited on collagenous fibers and demonstrated some specific crystallographic interactions with the HA or /3-TCP crystals of the ceramic (Fig. 30). The microcrystals associated with the CaP biomaterials were described as apatitic, similar to biological apatites based on TEM and electron diffraction studies (Tracy and Doremus, 1984), and were established as CHA, similar to biological apatite based on IR analyses (Daculsi et al., 1990a; Heughebaert et al., 1988; LeGeros et al., 1988a; LeGeros and Daculsi, 1990) (Fig. 31). These biological apatites associated with CaP ceramics were similar in spectral properties to those observed associated with HA ceramics implanted in nonosseous sites (Heughebaert et al., 1988) and those suspended in serum in vitro (Gregoire et al., 1987; LeGeros et al., 1987, 1991~).The observed formation of carbonate apatites in association with the CaP biomaterials, regardless of implant sites (osseous or nonosseous), indicated a process of calcification without cell differentiation. The presence of these microcrystals was interpreted by some investigators to show “osteogenic” properties of the HA ceramic (Frank et al., 1987). However, the histological properties of true bone formation were observed only in osseous sites, confirming the current general agreement regarding the nonosteogenic but osteoconductive properties of the CaP ceramics (LeGeros er al., 1988a; Amler, 1988). The CHA crystals associated with CaP ceramic materials form by the processes of dissolution and precipitation: (i) Partial dissolution of the HA or P-TCP crystals of the ceramic causes an increase in the supersaturation level of the immediate microenvironment of the CaP implant, subsequently leading to the precipitation of the new apatite crystals incorporating other ions (e.g., C 0 3 ,Mg, and H P 0 4 from the biological fluid) during its formation; and/or (ii) precipitation of the new apatite crystals with or without the dissolution of the ceramic crystals, the ceramic particles acting as nucleator or seeds, and epitaxic and heteroiepitaxic growing processes, was observed (Fig. 32).

D. Biological Significance of Carbonate Apatite Precipitation The coalescing interfacial zone of biological apatite and residual crystals (Fig. 31) provides a scaffold for boneicell adhesion and further bone ingrowth (Fig. 29) (Daculsi and Dard, 1994). The restoring process involves a dissolution of calcium phosphate crystals and then a precipitation of CHA needle-like crystallites in micropores close to the dissolving crystals. The formation of CHA may occur by secondary nucleation and an epitaxial

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growth process (Fig. 32). The dissolution of CaP ceramic or related materials (e.g., bioactive glass) and precipitation of CHA is one of the characteristics of bioactive materials (Hench, 1991; LeGeros et al., 1988a, 1991,1992). The coalescing zone constitutes the new biomaterial/bone interface that includes the participation of proteins and CHA crystals originating from the CaP materials but does not include the biomaterial surface. The following events of bone ingrowth and the newly formed bone progressively replaces the initially formed CHA from the CaP biomaterials. In the case of calcium carbonate biomaterial (coral), the dissolution/ precipitation process cannot be similar to that of the CaP biomaterials. The dissolving CaCO? crystals from the coral provide an environment enriched in calcium and bicarbonate ions but deficient in phosphate ions. Based on results of studies on the formation of carbonate apatite from synthetic systems, such an environment will permit the formation of highly carbonated apatite with very poor crystallinity and even amorphous calcium carbonate phosphate that will be readily soluble (LeGeros, 1967, 1981a, 1991b; LeGeros et al., 1973; LeGeros and Tung, 1983). The incorporation of large amounts of carbonate in apatite causes dramatic changes in morphology (size and shape) (LeGeros, 1967, 1981a, 1991b; LeGeros et al., 1967, 1971, 1986). This explains the observation that needle-like apatite crystals normally associated with crystals of CaP materials were not observed on the surfaces of the CaC03crystals of the coral implants (Richard et af., 1996).CaP layer bordering coral grains has been described by Damien et al. (1994). This layer may consist of highly carbonated apatite that are more sensitive to physiological dissolution during bone remodeling compared to those observed with implanted CaP materials. It is possible that, in the case of coral biomaterials, bone ingrowth takes place after a dissolution process has occurred. Further investigation on the new bone formation associated with CaC03 and CaP materials should clarify this issue.

E. Osseo-Coalescing interface: A Dynamic Process An apparent tight bonding of tissue to the calcium phosphate and related bioactive materials is the most important requirement for ceramic materials in order to be osteointegrated and described as bioactive (Hench, 1994; Hench and Wilson, 1984; Hench et al., 1971). Nevertheless, the exact nature of the bonekalcium phosphate interface is, at the present time, intensively discussed. Bone formation seems to start directly on the surface of bioactive materials, whereas it stays away from the surface of nonbioactive materials, resulting in fibrous tissue interposition. From studies in which the behavior of the blood clot and subsequent cellular reaction were studied by use of combined morphological methods it is known that the first cells colonizing


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the surface are macrophages (Van Blitterswijk et al., 1986; Muller-Mai et al., 1990). After the macrophages disappeared, the various steps of primary mineralization of bone begin, including bone cell proliferation and appearance of matrix vesicles, and then synthesis of extracellular matrix (ECM) consisting of collagen and glycoaminoglycans(Gross et al., 1991). The phenotypic expression of undifferentiated cells from the bone marrow on the surface of bioactive materials supports the osteogenic differentiation on the surface (Ohgushi et al., 1992). Interface reactions with marrow-derived cells, osteoblast-like cells, or bone tissue are dependent on the crystal structure (Daculsi et al., 1989a), degree of crystallinity (De Bruijn et al., 1992, 1993), and surface roughness and composition of the calcium phosphates (De Bruijn et al., 1992). According to Osborn and Neweseley (1980) interface processes are based on epitaxy, “apatite-protein affinity,” and structural osteotropism. Using HR TEM Daculsi et al. (1990~)have demonstrated epitaxial growing process and secondary nucleation of biological apatite on both H A and 0-TCP ceramic crystals (Fig. 32). LeGeros et al. (3988b, 1991, 1992; LeGeros and Daculsi, 1990) made a strong argument for the importance of the formation of carbonate apatite in reflecting the bioactivity of biomaterials and to the development of the biomaterial/ bone interface unique to bioactive materials. The following mechanism was proposed: (i) acidification of the microenvironment as a consequence of cellular interaction with the materials; (ii) dissolution of the CaP biomaterials followed by the formation of CHA associated with an organic matrix incorporating the carbonate (and Mg) ions from the biological fluid; (iii) production of extracellular matrix (collagenous and noncollagenous proteins); and (iv) simultaneous mineralization of the collagen fibrils and incorporation of the newly formed CHA crystals in the remodeling new bone. The relationship of CHA formation to bioactivity of materials (calcium phosphate materials and bioglass) has been supported in subsequent studies (Hench, 1994) and its importance in cellular activity has also been elucidated (Yamada et al., 1994). The process of cell colonization, adhesion, phagocytosis and osteoclastic resorption, ECM elaboration and mineralization, and bone ingrowth and bone remodeling associated with the biological apatite precipitation during CaP ceramics dissolution is continuously in progress. Consequently, the interface is not static but dynamic, in constant evolution, taking into account bone physiopathology, biomechanical factors, and bone maturation.

IX. Summary and Conclusion Dental and skeleton mineralization are natural phenomena involving complex interactions among cellular activities, extracellular components, and

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condition and composition of the biological environment. The type of calcium phosphate phase that forms depends on the conditions (e.g., pH) and composition (e.g., Ca/P, Mg/Ca, H C 0 3 / P 0 4 ,specific proteins, and macromolecules). The orientation and morphology of the apatite crystals in enamel is influenced by specific proteins and proteidmineral interactions. The stability of calcium phosphate phases in vitro and in vivo also depends on the presencc of some of the inorganic and organic molecules. In normal calcifications (enamel, dentin, and bone) the mineral phase consists principally of CHA, differing in crystal size and shape, orientation, and concentration o f minor but important elements, (C03and Mg). The crystal size and shape, and consequently the mechanical property of the tissue, and the physicochemical properties (specific surface area, ionic substitution, and dissolution properties) are adapted to the nature and function (mechanical or physiological) of the tissue. In pathological calcifications (e.g., dental and urinary calculi and soft-tissue calcifications) in human, several types of calcium phosphate phases (ACP, DCPD, OCP, j3-TCMP, and CHA) coexist, suggesting changing conditions of pH and composition of the biological environment. The incorporation of F, Mg, C 0 3 , H P 0 4 , Sr, or CI affects the crystal growth and crystal properties of synthetic apatites. The presence of some of these ions and of P 2 0 7in solution affects the formation of apatites and other biologically relevant calcium phosphates. The knowledge on biological crystals has involved the development of synthetic bone graft substitute. Commercial and laboratory prepared CaP materials (HA, P-TCP, BCP, and coralline HA) or calcium carbonate (natural coral) have gained acceptance as materials for bone repair, substitution, and augmentation. Results from studies demonstrated that all these materials are dissolved or biodegraded to a greater or less extent, depending on the physical (density, macro- and microporosity, and surface topography) and crystallographic (defects and crystallinity) properties and composition of the biomaterials and on the extracellular and intracellular activity (phagocytosis processes). The cells involved in the biological resorption processes are still unknown. However, fibroblasts, monocytes, and osteoclast-like and giant cells are observed. During these dissolution/transformation processes, bioactive ceramics have the same evolution and adaptation to the tissues: (i) Partial dissolution of the CaP ceramic macrocrystals causes an increase in the calcium and phosphate concentrations in the local microenvironrnent; (ii) formation of C H A (either by direct precipitation or by transformation from one CaP phase to another or by seeded growth) incorporating ions (principally, carbonate) from the biological fluid during its formation; (iii) association of the carbonate/apatite crystals with an organic matrix; and (iv) incorporation of these microcrystals with the collageneous matrix in the newly formed bone (in osseous sites). The events at the CaP biomaterial/bone interface represent a dynamic process, including


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physicochemical processes, CrystaUproteins interactions, cells and tissue colonization, and bone remodeling, finally contributing to the unique strength of such interfaces. Acknowledgment The individual and collaborative studies involving the authors were suppported by research Grant CJF 93-05 from the INSERM and Grant EP 59 from CNRS (Dr. G. Daculsi, Director) and Grants DE04123 and DE07223 from the National Institute for Dental Research of the National Institutes of Health and special Calcium Phosphate Research Funds (Dr. R. Z. LeGeros, Principal Investigator).

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The Biogenesis, Traffic, and Function of the Cystic Fibrosis Transmembrane Conductance Regulator Tamas Jilling and Kevin 1. Kirk Gregory Fleming James Cystic Fibrosis Research Center and Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMPactivated chloride channel that is encoded by the gene that is defective in cystic fibrosis. This ion channel resides at the luminal surfaces and in endosomes of epithelial cells that line the airways, intestine, and a variety of exocrine glands. In this article we discuss current hypotheses regarding how CFTR functions as a regulated ion channel and how CF mutations lead to disease. We also evaluate the emerging notion that CFTR is a multifunctional protein that is capable of regulating epithelial physiology at several levels, including the modulation of other ion channels and the regulation of intracellular membrane traffic. Elucidating the various functions of CFTR should contribute to our understanding of the pathology in cystic fibrosis, the most common lethal genetic disorder among Caucasians. KEY WORDS: Cystic fibrosis, Genetic disease, Ion channels, Membrane traffic, Protein processing.

1. Introduction The cystic fibrosis (CF) gene encodes a CAMP-activated C1- channel (i.e., the cystic fibrosis transmembrane conductance regulator; CFTR) that resides at the apical membranes of a variety of epithelial cells. The goals of this article are to review our current understanding of the biogenesis, intracellular processing, and traffic of the CFTR and to summarize our knowledge regarding its participation in epithelial cell function. Under~nr~wnurronsi nrVIPIV of

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TAMAS JlLLlNG AND KEVIN

L. KIRK

standing early events in CFTR biogenesis is becoming one of the prime interests of C F researchers because a defect in the posttranslational processing of CFTR is considered to be the predominant mechanism by which the most common mutation causes disease. The mechanism and regulation of CFTR traffic beyond the biosynthetic pathway are also of interest because of evidence that CFTR recycles between endosomes and the apical plasma membranes of epithelial cells. We discuss the emerging notion that CFTR is not a passive cargo molecule in intracellular organelles and transport vesicles but rather it possibly regulates the composition and traffic of these compartments. A more detailed understanding of such a putative intracellular function for this ion channel might provide insights into the pathogenesis of CF, which is a pleiotropic disease that affects many epithelial surfaces. Cystic fibrosis is the most common lethal, autosomal, and recessive hereditary disease among Caucasians. The morbidity is approximately 1: 2000 live births in the general Caucasian population and can be considerably higher among families of northern European origin and mormons in Utah. The disease is characterized by impaired mucociliary clearance in the lung, recurrent respiratory infections, intestinal obstruction, male infertility, and, in most cases, pancreatic insufficiency (Boat et al., 1989). The life-limiting aspect of CF is a loss of pulmonary function as a result of recurrent respiratory infections and inflammation. However, other aspects of the disease such as male infertility are becoming more relevant as improvements in clinical treatments have prolonged the mean life expectancy of CF patients from 10 years in the 1960s to 28 years in 1990 (Fitzsimmons, 1993). This improvement has been due primarily to the refinement of CF treatment methods that focus on alleviating symptoms such as pancreatic enzyme replacement therapy, treatment of recurrent infections with antibiotics, and reducing inflammation and airway obstruction with pharmacological and physical methods. Novel therapeutic approaches that attempt to correct or bypass the cellular defect caused by CFTR mutations including in vivo gene therapy are currently under development (Welsh, 1995). Streptococcus and hnemophilus bacteria are the predominant lung pathogens in the first 2 years of life. Subsequent colonization of airways by Pseudornonas ueruginosa provides the major clinical challenge (Rubio, 1986). Recent data indicate that, in addition to bacterial infections, an exaggerated inflammatory response also contributes to the deterioration of pulmonary tissue in CF. An elevated level of inflammatory mediators exists in the airways of CF infants prior to the development of bacterial infections (Khan et al., 1995). The finding that CF patients respond favorably to high doses of the anti-inflammatory drug ibuprofen indicate that inflammation plays an active role in CF pathogenesis. Maintenance of a plasma concentration of ibuprofen at 50-100 pg/ml €or 4 years resulted in a statistically significant reduction in the rate of lung function deterioration in CF patients (Konstan et al., 1995). The beneficial effect of ibuprofen was most

BIOGENESIS, TRAFFIC, AND FUNCTION OF CFTR

195

evident in patients who were enrolled in the study at less than 13 years of age before or during the early stages of pulmonary infection. As discussed below, a defect in the apical membrane C1- channel function of CFTR plausibly explains many of the cellular abnormalities in CF such as impaired mucociliary clearance. The relationship between this functional attribute of CFTR and the exaggerated inflammatory state of the CF lung is less clear. Studies addressing novel cellular roles of CFTR in epithelial cells, including a possible role in regulating membrane traffic and epithelial secretory processes (see Section VIII), might help us understand the link between CFTR and inflammation.

II. Cystic Fibrosis Transmembrane Conductance Regulator

A. CFTR: The Protein The CF gene encodes a protein that is 1480 amino acids (aa) in length (Riordan et aZ., 1989). CFTR splice variants of differing lengths have been reported (Chu et aZ., 1992; Strong et al., 1993), although no functional significance of alternative splicing has yet been described. Five functional domains were predicted on the basis of the deduced amino acid sequence at the time of the discovery of the CFTR gene (Riordan et al., 1989). This five-domain model of CFTR protein is now generally well accepted (Fig. 1). Four of the five domains form two symmetrical halves of the CFTR molecule, each being composed of a set of six membrane-spanning a helices and a cytoplasmic nucleotide binding domain (NBD). The two halves are separated by a cytoplasmic regulatory (R) domain that includes multiple phosphorylation sites for a number of protein kinases including cyclic AMPdependent protein kinase (PKA) and protein kinase C. The characteristic symmetrical arrangement of the membrane-spanning domains and the two NBDs places CFTR in the family of ATP binding cassette (ABC) transporters (Hyde er al., 1990). The R domain is a feature of CFTR that distinguishes it from other members of this family. Interestingly, several other members of the ABC transporter family serve as active transport pumps that couple ATP hydrolysis at one or both NBDs to solute transport (e.g., the bacterial oligopeptide permease, OPP; Hiles et al., 1987). No such active transport function for CFTR has been described.

B. CFTR: The CI- Channel The most well-accepted function of CFTR is that of a CAMP-regulated chloride channel. Although it was known for more than 50 years that an

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TAMAS JlLLlNG AND KEVIN L. KIRK

OUT

L C O O H

IN FIG. 1 A schematic diagram of the CFTR. The 12 putative membrane-spanning domains are indicated by stippled rectangles. NBD, nucleolide binding domain; R, R domain; AF508, approximate location of the AFS08 mutation. Asterisk denotes approximatc locations of two extraccllular glycosylation sites.

elevated sweat NaCl content is a hallmark of CF, it was Ouinton (1983) who first showed that a reduced C1- conductance in the sweat duct is responsible for the defect in NaCl reabsorption in the CF sweat duct. The laboratories of Frizzell (Schoumacher et al., 1987) and Welsh (Li et al., 19SS) subsequently used patch clamp techniques to establish that the regulation of C1- channels by PKA was defective in C F airway epithelial cells. After the gene was cloned in 1989 in a joint effort by the laboratories of Collins, Tsui, and Riordan (Kerem et al., 1989; Riordan et al., 1989; Rommens et al., 1989), several groups showed that the defective CI conductances of CF epithelial cells could be complemented by introducing the wild-type CFTR cDNA or gene into these cells (Rich et al., 1990; Drumm et al., 1990; Jilling et al., 1990). The most definitive evidence that CFTR is a CI- channel rather than a regulator of endogenous CI- channels is that purified CFTR protein, when reconstituted into liposomes and incorporated into artificial lipid bilayers, generates PKA-activatable CI- channel activity that is indistinguishable from that observed in patch clamp studies of CFTR-producing cells (Bear et ai., 1992). On the basis of the results of these patch clamp


197

and bilayer experiments, the following biophysical and pharmacological “fingerprint” of CFTR has emerged: (i) 5-10-pS single channel conductance (Tabcharani et al., 1991), (ii) linear I/V relationship under symmetrical ionic conditions; (iii) permselectivity of Br- 2 C1- > 1- > F- (Cliff and Frizzell, 1990; Cliff et al., 1992); (iv) activation by PKA phosphorylation and dependence of channel activity on cytosolic ATP (Bear et al., 1992; Cheng et al., 1991); (v) insensitive to the disulfonic stilbene DIDS, which blocks other CI- channel types (Cliff and Frizzell, 1990; Cliff et al., 1992); and (vi) blocked by the broad-spectrum CI- channel blocker, diphenylamine carboxylate (Schwiebert et al., 1994a), and the sulfonylurea, glibenclamide (Schultz et al., 1996), the latter of which also blocks ATP-dependent potassium channels in pancreatic p cells (Aguilar-Bryan et aZ., 1995). Figure 2 shows a typical patch clamp record of CFTR C1- channel activity stimulated by PKA plus ATP. The results of a large number of structure-function studies indicate that the C1- channel activity of CFTR is coordinately regulated by the R domain and the two NBDs. Channel activation requires both phosphorylation of the R domain and ATP binding by the NBDs. In most tissues the physiologically relevant kinase for CFTR phosphorylation is CAMP-dependent protein kinase (Li et al., 1988; Cheng et al., 1991), although the type I1 cGMPdependent protein kinase may also play a role in CFTR activation within the intestine (French et al., 1995). At least 11 different serine residues are phosphorylated by PKA in vivo, 10 of which occur within the R domain. Site-directed mutagenesis of these residues has revealed that all 11 phosphorylation sites and possibly others as well contribute to channel activation (Cheng et a/., 1991; Seibert et al., 1995). On the basis of these results, it has been suggested that CFTR C1- channel activity can be elevated in a graded fashion rather than all or none in response to stimulation of the cAMP pathway (Chang et al., 1993). Interestingly, deletion of the R domain (aa 708-835) from CFTR results in a C1- channel that is constitutively active in the absence of cAMP or PKA (Rich et al., 1991). This observation implies that phosphorylation of the R domain reverses an otherwise negative modulation of CFTR C1- channel activity by this structural domain. Conceivably, the minimally phosphorylated R domain occludes or blocks the conducting pore in a manner crudely analogous to the “ball and chain” mechanism for K+ channel inactivation originally described for the Shaker K+ channel in Drosophila (Zagotta et al., 1990). Each of the NBDs contains consensus sequences for ATP binding and hydrolysis (i.e., Walker A and B sequences) that are characteristic of the ABC transporters (Hyde et al., 1990). Each CFTR NBD binds ATP in vitro (Hartman et al., 1992; Randak et al., 1995), although only NBDl has been shown directly to possess ATP hydrolytic activity (KO and Pedersen, 1995). Several groups have argued on the basis of the results of mutagenesis

198


L. KIRK

Cell-attached

Excised

+ PKA & MghTP

0.3

PAL Is

FIG. 2 Representative current tracings of CFTR C1- channel activity in transl'ected mouse I, fibroblasts. Thc records shown were obtaincd from a single membrane patch that contained two active CFTR channels. The dashed lines represent the baseline or zero currcnt Icvcl. The pipette contained only impermeant cations in addition to CI so that the currents shown can only be due to the flow of CI- into the pipette. Forskolin (10 p M )was added to increase cellular CAMP and activatc the CFTR channels in thc cell-attached configuration approximately 100 s prior to the lirst record. Both channels inactivated within seconds following patch excision. Addition of protein kinase A (PKA, 150 unitslml) and Mg-ATP (300 p M ) to the solution bathing the cytosolic side of the excised patch immediately restored the channel activity. The holding potential (-60 mV) and the single channel current amplitude in the excised configuration (0.48 PA) yield an estimated unitary conductance of 8 pS, which is the cxpcctcd conductance of CFTR CI- channels at room temperature (22-25°C). The cells and experimental conditions were identical to those described by Venglarik ef ul. (1994), except for the lower temperature. The recordings were filtered at 100 Hi! (-3 dB attenuation) and sampled at 500 Hz. ~

experiments that both NBDs hydrolyze ATP, but with distinct functional consequences (Carson et al., 1995; Gunderson and Kopito, 1995; Wilkinson et ul., 1996).In particular, mutating the strictly conserved lysine residue in the Walker A box in N B D l (K464) reduced the rate of channel activation.


199

Conversely, the analogous mutation in NBD2 (K12.50) stabilized C1- channel activity. Neither mutation decreased azido-ATP binding to CFTR (Carson et d., 1995), whereas analogous mutations in other ATP-binding proteins decreased the rate of ATP hydrolysis (e.g., Tian et d., 1990). These results indicate that CFTR C1- channels are either activated or primed for activation by ATP hydrolysis at NBD1. The duration of channel activation is then controlled by ATP hydrolysis at NBD2. In this regard, Carson et al. (1995) have argued that the NBDs serve as timer switches to regulate the amount of time that CFTR C1- channels are open, by analogy to the function of GTPases to regulate the duration of activation of downstream effectors. According to this model, ATP hydrolysis at NBDl and at NBD2 opens and closes the channel, respectively. Gunderson and Kopito (1995) have proposed a somewhat different model in which ATP hydrolysis at NBDl primes CFTR for channel activation, whereas ATP hydrolysis at NBD2 provides the thermodynamic driving force for the cycling of CFTR between closed and open conformations. Despite these differences in interpretation, the available data clearly indicate that the NBDs are critical and functionally distinct modulators of CFTR C1- current activity. The membrane-spanning domains presumably play a role in forming the conducting pore and perhaps the selectivity filter for this pore. Anderson et al. (1991) have reported that the halide selectivity of CFTR is determined in part by positively charged residues in the first and sixth putative transmembrane domains. Namely, they observed that the mutants K95D, K335E, and R347E exhibited a reversed permeability ratio for 1- and C1- compared to wild-type CFTR ( P I > PClrather than Pa > P I )when analyzed by whole cell patch clamping in transfected HeLa cells. These results have since been called into question by Hipper et al. (199.5), who observed no such reversal of the permeability ratios for the same mutants produced in Xenopus oocytes. The reason for this discrepancy is unclear but probably relates to the different expression systems and electrophysiological techniques used by these two groups. Regarding the ion selectivity of CFTR, this channel also conducts bicarbonate (Paulsen et al., 1994) and has been reported to conduct ATP as well (Reisin et al., 1994). As a bicarbonate channel, CFTR could contribute to the regulation of luminal and perhaps intracellular pH. The notion that CFTR conducts ATP is more controversial; in particular, Reddy et al. (1996) failed to detect ATP currents through CFTR C1- channels in a variety of experimental systems. It is important to resolve this issue because several groups have argued that CFTR could regulate neighboring ATP-dependent ion channels by serving as a transmembrane conduit for ATP secretion into the luminal space (see Section V1,A). In summary, the C1- channel activity of CFTR is regulated by an interplay between phosphorylation of the R domain and ATP binding and/or hydroly-

200


sis at each of the NBDs. The available data indicate that the NBDs are not functionally equivalent CFTR domains, with several groups having proposed that NBDl and NBD2 serve as positive and negative regulators of channel activity, respectively (Carson et al., 1995; Baukrowitz et ul., 1994; Wilkinson et al., 1996). A certain degree of functional redundancy between the two halves of this bipartite molecule may also exist, however, as indicated by the analysis of a CFTR truncation mutant (D836X) by Sheppard et ul. (1 994). This mutant, which contains the first six transmembrane domains, NBD1, and the R domain, is capable of forming regulated C1- channels when expressed in HeLa cells. In cell lysates D836X protein cosedimented with mature CFTR on a sucrose gradient implying that this truncation mutant forms homodimers. D836X generates CI- channels that can be activated by CAMP and PKA and that have the same halide selectivity as wild-type CFTR. In several other respects D836X and wild-type CFTR differ; in particular, the truncation mutant exhibited a modest level of Clchannel activity in the absence of PKA phosphorylation and a considerably greater sensitivity to stimulation by Mg-ATP. These differences may be attributable to the proposed negative modulatory role of NBD2, which is lacking in D836X. The fact that D836X can form regulated C1 channels that are otherwise very similar to the wild-type protein implies that the two halves of this symmetrical molecule share functional similarities.

111. CFTR Mutations That Cause Disease A. Classes of Mutations Over 550 disease-causing mutations in CFTR have been reported, although only a small fraction of these have been functionally characterized (Zielenski and Tsui, 1995). Welsh and Smith (1993) have categorized CFTR mutations into four groups based on their influence on CFTR processing and function (Fig, 3). Class I mutants constitute nonsense, splice, and frameshift mutants that encode truncated or aberrant forms of CFTR (e.g., G542X). Such mutants constitute about one-half of the 550 different CF mutations that have been reported. A number of the mutants in this class associate with greatly reduced mRNA and protein abundance due presumably to instability of the altered transcripts. Many of these mutants associate with severe pathology (e.g., G542X) including pancreatic insufficiency, the most reliable indicator of disease progression in CF (see Section I). Interestingly, Howard et al. (1996) have reported that two premature stop mutations in the CFTR coding region (G542X and R553X) can be suppressed by treating cclls with modest doses of the aminoglycoside antibiotics G418 and gentami-

201


LUMEN

4 Class

2 (AF508)

Class 1 (G542X)

INTERSTITIUM FIG. 3 The four major classes of CFTR mutations. An example of each class of mutation i s shown in parentheses. MSD, membrane spanning domains. Adapted from Welsh and Smith (1991) with permission from Cell Press.

cin. HeLa cells that were transiently transfected with GS42X or R553X CFTR cDNAs produced full-length CFTR protein and exhibited CAMPactivated halide permeability when cultured in the presence of 0.1 mg/nil G418. These results are consistent with previous reports that aminoglycosides can suppress stop mutations in a wide variety of organisms ranging from bacteria to mammalian cells (Martin et al., 1989). If clinically appropriate aminoglycosides are capable of suppressing premature stop mutations in endogenous CFTR transcripts within airway epithelial cells, such drugs may be useful for treating CF patients who harbor such mutations, which account for approximately 5% of all CF alleles. Class 11 mutants are processing mutants that produce protein that is incompletely glycosylated because it is retained within the endoplasmic reticulum (ER). These mutants constitute the most prevalent diseasecausing alleles including the AF.508 mutation, which accounts for approxi-

202

TAMAS JILLING AND KEVIN L KIRK

mately two-thirds of all CF alleles. In general, class I1 mutations represent single amino acid substitutions or deletions in one of the two NBDs, although this feature is not unique to mutations in this class (see below). As expected, disease severity within this class correlates with the amount of mutant protein that can be released from the ER. For example, the AF508 mutation results in the production of full-length protein, but virtually none of this protein escapes the endoplasmic reticulum (Cheng et a]., 1990; see below for discussion of mechanism of E R retention). Consequently, there is a nearly complete failure of CFTR AF508 to mature beyond the E R glycosylated form of this protein, as can be monitored by one-dimensional SDS-PAGE analysis of immunoprecipitated protein (see Fig. 4). Not surprisingly, the AF508 mutation associates with severe disease (Johansen et al., 1991). In contrast, A455E and P574H are two processing mutants that associate with milder disease (Kerem et al., 1990; Kristidis et al., 1992). For each of these mutants some protein escapes the E R and becomes fully glycosylated at amounts that are intermediate between wild-type CFTR and the AFS08 mutant. Interestingly, the latter of these two mutants (PS74H) exhibits a compensatory increase in channel open probability relative to wild-type CFTR when produced as recombinant protein in HeLa or Vero cells (Sheppard et af., 1995; Champigny et af., 1995). This result provides additional evidence for the involvement of the first NBD and proline 574, in particular, in CFTR channel gating. The increased C1 chan-

WTCFTR

AF508CFTR

BZ"++

I

+

I

+

FIG.4 Characteristic profile of WT CFTR and AFS08CFTR visualized by SDS-PAGE analysis. LLCPKI cells stably transduccd with WT CFTR or AF508CFTR cDNAs under the transcriptional regulation of the Zn2'-inducible metallothionein promoter were subjected to CFI'R immunoprccipitation followed by in v i m phosphorylation. The fully processed form (band C) of WT CJTR protein can be detected even in the absence of exogenous Zn2' (is., the serum in the tissue culture medium contains some heavy metal), whcreas AF508CFTR remains virtually undetcctable under these unstimulated conditions. Following 25 h treatment with 100 gM Zn", an increase in WT CFTR band C and the appearance of hand B (i.e., ER form) in both the WT CFTR and AFS08CFTR immnnoprecipitates is obscrved.


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nel activity exhibited by P574H presumably contributes to the milder clinical phenotype in patients with this mutation. Class I11 and class IV mutants encode proteins that are fully glycosylated and that are targeted to the plasma membrane but are defective either in channel regulation (class 111) or in ion conduction (class IV). The class I11 regulatory mutants generally exhibit single amino acid substitutions in one of the NBDs (usually NBD1) that lead to reduced channel activation by ATP. The loss of channel activation by ATP can be partial (e.g., G551S) or virtually complete (e.g., G551D), which correlates with mild or severe disease, respectively (Anderson and Welsh, 1992; Strong et al., 1991; Cutting eta/., 1990). The conduction mutants (class IV) represent a small group of rare mutations in the membrane-spanning domains (e.g., R117H and R347P; Sheppard et al., 1993). These mutant channels exhibit reduced single channel conductances; namely, when open they conduct fewer ions per second than wild-type CFTR at the same electrochemical driving force (ca. 20 and 70% fewer for R117H and R347P, respectively). Not surprisingly, these partial loss-of-function mutants associate with milder clinical phenotypes such as pancreatic sufficiency (Kristidis et al., 1992). This categorization of CF mutations into four classes has heuristic value; however, it should be noted that some mutations can be included within multiple categories. For example, the AF508 mutation, which leads to profound defects in CFTR processing, also apparently results in a partially reduced open probability at the single channel level relative to wild-type CFTR under conditions when the production of fully glycosylated AF508 protein is artificially induced (Dalemans eta/., 1991; Denning et aL, 1992b, see below). In addition, because many CF patients (ca. 50%) are compound heterozygotes, some patients will possess mutant alleles from different classes that may associate with differing clinical severity. Compound heterozygotes that carry alleles associated with both severe and mild phenotypes typically exhibit the milder phenotype, given the recessive nature of this genetic disorder. Finally, there are reports of complex alleles in which second site mutations may influence the primary mutation. The R553Q mutant is a particularly interesting example of a second site mutation that has been found on an allele carrying AF508. The milder clinical pathology that associates with this complex allele implies that the R553Q mutation alleviates, or reverts, the severe phenotype that normally associates with the AF508 mutation (Dork eta/., 1991). This notion is consistent with the observation that the R553Q mutant serves as an intragenic suppressor of the AF508 mutation in a hybrid molecule containing a portion of CFTR NBDl that replaces an analogous region of the corresponding NBD of the yeast ABC transporter, STE6 (Teem et al., 1993).

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L. KIRK

B. Defective Biosynthetic Processing of the Most Common Mutant, AF508

What is the mechanism by which the most common mutant is retained within the endoplasmic reticulum? Interest in this question has been fueled by the observation that AFS08 protein is capable of eliciting CI- current activity, although probably not at wild-type lcvels (see previous section). On this basis it has been proposed that pharmacologic maneuvers that release AF508 protein from the E R and allow it to be targeted to the apical plasma membrane could be therapeutically beneficial (Denning er al., 1992b). No such maneuvers that are clinically appropriate have yet been identified in large part because of our limited understanding of the fundamentals of protein folding, retention, and degradation within the ER. However, certain aspects of the basic biology of protein folding in general and CFTR processing in particular are becoming clearer as a result of the pioneering studies of several laboratories, as discussed below. One of the most striking features of CFTR biosynthesis is that wild-type CFTR protein is also inefficiently processed within the ER. Typically, less than 20-30% of newly synthesized wild-type CFTR molecules escape from the E R to become fully glycosylated within the Golgi complex (Ward and Kopito, 1994; Lukacs et al., 1994). Instead, the majority of wild-type CFTR protein and virtually all AF508 protein is degraded within the ER with rclatively rapid kinetics (Tli2of 15-40 min depending on cell type). This inefficient processing of wild-type CFTR occurs both for native protein in epithelial cclls and for recombinant CFTR protein when heterologously expressed in nonepithelial cells (Ward and Kopito, 1994). Thus, it is a feature of the protein itself and not the epithelial tissues that normally cxpress it. The degradation of immature CFTR protein within the E R appears to involve in part the proteasome, a 26s cytosolic complex of multiple peptidases. The proteasome degrades a variety of short-lived cytoplasmic proteins that have been “tagged” by polyubiquitination, the covalent addition of a chain of ubiquitin molecules catalyzed by a series of ubiquitinating enzymes (see review by Ciechanover, 1994). Until recently there had been no compelling evidencc for the involvement of this cytosolic complex in the dcgradation of integral membrane proteins within the ER. However, two groups have now shown that the E R degradation of immature wildtype and AFS08 CFTR protein is slowed substantially by cell permeant inhibitors of the proteasome such as lactacystin (Jensen et nl., 1995; Ward et al., 1995). Polyubiquitinated forms of CFTR accumulate coincident with the lactocystin-induced inhibition of degradation within the ER. Moreover, the degradation of AFS08 CFTR within the E R is inhibited when this protein is coexpressed with a dominant-negative ubiquitin mutant (K48R)


205

in HEK cells or when wild-type protein or AF508 is expressed in a cell line that contains a temperature-sensitive mutant of the ubiquitin-activating enzyme E l (Ward et al., 199.5). These results provide strong evidence for an important role for ubiquitination in the degradation of CFTR within the ER. This covalent modification presumably targets CFTR for degradation by the proteasome, although the involvement of other proteolytic pathways cannot be ruled out on the basis of the protease inhibitor studies performed to date. It is perhaps not too surprising that this cytosolic protease complex could participate in CFTR degradation because approximately 80% of the mass of CFTR is predicted to be cytosolic (Fig. 1). Establishing that ubiquitination and the proteasome participate in CFTR degradation represents an important advance in our understanding of the cell biology of this protein. Unfortunately, neither blocking this degradative pathway at the level of CFTR ubiquitination nor inhibiting the proteasome itself using protease inhibitors releases AF.508 CFTR protein from the ER. For example. when AF508 CFTR is coexpressed with the dominant-negative ubiquitin mutant K48R, AF508 protein that escapes short-term degradation gradually accumulates as a pool of Triton-X 100 insoluble material rather than being converted to soluble, mature CFTR (Ward et a/., 1995). Thus, ubiquitination appears not to be the only factor that prevents AF508 protein from proceeding along the biosynthetic pathway. Other possibilities include interactions between AF.508 protein and two putative molecular chaperones-cytosolic hsp70 and the E R resident membrane protein, calnexin. Both are candidate facilitators of protein folding; the latter of which binds to the carbohydrate moieties of partially folded glycoproteins within the E R (Ora and Helenius, 1995; Ware et al., 1995). The results of pulsechase, immunoprecipitation, and cosedimentation experiments indicate that immature wild-type CFTR and AF.508 CFTR each associate with hsp70 (Yang et al., 1993) and calnexin (Pind et al., 1994) within the ER. Wildtype CFTR molecules that have progressed to the Golgi, which occurs within 30-45 min of synthesis, do not bind either chaperone. Conversely, the interactions between AF508 protein, calnexin, and hsp70 are long lived (>1..5-3 h). It has not been formally demonstrated that hsp70 and calnexin bind to those immature wild-type molecules that ultimately escape the E R and progress to the Golgi; for example, it could be that these chaperones bind only to that population of wild-type molecules that is targeted for degradation within the ER. However, if calnexin and hsp70 do indeed transiently bind to wild-type CFTR molecules that are destined to be completely processed, then the release of CFTR protein from hsp70 and calnexin would be an important step in the commitment of newly synthesized protein to further biosynthetic processing in the Golgi. The stable association of AF508 CFTR protein with calnexin and/or hsp70 is conceivably due to incorrect folding of the mutant protein. Indeed, one

206


of the proposed functions of calnexin is to retain partially folded proteins within the ER (Hammond and Helenius, 1994). These considerations lead to an important question: To what extent is AF.508 protein in the E R misfolded? Simply deleting phenylalanine 508 from bacterially produced NBDl has no obvious effect on nucleotide binding or on the solution structure of this recombinant polypeptide as determined by circular dichroism spectra (Hartman et af., 1992). Of course, this deletion could have more profound structural consequences in the context of the folding of full-length CFTR protein. In this regard, however, Pasyk and Foskett (1995) have shown quite elegantly that there exists AF508 protein within the ER that can fold sufficiently well to function as a CAMP-regulatable CI- channel. These investigators patch clamped the outer membranes of nuclei with attached E R that were isolated from Chinese hamster ovary (CHO) cells producing recombinant wild-type or AF508 CFTR. In either case single C1 channels were observed with properties identical to cell surface CFTR such as PKA dependence, DIDS insensitivity, and a single-channel conductance of about 8-10 ps. No such channels were observed for nuclei that were isolated from mock-transfected CHO cells. One cannot ascertain from these single-channel measurements the relative amount of AFS08 protein that is functionally viable, although CFTR CI- channels were observed with the same approximate frequency for nuclei that were isolated from AF508 CFTR-expressing cells compared to wild-type CFTR-expressing cells. The simplest interpretation of these results is that some AF508 protein can fold sufficiently well within the E R to perform the function of a regulatable ion channel. How can we incorporate these observations into a model that accounts for the inefficient processing of wild-type CFTR and the virtually complete retention of AF508 CFTR in the ER? Several groups have proposed such a model that is based on the relative kinetics of CFTR folding and degradation within the E R (Ward and Kopito, 1994; Lukacs el al., 1995). According to this model CFTR folding occurs with relatively slow kinetics due to the complicated and interrupted topology of this polytopic protein, which includes 12 membrane-spanning domains that are interrupted by two NBDs and by a unique R domain. Consequently, the kinetics of CFTR folding overlap with the kinetics of sorting incompletely folded proteins to the E R degradative pathway. As a result, a large fraction of newly synthesized CFTR becomes targeted for degradation before it can fold completely (Fig. 5). The AF508 mutation likely further retards the kinetics of CFTR folding without inducing gross misfolding, such that virtually all AF50X molecules are sorted to the degradative pathway before they can fold completely. The sorting of AF508 protein between the biosynthetic pathway and the degradative pathway presumably occurs upstream of ubiquitination; otherwise, blocking this covalent modification would lead to maturation of AF508 protein. The nature of this putative sorting event is currently unknown,

207


WTCFTR

AF508CFI’R

Time Deadline for folding

FIG. 5 Putative timeline for the processing and degradation of wild-type (WT) and AF508 CFTR. Shaded regions represent populations of newly synthesized WT CFTR or AF508CFTR molecules. The “threshold for transit to Golgi” represents an arbitrary state of folding that permits newly synthesized molecules to proceed from the E R to the Golgi for further processing. The “deadline for folding” represents an arbitrary time window beyond which molecules that have not reached the aforcrnentioned threshold are targeted for degradation. Maneuvers that allow some AF508 protein to escape the ER, such as reduced temperature and glycerol, could extend the deadline for folding, increase the rate of folding within the ER, and/or reduce the threshold for transit to the Golgi.

although the release of calnexin and hsp70 from CFTR protein may be a corollary to this event (see above). Jensen et al. (1995) have also provided evidence for an ATP-dependent step in the maturation of wild-type CFTR within the E R than can be blocked by the peptide aldehyde, MG-132. The relationships between this step and the interactions between CFTR, calnexin, and hsp70 are currently unclear. One of the major challenges t o workers in this field will be to define at a molecular level the nature of the event or events that sort newly synthesized CFTR protein between the biosynthetic and degradative pathways in the ER. Are there any circumstances in which AF.508 protein can mature into fully glycosylated protein? According to the model described previously, if the kinetics of CFTR folding could be accelerated relative to the kinetics

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of protein sorting to the degradative pathway, some AF508 protein might fold completely before being committed to degradation and thereby escape to the Golgi. Hope for this scenario comes from two observations. First, the AF508 mutant is a temperature-sensitive mutant. Denning et al. (1992b), following up the observation that CAMP-activated C1- channels appeared on the surfaces of AF508 CFTR-expressing SF9 insect cells and Xenopus oocytes cultured at reduced temperature, showed that a moderate amount of AFS08 protein escapes the ER when mammalian fibroblasts are cultured at 23-26°C for 2 or 3 days. The maturation of AF508 protein at reduced tcmpcraturc was evidenced both as the appearance of the fully glycosylated form of CFTR by SDS-PAGE analysis and by the appearance of PKAactivated CI- channels at the surfaces of AFS08-expressing fibroblasts. Presumably, by reducing temperature the kinetics of CFTR folding are accclcrated relative to the kinetics of protein sorting to the degradative pathway with the result that some AF508 protein can “beat the clock” and fold completely before being targeted for degradation. Second, Sat0 et al. (1996) have shown that a substantial amount of AFSOX protein can mature, be delivered to the cell surface, and generate CAMP-activated C1- currents when AF.508-expressingcells are grown in 10%glycerol. Glycerol treatment retarded the degradation of both immature AF508 and wild-type CFTR protein. Unlike the situation in which ubiquitination is blocked, a significant fraction of AFS08 protein that escaped short-term degradation progressed to the Golgi and becamc fully glycosylated. This polyol likely functions as a chemical chaperone that facilitates AF508 protein folding by analogy to its well-known effects on protein folding and stability in vitro. By facilitating AF.508 protein folding, glycerol would be expected to shift the balance between maturation and degradation in favor of the former, as predicted by the kinetic model outlined previously. The utility of reducing temperature or of glycerol treatment as therapeutic strategies for releasing AFS08 protein from the ER is obviously limited. Indeed, neither maneuver has been rigorously shown to augment AFS08 protein maturation and function in polarized epithelial tissues, the target tissues of interest. Nonetheless, these observations raise the possibility that clinically appropriate maneuvers to release AFS08 protein from the E R that are based on facilitating protein folding may be feasible.

IV. Physiological Role of CFTR as an Apical CI- Channel in Epithelial Tissues CFTR residcs in part at the apical membranes of epithelial cells that line the pancreatic duct (Marino el al., 1991), airways (Puchelle et al., 1992),

BIOGENESIS,TRAFFIC, AND FUNCTION OF CFTR

209

large intestine (Cohn et al., 1992), and reabsorptive sweat duct (Kartner et al., 1992). Most of the epithelial cells that express CFTR engage in regulated fluid and electrolyte secretion (Fig. 6). CFTR C1- channels at the apical surfaces of secretory cells serve as regulatable conduits for CI- efflux from the cell. This efflux is driven by an electrochemical potential gradient that favors passive C1- transport from cell to lumen, which in turn is generated by the operation of two parallel active transport processes at the basolateral membrane (Frizzell and Halm, 1990). Transepithelial CI- secretion electrically obliges Na' secretion via the generation of a lumen-negative transepithelial voltage and osmotically obliges fluid secretion via the generation of a transepithelial osmotic pressure difference. Apical CFTR CI- channels constitute the rate-limiting step in CAMP-stimulated electrolyte secretion by airway and intestinal epithelial cells, as evidenced by the defects in CAMP-dependent secretion exhibited by these cells in cystic fibrosis. CFTR C1- channels also come into play in cholera, another devastating disease of secretory epithelial cells. Cholera toxin promotes massive secretory diarrhea by markedly and chronically stimulating cAMP production in secretory epithelial cells within the colonic crypt (Field et al., 1972). Such elevations in cAMP activate apical CFTR C F channels and thereby fluid

4

4

+

Lumen

lnterstitium

FIG. 6 Model of secretory epithelial cell. CFTR C1- channels reside at the apical plasma membrane in series with three relevant basolateral transport processes: (i) the N a ' / K ' ATPase, (ii) the Na'l2CI-/K- cotransporter, and (iii) K' channels through which K ' that is transported into the cell by the Nat/Ki ATPase and the cotransporter recycles back into the interstitium. Water secretion is shown to be paracellular (i.e., between cells) in this schematic, although fluid secretion probably also occurs across the cells (i.e., via a transcellular pathway).

210


secretion within the crypt, as explained previously. On this basis it has been argued that there may exist a heterozygote advantage for subjects that harbor a single defective CF allele (Quinton, 1994; Guggino, 1994). Heterozygotes presumably exhibit a partial loss of CFTR functional activity and, therefore, may be less sensitive to cholera toxin and other factors that promote secretory diarrhea. This notion might help explain the relatively high carrier frequency of mutant alleles among populations whose ancestors experienced cholera epidemics in the Middle Ages. However, apparently conflicting results have been obtained in studies of the effects of cholera toxin on intestinal fluid and electrolyte secretion by transgenic mice that lack one or both CF alleles (Gabriel et al., 1994; Cuthbert et al., 1995). CFTR is also expressed in the human reabsorptive sweat duct in which it facilitates NaCl reabsorption from the primary sweat secretion (Fig. 7). An elevated sweat C1- concentration has been the most reliable clinical indicator of CF since the 1950s (Gibson and Cooke, 1959). Quinton (1083) established that the CF sweat duct exhibits a reduced transepithelial C1

Lumen

ci

1I

ci

N.a+

Na'

+

lnterstitium FIG. 7 Model of reabsorptive sweat duct cell. The sweat duct epithelium has a low water permeability such that NaCl is reabsorbed in excess of water and the final sweat secretion is dilute with respect to plasma. For simplicity the epithelium is depicted as a single cell layer, although the sweat duct consists of two layers that function as a syncytium because the two layers are connected by gap junctions (Jones and Quinton, 1989).


21 1

conductance that is attributable at least in part to a reduced CI- conductance of the plasma membrane ( Reddy and Quinton, 1989; Ram and Kirk, 1989). The C1- conductance of this tissue serves to shunt the transepithelial voltage that is generated by active Na' reabsorption. A reduced CI-conductance results in the development of a large transepithelial voltage that opposes Na' reabsorption by the CF sweat duct. As a result, the reabsorption of both ions is reduced and the NaCl concentration within the final sweat secretion, which is normally hyposmotic with respect to plasma, is elevated. One of the more interesting unresolved issues regarding the sweat duct is the observation that the CFTR-dependent CI- conductance of this tissue is tonically stimulated, unlike in other CFTR-expressing epithelia. To what extent this difference is attributable to tissue-specific variations in cyclic nucleotide metabolism versus differences in the CFTR molecule itself, such as alternative splicing, remains to be determined. Can a defective apical CI- channel in epithelial tissues explain the pathology in CF? For many but not all of the pathologies in CF there appears to be a relatively straightforward connection to a defect in apical C1conductance. Elevated sweat NaCl levels in CF subjects are easily explained by a reduced CIAconductance in the reabsorptive sweat duct, as noted previously. Pancreatic insufficiency, which associates with the more severe cases of CF, may be attributable to a defective apical CI- channel in pancreatic duct epithelial cells. CFTR C1- channels normally function in parallel with an apical CI-/HC03- cotransporter to secrete HC03- into the pancreatic duct lumen (Gray et al., 1988, 1994; Novak and Greger, 1988). CFTR channels may also participate in HC03- secretion by directly conducting HC03- ions across the apical membranes of pancreatic duct cells, as discussed in Section 11. HC03- secretion serves to neutralize acid emptied into the small intestine from the stomach and to provide an appropriate pH for pancreatic enzymes. A defect in HC03- secretion by the pancreatic duct could contribute to this aspect of the disease. Impaired mucociliary clearance in the Iung may be attributable at least in part to poor hydration of the mucous due to defective fluid secretion by airway epithelial cells. Despite the fact that certain pathologies in CF can be explained on the basis of a defective apical CI- channel in epithelial tissues, other clinical manifestations of this disease are not so easily explained in this way. These include an exaggerated inflammatory response in the CF lung in the apparent absence of infection (Section I), altered sialylation of secreted proteins in the meconium (Duthel and Revol, 1993), decreased ingestion of P. aeruginosa by airway epithelial cells (Pier et al., 1996), and elevated Na' reabsorption by CF airway epithelial cells (Boucher et al., 1986).The apparent lack of connection between these clinical manifestations and a defective apical CI- channel may simply reflect our incomplete appreciation of the physiological role that apical CI- channels play in modulating mucosal

212


homeostasis. However, there is accumulating evidence that the function of CFTR extends beyond that of an apical C1- channel in ways that may better explain some of these other clinical abnormalities. In Sections VI and VIII we discuss two other putative functions of CFTR: (i) CFTR as a regulator of other apical ion channels (Section VI), and (ii) CFTR as a regulator of organelle composition, membrane traffic, and the biosynthetic pathway (Section VIII).

V. Regulation of CFTR Function by the Cytoskeleton A. Microtubules The activity of CFTR C1 channels at the surfaces of colonic epithelial cells is dependent to some extent on the microtubule cytoskeleton. Long-term exposure (2.5-3 h) of TX4cells (Fuller et a/., 19Y4) and airway epithelial cells (Schwiebert et al., 1994b) to inhibitors of microtubule polymerization, such as nocodazole and colchicine, moderately reduces CAMP-dependent halide permeability. Microtubule disruption has no effect on Ca2' -activated halide permeability (Fuller et al., 1094); thus, the inhibition appears to be specific for the CFTR-dependent C1 permeability pathway. How such microtubule inhibitors reduce CFTR-dependent halide pcrmeability is currently unclear, although it is tempting to speculate that they disrupt CFTR delivery to the cell surface via the recycling and/or biosynthetic pathways. Microtubule disruption dramatically inhibits trans-Golgi network (TGN)to-apical membrane traffic in epithelial cells (Matter et al., 1090b). Thus, if these inhibitors disrupt CFTR delivery to the cell surface from the TGN and/or recycling endosomes (see Section VII), they would be expected to downregulate the numbers of CFTR C1- channels at the cell surface.

B. Actin The actin cytoskeleton regulates CFTR C1- channel activity in hcterologous expression systems, although the mechanistic basis for this regulation is somewhat controversial. Fischer et al. (1995) have reported that cytochalasin D, a disrupter of microfilaments, rapidly (

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