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•THE ROLE AND REGULATION OF GAP JUNCTIONS

IN UTERINE S~09TH MUSCLE

BY

© WILLIAM CRAWFORD COLE

AThesis'

Submitted to the School of Graduate Studies

in Partial Fulfillment of the Requirements

for the Degree

Doctor of Philosophy

McMaster University

August, 1985

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GAP JUNCTIONS IN

UTERINE SMOOTH 11USCLE"

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DOCTOR OF PHILOSOPHY (1985)

(Medical Sciences)

McMASTER UNIVERSITY

Hamilton, Ontario

TITLE: The Role and ~gulation 'ofGa~ Junctions in Uterine

Smooth Muscl e

AUTHOR: William Crawfo~d Cole, 8Sc. (University of Toronto)

.~Sc_. (University of Toronto)

SUPERyISOR: Professor Robert E. Garfield

NUM8ER OF PAGES: xvi, 224

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ABSTRACT I . •

Regular, coordinated contractions of uterine smooth muscle are

thought to facilitate the delivery of the fetus(es) at parturition.

The development of synchronous activity at term follows the

development of many, large gap junctions between uteri~e smooth muscle

cells. The objectives of this thesis were to test the hypothesis that

the formation of gap· junctions improves direct intercellular

communication between the muscle fibers and to determine whether the

functional properties of the junctions are regulated'! I.ncreased

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intercellular communication

synchronous,~ctivity during

in uterine tissues may facilitate"r' .

labor.and the regulation of such cell-cell

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interactions by modulating the function of the gap junctions may

participate in th~ control of uterine contractility dU~ing pregnancy

an~ partur-ition.

A technique was developed to study the diffusion of a small

radiolabelled glucose analog, 2-deoxyglucose, through small strips of

myometrium; Tissues with many gap junctions from rats in labor

demonstrated a significantly greater redistribution of tracer compared

to muscle removed from days 17-20 pregnant and days 2-3 post partum

animals not in lab.or which had few junctions. This movement of. tracer,- .

wa~ shown to be the result of intracell~lar and direct, cell-to-cell

diffusion. Thus, there is evidence for improved intercellular

communication in the myometrium during parturition when gap junctions

are present.iii

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The extent of intercellular communication in the parturient

myometrium.was reduced by elevating the i~tracellular concentrations

of ~a++ and'cyclic AMP ,in the absence b~ a change in the extent of gap

junctions. More significantly, howeve~, reduced communicption was. 0 .JI,

shown in tissues exposed to specific agents which are thought ~o ,play

a cole in the Tegulation of pregnancy and parturition. Thui, in

addition to 'pr.oviding for the incre'ase in gap junctions in the

myometrium at term, the hormonal a~terations which' precede and

accompany labor may also regulate the functional properties of these

cell-to-cell membrane channels.

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Acknowledgementsr

i wouldlik~ to express my gratitude to my supervisor, Dr. Bob

Garfield. ~or his support and encouragement during the course of this

study. Many opportun it ies were provided for my grqwth and for these I

am deeply grateful.

I cannot adequately return the friendship which Dr. M.S. Kannan

has extended ·to me ~his advice, criticsm and e~couragement wil." long'

be remembered as will hi's .support during myoral defense.

I am indebted to my 'supervisory .committee consisting of Dr. Denis~

Crankshaw, Dr. Jac.k Diamond, and ·Dr. Jack Kirkaldy for their interest

in me and this project. Their mpny helpful suggestions and crjticisms

were very appre"ci ated.

Many other individuals at McMaster University made my sojourn an- .

enjoyable experience. The stimulating companionship and advice of Dr.

P.K •. ~angachari was a highlight. I want to thank Jean Crankshaw for,t~. many cups of' tea that I stole. Colin Ike~on gave his time and

techn'ical expertise without reservation and I sh'all always be indebted",

to him for the manufacture of the apparatus used in this study. Thanks

also to Debbie Merrett, Elizabeth Welsh, and Peter Bowker who helpe~

out"with 'some qf the drUdgery in this project.

I am very thankful for the Research Traineeship award and.continued support granted ·to me by the Can~di~n Heart Foundation.

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The ever present support and love given to me by my parents made

this work worthwhile.

Mr. & Mrs. T.T. Gole provided the distractions necessary to put

lifQ into context and I thank them for this an~ for their patience

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when I wasn't around and when ~ was to busy to run.

Finally, my wife, Catherine, for her contributions to my studies,

thesis; and life ~ without the balloons I couldn't have made it.

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. .TABLE OF CONlENTS

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CHAPTER 1.0: I~TROOUCTION------:

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1.1.GENERAL INTRODUCTION AND OBJECTIVES ..•.............. 2

1.2 GAP JUNCTION STRUCTURE •••••••••••.•..•..••••.••• , •••• 4

~ 1.2.1 Thin Section Electron Microscopy of GJs 5

1.2.2 Freeze-fracture Microscopy of GJs ..•.•..•• : .•.•.. 5

1.2.3 Diffraction Analysis of GJs •.••..•..••..•..•••..• 6

1.3 DIRECT INTERCELLULAR COMMUNICATION 10, .' .1.3.1 Evidence for C.~ll-to-Cell Coupling , 12

1.3.2 Ch.aracteri st i cs of the Ce ll-to-Ce 11 Pathway :. 14

1.3.3 !ll1gulation of Cell-to-Cell Coupling 16

1.3'.4 Roles of Cell-to-Cell.coupli.n~ , 20,

1.4 UTERINE SMOOTH MUSCLE .••••.••.•••.••.••.••••..•••.••• 21

1.4.1 Structure of the Uterus a~d Myometrium••••••••••• 21

1.4.2 Alter'atjons in Myometrial GJs at Term....... ::... 22

1.4.3 Control ~f Myometrial GJs ••••••••••••••.••••••••• 25

1.4.4 El~ctrical Properties of Uterine ,Smooth.Muscle ••• 30

" 1.5 MECHANIS'M OF PARTURITION 34

__1.6 STRATEGY OF THE PRESENT STUDY •• : •.••..••.•••••••.•••• 39

CHAPTER 2.0:

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MET~S AND HAT~~IALS\

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2.1 TISSUES AN~ PREPARATION OF MUSCLE STRIPS FOR..... ,

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THE DIFFUSION EXPERIMENTS •••••••••••••• ~ •••••••.••••• 43

2.2 DIFFUSION APPARATUS AND TECHNIQUE •••••••••••••••••••• 46

2.3 ANALYSIS OF DIFFUSION DATA 47"

2.4 UPTAKE AND EFFLUX EXPERIMENTS 50

2.5 ELECTRON MICROSCOPY AND MO~PHDMETRIC ANALySIS •••••••• 51

CHAPTER 3.0: ALTERATIONS IN FUNCTIONAL COUPLING AND GAP

JUNCTIONS IN' UTERINE SMOOTH MUSCLE 55

3.1 INTRODUCTION .••••••••••••••••••• ~••••••••.••••..••••• 56

3.2 METHODS AND MATERIALS : : ~ 57

"3.3 RESULTS •• ~ ••••••••••••••••••• ~ •••••••••••••••.••••••. 58

3.3.1, Diffusion E'xp!lriments •••••••••••• ,•••••••••••••••• 58

3.3.~,DG diffusion in longitudinal myometrium.... 58\

3.3.1.2 Ex~racellular trace~ diffusion

in longitudinal myometrium 65

3.3.1.3 2-DG and sucrose diffusion in hepatic portal

vein longitudinal smooth muscle 66

3.3.2 Struct'ural Analysis 67

3.3~2.1 GJ area and muscle fiber size in

longitudinal myometrium 67

3.3.2.2 GJ area.in hepatic portal vein ••••••••••••• ,. 73

3.3.3 2-DG, Sucrose and Mannitol Uptake,and Efflux..... 77.

3.4 DISCUSSION ..................•...•..................... 82~ . ,

3.4,1 GJs Between Uterine Smooth Muscle Cells .......... 82. .3.4.2 Route of 2-DG Diffusion 84 C

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3.4.3 Role of GJs and Intercellular Communication...in Uterine Smooth Muscle 88

3.4.4 Calculation of a Theoretical Value of Da

for 2-DG 'Diffusion in Longitudina.l Myometrium•... 90

3.5 SUMMARy -; ;.......... 98

CHAPTER 4.0: REGULATION OF MYOMETRIAL GAP JUNCTION

PERMEABILITY BY [Ca++]i AND CALMODULIN 99

4.1 INTRboUCTION. _._ ..•. "•••..•. '\._ •..•.....•...••....•...•... 100

4.2 METHODS AND MATERIALS 101

4.3 RESULTS ••••••••••••·•••• ·.•••••••••.•••.••••••••• '•••••• 102

4.3.1 Diffusion Experiments ·: 102

- 4.3.1.1 Calcium ionophore reduces 2-DG diffusivity ••.•102-,'" . ' -.

·4.3.1.2 Calmotlulin antagonists block the

effects, of ionophora ' 104..A.3.2 Lack of an Effect of Ionophore and

Calmodulin Antagonist on St~ucturar Coupling..... 106

4.3.3 Lack of an Effect of Ionophore and'

Calmodulin Antagonist on 2-DG Efflux ••••••••.•••. 106\ .

DIS~USSION ••••••••••.••• .....................................)18

4.4.1 Mechanism for Altered 2:DG Diffusivity.:·•••••••• 118

4.4.2 Uncoupl.ing Induced by Elevated [C?++]i 121

4.4.3 Roles of Calmodulin in the Control

• i,' <if GJ Permeabil ity 124

4.5 SUMMARy ••••••••••••••••••••••••••••••••••••.••••••••• 132

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CHAPTER 5.0: REGULATION OF MYOMETRIAL GAP JUNCTION

PERMEABILITY BY [cAMP]i 133

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5.1 INTRODUCTION .•••••••••.•••.•• , ••••••.••••..••••.•••.. 134

5.2'METHDDS AND MATERIALS ~ ;: 135

5.3 RESULTS." •• ".""."""" ••••• """."." ••• "" •••. "••• "" •• "."" 135

5.3.1 Diffusion Experiments.: ., ;.~ 135

5.3.1.1 Db-cAMP and a-Bromo cAMP, reduce 2-DG

diffusivity •••••••••••.••••••••. ~ .••....•....• 135.

5.3.1.2 Forskolin and theophyll ine ~reduce

2-DG di"ffusivity : 139

5.3.2 Structural Analysis · · 144I

5.3.2.1 Lack of an effect of elevated [cAMP]i

,on GJ 'area" "." ""." " " ". ".. ". "." 144

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5.3.2.2 Forskolin induces an alterationI ,

~J morphology"" .".""" ".. ".""" .•. "":".,, 144

5.3.3 Lack of an E~t of Elevated [cAMP];. . ~

on 2-0G Efflu1'--" .. ".. "."" .. "" .. ""."."" .. """ .. "... 146

," 5.4 DISCUSSION .•.'."""." .. """."""" .•. "..• ".• "."""."." .• "." 151

5.4.1 Modulation of Functional Coupling by [cAMP]; ••• ~. 151

5.4.2 Possible Assqciation between GJs and

Adenylate Cyclase••••••••.••••••• ~ ••.•••.•.•••••• 154

5.5 SUMMARy."."." •• "•• """" ••• """"" ••• """"."".""""".".""". 157,

CHAPTER 6.0: EVIDENCE ,FOR 'PHYSIOLOGICAL REG.ULATIDN OF

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MYOMETRIAL GAP JUNCTION PERMEABILITY 159

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6.1 INTQCTION : ••• 160

6.2 METHODS AND'MATERIALS -161.6.3 RESULTS ~ .. · ~ ~ 'J ••• 162

6.3.1 Diffusion Experiments ,. 162

6.3.1.1 Agonists which elevate [cAMP]i reduce

2~DG diffus;vity , : _, 162

6.3.1:2 Effect of oxytocin, PG E2• and PG F2a on

2-06 diffusivity 162

6.3.1.3 Effect of indomethacin and ETYA

On 2~DG di ffus ; vi ty. " ~ .~~ " ..,.. ~66

6.3.2 Structural Ari·~lysis 172

6.3.2.1 Lack of any effect on GJ area 172

6.3.2.2 Alterations in GJ morphology •••••••••••••.••• 172

6.4 DISCUSSION ••••••••••••••••••••••••• ~ ••••••••••••••••• l 72

6.4.1 Control of functional Coupling by.'

Physiologically Relevant Agonists •••••••••••••• 172.

6.4.2 Possible Mechanisms for Enhancing....

Myometrial GJ Permeability .. : 176

.6'.4.3 Two D1fferent Mechanism~' Control· Functional

Coupl ing in .the Myometrium : 178

6.5 SUMMARy ••••.••••••••••••• .; ••••••••••.•••••••••••• "•••• 183

CHAiTER 7.0: SUMMARY AND CDNCCUSIONS

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7.1 GENERAL CONCLUSIONS AND FURTHER QUESTIONS •••...•.•••• 185

7.2 SUMMARy •••• , ••••••••••••••••••••••••••••••••••••••••• 188

REFERENCES .•..••..••.••... , ••..••..•.•.•....••.•.•••••..••. 191

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List of Tables

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1. TABLE 3.1:' The apparent. diffusion efficient (Da) for

2-DG in myometrial tissues from rats on various

days of pregnancy .•••....•..••••..•..•.••••••.•....•... 62

2. TABLE 3.2: Gap junctions in the myometrium and hepatic

portal vein in vivo and after the diffusion

experiments in vltro 74

3. TABLE 4.1: The apparen~ diffusion coefficient (Da)

for 2-DG in parturient myometrial tissues with

TE c ++ELEVA D [ a. ]i ••• : ••••••.••••••••••••.•..••••••.•..•. 105

4. TABLE 4.2: Gap junctions in parturient myometrium

after treatment with ionophore (Iono},

chlorpromazine (Chlor.), or Ca++ free solutions,

during the diffusion experiments ••••••••••••••••••••••• 115

5. TABLE 5.1: The apparent diffusion coefficient (Da)

for 2-DG in parturient myometrial tissues with

elevated [CAMP]i •.~•••.••••.••• ; 138

r 6. TABLE 5.2: Gap ~unctions in parturient myometrium

after treatment with dibutyryl cAMP (DBcAMP) or

forskolin (Forsk.) during the diffusion

experi,ments 145

7. TABLE·6.1: The apparent diffusion coeff!cient (Da)

for 2-DG in parturient myometrial tissues treated

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with prostaglandins, oxytocin, isoproterenol,

relaxin, indomethacin and 5,8,11,14

eicosatetraynoic acid (ElVA) .••••.•.....••...•... : •.... 145

8. lABLE 6.2: Gap junctions in parturient myometrium"

after treatment with isoproterenol (Isopr.),

relaxin (Relax.), carbacyclin (Carba.), or

5,8,11,14 eicosatetraynoic acid (ElVA) •••••••••••..•••• 171

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