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NAME OF AUTHOR _ mandish ©. Khatter.........--- ao) diel ae SIrLe OF THESIS ..Aifacts of. preaanalionic, stimilation and : : Hepad chro. tavtan NO... AOA chal ineraic mec. Wiliams. in & sympathetic sanglion... DEGREE POR WHICK THESTS was Peesenrey .. PRU...... WEAR THIS DecRES GRANTED .. SHNIAG. J97A..........-. Permiasicn ie hereby granted tc THE UNIVERSITY OF

ALBERTA LIBRARY to tepreduce single copies of

thesis and to lend or sell such copies for pr-vate,

= scholarly or weientific research purposes only. —,, ; j The author teaerves other publication rightea, and

netther the thesis nor extensive extracts from it may

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THE UNIVERSITY OF ALBERTA

EFFECTS OF PREGANGLIONIC STIMULATION AND HEMICHOLINIUM NO. 3 ON CHOLINERGIC MECHANISMS IN A SYMPATHETIC GANGLION

BY

(C) JAGDISH C. KHATTER

A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES AND RESEARCH IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

DEPARTMENT OF PHARMACOLOGY EDMONTON, ALBERTA SPRING, 1974

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THE UNIVERSITY OF ALBERTA FACULTY OF GRADUATE STUDIES AND RESEARCH

The undersigned certify that they have read, and recommend to the Faculty of Graduate Studies and Research, for acceptance, a thesis entitled Effects of Preganglionic Stimulation and Hemicholinium No. 3 on Cholinergic Mechanisms in a Sympathetic Ganglion, submitted by Jagdish C. Khatter in partial fulfilment of the requirements for the degree of Doctor of Philosophy.

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siaemagl ets stimulation of the cat ot 60/sec for 2 to 8 min reduced the oanelionte acetylcho) ine(ACh} content Dy about 20%. With continued stimulation,

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covered within 15 win. However, when oanalie were allowed to rest follow ing 4 min of stimulation at 60/sec not only was there a rapid restoratio of ACh Content, bUt the ACh levels rose to 130% of contro) after 10 mis

of rest. Under either of these experimental conditions the choline content “Increased transiently onty after the ACh stores had returned ‘te centre] values . The results sre consistent with tne concept that abou e-ti

Gr more Of the total ACh stores of » rested ganglion is in a form. ti : be readily mobilized for release. The observed rebound tncréase tn the ACh content probably means To my mite Hae cua ge capacity is ne mally Setureble and ghar Santa ars Kapil] and Manisht. a maintained within certain limits by 4 precise control of ACh sy thes Remicholinium.no.3 (HC-3) prevented not only the recovery of the ACh stores. but also the subsequent increase in both ACh and chol{ne levels induced

by 4 min of stimulation pivs 10 min of rest. These latter results ere in

accord with the proposed mechaniam of action of HC-3. sou vm Afvestigation with HC-3 (img/Kg) has revealed that the mictit- atina renbrane respons e to ers nerve stimletion may be an unre-

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ABSTRACT

Preaqanglionic stimulation of the cat superior cervical ganglia at 60/sec for 2 to 8 min reduced the ganqlionic acetylcholine(ACh) content by about 30%. With continued stimulation, the ACh stores gradually re- covered within 15 min. However, when ganglia were allowed to rest follow- ing 4 min of stimulation at 60/sec not only was there a rapid restoration of ACh content, but the ACh levels rose to 130% of control after 10 min of rest. Under either of these experimental conditions the choline content increased transiently only after the ACh stores had returned to control values. The results are consistent with the concept that about one-third or more of the total ACh stores of a rested ganglion is in a form that can be readily mobilized for release. The observed rebound increase in the ACh content probably means that the ACh storage capacity is not normally saturable and that under most physiological conditions the ACh levels are maintained within certain limits by a precise control of ACh synthesis. Hemicholinium no.3 (HC-3) prevented not only the recovery of the ACh stores, but also the subsequent increase in both ACh and choline levels induced by 4 min of stimulation plus 10 min of rest. These latter results are in accord with the proposed mechanism of action of HC-3.

Investigation with HC-3 (1mg/Kg) has revealed that the nictit- ating membrane response to cholinergic nerve stimulation may be an unre- liable index of the ability of HC-3 to inhibit ACh synthesis. The time- course effects of different doses of HC-3, in ganglia stimulated at 20/sec, indicate that 2 mg/Ka dose of HC-3 may be an optimal dose and appears cap- able of immediately and completely blocking ACh synthesis. When ganglia

are pretreated with this dose of HC-3 and stimulated at 20/sec, a 50% re-

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duction in the content of ACh occurs within 5 min. The data suggest

that about 50% of the total ACh stores of a ganglion can be rapidly mo- bilized for release and the rest is only slowly converted to a releasable form.

That HC-3 can deplete ACh stores without reducing the choline content of ganglia, was also observed during the above investigations.

The data indicate that HC-3 may not only block the uptake of extracellu- lar choline but may also impair the intracellular utilization of choline for ACh synthesis.

When the above conditions of stimulation and HC-3 were applied to investigate ultrastructural changes, variable results were obtained. While in some cases the reduction in number of agranular vesicles appeared to correspond to the depletion in ACh content, in other cases no such relationship was evident. Also there was no increase in number of agran- ular vesicles corresponding to the rebound increase in ACh content. Under most conditions siqnificant reduction in the density of agranular vesicles occurred mainly in the areas away from the synaptic region. These results do not necessarily negate the vesicle hypothesis and can be explained if One assumes that agranular vesicles stores variable amounts of ACh. The data also suggest that the vesicles from the non-synaptic zone may be mobil- ized to replace those depleted in the synaptic region during stimulation.

The number of large dark-core vesicles, of unknown content, also appeared to decline under the conditions which reduced the number of agranular vesicles. These observations indicate that the less numer- ous dark-core vesicles may also deplete their content during preganglionic stimulation. However, their content and the vhysioloaical significance of

this observation is not known.

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ACKNOWLEDGEMENTS

To my supervisor, Dr. A.J.D. Friesen, I would like to express my sincere gratitude for his guidance, constructive criticism and en- couragement which enabled me to complete this work. My thanks are also due to Miss Pat Heyland for her valuable technical assistance. In addition, I wish to express my appreciation to Mr. G. Duchon and Mr. F. Loeffler for their assistance.

To my friends and relatives I am indebted for their encour- agement and support throughout the undertaking of this project.

I also wish to acknowledge with thanks the financial support

from the Medical Research Council of Canada.

vii

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TABLE OF CONTENTS

HISTORICAL DEVELOPMENT------------------------------------------- Ree rvidencesot chemical 1ransmissioi=-----~----------------~ B. Chemical Transmission in Sympathetic Ganqlia------------ C. ACh Turnover in Sympathetic Gang]lia--------------------- POUL CE OT INO) | ieee ama a ee E. Mode of Action of Hemicholinium No. 3------------------- F. Synaptic Vesicles and ACh Storage in Cholinergic

Nerve Endings-------------------------------------------

RATIONALE FOR PRESENT RESEARCH ---------------------------------- A. Acetylcholine Content and the Storage Capacity of Superior Cervical Sympathetic Ganglia of Cats----------- B. The Time Course Effects of Hemicholinium No. 3 on the Estimation of the Size of Readily Releasable Pool of Acetylcholine in Sympathetic Ganglia of Cat---------- C. Ultrastructure of Presynaptic Endings in Relation

to the Ganqlionic Stores of Acetylcholine---------------

MATERIALS AND METHODS ------------------------------------------- A. Druas and Other Solutions------------------------------- B. Surgical Procedures------------------------------------- C.-- Stimulation Parameters----------+--<--.-2-2026--.-2 650-0 D. Extraction Procedure-----------------.----6-45..--<-.--- E. Bioassay Procedure-------------------------------------- F. Measurements of Choline in Plasma-----------------------

G. Procedures Involving Ultrastructure Studies-------------

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RESULTS------------------------------------------------------------ 38 A. Acetylcholine and Choline Content of Control Resting Ganglia--------------------------------------------------- 38 B. Effects of Pregqanglionic Stimulation on Acetylcholine and Choline Content of Ganalia---------------------------- 38 C. Rate of Recovery of ACh Content During a Resting Period after Preganglionic Stimulation-------------------- 43 D. Effects of Hemicholinium No. 3 on Acetylcholine and Choline Levels in Ganalia Stimulated at Different Frequencies----------------------------------------------- 45 E. Effects of Preganglionic Stimulation and HC-3 on the Ultrastructure of Ganglia and the Relationship of These Effects to the Acetylcholine Content---------------- 62 1. Electron micrographs of preganglionic nerve terminals of resting (unstimulated control ganalia)-- 64 2. Effects of preganglionic stimulation at 60/sec on the number of agranular vesicles in relation to the ACh content of ganglia------------------------------- 68 3. Effects of hemicholinium No. 3 (2 mg/Kg) and pre- ganglionic stimulation at a frequency of 20/sec on * the number of agranular vesicles--------------------- 74 F. Ultrastructural Changes in Dark-core Vesicles in Relation to the Alterations in Ganglionic Content of ACh----------- 84 1. Dark-core vesicles in nerve terminals of resting (unstimulated) ganglia------------------------------- 84 2. Frequency distribution of the number of dark-core vesicles per u* different nerve endinas-------------- 86

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3. Effects of preaanglionic stimulation at 60/sec on the density of dark-core vesicles in the preqana- lionic nerve terminals------------------------------

4. Influence of preganglionic stimulation at 20/sec, in absence or presence of Hemicholinium No. 3 on the

population of dark-core vesicles--------------------

DISCUSSION-------------------------------------------------------- A. ACh Turnover During Activity in Superior Cervical Ganaql ion------------------------------------------------- B. Readily Releasable Fraction of ACh in Superior Cervical Sympathetic Ganalia of Be ae a eer eee aren a eee C. Choline Metabolism--------------------------------------- D. Mode of Action of Hemicholinium No. 3 to Prevent Synthesis of Acetylcholine in Sympathetic Ganglia of the Cat-------------------------------------------------- E. Aqranular Vesicles in Relation to the Storage and Release of ACh in the Preganalionic Nerve Terminals-------------- F. Influence of Experimental Conditions, Which Alter Gang- lionic ACh stores on the Density of Dark-Core Vesicles in the Preganal ionic Nerve ,lerminalis ------------~--------- SLE Fi a a a a gees gucci ene aoa aaRain is ee REFERENCES =-<=6 sane ea ae nse aan ae AS ese aa Senn Renee eee eee nee

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LIST OF TABLES

Description _Page

Effects of preganglionic stimulation at 20 and 60/sec,

for 2 and 8 min, on the aanglionic ACh content------------- 4] Comparison of the effects of stimulation at 60/sec on

ganalionic ACh content with effects seen in ganglia which

have been stimulated at 20/sec prior to switching to the frequency of 60/sec----------------------------------------- 42 The effects of stimulation at different frequencies for 4

min followed by 10 min of rest on the ganalionic ACh content----------------------------------------------------- 47 Effects of HC-3 on the ACh and choline content of ganglia stimulated at a frequency of 20/sec------------------------- 55 Effect of HC-3 and preganalionic stimulation at a frequency

of 20/sec on the ACh content-------------------------------- 59 The Ach and choline content of resting ganqlia exposed to

HC-3 for 35 min--------------------------------------------- 60 Effect of HC-3 and preganglionic stimulation at a frequency

of 20/sec on the choline content---------------------------- 61

Effect of HC-3 on the concentration of choline in cat

Effects of preganglionic stimulation at 60/sec on the aaran-

ular vesicles of total, synaptic and non-synaptic regions

of nerve endings-------------------------------------------- 7\

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Table Description Page

X. Effect of HC-3 (2 mq/Kq) and preaanglionic stimulation at 20/sec on the agranular vesicles of total, synaptic, and non-synaptic reaions of nerve endinas------------------ 82 XI. Effects of preganglionic stimulation and HC-3 on the area of nerve endings-------------------------------------- 85 XII. Relationship between the number of dark-core and aaranular vesicles in the same nerve endings-------------------------- 88 XIII. Number and distribution of dark-core vesicles in pregang- lionic nerve endings during resting conditions and after stimulation at AN/sec-------------------------------------- 90 XIV. Effect of preganglionic stimulation at 60/sec on the density of agranular and dark-core vesicles in the pre- ganglionic nerve endings of superior cervical sympathetic ganglia of the cat------------------------------------------ Fe) XV. The effects of preganglionic stimulation at 20/sec and HC-3 (2 mg/Kg) on the number and distribution of dark-core vesicles in preganglionic nerve endinas of sympathetic ganglia----------------------------------------------------- 94 XVI. Effects of preaganalionic stimulation at 20/sec and HC-3 on

the density of aqranular and dark-core vesicles in the pre-

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10.

LIST OF FIGURES Page

The effects of continuous preaanglionic stimulation at

60/sec on the ACh and choline content of cat superior

cervical ganglia------------------------------------------- 39 Rate of recovery of ACh content from 4 min of stimulation

at 60/sec during a subsequent rest veriod------------------ 44 Effects of HC-3 (1 mg/Kg) on the ACh and choline content

of cat SCG stimulated at 60/sec for 4 min followed by

Effects of HC-3 (1 ma/Ka) on the ACh and choline levels

of aanaglia stimulated for 30 min at different frequencies------------------------------------------------ 49 Effects of preganglionic stimulation at different fre-

quencies and HC-3 (1 mq/Ka) on the isometric contractile response of the nictitating membrane----------------------- 90 Time-course effects of HC-3 (1 ma/Ka) in ganglia stimul-

ated at 20/sec on ACh and choline content------------------ oI Time-course effects of HC-3 (1 mq/Ka) in ganalia stimul-

ated’ at 2 and 20/sec on acetylcholine and choline content-- 53 Time-course effects of HC-3 (2 mg/Ka) in ganglia stimul-

ated at 20/sec on ACh and choline content------------------ 96 The electronmicrograph represents a preaanalionic nerve

ending of a restina control ganalion----------------------- 65 The electronmicroqraoh represents a preganglionic nerve

ending of a resting control ganalion----------------------- 66

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Figure Page

11. Frequency distribution of the number of aqranular ves-

icles per u* in different nerve endinas------------------- 67 12. The electronmicrograph shows the ultrastructural chanae

in agranular vesicles in preganglionic nerve terminal,

when stimulated for 4 min at 60/sec----------------------- 69 13. The effects of 4 min of preqanalionic stimulation at

60/sec on the ultrastructure of presynaptic mitochondria

are depicted---------------------------------------------- 70 14. Effects of preganglionic stimulation at 60/sec on the

number of aqranular vesicles and preganglionic acetyl-

choline content------------------------------------------- 72 15. Influence of preganalionic stimulation for 4 min at 60/sec

on the density of agranular vesicles in different re-

gions of nerve endinas------~------------------------------ 19 16. Effects of HC-3 (2 mq/Ka) and preganglionic stimulation

at 20/sec, on the population of aqranular vesicles in

the nerve endings, and on ganglionic ACh content---------- 76 17. Influence of HC-3 (2 maq/Ka) and preganglionic stimulation

at 20/sec on synaptic and non-synaptic vesicles----------- 78 18. Effects of HC-3 (2 ma/Ka) and 30 min stimulation at 20/sec

on the density of aagranular vesicles in preganglionic

nerve endingS--------------------------------------------- 79

19a. The electron micrograph represents ultrastructural changes in agranular vesicles in preganalionic nerve terminal,

pretreated with HC-3 and stimulated at 20/sec for 30 min-- 80

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the ultrastructure of presynaptic mitochondria------------ 8] 20. Influence of hemicholinium no. 3 and 30 min preqanglionic

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vesicles in preqanalionic nerve endinas------------------- 83 21. Frequency distribution of dark-core vesicles per uy? in

different nerve endinas----------------------------------- 87 22. Influence of preaanalionic stimulation at 60/sec on the

number of dark-core vesicles in different regions of

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HISTORICAL DEVELOPMENT A. Evidence of Chemical Transmission

It is now almost a century since pu Bois-Reymond (1877) suggested that the motor nerves secreted a substance which activated the muscle. Lewandowsky (1898) and Langley (1901) noted independently the similarity between the effects of injection of extracts of adrenal glands and stimulation of synpathetic nerves. A few years later, in 1904, T.R. Elliot, while a student of Physiology at Cambridge, England, extended these observations and postulated that sympathetic nerve impulses release minute amounts of an epinephrine-like substance on immediate contact with effector cells.

Several years later, Dixon (1907) noticed the correspondence between the effect of muscarine and the response to vagal stimulation and in 1909 Dixon and Hamil advanced the hypothesis that the vagus nerve 1ib- erated a muscarine-like substance that acted as a chemical transmitter of its impulses. They suggested that excitation of nerve induces the local liberation of a hormone which causes specific activity by combina- tion with some constituent of the end oragan, muscle or gland. Dixon's theory met with universal skepticism which discouraged him from following this promising field of investigation.

In 1914 Dale made a thorough study of the pharmacological pro- perties of acetylcholine (ACh) and some other choline esters. He observed that these drugs produced effects that were analogous to those observed after electrical stimulation of certain peripheral nerves. He was so impressed with the remarkable fidelity with which these druas reproduced

the responses to stimulation of parasympathetic nerves that he introduced

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the term parasympathomimetic to characterize their effects.

Loewi (1921) established the first real proof for chemical mediation of nerve impulses. In his ingenious experiment he stimulated the vaqus nerve of a perfused (donor) frog heart and allowed the perfusion fluid to perfuse a second (recipient) frog heart used as a test object. The contraction of the second heart was inhibited by this fluid. With this experiment he demonstrated that a substance was liberated upon stim- ulation of the:vaqus trunk of the donor heart; this substance in turn in- hibited the recipient heart producing an effect analogous to that of vagal stimulation. He called this substance “vagustoff". Loewi and coworkers subsequently reported that vagustoff had many properties in common with ACh (Loewi, 1932, 1933).

Many other investigators established quite conclusively that a chemical mediator is instrumental in the activity of all peripheral efferent nerves. The concept of chemical transmission of nerve impulses has been universally accepted at most synaptic junctions.

The evidence for chemical transmission of nerve impulses in sym-

pathetic ganglia is discussed in the following section of the thesis.

B. Chemical Transmission in Sympathetic Ganglia

The evidence of chemical transmission in autonomic ganglia has been obtained chiefly from experiments on perfused ganglia. Kibjakow (1933) described a method for perfusing the superior cervical ganglion (SCG) of the cat with Locke's solution. The perfusate collected during electrical stimulation of the preganglionic nerve fibres was reinjected into the arterial supply of the SCG. A contraction of the nictitating

membrane was observed similar to that caused by nerve stimulation. How-

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ever, when the perfusate was collected in the absence of nerve stimulation no such contraction of the membrane was seen.

Feldberg and Gaddum (1934) repeated Kibjakow's experiment and found that the active substance could only be recovered in the perfusate if an anticholinesterase was present in theLocke's solution. They iden- tified the active substance in the perfusate on the basis of its behavior or activity when subjected to five different pharmacological and biolo- gical tests. These observations supported the theory that the mechanism by which each nerve impulse normally passes the synapse consists in the liberation of small quantities of ACh. Feldberg and Vartianen (1935) ob- served that ACh appears in perfusion fluid after preganglionic but not antidromic stimulation. They also reported that physostigmine potentiates the effect of submaximal electrical stimulation as well as the ganglionic stimulant action of injected ACh. These findings further supported the chemical transmission theory in SCG.

Nicotine (Feldberg and Vartianen, 1935) and curare (Brown and Feldberg, 1936a) were shown to block transmission in the perfused SCG without impairing the release of ACh-like substance. Both of these block- ing agents also prevented the effects of injected ACh on the ganglion.

The results of these experiments added support to the contention that transmitter’released on nerve stimulation in SCG is a choline ester.

The presence of enzyme for synthesizing ACh in SCG of cat was suggested by Brown and Feldberg in 1936b. They observed that the total amount of ACh liberated in stimulated ganglia perfused with choline-Locke's solution was several times of that obtainable from the contralateral un- stimulated ganglion by extraction.

Bannister and Scrase (1950) used a relatively crude enzyme pre-

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