8214146

Snider, Ray Michael

AMPHETAMINE AND METHYLPHENIDATE EFFECTS ON NEUROMUSCULAR TRANSMISSION IN THE RAT PHRENIC NERVE - DIAPHRAGM PREPARATION: MECHANISTIC STUDIES CORRELATING MUSCLE CONTRACTION, BIOCHEMICAL AND ELECTROPHYSIOLOGICAL RESULTS

The Ohio State University PH.D. 1982

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University Microfilms International AMPHETAMINE AND METHYLPHENIDATE EFFECTS ON NEUROMUSCULAR

TRANSMISSION IN THE RAT PHRENIC NERVE-DIAPHRAGM PREPARATION:

MECHANISTIC STUDIES CORRELATING MUSCLE CONTRACTION,

BIOCHEMICAL AND ELECTROPHYSIOLOGICAL RESULTS

DISSERTATION.

Presented in P a rtia l Fu lfillm en t o f the Requirements for

the Degree Doctor of Philosophy in the Graduate

School o f The Ohio State University

By

Ray Michael Snider; B.A.

*****

The Ohio State University

1982

Reading Committee: Approved By Michael C. Gerald

Dennis R. F e lle r

Norman J. Uretsky Adviser Lane J. Wallace College o f Pharmacy ACKNOWLEDGEMENTS

To Mary Ellen and my parents, Robert and Dorothy, whose -

loving devotion and encouragement has been a source of

strength to me, and to who I owe a great deal.

To Dr. Michael C. Gerald with whom I have shared a productive,

professional relationship and who has been profoundly

instrumental in shaping my career.

To the Faculty o f the Division of Pharmacology.

I dedicate this work to Dr. Lauren K. Gerbrandt; without his

influence on my l i f e I would not be where I am today. VITA

December 28, 1948 ...... Born - Los Angeles, Calif.

1974 ...... B.A. in Psychology, C alifornia State University Northridge, Northridge, California

1974-1975 ...... Technician in the Psychobiology Research Laboratory, V.A. Hospital, Sepulveda, California

1975-1976 ...... Teaching Assistant, Department of Psychology, California State University Northridge, Northridge, C alifornia

1976-1981 ...... Research Assistant, College of Pharmacy, The Ohio State U niversity, Columbus, Ohio

PUBLICATIONS

Gerbrandt, L.K., Weyand, T.G. and Snider, R.M.: Afferent control o f hippocampal theta rhythms. Soc. Neurosci. Abstr. JL_: 548, 1975.

Gerald, M.C., Gupta, T.K. and Snider, R.M.: Tolerance to amphetamine- induced impairment o f rotarod performance in rats . Psychopharmacology 61: 317-318, 1979.

Snider, R.M. and Gerald, M.C.: Electrophysiological analysis of (+)-amphetamine effects on neuromuscular transmission in the rat. Pharmacologist 22: 165, 1980.

Snider, R.M., Meldrum, M.J. and Gerald, M.C.: Effects of methyl- phenidate on neuromuscular transmission and muscle contraction in v it r o . Fed. Proc. 4(h 278, 1981.

Gerald, M.C. and Snider, R.M.: Amphetamine effects on muscle contraction in the incannulated rat phrenic nerve-diaphragm preparation. Fed. Proc. 40: 278, 1981.

i i i Snider, R.M. and Gerald, M.C.: Amphetamine effects on acetylcholine release and mammalian neuromuscular transmission. L ife Sci. 29: 1661-1668, 1981.

Snider, R.M. and Gerald, M.C.: Amphetamine increases acetylcholine release from the phrenic nerve: Evidence fo r a noradrenergic- mediated response. Soc. Neurosci. Abstr. 7_: 795, 1981.

Meldrum, M .J ., Snider, R.M. and Gerald, M.C.: Cholinergic- independent effects of amphetamine on mammalian skeletal muscle contractions. Neuropharmacology, in press.

Snider, R.M. and Gerald, M.C.: Studies on the mechanism of (+)- amphetamine enhancement o f neuromuscular transmission: Muscle contraction, electrophysiological, and biochemical studies. J. Pharmacol. Exp. Ther., submitted fo r publication, 1981.

Gerald, M.C. and Snider, R.M.: -Adrenoceptor enhancement of acetylcholine release from motor nerves and effects on muscle contractions by amphetamine and norepinephrine. Fed. Proc., submitted for publication, 1981.

FIELDS OF STUDY

Major Field: Pharmacology

Neuropharmacology

Neurobiology

Neuromuscular Junction Pharmacology

Professor, Michael C. Gerald TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS...... i i

VITA...... i i i

LIST OF TABLES...... ix

LIST OF FIGURES...... x ii

Chapter

I . INTRODUCTION ...... 1

Overview ...... 1 Physiology o f Neuromuscular Transmission and Muscle Contraction ...... 3 Neuromuscular transmission ...... 3 ACh synthesis ...... 9 ACh storage ...... 10 ACh release ...... 11 Methodological considerations ...... 12 Termination of ACh action ...... 18 Muscle contraction ...... 19 Pharmacology o f Neuromuscular Transmission ...... 23 Presynaptic Effects ...... 24 Hemicholinium and related compounds that inhibit ACh synthesis ...... 24 Agents which in h ib it the release o f ACh . . . 24 Agents which enhance the release o f ACh . . . 25 Postsynaptic Effects ...... 27 Competitive antagonists ...... 27 Noncompetitive or depolarizing blockers . . . 30 Agents which enhance postsynpatic effects o f A C h...... 31 Amphetamine Effects on Physical Performance in Humans ...... 32 Overview ...... 32 Laboratory studies of amphetamine's effects on physical performance in humans ...... 33

v Page

Amphetamine Effects on Physical Performance in Laboratory Animals...... 39 Enhancement o f performance ...... 40 Impairment of performance ...... 41 Amphetamine Effects on Isolated Mammalian Skeletal Muscle ...... 42 Amphetamine Effects on D irectly Stimulated Muscle Contractions ...... 52 Neuropharmacological Mechanism o f Amphetamine Action...... 53 Adrenergic Mechanisms ...... 53 Release o f catecholamines ...... 54 Inhibition of reuptake ' ...... 54 In h ib itio n of monoamine oxidase (MAO). . . . 55 Direct adrenoceptor effects ...... 55 Amphetamine Effects on Cholinergic Systems .... 55 Effects on autonomic ganglia ...... 55 Effects on CNS preparations ...... 57 Adrenergic-Cholinergic Interactions ...... 57 Adrenergic effects on neuromuscular transmission ...... 58 Adrenergic effects on autonomic and central nervous system cholinergic preparations . 62 Mechanisms of NE effects on ACh release. . . 65 Methyl pheni date ...... 67 Neuropharmacology o f methylphenidate .... 67 Methylphenidate, performance and neuro­ muscular transmission ...... 69

STATEMENT OF THE PROBLEM...... 72

Chapter

I I . MATERIALS AND METHODS...... 76

Animals ...... 76 Muscle Contraction Studies ...... 76 General procedure ...... 76 Electrophysiological Studies ...... 84 Biochemical Studies ...... 90 Collection of samples...... 90 Extraction of ACh from Krebs' solution . . . 91 Enzymatic assay for ACh ...... 93 Separation o f [ 32P]-phosphorylcholine from [ 32P ]-A T P ...... 95 Quantitation of ACh ...... 96

vi Page

Drugs and Chemicals ...... 97 Treatment of Data ...... 97

III. RESULTS...... 101

Amphetamine Effects on Nerve- and ACh-Stimulated Muscle Contractions ...... 101 Nerve stimulated contractions in the noncannulated preparation ...... 101 Contractions in the cannulated diaphragm preparation ...... 102 Electrophysiological Studies ...... 113 ACh Release S tu d ie s ...... 121 Amphetamine effects on ACh release ...... 127 Amphetamine-Catecholamine Interaction Experiments . 127 Effects of catecholamine modifying on amphetamine's enhancing e ffe c ts ...... 127 Effects of a-adrenoceptor antagonists on amphetamine's enhancing e ffe c ts ...... 138 Noradrenergic Effects on ACh Release and Nerve Stimulated Contractions ...... 146

Methylphenidate Results ...... 157

Overview ...... 157 Muscle contraction studies ...... 157 Methylpheni date-catecholami ne interaction studies ...... 163 Electrophysiological studies ...... 163 Failure of methylphenidate to protect against irreversible a-bungarotoxin blockade . . . 167 ACh release studies ...... 171

IV. DISCUSSION...... 172

Overview o f Discussion ...... 172 Methodological considerations employed in this investigation ...... 173 Overview of possible sites of amphetamine action at the neuromuscular junction . . . 174 Postsynaptic Effects ...... 174 Amphetamine effects on the postsynaptic ACh re c e p to r ...... 174 Inhibition of acetylcholinesterase (AChE) . . 178 Cholinergic-independent effects of amphetamine on muscle contraction ...... 179

v ii Page

Presynaptic Effects ...... 180 Amphetamine enhancement o f ACh release . . . 180 Amphetamine in h ib itio n of ACh release. . . . 181 Mechanism o f Amphetamine Enhancement o f ACh R elease ...... 182 Amphetamine-catecholamine interactions . . . 183 Location of endogenous NE in this preparation ...... 185 Noradrenergic effects on neuromuscular transmission ...... 186 Comparison o f amphetamine and NE effects on neuromuscular transmission ...... 187 Characterization of the a-adrenoceptor type involved in amphetamine's enhancing e f f e c t s ...... 190 Methylphenidate Effects on Neuromuscular Transmission ...... 192

V.SUMMARY AND CONCLUSIONS...... 198

Amphetamine Effects on Neuromuscular Transmission. 198 Methylphenidate Effects on Neuromuscular Transmission ...... 199

APPENDIX A ...... 201

LIST OF REFERENCES...... 205 LIST OF TABLES

Table Page

1 Agents Which Reduce the Spontaneous and/or the Evoked Release o f ACh from Mammalian Neuromuscular Preparations ...... 26

2 Agents Which Enhance the Spontaneous and/or Evoked Release of ACh from Mammalian Neuromuscular Preparations ...... 28

3 Comparison of Nondepolarizing (d-Tubocurarine) and Depolarizing ( Decamethoniurn) Neuromuscular Blockers ...... 45

4 Schematic Flow Diagram o f the Procedure Used to Assay fo r ACh ...... 94

5 Drugs, Chemicals and Radionuclides Used in the Present Study ...... 98

6 Effects o f (+ )-Amphetamine on Noncannulated Nerve Stimulated Diaphragms ...... 105

7 Control ACh-Induced Muscle Contractions in Two Cannulated Diaphragm Preparations ...... 106

8 Effects o f Amphetamine on Nerve-Stimulated (NS) and ACh-Stimulated (AChS) Contractions in Cannulated Diaphragm Preparations ...... 112

9 E ffect o f (+ )-Amphetamine on the Resting Membrane Potential (RMP) ...... 114

10 Effects of (+)-Amphetamine on Miniature Endplate Potential (MEPP) Frequency and Amplitude ...... 117

11 Effects o f (+ )-Amphetamine on the Nerve Stimulated Endplate Potential ...... 124

ix Page

12 Nerve Stimulated Release o f ACh in 2 Control Preparations...... 125

13 Amphetamine Effects on Nerve Stimulated Release o f ACh from the Phrenic Nerve ...... 130

14 Effects of Animal Pretreatment with Catecholamine- Modifying Agents on Amphetamine-Induced Enhancement of Muscle Twitch ...... 134

15 Effects of Animal Pretreatment with Catecholamine- Modifying Agents on Amphetamine-Induced Enhancement of ACh Release ...... 137

16 Effects of a-Adrenoceptor Antagonists on Amphetamine-Induced Modification of Nerve- Stimulated Twitch ...... 142

17 Effects of a-Adrenoceptor Antagonists on Amphetamine-Induced Enhancement of ACh Release. . . 145

18 Effects of a-Adrenoceptor Agonists and Antagonists on ACh Release ...... 149

19 Effects of Norepinephrine Alone and with Selective Antagonists on Nerve Stimulated Contractions. . . . 152

20 Effects of Selective a-Adrenoceptor Agonists on Nerve Stimulated Contractions ...... 156

21 Effects o f Methylphenidate on Nerve Stimulated Muscle Contractions ...... 160

22 Failure of Animal Pretreatment with Reserpine or a-MT to Modify Methylphenidate-Induced Enhancement o f Nerve Stimulated Muscle Twitch ...... 164

23 Effects on Methylphenidate on the Resting Membrane Potential (RMP) ...... 165

24 Effects of Methylphenidate on Miniature Endplate Potential (MEPP) Frequency and Amplitude ...... 166

25 Effects of Methylphenidate on the Nerve- Stimulated Endplate Potential (EPP) ...... 168

x Table Page

26 Comparison of Amphetamine and Norepinephrine Effects on Mammalian Neuromuscular Transmission. . . 189

27 Comparison of Methylphenidate and Amphetamine Effects on Mammalian Neuromuscular Transmission. . . 194

x i LIST OF FIGURES

Figure Page

1 Diagrairmatic representation o f the neuromuscular ju n c tio n ...... 5

2 Diagrammatic representation o f the anatomical configuration of the neuromuscular junction ...... 7

3 Schematic diagram o f the muscle sarcomere (contained between the Z lines) in relaxed and contracted states showing sliding of the actin filametns (I band) into the channels between the myosin filaments (A Band). Illustration of crossbridge formation and active contraction mechanism b.y which actin (thin) filaments slide along myosin (thick) filaments . . . 20

4 Diagrammatical representation of the organization of myofibrils, sarcoplasmic reticulum and T-tubules within the muscle fiber ...... 21

5 Excitation-contraction coupling in the muscle, showing an action potential that causes release of Ca++ from the sarcoplasmic reticulum and then reuptake o f the Ca++ by an ATP-dependent pump. . . . 22

6 Schematic representation of the ra t phrenic nerve- diaphragm mounted on the Palmer Phrenic Nerve- Diaphragm Electrode ...... 79

7 Schematic representation o f the cannulated diaphragm preparation ...... 82

8 Photograph of the electrophysiological recording chamber ...... 85

9 Photograph of cannulated vascular perfused diaphragm mounted in the chamber used for the biochemical studies ...... 92

x ii Figure Page

10 Effects of (+ )-amphetamine on muscle contractions elicited by nerve stimulation in the noncannulated diaphragm preparation ...... 103

11 Representation experiment illustrating the d iffe re n tia l effects o f (+ )-amphetamine on nerve stimulated (NS) and ACh (A) induced twitch in the cannulated diaphragm preparation ...... 108

12 Time response of (+)-amphetamine effects on muscle contractions elicited by nerve stimulation and by pulsed ACh (0.8-8 nmoles) injections in the cannulated diaphragm preparation ...... 110

13 Amphetamine effects on miniature endplate potential (MEPP): top, frequency; bottom, amplitude ...... 115

14 Amphetamine effects on MEPP amplitude and frequency. . 119

15 Amphetamine effects on the nerve stimulated endplate potential (EPP) ...... 122

16 Amphetamine effects on nerve evoked release o f ACh from the phrenic nerve ...... 128

17 Effects of animal pretreatment with catecholamine- modifying agents on amphetamine (270 yM) enhancement of nerve-stimulated contractions ...... 132

18 Effects o f animal pretreatment with catecholamine- modifying agents on amphetamine (100 yM) enhancement of ACh release ...... 135

19 Effects o f a-adrenoceptor antagonists on amphetamine- induced m odification o f nerve-stimulated contractions. 140

20 Effects of a-adrenoceptor antagonists on amphetamine- induced enhancement o f ACh release ...... 143

21 Effects of a-adrenoceptor agonists and antagonists on ACh re le a s e ...... 147

22- Effects of norepinephrine (NE) alone and in combination with selective adrenergic antagonists on nerve stimulated muscle contractions ...... 150

x i i i Figure Page

23 Effects of selective a-agonists on nerve stimulated contractions ...... 154

24 Effects of methylphenidate on nerve stimulated muscle contractions ...... 158

25 Concentration-dependent effects of methylphenidate on nerve stimulated muscle contraction following 10 min of drug incubation ...... 161

26 Failure of methylphenidate (MP) to inhibit the irreversible binding of a-bungarotoxin (a-BGT). . . . 169

27 Overview of possible sites and mechanisms by which amphetamine might potentiate neuromuscular transmission ...... 175

xiv CHAPTER I

INTRODUCTION

Overview NH0

------Amphetamine (1-phenyl-2-aminopropane, , (o^-CHg-CH ^ -CHg) 1 has V been reported to enhance physical performance in humans and animals.

Moreover, using isolated in vitro nerve-skeletal muscle preparations, a similar enhancement of nerve stimulated muscle contractions is evident, thus suggesting that a peripheral component may contribute to this e ffe c t. In an attempt to c la rify the possible sites and mechanism(s) of amphetamine action in the isolated nerve-muscle preparation, a clear understanding of the sequence of events leading from nerve stimulation to muscle contraction is necessary. Consequently the physiology and pharmacology o f nerve conduction, neuromuscular transmission and muscle contraction will be discussed. In that amphetamine is hypothesized in this study to enhance cholinergic transmission as a major effect mediating enhancement of contractility, special emphasis w ill be placed on an understanding of the mechanism and pharmacological modification of acetylcholine (ACh) release.

High amphetamine concentrations produce muscle weakness in vivo and neuromuscular blockade in v it r o . This biphasic response of amphetamine makes it necessary to understand the nature of

1 neuromuscular blocking agents. Thus, the pharmacology o f neuromuscular

blockers as well as a comparative discussion of amphetamine as a

neuromuscular blocker w ill be presented.

Numerous reports have appeared 1n the lite ra tu re regarding the

effects of amphetamine on physical performance and endurance in both

humans and animals. The well known central stimulatory effects of

amphetamine are commonly viewed as being o f primary importance in the

performance enhancing effe c ts . Nevertheless, a c ritic a l survey of

many in Vivo studies concerning the effects of amphetamine on physical

performance w ill be reviewed to examine the p o s s ib ility o f the

involvement of a peripheral component. A limited number of reports

have appeared on the effects of amphetamine on isolated in vitro and

in situ nerve-skeletal muscle preparations. As the present work is most closely related to these studies, they will be more extensively

reviewed.

The molecular mechanism of amphetamine action w ill be discussed

relative to adrenergic and cholinergic neurotransmission. Special

emphasis w ill be given to presenting the evidence that amphetamine is

capable o f enhancing cholinergic neurotransmission as th is most closely

relates to the working hypothesis in the present work. Evidence is

presented suggesting th at amphetamine's enhancing effects on neuromuscular transmission may be in d ire c t, mediated via endogenous

catecholamines. The neuropharmacological evidence underlying this mechanism w ill be evaluated with respect to adrenergic and cholinergic neurotransmission. Finally, literature examining the effects of methylphenidate on physical performance and neuromuscular transmission w ill be reviewed.

In that methylphenidate and amphetamine both produce behavioral arousal and central nervous system stimulation by a mechanism mediated via catecholamines, a comparison of these two drugs on the neuromuscular system w ill be presented.

Physiology of Neuromuscular Transmission and Muscle Contraction

This section will review the basic physiology underlying the sequence of events that transpire from the initiation of an action potential in a motor neuron to contraction of skeletal muscle. Since a major hypothesis of the present work involves modification of cholinergic neurotransmission, special emphasis will be given to an understanding of the molecular basis of ACh release.

Neurotnuscular tranSmission. The molecular mechanism by which a nerve impulseis carried through a motor neuron (or muscle c e ll) have been elegantly investigated by A. L. Hodgkin, A. F. Huxley and coworkers (see Katz, 1966). B rie fly , the membrane at rest has an internal negative charge (-70 mV) and a large (10-12 fold) excess of sodium ions (Na+) e x tra c e llu la rly . These two factors produce a chemical and potential gradient for Na+which results in Na+ tending to go down this gradient and into the cell. Following any depolarizing event, which results in the membrane becoming less negative in tra c e llu la rly , the Na+ conductance or permeability is increased. + As Na enters the c e ll, i t carries with i t a positive charge which further depolarizes the membrane and produces a further increase in Na+ conductance. This sequence of events is the driving force for the propagation of the action potential. Repolarization occurs less than 1 msec following the peak of the Na+ conductance due to an increase in potassium ion (K+) perm eability. Potassium ions are* under resting conditions, in large excess (40-fold) intracellularly. The repolarization current which is necessary to reestablish the internal negative potential of the cell, is accomplished by K+ efflux. Following repolarization, the resting membrane ionic environment is reestablished by Na+/K+ exchange which is ATP-dependent.

The initiation of muscle contraction (as is necessary for bicycle riding) involves the transduction of the action potential in the motor neuron to an action potential in the muscle fiber. This transduction step occurs by chemical synaptic transmission at the neuromuscular junction. The neuromuscular junction is a specialized area which consists o f unmyelinated endings o f the motor nerves (the motornerve terminals), the synaptic (junctional) cleft, and the motor endplate of the muscle. The neuromuscular junction is illu s tra te d in functional

(Fig. 1) and anatomical (Fig. 2) diagrams.

The chemical nature of this transduction process was elucidated by the work of S ir Henry Dale and his collaborators (Dale et a l . , 1936;

Brown et a l. , 1936), who showed that ACh is released by stimulation of the motor nerve and that this chemical is a potent stimulant of skeletal muscle. Figure 1. Diagrammatic representation of the neuromuscular junction. Acetylcholine (ACh) is synthesized in the nerve terminal from choline and acetyl CoA via the enzyme choline acetyl transferase (CAT). The ACh is stored in the nerve terminal in both vesicles and unbound in cytosol. ACh is released into the junctional (synaptic) cleft in quantal form via a Ca -dependent process. Released ACh diffuses across the synaptic cleft, interacts with ACh receptors (AChR) located postsynaptically in the junctional folds and causes changes in the ionic (Na and K+) permeability in the endplate region. When the resulting depolarization reaches threshold, further permeability changes occur in the muscle fib e r and causes muscle contraction via myofilaments. Termination o f ACh action occurs by hydrolysis of ACh by the enzyme acetyl­ cholinesterase (AChE) located in the junctional folds. Choline formed by ACh hydrolysis is taken up and reused for ACh synthesis.

5 6

Choline

Acetyl CoA

ACh

++ ++ Choline

Acetate ACh

• AChR

Depolarization

Muscle Contraction ^ ------1------^ EPPs and MEPPs Figure 2. Diagrammatic representation of the anatomical configuration of the neuromuscular junction. The nerve terminal is packed with vesicles containing ACh which, when released into the junctional (or synaptic) cl eft,interact with ACh receptors in the junctional folds to produce muscle contractions. Following the action of ACh, it is hydrolyzed by the enzyme AChE, also located in the junctional folds. Adapted from copyrighted material (Bowman and Rand, 1980).

7 *

&

f Mitochondrion A nucleus of the sole plate reaion Myofilaments 9

ACh synthesis. ACh is synthesized in the motor nerve terminal by the soluble enzyme choline acetyl transferase (E C 2.3.1.6) (Nachmansohn and Machado, 1943). ACh synthesis occurs most a c tive ly in the region of motor nerve terminals where the highest concentrations o f choline acetyl transferase are located (Hebb et a l., 1964).

While the precise mechanism by which ACh synthesis is regulated is not known, it is believed that the synthesis and release of ACh are closely linked. During periods of rest, ACh synthesis is slow but increases during nerve stimulation to meet the demand for release

(P o tter, 1970). A ll o f the hypotheses proposed to explain the regulation of ACh synthesis involve the concept of mass action. Simply stated, the rate of ACh synthesis depends on the relative levels of the substrates choline and acetyl-CoA, and products, ACh and CoA, and the equilibrium constant for choline acetyl transferase (Macintosh and

Collier, 1976).

Choi ine + Acetyl CoA ^ACh + CoA

To explain the regulation of ACh synthesis, hypotheses favoring availability of substrates as well as product inhibition have been proposed. The data, however, do not strongly favor the hypothesis that

ACh regulates its own synthesis by product inhibition of choline acetyl transferase. ACh concentrations as high as 0.1 M inhibited this enzyme by only about 50% (Potter et a l., 1968; Glover and Potter, 1971).

Based on several indirect observations, the availability of substrates, specifically choline, appears a likely possibility for serving as the chief regulatory factor of ACh synthesis. This concept 10

is supported by the observation that the amount of ACh synthesized is

linearly related to the amount of choline transported into the nerve

terminal and is capable of meeting the synthesis requirements at

stimulation frequencies up to about 20 Hz (Guyenet et a l . , 1973).

Moreover, the most effective inhibitor of ACh synthesis is

hemicholinium-3, a choline uptake blocker (Potter, 1970; Guyenet et a l.,

1973). The main source of choline fo r ACh synthesis is obtained from

the re-uptake of choline from previously released ACh, following its

hydrolysis. Quantitatively, using [^C]-labeled ACh, Potter (1970)

has demonstrated that in the isolated ra t diaphragm about 50% of the

choline produced by ACh hydrolysis is recaptured and used fo r ACh

synthesis. Moreover, several investigators have reported that this choline uptake mechanism is greatly accelerated during nerve activity and have hypothesized that this mechanism helps to maintain effective chemical neurotransmission when synaptic a c tiv ity is prolonged or of

high frequency (C o llie r and Macintosh, 1969; Potter, 1970; C ollier and Katz, 1974).

ACh storage. The nature of ACh storage varies depending on the type of tissue; for the purpose of the present report, only skeletal muscle will be considered. In the rat diaphragm, the only muscle that has been carefully examined, almost a ll the ACh is located in the motor endplate region (Hebb et a l., 1964; Potter, 1970). Using 14 [ C] choline to label the ACh stores in diaphragm muscle, Potter

(1970) concluded that two pools o f ACh e x is t. About 80% of the ACh located in nerve terminals is releasable and has been termed "depot"

ACh. The remaining 20%, termed "stationary ACh",is labeled much more 11

slowly and is not released even when the nerve is stimulated for

prolonged periods in the presence o f hemicholinium-3. Electro-

physiological observations, based on changes in EPP amplitude (see

bel ow),al so support a two-pool system in that the fractional release

rate falls from an initial value (higher EPP amplitudes) to a lower

steady value (Ginsborg and Jenkinson, 1976). Results from biochemical

and neurophysiological studies both indicate that newly synthesized

neurotransmitter is preferentially released over pre-formed ACh from motor neurons (P o tter, 1970).

ACh release. The mechanism underlying the release of ACh

remains poorly understood. It is clear, however,that ACh is released in

quanta! form (i.e . in multimolecular 'packets') and is calcium

dependent (see Hubbard, 1973; Macintosh and C o llie r, 1976). The

question remains as to whether ACh is released from vesicles or from

the nerve terminal cytoplasm. Following the discovery that ACh was

stored in presynaptic vesicles, i t was concluded that the amount of

ACh within a vesicle (15,000-40,000 molecules) was equal to a quanta.

Vesicles were believed to fuse with the inside of the presynaptic membrane and release their contents into the synaptic cleft (junction c le ft in Fig. 2) (Heuser and Reese, 1973).

The alternative to the "vesicle hypothesis" is that ACh is released in free form from the nerve terminal cytosol (see Macintosh and C o llie r, 1976). Quantal release is then postulated to result from discrete changes in the permeability of the nerve terminal membrane at specific release sites. Thus, "ACh gates" are opened for a specific amount of time allowing the diffusion of ACh quanta into the synaptic cleft. Perhaps the most damning evidence against the vesicle hypothesis, and in favor of the cytosol hypothesis was obtained by Tauc et a l.

(1974). After injecting purified AChE into the cell body of giant cholinergic neurons in Apl.ysia, cholinergic synaptic transmission was abolished. This suggests strongly th a t, ACh is not released d ire c tly from a vesicle into the synaptic cleft but must have been free in the cytosol before release. The authors argue that the injected enzyme would not be expected to reach the vesicle interior. No attempt will be made to present all the evidence, for and against each of these theories. The interested reader should consult the following review articles: Hubbard (1973) who presents evidence mostly in favor of the vesicle hypothesis; Cooper et al . (1978) who discuss the evidence which favors the cytosol proposal; and Macintosh and Collier (1976) who present a most complete and balanced presentation of the relevant

1iterature.

Methodoloqical considerations. ACh release is generally assessed by one of two possible techniques: directly, by the collection and assay of released ACh; or indirectly, by recording electrophysio- logically postsynaptic events with intracellular microelectrodes.

Since both of these methods were used in the present investigation, it is useful to present the reader with a clear understanding of the advantages and limitations of these techniques. The direct assay of released ACh gives clear answers to questions about whether presynaptic factors are involved in a drug effect on release. This technique, however, is not responsive to changes in postsynaptic sensitivity to 13

released ACh. Electrophysiol ogical methods are employed to examine

drug effects on postsynaptic receptor sensitivity to ACh.

After its release from a presynaptic motor neuron, ACh diffuses across the synaptic cleft and, after interacting with postsynaptic

nicotinic ACh receptors, electrical potentials in the endplate region of the muscle fib e r are produced. These e le c tric a l potentials can be recorded by an intracellular microelectrode in the body of the muscle fiber near the endplate region. Two types of endplate potentials have been studied, namely, the endplate potential and miniature endplate potential. Stimulation of motor nerve results in the evoked release of many quanta or 'packets' o f ACh and is termed the 'endplate potential' or EPP (Fatt and Katz, 1951). In addition to the EPP, which is an evoked event, spontaneous activity is also recorded in the endplate region and is termed "miniature endplate potentials' or

MEPPs (F att and Katz, 1952). MEPPs correspond to the spontaneous release of single quanta of ACh from the nerve terminal. The physiological significance of MEPPs is not known but it has been suggested that the continual secretion of ACh may have a trophic influence which serves to regulate the excitability of the muscle fiber (Milidi, 1963).

The EPP and MEPP were firs t described in detail in frog sartorius muscle preparations (Fatt and Katz, 1951; 1952). Subsequently, intracellular analysis of endplate events (EPPs and MEPPs) was reported in mammalian preparations, namely, the ra t diaphragm (L ile y , 1956a;

1956b) and cat tenuissimus preparation (Boyd and Martin, 1956a; 1956b). 14

A summary of the characteristics of the EPP and MEPP provide clear evidence of cholinergic neurotransmission. These endplate electrical events are characterized by an initial depolarization of 1-5 mV in

EPPs and less than 1 mV for MEPPs, a rise time o f less than 1 msec, and an exponential decay back to the resting potential within 3-5 msec. Unlike the action potential, the EPP and MEPP are not propagated,and their amplitude and time course declines as the recording electrode is moved away from the endplate region (Fatt and

Katz, 1951; Boyd and Martin, 1956a). Moreover, these endplate events are attenuated by nicotinic blockers ((+)-tubocurarine), augmented by anticholinesterase drugs (physotigmine), and abolished by denervation, thus clearly demonstrating the presynaptic, cholinergic neurotransmission of these events.

A d is tin c t advantage of employing electrophysiological methods is that information about both pre- and postsynaptic effects can be gained from the same preparation. For example, the amplitudies of the EPP and MEPP are very good indicators of the postsynaptic sensitivity to released ACh (e.g., curare-like agents produce a decrease in amplitude). MEPP frequency analysis provides clear evidence of presynaptic function, an increase or decrease in frequency directly corresponds to presynaptic release. The EPP amplitude can also provide evidence about presynaptic function. An increase in EPP amplitude could result from an increase in either presynaptic ACh release or postsynaptic sensitivity to released neurotransmitter.

However, i f the EPP amplitude is increased while MEPP amplitude is not, the increase must be due to enhanced presynaptic release. A major 15 lim itation of these electrophysiological methods arises when attempting to analyze drugs with curare-like properties (like amphetamine). Because the recording electrode is located postsynaptically, if the drug increases ACh release, but also decreases the postsynaptic receptor sensitivity, the former effect could be masked. Moreover, at higher drug concentrations the ability to record postsynaptic electrical events is totally blocked due to drug-induced attenuation of receptor s e n s itiv ity to released neurotransmitter. However, by employing both electrophysiological and direct assays to monitor ACh release and action, clear results can be obtained concerning drug effects on both pre- and postsynaptic phenomena.

Many factors can affect ACh release and action. Pharmacological agents and the mechanisms by which they modify neuromuscular transmission w ill be covered in the following section. A thorough review of the physiological mechanisms underlying the neurotransmitter release process w ill be f ir s t presented to aid in the understanding o f how drugs act in th is system.

The single most important factor influencing the quantity of ACh release is the extracellular Ca concentration, or more specifically the Ca /Mg ratio. Extensive electrophysiological studies have been conducted examining the effects of varying the Ca++ and Mg++ concentrations on both spontaneous (MEPP) and evoked (EPP) ACh release in mammalian preparations (Hubbard et a l., 1968a; 1968b).

In these experiments, ACh release was assessed by monitoring changes in EPP amplitude and MEPP frequency. ACh release generally was found to vary directly with increasing Ca++ and inversely with Mg++ 16 concentrations in a manner suggesting competition between these two cations for participation in the release process. Specifically, for ++ optimum ACh release,3 Ca ions in teract to form a complex CagX between

- f . i, Ca and a membrane component ' X1. Mg can also form a sim ilar complex but transmitter release does not result. The major finding of these studies is that release varies directly with the third power of the external Ca + + and inversely with the Mg concentrations. + 4* For the biophysical equations and derivations supporting these findings, see

Hubbard et a l . (1968b), Cooke et a l . (1973) or Ginsborg and

Jenkinson (1976).

In general, any agent which, like Mg++, inhibits the action or a v a ila b ility o f Ca++ w ill reduce the release o f ACh from nerve

4"4* terminals. Although the concept of an action involving Ca is a predominant one in many hypotheses of the mechanism o f drug action on

ACh release from motor neurons, d irect evidence of Ca++ involvement is virtually nonexistent. Some investigators have attempted to study ++ 45 Ca movement using iso to p ically labeled Ca at the neuromuscular junction (Evans, 1973). Although this study reported the selective accumulation of calcium at the endplate region, much of the Ca uptake was post- rather than presynaptic. The fact that skeletal muscle appears to have multiple Ca++ pools (both bound and free) is a recurrent, seemingly insurmountable obstacle for those interested in designing experiments to clearly establish the role of Ca++ in neuromuscular transm itter release. 17

The degree of nerve terminal polarization also plays an important role in determining the quantity of ACh release. Nerve terminal depolarization induced e ith e r by e le c tric a l means (del C astillo and

Katz, 1954; L ile y , 1956c; Katz and M iled i, 1967) or by high potassium

(Liley, 1956c) greatly increases the spontaneous release of ACh. This effect is reflected by an increase in MEPP frequency. The conclusion based on these studies is that spontaneous release increases exponentially with nerve terminal depolarization.

In contrast to the effects of nerve terminal depolarization on spontaneous release, evoked release is decreased by depolarization induced by externally applied current (Hubbard and W illis , 1968). The authors suggest that th is reduction results from an attenuation o f the nerve terminal action potential. An alternate explanation is that the readily releasable store of ACh is depleted due to an enhanced MEPP release, thus decreasing the number of ACh quanta available for evoked release. This effect of depolarization (decreased ACh release) ++ can be reduced by increasing the Mg concentration, or by a preceeding hyperpolarization of the nerve terminal. No compelling explanation o f these effects is given but the authors suggest that magnesium and hyperpolarization may be acting at some other stage of the release process (Hubbard and W illis , 1968). I t is possible that the effect of a preceding hyperpolarization is physiological antagonism,in that hyperpolarization is reportedly capable of greatly enhancing evoked release of transm itter (del C as tillo and Katz, 1954;

Hubbard and W illis, 1962). No explanation for the mechanism of the 18

effect of hyperpolarization is given, but it is apparently not linked

to any effect on spontaneous release as hyperpolarization of nerve

terminals does not produce any e ffe c t on MEPP frequency (Hubbard and

W illis, 1962).

Termination of ACh action. Following the postsynaptic effects of

released neurotransmitter, its action is rapidly terminated by the

enzyme acetylcholinesterase (AChE). This cholinesterase is found in

high concentrations associated with the postsynaptic area of the

endplate where its presence has been demonstrated by histochemical methods (Koelle and Friedenwald, 1949). Pseudocholinesterase, an

isoenzyme with similar function to AChE, is present in the serum of mammals and is also capable of terminating the action of ACh (Mendel

and Rudney, 1943).

In summary, the sequence of events during neuromuscular transmission has been established to occur as follows:

1) Action potentials in the motor neuron arrive at the nerve

terminal and resu lt in Ca++ dependent release o f ACh;

2) ACh combines with postsynaptic nicotinic receptors

leading to a selective increase in ionic permeability

which causes depolarization of the endplate region of

the muscle fib e r;

3) The resulting depolarization, when threshold is reached,

produces a propagated action potential in the muscle

fib e r and muscle contraction ensues;

4) ACh action is terminated by hydrolysis. Muscle contraction. The biochemistry of muscle contraction is explained by considering the interactions of several structural

proteins, which are the main constituents of skeletal muscle. These

proteins are termed a c tin , myosin, troponin and tropomyosin. Actin and mysoin, thin and thick filaments, respectively, are arranged in

parallel and overlap each other such that when activated they slide

past each other thus producing muscle shortening (Fig. 3 ). This

"sliding filament" model of muscle contraction is generally accepted as the mechanism of muscle contraction and is described in detail by

Z ie rle r (1974). B rie fly , the process of contraction results from the formation of cross bridges between the globular heads of the thick filaments and the globular portion of the thin filaments. Active shortening results from the rapid making and breaking of successive cross-bridges, each detaching from its site on the thin filament and reattaching to another site further along, and so on, resulting in the thin filaments slid in g along the thick filaments (F ig. 3 ). Calcium and metabolic energy in the form of ATP are required. The sequence of events in excitation-contraction-relaxation are as follows (refer to

Figures 3-5):

1) Action potential in muscle cell;

2) E lectrical disturbance conducted along T-tubule system

to interior of muscle;

. 3) Small amounts of "trigger Ca++" released from tria d ic

junctions;

4) Large amounts of 'a c tiv a to r Ca++I are released from

lateral sacs and sarcoplasmic reticulum; I A I ,— <— ,,------«------i r - " - Z Z

RELAXED

CONTRACTED

Actin Filament MOVEMENT Active sites :

POWER STROKE Jv* HINGES

Myosin Filament

Figure 3. Top: Schematic diagram o f the muscle sarcomere (contained between the Z lin es) in relaxed and contracted states showing sliding of the actin filaments (I band) into Plasme*mwnBrene*

Myofibrils

Longitudinal tubules of 'Sarcoplasmic reticulum

T-lubule

Lateral sacs — of sarcoplasmic reticulum

Figure 4. Diagrammatical representation of the organization of myofibrils, sarcoplasmic reticulum and T-tubules within the muscle fib e r. Adapted from copyrighted m aterial (Bowman and Rand, 1980). 22

T-tubule ACTION POTENTIAL

Sarcolemma .sarcopl asmic CALCIUM PUMP r e ti culum 1 “ T CO •«£

* AT.P A [ required i Ca Ca

Myosin Filaments Actin Filaments

Figure 5. Excitation-contraction coupling in the muscle, showing an action potential that causes release of Ca++ from the sarco­ plasmic reticulum and then reuptake of the Ca++ by an ATP-dependent pump. Adapted from copyrighted material (Guyton, 1976). 23

5) Troponin binds Ca++ and removes in h ibitio n of the ac tin -

myosin complex;

6) Myosin ATPase is activated and ATP is hydrolysed;

7) Actin and myosin form cross-bridges and active contraction

of thin filaments results;

8) Sarcoplasmic reticulum and mitochondria actively (i.e .,

ATP-dependent) sequester Ca++, thus decreasing the

• | " + intracellular [Ca ];

9) Ca -free troponin-tropomyosin complex inhibits formation

of cross-bridges;

10) Actin and myosin filament separate and relaxation occurs.

For more detailed analysis of the events in excitation-contraction coupling, the reader is referred to excellent review articles by

Huxley (1965) and Murray and Weber (1974).

Pharmacology Of Neuromuscular Transmission

Pharmacologically, neuromuscular transmission can be altered presynaptically, either by increasing or decreasing the release of ACh from motor neurons, or postsynaptically by a lterin g the s e n s itiv ity of the motor endplate to released neurotransmitter. This section will deal with classical agents in pharmacology which are known to produce effects by interactions at one or more of these sites. 24

Presynaptic Effects

Hemicholinium and related Compounds that inhibit ACh synthesis.

Hemicholinium-3 is the prototype agent for drugs that decrease the synthesis and release of ACh. The mechanism responsible for this e ffe c t appears to involve in h ib itio n of choline uptake into cholinergic nerve terminals (Macintosh et a l., 1956). Since choline availability is likely to be the main determinant of ACh synthesis (see above section on synthesis of ACh) , the net result is a progressive reduction in the amount of ACh released per quanta. Other agents which have a similar mechanism of action include triethylcholine, sulphocholine and AH 5183 (Bowman and Rand, 1980).

Agents which in h ib it the release of ACh. Botulinum toxin, a classical inhibitor of ACh release (Burgen et a l., 1949), irreversibly blocks ACh release from motor nerve terminals by a mechanism that is not well understood. However, much is known about the phenomenon of botulinum poisoning, and the physiological and anatomical changes that are associated with it (see Bowden and Duchen, 1976). ++ ++ The importance o f the mechanism by which Ca and Mg e ffe c t the spontaneous and evoked release of ACh was presented above. In general, any drug, like Mg++, which acts, to inhibit the participation ++ o f Ca in the release process w ill decrease ACh release. Another mechanism by which evoked ACh release can be reduced or abolished is by an action on the motor nerve it s e lf . Thus, local anesthetics and tetrodotoxin, which act primarily by preventing the action potential via inhibition of Na+ movement in the nerve, can prevent the nerve evoked release of ACh. In contrast to the effects of 25 tetrodotoxin on evoked release, this agent reduces, but does not abolish the spontaneous release o f ACh; MEPP frequency is decreased about 40% (Landau, 1969). Tetrodotoxin does not modify the post­ synaptic action of ACh, either nerve evoked or iontophoretically applied (Elmquist and Feldman, 1965).

Many agents have been found which are capable of reducing the spontaneous and/or the evoked release of ACh from neuromuscular preparations. A summary of many of these agents is presented in

Table 1. For the purpose o f th is introduction, only compounds found to be active in mammalian preparations are listed and no detail of the precise mechanism or conclusions of each study is presented. For references the reader is referred to Macintosh and C o llie r (1976) and Ginsborg and Jenkinson (1976).

Agents which enhance the release Of ACh. The same concepts describing the physiology of ACh release presented above are applicable to understanding the pharmacology of agents which augment ++ neuromuscular transmission. S p e c ific a lly , agents which enhance Ca participation in the release process would be also expected to increase ACh release, as would agents which depolarize (+ spontaneous release) or hyperpolarize ( f evoked release) the nerve terminal membrane. Another physiological factor related to nerve terminal depolarization, which can markedly modify neurotransmitter release, is the duration of the action potential within the nerve terminal.

For example, agents such as tetraethyl ammonium act by inhibiting the efflux Of K+, which is responsible for repolarizing the nerve cell 26

TABLE 1

Agents Which Reduce the Spontaneous and/or the Evoked Release of ACh from Mammalian Neuromuscular Preparations9

A. Agents which decrease spontaneous ACh release (4-MEPP frequency)

Mg Ions

Tetrodotoxin 3-Bungarotoxin Toxins Botulium toxin y-Aminobutyrate D Adenosine and phosphate analogues (AMP, ADP, ATP) a

B. Agents which decrease evoked ACh release (+EPPs)

.. ++ Mg Ions

Botulium toxin 3-Bungarotoxin Tetanus toxin Toxins Black widow spider venom Batrachotoxin and analogues

Streptomycin and neomycin Hemicholinium-3 Metabolic inhibitors (2,4-dinitrophenol) Drugs Adenosine and phosphate analogues (AMP, ADP, ATP) ACh, carbachol

a Adapted from Ginsborg and Jenkinson (1976). 27 following Na+ influx and the action potential. The result is an extension of the duration of the action potential within the nerve terminal, resulting in a lengthening of the duration of active neurotransmitter release (for references see Macintosh andiCollier,

1976).

Many types o f pharmacological agents are capable of enhancing spontaneous and/or evoked release of ACh (Table 2 ). As was outlined above, only compounds found to be active in mammalian preparations are listed. Virtually all of these agents are hypothesized to act by one or more of the mechanisms previously described. For references fo r each agent lis te d see Macintosh and C o llie r (1976) and Ginsborg and Jenkinson (1976).

Of special interest among the agents listed in Table 2 that enhance the release of ACh are the sympathomimetic amines. Because amphetamine is believed to act by releasing endogenous catecholamines

(see below), it is of great interest to determine whether amphetamine acts on neuromuscular transmission by a catecholamine mediated action.

For th is reason, the mechanism o f sympathomimetic action on neuromuscular transmission w ill be presented in detail in a following section.

Postsynaptic Effects

. Competitive antagonists. Classically, the drugs included in this class are (+)-tubocurarine, gallamine and pancuronium, although many other compounds containing a quaternary nitrogen atom also produce the same e ffe c t (Maclagan, 1976). These drugs combine with ACh receptors 28

TABLE 2

Agents Which Enhance the Spontaneous and/or Evoked Release of ACh from Mammalian Neuromuscular Preparations

A. Agents which increase spontaneous ACh release (+MEPP frequency)

Ca++ Ba Cardiac Glycosides Theophylline Dibutyryl cyclic AMP ++ Noradrenaline (norepinphrine) Enhance Ca Adrenaline (epinephrine) induced release Catechol Alcohols (methanol, ethanol, propanol) Methohexital, pentobarbital Chi orpromazine Ether, chloroform

Veratrine and derivatives Depolarize

B. Agents which enhance evoked ACh release (tEPPs)

K+

act1°" I-WnS'ne P°tant1al Guanidine and derivatives

Caffeine, theophylline, aminophylline Di butyryl cycl i c AMP Noradrenaline r . _ ++ Adrenaline f n* a"ca Ca, ‘ Dopamine induced release Catechol Ethanol

a Adapted from Ginsborg and Jenkinson (1976), Macintosh and C o llier (1976) and Bowman and Rand (1980). 29

on the postsynaptic membrane of motor endplates but possess no agonist

activity. As a result, the effects of ACh released from nerve

terminals is inhibited. Because these agents are competitive

antagonists, the blocking action can be overcome by any procedure that

increases the amount o f ACh present a t the neuromuscular junction.

Curare-like blockade is analogous to the competitive in h ib itio n of

enzymes, with ACh being the normal substrate and curare the

competitive inhibitor. The antagonistic relationship is quantitatively

isomorphic with Michael is-Menten kin etic equations (Jenkinson, 1960).

(+)-Tubocurarine has been reported to increase, decrease and have

no effect on ACh release (for references see Hubbard and Wilson, 1973).

It is generally believed that curare and some other competitive

antagonists may have some minor presynaptic effect but this has been

d if f ic u lt to demonstrate and is o f questionable pharmacologic

relevance in the face of a simultaneous massive postsynaptic e ffe c t

(Ginsborg and Jenkinson, 1976; Maclagen, 1976).

As a result of competitive antagonism, the postsynaptic

depolarizing action of ACh is reduced. This is manifested as a

depression in the amplitude of postsynaptic events such as the EPP and

MEPP and the response to iontophoretically applied ACh; by contrast, no

change in resting membrane potential or MEPP frequency are seen (Fatt

and Katz, 1951; 1952; del C astillo and Katz, 1957). I f the

concentration of curare is increased, the amplitudes of postsynaptic events are further reduced until they become undetectable (Katz, 1966).

Amphetamine has been reported to possess curare-1 ike a c tiv ity 30

(Anderson and Ammann, 1963; Peterson et a l . , 1964; Gerald and Hsu,

1975; Skau and Gerald, 1978a, 1978b). These studies will be analyzed

in detail in a subsequent section of this introduction.

Noncompetitive or depolarizing blockers. Drugs of this type

include decamethonium and succinylcholine. Structurally, these agents

are bisquaternary ammonium salts and contain intramolecular moieties

that resemble ACh. The mechanism of action of these agents involves

their ability to activate the postsynaptic nicotinic ACh receptor

but, since they are resistant to inactivation by AChE, they produce

persistent depolarization o f the motor endplate followed by subsequent

blockade o f neuromuscular transmission (Burns and Paton, 1951). The

concept of blockade by persistent depolarization is not entirely

accurate for all muscle types in all species. In many mammalian

preparations the in it ia l depolarization blockade is transient and is

followed by a long lasting blockade in which the muscle endplate and

fibers are repolarized but s till unresponsive to released ACh. This

second phase of neuromuscular block is one o f the least understood

areas in the fie ld o f neuromuscular pharmacology. The blockade of

th is type is a lte rn a te ly viewed as more closely related to

depolarization blockade followed by eith er competitive (c u rare-lik e)

blockade or desensitization. Desensitization (or receptor

inactivation) is defined as a condition in which application of a

depolarizing drug has rendered the receptors of the endplate

refractory to chemical stim ulation. Moreover, the desensitized state may or may not be accompanied by persistent depolarization. This 31 topic is considered in great detail by Elmquist and Thesliff (1962),

Nastuk e t a l . (1966) and Rang (1975).

Agents which enhance postsynaptic effects of ACh. Anticholines­ terases o f the carbamate (physostigmine and neostigmine), organo- phosphate (DFP) and anilinium ion (edrophonium) types potentiate skeletal muscle twitch elicited by indirect, low frequency stimulation both in vivo and in vitro (Koelle and Gilman, 1949). The mechanism of this effect on low frequency nerve stimulation involves a prolongation of the effects of released ACh on normal postsynaptic receptors due to an inhibition of the inactivation of ACh by AChE. As a re s u lt, in the presence o f these drugs, a single in d irect stimulus to the nerve produces a burst o f muscle action potentials and causes the muscle to respond as i f i t were being stimulated a t high frequencies (Brown et a l., 1936). Higher frequencies of stimulation, in the presence of AChE inhibitors, result in an accumulation of ACh a t the endplate region and produce neuromuscular block of the depolarization type (Burns and Paton, 1951).

In summary, neuromuscular transmission is modified by agents that interact presynaptically to enhance or attenuate neurotransmitter release, and postsynaptically by either altering the sensitivity of the receptors to released ACh or by altering the normal time course of ACh action. 32

Amphetamine Effects on Physical Performance in Humans

Overview. The lite ra tu re describing amphetamine's a b ility to produce performance enhancement in humans is inconclusive and controversial. Polarized elements exist within the scientific (and athletic) community who do not agree whether or not these drugs are capable o f enhancing physical performance. The concept is advanced th at amphetamines can enhance physical or endurance performance in humans and is supported by subjective data gathered from professional football players (Mandell, 1979). Mechanisms proposed to mediate these effects include: increased running speed, rage and analgesic effects o f amphetamine (Mandell, 1979). In agreement with Mandell, others also believe that the ab ility of amphetamine to enhance a th le tic performance is firm ly established (Laties and Weiss, 1981) and theysuggest that for athletic purposes, amphetamine is called

"speed" fo r good reason.

The altern ative view is that amphetamine may be capable of enhancing performance, but only by a psychological or "placebo" action

(Williams, 1974). In support of this latter concept, Leitao (1971) confirmed the existence of a placebo effect in studies attempting to quantify drug effects on physical performance. He found that 63% of the subjects s ig n ific a n tly improved th e ir performance in a strength te st (manual dynamometer) a fte r receiving a placebo but being informed that they were given a performance enhancing drug. 33

Laboratory studies o f amphetamine's effects on physical performance 1n humans. The lite ra tu re discussed in this section w ill emphasize only those studies in which drug effects were examined with respect to physical performance or endurance. Any studies which a ttrib u te drug effects solely to central components such as mood, m otivation, learning or judgment,as well as poorly designed studies with inadequate controls or small sample size (N = 2 - 3) w ill not be discussed in detail.

Foltz et a l. (1943a) compared the effects of (jO-amphetamine*

(10-15 mg), methamphetamine (5 mg), caffeine (500 mg) and placebo on trained subjects worked to exhaustion on a bicycle ergometer task, given a 10 min rest, and again worked to exhaustion on the same task.

Drugs were administered intravenously between 30 sec and 30 min before the f ir s t exercise period. Whereas (+ )-amphetamine had no e ffe c t on work output, methamphetamine pretreatment produced a 25% enhancement and caffeine produced intermediate improvement between the other groups. This study lacked statistical analysis and only 2 subjects received amphetamine whereas 4 subjects were included in the other drug groups. The same authors examined the effects of (+)-amphetamine on 23 untrained subjects on a rapidly exhausting task (Foltz et a l.,

Differences in potency of the amphetamine isomers exist in causing central stimulation. While it is not known whether a similar potency effect exists in the periphery, it is necessary to attempt to equate the dosages of the isomers to evaluate the studies in this section. Potencies are: 10 mg (+)-amphetamine a 5 mg'(+)- amphetamine = 2.5 mg methamphetamine. 1943b). In this study, subjects wore a load equal to one-third their

body weight in a knapsack and stepped up and down a 16-inch step every

3 seconds. Exhaustion was said to occur when the subject could no

longer maintain this rate. After a rest period, the subject worked

again to exhaustion for a total of 3 work periods on each experimental

day. (+)-Amphetamine (10 mg) was given orally one hr before the firs t

work period. Although no significant differences were found between

treatment groups, subjects improved so much from one trial to the

next, th at any e ffe c t due to drug treatment may have been masked.

Alles and Feigen (1942) investigated the effects of (+)-

amphetamine (10, 20 and 40 mg o ra lly ) on muscle endurance performance

of six subjects on a Mosso finger ergograph. While a 10 mg dose

maintained performance at pre-drug lev els , an increased work capacity

was noted with 20 and 40 mg. These authors attributed this effect to

a central or motivational component.

Cuthbertson and Knox (1947) confirmed the ability of (+)-

amphetamine (10-15 mg) and methamphetamine (10 mg) to enhance the

endurance performance of fatigued subjects on a bicycle ergometer

task by 25-30%. Another aspect of endurance performance reported by

Cuthbertson and Knox, possibly related to athletic performance, was

the ability of subjects to ignore painful foot blisters after being

given amphetamine. Supportive evidence that amphetamine might have

analgesic effects in humans was also reported from data gathered by

Mandell (1979) from professional football players. Thus, i t is 35 possible that amphetamine could be enhancing the endurance of subjects, in part, by masking the pain associated with normal fatigue.

The most extensive and widely cited studies on the effects of

(+ )-amphetamine on a th le tic performance were conducted by Smith and colleagues (Smith and Beecher, 1959; 1960; Smith e t a l . , 1963). These studies,employing double-blind techniques with adequate placebo controls, examined the effects of amphetamine (14 mg/70 kg, o ra lly

2-3 hrs prior to testing) on the performance of both trained and untrained swimmers, runners and shot-putters. Ninety-three percent

(14 out of 15) o f the subjects in the swimming experiments performed faster with amphetamine than with placebo. A sim ilar degree of enhancement (0.59 - 1.16%) was observed for the trained and untrained groups, but the findings were more often statistically significant in the trained subjects because of smaller standard errors in this group.

Similarly, in running events ranging from 600 yds to 12.7 miles, amphetamine enhanced performance by an average o f 1.5%. Smith and

Beecher reported greater v a ria b ility in the running than in the swimming events, possibly because the events ranged from 600 yd to

12.7 mi and because the events took place outside, where conditions such as wind velocity and temperature could not be controlled between trials. Nevertheless, they reported that 73% (19 of 26 subjects) of the subjects improved their running times after amphetamine.

Thirteen weight-throwers (35-lb) and shot-putters (16-lb shot) improved both maximum and mean distances thrown a fte r amphetamine by approximately 4.4%. This study is particularly relevant to the work 36 conducted by the author (R. M. Snider) in that th is task involves use of specific muscles in an acute expenditure of energy (most like a twitch response in an isolated nerve-muscle preparation), rather than a prolonged endurance e ffo rt. While the level of performance augmentation noted in these studies was re la tiv e ly small by absolute standards, Smith et al. point out that athletes may train for years to achieve an increase o f th is magnitude (1-2%).

In a recent study Chandler and Blair (1980) investigated the effects o f (+)-amphetamine (15 mg/70 kg, 2 hr) in nontrained, former athletes on physiologic components involved in athletics but unrelated to s k ill. The research design involved repeated observations on the same subjects on six consecutive Fridays; three each under placebo and treatment conditions, with amphetamine administered orally in a double blind fashion two hr prior to testing. The test battery and order of testing was as follows: (1) muscular strength (cable tensiometer; tests fo r elbow flexion and knee extension strength); (2) muscular power

(bicycle ergometer); (3) running speed (30 yard sprint); (4) accelera­ tion (10 yard sprint); (5) aerobic power (treadmill test while monitoring oxygen consumption; (6) anaerobic capacity (la c tic acid accumulation after exhaustion on a treadmill). Critical analysis of the results revealed significant (p < 0.01) enhancement in knee extension strength, anaerobic capacity (maximum lactic acid tolerance) and time to exhaustion on a treadmill, analyzed as the mean performance of each individual on the 3 trials after placebo compared with 3 t r ia ls a fte r amphetamine. Acceleration was s lig h tly decreased following amphetamine but the authors attributed this effect 37 to intrasubject v a ria b ility . Chandler and B la ir concluded that the subjects were better able to maintain performance under exhaustive, anaerobic conditions. They supported this conclusion baseid on the ability of amphetamine to increase lactic acid tolerance and time to exhaustion, without producing any change in aerobic capacity. They suggested that the drug may mask the effects o f fatigue by in terferin g with the body's fatigue-alarm system ( i . e . , pain), and could thus be dangerous to the individual. They also concluded that the effects o f amphetamine on pre-exercise and maximum heart rate could have undesirable consequences. Analysis of the results that amphetamine enhanced muscle strength centered on the possibility of a peripheral drug action. S p e c ific a lly , the authors suggested th at amphetamine may interact directly with muscle fibers involved in movement and render muscle groups more capable o f executing power movement. This study is of particular interest to the present work because it supports the hypothesis that amphetamine may have a peripheral site of action in humans that results in enhancement of physical performance.

Moreover, direct evidence was presented that amphetamine is capable of masking pain, supported by the data that subjects could tolerate higher lactic acid levels after amphetamine than after placebo. This la tte r observation supports the concept of an analgesic action of amphetamine contributing to enhanced endurance performance as presented by Mandell (1979) and Cuthbertson and Knox (1947).

Not all studies which have attempted to examine the effects of amphetamine on physical performance have found enhancement. Karpovich

(1959) studied running, swimming and various track events and found 38 no improvement a fte r amphetamine (10 mg, 1 hr or 20 mg, 30 min).

Similarly, Haldi and Wynn (1946) failed to show any effect of amphetamine (5 mg, 90 min) on swimming performance in untrained

subjects. More recently, Borg et al. (1972) studied the effects of amphetamine (10 mg unstated isomer, 30 min) on cycling strength and endurance. These latter authors reported that while no enhancement of initial trials was observed, the final trials on the test day

(2-3 hr after amphetamine ingestion) were significantly increased, leading to the conclusion th at the drug may postpone the onset of fatigue. An alternate possibility, based on the findings of Smith and Beecher (.1959) is that the onset of amphetamine’ s effects (2-3

hr) caused the enhancement of later, but not of the earlier trials.

Others have also reported no improvement in any of the performance variables being tested (Blyth et a l . , 1960; Akai and Steinhaus, 1961;

Golding and Barnard, 1963; Williams and Thompson, 1973). Although these investigators failed to detect any effects, these studies often

used low doses of amphetamine, and untrained in addition to re la tiv e ly

few subjects thereby making statistical analysis difficult. Untrained subjects are problematic to analyze because of large intrasubject variability.

In summary, many of the above studies suggest that amphetamine

is active in enhancing physical performance above baseline levels and extending the time individuals can perform exhausting work. It is

important to note (for the present report) that amphetamine can augment performance in tasks suchas weight throwing and knee 39 extension strength in which i t is less lik e ly th at endurance, fatigue or psychological factors would play a major role. In evaluating the literature, it is of utmost importance to consider the time and dose of drug adminstered, in addition to the level of training of the subjects in order to show significant drug effects. In general, those studies using lower doses (5-10 mg) and shorter times between oral administration and testing (< 1 hr) failed to show drug enhancement on performance.

Amphetamine Effects on Physical Performance in Laboratory Animals

Amphetamine has been studied in laboratory animals with respect to both fa c ilita tio n of physical or endurance performance as well as for decremental effects on performance. Animal studies on the performance enhancing effects of amphetamine provide support fo r the hypothesis that these effects could be mediated by a peripheral component. Animal studies may employ more stringent controls and are less likely to be influenced by psychological (i.e . placebo) effects, than are human studies. Therefore, i f amphetamine enhances performance in animal studies, and control animals receive the appropriate vehicle injection, it supports the possibility that a physiological component

(centrally and/or peripherally mediated) is involved. Performance may be evaluated in rodent species by monitoring running (treadmill) or swimming times to exhaustion following drug administration. Enhancement o f performance. In studies employing swimming

endurance in ra ts , amphetamine (4 mg/kg) produced a sig n ifican t 26%

increase.in swimming time. Lower doses fa ile d to produce sig n ifican t

enhancement and, when a 5 gm load was attached to the animal's t a i l ,

the same dose (4 mg/kg) impaired swimming endurance by 35% (B attig ,

1963). Bhagat and Wheeler (1973) used rats,previously swim-trained

in 21°C water,with a 5% load attached to their tails to prevent

flo a tin g . Amphetamine isomers (dextro, levo and racemic) a t doses

below 5 mg/kg were ineffective in prolonging times to exhaustion. Both

optical isomers, however, s ig n ific a n tly enhanced endurance times a t 10 and 20 mg/kg (46-52% and 117-127% increase, resp ectively). The

racemic compound was less e ffe c tiv e than eith er isomer (63% increase with 20 mg/kg). The authors suggested that a peripheral component could be, in p a rt, responsible for amphetamine's effects on physical

performance. Bhagat and Wheeler (1973) based th is conclusion on the

fa c t that while there was no difference between isomers ((+ ) versus

(-)-amphetamine) on swimming performance, the (+)-isomer is more

potent than the (-)-isom er in enhancing centrally-m ediated locomotor activity (Taylor and Snyder, 1970).

(+)-Amphetamine has also been reported to enhance the endurance performance of rats run to exhaustion on a treadm ill (speed 18.8 m/min,

8° incline) (Gerald, 1978). Low doses of amphetamine (0.31 - 5.0 mg/ kg) increased the endurance time o f untrained rats by 24 to 67% as compared to control animals, while higher doses (7.5 and 10 mg/kg) reduced endurance by 18 and 47%, respectively. In the same report,

(+)-amphetamine was also shown to enhance the endurance o f fatigued 41 rats on a treadm ill (speed 26.8 m/min, 8° in c lin e ). Two groups of rats were run to exhaustion, then injected with saline or (+)- amphetamine (1.25 mg/kg) and returned to the treadm ill and run to exhaustion a second time. The group injected with amphetamine showed a 44% greater endurance than the saline injected group. This author also presents the hypothesis that while central activation may be responsible fo r the enhanced endurance noted, a peripheral component involving drug mediated effects on neuromuscular transmission, metabolism or fatigue biochemistry could not be excluded (Gerald, 1978).

Impairment of performance. In contrast to the fa c ilita to ry effects produced by low doses o f amphetamine, toxic doses can produce a syndrome characterized by muscle weakness and hindlimb paralysis; most animals however, die in convulsions (Ehrich et a l ., 1939).

Although it is not possible to specify the mechanism reponsible for muscle weakness or paralysis in intact animals (central and/or peripheral), the animals injected with very high doses o f amphetamine

(up to 500 mg/kg sc) appear to "fade away" rather than dying in convul sions.

Studies conducted in mice by Htihn and Lasagna (1960) showed that at room temperature (25°C) death due to high doses o f amphetamine

(150-200 mg/kg) is accompanied by convulsions. By contrast, mice given amphetamine (10-100 mg/kg) as well as those injected with higher doses (150-200 mg/kg) at environmental temperatures of only

10°C were described as having "faded away" and exhibited hindleg paralysis and muscle weakness as opposed to death in convulsions. The authors concluded that d iffe rin g mechanisms underlie death at low and 42

high temperatures but offer no specific explanation (Hflhn and Lasagna,

1960). Muscle weakness following toxic doses o f amphetamine has been observed by others (Askew, 1961; Moore, 1963) but again, no explanations

have been presented.

Amphetamine toxicity is greater in animals forced to exercise

than in sedentary animals (Hardinge and Peterson, 1964). Exercised mice exhibit an 80% m ortality at a dose of 8 mg/kg while 100 mg/kg

produces only 60% mortality in isolated sedentary animals. As noted above, two types o f le th a lity syndromes were observed; most animals died in convulsion, but many animals receiving the higher doses of amphetamine seemed to be paralysed. The investigators concluded that increased motor activity, and an associated rise in body temperature as was observed with amphetamine in to xicatio n , may lead to the enhanced lethality of the drug (Hardinge and Peterson, 1964).

In summary, amphetamine has been reported to have a biphasic e ffe c t on physical performance in in tact animals. Low doses fa c ilita te swimming and running to exhaustion in controlled animal studies, while higher doses impair animal performance. Toxic doses of amphetamine have been shown under certain experimental conditions to produce a syndrome characterized by muscle weakness and hindlimb paralysis prior to death.

Amphetamine Effects on Isolated Mammalian Skeletal Muscle

The effects o f amphetamine on mammalian isolated nerve-skeletal muscle preparations will be discussed in detail in this section.

Although emphasis w ill be placed on those studies employing in d irect 43

(as opposed to direct) stimulation of muscle contractions, all effects of amphetamine on these preparations w ill be presented. These studies are of paramount importance in establishing an argument in favor of a peripheral component being involved in the performance enhancing effects o f amphetamine, as no central influences exist in isolated preparations. Thus, discrete questions regarding mechanism of drug action can be posed in these studies.

The effects o f amphetamine and a series of monoamine oxidase

(MAO) inhibitors on the muscle contractions of directly- and indirectly- stimulated rat phrenic nerve-diaphragms were reported by

Anderson and Ammann (1963). All of the MAO inhibitors tested (e.g., tranylcypromine,iproniazid, and harmaline) as well as amphetamine and methamphetamine were observed to produce an in it ia l increase in the height of nerve stimulated contractions. This effect was transient and was followed by neuromuscular blockade within 15 min with amphetamine a t concentrations of 400 and 800 yM (4 and 8 x 10"^ M).

Neuromuscular blockade was reversible a fte r washing the preparation with fresh Krebs' solution. Amphetamine (at an unstated concentration) blocked the contraction produced by ACh (15 yM) on chronically denervated (2 week) diaphragms. The in a b ility of amphetamine to block neuromuscular transmission when applied only to the phrenic nerve eliminated potential local anesthetic activity. Enhancement of contractions elicited by direct stimulation of the diaphragm was also noted. However, the investigators did not block the effects of normal neuromuscular transmission (ACh release) with a curare-like drug, making a clear analysis of the mechanism of the latter effect 44 d ifficu lt. The authors concluded that amphetamine inhibited nerve stimulated muscle contractions by an interaction at the neuromuscular junction. They suggest that the effects are due to a blockade o f the action of ACh and not due to local anesthetic activity, nor to MAO inhibition. The enhancement of directly stimulated contractions was attributed to direct muscle effects while no explanation was offered fo r amphetamine's enhancing effects on nerve stimulated contractions.

Peterson et al. (1964) have also studied the effects of (+)- amphetamine (50-200 yM) on the isolated rat phrenic nerve-diaphragm preparation. In contrast to the work of Anderson and Ammann (1963), no enhancement of muscle contractions (either direct- or indirectly elicited) were noted by this group. This report was the first attempt to characterize the nature of the neuromuscular blocking properties o f amphetamine. This was done by examining the influence o f various physiologic and pharmacologic conditions (detailed below) on the nature o f the neuromuscular blockade produced by amphetamine. A summary of the characteristics of depolarizing and nondepolarizing blocking agents is presented in Table 3. At 35, 37, 39 and 41°C, the concentrations o f amphetamine required to produce 50% blockade within

30 minutes were 217, 179, 146 and 103 yM, respectively. Thus, an inverse relationship between temperature and blockade was seen which is a characteristic of nondepolarizing (curare-like) blockers. The inhibition of nerve-stimulated contractions was readily reversible upon washing the preparation with Krebs' solution. The authors report no local anesthetic activity, which is consistent with the findings o f Anderson and Ammann (1963). Amphetamine-induced blockade of 45

TABLE 3

Comparison of Nondepolarizing (d-Tubocurarine) and Depolarizing (Decamethonium) Neuromuscular Blockers

Effect Nondepolarizing Depolarizing

Pretreatment with Additive blockade Antagonism of blockade d-TC

Pretreatment with No effect or Tachyphyaxis decamenthonium increased blockade

Transient excitatory e ffe c t on muscle No Yes

Contracts chronically denervated muscle No Yes

Blockade is reversed by anticholinesterase Yes No drugs

Blockade is reversed Yes No by KC1

S hifts ACh concentration-response curve of denervated Yes No muscle to rig h t

Effect on motor Decreases s e n s itiv ity Persistent endplate to ACh depolarization

Effect of lowering Antagonism o f Enhancement of temperature on blockade blockade bl ockade

a Based on Paton and Zaimes (1949, 1952) and Skau (1977). 46 indirectly stimulated contractions was enhanced by (+)-tubocurarine

(0.21 yM), succinylcholine (0.4 yM) and phenoxybenzamine (107 yM); conversely, the blockade by each of the above drugs was potentiated by amphetamine (73.5 yM). Whereas amphetamine blockade was antagonized by 10 yM neostigmine (consistent with curare-like blockers) it was potentiated by 0.18 yM carbachol (consistent with depolarizing blockers). Increased concentrations of K+ (to 7.0 mM) and Ca++ (to

3.8 mM) antagonized amphetamine blockade while increasing Mg++ levels

(to 0.75 mM) enhanced blockade. The authors also measured amphetamine levels in thigh muscles and the rectal temperature in animals (e ith e r forced to exercise or sedentary) injected with lethal doses of amphetamine (125 mg/kg). Confirming the reports of enhanced toxicity of amphetamine in exercised animals (Hardinge and Peterson, 1964), the rectal temperature of exercised rats rose to 41.1°C versus 38.7°C in sedentary animals. Muscle amphetamine concentrations were approximately 50% lower in exercised than sedentary rats, in spite of the injection of two to five times the dose to the non-exercised animals. These investigators also reported on the neuromuscular blocking activity of a series of adrenergic agonists and antagonists. It should be pointed out th at these adrenergic agents, as well as the

MAO inhibitors studied by Anderson and Ammann (1963), have the chemical s im ila ritie s o f a nonquaternary nitrogen group that is protonated (thus positively charged) at physiological pH. Peterson et a l. (1964) concluded that amphetamine blocks neuromuscular transmission via a curare-like mechanism. Consistent with this conclusion is the enhancement of curare and antagonism by neostigmine 47 of the neuromuscular blockade produced by amphetamine. By contrast, potentiation o f (+)-amphetamine blockade by succinylcholine and carbacol are more consistent with depolarizing blockers (Table 3).

The amphetamine isomers (+ and - ) and methamphetamine have also been studied and compared on neuromuscular transmission in the ra t phrenic nerve-diaphragm preparation (Gerald and Hsu, 1975a; b). A biphasic effect of the amphetamine isomers was observed, with low (135 and 270 pM) and higher (405-810 pM) amphetamine concentrations producing potentiation (maximum of 23%) and blockade o f nerve stimulated twitch, respectively. The concentrations required to block indirect muscle contractions by 50% (IC50), 10 min following drug addition were 405-444 pM for the amphetamine isomers and 780 pM for methamphetamine. Reserpine pretreatment o f animals (3 mg/kg, 24 hr) reduced both the enhancing and blocking effects of ( - ) -amphetamine, while no effects were reported for the (+)-isomer. Since reserpine depletes an amphetamine-insensitive pool of norepinephrine ( see below), no specific information can be gained from this study with respect to cbtecholamine-amphetamine interactions. In curare (38 pM)-pretreated, directly stimulated preparations, the amphetamine isomers (270-810 pM) and methamphetamine (470 pM) reportedly enhanced contractions by 14-74 and 21%, respectively; With a curare concentration of 100 pM, the amphetamine isomers (270-810 pM) enhanced d ire c tly stimulated contractions by 308-756%, w hile a t 25°C, methamphetamine (470 pM) produced a 104% enhancement. I t was noted that with high curare 48

concentrations (100 yM) the baseline was depressed, thus amphetamine-

induced enhancement o f contractions was o f re la tiv e ly greater magnitude

(M. C. Gerald, personal communication).

Amphetamine';s effects were studied in combination with treatments

known to modify neuromuscular function (e .g ., curare, succinylcholine,

high K+, physostigmine and temperature change) in an attempt to

characterize the nature of neuromuscular blockade produced (Gerald

and Hsu, 1975a). (+)-Amphetamine blockade was enhanced by (+)-tubo-

curarine and succinylcholine and, conversely, blockade by these

agents was increased by (+)-amphetamine. Reducing the bath temperature

from 37 to 25°C slowed the development of a 50% reduction in twitch

height for (+)-amphetamine (470 yM) blockade from 5.4 to 12.9 min. An

increase in the K+ concentration (from 4.6 to 7.5 mM) antagonized block­

ade, while a 3-fold increase in the Mg++ or 3-fold decrease in the Ca++

concentration enhanced the development o f neuromuscular blockade

produced by amphetamine. Depression o f nerve-stimulated twitch by

amphetamine (470 yM) was enhanced by preincubation with physostigmine

(0.03-24 yM fo r 10 m in). S im ila rly , amphetamine reversed physo­

stigmine enhancement of tw itch, an e ffe c t not in agreement with

Peterson et a l. (1964). The results of Gerald and Hsu do not clearly

support a single mechanism underlying the neuromuscular blocking actions of amphetamine. The antagonism of blockade by lowering the

temperature or by raising [K+] and the mutual enhancement o f blockade

with (+)-tubocurarine are consistent with a nondepolarizing blockade

(Table 3). By contrast, the reported mutual enhancement of blockade

with amphetamine and both succinylcholine or physostigmine is more 49 indicative of depolarizing blockade. The authors conclude that the effects o f amphetamine on neuromuscular function may resu lt from interactions with adrenergic (based on results with reserpine pretreatment) and/or cholinergic systems, but that the precise mechanisms involved were unclear.

In a related report, the same group of investigators reported that (+)-amphetamine (250 yM) enhanced the nerve-stimulated (0.2 Hz) release of acetylcholine by 2.5 fold from the isolated phrenic nerve- diaphragm preparation (Hsu and Gerald, 1973). This suggests a possible cholinergic mechanism may be responsible for the enhancement of nerve- stimulated muscle contractions as i f is known that ACh is the neuro­ transmitter primarily responsible for muscle twitch.

A biphasic effect of (+)-amphetamine was also reported by Skau and Gerald (1978a) fo r the same concentration range and employing the same twitch preparation as Gerald and Hsu (1975a). Skau and

Gerald (1978a) reported a difference in the blocking activity of the amphetamine isomers, with the (+) isomer approximately twice as potent as the (-)-isomer. Tissue pretreatment for 10 min with dopamine antagonists or a- or 3-adrenoceptor antagonists prior to amphetamine fa ile d to modify the biphasic response o f(+ )-amphetamine

(405 yM). Animal pretreatment with reserpine (3 mg/kg,24 hr) also fa ile d to modify the biphasic e ffe c t of (+ )-amphetamine, a result in contrast to that reported earlier by Gerald and Hsu (1975a). Since amphetamine is thought to act on catecholaminergic systems by releasing a newly synthesized, reserpine-resistant pool of norepinephrine (see below), the use of reserpine as an experimental tool in the analysis 50 of amphetamine's effects is o f lim ited value, but can be useful when used in combination with other treatments. In order to make an unambiguous assessment of a catecholaminergic involvement in amphetamine's e ffe c ts , pretreatment with the tyrosine hydroxylase inhibitor, a-methyl-p- tyrosine would be preferred (see below).

In denervated diaphragms (13-17 days p rio r to experimentation), amphetamine concentrations o f 270 and 405 yM (which produce enhancement of nerve-stimulated twitch in normal tissues) failed to cause contractures, but did shift the ACh concentration-response curve to the rig h t indicating a postsynaptic s ite of action. Physostigmine

10 yM) partially and temporarily reversed the blockade produced by amphetamine a t 540 yM as well as slowed the rate o f blockade by

810 yM, in contrast to the results of Gerald and Hsu (1975a). KC1

(11 mM) antagonized the inhibition of nerve stimulated contractions produced by (+)-amphetamine (540 yM), (-)-amphetamine (810 yM) and

(+)-tubocurarine (0.5 yM); but not by succinylcholine (10 yM). The major conclusion based on the above findings is that amphetamine produces blockade o f the postsynaptic ACh receptors by a cu rare-like

(nondepolarizing) rather than a depolarizing mechanism (Skau and

Gerald, 1978a).

Tissue incubation with a-bungarotoxin (a-BGT) results in irrever­ sible blockade o f nerve stimulated muscle contractions. Experiments employing th is neurotoxin were conducted to assess the a b ility of amphetamine to protect the postsynaptic cholinergic receptor against irre v e rs ib le a-BGT blockade (Skau and Gerald, 1977; 1978b). 51

(+)-Amphetamine and (+)-tubocurarine inhibited the specific binding o f 125 C I]-a-BGT to the rodent diaphragm in a concentration-dependent manner;

(+)-amphetamine (135-3000 yM* IC50 = 338yM) reduced binding by 34-88% and (+)-tubocurarine (0.1-1000 yM, IC50 = 5 .2 yM) reduced specific binding by 14-88%. These results elegantly and clearly confirm the hypothesized ab ility of amphetamine to interact with the postsynaptic nicotinic ACh receptor (Skau and Gerald, 1977).

In a more detailed subsequent report, Skau and Gerald (1978b) compared the in h ib ito ry effects of the amphetamine isomers and 125 related nonquaternary compounds on the specific bindingof [ I]-a-BGT to ra t and mouse hemidiaphragms. The IC50 values for in h ib itio n of 125 specific [ I3 -a - BGT binding for (+) and ( - ) -amphetamine were 338 and 695 yM, respectively. Chemically related compounds, including the four ephedrine isomers, methamphetamine, propylhexedrine and 2-amino- octane; were also capable of in h ib itin g specific a-BGT binding.

Increasing the pH of the incubation medium from 6.8 to 8.1 reduced the inhibition of a-BGT binding produced by (+)-amphetamine (405 yM) from 33 to 16%. At physiological pH (7 .4 ), amphetamine with a pKa of

9.9 (Vree et a l., 1969) would exist predominantly as the protonated quaterinary species. Increasing the pH of the incubation medium would decrease the concentration of amphetamine existing as the protonated, thus quaternary, compound. The authors concluded that the pronated nitrogen of the compounds tested is important in the neuromuscular blocking effects by binding to anionic sites on the nicotinic receptor. 52

Amphetamine Effects on Directly Stimulated Muscle Contractions

In addition to its effects on nerve stimulated preparations,

amphetamine has also been found to modify contractions e lic ite d by

d irec t muscle stim ulation (Anderson and Ammann, 1963; Peterson e t a l .,

1964; Gerald and Hsu, 1975a). In agreement with the studies cited

above, Skau (1977) reported that in tissues pretreated with (+)-

tubocurarine (50 yM, 15 min) a-BGT (1 yg/m l, 2 hr) or 3-BGT (1 ug/ml,

3 h r), amphetamine (270-1080 yM) produced a concentration-dependent

enhancement of contractions. These results suggest that in directly

stimulated tissues, unlike nerve stimulated preparations, enhancement

of contractions is independent of cholinergic factors. A detailed

analysis of the mechanism by which amphetamine modifies d ire c tly

stimulated muscles (pretreated with a-BGT 1 mg/ml, 2 hr) was conducted

by Meldrum (1980). A biphasic response of amphetamine was noted with

lower (270-1080 yM) and high (1350-2160 yM) amphetamine concentrations

enhancing (up to 22%) and completely blocking directly stimulated

contractions, respectively. The major conclusions from these studies

were that amphetamine may be enhancing postsynaptic ionic movement

at low concentrations and producing ion channel blockade at high

concentrations.

In summary, the available literature suggests that amphetamine

produces a biphasic effect on nerve-stimulated skeletal muscle

preparations in vitro. Low concentrations enhance, while higher concentrations depress neuromuscular function. Enhancement o f muscle

contractions by amphetamine has been hypothesized to resu lt from

increased presynaptic release of ACh (Hsu and Gerald, 1973). The 53 neuromuscular blockade produced by higher concentrations of amphetamine appears to be the consequence of binding to postsynaptic n ico tin ic cholinergic receptors and a cu rare-like blockade (Skau and

Gerald, 1978a, b). Amphetamine also produces a biphasic effect on directly stimulated muscle contractions by a cholinergic-independent mechanism. Based on the available evidence, it was hypothesized that amphetamine interacts with postsynaptic ion channels producing enhancement and blockade o f ion movements at low and high concentrations, respectively (Meldrum, 1980).

NeuropharmacQlogical Mechanism o f Amphetamine Action

This section will present the current thought relative to the mechanism o f action of amphetamine in the central and peripheral nervous systems. Because both adrenergic and cholinergic neuro­ transm itters are known to produce effects on neuromuscular transmission, special emphasis will be given to amphetamine-interactions with these neurobiological systems.

Adrenergic Mechanisms

Amphetamine is thought to produce sympathomimetic effects,both centrally and peripherally,by one or more of the following mechanisms:

(1) enhancement of release of catecholamines from nerve terminals;

(2 ).inhibition of neurotransmitter reuptake by the nerve terminal;

(3) inhibition of the catecholamine-degrading enzyme, monoamine oxidase (MAO); and (4) possible direct activation of adrenoceptors. 54

Release of catecholamines. Amphetamine is capable o f enhancing the release of catecholamines from nerve terminals in the brain

(Glowinski and Baldessarini, 1966; Carlsson et a l., 1966) and in the periphery (Carlsson et a l., 1966; Carlsson and Waldeck, 1966).

Following animal pretreatment with the amine-degranulating agent reserpine, amphetamine continues to produce its pharmacological effects

(see Carlsson, 1970). By contrast, following animal pretreatment with a-methyl-p-tyrosine, an inhibitor of tyrosine hydroxylase (the rate lim iting enzyme in catecholamine synthesis), the pharmacological effects and the release of catecholamines by amphetamine are blocked

(Weissman e t a l . , 1966). These results suggest th at amphetamine acts by releasing newly synthesized extragranular stores of catecholamines from nerve termins (see Carlson, 1970).

In h ib itio n of reuptake. Amphetamine has been shown to in h ib it the uptake of norepinephrine into noradrenergic nerve terminals in peripheral tissues (Axelrod et a l., 1961) and brain (Glowinski et a l.,

1966). The inhibition of uptake is reported to be independent of the releasing properties of amphetamine (Carlsson et a l., 1969), but occurs in the same range o f concentrations that promote catecholamine release

(1-10 yM) (H eikkila e t a l . , 1975; Taylor and Ho, 1978). Blockade of reuptake is suggested to involve in h ib itio n o f the membrane c a rrie r for catecholamines (see Carlsson, 1970). 55

In h ib itio n o f monoamine oxidase (MAO). Amphetamine was f ir s t suggested to possess MAO in h ib ito ry a c tiv ity by Mann and Quastel (1940).

While this observation has been confirmed, amphetamine is a relatively weak MAO inhibitor (Glowinski et a l., 1966); both in vitro and in vivo high amphetamine concentrations (20 mg/kg in ra ts ) produce only modest

(*20%) inhibition (Miller et a l., 1980). Amphetamine is 1000-5000 times less potent than tranylcypromine and other MAO inhibitors

(Biel, 1970). Therefore, MAO inhibition is likely not a major component o f amphetamine actions.

Direct adrenoceptor effects. It is now well accepted that amphetamine produces effects indirectly by releasing extragranular, newly synthesized catecholamines, and by inhibiting reuptake; it has no direct agonist activity on adrenoceptors (Carlsson, 1970; Hollister et al., 1974).

Amphetamine Effects on Cholinergic Systems

In addition to the effects of amphetamine on cholinergic transmission in neuromuscular preparations as outlined above, several investigators have studied amphetamine's effects on other systems.

This section will present the findings relative to cholinergic effects o f amphetamine in autonomic (ganglia) and central nervous system preparations.

. Effects on autonomic ganglia. The first report of an action of amphetamine on autonomic ganglia was presented by Luco et a l . (1949), who showed that doses o f 0 .1 -0.5 mg/kg in anesthetized cats produced an in itial enhancement of ganglionic transmission (superior cervical 56 ganglia) followed by ganglionic blockade. Amphetamine doses greater than 0.5 mg/kg produced no enhancement, but only blockade of transmission. A biphasic effect of amphetamine in the same preparation has also been reported by Kewitz and Reinert (1954). Gold and Reinert

(1960) reported this action to be identical to the action of nicotine in ganglia; initial stimulation followed by depolarization type blockade.

Amphetamine facilitates transmission through the isolated superior cervical ganglion of the ra t (Downing, 1972). Postganglionic action potentials were recorded electrophysiologically following preganglionic stimulation. At low rates of stimulation (0.1 Hz), amphetamine (27-

860 yM) had only a depressant effect on transmission. By contrast, amphetamine (27 yM) caused a partial reversal of the depression of transmission seen with high frequency stimulation (>4 Hz). This e ffe c t was mimicked by epinephrine (30 yM) and by norepinephrine

(96 yM) but not by isoproterenol (18 yM). These effects were prevented by the a-adrenoceptor antagonist phenoxybenzamine (5.8 yM), but not by the e-antagonist propranolol. Reserpine pretreatment did not alter the effects of amphetamine, a treatment of questionable value in determining a catechol amine-amphetamine interaction (see above). Downing (1972) concluded that amphetamine enhanced ganglionic transmission by a direct action, not mediated by endogenous catecholamine release, on a-adrenoceptors - located at preganglionic si tes. 57

Effects on CNS preparations. Amphetamine-induced enhancement of

ACh release has also been demonstrated in the central nervous system

(Beani et a l . , 1968; Pepeu and B a rto lin i, 1968). ACh was measured after collection of a perfusate through epidural cortical cups in mammals (ra b b it, ca t, guinea pigs ) . Although local administration of amphetamine (10 yg/ml) into the cortical cup did not alter ACh release, when administered intravenously amphetamine (1.5-5.0 mg/kg) was found to enhance ACh release by 0.75-3 fold (Beani et a l., 1968;

Pepeu and B a rto lin i, 1968). In subsequent reports by the same groups of investigators, it was reported that the effects of amphetamine could be prevented by animal pretreatment with a-methyl-p-tyrosine

(Nistri et a l., 1972; Beani and Bianchi, 1973). The ACh release enhancing effects of amphetamine were prevented by a-adrenoceptor antagonists (e.g., phentolamine) (Pepeu and Bartolini, 1968), which suggests that endogenous catecholamines are involved in this effect.

I t was concluded from these studies that amphetamine increases ACh output via the release of catecholamines from adrenergic nerve endings, which in turn stimulate a cholinergic pathway which ascends to the cerebral cortex (Nistri et a l., 1972; Beani and Bianchi, 1973).

Adrenergic-Cholinergic Interactions

Many o f the results presented above suggest that amphetamine produces effects on cholinergic transmission indirectly, via the release of catecholamines from nerve terminals. If this hypothesis is valid, it is logical to assume that norepinephrine and other 58 sympathomimetic amines should produce effects sim ilar to amphetamine.

This section w ill present a summary of the effects of adrenergic agents on cholinergic neurotransmission.

Adrenergic effects on neuromuscular transmission. Epinephrine

(EPI) and norepinephrine (NE) produce a biphasic e ffe c t o f neuro­ muscular transmission; namely, an initial facilitatory effect of relatively rapid onset followed by a slowly developing depressant e ffe c t (Bowman and N ott, 1969; Bowman, 1981). EPI and NE increase the EPP amplitude by approximately 50% (26-67%) in both frog and mammalian neuromuscular preparations in which transmission is

1 1 partially blocked by (+)-tubocurarine or high Mg (Hutter and

Lowenstein, 1955; Krnjevic and M iledi,1958; Jenkinson et a l . , 1968;

Kuba, 1970). This effect, coupled with a reported increase in MEPP frequency (from 1.32 to 2.92 Hz) with no increase in MEPP amplitude

(Krnjevic and Miledi, 1958; Kuba, 1970) strongly supports the hypothesis that the facilitatory effects of EPI and NE are due to an enhanced prejunctional release o f ACh. This enhancement has been termed an "anticurare" e ffe c t and has been demonstrated in in vivo and in v itro preparations; some investigators, however, have not been able to demonstrate th is response in v itro (see Bowman and N ott, 1969).

The anti curare actions of EPI and NE are blocked by the a-adrenoceptor blocking drugs phentolamine and phenoxybenzamine (Bowman and Raper,

1966; Kuba, 1970), but not by the e-adrenoceptor antagonist pronethalol

(Kuba, 1970). Kuba concluded that NE interacts with a-adrenoceptors on nerve endings to increase transmitter release. 59

Other phenolic compounds, such as phenol and catechol, also

enhance neuromuscular transmission by augmenting the amount o f ACh

released from prejunctional sites by nerve impulses. Like EPI or

NE, phenol and catechol increase the frequency of MEPPs without

effecting their amplitude or time course; EPPs evoked by nerve

stimulation are enhanced but not the endplate depolarizations produced

by iontophoretically applied ACh (Blaber and Gallagher, 1971; Gallagher

and Blaber, 1973). Although the presynaptic effects of NE and EPI are

practically identical with those of catechol and phenol, since the

effects of the latter compounds are not blocked by a-adrenoceptor

antagonists,it appears that different receptors are responsible for

the effects. Gallagher and Blaber (1973) present the possibility that

catechol represents the simplest a-adrenoceptor activating agent and

may be acting solely at a prejunctional site. To explain the inability

of a-blockers to antagonize the effects of catechol, they suggest

that the prejunctional a-adrenoceptor contains 2 sites, one occupied

by the catechol group and the other by the amino moiety, and that

antagonists occupy primarily the latter site. Thus, catechol could

s t i l l stimulate the prejunctional receptor in the presence of the

antagonist but a catecholamine would not.

The late developing "curare-potentiating" ieffects of catechola­ mines (i.e . the ability of EPI, NE and isoproterenol to enhance curare

blockade are mediated by a distinctly different mechanism than

the anticurare e ffe c t. I t is suggested that th is e ffe c t is due to a

decrease in the postsynaptic excitability of the muscle fiber membrane

(Krnjevic and Miledi, 1958). Moreover, the curare-potentiating effects 60 are seen most predominantly with the B-adrenoceptor agonist

isoproterenol and to a much lesser extent with EPI and NE. The ability of the nonselective B-antagonist propranolol to block this effect is also consistent with B-adrenoceptor mediation (Bowman and Raper, 1966).

In more recent unpublished experiments (reported by Bowman, 1981), the curare-potentiating action is reproduced by salbutamol and rimiterol ( 32-adrenoceptor agonists) and that these effects are

blocked by (+)-propranolol (Bj and b2 antagonist) but not by the selective Bj_ antagonists (+)-propranolol or practolol. These results strongly suggest that the curare-potentiating effects are mediated by

B2-adrenoceptor interactions. Neither the anticurare nor the curare- potentiating effects of catechol aminergic drugs are dependent on concomitant blood flow changes that catecholamines produce via effects on vascular resistance (Bowman and Raper, 1966).

The precise mechanism by which catecholamines enhance the release of ACh from motor nerve endings has yet to be fu lly elucidated

(Bowman, 1981). I t was f ir s t proposed by Krnjevic and Miledi (1959) that the EPI-induced increase in ACh release from mammalian motor nerve terminals was the result of nerve terminal hyperpolarization

(see above for discussion of hyperpolarization). However, Kuba (1970), by directly measuring the action current of the nerve terminal by electrophysiological methods, found that NE did not a ffe c t the membrane potential of the nerve term inal. A lte rn ate ly, Kuba and Tomita (1971;

1972) have provided electrophysiological evidence that NE increases the probability of ACh release from the nerve terminal. In addition, release can be further augmented by NE i f some other means of 61

increasing the probability of release is added to the NE effect (e.g. ++ increase Ca , depolarization of the nerve terminal with external

current or action potential or decreased Mg++) . They proposed a model

that the effect of NE is similar to excess Ca++ (Kuba and Tomita, 1972).

According to their hypothesis, a-adrenoceptor agonists augment the ++ kinetics of the Ca complex responsible for releasing ACh from motor

nerve term inals.

Further evidence supporting a presynaptic action of EPI and NE

was gained in in vivo experiments. Bowman and Raper (1966) found that

while EPI (5 yg/kg i.v ) enhanced the nerve stimulated contractions of

the in situ cat tibialis anterior, contractions elicited by close-

arterial injections of ACh, or the endplate depolarizations produced

by succinylchol ineswere sl ightly depressed. These results suggest that

facilitation of nerve stimulated transmission can occur in spite of,

or simultaneously with a small decrease in the postsynaptic sensitivity

to ACh (Bowman and Nott, 1969).

This latter observation that enhanced presynaptic activity can

fa c ilita te contractions in spite o f depressed postsynaptic s e n s itiv ity

to ACh is of great importance in the analysis of the effects of amphetamine on neuromuscular function. Amphetamine is known to

produce a biphasic effect on muscle contractions; low and high concentrations enhance and depress twitch, respectively. The ability of amphetamine to interact with the postsynaptic ACh nicotinic

receptor has been characterized as leading to a curare-like blockade

(Skau and Gerald, 1978a; 1978b). The hypothesis under evaluation in the present study (R. M. Snider) is th at the amphetamine-induced 62 enhancement of muscle contractions is the result of a potentiation of the presynaptic release of ACh. Thus, the net effect of amphetamine at any given time a fte r drug administration appears to represent a balance between an enhanced presynaptic output of ACh, and a depressed postsynaptic sensitivity to this neurotransmitter. It is possible that at low concentrations o f amphetamine, presynaptic enhancement pre­ dominates which high concentrations result in a dominant cu rare-like e ffe c t.

Adrenergic effects on autonomic and central nerVOUs system cholinergic preparations. In order to provide further support for the hypothesis that adrenergic agents are capable of modifying cholinergic neurotransmission, an analysis of the effects of EPI and NE on other cholinergic systems will be presented.

In contrast to the enhancement of cholinergic transmission by catecholamines noted above in the somatic nervous system, the predominant e ffe c t of these agents is to reduce or in h ib it ACh release in mammalian autonomic and central nervous system preparations. In a c a re fu lly designed study employing in tra c e llu la r electrophysiological analysis of postganglionic excitatory and inhibitory potentials in the guinea pig by Dun and Karczmar (1977), NE (1-10 yM) suppressed the excitatory effects of preganglionic stimulation without effecting the postganglionic response to iontophoretically applied ACh.

Pretreating the ganglion with phenoxybenzamine (10 yM) completely prevented, whereas propranolol (30 yM) failed to antagonize the ganglionic depressant action of NE. Moreoever, the frequency but not 63 the amplitude of miniature excitatory postsynaptic potentials was reduced (by 42-59%) by NE (1-10 yM) which also supports the conclusion that NE inhibits ganglionic transmission by reducing the output of ACh from the presynaptic nerve terminal. This effect of NE on ganglionic transmission, resulting from an inhibition of preganglionic release o f ACh, has also been observed in the isolated rab b it superior cervical ganglion (Chirst and Nishi, 1971; Dun and Nishi, 1974). In contrast,

Birks and Macintosh (1961) reported that NE (1-10 yM) increased ACh release from the in situ cat superior cervical ganglion. This opposite effect reported in the cat is contrary to the majority of work conducted in th is fie ld with guinea pigs and rabbits. I t is possible that the contradictions reported could be the result of methodological differences. Birks and Macintosh (1961) collected and measured ACh from perfused ganglia in the presence of an AChE in h ib ito r, whereas the other studies examined ganglionic transmission by electrophysiological methods in the absence of other pharmacological agents. Thus, the reported increase in ACh release by NE could be the resu lt o f the presence of the cholinesterase in h ib ito r, as AChE inhibition results in much higher levels of ACh than normally exist in vivo. In support of this concept, deGroat and Voile (1966), employing electrophysiological methods, found that NE decreased preganglionic cholinergic neurotransmission in the cat superior cervical ganglion.

An effect similar to that in ganglia has also been reported in other autonomic preparations. NE and EPI (1-10 yM) reduced by as much as 80% the output of ACh in the guinea-pig ileum longitudinal s trip (Paton and V iz i, 1969; K osterlitz et a l . , 1970) and in the guinea pig colon (Beani et a l., 1969). This diminution of ACh release by exogenously applied NE is mimicked by transmural stimulation to release endogenous catecholamines. All of the above studies noted that this decrease in ACh release by catecholamines is inhibited by a-adrenoceptor antagonists while 3-adrenoceptor antagonists failed to antagonize this effect.

Concerning the influence of NE on ACh release in the brain, the reports are conflicting: Singer et al . (1971) and Ho et al . (1975) found that repeated NE injections into the rat brain increased choline acetyl transferase activity (ACh synthesis and release are closely related), and this was interpreted as NE stimulation of central cholinergic neurons. Others, by contrast, have reported that NE reduces the ACh release from brain tissue in vivo (perfused ventricle preparation) and in vitro (tissue slices) measured by bioassay procedures a fte r collection of samples in the presence of a cholinesterase inhibitor (for references see Beani et a l., 1978). Due to the c e llu la r and neurochemical complexity of central nervous system preparations, it is very difficult to clearly evaluate, much less reconcile the different conclusions reached by the above investigators, especially in the case of vastly different techniques and measurements being used to answer a given question. It is apparent that if a clear answer to the question, "Does NE modify ACh release?" is to be obtained, i t is of utmost importance to use as simple a biological system as possible in the investigation. The isolated motor nerve- skeletal muscle and/or the isolated ganglia preparations are of much 65 simpler complexity than any type of central nervous system

preparation. The problem remains, however, whether information gained

from these simpler analogous preparations are d ire c tly applicable to

our knowledge o f how the CNS functions.

Mechanisms of NE effects on ACh release. Ahlquist (1948)

classified adrenergic receptors as a and 8 according to the

hemodynamic effects of sympathomimetic catecholamines. The

8-adrenoceptors were later subdivided into 8^ and $2 on basis of organ selectivity of a series of g-adrenoceptor agonists (Lands et a l.,

1967). The modulation of transmitter (ACh) release by catecholamines

has been shown to result from an a-adrenoceptor interaction in the

somatic and autonomic nervous systems (see above). Moreoever, a-adrenoceptors are also located on presynaptic neurons which release

NE, and are termed autoreceptors (i.e ., receptors which effect their own release). In addition, there are a-receptors that are located on postsynaptic sites in the autonomic and central nervous systems which mediate the physiological effects of released NE. Recent developments

in the area of presynaptic autoreceptors, mainly studied in the autonomic nervous system, have d iffe re n tia te d a-adrenergic receptors

into two classes, a^ (generally postsynaptic) and a2 (generally presynaptic) (Langer, 1977; Berthe!sen and Pettinger, 1977). The existence of distinct structure-activity relationships for pre- versus postsynaptic a-adrenoceptors, and the existence of relatively selective antagonists and agonists for these two receptor types, suggests that this subclassification is both anatomical and functional. In contrast to the in h ib itio n of transm itter release observed at neuroeffector junctions in the autonomic nervous system, stimulation of presynaptic a-adrenoceptors located on somatic nerve terminals leads to an increase in neurotransmitter release as was discussed in detail above. In order to characterize the nature of the presynaptic a-receptors located on motor nerve terminals, Malta et a l. (1979) compared a series of a-agonists and antagonists on a classical autonomic post-junctional response (hind-1imb vascular resistance) with the ability of these agents to enhance presynaptic function of motor neurons (reversal of p a rtial (+)-tubocurarine neuromuscular blockade) in the same in Vivo cat preparation.' From the results obtained with the series o f agonists (NE, EPI, phenylephrine, methoxamine, oxymeta- zoline and clo nidine), i t was concluded that the post-junctional vascular a-receptors and the prejunctional motor neuron a-receptors possess very similar characteristics (i.e. of the a^-type). Clonidine

(selective a2 agonist) was the only agonist which produced only small or inconsistent effects on the parameters being evaluated. Results from in vitro experiments indicate clonidine possesses selective presynaptic agonist a c tiv ity in the autonomic nervous system (a2- agonist), although its selectivity in vivo is not as clear (see Malta et al., 1979 for references). The results of studies with the series of a-antagonists (phentolamine, thymoxamine and tolazoline) gave results consistent with those obtained with the agonists. Since the prejunctional a-receptors at the motor nerve terminal are pharmacologically similar to the postsynaptic autonomic a-receptors, 67

these prejunctional receptors would be classified as aj-type

adrenoceptors based on the established terminology (Langer, 1977).

Methylphenidate

Neuropharmacology of methylphenidate. Methylphenidate <2X ( \.^ CHCOOCHg, a-phenyl-2-piperidineacetic acid methyl ester) H produces amphetamine-like effects in vivo; both agents produce central

stimulation in rats characterized by increased locomotor activity and

stereotyped behavior (Scheel-Krllger, 1971). Amphetamine produces

central stimulation by enhancing the release of newly synthesized

extragranular catecholamines; this stimulatory effect can be blocked

by animal pretreatment with a-methyl-p-tyrosine (Weissman et a l., 1966).

By contrast, the effects of methylphenidate are blocked by reserping

pretreatment but not by a-methyl-p-tyrosine (van Rossum et a l., 1962;

Scheel-Krllger, 1971) suggesting that the granular pool of catecholamines

is necessary for methylphenidate effects. Moreover, methylphenidate

preferentially releases catecholamines from reserpine-sensitive storage pools in vivo (Chiueh and Moore, 1975).

This classical concept has recently been questioned and it is currently believed that methylphenidate's effects may actually be mediated solely by inhibiting catecholamine uptake. By contrast, amphetamine acts both by promoting catecholamine release and by inhibiting reuptake (Heikkila et a l., 1975). The hypothesis that methylphenidate acts by inhibiting reuptake was examined by noting that the effects of amphetamine are antagonized by inhibitors of catecholamine reuptake, including methylphenidate (Ross, 1977).

9 68

Moreover, reserpine antagonizes the behavioral effects of

methylphenidate but not those produced by amphetamine, as previously

stated. Thus, i f methylphenidate produces effects in Vivo via uptake

inhibition, it should antagonize the behavioral effects of amphetamine

in reserpinized animals. Ross (1979) confirmed this mechanism by

showing that methylphenidate is a potent catecholamine uptake inhibitor

in vitro and that it inhibits (by up to 65%) the ability of amphetamine

to produce behavioral effects in reserpinized mice in Vivo.

In addition to its effects on catecholaminergic systems,

methyl phenidate is also reported to augment nicotinic cholinergic

transmission. Shih et a l . (1976; 1977) examined the mechanism by which

methylphenidate may act in alleviating the symptoms of hyperkinetic

children. The model used in these studies was an evaluation of the

neuronal discharge rates of cells in the mesencephalic reticular

formation of immobilized rats. This brain structure is thought to be

involved in the mediation of perception, attention and arousal,and it has

been proposed that this brain area is hyperactive in children with

hyperkinetic behavioral problems (Werider, 1974). Shih and coworkers

found that methylphenidate (1-2 mg/kg i.v .) markedly attenuated the

discharge rate of these neurons by 40-50%. Moreover, these effects

of methylphenidate are mimicked by oxotremorine (0.5 mg/kg) or nicotine (0.125 mg/kg), both agents which enhance central nicotinic activity,and abolished by mecamyl amine, a central nicotinic antagonist.

The authors suggest that cholinergic mechanisms might mediate the electrophysiological effects of methyl phenidate and may also contribute to the therapeutic effects of this drug in hyperactive children. 69

Using a behavioral paradigm, Bryan and Ellison (1975) reached a similar conclusion. In rats given (+)-amphetamine or methylphenidate

(both 1 mg/kg), rearing responses of high frequency-short duration and low frequency-long duration are produced, respectively. In evaluating a possible cholinergic component of these effects, the cholinergic agent physostimine was found to further potentiate the effects of methylphenidate while scopolamine produced effects sim ilar to amphetamine. The authors concluded that methylphenidate's central actions may be mediated by cholinergic mechanisms. This conclusion seems highly simplistic and, while consistent with the data presented, numerous alterate hypothesis may be equally appropriate.

The studies reported above (Shih et a l., 1976; 1977; Bryan and

E lliso n , 1975) were using a central nervous system (electrophysiological or behavior) model which, as was noted above, has both m ultiple cell and receptor types which mediate any CNS response that is recorded.

Methylphenidate is known to have effects on central aminergic neurotransmitter systems similar to amphetamine. Therefore, before any direct link between methylphenidate and cholinergic function is established, it would be beneficial to provide evidence in a less complicated nicotinic system, lik e the neuromuscular junction or autonomic ganglia, that methylphenidate is indeed active .

Methylphenidate, performance and neuromuscular transmission.

Methylphenidate produces central stimulation with a somewhat lower potency than amphetamine., Nevertheless no report could be found in the literature concerning the influence of methylphenidate on physical performance or endurance performance in animals or humans. There is 70

one report of an assay for methylphenidate metabolites in the urine of

racing greyhounds in Australia (Lewis, 1979). It is logical

(R. M. Snider) to assume that if methylphenidate's detection in racing

animals is important, then it may be used in these animals to enhance

their performance.

One report was located in the literature on the effects of

methylphenidate on the isolated ra t phrenic nerve diaphragm preparation

(Ndika, 1966). Ndika states "methylphenidate (2 x 10"®) reduced the

contractile responses of rat diaphragms to indirect stimulation", and

"with concentrations of methylphenidate less than 1 x 10“® no block

of the muscle responses e ith e r to in d irect or to d ire c t stimulation was demonstrated but rath er, some sustained increase in these responses

was observed." This study was poorly conducted and d if f ic u lt to

analyze because no data was presented to support these statements, the

author did not clearly specify which concentration units were used

(M, yg/ml, etc.) nor did he state whether methylphenidate base or a

s a lt was employed. Assuming that the hydrochloride s a lt was used

and the concentrations employed are in molar units, the following e ffec ts may be assumed: (1) Low concentrations (<10 yM) enhanced

contractions to nerve stimulation (degree of enhancement not reported);

(2) Higher concentrations (>30 yM) blocked both direct and indirectly- elicited contractions although again no data was given; (3) Methyl- phenidate (37-74 yM) enhanced neuromuscular blockade produced by

(+)-tubocurarine, decamethonium and ACh (220 mM); (4 ) Methylphenidate

(3.7 mM) reduced the amplitude of action potentials in the isolated 71 rabbit sciatic nerve, while 1/10 this concentration produced no effect.

The author concluded that the neuromuscular blockade produced by methyl phenidate is neither of the depolarizing or nondepolarizing type but rather due to local anesthetic activity. This is a rather questionable conclusion in that concentrations of methylphenidate

100 times higher than those reported to produce neuromuscular blockade were necessary to reduce the frog sciatic nerve action potential amplitude, a measure of local anesthetic activity.

In summary, methylphenidate poses interesting questions for the investigator interested in the effects of amphetamine-like drugs on physical performance and cholinergic neurotransmission. There is evidence that methyl phenidate potentiates cholinergic as well as adrenergic neurotransmission centrally, and a biphasic effect has been reported in the isolated nerve-muscle system, although the details of this latter report are questionable. Methylphenidate, chemically, possesses both an amphetamine-like moiety and an ester linkage and, therefore, it is of interest to determine whether it interacts in a similar nanner to amphetamines with the postsynaptic nicotinic receptor, and whether the ester moiety is capable of in teracting with acetylcholinesterase. These questions remain to be answered. STATEMENT OF THE PROBLEM

(+ )-Amphetamine, at low to moderate doses, can enhance the

physical performance of humans and animals,while toxic doses in rodents

produce muscle weakness and hindlimb paralysis. The re la tiv e

importance of ce n trally (central nervous system) versus peripherally

(e .g ., metabolic or neuromuscular)-mediated mechanisms remains to be

determined. The central stimulatory effects of amphetamine are commonly viewed as prim arily responsible fo r its performance enhancing

effects, although it is possible that peripheral mechanisms may also be

important. In support o f th is concept, amphetamine has been shown to

produce a similar biphasic effect in the isolated mammalian nerve-

skeletal muscle preparation. Low and high amphetamine concentrations enhance and depress nerve stimulated contractions, respectively. These observations suggest th at a s ite of amphetamine action may be at the somatic nerve, the neuromuscular junction or the skeletal muscle fib ers.

Enhancement of nerve stimulated muscle contractions might be mediated presynaptically, via enhanced release of acetylcholine (ACh), postsynaptically, by an increase in receptor sensitivity to this neurotransmitter or by a mechanism independent of cholinergic transmission (e.g., ionic movements across muscle fibers). Conversely, neuromuscular blockade could be caused by a decrease in ACh release

72. 73 presynaptically, a decrease in receptor sensitivity to released ACh or by a cholinergic-independent mechanism such as ion channel blockade.

The studies reported herein concern only cholinergic-dependent effects.

The strategy employed in elucidating the mechanism of these effects on neuromuscular transmission begins with establishing the sites (pre- and/or postsynaptic) at which amphetamine acts. By employing d is tin c t methodological approaches in the same preparation

(the isolated rat phrenic nerve-diaphragm), and correlating the results obtained by the different methods, specific insight is gained relative to s ite and mechanism of drug action. These methods include: nerve stimulated muscle contractions; ACh stimulated contractions; intracellular electrophysiological analysis of end plate events; and direct biochemical analysis of ACh released from motor neurons.

Amphetamine is classified as an adrenergic agent, and i t produces most of its characteristic effects by releasing newly synthesized stores of catecholamines and by inhibiting catecholamine reuptake. If amphetamine's effects on neuromuscular transmission are mediated indirectly, via catecholamines, depletion of newly synthesized stores should markedly attenuate the effects o f amphetamine.

Norepinephrine (NE) is known to enhance the release of ACh from somatic nerve terminals, and this effect is prevented by a-adrenoceptor antagonists. In recent years, the a-adrenoceptor has been subclassified into a j and ag-types based on the responses o f agonists and antagonists in different a-adrenergic systems (e.g., vas deferens, aorta, spleen).

Anatomically, the a^- and ag-adrenoceptor types are located at post- and 74

presynaptic site s , respectively. By employing selective a^- or ctg-

adrenergic agonists and antagonists, an attempt is made in the present

investigation to characterize the nature of the presynaptic a-adreno-

ceptor type on motor nerve terminals. If the effects of amphetamine

are mediated by the release of NE (acting on presynaptic a-adrenoceptors

on motor nerve term inals), selective a-adrenoceptor agonists and

antagonists should modify in a qualitatively similar manner the effects o f amphetamine and NE.

Methylphenidate, an agent which has properties similar to amphetamine in vivo (e.g., behavioral arousal), may also modify muscle

contractions and neuromuscular transmission. An attempt is made to

characterize the a c tiv ity of methyl phenidate in this system, examine the mechanism responsible for its e ffe c t, and compare such effects with those produced by amphetamine.

In summary, the specific goals of this study include the following:

( ! ) Examination of the time course of amphetamine enhancement of nerve stimulated muscle contractions.

(2) Determination of the site of action (pre- or postsynaptic) of the facilitatory and inhibitory effects of amphetamine. More s p e c ific a lly , I w ill te s t the hypothesis that the enhancing effects result from a presynaptic increase in ACh release and the inhibitory effects result from a postsynaptic inhibitory effect.

(3) Determination of whether the effects of amphetamine are direct, or indirectly mediated via the release of catecholamines.

(4) Characterization of the presynaptic a-adrenoceptor type mediating the effects of NE at the somatic nerve terminal and determine whether amphetamine acts in an analagous manner as NE.

(5) Characterization of the nature of methyl phenidate's effects on neuromuscular transmission. CHAPTER I I

MATERIALS AND METHODS

Animals

Male, albino Sprague-Dawley rats weighing 175-300 gm were obtained from Lab Supply (Indianapolis, IN) and Harlan Industries, Inc.

(Indianapolis, IN), housed in groups of 5-6 per cage and provided free access to food (Purina Rat Chow) and water. The rodent fa c ilit ie s were maintained on a 12-hour light-dark cycle (700-1900 hrs).

Muscle Contraction Studies

General procedure. A ll studies were conducted using the le f t phrenic nerve-diaphragm which was dissected as described by BUI bring

(1946). Animals were anesthetized with diethyl ether and secured with limbs extended on to a frog dissecting board. The skin and muscles overlying the le ft thoracic cavity were removed and a lateral cut was made distal to the rib cage from flank to flank on the ventral side o f the animal . The thoracic cavity was then opened from the base of the sternum laterally to the animal^ left flank, then rostrally to the neck followed by removal of the entire chest wall over the left side of the animal. The left lung was held to the side with needle holders allowing a clear view of the left phrenic nerve. The nerve was then

76 77 isolated by tying a length o f suture thread around the nerve in the region of the thymus, followed by severing the nerve rostral to the tie and dissecting it free from the adhering pleural membrane down to within 5 mm of the muscle. Extreme care was taken to avoid stretching the nerve and to manipulate the nerve only by the suture thread. The l e f t hemidiaphragm was then removed from the animal by cutting the muscle along the midline as far dorsally as possible into the tendon, then laterally to the left through the remaining ribs. The tissue was gently lifted free of the animal and placed in a petri dish containing

Krebs' solution (described below) at room temperature. The petri dish had a 1 cm coating of Sylgard (Dow-Corning) silicone-elastomer into which the tissue could be pinned to fa c ilita te trimming. Most of the thoracic tissue was trimmed leaving a thin strip (approximately

5 mm) of ribs at the base of the diaphragm. The muscle was then trimmed to about 2 cm a t the base or rib end and to 1 cm at the top or tendon end.

For studies using a cannulated diaphragm (cannulated twitch and

ACh release studies), the procedure fo r removing the diaphragm was slightly modified. Animals received sodium heparin (10,000 U/k^i.p.) 15 min before ether. The animals were bled by cutting the jugular vein when removing the skin and muscles overlying the thoracic cavity.

The abdominal cavity was opened as described above, the diaphragmatic vein located and a small cut made on each side of the vein at a point where the vein intersects the abdominal vena cava. Two suture threads, passed around the diaphragmatic vein and lightly tied, were 78 used as anchoring tie s fo r the cannula. The cannula (PE 10 polyethylene tubing, 30-40 cm in length) was positioned into the diaphragmatic vein through a small cut in the abdominal vena, cava between the two dorsal lobes of the liver. Care was taken to completely fill the cannula with Krebs' solution to remove all air from the cannula prio r to inserting i t into the vein. The tip o f the cannula was positioned to rest approximately 1 mm from the venous bifurcation in the le ft side of the diaphragm and securely anchored.

The diaphragm was then carefully dissected and trimmed, as described above and the adequacy of the cummulation procedure examined. This was accomplished by slowly infusing Krebs' solution through the cannula and observing the movement of blood out of the diaphragm. I f any major large vessels were cut during the dissection procedure, these were electrolytically ligated (National Cauterizer, National

Electric Instruments Co., NY) to prevent any low resistance leakage of the perfusion fluid or injected ACh.

The tissues were affixed by two suture threads to thestable base of a phrenic nerve-diaphragm electrode (C. F. Palmer, Bucks,

England, Fig. 6 ), and the phrenic nerve was c a re fu lly passed through the nerve-stimulating platinum wire electrodes. The preparation was then placed in a 30 ml jacketed organ bath containing modified Krebs' solution of the following composition (mM): NaCl, 125; KC1, 5.0;

CaClgj 2.0; MgClg, 1.0; NaHgPO^, 1.0; NaHCOg; 15 and glucose, 11.

The resulting solution was gased with 95% Og + 5% COg* had a pH =

7.3 + 0.1 and the temperature was maintained at 32 +_ 0.5°C. Figure 6. Schematic representation of the ra t phrenic nerve-diaphragm mounted on the Palmer Phrenic Nerve-Diaphragm Electrode. The rib portion of the diaphragm was tied by two threads to the base of part A. The phrenic nerve was threaded through the exposed wires at the base o f part B. Nerve stimulation occurred by attaching the stimulator to leads 1 (cathode) and 2 (anode). Bath volumes were always kept above the top of the tissue.

79

81

Nerve stimulation of muscle contractions was achieved with a Grass

S 88 stimulator (Grass Instruments, Quincy MA) at 0.2 Hz with monophasic 5V square wave pulses of 0.4 ms duration. In some experiments (cannulated diaphragms), pulsed injections of ACh were used to e lic it muscle contractions as described by Wright and Collier

(1977) and compared with twitches elicited by nerve stimulation in the same tissue (Fig. 7). The amount of ACh injected into the muscle through the vein was varied so as to give a nearly equal pen deflection as that elicited by nerve stimulation (25-35 mm). This was accomplished by varying eith er the volume (15-40 p i) or the concentration of ACh (50-200 yM) solution injected into the vein.

The rate of injection was kept constant by using a spring-loaded syringe operated by a push button (Hamilton CR700-200). Injection of

ACh-free Krebs' solution was tested to verify that the injection procedure alone would not elicit a contraction (i.e ., less than 5 mm pen deflection). Injections of ACh were made no more often than every

10 min and the bath solution was changed after every 2-4 injections to prevent endplate desensitization.

Muscle contractions were recorded by attaching the muscle with

500 mg resting tension to a FT.03C isometric transducer coupled to a Model 7 Polygraph (Grass Instruments, Quincy, MA). Tissues were allowed a 45-60 min equilibration period before experiments were started and Krebs' solution was changed at 10-15 min intervals during this period. Figure 7. Schematic representation of the cannulated diaphragm preparation. By cannulating the diaphragmatic vein, the preparation was perfused by its own vascular system. This preparation allowed muscle contractions to be elicited by nerve stimulation or by injecting a pulse of ACh. Alterna­ tively, the perfusate could be collected and analyzed for released products such as ACh.

82 Injection Syringe (or) Perfusion Pump

Phrenic Nerve 84

Electrophysiological Studies

The isolated nerve-muscle preparation, dissected as described above, was positioned horizontally and pinned in Syl garcf® ( Dow

Corning) to the bottom of a plexiglass chamber of approximately 12 ml capacity (Fig. 8). The nerve was passed under a sliding partition to a second, adjoining chamber, passed around two platinum wire nerve-stimulating electrodes, and the en tire nerve covered with heavy mineral o il. The temperature in the muscle chamber was constantly monitored (Telethermometer, Yellow Springs Instruments, Inc., Yellow

Springs, OH) and was maintained at 32 + 1.0°C by superfusing the tissue with warmed, aerated Krebs' solution at a rate of 3 ml/min. The solution was removed at the same rate by suction created by a negative pressure pump (FMI Lab Pump, Fluid Motering Inc., Oyster Bay, NY).

The tissues were allowed at least a 30 min equilibration period in the chamber before the experiment was started.

In tra c e llu la r recording techniques were conducted as described by Fatt and Katz (1951). Glass capillary microelectrodes filled with

3 M KC1 were fabricated with a vertical pipette puller (Model 700C;

David Kopf Instruments, Tujunga, CA) to have an internal resistance between 6-25 Mft (heater setting of 19A, solenoid setting of 55).

Electrode resistance was the most important single variable in establishing an adequate intracellular recording. Groups of 15-20 electrodes were fabricated at a time and the resistance determined, one at a time, using a dual microprobe electrometer (WP Instruments

In c ., New Haven, CN). Only those electrodes that tested in the range 85

Figure 8. Photograph of the electrophysiological recording chamber. The tissue was mounted horizontally and pinned to a Sylgard base in the bottom of the chamber. The nerve passed under a sliding partition to the nerve-stimulation chamber, and covered with mineral o il to reduce stimulus a r tifa c t. 86 of 6-25 Mfi with noise values less than 150 yV were kept for intracellular studies. Electrodes with resistance lower than 6 Mfi generally would not penetrate the cell and provide stable recordings, while electrodes with resistance greater than 25 Mn were too noisy

( i . e . noise levels higher than 150 yV).

Placement of the microelectrode was fa c ilita te d by viewing the tissue through a dissecting microscope at 15-40X magnification

(American Optical) and movements of the electrode were accomplished with a micromanipulator (Brinkman). The placement of the microelectrode, as well as the stability of the recording was constantly monitored by listening to the noise generated by the preamplifier signal as the microelectrode entered the cell with the aid of an audio monitor (Model AM8, Grass Instruments, Quincy, MA).

Recording of the resting membrane potential (RMP), endplate potential

(EPP) and miniature endplate potential (MEPP) was accomplished using differential amplification of the membrane signal (Model 750 dual microprobe, WP Instruments, New Haven, CT) and passing the output to a dual beam storage oscilloscope (Tektronix Model 5113, Beaverton,

OR). RMPs were determined by recording the microelectrode signal on the storage oscilloscope (DC coupled) and noting the trace deflection as the microelectrode penetrated the membrane. MEPPs (which correspond to the-spontaneous release of ACh quanta) were recorded from muscle cells in the endplate region and were analyzed by collecting data before and after drug additions; thus each cell was used as its own control. Amplitude analysis was performed by storing a series of oscilloscope traces and photographing the trace on Polaroid film

(Type 7 Pack Film) with an oscilloscope camera (Tektronix Model C5A

Camera, Beaverton, OR). Each MEPP was physically measured using a

vernier caliper and recorded, thus generating an amplitude profile for

each cell both before and after drug addition. MEPP frequencies were

evaluated in two ways: by counting the MEPPs that were photographed

on film, thus each amplitude profile had a frequency associated with it

or by further amplifying the signal from the electrometer with a

separate amplifier (Tektronix AM502 differential amplifier) and

analyzing the signal for MEPPs during successive 1 min intervals before

and during drug infusion using a signal counter (Hewlett Packard 5302A

Universal Counter). Only c e lls having rise times of <1 msec and h a lf

decay times of <1.5 msec during the control period were used for data

collection. If the RMP decreased by more than 10 mV (toward the

depolarized direction), the cell was not included as data. On the average,about 1 in every 4 cells from which data was collected met

these criteria by the end of the data collection period (i.e . 10-15 min of drug infusion plus 10 min of control data). The optimal area on the muscle surface for recording endplate events is within a 5-7 mm radius of where the phrenic nerve enters the muscle. The best method of locating a cell from which to record is simply to move the electrode within the area specified, impale a cell, and determine whether it meets the criteria established for acceptance. Many times control data would be collected and after 5-10 min of monitoring MEPPs, the cell's

RMP would drop and the cell would "die". It is important to be confident that a "good" cell is well established before drug addition as, if the cell dies before the end of the data collection period in 88 the presence of drug, the tissue must be discarded and a fresh tissue set up.

EPPs, which correspond to the nerve stimulated release of ACh, were recorded from the same area in the diaphragm as MEPPs and in a similar manner with few exceptions. To record the EPP the nerve must be stimulated and the muscle treated to reduce neuromuscular transmis- ? sion and muscle contractions. If the muscle twitches, or moves in any

Muscle contractions are blocked in electrophysiological studies using (+)-tubocurarine, raised Mg++ concentrations or by using a cut- muscle preparation. (+)-Tubocurarine acts to block muscle contrac­ tions by reducing the sensitivity of the postsynaptic ACh receptor to released neurotransmitter. Thus, released ACh interacts with the receptor sites to produce the EPP, but the threshold for a propagated muscle action potential is not reached. A limitation ofusing curare blockade is the possibility that this drug effects presynaptic neuro­ transmitter release (as discussed above). Raised Mg++ concentrations (from 1.0 to 12.5 mM) is a second commonly used method to abolish the muscle action potential and tw itch . The mechanism by which Mg++ acts has been discussed in detail above and involves a decrease in the amount of ACh release from the presynaptic nerve without a major effect on postsynaptic receptor s e n s itiv ity . Both high Mg++ and curare blockade have the disadvantage of recording electrophysiological events under pharmacologically modified conditions. Thus, data collected could result, in part, from the effect of the blocking agent, or an interaction of the blocking agent with the drug under investigation. To avoid the need to use a blocking agent in order to abolish the muscle twitch and allow recording of the EPP, the cut-muscle preparation may be used (Barstad and Lilleheil, 1968). This procedure involves cutting of the distal 2-5 mm of muscle fibers on each side of the endplate region which prevents tissue contractions. Major limitations of this preparation have been noted including the deteri­ oration of the RMP to as low as -17 mV (from the normal -70 mV). Moreover, it is proposed that presynaptic function is also affected, supported by the observation that normal K+ concentrations (5 mM) induces nerve block in the c u t-fib e r ra t diaphragm preparation (Randic and Straughan, 1964). Thus, the c u t-fib e r preparation, while pharmacologically uncontaminated, has disadvantages based on the physiologic disruption of the tissue. The hypothesis under investigation in the present study is that amphetamine enhances the presynaptic release o f ACh. For th is reason raised Mg++ was chosen as the method to block muscle contractions. While (+)-tubocurarine and the cut-fiber preparation may produce some effect on presynaptic function, high Mg++ blocked tissues are well characterized as having a consistent effect on neurotransmitter release and thus, was deemed the most appropriate method for use in the present investigation. 89 manner while an electrode is in contact with the tissue, the glass pipette will move and create an electrical "movement artifact", or worse, the pipette will break.

The EPP was elicted by nerve stimulation (Grass Model S88

Stimulator with Model SIU-5 Stimulus Isolation Unit; Grass Instruments

Inc,, Quincy, MA) at 0.2 Hz with the voltage and stimulus duration varied (0.5-10 V; 0.01-0.5 msec) to be of sufficient intensity to e lic it an EPP but kept barely supramaximal to minimize the stimulus a r tifa c t. Neuromuscular transmission was inhibited by raising the Mg++ concentration from 1.0 to 12.5 mM, with appropriate adjustments in the

NaCl concentration to maintain the osmolarity (NaCl concentration reduced from 135 to 116 mM).

EPPs were recorded by noting the amplitude of each potential

(in mV) as i t appeared on the oscilloscope screen. Predrug amplitudes were collected fo r 5 min (60 EPPs) and averaged to give a control value, followed by drug addition and continuous monitoring of the EPPs generated for 10-15 min. Control and treatment values were obtained in the same c e ll. Many of the same problems outlined above for MEPPs also exist in attempting to record EPPs. Electrophysiological recording of endplate events is technically very difficult. The two most important factors in establishing a productive preparation are: maintaining the preparation absolutely motionless (vibrations from a water heater/pump can be a source o f vibrational 'n o is e '), and using electrodes of the proper resistance (6-25 Mn). However, even with a great deal of care and experience in this technique, for reasons that are not understood, the preparations sometimes do not work, while at 90 other times,data is collected with relative ease (D. F. Wilson, Miami

U niv., Oxford, OH, personal communication). In my experiments, 10-20% o f the tissues which were dissected produced usable results, the rest failing for one or more technical reasons. I found the experience of conducting intracellular electrophysiological studies enjoyable and challenging, but at times very frustrating and tedious.

Biochemical Studies

Collection Of Samples. The cannulated diaphragm preparation was used for these experiments as described above with the following exceptions. The tissue was mounted vertically into a specially designed chamber (Bierkamper and Goldberg, 1977) and perfused with

Krebs' solution containing 30 yM physostigmine su lfate (9.8 mg/L) to prevent ACh hydrolysis. The tissue was perfused at a rate of

40 yl/m in , compared to 34 yl/m in used by Bierkamper and Goldberg

(1977), using a peristaltic tubing pump (Sage Instruments Model 375A,

Cambridge, MA). These experiments were conducted at room temperature

(23 + 1,0°C) and the tissues were perfused for 1 hr before experiments began. The nerve was stimulated by two platinum wire electrodes at

10 Hz,5Vs 0.4 msec duration for a 10 min interval during which time the perfusate was collected for ACh assay. Thus a total volume of 400 yl was collected for each data interval. Five min rest periods (with no nerve stimulation) were allowed between stimulation periods. Each tissue was subjected to a total of 7 ten min stimulation periods; the perfusate for the first 10 min interval was not collected; 2 control in tervals; 2 periods in the presence o f drug perfusion; and 2 wash-out 91 periods. At the end of most experiments, a permeating dye (red physiograph ink) was perfused through the preparation to confirm complete and uniform perfusion of the tissue (Fig. 9).

Extraction of ACh from Krebs1 solution. Following collection of the perfusate,ACh was separated from the Krebs' solution using liq u id - liq u id cation exchange chromatography (Fonnum, 1969). The Krebs' perfusate (400 y l ) was vigorously mixed (Vortex-Genie, Scientific

Industries, Springfield, MA) with 250 yl of tetraphenylboron-heptanone in the same 15 ml glass centrifuge tubes in which the perfusates were collected. A fter centrifugation to break the emulsion into two phases

(5 min at setting 5; Safety Head Centrifuge, Clay Adams Inc.,

Parsippany, NJ), 175 yl of the organic (upper) phase was transferred to a fresh 15 ml glass centrifuge tube containing 175 yl of 1 N HC1 and vigorously shaken as above. The residue formed at the interface of the two phases during the firs t centrifugation was discarded.

Following a brief (30 sec) centrifugation to break the emulsion, the organic (upper) phase was removed by suction using vacuum filtr a tio n into a liquid trap. The acid phase (150 y l ) was transfered to a 6 x 50 mm glass test tube, placed in a larger 12 ml centrifuge tube, and the sample evaporated to dryness (a fin e white residue) under a gentle stream of nitrogen gas a t 0-5°C (N-Evap Analytical Evaporator;

Organomation Associates, Northborough, MA). This drying process was usually completed in 1.5-2 hrs. The residue formed was stored at -10°C until assayed, no longer than 12 hr later. Using 14C-labeled ACh, the recovery of ACh by the above procedure was found to be 83 + 5% (N = 5 ). 92

Figure 9. Photograph o f cannulated vascular perfused diaphragm mounted in the chamber used for the biochemical studies. The dark areas in the diaphragm illu s tra te s the complete and uniform perfusion of the tissue in these studies. 93

En2ymatic assay fo r ACh. A radioenzymatic assay fo r ACh was

employed (Goldberg and McCaman, 1973; McCaman and S te ltze r, 1977). 32 This method is based on the conversion of ACh to P-labeled phosphoryl- 32 choline by choline kinase and P-ATP in the presence of AChE. Any

choline present is removed by a prior incubation with choline kinase

and unlabeled ATP. (See Table 4 for Schematic.) Major reactions in

this assay include:

(1) choline + ATP ch6l1|jg4.+i-- — > phosphorylcholine

(2) ACh + AChE Mg=R:------^ choline + acetate

(3) choline + ^P-ATP —c—^ 6^ ^P-phosphorylcholine

The ACh-containing residue from the nitrogen evaporation was

dissolved in 30' yl of a cold (0-5°C) reaction mixture containing ATP

(0.8 mM), MgClg (10 mM), dithiothreitol (5 mM) and choline kinase

(1.5 yg/yl; 0.46 U/mg, Sigma Chemical, St. Louis, MO) in the 50 mM

Sorensens' phosphate buffer (pH = 8 .0 ). The samples were vortexed

gently to mix the reaction mixture then placed in a shaking incubator

(Dubnoff Metabolic Shaking Incubator, Precision Instruments, Chicago

IL) set at 100 oscillations/min, 37°C for 15 min. The samples were

then returned to an ice bath and 6 yl of a solution containing acetylcholinesterase (5 U, 1.25 U/yl; eel Type V, Sigma Chemical, St.

Louis, M0) and [6-^P]-ATP (0.8 yCi, 20-40 Ci/mmole) was added to each

sample. The tubes were then returned to the incubator fo r 15 min

at the same settings as above. The reaction wa;s terminated by

returning the samples to an ice bath and maintained at 0°C until 32 separation o f the phosphorylcholine from unchanged [ P3-ATP. 94 TABLE 4

Schematic Flow Diagram o f the Procedure Used to Assay fo r ACh

aqueous (discard) Krebs' solution vortex, containing ACh and , then centrifuge physostigmine (30 yM) 3-heptanone with (400 yl) tetraphenyl boron ^1 M 7 K -w (ACh + Choiine) 10 mg/ml (250 y l ) or9anic (175 yl}

extract with 175 yl 1 N HC1

centrifuge

dr.y under Residue <- ■ aqueous (acid) organic (discard) N0 stream (ACh + Choline) phase (150 y l ) 0-5 C I (ACh + Choline) Dissolve in 30 yl of solution containing incubate to convert ATP (0.8 mM), MgCl? 37°C any choline present (10 mM), choline 15 min to phosphorylcholine kinase (45 yg), DTT (5 mM), in 50 mM phosphate buffer (pH = 8.0) a t 0°C return to ice bath \J/ Add 6 yl of a solution Separate Incubate to 32 containing AChe «C5U> P-phosphoryl- convert 1.25 U/yl) and ^ P - choline-from ^ choline $rom 37°C - ATP (0.8 yCi, 20-40 other x p- ACh to " p - 15 min Ci/mmole) in 50 yM products on phosphoryl- phosphate buffer Dowex 1 x 8 choline (pH = 8.0) a t 0°C column \ i Count using liquid scintillation spectrometry 32 32 Separation of C Pj-phosphorylcholine from [ P]-ATP. The separation was carried out using anion exchange column chromatography

(McCaman and S te ltz e r, 1977). Ion exchange columns were prepared fresh for each separation in Pasteur pipettes containing a small plug 3 ? o f glass wool (3 mm ) ju s t below the shank. A slurry (100 g resin /

100 ml d is tille d HgO) of anion exchange resin (Dowex 1x8, 200-400 mesh, Sigma Chemical, St. Louis, MO) was added to give a bed volume of approximately 0.7 ml (6 x 25 mm) and columns drained by gravity prior to the addition of the next wash in each case. The columns were washed twice with 1 ml aliquots of distilled water, followed by 2 x 1ml washings with 2 Nammonium formate in 5 M formic acid, and finally with

4 x 1 ml rinses with distilled water. The columns were prepared in advance and stored wet, under distilled water at this stage.

Immediately prior to use, the columns were washed with 50 mM NaOH

(prepared fresh from 5 N stock NaOH, 1 to 100) until the pH of the effluent was approximately 12, as determined by pH paper,and the columns drained prior to addition of samples. The resin becomes darker in color as the pH increased, thus facilitating monitoring the progress of the final wash. Scintillation counting vials were then placed under the columns to collect the effluent after applying samples to the columns.

Samples were carefu lly applied to the prepared columns and washed on with 2 x 100 yl of 50 mM NaOH and allowed to drain. Elution of 32 C P3-phosphorylcholine was accomplished with 3 x 0.5 ml addition of

50 mM NaOH (allowing each 0.5 ml addition to drain before adding the 96 next) for a to tal elution volume of 1.7 ml. The columns were removed and 13 ml of aqueous scintillation cocktail (Thrift-Solve, Kew

S c ie n tific , Columbus, OH) was added and the ra d io a c tiv ity measured in a liquid scintillation counter (Beckman 8100 or equivalent) in an 32 14 open ( P) window. Recovery of C-labeled phosphorylcholine from these columns was determined to be 90 + 3% ('n = 5 ).

Quantitation of ACh. In all experiments, ACh-free blanks as well as samples containing known amounts of ACh were processed in an identical manner as tissue perfusates. This standard curve was generated by making a fresh stock solution of ACh (3 pmoles/yl) by adding 9.1 mg ACh to 10 ml of distilled water and diluting 60 yl of th is solution up to 100 ml with Krebs' solution containing 30 yM physostigmine (sulfate salt, 9.8 mg/1). Known volumes of this ACh- containing Krebs' 8, 25, 70, 150 and 250 yl were added to give 24,

75, 210, 450 and 750 pmoles ACh, respectively. For each sample,the volume was brought to 400 yl (as with tissue perfusates) with ACh- free Krebs'. Blanks contained only ACh-free solution generally gave

150-350 CPM. The average of duplicate blank determinations was subtracted from all measurements for the standard curve and tissue perfusates thus giving a NET CPM (to ta l CPM-blank). The lim it of sensitivity for an individual assay was taken as twice the total CPM measured in blanks. Limit sensitivity of this assay in my hands was generally 3-8 pmoles which is in agreement with others using the same method (Kato et al., 1975). Quantitation of ACh in unknown samples was calculated d ire c tly from the standard curve generated from the known samples on a Texas Instruments TI 55 calculator by a lin e a r 97

regression analysis. Correlation coefficients of pmoles plotted

against NET CPM were always greater than 0.99 and often greater than

0.9999 verifying the accuracy and linearity of this assay. A

representative standard curve and other methodological considerations

o f th is assay are provided in Appendix A.

Drugs and Chemicals

Drugs and chemicals with th e ir sources are shown in Table 5. All

solutions were prepared in demineralized double distilled (DDD) water except reserpine and 6-hydroxydopamine. Reserpine (25 mg) was

dissolved in 0.5 ml glacial acetic acid, then diluted to volume (5 ml) with DDD water. 6 -Hydroxydopamine was dissolved in 0.9% NaCl

containing 60 mg/100 ml ascorbic acid. All standard laboratory

chemicals (e.g. NaCl, HC1, e tc .) were o f reagent grade.

Treatment of Data

In all experimental procedures, data were collected in the same tissue (same cell for the EPP and MEPP analysis) both before and after drug treatment; thus each tissue (c e ll) was used as its own control for statistical analysis. Statistics were performed on the raw data using the paired Student's t-test for results (biochemical and electrophysiological) in which a treatment value was compared to its pre-drug control. For some experiments, multiple treatment values were compared to a single control value by the use of Dunnett's test, which is a modification of Student's t-test (Steel and Torrie, 1960). Values were considered significant if p <_ 0.05. For purposes o f graphic 98

TABLE 5

Drugs, Chemicals and Radionuclides Used in the Present Study

Drugs and Chemicals Source

(+ )-Amphetamine sulfate Smith Kline and French (Philadelphia, PA) (+ )-Methyl phenidate HC1 CIBA-Geigy (Summit, NJ) (R italin R ) Acetylcholine chloride Sigma Chemical Co. (S t. Louis, MO) Acetylcholinesterase, Type V Sigma Chemical Co. (S t. Louis, MO) Adenosine 5 1-triphosphate Sigma Chemical Co. (S t. Louis, MO) a-Methyl-p-tyrosine Sigma Chemical Co. (S t. Louis, MO) methyl ester Choline kinase Sigma Chemical Co. (S t. Louis, MO) Cl onidine HC1 Boehringer Ing. (W. Germany) Dithiothreitol Sigma Chemical Co. (S t. Louis, MO) Heparin sodium (10,000 U/ml) Riker Laboratories (Northridge, CA) 3-Heptanone Aldrich Chemical Co. (Milwaukee, WI) 6-Hydroxydopami ne Sigma Chemical Co. (S t. Louis, MO) (-)-Norepinephrine bitartrate Sigma Chemical Co. (S t. Louis, MO) Phentolamine mesylate CIBA (Summit, NJ) ( - ) -Phenylephrine HC1 Sigma Chemical Co. (S t. Louis, MO) Physostigmine sulfate Sigma Chemical Co. (S t. Louis, MO) (+)-Propranolol HC1 Ayerst Laboratories (New York, NY) Reserpine Sigma Chemical Co. (S t. Louis, MO) Sodi urn tetraphenyl boron Aldrich Chemical Co. (S t. Louis, MO) WB-4101 HCl W. L. Nelson (University of Washington, College of Pharmacy, Seattle, WA) Yohimbine HCl ICN (C ity of Industry, CA). 99

TABLE 5

(Continued)

Radionucl ides Source

32 [y- P]-adenosine 5'-triphosphate New England Nuclear (Boston, MA) te tra (trie th y l ammonium) s a lt [methyl-^C]-phosphorylcholine New England Nuclear (Boston, MA) chloride [l-^C]-acetylcholine chloride Amersham (Arlington Heights, IL) and tabular comparison of the results, data were converted to percentage of pre-drug control values and are presented as mean

+ S.E.M. Slopes and their deviations from parallelism were determined using methods in Sohal and Rohlf (1969). CHAPTER I I I

RESULTS

This chapter is subdivided into results of studies with amphetamine

and methylphenidate. Within each subsection, findings relative to drug

effects on muscle contraction, electrophysiological eventsand ACh

release are presented. The same experimental protocols were employed

for both amphetamine and methyl pheni date in order to fa c ilita te a

c ritic a l comparison of the neuromuscular actions of these agents (see

Discussion).

Amphetamine Effects on Nerve- and ACh-Stimulated Muscle Contractions

This series of experiments was designed to evaluate the phenomenon

o f amphetamine's enhancing and blocking effects on neuromuscular

transmission as measured by muscle contractions. The cannulated

phrenic nerve-diaphragm preparation was used to more clearly

d iffe re n tia te presynaptic versus postsynaptic drug e ffe c ts . This

d istin ctio n was accomplished by comparing nerve stimulated muscle contractions to those elicited by a pulsed injection of ACh in the

same preparation.

.Nerve stimulated contractions in the noncannulated preparation.

In these experiments, the time-response and concentration-response relationships of amphetamine on nerve stimulated muscle contractions

101 102

were established. In the absence of drug addition, nerve stimulated

muscle contractions were very stable with time; over a 120 min period,

the contraction height decreased by approximately 5% (Fig. 10, Table 6).

Because the focus of th is investigation was on the enhancing properties

o f amphetamine, only re la tiv e ly low concentrations o f amphetamine were

tested ( i.e . <540 yM); higher concentrations (>540 yM) produced blockade

of nerve stimulated contractions within 30 min (Skau and Gerald, 1978a).

The lowest amphetamine concentrations tested (135 yM) produced a

s ig n ifican t 10-12%, long-lasting (90 min) enhancement of contractions

(Fig. 10, Table .6). While 270 yM amphetamine produced a more pronounced

early enhancement of contractions (23% at 30 min), significant

enhancement lasted fo r only 45 min, followed by neuromuscular blockade which reduced contraction height below control values a fte r 75 min of

incubationand s ig n ific a n tly reduced contractions a fte r 105 min (Fig. 10,

Table 6).

Contractions in the cannulated diaphragm preparation. The

twitch response in the cannulated preparation more clearly depicts

amphetamine enhancement and blockade with time. These studies were

designed to differentiate between the pre- and postsynaptic effects

o f amphetamine by comparing the twitch responses evoked by nerve stimulation with those produced by a pulsed injection of ACh. In the absence of drug treatment, the twitch response induced by ACh

injections varied by up to + 19% about the mean over a 110 min

interval in two control preparations (Table 7). Representative results Figure 10. Effects of (+)-amphetamine on muscle contractions e lic ite d by nerve stimulation in the noncannulated diaphragm preparation. • = Control. (+)-Amphetamine: ■ = 135 yM; A = 270 yM. Each point represents the mean height of muscle contractions (+ S.E.M.) of 3 preparations expressed as a percentage of pre­ drug baseline. Values for drug treatment significantly different than pre-drug control (P < 0.05) are denoted by * .

103 Contraction height (% pre-drug baseline) ooooooooouicn-jaDcoo — f\)oj

OJ

u i ■ H

IH

ro

SOI 105

TABLE 6

Effects of (+)-Amphetamine on Noncannulated Nerve Stimulated Diaphragms

Time After Contraction Height (% of Pre-Drug Baseline)3

Addition ------(t)-Awiphetamine ------(min) Time Control 135 yM 270 yM

0 100 100 100

10 101.7 + 1.3 106.7 + 0.7 112.7 + 0.7

20 101.0 + 2.0 110.7 + 2.4 120.3 + 1.5

30 100.0 + 3.0 111.3 + 2.7b 123.0 + 2.5

45 99.7 + 2.9 112.3 + 3.2b 117.7 + 7.1

60 97.7 + 2.3 110.7 + 2.3 107.3 + 9.8

75 96.0 + 2.7 110.3 + 2.6 95.3 + 12.4

90 95.3 + 2.6 108.0 4.0 83.3 + 12.8

105 94.7 + 2.0 105.7 + 5.5 70.3 + 12.7

120 95.0 + 2.9 103.3 + 6.1 61.3 + 11.7

3 Each value represents the mean + SEM of 3 tissues. b Values significantly different from pre-drug control (contraction height at 0 min) (p < 0.05), using Dunnett's procedure (one-sided) for each concentration test. Dunnett's test is a modification of Student's t-test and is designed to compare more than one treatment to a single control. Statistical significance is more difficult to show using Dunnett's than Student's procedure, however, and thus Dunnett's test is considered a conservative estimate of differences between treatments and a single (pre-drug) control. 106

TABLE 7

Control ACh-Induced Muscle Contractions in Two Cannulated Diaphragm Preparations

Tissue 1 Tissue 2

Twitch Height Twitch Height (mm) % o f X (mm) % o f X

29 102 26 89 34 119 28 96 31 109 28 96 30.5 107 32 109 29 102 29 99 26 91 27 92 31 109 31 106 26 91 31 b 106 24 84 23 81 27 92 30 105 34 116

X = 28.5 X = 29.3 Range = 23-34 Range = 26-34 S. E. M. = 1.0 S.E.M.= 0.8 ACh = 1 nmole in 20 y l/in je c tio n ACh = 2 nmole in 20 y l/in je c tio n

Experiments were conducted as described in METHODS. ACh-induced contractions were elicited at 10 min intervals for 110 min in the absence of drug treatment. b Syringe slipped out of cannula, thus the total amount of ACh was not delivered to the muscle. 107 of a typical experiment with amphetamine are reproduced in Fig. 11 with the time-response summary of 3-4 diaphragms presented in Fig. 12 and

Table 8. Nerve-stimulated twitches were similar in both the cannulated

(Fig. 12, Table 8) and noncannulated (Fig. 10, Table 6) preparations.

Amphetamine (135 yM) enhanced twitch by 8 + 2% and 11 + 3% and (270 yM)

28 + 5% and 23 ± 3% in the cannulated and noncannulated preparations, respectively, after 30 min of incubation.

In marked contrast to the facilitatory effects of amphetamine on nerve stimulated contractions, only blockade of twitch height was observed to ACh-stimulated contractions following amphetamine. Within

5 min a fte r addition o f amphetamine (135 yM), the response to a pulsed

ACh in jection was reduced by 23 + 4%; th is decrease in the ACh-induced tw itch response progressed to as much as a 75 + 3% blockade a fte r 45 min of incubation (Fig. 12, Table 8). Incubation with higher amphetamine concentrations (270 and 540 yM) produced a more marked and rapid blockade (>90%) within 15 min a fte r amphetamine addition, resulting in a complete suppression o f ACh-induced contraction within

45 min. Blockade of contractions was also observed to nerve stimulation following a higher amphetamine concentration (540 yM); contractions were briefly enhanced following drug addition (increased 5%, 3-5 min following drug a d d itio n ), but a pronounced blockade quickly developed and reduced twitch height by 14, 67 and 95% following 10, 20 and 30 min of drug incubation, respectively (Fig. 12, Table 8). The blocking actions of the higher amphetamine concentrations was reversible upon washing the preparation with drug-free Krebs' solution. Generally, Figure 11. Representative experiment illustrating the differential effects of (+)- amphetamine on nerve stimulated (NS) and ACh (A) induced twitch in the cannulated diaphragm preparation. Following a control period, (+)- amphetamine (135 yM) was added, NS (5V, 0.2 Hz, 0.4 msec) and A (1.5 nmoles) induced twitch were recorded. Whereas NS twitch was potentiated up to 12% after 30-40 min, ACh elicited twitch was progressively reduced by up to 78% after 5 min.

108 A A Amph A A A A A 135/iM 5 min 15 min 25 min 35min 4 5 min Control------1 Figure 12. Time response o f (+)-amphetamine effects on muscle contractions elicited by nerve stimulation and by pulsed ACh (0 .8-8 nmoles) injections in the cannulated diaphragm preparation. Experiments were conducted as detailed in METHODS. Each bar represents the mean height of muscle contractions (+ S.E.M.) of 3 or 4 preparations as a percentage of pre-drug baseline. All values for drug treatment are significant for the ACh elicited twitch (open bars). S ign ificant enhancement of NS twitch (hatched bars) was noted a fte r 25-30 min with 135 pM and a fte r 15-20 min o f 270 pM amphetamine. By contrast, depression of NS contractions was observed a fte r 5-10 min with 540 pM (p < 0 .0 5 ).

110 U1 ( “p „.j - * it b m r n m m m m . ,r . _.r j ( .. , ro js a> to o i\> to , a> js ro 45 A 1 1 ro »—4—« * 3 * ■ ■ l a ------O Contraction height (% pre-drug baseline) 1 1 (ji " r " i i 'ii i i i i i t i i l i i\) cn ao O f\j .r* r\> cji a) o pj o pj a) .r* r\> cji f\j O ao i\) cn b I ------OOOOOOOOOOOOOOOOOOOOOOOACh NS 5-10 15-20 25-30 35-40 4 5 -5 0 Control Time after (+)-am phetam ine TABLE 8

Effects of Amphetamine on Nerve-Stimulated (NS) and ACh-Stimulated (ACh'S) Contractions in Cannulated Diaphragm Preparations

Contraction Height (% of Pre-Drug Baseline)3

(+ )-Amphetamine Concentration ______Time After 135 uM (n=4) 270 uM (n=3) 540 pM (n=3) Drug Addition (min) NS AChS NS AChS NS AChS

0 . 100 100 100 100 100 100

5 77.2 + 3.8 L. 26.7 + 7.8 36.7 + 5.3 10 102.6 + 0.4° 108.0 + 1.7 85.7 + 8.0bc

15 L 52.2 + 5.4 8.3 + 4.4 6.0 + 1.5 20 106.1 + 1.2 119.0 + 3.0 33.1 + 6.5 25 38.5 + 3.5 3.3 + 3.3 3.0 + 0.9 30 108.1 +1.9 128.3 + 4.5 5.4 + 1.4 35 33.9 + 1.4 0 0 40 109.3 + 1.3 122.1 + 6.2 0 45 25.4 + 3.1 0 50 109.4 + 1.6 115.0 + 8.0°

55 23.5 + 2.4 L 0 60 108.9 + 3.1 107.3 + 9.8° a Each value represents the mean + S.E.M. b Values not sig nifican tly different from pre-drug controls (p > 0.05) by Dunnett's procedure (one­ sided).

In it ia lly an enhancement of NS twitch was noted. Peak height of muscle contractions was 105 + 4.0% * ro 3-5 min following drug addition. 113

3 such washes at 5 min intervals produced complete recovery o f nerve stimulated contractions and approximately 75% return in ACh-induced twitches towards pre-drug baseline.

Electrophysiological Studies

These studies were designed to examine the effects o f amphetamine on individual skeletal muscle fib ers in the neuromuscular junction.

Intracellular electrophysiological analysis of both spontaneous (MEPP) and evoked (EPP) endplate events can answer discrete questions regarding site and mechanism of drug action. In this case, it was of value to gain insight as to whether amphetamine affects neuromuscular transmission presynaptically, postsynaptically, or both.

(+)-Amphetamine effects on the resting membrane potential (RMP) are depicted in Table 9. Tissue incubation for 20-30 min with 135 or

540 pM amphetamine failed to modify the RMP, while the highest concentration tested (1080 pM) produced a 5.5 mV (8%) decrease in the

RMP (p < 0 .0 5 ).

Analysis of endplate events (MEPPs and EPPs) indicates that both

MEPP and EPP amplitude as well as MEPP frequency are affected by amphetamine. Amphetamine concentrations ranging from 34-810 pM produced a biphasic e ffe c t on MEPP frequency, while a monophasic, concentration-dependent inhibition of MEPP amplitude was observed over the same concentration range (Fig. 13, Table 10).

The lowest amphetamine concentration tested (34 pM) produced a modest (8%) decrease in MEPP frequency, while concentrations of 68 and

135 pM enhanced MEPP frqeuency by 11 and 43%, respectively. Thisincrease TABLE 9

E ffect o f (+ )-Amphetamine on the Resting Membrane Potential (RMP)

RMP (X + S.E.M.)

DnV] Controla -71.31 + 0.31

(+)-Amphetamine 135 yM -71.18 + 0.42 540 yM -70.78 + 0.58 1080 yM -65.78 + 0.49b

Control RMP data were collected, followed by a 20-30 min drug incubation, at which time treatment data were collected. Control values represent the T + S.E.M. for 90 endplates take£ from 6 tissues. Treatment values are the X + S.E.M. for 45 endplates from 3 tissues for each drug concentration. b Value significantly different from pre-drug control (p < 0.05), unpaired Student's t- test (two-tailed). Figure 13. Amphetamine effects on miniature endplate potential (MEPP): top, frequency; bottom, amplitude. Control and treatment values were obtained in the same fiber by monitoring the response a fte r a 10-15 min exposure to drug. Control values: MEPP frequency, range = 0.91-7.36 Hz (20/22 fibers < 3.3 Hz), J = 2.43 + 0.31 Hz; MEPP amplitude, range = 0.30-0.86 mV, X = 0.45 + 0.03 mV. Each value represents the average of 3-5 preparations; N = 22 muscle fiber preparations; mean values represent the average of 75 even ts/fib er. Values s ig n ific a n tly different from pre-drug control are denoted by closed symbols.

115 MEPP Amplitude (% pre-drug baseline) MEPP Frequency (% pre-drug basepne) 100 140 120 100 80 60 20 40 60 80 0 3 30 mhtmn [x ] [/xM Amphetamine - ) + ( 100 100 1000 1000 116 117

TABLE 10

Effects of (+)-Amphetamine on Miniature Endplate Potential (MEPP) Frequency and Amplitude

MEPPs (,% of Pre-Drug Control at 10-15 min)a

(+)-Amphetamine ~X + S.E.M. Concentration (yM) N Frequency Amplitude

34 3 92.3 + 8.9 97.7 + 4.2

68 3 110.5 + 11.7 95.7 + 6.8

135 4 142.7 + 13.0b 100.1 + 14.7

270 4 111.3 + 3.7b 74.4 + 7.9b

540 5 114.4 + 14.1 54.5 + l . l b

810 4 71.7 + 5.6 71.8 + 8. lb

Data were collected in each cell before and continuously following drug addition with the values presented representing the mean (+_ S.E.M.) response 10-15 min a fte r drug as a percentage of pre­ drug values. Control values are based on an average of 75 MEPPs collected from each fib e r: MEPP frequency, range = 0.91-7.36 Hz, with 20/22 < 3.3_Hz, X = 2.43 + 0.31 Hz; MEPP amplitude, range = 0.30-0.86 mV, X = 0.45 + 0.03 mV (n = 22 fibers). b Values significantly different from pre-drug control (p < 0.05) by paired Student's t-test (two-tailed) performed on the raw data before percentile transformation. 118

in MEPP frequency re fle c ts enhanced spontaneous release o f ACh. Higher

concentrations (270 and 540 yM) reduced this fa c ilita to ry e ffe c t to a

10-15% increase in frequency, while 810 yM reduced MEPP frequency to

28% below pre-drug levels (Fig. 13, Table 10).

Amphetamine (34-135 yM) produced no effects on MEPP amplitude.

Higher concentrations of amphetamine ranging from 270-810 yM produced

a concentration and time-dependent blockade of MEPP amplitude, reducing

amplitudes by 25-45%. Exposure to these higher amphetamine

concentrations, notably above 540 yM, completely suppressed MEPP

amplitude into the baseline noise level of the recording instruments

(150 yV) (Fig. 13, Table 10).

Analysis o f representative MEPP amplitude histograms (Fig. 14)

suggests that MEPP amplitude and frequency may be in terre late d at

higher (810 yM) but not lower (135 yM) amphetamine concentrations. Low

concentrations (Fig. 14, top),while not significantly modifying MEPP amplitude (91% of control), potentiated the MEPP frequency by 51%. By

contrast, tissue exposure to 810 yM (Fig. 14, bottom) fo r 10 min caused a marked shift to the le ft in the amplitude profile resulting in a decreased MEPP amplitude and frequency by 35 and 36%, respectively

(Fig. 14). The decrease in MEPP frequency produced by the higher amphetamine concentrations (F ig. 13) may actu ally be the consequence of a shift to the left in the amplitude profile (Fig. 14) resulting in the lower amplitude MEPPs being quenched into instrument noise levels

(below 150 yV). This suppression of MEPP amplitude suggests that

postsynaptic s e n s itiv ity to ACh is reduced (e .g ., possibly by a cu rare-like e ffe c t). In support of th is notion, tissue exposure to Figure 14. Amphetamine effects on MEPP amplitude and frequency. Top and bottom panels represent the response of representative endplates prior to and 10 min after exposure to 135 and 810 yM amphetamine, respectively. At 135 yM, frequency was increased by 51% with no change in amplitude. By contrast, 810 yM reduced both amplitude and frequency by 35 and 36%, respectively. The hatched bars represent instrument noise levels ('v.l50 yV). Vertical dashed lines represent mean MEPP amplitudes.

119 # of Observations 40 40 20 30 20 30 10 10 F3 z:C z £ z £ z

. 03 . 05 0.6 0.5 0.4 0.3 0.2 . 03 . 05 . 02 . 04 . 0.6 0.5 0.4 0.3 0.2 0.6 0.5 0.4 0.3 0.2 11—T il r-rg otdu ( min) 0 (1 Post-drug Pre-drug Pre-drug TTW +-MHTMN (8I0MM) (+)-AMPHETAMINE +)APEAIE (135 )-AMPHETAMINE (+ MEPP Amplitude (mV) ti J 0.46 mV 5.20 Hz 0.32 mV l j 1.08Hz L

r-^7 0 4 40 20 30 20 30 10 10 Z X Z Z Z Z Z Post-drug (10 min) (10 Post-drug . 03 . 05 0.6 0.5 0.4 0.3 0.2 fM) CL -m ^ r T L J 0.29 mV 0.30 mV 3.34 Hz 1.63Hz

o ro 121 the higher amphetamine concentrations (above 540 yM) fo r longer than

15-20 min resulted in the elimination of MEPP frequency and amplitude.

This depressant effect was reversible upon washing the tissue with drug- free Krebs' solution.

A sim ilar biphasic e ffe c t of amphetamine was observed in the amplitude of nerve stimulated EPPs. Amphetamine concentrations of 68 and 135 yM elevated EPP amplitude by 24 and 16%, respectively (Fig. 15,

Table 11). Higher concentrations (270-810 yM) produced a concentration- dependent 29-53% reduction in the EPP amplitude. As was observed with

MEPPs, prolonged exposure to higher concentrations (above 540 yM) completely suppressed the EPP amplitude into instrument noise levels

(150 yV), an effect reversible upon washing with fresh Krebs' solution.

ACh Release Studies

These studies were designed to directly examine the hypothesis that amphetamine enhances the nerve-evoked release of ACh. Preliminary experiments were conducted to establish a nerve stimulation frequency to e lic it sufficient ACh release; 0.2 and 2.0 Hz did not release measurable levels of ACh (i.e ., below 1.0 pmoles/min), while 7 and 10 Hz were found to release enough ACh to measure. Preliminary control experiments were conducted at 10 Hz in the absence o f drug treatment and were determined to evoke relatively consistent levels of

ACh release throughout the 6 (10 min) stimulation periods (Table 12).

Intertissue v a ria b ility , however, was rather high. Pre-amphetamine release in these experiments ranged from 1.0-13.1 pmoles/min (X = 4.3

+0.2 pmoles/min, n = 94 preparations). The average total amount of Figure 15. Amphetamine effects on the nerve stimulated endplate potential (EPP). Transmission was p a rtia lly blocked by raising the Mg++ concentration to 12.5 mM. Control and treatment values were obtained in the same fiber, and the EPP response was determined over a 5 min period 10-15 min after drug. Low amphetamine concentrations (68 and 135 yM) enhanced EPP amplitude,while higher concentrations produced depression. Each value represents the average of 3-6 preparations. Control value = 1.08 mV + 0.08 mV, n = 23 muscle fiber preparations. Individual EPP amplitudies represent the average of 60 EPPs/fiber recorded over 5 min for each control and treatment period. Values significantly different from pre-drug control are denoted by closed symbols.

122 EPP Amplitude (% pre-drug baseline)

00 o ro O O O o o o

I > 3 TD=r CD o Q o 3 D CD

o o ro o CO 124

TABLE 11

Effects of (+ )-Amphetamine on the Nerve Stimulated Endplate Potential (EPP)a

EPP Amp!itude (+)-Amphetamine 10-15 min Concentration Following Drug Addition (yM) N (% of Pre-Drug Control)

34 3 96.0 + 11.1

68 6 124.2 + 6.1

135 5 116.0 + 4.6

270 5 70.6 + 1.8

540 3 57.9 + 11.4

810 3 47.5 + 9.01

To prevent muscle tw itching, neuromuscular transmission was partially blocked by raising the [Mg++] to 12.5 mM. Data were collected in each cell before and continuously following drug ad dition. Values presented represent the response 10-15 min after drug as a percentage of pre-drug control. Control value X = 1.08 +_ 0.08 mV (range = 0.44 - 2.18 mV), n = 23 muscle fib e r preparations. Individual EPP amplitudes represent the average of 60 EPPs/fiber recorded over 5 min for each control and treatment period. k Values s ig n ific a n tly d iffe re n t from pre-drug control (P < 0.05) by paired Student's t-test (two-tailed) run on the raw data before percentile transformation. TABLE 12

Nerve Stimulated Release of ACh in 2 Control Preparations®

Tissue 1 Tissue 2

ACh Release ACh Release (pmoles/10 min) (pmoles/10 min)

64.3 49.6 75.2 34.8 56.8 31.0 65.2 50.2 60.2 28.2 62.7 27.5

X = 64.1 X = 36.9 Range = 56.8 - 75.2 Range = 27.5 - 50.2 S.E.M. = +2.5 S.E.M. = +4.2

Experiments were conducted as described in METHODS. Nerves were stimulated a t 10 Hz fo r six successive 10 min intervals, interspersed with 5 min rest periods. 126 ACh released in these studies was siightly lower than has been reported by others. Bierkamper and Goldberg (1978), using the same vascular perfused diaphragm preparation employed in the present studies, reported ACh release values ranging from 3.0-8.0 pmoles/min.

Methodological differences, however, make a d irec t comparison of my experiments with those of Bierkamper and Goldberg (1978) d if f ic u lt .

They used continuous 7 Hz stimulation, and perfusion with HEPES buffer

(unspecified pH) containing 30 yM choline in the absence of an AChE inhibitor, whereas my protocol employed 10 Hz stimulation periods

(interspersed with 5 min rest periods), and perfusion with Krebs' solution containing 30 yM eserine but no choline. Employing bioassay procedures, others have reported nerve stimulated (10-12 Hz) ACh release values from the rat phrenic nerve of 5.5-11.1 pmoles/min

(Straughan, 1960), and 6.6 -15 .0 pmoles/min (Krnjevic and M itch ell,

1961). No explanation is given for the somewhat low ACh release values gathered under the experimental conditions employed in the present study. However, intratissue release was found to be consistent from one stimulation period to the next (Table 12).

Moreover, the average of the two control and drug treatment periods were used fo r s ta tis tic a l comparison o f drug effects (see METHODS) and many (3-11) tissues were used to demonstrate each drug e ffe c t on ACh release. Therefore, the results obtained concerning drug effects on

ACh release in the present studies should be considered reliable and are presented as a percentage of pre-drug values to facilitate comparison and in terp reta tio n . 127 Amphetamine effects on ACh release. Tissue perfusion with

amphetamine (30-300 yM) s ig n ific a n tly enhanced ACh release by 0 .5 -4 .8

fold in a concentration dependent manner; the highest amphetamine

concentration tested (1 mM) reduced ACh release by 50% (F ig. 16, Table

13). While this experiment demonstrated that low amphetamine

concentrations augment ACh release, no insight could be gained

regarding a possible mechanism for this effect.

Amphetamine-Catechol amine Interaction Experiments

A ll subsequent groups of experiments (catecholamine interactions

and a-adrenoceptor effects) were conducted employing ACh release

studies in parallel with muscle contraction experiments in order to

examine the mechanism responsible fo r the above e ffe c t of amphetamine

on ACh release.

Effects of catecholamine modifying agents on amphetamine's

enhancing e ffe c ts . In this series of experiments, animals were

pretreated with the tyrosine hydroxylase inhibitor, a-methyl-p-tyrosine methyl ester (a-MT), the catecholamine depleting agent, reserpine, or

the peripheral catecholamine nerve terminal destroying agent 6- hydroxy-

dopamine (6-OH-DA, administered i .v.). These experiments were designed

to determine whether the effects of amphetamine on nerve evoked twitch

and ACh release are mediated indirectly, via catecholamine release.

Prior to sacrificing the animals treated with a-MT, reserpine or

6-OH-DA, i t was noted that they displayed signs o f catecholamine

depletion (e.g., ptosis, diarrhea, hypothermia and generalized

behavioral depression). Any animals so pretreated which failed to Figure 16. Amphetamine effects on nerve evoked release of ACh from the phrenic nerve. Experiments were conducted as described in METHODS; b rie fly , diaphragms were perfused with Krebs' solution containing eserine (30 uM) at the rate of 40 ul/min. Tissue perfusates were collected and assayed fo r ACh following nerve stim ulation (10 Hz) for successive 10 min intervals (interspersed with 5 min rest periods).

128 129

8 0 0

700

o 600 - CP 2 500 -

0.03 0.1 0.3 ( + ) - Amphetamine (mM) 130

TABLE 13

Amphetamine Effects on Nerve Stimulated Release o f ACh From the Phrenic Nerve3

Amphetami ne Concentration . (yM) ...... ACh.Release0 ...... N

30 144 + 4.6 4

100 417 + 95.0 4

300 578 + 188 5

1000 49.5 + 12.8 4

a The cannulated, vascular perfused diaphragm was stimulated at 10 Hz. Samples were collected and assayed as described in METHODS. k Values represent the mean (+ S.E.M.). ACh release values are expressed as a percentage of pre-drug release with each tissue used as its own control. Pre-drug ACh release in these tissues ranged from 1.0 -7.5 pmole/min (X = 3.6 +0.5 pmole/min). All values are significantly different from pre-drug control (p < 0.05) by paired Student's t-test (two-tailed) performed on the raw data before percentile transformation. 131 exhibit any of these symptoms were not employed in the studies presented below.

Amphetamine (270 yM),when administered to control tissues, s ig n ific a n tly enhanced muscle contractions by as much as 23%. However, the same amphetamine concentration (270 yM) failed to significantly potentiate twitch in diaphragms obtained from animals pretreated with a-MT (Fig. 17, Table 14). In contrast to the effect of a-MT pretreatment, animal pretreatment with reserpine did not alter the potentiating effects of amphetamine on nerve stimulated contractions

(Fig. 17, Table 14). These results suggest that amphetamine-induced enhancement of muscle twitch may be mediated via the release of newly synthesized catecholamines (i.e., norepinephrine). In order to further test this hypothesis, animals were pretreated peripherally

(i.v.) with 6-hydroxydopamine (6-OH-DA) by a treatment schedule shown to destroy peripheral catecholaminergic nerve terminals (Thoenen, 1971).

Diaphragms obtained from such animals responded to amphetamine (270 yM) in a manner similar to animal pretreatment with a-MT (Fig. 17, Table

14), supporting the hypothesis th at amphetamine's effects on muscle contraction may be indirect, mediated via catecholamine release from aminergic nerve terminals.

Parallel experiments were conducted to test whether these treatments modify the ab ility of amphetamine to enhance ACh release.

Amphetamine alone (100 yM) enhanced release of ACh to 417% of control

(Fig. 18, Table 15). Following animal pretreatment with a-MT or

6-OH-DA amphetamine produced no enhancement of ACh release (89.1 and

117% o f co ntrol, resp ectively). By contrast following animal Figure 17. Effects of animal pretreatment with catecholamine-modifying agents on amphetamine (270 yM) enhancement of nerve-stimulated contractions. Pretreatments: X, control vehicle; □ , reserpine (5 mg/kg, i.p., 18 hrs); 0, a-m ethyl-p-tyrosine (250 mg/kg, 1.p., 18 hrs); A, 6-hydroxydopamine (50 mg/kg, i.v., on days 1 and 2, determination on day 4). Each point represents the mean contraction height (+ S.E.M.) of 3 tissues expressed as a percentage o f pre­ drug baseline.

132 Contraction height (% pre-drug baseline) 140 120 0 8 0 10 20 ie (min) Time 0 3 5 4 0 6 TABLE 14

Effects of Animal Pretreatment with Catecholamine-Modifying Agents on Amphetamine^Induced Enhancement of Muscle Twitch

Time After Contraction Height (% of Pre-Drug Baseline) ______Amphetami ne (270 yM) ______Treatment ______Addi ti on . . (min) Control a-MTc Reserpine 6-0H-DAe

0 100 100 f 100 100 , 10 112.7 + 0.7 103.7 + 0.8* 111.0 + 1.4 99.5 + 1.1* 20 120.3 +1.5 106.3 + 2.8* 120.4 + 2.8 101.1 + 1.7* 30 123.0 + 2.5 104.4 + 3.7L 126.0 + 3.4 103.7 + 0.8* 45 117.7 + 7.1 96.3 + 2.8* 126.9 + 4.8 101.6 + 1.6* 60 107.3 + 9.8 85.6 + 3.8* 110.1 + 5.3 99.6 + 1.5

3 Each value represents the mean + S.E.M. of 3 tissues. ^ Represents vehicle control treatment; amphetamine (270 yM) was added to the nerve-muscle bath and twitch followed over time. c a-Methyl-p-tyrosine methyl ester, 250 mg/kg, i.p., 18 hr pretreatment. ^ 5 mg/kg, i.p., 18 hr pretreatment. e 6-Hydroxydopamine, pretreatment schedule: 50 mg/kg, i.v ., on days 1 and 2, with animals sacrificed on day 4. f Denotes values significantly (p < 0.05) different from vehicle control (column b) compared at the same time a fte r amphetamine addition by Dunnett's procedure (one-sided). Figure 18. Effects of animal pretreatment with catecholamine-modifying agents on amphetamine (100 yM) enhancement of ACh release. Pretreatments: reserpine (5 mg/kg, i . p . , 18 hrs); a-methyl-p- tyrosine (a-MT; 250 mg/kg, i . p . , 18 h rs ); 6-hydroxydopamine (6-OH-DA; 50 mg/kg, i . v . on days 1 and 2, with animals sacrificed on day 4 ).

135 500

Q) <0 0 400 JQ c n 3 w “D 300 1 a> Q. s P 200

a> o Q) 4 * io o

Effects of Animal Pretreatment with Catecholamine- Modifying Agents on Amphetamine-Induced Enhancement o f ACh Release

N Treatment ACh Release9

Amphetamine alone 417 + 95 .0e 4 (100 yM)

+ a-Methyl-p- 89.1 + 8.9 4 tyros i neD

+ Reserpinec 228 + 35. l e 8

+ 6-Hydroxydopamined 117 + 11.9 3

Values represent the mean (+ S.E.M.) compared to pre­ drug release expressed as a percentage of pre-drug control. Pre-drug ACh release iri these tissues ranged from 1.4-10.2 pmole/min (X = 4.4 + 0.6 pmoles/ m in). b 250 mg/kg, i . p . , 18 hr pretreatment. c 5 mg/kg, i . p . , 18 hr pretreatment. d 50 mg/kg, i . v . , on days 1 and 2, with animals sacrificed on day 4. e Values s ig n ific a n tly d iffe re n t from pre-drug control (p < 0.05), paired Student's t-test (two-tailed). 138 pretreatment with reserpine, amphetamine significantly enhanced ACh 3 release to an average of 228% of control;a 60% reduction compared to amphetamine alone with no pretreatment (417% of control). It should be noted that tissue responses following reserpine pretreatment were highly variable; of 8 tissues tested, ACh release was enhanced in 6

(range = 160 - 394% of co n tro l), depressed in 1 (80% of control) and not changed in 1 (106% of co n tro l).

Effects of g-adrenoceptor antagonists on amphetamine's enhancing e ffe c ts . Based on the a b ility of a-MT or 6-hydroxydopamine pretreatment to abolish the potentiating effects of amphetamine on

ACh release and muscle contractions, catecholamines were hypothesized to contribute to these effects. This series of experiments were designed to evaluate whether a catecholamine interaction with presynaptic a-adrenoceptors located on motor nerve terminals underlies the enhancing effects o f amphetamine on ACh release and muscle tw itch.

Moreover, an attempt was made to determine the a-adrenoceptor type

(a^ or otg) which mediates these effec ts. This was accomplished by employing selective a-adrenoceptor antagonists (phentolamine, WB 4101 and yohimbine) at concentrations which produce no effects, when employed alone, on the parameters under investigation. ACh release did not vary by more than + 5% following incubation with these agents

(Table 18). S im ilarly, the time responses of muscle contractions in

3 This value was obtained using the following formula: (1 % change o f amphetamine -f reserpinex , 0Q % change of amphetamine alone ' where % change = % of control - 100. 139 the presence of these antagonists did not differ significantly from the time response with no drug added (maximum decrease of 7.5% a fte r

60 min of incubation).

Phentolamine (nonselective and a2-adrenoceptor antagonist) abolished the a b ility of amphetamine to enhance muscle contractions

(Fig. 19, Table 16) and ACh release (Fig. 20, Table 17). Inhibition o f muscle contractions (p < 0.05) was observed 45 min a fte r phentolamine

(10 yM) incubation with amphetamine. The selective a^-adrenoceptor antagonist WB 4101 also blocked the a b ility o f amphetamine to enhance nerve stimulated contractions and ACh release. Inhibition of muscle contractions (p < 0.05) was observed 60 min a fte r WB 4101 (3 yM) incubation with amphetamine (Fig. 19, Table 16). In contrast to the above effects employing agents which block a-^-adrenoceptors (WB 4101 and phentolamine), the selective ^-an tag o n ist yohimbine did not s ig n ific a n tly reduce the a b ility o f amphetamine (270 yM) to enhance muscle contractions (Fig. 19, Table 16), or to enhance ACh release

(Fig. 20, Table 17). Yohimbine decreased the amphetamine induced enhancement of ACh release by 39%^, from 417% to 293% of control

(p > 0.05).

This value was obtained based on the following formula:

M _ % change o f amphetamine + yohimbine* , nn % change o f amphetamine alone 1 where % change = % of control - 100. Figure 19. Effects of a-adrenoceptor antagonists on amphetamine-induced modification of nerve-stimulated contractions. Tissues were pre-incubated with antagonist for 20 min prior to the addition of amphetamine (270 yM). X, amphetamine alone; 0, yohimbine (10 yM) ; □ , WB 4101 (3 yM); A, phentolamine (10 yM). Each point represents the mean contraction height (+ S .E .M .) of 3 tissues expressed as a percentage of pre-drug baseline.

140 Contraction height u JD vO | | ) a o co a> a. a. D I 120 100 40 0 6 - 0 8 0 3 0 2 ie (min) Time TABLE 16

Effects of a-Adrenoceptor Antagonists on Amphetamine-Induced Modification of Nerve-Stimulated Twitch

Time After Contraction Height [% of Pre-Drug Baseline)* Amphetami ne (270 yM) Treatment Addition (min) Control Phentolamine WB 4101 Yohimbine

0 100 100 100 100

10 112.7 + 0.7 101.7 + 1.7d 104.1 + l . l d 111.1 + 1.1

20 120.3 + 1.5 102.3 + 2.4d 108.9 + 0.7d 118.3 + 2.5

30 123.0 + 2 .5 94.3 + 4.7d 106.2 + 1.4d 119.0 + 2.5

45 117.7 + 7.1 61.3 + 6.5d 94.6 + 6.7d 110.6 + 2.3

60 107.3 + 9.8 42.0 + 7.3d 69.0 + 9.5d 95.3 + 4.7

a Each value represents the mean + S.E.M. of 3 tissues. k Represents no tissue pretreatment, amphetamine concentration of 270 yM was added and followed over time for a ll treatments. Tissues were pre-incubated for 20 min prior to the addition of amphetamine with phentolamine (10 yM), yohimbine (10 yM) or WB 4101 (3 yM). d Values significantly different from control (column b, no tissue treatment) compared at the same t time after amphetamine addition (p < 0.05), Dunnett's t-test (one-sided). j Figure 20. Effects of a-adrenoceptor antagonists on amphetamine-induced enhancement of ACh release. Following collection of control (pre-drug) samples, tissues were perfused with amphetamine alone (100 yM) or in combination with phentolamine, WB 4101 or yohimbine (a ll 10 yM), with perfusates collected and analyzed for released ACh.

143 ACh Release (% pre-drug baseline) 0 0 4 0 0 2 0 0 3 0 0 5 100 - - - - - mhtmn Apeaie mhtmn Amphetamine Amphetamine Amphetamine Amphetamine (10 0/tM ) + Phentolamine +WB 4101 + Yohimbine + 4101 +WB Phentolamine + ) 0/tM (10 TABLE 17

Effects of a-Adrenoceptor Antagonists on Amphetamine-Induced Enhancement o f ACh Release

Treatment ACh Release3 N

Amphetamine alone 417 '+ 95b 4 (100 yM)

+ Phentolamine 127 + 35.5 3 (10 yM)

+ WB 4101 (10 yM) 123 + 12.5 4

+ Yohimbine (10 yM) 293 + 4 8 .8 bc 4

Values represent the mean (+ S.E.M.) compared to pre­ drug release expressed as a percentage of pre-drug control. Pre-drug ACh release in these tissues ranged from 1 .8 -6 .8 pmole/min (X = 3.5 + 0 .4 pmole/min). b Values significantly different from pre-drug control (p < 0.05), paired Student's t-test (two-tailed).

Values not s ig n ific a n tly d iffe re n t (p > 0.05) from amphetamine alone. 146

Noradrenergic Effects on ACh Release and Nerve Stimulated Contractions

Since a catecholaminergic interaction with a-adrenoceptors may be underlying the effects of amphetamine, i t was logical to hypothesize that NE could have effects similar to amphetamine. Therefore, as an internal control, NE (50 pM) was tested and determined to enhance ACh release to 283% of pre-drug control values (Fig. 21, Table 18).

Moreover, this effect of NE was abolished by simultaneous incubation with phentolamine or WB 4101. Yohimbine treatment reduced the NE- induced enhancement o f ACh release by 27% (calculated as outlined above), from 283 to 233% o f control (Fig. 21, Table 18). Yohimbine produced a sim ilar degree of inhibition(39% ) on amphetamine-enhancement o f ACh release (from 417 to 293% of control, Table 17).

It was also of interest to examine the effects of NE and other more selective agonists and antagonists on nerve stimulated contractions 5 in the isolated diaphragm preparation. NE, when added cumulatively , produced a concentration-dependent enhancement in nerve stimulated muscle contractions (Fig. 22, Table 19). Pre-incubation with phentolamine (10 pM) resulted in a modest but sig nifican t 4-6% decrease in NE enhancement in twitch; WB 4101 pretreatment (3 pM) produced a similar 5-6% depression but only at the highest NE concentrations

(3 x 10"4 - 10'^M). By contrast, yohimbine pretreatment (10 pM)

5 In preliminary experiments, cumulative addition of NE at 5 min intervals was determined to be optimum for assessing changes in twitch. Following NE addition, maximum twitch enhancement occurred within 2-4 min and, if no higher NE concentration was added, the twitch response began to decay. All experiments with NE and other adrenergic agonists were conducted using cumulative 5 min drug additions. Figure 21. Effects of a-adrenoceptor agonists and antagonists on ACh release. Following collection of control (pre-drug) samples, tissues were perfused with NE (50 yM), clonidine (30 yM) or NE + phentolamine, WB 4101 or yohimbine (a ll at 10 yM), with perfusates collected and analyzed for released ACh.

147 CD = 300 H CD If) a

CP =3 XJ 2 0 0 - I S> Q. a> 100- ■ £ o Q> tr(D SI

Effects of c*-Adrenoceptor Agonists and Antagonists on ACh Release-

Treatment ACh Release9 N

NE (50 yM) 283.1 + 41.5b 11

+ Phentolamine (10 yM) 102.0 + 7.8 4

+ WB 4101 (10 yM) 93.9 + 7.7 4 o o

+ Yohimbine (10 yM) 233.0 + • 4

Clonidine (30 yM) 87.4 + 15.5 3

Phenotolamine (10 yM) 101.9 + 5.5 4

WB 4101 (10 yM) 101.7 + 6.2 3

Yohimbine (10 yM) 98.5 + 16.8 4

Values represent the mean (j^ S.E.M .) compared to pre­ drug release expressed as a percentage of control. Pre­ drug ACh rejhease in these tissues ranged from 1.1-13.1 pmole/min (X = 4.6 + 0.4 pmoles/min). b Values significantly different from pre-drug control (p < 0.05), paired Student's t-test (two-tailed). Figure 22: Effects of norepinephrine (NE) alone and in combination with selective adrenergic antagonists on nerve stimulated muscle contractions. Tissues were preincubated with antagon­ ists for 20 min prior to the addition of NE. NE was added in a cumulative manner at 5 min in tervals: X, NE alone; ■, yohimbine (10 yM); • , phentolamine (10 yM); A, WB 4101 (3 yM); ♦, propranolol (10 yM). Each point represents the mean (± S.E.M.) contrac­ tion height of 3 tissues expressed as a percentage o f pre-NE baseline as assessed 5 min a fte r each NE addition. Standard errors ranged from 0.2-3.6% (X = 1.1%, Table 19).

150 Contraction height (% pre-drug baseline)

o ro oj o O O O -ji

o.I o>

CD ■g z > ’ CD •o Oi Ul CD

o,

I

ISI TABLE 19

Effects of Norepinephrine Alone and with Selective Antagonists on Nerve Stimulated Contractions

______Contraction Height (% of Pre-Drug _Baseline)9 ______

______NE +_Antagonist^ ______Cumul ative Total NE Phentolamine WB 4101 Yohimbine Propranolol [NE] (M)c Alone (10 yM) (3 yM) (10 yM) (10 yM)

Control 100 100 100 100 100 10-7 101.4 + 0.7 99.7 + 0.3 99.3 + 0.7 100 + 0 100 + 0 3 x IQ"7 101.1 + 0.7 98.3 + 0.9. 99.7 + 0.3 100.2 + 0.2 . 102.2 + 0 .7 IO"6 _ 103.4 + 1.2 99.6 + 0.4 101.0 + 0.6 100.4 + 0.4° 103.3 + 0.6 3 x IQ"6 105.7 + 0.4 102.8 + 1.5 104.3 + 1.8 100.4 + 0.4° 104.3 + 0.1. io -5 111.0 + 0.6 106.7 + 1.5° 109.0 + 2.1 103.0 + 1.9° 104.8 + 0.2^ 3 x io -5 115.9 + 0.5 110.7 + 1.9° 113.0 + 1.7. 111.0 + 1.8° 105.8 + 0.6j 10"4 120.7 + 0.7 114.1 + 1.0° 114.3 + 2.3° 117.3 + 2.3 104.9 + 0.3° 3 x 10~4 121.5 + 0.8 117.5 + 1.3d 116.3 + 2.3° 123.6 + 3.6 104.9 + 0 .3 . 10-3 122.4 + 1.2 118.1 + 0.7° 117.7 + 1.9° 127.7 + 3.5 106.1 + 0.8°

a Each value represents the mean (+ S.E.M.) of 3 tissues, k Tissues were preincubated with antagonists for 20 min prior to NE addition. c NE was added in a cumulative manner at 5 min intervals. ^ Values significantly different from NE alone at the same NE concentration (p < 0.05) Dunnett's procedure (one-sided). ^ CXI I\3 s ig n ific a n tly reduced the potentiating effects of lower NE -6 -5 concentrations (10 - 3 x 10 M), but did not reduce the maximum -3 enhancement observed to NE (10" M); indeed, a modest (5.3%) increase was noted. An approximate 0.5 fold shift to the right in the NE concentration-response curve was observed following yohimbine preincubation, while phentolamine and WB 4104 produced a lesser shift

(Fig. 22, Table 19). While propranolol (3-blocker) did not alter the enhancement of contractions produced by lower NE concentrations (up to

3 x 10”6 M), a significant reduction was produced in the twitch enhancement of higher NE concentrations (10”^ - 10"^ M), from 11-22% to 5-6% (Fig. 22, Table 19).

To further investigate the nature of the presynaptic a-adrenoceptor hypothesized to mediate enhanced ACh release from motor neurons, the selective a-adrenoceptor agonists phenylephrine and clonidine were compared with NE for their ability to enhance nerve- stimulated contractions. The selective a 2-adrenoceptor agonist clonidine (30 yM) failed to modify the nerve stimulated release of

ACh (Fig. 21, Table 18). Consistent with the failure of clonidine to enhance ACh release was the finding that this agent did not enhance nerve stimulated contractions. Clonidine produced significant O inhibition of twitch at high concentrations (31% decrease at 10 M)

Fig. 23, Table 20). By contrast, phenylephrine, a selective a^- adrenoceptor agonist, produced a concentration-dependent increase in nerve-stimulated contractions. Although this agent produced significantly less enhancement than NE at concentrations ranging from -5 -4 10 - 3 x 10 M, the maximum response to phenylephrine did not d iffe r from that with NE (119.5 vs. 122.4%) (Fig. 23, Table 20). Figure 23. Effects of selective a-agonists on nerve stimulated contractions. Agonists: X, NE; • , phenylephrine; ■ , clonidine were added in a cumulative manner at 5 min intervals. Each point represents the mean (± S.E.M.) contraction height of 3 tissues, expressed as a percentage of pre-drug baseline, and assessed 5 min after each cumulative addition. Standard errors ranged from 0.3 to 1.9% with the exception of clonidine at 10-3M (3.2%).

154 oo o o (0 o o CD Contraction height (% pre-drug baseline) j -> o I i i I a> Ol o o O O o

[Agonist] (M ) 9 SI 9 156

TABLE 20

Effects of Selective a-Adrenoceptor Agonists on Nerve Stimulated Contractions

Contraction Height (% Pre-Drug Baseline)* [Agonist] NE Phenylephrine Cl onidine

Control 100 100 100

10" 7 101.4 + 0.7 99.7 + 0.3 100.2 + 1.2

! X IO"7 101.1 + 0.7 100.5 + 0.5 100.5 + 1.4 1 o>

o 103.4 + 1.2 102.0 + 0.5 99.0 + 1.1

i x 10"6 105.7 +0.4 102.6 + 1.1 98.6 + 0.9C

10"5 111.0 + 0.6 103.8 + 2.0C 96.8 + 1.0°

; x 10"5 115.9 + 0.5 106.2 + 1.9C 96.9 + 0.9C 1 -P*

I—* O 120.7 + 0.7 112.3 + 1.5C 98.9 + l.lc

x 10"4 121.5 + 0.8 115.5 + 1.0C 103.8 + 1.9C

10" 3 122.4 + 1.2 119.5 + 0.3 69.3 + 3.2C

a Each value represents the mean (+ S.E.M.) of 3 tissues.

^ Agonists were added in a cumulative manner at 5 min intervals. c Values s ig n ific a n tly less than NE at the same [NE] (p < 0 .0 5 ), Dunnett's t-test (one-sided). 157

Methylphen i date Res ul t»s

Overview

This series of experiments was designed to evaluate the effects of methylphenidate on neuromuscular transmission under the same experimental conditions as was employed for the evaluation of amphetamine's effec ts. Thus, th is provides the basis fo r a systematic comparison of the effects of these two drugs on neuromuscular transmission.

Muse!e contraction studies. Methylphenidate produced a biphasic effect on nerve stimulated muscle contractions. Low concentrations

(30 and 100 yM) enhanced twitch by 4-13% and 23-49%, respectively, over a 2 hr incubation and 300 yM produced a profound 34-106% increase in twitch height over the same time period. Higher methylphenidate concentrations (600 and 1000 yM) produced a pronounced early enhancement of twitch by 70 and 52%, respectively, 5-10 min following drug addition, followed by a dominant blocking action th at completely inhibited contractions within 20-30 min (Fig. 24, Table 21).

By plotting methylphenidate concentration versus contraction height 10 min following drug addition, a biphasic response is c le arly observed (F ig. 25, from data in Table 21). Low concentrations (30-

300 yM) produced a concentration-dependent enhancement of nerve- stimulated contractions by 10-90%, while higher concentrations reduced Figure 24. Effects of methylphenidate on nerve stimulated muscle contractions. X, time control; methylphenidate: •, 30 y M ;*, 100 yM; A, 300 yM; ♦ , 600 yM; t , 1000 yM. Each point represents the mean (+ S.E.M.) height of muscle contractions of 3 preparations expressed as a percentage of pre-drug baseline followed over time.

158 o o o oo oo o ro [\> [\> 4* O O _ _ _no _ _ iv> — o oo Contraction height (% pre-drug baseline) _ ro - ro ai “ o “ ai ro o ~

Time (min) 6 SI 6 TABLE 21

Effects of Methylphenidate on Nerve Stimulated Muscle Contractions

Contraction Height {% Pre-Drug Baseline)9

Time After Methylphenidate (pM) Drug Additior i Time (min) Control 30 100 300 600 1000

0 100 100 100 100 100 100

5 - 105.0 + 1.2b 123.4 + 5.5 165.2 + 2.1 170.0 + 2.0 151.6 + 2.4 10 101.7 + 1.3 109.5 + l.tf* 135.9 + 5.5 189.5 + 5.2 158.0 + 3.0 29.3 + 2.8 20 101.0 + 2.0 112.7 + 2.8b 144.0 + 3.5 206.2 + .7.0 31.0 + 8.0 0 30 100.0 + 3.0 112.5 + 2.8b 144.1 + 1.8 204.9 +8.3 0 45 99.7 + 2.9 111.3 + 1.2 149.1 + 4.5 197.1 + 5.1 60 97.7 + 2.3 110.0 + 1.3 148.9 + 5.6 181.0 + 6.6 75 96.0 + 2.7 108.5 + 1 .3b 149.2 + 6.6 169.0 + 9.5 90 95.3 + 2.6 106.3 + 0.9b 149.2 +6.6 157.9 + 11.0 105 94.7 + 2.0 105.3 + 0.7b 146.7 + 7.2 145.3 + 11.lb 120 95.0 + 2.9 104.3 + 0.8b 145.0 + 6.6 133.7+ 9.8b

a Each value represents the mean (+ S.E.M.) of 3 tissues. b Values not significantly different from pre-drug control (p > 0.05), Dunnett's procedure (one-sided). Figure 25. Concentration-dependent effects of methylphenidate on nerve stimulated muscle contraction following 10 min of drug incubation. Each point represents the mean (+ S.E.M.) contraction height of 3 tissues expressed as a percentage of pre-drug control (data taken from Table 21 at 10 min).

161 Contraction height (% pre-drug baseline)

CD nr

“O = 5" CD 2. CL Q CD 3

• - i 163 this enhancement (600 yM) or resulted in neuromuscular blockade

(1000 viM) 10 min following drug addition (Fig. 25).

Methylphenidate-catecholamine interaction studies. To evaluate a possible catecholaminergic mediation of the enhancing effects of methyl phenidate noted above, studies were conducted employing animal pretreatment with reserpine or a-methyl-p-tyrosine. The rational for these pretreatments was identical to that proposed for amphetamine.

In contrast to the effects of these catecholamine-modifying agents in blocking the enhancing effects of amphetamine on muscle twitch (Fig.

17, Table 14), no modification of methylphenidate's (300 yiM) effects on muscle contractions was observed with eith er drug (Table 22 ). This suggests that catecholamines do not mediate the effects of methylphenidate on neuromuscular transmission.

Electrophysiological studies. As was conducted with amphetamine, these studies were designed to examine the effects of methylphenidate on single skeletal muscle fibers. Methylphenidate concentrations of

270 and 1080 yM, low and high concentrations, respectively, produced no effects on the RMP (Table 23).

Methyl phenidate concentrations ranging from 100-1080 yM were found to produce no significant effects on MEPP frequency (Table 24).

Lower concentrations of methylphenidate (100-540 yM) nonsignificantly reduced MEPP amplitude, while higher concentrations of 810 and 1080 yM significantly reduced MEPP amplitude by 47 and 59%, respectively

(Table 24). Incubation with the higher concentrations (540-1080 yM) for longer intervals of time (>15 min) resulted in a complete suppression of MEPP amplitude into instrument noise levels, as was 164

TABLE 22

Failure o f Animal Pre-treatment with Reserpine or a-MT to Modify Methyl phenidate-Induced Enhancement of Nerve Stimulated Muscle Twitch

Contraction Height (% of Pre-Drug Baseline)3 Time A fter Methylphenidate Treatment (300 yM) c d Addition (min) Control*3. Reserpine a-MT

0 100 100 100 5 165.2 + •2.1 162.7 + 6.4 163.7 + 9.4 10 189.5 + 5.2 184.8 + 7.0 190.0 + 12.6 20 206.2 + 7.0 200.8 + 7.5 208.3 + 16.9 30 204.9 + 8.3 204.0 + 6.1 216.0 + 16.3 45 197.1 + 5.1 196.8 + 5.1 209.5 + 15.0 60 181.0 + 6.6 180.2 + 6.8 200.3 + 13.3 75 169.0 + 9.5 168.5 + 8.6 188.8 + 8.3 90 157.9 + 11.0 154.7 + 11.0 175.7 + 5.8 105 145.3 + 11.3 140.3 + 10.9 158.3 + 1.6 120 133.7 + 9.8 123.7 + 9.7 144.3 + 0.7

a Each value represents the mean + S.E.M. of 3 tissues. b Control represents vehicle treatment of animals. Methyl phenidate (300 yM) was added to the nerve-muscle preparation and twitch followed over time for all treatments. Treatment values are not different from control (p > 0.05). c 5 mg/kg i.p ., 18 hr-pretreaitment. d a-M ethyl-p-tyrosine methyl es ter, 250 mg/kg i . p , , 18 hr-pre- treatm ent. TABLE 23

Effects on Methylphenidate on the Resting Membrane Potential (RMP)a

RMP (X + S.E.M.)

Control -71.7 + 0.4 mV

Methylphenidate

270 yM -7 1 .0 + 0.5 mV

1080 yM -71.4 + 0.6 mV

Control RMP data were collected, followed by a 20-30 min drug incubation, at which time treatment data were collected. _Control and treatment values represent the X + S.E.M. of 90 and 45 fibers taken from 6 and 3 tissues, respectively. 166

TABLE 24

Effects of Methylphenidate on Miniature Endplate Potential (MEPP) Frequency and Amplitude

MEPPS (% of pre-drug control at 5-10 min) Methyl phenidate v T T T T "m Concentration X + S.E.M. (yM) N . Frequency ...... Amplitude

100 4 94.0 + 6.9 100.0 + 1.1

270 4 95.5 + 4.2 94.0 + 5.2

540 3 94.7 + 9.9 85.6 +12.1

810 3 88.5 +19.2 53.3 + 4.3b

1080 4 93.9 + 8.4 40.2 + 8.3b

Data were collected in each cell before and continuously following drug addition with the values presented representing the X (+ S.E.M.) response 5-10 min a fte r drug expressed as a percentage of pre-drug control. Control values are based on an average of 63 MEPPs collected from each_fiber before drug addition: MEPP frequency, range = 0.97- 4.72 Hz, X = 2.54 + 0.26 Hz; MEPP amplitude, range = 0 .3 5 -0 .9 0 mV, K = 0.52 + 0.06 mV (n = 18 preparations). b Values significantly different from pre-drug control (p <0.05) by paired Student's t- te s t (tw o -tailed ) performed on the raw data before percentile transformation. 167 described above fo r amphetamine. These depressant effects were reversed upon washing the preparation with drug-free Krebs' solution.

Methylphenidate concentrations of 135 and 540 yM enhanced EPP amplitude by 9 and 14%, respectively, while 1080 yM significantly reduced the EPP amplitude by 26% (Table 25). As was observed with high amphetamine concentrations, prolonged (>15 min) exposure to the higher methylphenidate levels (>540 yM) produced depression in the

EPP amplitude which eventually completely suppressed these events into baseline noise of the recording apparatus. This inhibitory effect was also completely reversible upon washing the preparation with fresh

Krebs' solution.

Failure Of methylphenidate to protect against irreversible q-bungarotoxin blockade of contractions. Amphetamine inhibits irreversible specific a-bungarotoxin binding and protects the isolated nerve-skeletal muscle preparation against neuromuscular blockade produced by this toxin (Skau and Gerald, 1977; 1978b). This resu lt suggests th at amphetamine interacts with the postsynaptic nicotinic ACh receptor and may produce curare-like blockade.

Methylphenidate was tested in a sim ilar experimental paradigm to examine if it also interacts with the postsynaptic nicotinic ACh receptor in an analogous manner to amphetamine. Methylphenidate concentrations o f 540 and 1080 yM failed to protect the preparation against the irre v e rs ib le neuromuscular blockade produced by a-BGT(Fig. 26). 168

TABLE 25

Effects of Methylphenidate on the Nerve-Stimulated Endplate Potential (EPP)a

EPP Amplitude Methylphenidate 5-10 min Following Concentration Drug Addition (yM) N (% of drug co ntrol)

135 4 109.0 + 8.5

540 4 114.0 + 3.6b

1080 4 73.8 + 9 .6 b

To prevent muscle tw itching, neuromuscular transmission was p a rtia lly blocked by raising the [Mg*+] to 12.5 mM. Data were collected in each cell before and continuously following drug addition. Values presented represent the EPP amplitude 5-10 min after drug addition as a percentage of pre-drug control. Control values; X =1.11 + 0.13 mV (range = 0.64 - 2.13 mV), n = 12 muscle fib e r preparations. Individual EPP amplitudes represent the average of 60 EPPs/fiber recorded over 5 min for each control and treatment period. k Values significantly different from pre-drug control (P < 0.05) by paired Student's t-test (two-tailed) run on the raw data before percentile transformation. Figure 26. Failure of methylphenidate (MP) to inhibit the irreversible binding of a-bungarotoxin (ct-BGT). The upper tracing demonstrates the re v e rs ib ility o f blockade of nerve stimulated contractions produced by MP (540 yM). In the lower tracing MP blockade is followed by a 2-hour incubation with a a-BGT. No return of nerve stimulated contractions was observed upon washing the tissue at 5-10 min intervals for one hour, suggesting that methylphenidate does not protect the nicotinic receptor from a-BGT binding.

169 Hethylphenldate MO pH

Hetliylphentdate S4U fM

'-J O This result was not expected at the time and suggests that methylphenidate in h ib its neuromuscular transmission by a mechanism d is tin c tly d iffe re n t from amphetamine.

ACh release studies. Methylphenidate, at concentrations which markedly potentiated nerve-stimulated contractions (100 and 300 yM,

Table 21) were examined to determine whether, like amphetamine, this enchancement o f twitch may be due to an increased release of ACh.

These methylphenidate concentrations were found to produce no effect on ACh release.

...... C Methylphenidate (pM) ACh release (% pre-drug baseline) N

100 103.1 + 6.1 4

300 105.0+8.6 4

These results suggest that methylphenidate induced enhancement o f contractions is mediated by a d iffe re n t mechanism than amphetamine.

Values represent the mean (+ S.E.M.) compared to pre-drug release expressed as a percentage of control. Predrug ACh release in these tissues ranged from 2 .6 -4 .7 pmoles/min (X = 3.7 + 0.3 pmoles/min). CHAPTER IV

DISCUSSION

Overview o f Discussion

(+)-Amphetamine produces a biphasic e ffe c t on nerve stimulated skeletal muscle contractions in the isolated rat phrenic nerve diaphragm preparation (Anderson and Ammann, 1963; Gerald and Hsu, 1975a;

Skau and Gerald, 1978a). Low concentrations (135-270 yM) enhance, while higher concentrations (405-810 yM) depress contractions in a concentration- and time-dependent manner (Skau and Gerald, 1978a).

The present study has extended these observations to examine, in d e ta il, the mechanism by which amphetamine enhances neuromuscular transmission. Specifically, this investigation attempted to test the hypothesis that amphetamine enhancement o f nerve stimulated muscle contractions in this preparation results from enhanced release of ACh from the motomeuron.

In other neuropharmacological systems (e.g.,CNS), amphetamine is thought to act by promoting the release of catecholamines. A second goal of this investigation was, therefore, to determine if the amphetamine-induced effec ts in th is peripheral (nerve-skeletal muscle) system are also mediated via endogenous catecholamine release.

Thirdly, we sought to characterize the nature of the presynaptic 173 ex-adrenoceptor type which, when activated, results in enhanced ACh release. Fourth, an attempt was made to further characterize the nature and type o f neuromuscular blockade produced by amphetamine. F in a lly , methlyphenidate, a drug that produces amphetamine-like effects in the

CNS, was compared with amphetamine fo r its effects on neuromuscular transmission.

Methodological considerations employed in this investigation. This study attempted to differentiate presynaptic drug effects (e.g. changes in ACh release) from postsynaptic changes in neurotransmitter (ACh) receptor s e n s itiv ity . This was accomplished by employing several methodologies, each designed to contribute to a detailed understanding o f amphetamine's effects at the neuromuscular junction. Precise information regarding presynaptic drug effects was gathered in studies in which ACh release was measured biochemically. Electrophysio- logical studies provided information relative to both pre- and post­ synaptic drug effects: MEPP frequency analysis provided an indication of presynaptic ACh release, while MEPP and EPP amplitude analysis gave information concerning postsynaptic effects altering receptor s e n s itiv ity to released ACh. Muscle twitch studies were conducted to establish the net effect of pre- and postsynaptic factors in determining muscle contractility. Moreover, muscle twitch analysis provided a more direct linkage between drug effects on isolated tissues with those in vivo (e.g . performance enhancing effects o f amphetamine).

An attempt was made, using the cannulated diaphragm preparation, to differentiate between pre- and postsynaptic drug effects on in vitro muscle contractions. 174

Overview of possible sites of amphetamine action at the neuro­ muscular junction. Several potential sites exist within the neuro­ muscular junction at which amphetamine might in teract to augment muscle contractions (Fig. 27). Potentiation of nerve stimulated muscle contractions by amphetamine could resu lt from enhanced ACh release presynaptically, an increase in postsynaptic response to released ACh

(as would be expected i f AChE was inhibited, or the sensitivity of the receptor was augmented in some manner), and/or to an e ffe c t on muscle contractility by a cholinergic-independent mechanism. A systematic analysis of these possibilities (see Fig. 27) should reveal whether one or more of these mechanisms contribute to the biphasic neuro­ muscular effects of amphetamine.

Postsynaptic Effects

Amphetamine effects on the postsynaptic ACh receptor. It is possible that the potentiation of contractions produced by amphetamine result from an increase in the sensitivity of the postsynaptic nicotinic

ACh receptor to released neurotransmitter. However, several lines of evidence suggest that amphetamine decreases, rather than increases the

ACh receptor sensitivity. Nerve stimulated muscle contractions were enhanced by amphetamine (135 and 270 yM) in both the cannulated and noncannulated diaphragm preparations (Fig. 10 and 1 2 ). However, muscle contractions in the cannulated preparation elicited by a pulsed in jectio n of ACh were never enhanced, and in fa c t, were inhibited in a concentration- and time-dependent fashion, suggesting a depression in Figure 27. Overview of possible sites and mechanisms by which amphetamine might potentiate neuromuscular transmission. These mechanisms include: (1) enhanced nerve terminal excitability (e.g. hyperpolariza- tion); (2) increase in ACh synthesis; (3) potentiation of ACh release from the nerve terminal; (4) increased postsynaptic receptor sensitivity to ACh; (5) AChE inhibition; (6) potentiation of ion movement through the muscle membrane; (7) effects on muscle contractility; or (8) ct,-adrenoceptor activation, mediated by endogenous catecholamines which amphetamine releases.

175 176

Choiine

Acetyl CoA Blood vessel vasculature

++

Choiine

Acetate

Amphetami ne AChR Ion releases hanne

© Muscle C o n tra c tility 177

receptor sensitivity. Moreover, higher amphetamine concentrations

(540 yM) depressed nerve stimulated muscle contractions as well (Fig.

12).

I f amphetamine enhanced the s e n s itiv ity o f the ACh receptor, i t would be reflected by an increase in the electrophysiologically

recorded response to released ACh. Such an e ffe c t would be expected to resu lt in an increase in MEPP and EPP amplitudes. No amphetamine concentration tested (34 -810 yM) increased MEPP amplitude, rather a concentration-dependent decrease was observed (Fig. 13). EPP amplitude was increased by amphetamine (68 and 135 yM), while higher concentrations (270-810 yM) produced a significant depression in EPP amplitude (Fig. 15). Augmentation o f EPP amplitude could be the result of an increase in the receptor sensitivity'to released ACh or from enhanced evoked release o f ACh. I f enhanced receptor s e n s itiv ity to released ACh accounted for the increase in EPP amplitude, it would be expected that MEPP amplitude would also be increased. Moreover, an enhanced twitch response to a pulsed ACh in jectio n would also be expected. However, because no enhancement was observed, i t is concluded that the increased EPP amplitude results from presynaptic

(enhanced ACh release) rather than postsynaptic factors. An analysis of the presynaptic effects of amphetamine w ill be presented in the following section.

The conclusion that amphetamine decreases the postsynaptic sensitivity to ACh is consistent with the work of others using the rat phrenic nerve-diaphragm preparation. To summarize these reports I

178

(d etailed in Introduction) i t was found that amphetamine reduces the

postsynaptic response to ACh not by a local anesthetic e ffe c t

(Anderson and Ammann, 1963; Peterson e t a l . , 1964), but rather by

a curare-like interaction at the nicotinic ACh receptor (Peterson et al.,

1964; Gerald and Hsu, 1975a; Skau and Gerald, 1978a). Moreover, this

interaction with the n ico tin ic ACh receptor was elegantly demonstrated

by amphetamine-induced in h ib itio n of specific a-bungarotoxin binding

and protection against irreversible blockade of muscle contractions by

this toxin (Skau and Gerald, 1977; 1978b).

The results gathered in the present study confirm the finding

that amphetamine decreases the postsynaptic response to ACh.

Specifically, this effect was demonstrated in single muscle fibers by

amplitude analysis of EPPs and MEPPs (Fig. 13 and 15). The amplitude

o f these events were attenuated by amphetamine (>270 yM) in a concen­

tra tio n and time-dependent manner. Moreover, the twitch response in the

cannulated diaphragm was decreased in a s im ila r manner (Fig. 12). While

none of the results obtained in the present investigation provide a

specific mechanism for this inhibitory effect (curare-like or

su ccin ylch o lin e-like), i t is clear that the postsynaptic response to

ACh is reduced.

Inhibition of acetylcholinesterase (AChE). AChE inhibitors, such

as physostigmine, are capable o f enhancing nerve stimulated muscle

contractions in vivo and in vitro (Koell and Gilman, 1949). Thus, the

p o s s ib ility exists that amphetamine may enhance contractions by

in h ib itin g AChE. Our results however, argue against such a 178 p o s s ib ility . Namely, an AChE in h ib ito r would be expected to enhance

MEPP and EPP amplitude as well as potentiating contractions elicited by ACh injections in the cannulated diaphragm; none of these effects were observed. This conclusion is supported by Ho and Gershon (1972) who found th at amphetamine (10"® - 10~2 M) does not in h ib it AChE isolated from ra t brain.

In experiments conducted on AChE isolated from rat muscle, Skau

(K. A. Skau, Univ. o f Utah, personal communication, November, 1981) determined that amphetamine (100-1000 yM) produced no more than 23% inhibition. In these studies,- Skau investigated drug effects on the three molecular forms of AChE that were separated by sucrose density gradient centrifugation, as described by Skau and Brimijoin (1981).

The assay used was a radiometric procedure in which the enzyme is incubated with [^C-II (acetate)-ACh and drug, with enzyme activity 14 determined by liquid scintillation counting of hydrolyzed C-acetate

(P otter, 1967). Skau concluded th a t, because at least 75% of the AChE must be inhibited before effects are observed on neuromuscular transmission, amphetamine does not produce effects via AChE in h ib itio n .

Cholinergic-independent effects of amphetamine on muscle contraction. In addition to its effects on nerve stimulated twitch, amphetamine has also been reported to produce a biphasic e ffe c t on muscle contractions elicited by direct muscle stimulation in diaphragms pretreated with (+)-tubocurarine or a-bungarotoxin to preclude cholinergic effects (Gerald and Hsu, 1975a; Mel drum and

Gerald, 1980; Meldrum et a l., in press). This noncholinergic effect requires higher amphetamine concentrations than those which affect 180

nerve stimulated contractions; the maximum twitch enhancing concentra­

tions in nerve stimulated and directly stimulated tissues were 270 and

810 yM, respectively. Moreover, amphetamine concentrations of 540 yM

or greater completely inhibit nerve stimulated twitch within 15 min

(Fig. 12). Therefore, it is unlikely that the noncholinergic effects

of amphetamine play a major role in enhancing nerve stimulated

contractions. The major conclusion drawn from studies examining the

cholinergic-independent e ffe c t o f amphetamine was th at enhancement and

blockade of directly stimulated skeletal muscle results from a

facilitation and inhibition of Na+ flux, respectively, by interacting

with muscle membrane ion channels (Meldrum, 1980; Meldrum et a l. ,

1981).

Presynaptic Effects

Amphetamine enhancement o f ACh release. The results gathered in

the present investigation are consistent with the hypothesis that

amphetamine enhancement of nerve stimulated contractions is mediated by

an increase in ACh release from the motor nerve. Amphetamine (30-

300 yM) enhanced ACh release 0.5-4.8 fold (Fig. 16). This result is in

agreement with th at of Hsu and Gerald (1973) who, employing a bioassay

procedure, determined that (+ )-amphetamine (250 yM) enhanced ACh

release from the stimulated phrenic nerve 2.5-fold. Moreover, the

results of muscle contraction and electrophysiological studies also

support the conclusion th at amphetamine enhances ACh release.

Amphetamine potentiated MEPP frequency by up to 42%, and EPP amplitude was also significantly potentiated by 16-24% (Fig. 13 and 15). 181

Moreover, nerve stimulated contractions, but not those elicited by

ACh in je c tio n s , were s im ila rly enhanced by amphetamine (135-270 yM)

(F ig . 10 and 12'). As was noted above, the results from EPP and muscle

contraction studies support the conclusion that amphetamine enhances

the evoked release of ACh. The mechanism by which amphetamine enhances ACh release w ill be considered in the next major section.

Amphetamine in h ib itio n of ACh release. In addition to amphetamine's

ability to increase ACh release, high concentrations (1 mM) decreased evoked release by 50% (Fig. 16). While no attempt was made to

determine the mechanism of this inhibition, several possibilities may be considered. Local anesthetics decrease ACh release by depressing nerve e x c ita b ility . This altern ative is unlikely because amphetamine, at these concentrations, has been shown, to be devoid of local anesthetic properties (Anderson and Anmann, 1963; Peterson e* j f L , 1964;

Meldrum, 1980). Amphetamine possesses curare-like properties (Skau and Gerald, 1978a), and (+)-tubocurarine has been reported by some to produce presynaptic in h ib itio n of ACh release (Standaert, 1964; Hubbard et a l., 1969) although not by others (see Hubbard and Wilson, 1973 for references). For this reason, it does not seem justifiable to attribute the ability of amphetamine to decrease ACh release solely to a curare-like effect. Finally, the inhibitory effects of this high amphetamine concentration may be a nonspecific toxic effect on neurotransmitter release. It is possible that amphetamine inhibits

ACh release by disrupting the release process, possibly by interfering with Ca++. 182

Mechani sm of Amphetamihe Enhancement o f ACh Release

A major emphasis of this investigation was to examine the

mechanism by which amphetamine augments ACh release. I should point out

that these studies did not attempt to examine the molecular mechanism

of ACh release, but only the mechanism by which amphetamine might act

to promote release. Several mechanisms within the motor nerve terminal

are potential sites for amphetamine interaction (see Fig. 27). These mechanisms (presented in detail in the Introduction) include enhanced

nerve terminal excitability (e.g. hyperpolarization), augmentation

of ACh synthesis, or enhanced Ca++ involvement in the neurotransmitter

release process. No direct information was obtained in the present

investigation which would cause us to prefer one or more o f these

mechanisms. However, amphetamine produces neuropharmacological effects

via the release of catecholamines, and NE is believed to enhance ACh

release by promoting Ca++ activity in the nerve terminal (see

Introduction for details).

This section is organized systematically to address the hypothesis that amphetamine acts to release a catecholaminergic pool which, in

turn, acts on motor nerve terminal a^-adrenoceptors to promote ACh

release. This discussion begins by analyzing amphetamine-catecholamine

interactions in neuromuscular and other neurobiological systems and by considering the m odification o f amphetamine's e ffe c t by catecholamine depletion. Secondly, i f NE mediates the effects o f amphetamine, at

least a brief consideration of the source of catecholamine in this neuromuscular system is warranted. This is followed by a detailed, 183

mechanistically-oriented discussion of NE effects on neuromuscular

transmission and a comparison of these effects with those of

amphetamine. Finally, an analysis is presented of the available

evidence in characterizing the nature of the presynaptic a-adrenoceptor

located on somatic nerve terminals.

Amphetamine-catechblamine in teraction s. A series o f experiments

were conducted to evaluate the a b ility of catecholamine depleting agents

to modify amphetamine potentiation of ACh release and muscle tw itch.

Animal pretreatment with reserpine did not modify amphetamine (270 yM)

potentiation of twitch (Fig. 17), confirming a similar finding by Skau

and Gerald (1978a). In p arallel experiments, however, this treatment

decreased amphetamine-induced (100 yM) potentiation of ACh release by

60% (Fig. 18). The ability of reserpine pretreatment to attenuate

amphetamine's effects has been reported to d iffe r in central versus

peripheral systems. Whereas amphetamine continues to produce its

typical behavioral effects (increased locomotor activity and stereo­

typic behavior)in reserpinized animals (Carlsson, 1970), reserpine

pretreatment decreases by about 50% pharmacological responses in

peripheral autonomic preparations (cat n ic tita tin g membrane and blood

pressure) (Trendelenburg et a l., 1962).

There s t i l l e x is ts , however, a discrepancy in the present report; while reserpine pretreatment diminished the ab ility of amphetamine to enhance ACh release, i t did not affect the response oh muscle

contractions (Fig. 17 and 18). Perhaps this results from inherent

differences between the two preparations intended to assess muscle contraction and ACh release. Whereas an AChE inhibitor (physostigmine) was present in the perfusion media in the ACh assay studies, none was

added to the muscle twitch preparations. Moreover, release studies were conducted at 23°C while twitch experiments at 32°C. Perhaps a more fundamental question need be considered; what quantitative

relationship exists between modification of ACh release from the motor

nerve and a corresponding change in muscle contractions? Thus, i t is possible that a treatment which produces a large change in ACh release may only slightly modify muscle contractions. It would be of interest to attempt a quantitative investigation of parallel changes in ACh

release and muscle tw itch.

In contrast to reserpine, animal pretreatment with the tyrosine hydroxylase inhibitor a-methyl-p-tyrosine effectively eliminates the newly synthesized (amphetamine-releasable) pool of catecholamines

(Weissman e t a l . , 1966), while 6-hydroxydopamine produces the same effect by destroying peripheral catechol aminergic nerve terminals

(Thoenen, 1971). The results obtained in parallel experiments were unequivocal; these treatments completely blocked amphetamine enhancement o f ACh release and muscle twitch (Fig. 17 and 18) leading to the inevitable conclusion that amphetamine potentiates these parameters in d ire c tly ,v ia the release of newly synthesized catecholamines.

Amphetamine has been reported to enhance cholinergic transmission in other neuropharmacological systems. In the isolated ra t superior cervical ganglia, amphetamine (27 yM) was found to partially reverse 185 the depression of ganglionic transmission following high rates of stim ulation (Downing, 1972). Downing concluded that amphetamine acted directly on the preganglionic nerve terminal to enhance the release of

ACh, supported by his finding that reserpine pretreatment did not modify the effects of amphetamine. It would be of interest to quantitatively investigate ganglionic effects of amphetamine following a-MT pretreatment, which, unlike reserpine, would eliminate a possible catechol aminergic involvement.

Amphetamine-induced enhancement of ACh release has also been demonstrated in the CNS. Amphetamine (1.5-5.0 mg/kg, i.v .) increased

ACh release from epidural cortical cups in cats by 0.75-3.0 fold

(Beani et a l . , 1968; Pepeu and B a rto lin i, 1968), an e ffe c t prevented by a-MT (N is tri et a l . , 1972; Beani and Bianchi, 1973) or a-adreno- ceptor antagonists such as phentolamine (Pepeu and Bartolini, 1968).

These findings add further support to the hypothesis that amphetamine may be acting to enhance cholinergic neuromuscular transmission in d ire c tly , via catecholamine release and action on motor nerve term inals.

Location of endogenous NE in this preparation. To provide support for the hypothesis that the effects of amphetamine are mediated by the release of endogenous catecholamines, it is necessary to establish the existence of such an endogenous pool. Histologically, skeletal muscle is known to receive a copious vascular supply, and the walls of the blood vessles receive dense postganglionic sympathetic (NE- containing) innervation (Copenhaver e t a l . , 1978). The concentration 186 of catecholamines (EPI and NE) in rat skeletal muscle has been reported to be 0.52 pg/g tissue (Govyrin, 1965). A complete survey of the NE content in various muscle preparations has been summarized by

Holzbauer and Sharman (1972).

Noradrenergic effects on neuromuscular transmission. I f the above hypothesis suggesting that endogenous NE mediates the effects of amphetamine is valid, it is logical to predict that exogenously applied

NE should produce sim ilar effe c ts . The experiments conducted using

NE in th is investigation served two purposes. F irs t, to provide support fo r the hypothesis that endogenous NE might mediate amphetamine's effects. In effect, these experiments served as an internal control.

Rather than relying on indirect evidence, such as the ability of a-MT to prevent amphetamine's e ffe c ts , p o sitive, d irec t results were obtained that NE could produce such effe c ts . Second, the study of noradrenergic influences on neuromuscular transmission are of in terest and have been extensively studied (see Bowman, 1981; also detailed in

Introduction). To summarize, based on the in vivo and in v itro lite r a tu r e , NE enhances neuromuscular transmission by increasing ACh release from motor nerve term inals. This e ffe c t is thought to occur by NE interacting with presynaptic a-adrenoceptors which, when activated, augment the probability of ACh release by increasing Ca++ activity in the nerve terminal (Kuba and Tomita, 1971; 1972).

A major goal o f the NE experiments was to confirm by direct chemical measurement the ability of NE to stimulate ACh release; all previous studies have used electrophysiological or muscle contraction 187 parameters in reaching this conclusion. NE (50 pM) enhanced ACh release by 1.8 fold (Fig. 21) and produced a concentration-dependent enhancement of nerve stimulated contractions (Fig. 22). Also consistent with the literature (see Bowman, 1981) is the finding that the a- adrenoceptor antagonist phentolamine inhibited the NE-induced enhancement of ACh release (Fig. 21). Similarly, phentolamine inhibited the a b ility of amphetamine to augment ACh release and twitch height (Fig. 19 and 20); this is in contrast to the results of Skau and Gerald (1978a). This discrepancy may be resolved by noting the difference in amphetamine concentrations used in the two studies.

Skau and Gerald (1978a) were interested in both the enhancing and blocking effects of amphetamine, thus they used a concentration that produces both e ffec ts (405 pM). In the present studies, only relatively low amphetamine concentrations were employed (135-270 pM) to more clearly study the potentiating effects. As was noted above, amphetamine (>270 pM) is known to augment directly stimulated twitch.

Thus, the inability of phentolamine to inhibit amphetamine (405 pM) potentiation of contractions may be due to this direct effect of amphetamine on muscle. In contrast, this noncholinergic, nonadrenergic potentiation of twitch does not appear to play a major role in the present studies with lower amphetamine concentrations.

Comparison of amphetamine and NE effects on neuromuscular transmission. The majority of the results discussed above support the hypothesis that amphetamine's effects are mediated by norepinephrine.

This is perhaps best illu s tra te d by d ire c tly comparing the neuromuscular effects of amphetamine and NE (Table 25). Both agents potentiate MEPP frequency and EPP amplitude but do not augment MEPP amplitude. While d iffe re n t methods were used to assess drug effects on postsynaptic sensitivity to ACh, both agents produced similar resu lts; NE produced no enhancement of ACh (iontophoretically applied) potentials (Kuba, 1970), and amphetamine reduced the twitch response to ACh injections in the cannulated diaphragm preparation (Fig. 12).

The results of both groups o f experiments were interpreted to suggest th at enhancement of neuromuscular transmission was due to an increase in ACh release presynaptically rather than by an augmentation of postsynaptic ACh s e n s itiv ity . Moreover, both agents augment nerve stimulated muscle contractions and ACh release as measured biochemically.

In groups of experiments conducted to investigate the nature o f the a-adrenoceptor which mediates the effects of these agents (see below) it was found that c^-antagonism blocked while ag-antagonism only s lig h tly decreased the effects of both NE and amphetamine, thus adding fu rth er support to the hypothesis that amphetamine's effects are mediated via NE.

It is possible that quantitative differences exist between amphetamine and NE-induced enhancement of ACh release. Amphetamine

(30, 100 and 300 yM) increased ACh release by 0.5, 3.2 and 4.8 fold, respectively, while NE (50 yM) produced a 1.8 fold increase (Fig. 16 and 21). This concentration of NE was chosen based on its reported ability to potentiate MEPPs and EPPs (Kuba, 1970). In order to accurately compare the potency difference between NE and 189

TABLE 26

Comparison of Amphetamine and Norepinephrine Effects on Mammalian Neuromuscular Transmission

Effect

Parameter Measured Amphetamine3 Norepinephrine

Nerve stimulated twitch height twitch height contractions increased increased9

MEPP frequency increased increased*5

MEPP amplitude no change with low, no change1* decreased by high concentrations

EPP amplitude increased with low increased*5 decreased by high concentrations

Response to exo­ decrease in twitch no change in genously applied height to ACh-induced iontophoretic ACh ACh contractions in the potentials*5 cannulated preparation

ACh release increased increased3 a-j-Adrenoceptor inhibits potentiation inhibits potentiation antagonism of ACh release and of ACh release and twitch twitch9 a«-Adrenoceptor slight decrease in ACh slight decrease in antagonism release no effect ACh release, no on twitch effect on twitch9

Present work, k From Kuba (1970); Kuba and Tomita (1971). amphetamine, i t is necessary to construct a complete concentration- response relationship fo r NE. However, in the absence o f such a relationship, it is interesting to speculate on why amphetamine might produce greater enahncement o f ACh release than NE. The reuptake of

NE into adrenergic nerve terminals is believed to be a major route of

NE in activatio n , and amphetamine in h ib its th is reuptake process at concentrations used in the present study (Carlsson et a l., 1970). Thus, amphetamine might produce greater enhancement of ACh release by inhibiting NE inactivation or by allowing it to act at the motor nerve terminal area for a longer duration. To examine this hypothesis, i t would be of interest to quantitate NE-induced potentiation of ACh release in the presence versus the absence of an uptake in h ib ito r such as cocaine or desmethylimipramine. It is predicted that ACh release would be greater for a given NE concentration in the presence of the inhibitor. Finally, to verify the involvement of catecholamines in mediating amphetamine's effects on ACh release, the release of NE or its metabolites could be directly measured from the cannulated diaphragm preparation.

Characterization of the a-adrenoceptor type involved in amphetamine's enhancing e ffe c ts . Post- and presynaptic adrenergic receptors have been classified as and c^, respectively (Langer, 1977;

Berthelsen and Pettinger, 1977). Using selective a-adrenergic agonists and antagonists, an attempt was made to characterize the a-adrenoceptor type mediating amphetamine (and NE)-induced enhancement in ACh release. 191

Phentolamine (both a ^ and (^-antagonistic properties) and the

selective aj-antagonists WB 4101 inhibited the ability of amphetamine

to enhance ACh release (Fig. 20) and muscle contractions (Fig. 19).

S im ila rly , these antagonists blocked the NE-induced enhancement of ACh

release (Fig. 21) and decreased to an equal degree the NE-induced enhancement in nerve stimulated twitch (Fig. 22). By contrast, the

selective ^ -an tag o n is t yohimbine did not reduce the e ffe c t of

amphetamine or NE on nerve stimulated contractions (Fig. 19 and 22,

respectively). I t should be noted, however, that yohimbine reduced

the a b ility of amphetamine (by 38%) and NE (by 29%) to enhance ACh

release (Fig. 20 and 21, respectively). This latter effect might be

subject to at least two interpretations. First, it is possible that there is an a2-adrenoceptor component in the ACh release process that

is inhibited by yohimbine. Secondly, and more likelv, since yohimbine antagonizes both aj and a2-adrenoceptors, with only about a 25-fold

greater selectivity for a2 (Ruffolo et al., 1981), it is possible that, at the yohimbine concentration employed (10 yM), some antagonism occurred. Thus , we b e lie v e that this inhibitory effect of yohimbine on

ACh release resulted solely from its c^-antagonistic activity.

Further support for the involvement of (but not a2) adrenoceptor mediation of amphetamine and NE effects were obtained from experiments using selective a-adrenoceptor agonists. Clonidine, a selective a2-agonist failed to enhance ACh release (Fig. 21) or nerve stimulated contractions (Fig. 23). If a minor a2 component were involved in mediating the potentiation of twitch and ACh release, 192 clonidine would have been expected to augment these effects. Moreover,

the selective aj-agonist phenylephrine was found to potentiate nerve

stimulated contractions, exhibiting slightly less potency than NE

(Fig. 23). Wikberg (1978) has proposed that a-adrenoceptors be

classified according to the relative potency of phenylephrine and

clonidine and that when phenylephrine is of greater potency than

clonidine (as was found in the present experiment), the receptors are

of the aj-type. This classification has been verified based on data

accumulated in other systems (e.g . vas deferens, aorta, spleen) as well

(Berthelsen and Pettinger, 1977; Ruffolo et a l . , 1980). Thus, based on

the relative potency of selective a-adrenoceptor agonists and

antagonists in modifying the neuromuscular effects of amphetamine and

NE, the presynaptic a-adrenoceptor located on motor nerve terminals

appear to be of the a1 subtype.

Malta and coworkers (1979) reached the same conclusion based on

in vivo experiments in cats using selective a-adrenoceptor agonists

(clonidine, methoxamine, epinephrine and phenylphrine) and antagonists

(phentolamine, to lazo lin e and thymoxamine) by comparing these agents

potency in modifying neuromuscular transmission with an autonomic vascu­

lar response. Their results indicate that the prejunctional a-adreno­

ceptors involved in potentiating neuromuscular transmission are similar to postjunctional a-adrenoceptors in blood vessels ( i.e ., aj-adrenoceptors).

Methylphenidate Effects on Neuromuscular Transmission

This section will discuss the effects of methylphenidate on neuro­ muscular transmission and compare the effects with amphetamine. Methyl- phenidate produced a biphasic e ffe c t on neuromuscular transmission in the 193

same concentration range.as amphetamine. Low (30-300 yM) and high (6 0 0 -

1000 yM) concentrations enhance and inhibit nerve stimulated contractions, respectively (Fig. 24). A critical comparison of the neuromuscular effects of methylphenidate and amphetamine reveals major differences in the mechanisms of action (Table 27).

Analysis of neuromuscular effects of these two agents reveals that while both produce a biphasic effect on muscle contractions, different mechanisms mediate both phases o f these e ffe c ts . The mechanism of amphetamine enhancement o f muscle contractions is lik e ly due to an increase in ACh release from presynaptic sites,mediated by catechola­ mine release and involving an action on somatic nerve terminal ct^- adrenoceptor sites. By contrast, methylphenidate (1 0 0 -3 0 0 yM) does not enhance ACh release at concentrations which markedly augment twitch.

Furthermore, the neuromuscular effects of methylphenidate (300 yM) were not modified by animal pretreatment with either a-MT or reserpine, indicating the lack of adrenergic influence in the biphasic effect

(Table 22). This lack of adrenergic effect is best explained by considering methylphenidate's mechanism of adrenergic action and how it d iffe rs from amphetamine. Whereas amphetamine is capable of releasing nerve terminal catecholamines, methylphenidate's action is dependent upon previous release of this neurotransmitter (see Introduction for d e ta ils ). Thus, in the phrenic nerve stimulated diaphragm preparation, the stimulus for evoking neurotransmitter release is applied only to the nerve, consequently, there is no evoked release of NE (from 194

TABLE 27

Comparison of Methylphenidate and Amphetamine Effects on Mammalian Neuromuscular Transmission

Effect

Parameter Measured Amphetamine Methylphenidate

Nerve stimulated twitch biphasic e ffe c t biphasic effect

MEPP frequency increased no change

MEPP amplitude decreased at high decreased at high [amphetamine] [methylpheni date]

EPP amplitude increased increased

ACh release increased no change

Twitch height modified enhancement prevented no change by a-MT or reserpine by a-MT

Protects against yesa no irreversible a-BGT blockade o f twitch

a From Skau and Gerald (1977). 195

vascular sympathetic nerve terminals). As a result, methylphenidate is

unable to exert its adrenergic effects, via inhibition of reuptake.

Perhaps the most surprising finding in this investigation was

the failure of methylphenidate to protect the diaphragm preparation

against irreversible a-BGT blockade of twitch (Fig. 26). It was

expected because o f the structural s im ila ritie s between the two drugs,

that methylphenidate (like amphetamine) would protect the preparation

from blockade. Thus, i t is apparent that methylphenidate blockade of

contractions is not mediated by a mechanism involving interactions with

the postsynaptic nicotinic receptor (e.g. curare-like or succinylcholine-

1 ik e ).

The mechanism responsible for the enhancing effects of

methylphenidate is also unknown. It is clear from the present study

that the twitch enhancement is not due to presynaptic increase in ACh

release. Moreover, endogenous catecholamine release does not play a

role in mediating the effects. Thus, having ruled out presynaptic

factors as contributing to the twitch enhancing effects of methylpheni­

date, several postsynaptic sites might be involved in the enhancing

(and blocking) effects (Fig. 27). Methylphenidate might interact with

the enzyme AChE. By inhibiting AChE, either by competitive inhibition

or by acting as an alternate substrate (methylphenidate is an ester),

released ACh could interact with the nicotinic receptor for a longer

duration causing enhancement of nerve stimulated contractions.

However, methylphenidate (250-2500 yM) has recently been shown not to

act as a competitive AChE inhibitor, nor does it act as an alternate 196

substrate fo r the enzyme (K. A. Skau, personal communication,

November 1981, employing the experimental design described above for

amphetamine).

Other postsynaptic, noncholinergic sites of action at which

methylphenidate might act to biphasically modify contractions are

presented in Figure 27. These include interactions with the ionic

channel or directly with the molecular structures of muscle contractil­

ity (e.g., by increasing Ca++ movement). Noncholinergic effects of

methylphenidate have not been investigated in the present study.

However, Mel drum (1980) reported that methylphenidate produces a

biphasic e ffe c t on a-BGT pretreated, d ire c tly stimulated diaphragms,

and that methylphenidate (300 yM) enhanced muscle contractions by

about 50%. In the present study, methylphenidate (300 yM) produced

a much greater degree of potentiation of nerve stimulated twitch

(106% increase, Fig. 24). Thus, it is possible that a noncholinergically-

mediated, direct effect on muscle contractility or ionic movement

(Meldrum, 1980) might contribute to the marked enhancement of nerve

stimulated contractions observed following methylphenidate.

The mechanism responsible for the blockade of contractions by methylphenidate is also an area of conjecture. It is clear that the

neuromuscular blockade is not due to a cu rare-like e ffe c t or by

interaction with the postsynaptic nicotinic receptors. It is possible that this blockade is due to local anesthetic activity of methylphenidate as reported by Ndika (1966). As noted above, methylphenidate (>700 yM) also produced blockade of directly stimulated diaphragms (Meldrum, 1980). 197

In summary, methylphenidate produced a biphasic effect on nerve stimulated muscle contractions. The effects produced appear to be independent of adrenergic and cholinergic mechanisms and may involve effects on ionic channels or on muscle contractility. CHAPTER V

SUMMARY AND CONCLUSIONS

Amphetamine Effects on Neuromuscular Transmission

The results presented above suggest that amphetamine enhance­ ment of nerve stimulated contractions is due to an increase in acetylcholine (ACh) release. Moreover, this enhancement is mediated via the release of endogenous catecholamines which activate aj-adrenoceptors located on motor nerve terminals and promote enhanced ACh release. Amphetamine concentrations higher than those which f a c ilit a te neuromuscular transmission produce blockade of nerve stimulated contractions. Results are presented suggesting that this blockade may be due to a decrease in the postsynaptic receptor sensitivity to ACh or, less likely, to an inhibition of ACh release. Thus, the effect of amphetamine on nerve stimulated contractions may represent the net response of the tissue to presynaptic stimulation and postsynaptic blockade.

It is conceivable that these in vitro effects contribute to amphetamine's reported a b ility to enhance physical performance at low doses and produce muscle weakness at high doses.

These conclusions are based on the following observations:

1. Low amphetamine concentrations (135 - 270 pM) produced a signi­ ficant potentiation of nerve stimulated skeletal muscle contractions.

By contrast, these same concentrations produced only depression of ACh-induced contractions in the cannulated diaphragm and higher 198 concentrations (540 yM) also inhibited nerve stimulated twitch.

2. MEPP frequency and EPP amplitude were increased by low concentrations (< 270 pM), while higher drug levels resulted in the blockade of EPP and MEPP amplitude.

3. Amphetamine (30 - 300 pM) enhanced ACh release, measured biochemically, by 0.5 - 4.8 fold, while 1 mM decreased by 50%.

4. a-Methyl-jp-tyrosine (a-MT) or 6-hydroxydopamine pretreat­ ment of animals abolished the stim ulatory effects ofamphetamine on ACh release and muscle contractions. Reserpine pretreatment reduced amphetamine augmentation o f. ACh release but did not modify the effects on muscle contractions.

5. Norepinephrine (NE) increased nerve stimulated muscle twitch and ACh release. These effects were reduced by aj-adrenoceptor antagonism (with phentolamine or WB 4101) but not by ag-antagonism

(yohimbine).

6. Similarly, while a^-antagonists depressed the ability of amphetamine to potentiate ACh release and muscle contractions, a2-antagonism failed to significantly modify either parameter.

Methylphenidate Effects on Neuromuscular Transmission

The results presented above indicate that methylphenidate produces a biphasic effect on neuromuscular transmission, with low and high drug concentrations potentiating and depressing nerve stimulated muscle contractions, respectively. Unlike amphetamine, however, the biphasic effects of methylphenidate appear to be

independent of cholinergic and adrenergic mechanisms.

These conclusions are based on the following observations:

1. In a concentration-dependent fashion, methylphenidate potentiated nerve-evoked twitch with low (30-300 yM) and inhibited

contractions with high (600-1000 yM) concentrations, respectively.

2. EPP amplitude was increased by low concentrations (<540 yM), while higher levels reduced both EPP and MEPP amplitude.

3. Methylphenidate (100-300 yM) did not modify the nerve stimulated release of ACh, nor did pretreatment with a higher concentration

(540-1000 yM) protect the tissue from irreversible a-bungarotoxin blockade of twitch.

4. Animal pretreatment with reserpine or a-MT did not modify the effects of methylphenidate (300 yM) on nerve stimulated twi tch. APPENDIX A

Methodological considerations regarding the ACh assay

While attempting to set up this assay, several difficulties were encountered necessitating modifications in the published procedure. Specifically, three changes were made: addition of dithiothreitol (5mM) to the first (choline kinase-containing) reaction mixture; increasing the HC1 concentration in the

Krebs' extraction from 0 .4 -1 .0 N; and using 5 units of AChE per sample rather than 1.0 pg (0.8 units) in the second reaction mixture. These difficulties resulted in a loss of ACh from the Krebs' solution to the final amount of 32p-phosphoryl- choline produced (i.e . recovery was decreased). The use of

14c- ACh in helping to locate and solve the problems was invaluable.

By monitoring the amount of radioactivity (l^C) passing from

Krebs' solution to residue to assay, detection of where losses occurred was facilitated. A major problem, which, when corrected allowed the assay to perform properly was the change in acid strength. Using 0.4N HC1, recovery of (l^Cj-ACh was 45-50%, whereas changing to 1.0N HC1 improved recovery to 83 + 5%. A second major change was increasing the AChE to 5 units from 0.8 un its, resulting in a vast improvement in the assay working from one day to the next. The other changes were less consequential to the performance of the assay. Sigma Chemical Co. (suppliers o f choline kinase) suggested that d ith io th re ito l be used to improve

2 0 1 ' recovery of ACh thru the first stage of the reaction. In that th is change was made before the assay was working, I cannot comment on the degree o f improvement which resulted. Changing the te tra - phenylboron concentration between 5-10 mg/ml 3-heptanone did not 14 affect C-ACh recovery from Krebs'. In my experience, 3-heptanone was superior to 2-heptanone, increasing recovery by 5-8%.

Finally, the sensitivity of the assay can be improved (provided all else is performing properly) by increasing the amount of

Op y ’P-ATP added to the second reaction mixture. This concept was elegantly demonstrated by McCaman and Stetzler (1977). Basically, the higher the 3^P-ATP/ATP ratio in the second reaction mixture, the more 32p-phosphorylcholine w ill be formed. In early successful 32 assays, I was using 0.4 uCi P-ATP/sample and my lim it s e n s itiv ity was never better than 15 pmoles ACh. However, upon doubling the q p JCP-ATP added to 0.8 uCi/sample, my lim it sensitivity was reliably

3-8 pmoles. This ACh assay is very re lia b le and lin e a r, with cor­ relation coefficients for standard curves greated than 0.99 thru the range of 24-750 pmoles ACh. In my experience, once the "bugs" were worked out, no problems were encountered in restarting ACh assay experiments after a period in which none were performed.

Care should be exercised, however, in planning these experiments 32 well in advance so that when the P-ATP arrive^, all the studies can be conducted in the next 3-4 weeks. The longer the s tu ff sits around, the less of it you have to work with (^=14.3 d, and

A=Ao-e“n ). Stock solutions employed in the ACh assay.

HC1, 1.0N (83 ml of concentrated HC1, q.s. to 1L), and stored at room temperature.

Tetraphenylboron sodium in 3-heptanone (10 mg/ml), 50 ml is enough to prepare at any one time.

Dithiotreitol (32.4 mM; 25mg in 5ml DDW): for 5 mM in reaction mixture, use 4.6 jil/sample.

ATP, sodium sa lt (lOmM; 253.6 mg/50ml DDW, neutralized to pH=7.0) for 0.8 mM in reaction mixture, 2.4 /jl/sample.

MgCl2*6H20 (500 mM; 10.2g/100ml), for 10 mM in reaction mixture, use 0.6 jul/sample.

(above solutions stored in re frig e ra to r)—

AChE (Type V, Sigma Chemical Co.), stored frozen in 5 mM Sorensen's phosphate buffer (pH=8.0) a t a concentration of 1.25 U/pl. 1000 U AChE dissolved in 800 jjI buffer. For 5 U/sample, use 4 ul/sample.

32P-ATP, handle ca re fu lly and store frozen in NEN packing vial inside lead storage "pig". Use 0.8 uCi/sample, you must calculate how many |il to use based on the specific activity of the and the concentration (information provided by NEN with each shipment). Representative Standard Curve

pmoles ACh ______CPM______NET CPM (CPM-blank)

blank - 0 200 blank - 0 178 X = 189

24 890 701 75 2301 2112 210 6131 5942 450 13316 13127 750 21875 21686

correlation coefficient = 0.9999 lim it sensitivity ( 2X blank ) = 8 pmoles

CO o 12

X

C l . O

24 75 210 450 750

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