A Pharmacological Evaluation of New Calcium Antagonists: 2-Substituted 3

INFORMATION TO USERS

This material was produced from a microfilm copy of the original document. While the most advanced technological means to photograph and reproduce this document have been used, the quality is heavily dependent upon the quality of the original submitted.

The following explanation of techniques is provided to help you understand markings or patterns which may appear on this reproduction.

1.The sign or "target" for pages apparently lacking from the document photographed is "Missing Page(s)". If it was possible to obtain the missing page(s) or section, they are spliced into the film along with adjacent pages. This may have necessitated cutting thru an image and duplicating adjacent pages to insure you complete continuity.

2. When an image on the film is obliterated with a large round black mark, it is an indication that the photographer suspected that the copy may have moved during exposure and thus cause a blurred image. You will find a good image of the page in the adjacent frame.

3. When a map, drawing or chart, etc., was part of the material being photographed the photographer followed a definite method in "sectioning" the material. It is customary to begin photoing at the upper left hand corner of a large sheet and to continue photoing from left to right in equal sections with a small overlap. If necessary, sectioning is continued again — beginning below the first row and continuing on until complete.

4. The majority of users indicate that the textual content is of greatest value, however, a somewhat higher quality reproduction could be made from "photographs" if essential to the understanding of the dissertation. Silver prints of "photographs" may be ordered at additional charge by writing the Order Department, giving the catalog number, title, author and specific pages you wish reproduced.

5. PLEASE NOTE: Some pages may have indistinct print. Filmed as received.

University Microfilms International 300 North Zeeb Road Ann Arbor, Michigan 48106 USA St. John's Road, Tyler's Green High Wycombe, Bucks, England HP10 8HR 7819651 PXASCIK, MARY FAUST A pharmacological e v a l u a t i o n OF n e w c a l c i u m ANTAGONISTS* P-SUBSTITUTED 3-oimethylam ino - 5 , 6-methylenedioxyinoenes, THE OHIO STATE UNIVERSITY, PHtDt, 1978

(®) Copyright by

Mary Faust Piascik A PHARMACOLOGICAL EVALUATION OF NEW CALCIUM ANTAGONISTS:

2 -SUBSTITUTED 3-DIMETHYLAMINO-5, 6-METHYLENEDIOXYINDENES

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of The Ohio S ta te U n iv ersity

"by

Mary Faust Piascik, B.S.

*****

The Ohio State University

1978

Reading Committee: .pproved

A llan M. Burkman Duane D. M iller H alf G. Rahwan Norman J. U retsky Adviser College of Pharmacy Division of Pharmacology This dissertation is dedicated to:

My parents, who always encouraged me to use my God-given talents to their fullest potential and showed me through their own lives the value of hard work and persistence to achieve a goal.

My hushand, Michael, who made the last four years happy ones for me • when they might have heen very difficult and who always challenged me by his own example to strive to be the best at whatever I under took.

My adviser, Dr. Ralf Rahwan, for his guidance and faith (which often exceeded my own) in my ability to complete this undertaking.

--Thanks to all of you for being the kind of people I want to be proud of me.

ii ACKNOWLEDGMENTS

I would like to gratefully acknowledge the constructive criticism and h e lp fu l comments o f th e members o f my Ph.D. reading committee: Brs. A llan M. Burkman, Duane D. M ille r, and Norman J. U retsky.

iii VITA

October 1, 1951 B o rn -B e llev ille , I l li n o i s

1 9 7 4 ...... B.S.Fharm., Ohio Northern Univ e r s ity , w ith high honors, Ada, 0.

1^74-1975 University Fellow, Ohio State 1977-1978 . o ...... University, Columbus, Ohio

I 97Z1—I 9 7 6...... Teaching Assistant, College of Pharmacy, The Ohio State Univer s ity , Columbus, Ohio

1975-1977 ...... Fellow of the American Foundation for Pharmaceutical Education

1976-1978 ...... Research Assistant, College of Pharmacy, The Ohio State Univ ersity, Columbus, Ohio

PUBLICATIONS

Pharmacological evaluation of new calcium antagonists: 2-substituted 3-dimethylamino-5,6-methylenedioxyindenes. The Pharmacologist. 18: 234, 1976. Pharmacological evaluation of new calcium antagonists: 2-substituted 3-dimethylamino-5»6-methylenedioxyindenes. J. Pharmacol. Exp. Ther. 201: 125-137. 1977. Pharmacological evaluation of new calcium antagonists: 2-substituted 3-dimethylamino-5»6-methylenedioxyindenes. Effects on adrenomedullary catecholamine secretion. The Pharmacologist. 1£: 185, 1977*

Pharmacological evaluation of new calcium antagonists: 2-substituted 3 -dimethylamino- 5, 6-methylenedioxyindenes. Effects on adrenomedullary catecholamine secretion. J. Pharmacol. Exp. Ther. 205: in p re ss, 1978.

iv Pharmacological evaluation of new calcium antagonists: 2-substituted 3-dimethylamino-5,6-methylenedioxyindenes. Coronary and cardiac effects. The Pharmacologist. 20: in press , 1978.

Pharmacological evaluation of new calcium antagonists: 2-substituted 3-dimethylamino-3, 6-methylenedioxyindenes. Coronary and cardiac effects. J. Pharmacol, Exp. Ther., submitted.

Recent advances in aminoindene research. Abstract, The American Chem ical Society, 16th National Medicinal Chemistry Division Symposium, Kalamazoo, Michigan, June, 1978.

FIELDS OF STUDY

Major Field: Pharmacology

Calcium antagonists

Excitation-contraction coupling and stimulus-secretion coupling

Endocrine pharmacology

Genetics—particularly mechanisms of drug-induced mutagenesis

v TABLE OF CONTENTS

Page

DEDICATION...... i i

ACKNOWLEDGEMENTS ...... i i i

VITA ...... iv

LIST OF TABLES...... v i i i

LIST OF FIGURES...... ix

CHAPTER

I INTRODUCTION

ROLE OF CALCIUM ANTAGONISTS IN EXCITATION-CONTRACTION 1 COUPLING Historical Perspective and Skeletal Muscle ..... 1 Smooth Muscle ...... 5 Cardiac Muscle ...... 8

ROLE OF CALCIUM IN STIMULUS-SECRETION COUPLING .... 10 Historical Perspective ...... 11 Adrenal Medullary S ecretion ...... 13

POTENTIAL THERAPEUTIC USES OF CALCIUM ANTAGONISTS . . 16 Skeletal Muscle Relaxants ...... 16 Local Anesthetics ...... • 17 Anticonvulsants ...... 18 Antiarrhythraic A gents ...... 20 Agents used to treat "both Coronary Heart Disease and 22 Arrhythmias Antihypertensive Agents ...... 25 Miscellaneous Drugs ...... 2?

STATEMENT OF THE PROBLEM...... 35

vi Page

I I MATERIALS AND METHODS

Phaxmacological Evaluation of New Calcium Antagonists: 39 2-Substituted 3-Diraethylamino-5» 6-Methylenedioxyindenes. E ffe cts on Smooth Muscles. Pharmacological Evaluation of New Calcium Antagonists: 43 2-Substituted 3-Dimethylamino-5i6-Methylenedioxyindenes. Effects on Adrenomedullary Catecholamine Secretion. Pharmacological Evaluation of New Calcium Antagonists: 4?' 2-Substituted 3-Dimethylamino-5,6-Methylenedioxyindenes. Coronary and Cardiac Effects.

I l l RESULTS Pharmacological Evaluation of New Calcium Antagonists: 51 2-Substituted 3-Dimethylamino-5»6-Methylenedioxyindenes. E ffe c ts on Smooth Muscles. Pharmacological Evaluation of New Calcium Antagonists: 63 2-Substituted 3-Diinethylamino-5> 6-Methylenedioxyindenes. Effects on Adrenomedullary Catecholaine Secretion, Pharmacological Evaluation of New Calcium Antagonists. 75 2-Substituted 3-Dimethylamino-5,6-Methylenedioxyindenes. Coronary and Cardiac Effects.

IV DISCUSSION Pharmacological Evaluation of New Calcium Antagonists: 93 2-Substituted 3-Dimethylamino-5»6-Methylenedioxyindenes. E ffe cts on Smooth Muscles. Pharmacological Evaluation of New Calcium Antagonists: 97 2-Substituted 3-Diraethylamino-5>6-Methylenedioxyindenes. Effects on Adrenomedullary Catecholamine Secretion. Pharmacological Evaluation of New Calcium Antagonists: 101 2-Substituted 3-Dimethylamino-5>6-Methylenedioxyindenes. Coronary and Cardiac Effects. Pharmacological Studies from other Laboratories ..... 104 Proposed Future Studies with the Aminoindenes ...... 106

BIBLIOGRAPHY ...... 108

vii LIST OF TABLES

Table Page

1. Effect of 2-n-propyl 3-dimethylamino-5» 6-methylenedioxyindene on the spasmogenic effect of several a g o n i s t s ...... 61

2. Effect of 2-n-butyl 3-dimethylamino-5,6-methylenedioxyindene on the spasmogenic effect of several ag o n ists ...... 62

viii LIST OF FIGURES

Figure Page

1. Chemical structures of the 2-substituted 3-diraethylamino- 5,6-methylenedioxyindene hydrochlorides ...... 37

2 . Antagonistic effect of 2-n-propyl 3-|iimethylamino-5i 6- methylenedioxyindene hydrochloride on uterine contrac tions induced by prostaglandins and oxytocin ...... 52

3. Antagonistic effect of 2-n-butyl 3-dimethylamino-5»6- methylenedioxyindene hydrochloride on uterine contrac tions induced by prostaglandins and oxytocin. .... 54

4. Concentration-response curves for PGF^Q! -induced uterine contractions in presence of the 2-substituted 3-dimethylamino-5,6-methylenedioxyindene hydrochlorides ...... 56

5. Concentration-response curves for oxytocin-induced uter ine contractions in presence of the 2-substituted 3-dimethylamino-5,6-methylenedioxyindene hydrochlorides. . . 59

6. Effect of extracellular calcium on the antagonism of acetylcholine-induced uterine contractions by the 2-sub stituted 3-dimethylamino-5j6-methylenedioxyindene hydro chlorides...... 64

7. Effect of extracellular calcium on the antagonism of barium-induced uterine contractions by the 2-substituted 3-dimethylamino-5»6-methylenedioxyindene hydrochlorides. 66

8. Cumulative concentration-effect curves for carbachol- evoked catecholamine secretion from the isolated bovine adrenal gland in the presence and absence of 2-n-propyl aminoindene, ...... 68

9. Cumulative concentration-effect curves for carbachol- evoked catecholamine secretion from the isolated bovine adrenal gland in the presence and absence of 2-n-butyl aminoindene...... 71

ix Figure Page

10. Cumulative-concentration curves for acetaldehyde- induced catecholamine secretion from the isolated bo vine adrenal gland in the presence and absence of 10”^M 2-n-propyl or 2-n-butyl aminoindenes ...... * . 73

11. Effect of 2-substituted aminoindenes on washout of -'Ca 4*5 from the isolated bovine adrenal gland ...... 76

12. Effects of 2-substituted 3-dimethylamino-5,6-methylenedioxyindenes on potassium-induced contractions of bovine coronary strips • 78

13* The reversible effects of 2-substituted 3-dimethylamino- 5,6-methylenedioxyindenes and prenylamine on potassium- induced contractions of bovine coronary strips. . . . 81

14-. Effects of prenylamine and theophylline on coronary flow, force of myocardial contraction, and heart rate in iso lated perfused rabbit hearts ...... 83

15. The effects of 2-n-propyl 3-dimethylamino-5,6-methylenedioxyindene on coronary flow, force of myocardial contrac tion, and heart rate in isolated perfused rabbit hearts . 86

16. The effects of 2-n-butyl 3-dimethylamino-5,6-methylenedioxyindene on coronary flow, force of myocardial contrac tion, and heart rate in isolated perfused rabbit hearts . 88

17. The effect of 2-n-propyl 3-<3.imethylamino-5,6-methylenedioxyindene on ouabain-induced arrhythmia in the dog. . 90

x CHAPTER I

INTRODUCTION

ROLE OF CALCIUM ANTAGONISTS IN EXCITATION-CONTRACTION COUPLING

Calcium antagonists are valuable pharmacological tools for the study of excitation-contraction coupling and stimulus-secretion coup ling mechanisms. In order to understand how calcium antagonists can affect these processes, it is first necessary to discuss the role of calcium in the normal functioning of these processes.

Historical Perspective and Skeletal Muscle

Since Sidney Ringer (I 883) first demonstrated the importance of calcium in sustaining myocardial contractility, it has been recognized that this ion is essential in normal muscle responsiveness. Calcium involvement is complex and may occur at several different points in the contraction sequence, this involvement beginning in skeletal muscle, at the generation of the action potential since muscle equilibrated in calcium-free Ringer's solution does not respond electrically or mech anically to an electrical stimulus (Edman and Grieve, 1963). Calcium was first shown to be a unique specific activator of contraction by Heil- brunn (19^0) and Heilbrunn and Wiercinski (19^-7) when micropipette in jections of various ions demonstrated that only calcium could produce contraction (at a concentration of approximately 0,2 mM).

It is now known that the calcium ions which activate the contractile process are released not at the surface of the fiber as suggested by 1 Heilbrunn and Wiercinski (1947) but throughout the interior of the muscle cell due to a signal which is spread inwardly by the transverse tubular system. This release occurs at a distance of 0,5 to 1 micron from the myofibril to be activated (Sandow, 1 965)0

Weber et al. (1963)1 using a model system composed of extracted actomyosin or myofibrils in the presence of ATP and Mg , found that their system could simulate contraction or relaxation by properly in creasing or decreasing, respectively, the calcium concentration in the medium. Since contraction can occur at calcium concentrations of 0.2-

0,3^M in the medium, the myoplasmic calcium concentration of muscle musii’ be considerably less than this concentration during relaxation.

The sarcoplasmic reticulum is apparently capable of reducing the calcium concentration to 0.1 p,M or less (Weber et a l. , 1964), In fact, almost all of the calcium is sequestered during relaxation, lowering the cal cium level in the myoplasm to almost zero (0.1n,M or less).

Marsh (1952), using a minced muscle system, concluded that a re laxing factor (RF) exists which could reverse myofibrillar syneresis, suppress actomyosin ATPase activity and relax contracted glycerinated muscles. This relaxing factor was recognized by Ebashi (Kumagai et a l.,

1955)\ to be a particulate Mg ■H -activated1 ATPase, The ATPase is activa ted by calcium only for transient periods since the calcium is seques tered following the activation phase (Hasselbach and Makinose, 1961).

Not all actomyosin systems require calcium for contraction. Pure actin and myosin mixtures as well as tryptic digest of myofibrils do not, The calcium requirement is conferred by the presence of the pro tein component, troponin (Ebashi and Endo, 1968). Actually the troponin component is complexed with tropomyosin. This complex inter feres with the interaction between myosin, actin and ATP thus preventing

Mg -activated contraction and ATPase activity. Calcium released from the sarcoplasmic reticulum has a special affinity for troponin. Bind ing to troponin releases actin, and contraction ensues. Calcium, there fore, is the ultimate controlling factor in this process.

Bianchi ( 1969) has described three calcium pump sites by which intracellular calcium concentration is regulated, either via seques tration of calcium intracellularly or regulation of calcium efflux from the cell. The sarcoplasmic reticulum and mitochondria can utilize ATP to sequester calcium while the calcium pump located in the surface membrane appears to be ab le to use th e sodium g rad ie n t across th e mem brane to transport calcium from the cell. Calcium stored in the mito chondria is unlikely to be available for release during the action potential, The mitochondria may serve as a reserve sink of calcium when the sarcoplasmic reticulum sequestering sites cannot maintain sufficiently low myoplasmic calcium concentrations for relaxation.

Bianchi (19^9) has termed skeletal muscle (frog sartorius) an in directly coupled system since the action potential requires intact triad structures to be present for coupling of excitation to contrac tion. When depolarization occurs (or calcium removal from the stabil iz in g s ite s on the o u ter membrane su rfa ce , a conform ational change oc cu rs in membrane s tru c tu re . Membrane p erm eab ility in creases and c a l cium is released from the inner aspect of the membrane, This calcium

(which is low in skeletal muscle) can bind at the troponin sites of the myofibrils allowing contraction to occur. Also, in skeletal muscle, release of calcium into the myoplasm is presumed to occur additionally from the terminal sacs as well as the transverse tubule element.'

In summary then the follow ing events a re presumed to occur. At rest, calcium is sequestered in the sarcoplasmic reticulum. Actin is

therefore suppressed by the troponin-tropomyosin complex. When activa

tion occurs, the surface impulse spreads throughout the transverse tu bules and induces a change in the sarcoplasmic reticulum which releases

calcium ions into the myoplasm. Transiently attaining the threshold

concentration, calcium reaches the myofilaments and binds troponin.

Actin is released to form actomyosin and contraction ensues with split- 4*4* ting of ATP by Mg -activated actomyosin-ATPase. Calcium ions are then actively sequestered again into parts of the sarcoplasmic reticulum— not necessarily the same parts in which it will eventually be stored

at rest (Mommaerts, 1971)—thus allowing for relaxation.

Skeletal muscle differs from smooth and cardiac muscle in having

the highest requirement for calcium intracellularly in the region of

the contractile apparatus. Skeletal muscle also contains the least

amount of su rface membrane a rea (ex clu siv e of th e tran sv erse tubule

system ).

Excitation-contraction coupling in skeletal muscle is less sensi

tive to changes in extracellular calcium concentration or transmembrane

calcium conductivity than the other two types of muscle, because the

large intracellular stores found in skeletal muscle provide quantities

of calcium capable of fully activating the contractile system (Pleck-

enstein, 1977)* This is the most likely explanation for the relatively

greater resistance of skeletal muscle to pharmacological interventions

by calcium antagonistic drugs. Smooth Muscle

In th e ir 1970 symposium, Somlyo and Somlyo discussed th e physiol ogy and pharmacology of smooth muscle in terms of their own classifica tion of excitation-contraction coupling into electromechanical and pharmacomechanical coupling. This model assumes that the most important source of activator calcium mediating drug-induced contraction is ul timately extracellular calcium, and also that the primary determining factor in contraction is the increase or decrease in intracellular free calcium. However, at a given calcium concentration, actomyosin ATPase activity can vary with other factors such as pH, temperature, or mag nesium concentration.

Electromechanical coupling involves membrane depolarization, a change in membrane p erm eability and a subsequent in flu x of calcium by displacing activator calcium across the cell membrane. This activa tor calcium may be due to influx of extracellular calcium or translo cation of calcium bound at the surface membrane. Activator calcium may also come from a storage pool of calcium at an intracellular site such as the sarcoplasmic reticulum. The calcium sinks are thought to be re gions of high electrochemical potential requiring energy expenditure for sequestration of calcium during relaxation (Bianchi, 1969)• The assumption that excitatory drugs act through permeability changes of the plasma membrane to calcium implies that the main source of activa tor calcium is extracellular in nature.

Pharmacomechanical coupling differs from electromechanical coupling in th a t in the former no change occurs in membrane p o te n tia l. The drug enters the cell and releases calcium from storage sites, or pre- vents calcium sequestration into such sites. Activator calcium may arise from the inner aspect of the plasma membrane itself, or from storage pools located intracellularly, and is replenished eventually from the extracellular fluid.

Somlyo and Somlyo (1970) have c ite d convincing evidence fo r th e ir hypothesis of pharmacomechanical coupling, l) The same blocking and potentiating agents are effective both in polarized and depolarized smooth muscle. 2) High potassium solutions may have a greater depo larizing effect than certain drugs which act by pharmacomechanical coupling, but the drugs can produce a greater maximal contraction,

3) Drug-induced depolarization may be sustained in smooth muscle which responds to potassium-induced depolarization in a transient manner.

U-) Certain spasmogens can further contract a muscle maximally depolar ized with potassium. 5) Relaxing agents can relax polarized smooth muscle without evidence of hyperpolarization. 6) In polarized smooth muscle, dissociation of electrical and contractile effects of drugs is readily seen. Similar mechanisms to electromechanical and pharmacomechanical coupling in smooth muscle may exist in skeletal and cardiac muscle (Sandow, 1965; Haugaard et a l., I 969).

An alternative to the pharmacomechanical coupling hypothesis was proposed by Van Breemen and Daniel ( 1966), Two separate calcium sites in parallel would exist in which calcium displaced by potassium (for example) from a superficial site would be replenished from a deep site rather than the extracellular fluid.

Inhibitory stimuli producing relaxation may cause either a de crease in membrane p erm eab ility to calcium in flu x , an enhanced operation of the calcium extruding pump, increased sequestration of intracellular calcium, decreased mobilization of calcium from storage pools, or a combination of any of these mechanisms.

Hurwitz and Joiner ( 1969) have proposed that, in smooth muscle, extracellular calcium reaches the myoplasm by two parallel pathways.

Ions migrating along one path are taken up into a cellular compartment relatively inaccessible to the contractile proteins. Then a portion of these ions are released to migrate into the myoplasm where con traction can occur. When the depot is partially loaded with calcium, muscle tone can be su stain ed in a calcium -free medium. Secondly, ex tracellular calcium may reach the myoplasm by bypassing the depot.

This source of calcium could either diffuse down an electrochemical gradient or may involve another cellular compartment where calcium ions are loosely bound.

Unlike skeletal muscle, in smooth muscle calcium is required for discharge of propagated action potentials as well as for excitation- contraction coupling. Therefore, calcium antagonism could produce relaxation directly by inhibiting contractility or indirectly by sup p ressin g membrane e x c ita b ility . Smooth muscle has th e low est re q u ire ment among muscle types for the amount of calcium necessary at the contractile apparatus to produce contraction. Since smooth muscle is particularly unspecialized in terms of the development of large sarcoplasmic calcium pools, excitation-contraction coupling is sus ceptible to changes in extracellular calcium concentration or trans membrane calcium co n d u ctiv ity and th e re fo re i s su sc e p tib le to phar macological agents or changes in environmental calcium (Fleckenstein,

1977). Cardiac Muscle

In 1913» Mines demonstrated that withdrawal of calcium impairs mechanical performance in cardiac muscle but not the ability to pro duce normal action potentials. Calcium serves not only as an initia tor of the contractile process but also can control in a quantitative manner the amount of ATP metabolized and subsequently the output of mechanical tension (Fleckenstein, 1963)1

The sudden influx of calcium across the excited cardiac muscle cell is electrogenic. That is, calcium ions, as well as sodium and potassium , c o n trib u te to th e changes in membrane p o te n tia l during c ar diac activity. In special circumstances, calcium may even substitute for sodium as the transmembrane charge carrier in the electrogenesis of the action potential. However, in general, sodium and calcium use sep arate transmembrane c a r r ie r systems in e x cite d mammalian m yocardial fibers. These have been termed a fast channel for sodium and a slow channel for calcium (Beeler and Reuter, 1970). Fleckenstein (1970) has shown that selective drug actions may inhibit predominantly the sodium influx, reducing myocardial excitability, or the slow calcium channel, decreasing myocardial contractility,

Alterations in the extracellular calcium concentration will lead to parallel changes in the amount of ATP consumed by the contractile apparatus, the magnitude of mechanical tension developed, and extra uptake of oxygen related to the contractile force generated. However, the resting myocardial respiration rate remains stationary despite changes in calcium (Fleckenstein, 1963). Therefore, other ATP-con-

suming processes, not concerned with mechanical activity are insen

sitive to calcium levels. The importance of calcium in determining cardiac metabolic ac

tivity can be demonstrated by examining the role of calcium in the

actions of agents with a positive inotropic effect on the heart. For

example, beta adrenergic agonists promote transmembrane calcium influx

through the slow channel (Fleckenstein, 1977)* The cardiac glycosides

also facilitate access of extracellular calcium to intracellular com

partments where contraction is activated (Fleckenstein et a l., 1976),

but by a different mechanism of action involving inhibition of the out wardly directed Na-K pump,

In addition to impairing mechanical performance, calcium de

ficiency inhibits cardiac pacemaker activity. Calcium antagonistic

divalent cations—cobalt, nickel, and manganese—suppress myocardial

contractility and pacemaker activity equally (Kohlhardt et a l.. 1976).

Fleckenstein (1977) has concluded that cardiac pacemaker activity—

impulse generation and propagation—-require calcium. An apparently

similar or even identical calcium transport system operates as the slow

calcium channel in both myocardial fibers and nodal cells.

B ianchi ( 1969) has described a directly coupled system for

cardiac muscle (as opposed to the indirectly coupled system in skeletal

muscle) in which membrane depolarization directly results in the re

lease of calcium from binding sites present on the inner surface of the

cell membrane. This release is necessary to account for the amount of

calcium needed in the contraction process,

Haugaard et al, (1969) have proposed a possible role for mito

chondria in calcium movements in the heart, thus regulating relaxation,

based on th e follow ing evidence. The calcium content of the 10 mitochondria in the heart depends on the content of the perfusing me dium. Most of the calcium content of the heart is contained in the mitochondria, and the mitochondria lie in close proximity to the myo fibrils, the contractile machinery of the heart. They further specu lated that phosphate formed during breakdown of ATP could possibly effectively stimulate the removal of cytoplasmic calcium into the mitochondria thus promoting relaxation.

Cardiac muscle is much more dependent on extracellular calcium as a source of internal calcium than skeletal muscle with its large internal stores of calcium. Calcium in the perfusing solution is re quired in heart fibers, but not twitch fibers. Also, calcium increases the threshold of tension in skeletal muscle but not cardiac muscle.

Since myocardial fibers are less specialized in the development of large sarcoplasmic calcium pools than skeletal muscle (as has been previously noted to apply to smooth muscle also), myocardial contrac tility is very susceptible to changes in environmental calcium or to pharmacological agents which may affect calcium in a variety of ways.

ROLE OF CALCIUM IN STIMULUS-SECRETION COUPLING

Similarities in the requirements for calcium in excitation-con traction and stimulus-secretion coupling have been discussed by Rahwan and Borowitz (1973) and Rubin (1970). For example, secretory systems are also critically dependent on the presence of either extracellular or intracellular calcium with only limited exceptions (O'Neill and

Rahwan, 1975)• Furthermore, the discharge of stored product in many systems will not occur if ATP synthesis is blocked or granular ATPase 11

Is inhibited. Finally, a number of secretory cells have been demon strated to contain an actomyosin-like contractile protein, Borowitz

(1967) has demonstrated that calcium is bound to the subcellular organ elles of the adrenal medulla, in analogy to the way in which the free intracellular calcium levels in muscle cells are reduced by an ATP- activated calcium uptake system in the sarcoplasmic reticulum. Pois- ner and Hava (1970) have described an ATP-activated calcium uptake by the mitochondria of adrenal chromaffin cells similar to that pre viously discussed for cardiac muscle (Haugaard et a l., 1969)1

Historical Perspective

That calcium is required for the release of neurotransmitters from nerve endings was first demonstrated by Harvey and Macintosh

(1940; cholinergic) and Burn and Gibbons (1965; adrenergic). Rubin

(1970) stated that calcium influx seems to be a critical step between depolarization of the nerve terminal and transmitter release at periph eral cholinergic and adrenergic synapses.

Jaanus and Rubin (1971) have shown that an intracellular calcium pool presumed to exist at the inner surface of the cell is probably responsible for AGTH-induced corticosteroid secretion from the adre nal cortex rather than an extracellular source. This conclusion was based on the finding that ACTH stimulation of the adrenal changes the intracellular distribution of radiolabeled calcium but not the overall calcium content of the organ.

The requirement for extracellular calcium in oxytocin and vaso pressin release from the posterior pituitary was first demonstrated by Douglas ( 1963) and Douglas and Poisner (1964a, 1964b). There is 12 evidence to suggest that depolarization of neurosecretory terminals followed by calcium influx is the cause of hormone release (Rubin, 1970).

Adenohypophyseal secretion also appears to depend on extracellu lar calcium. Calcium-free incubation diminished or abolished release of thyrotropin (Vale et a l., 1967), luteinizing hormone (Samli and

Geschwind, 1968), ACTH (Kraicer et a l., 1969), and prolactin (Parsons,

(1969). Insulin secretion in response to numerous stimuli can be abolished by eliminating extracellular calcium (Curry et a l., 1968; Hales and

Milner, 1968; Milner and Hales, I 967).

The interaction of calcium with parathyroid hormone and calcito nin is quite complex. Although these two hormones play a critical role in maintaining calcium homeostasis, their rate of release is gov erned by the very calcium levels in the extracellular fluid which they attempt to control, Perfusion of isolated parathyroid glands with calcium-free medium increases the level of parathyroid hormone release.

This unusual event may possibly be explained by the fact that biosyn thesis of the hormone may be the limiting factor in determining secre tion rate and calcium may exert its effect at this level (Rubin, 197*0•

Calcitonin release from cells in organ culture has been shown to be d ir e c tly p ro p o rtio n al to the calcium concentration of the medium. I t appears that calcitonin secretion induced by calcium may require no other stimulus. If true, this would represent a unique system, since other glands require some prior stimulation preceeding calcium- mediated secretion, Non-hormone secretory substances also require calcium for release.

Hydrochloric acid secretion from the parietal cell in vitro has been demonstrated to require calcium (Jacobson et a l., 19 65). Histamine and serotonin also have shown a secretory requirement for calcium in some tissues. Mongar and Schild (1958) demonstrated that the release of histamine in anaphylaxis was markedly inhibited by lack of calcium, but neither potassium or sodium were essential requirements for his tamine release.

Calcium ions can affect the release of transmitter substances from nerve tis s u e —dopamine (Bustos and Roth, 1972), gamma-amino bu tyric acid (Otsuka et ad., I 966), glutamate (Usherwood et a l,, 1968) as well as acetylcholine and norepinephrine as previously discussed.

Excitatory and inhibitory synaptic transmission depends on the pres

ence of calcium in the extracellular fluid. Release of transmitter appears to be proportional to external calcium concentration and is an

tagonized by the presence of magnesium (Rubin, 197*0*

Adrenal Medullary Secretion

Since much of the present knowledge about stimulus-secretion coupling resulted from studies using the isolated adrenal medulla, this

organ will be discussed in greater detail.

Calcium-free perfusion of the adrenal medulla can diminish or

abolish evoked secretion (see Rahwan et a l., 1973*0. Many secreta-

gogues a re known to d ep o larize the chrom affin c e ll membrane (Douglas

e t a l . , I 967). Furthermore, an increase in radiolabeled calcium influx

into chromaffin cells occurs during stimulation (Borowitz, 1969; Doug

las and Poisner, 1962; Rubin et al., 1967)* Depolarization may not 14 necessarily be coupled tightly with secretion as long as calcium is supplied (Douglas, 1966; Douglas and Rubin, 1963)* Douglas et al.

(1967) showed that acetylcholine or excess potassium still could de polarize the chromaffin cell without evoking catecholamine secretion if the perfusion solution was calcium-free or contained excess magne sium (which antagonizes calcium).

Despite the importance of extracellular calcium to stimulus-secretion coupling, some drugs (e.g. caffeine, amphetamine, and chlorpromazine) are capable of releasing catecholamines from the adrenal in the absence of calcium in the perfusion solution (Rahwan et ad., 1973b).

Borowitz et al. (19&5) demonstrated a high content of calcium in the various intracellular organelles which could be involved in catechol amine secretion from adrenal chromaffin cells, as Sandow ( 1965) has proposed for skeletal muscle, In fact, mitochondrial and endoplasmic reticulum calcium have been demonstrated to be involved in catecholamine secretion induced by chlorpromazine and amphetamine respectively

(Rahwan et ad,, 1973b)* A nonutilizable calcium pool has been identi fied as being located in the mitochondria (Carafoli,' 1967).

Chromaffin granules may play a role in terminating the secretory response. A Mg -dependent ATP pump which concentrates calcium in the granule resembles that seen in muscle (Taugner and Hasselbach, I 966),

The electromechanical-pharmacomechanical coupling classification developed by Somlyo and Somlyo (1970) fo r muscle tis s u e has been ap plied to the adrenal medullary system by Rahwan et al. (1973b). V/hile electromechanical coupling is dependent on extracellular calcium, pharmacomechanical coupling depends on intracellular calcium and could 15 occur in several ways: release of calcium from or inhibition of calcium uptake into the endoplasmic reticulum or mitochondria, release of calcium from or in h ib itio n of calcium binding to c e l l membrane sto rag e s i t e s , and inhibition of the outwardly directed sodium-calcium membrane pump.

In order to clearly understand the role of calcium in regulating stimulus-secretion coupling, the mode of release of the secretory prod uct should be discussed. However, no single theory is accepted to reflect the true nature of secretion in all systems. Catecholamine release from the adrenal medulla has been proposed to occur via either exocytosis (first proposed by DeRobertis and Vaz Ferreira, 195?) "the existence of microtubules (Schmitt, I 968). Poisner and Trifaro ( 1967) developed a model in which ATP and ATPase in te r a c t (th e in te ra c tio n being promoted by calcium) to bring about attachment of the granule membrane to the plasmalemma. Membrane fu sio n occurs w ith a subsequent conformational change in the granule membrane, and the granule contents are released. Simpson ( 1968) has proposed that the calcium influx re sulting from chromaffin cell membrane depolarization may allow a colli sion between the chromaffin granule (ganglioside in nature) and the plasma membrane (organic phase) to persist. Calcium could activate membrane phospholipase A or granule ly s o le c ith in f a c i li t a t i n g fu sio n of the granule to the plasma membrane.

Rahwan et_ al, (I973a) have advanced a theory for secretion in which hormones exist as intratubular droplets in the chromaffin cell.

Influx of extracellular calcium or translocation of intracellular cal cium, by increasing the free cytoplasmic calcium concentration, would activate an actomyosin-1ike protein in the contractile membranes of 16

the secretory tubule, perhaps by binding to troponin. This would

ultimately result in hormone release.

Finally, Jahn and Bovee (I 969) have proposed a cytoplasmic mo

tility theory of secretion. The granules would be propelled by proto

plasmic movement, either rotational or circulatory, forming a inicro-

stream. M otility would be provided by ATP splitting by an actomyosin-

like protein. This energy would cause a conformational change in the myosin cross-bridge allowing actin and myosin to slide as in the

sliding filament theory of muscle contraction.

In summary, i t i s p o ssib le th a t, ju s t as the mode of se c re tio n may vary in different secretory systems, the role of calcium may also vary with the particular secretory system under discussion.

POTENTIAL THERAPEUTIC USES OF CALCIUM ANTAGONISTS

Although very few clinically useful drugs have calcium antagonism as their main pharmacological property, many drugs exhibit calcium an tagonistic effects in addition to their primary actions. This sec tion will discuss the calcium antagonistic effects of these drugs and the possible relationship between their calcium antagonistic properties and therapeutic usefulness.

Skeletal Muscle Relaxants

As discussed previously, skeletal muscle is relatively insensi tive to pharmacological intervention due to the large intracellular calcium stores of the sarcoplasmic reticulum. Therefore, most skel etal muscle relaxants presently in clinical use act through the central nervous system, However, one direct-acting skeletal muscle relaxant, 17 dantrolene, Is presently in use (Snyder et al,, 196?). This drug has no central effect and little or no effect on smooth or cardiac muscle

(Ellis et a l., 1973)• It has teen hypothesized that the skeletal mus cle relaxant effect of dantrolene is due to a direct calcium antagonism, perhaps by decreasing either calcium influx into the cell or calcium release from the sarcoplasmic reticulum (Ellis and Carpenter, 1972,

1974-; Putney and Bianchi, 197^)* This would effectively decrease the calcium available to bind troponin, thereby allowing muscle relaxation to occur.

Local Anesthetics

Calcium ions, bound to the external surface of the cell membrane, may exert a regulatory role on sodium ion movements across the nerve cell membrane. It has been suggested that the initial step in nerve cell depolarization may be the release of externally bound calcium causing an in creased membrane p erm eab ility to sodium (B lau stein and

Goldman, 1 966). Therefore, the local anesthetics may exert their ef fect, at least in part, by an interaction with calcium (Bondani and Kar- ler, 1970; Papahadjopoulos, 1970). Aceves and Machne (19^3) have pro posed that local anesthetic molecules may compete with calcium for a s i t e on the nerve membrane. The lo c a l a n e sth e tic in h ib itio n o f sodium conductance is accentuated by decreasing calcium concentration and re versed by increasing calcium concentration. It is presently proposed that local anesthetic agents act by replacing calcium ions at a recep tor site located at the cell membrane. In addition to inhibiting cal cium influx across the cell membrane, local anesthetics also inhibit 18 calcium release from sarcoplasmic reticulum stores (Feinstein and

Paimre, 1969). Calcium antagonism is also responsible for the ability of local anesthetics to depress smooth muscle contraction (Feinstein, I 966) and prevent catecholamine release from the adrenal medulla (Jaanus et al. , 196?). The local anesthetic effect on vascular smooth muscle is actually biphasic. Since a competitive antagonism exists between lo cal anesthetics and calcium, the in itial displacement of calcium from binding sites on the outer surface of the membrane may temporarily increase intracellular calcium concentrations by releasing calcium bound to the in n er su rface of the membrane leading to v a so co n stric tio n .

Ultimately, however, the displacement of calcium will decrease cyto plasmic calcium levels by inhibiting calcium influx and muscle relaxa tion, or vasodilation, will occur (Somlyo and Somlyo, 1970).

Anticonvulsants

Since epileptic seizures are characterized by the existence of an epileptogenic focus which produces a chronic, spontaneous, recurring seizure, it would seem that any agent which can either reduce the rate of discharge of the focus neurons or prevent the subsequent spread of the discharge to normal neurons would have usefulness as an anticon vulsant agent. It is conceivable, therefore, that calcium antagonistic drugs could serve as antiepileptic agents by their ability to stabil ize nerve and muscle membranes.

In fact, two agents (phenytoin and barbiturates), which are high ly effective anticonvulsants, are now known to diminish calcium perme ability of membranes. In 1973» Sohn and Ferrendelli demonstrated inhibition of calcium transport into rat brain synaptosomes by pheny toin, suggesting that a major pharmacological action of phenytoin is inhibition of calcium transport into stimulated neural tissue. They further noted the ability of phenytoin to suppress post-tetanic poten tiation (PTP) which may be related to the inhibition of calcium trans port since calcium is proposed to play a role in the development of PTP

(Raines and Standaert, 1967). Pincus and Lee (1973) proposed the mechan ism of action of phenytoin to be an interaction with calcium to limit

PTP and thereby prevent the spread of discharge from an epileptic fo cus.

Opposing actions of phenytoin (decrease) and calcium (increase) on the level of radiolabeled phosphate incorporated into rat and human brain proteins have been demonstrated (DeLorenzo, 1977). Phosphopro- tein synthesis may play a role in neurotransmitter release and convul- sant activity. The antagonism seen in this study would be consistent with the hypothesis that the anticonvulsant actions of phenytoin may be mediated by stabilizing neuronal tissue and inhibiting seizure dis charge via decreased phosphoprotein synthesis.

A dilantin-calcium interaction involved with active sodium trans port in frog skin was noted by Riddle et al. (1975)* The antagonism was likened to the type of antagonism which exists between barbitu rates and calcium.

Phenobarbital and phenytoin are capable of inhibiting calcium uptake into rabbit neocortex synaptosomes whereas ethosuximide does not (Sohn and Ferrendelli, 1976). This inhibition is proposed to occur v ia membrane s ta b iliz a tio n as suggested by Hasbani e t a l, (197*0» 20 limiting seizure spread in two ways. First "by limiting sodium conduc tance, the action potential would be impaired. Secondly, inhibition of calcium uptake at presynaptic terminals would decrease neurotransmitter re le a s e .

Blaustein (1976) demonstrated that pentobarbital and thiopental block the extracellular calcium uptake by stimulated and potassium- depolarized rat sympathetic ganglia, Barbiturates may block calcium conductance not only at presynaptic terminals but also at other sites such as vertebrate cardiac and invertebrate skeletal muscle fibers

(Reuter, 1973)* Antiarrh.ythmic Agents

A definite crossover can be noted in drugs useful in convulsive and arrhythmic states (e.g. phenytoin). This lends support to Yahr's hypothesis (1971) comparing the genesis of epileptic states in neural tissue with arrhythmias in cardiac tissue. Both syndromes have foci of rapid repetitive discharge and both may be initiated either by drugs or electrical stimulation.

The involvement of calcium in cardiac pacemaker activity—impulse generation and propagation—has been previously discussed. Any agent capable of preventing tranSlocation of calcium ions across the cardiac c e ll membrane o r in h ib itin g re le a s e o f calcium from in tr a c e llu la r stores could effectively decrease the frequency of excitation and con traction of the cardiac cell. Several possible mechanisms of antiarrhythmic action involve calcium antagonism—hyperpolarization of the card iac muscle c e ll membrane thereby decreasing p erm eab ility to e x tra cellular calcium, increased uptake by or decreased calcium release 21 from the sarcoplasmic reticulum, increased uptake of calcium into or decreased release of calcium from mitochondria, stimulation of the sodium pump promoting calcium efflux, decreased calcium influx into myocardial cells via the slow calcium channel, or direct interaction with contractile proteins decreasing the sensitivity to intracellular calcium .

Antiarrhythmic agents tend to he a very heterogeneous group of drugs. However, a common property of all these agents—quinidine, procainamide, lidocaine, propranolol, etc.—is a local anesthetic ef fect which has been previously discussed.

Although propranolol is essentially a beta adrenergic blocking agent, its antiarrhythmic actions are not entirely due to its antiadrenergic effects (Moss and Patton, 1973)* This can be demonstrated by the fact that d-propranolol, a very weak beta blocker, is equieffective with 1-propranolol as an antiarrhythmic agent (Somani and Lum, 1963)*

The ability of propranolol to uncouple excitation-contraction coupling

(an effect shared by barbiturates) is considered a side effect which occurs at supranormal doses (Fleckenstein, 1977). These effects are rapidly and completely reversed by administration of calcium chloride or calcium-synergistic promoters of excitation-contraction coupling such as beta adrenergic agonists or cardiac glycosides.

In addition to the local anesthetic type of antiarrhythmic agents characterized by quinidine which blocks both the fast depolarizing sodium current and the slow calcium current in heart muscle, a class of drugs is now known which can specifically inhibit the slow calcium 22 current (Fleckenstein, 1977)• These drugs are all primarily calcium antagonists and w ill he discussed below.

Agents used to treat both Coronary Heart Disease and Arrhythmias

It is presently thought that an effective coronary therapeutic agent reduces or abolishes the imbalance between oxygen supply and the oxygen consumption of an ischemic heart (Vater et a l., 1976). Oxygen consumption may be reduced thragh a direct depressant action on the myocardium (Fleckenstein et a l., 1976), or by inhibition of adrenergic influences in the myocardium (Raab, 1962, 1963)* Increased oxygen sup ply can be achieved through coronary vasodilation. From the previous discussion of excitation-contraction coupling, it can be seen that a compound with calcium antagonistic properties could potentially increase oxygen supply to, as well as decrease oxygen consumption by, the myo cardium.

Nitroglycerine and related nitrites were originally presumed to relieve angina pectoris via coronary dilation (Winbury, I 967).

Actually the vasodilator action of nitroglycerine is characterized by rapid onset, incomplete relaxation, and spontaneous recovery of coro nary tone. Therefore, although nitroglycerine is capable of inter

fering with the calcium-dependent processes involved in excitation- contraction coupling, in vascular smooth muscle, the beneficial ther

apeutic effect is now proposed to occur, at least in part, via a de

crease in myocardial oxygen consumption. This may not be a calcium-

dependent process since, as Needleman and Hunter (1966) have proposed,

nitro-derivatives may interact with sulfhydryl groups in the cardiac mitochondria, leading to the uncoupling of phosphorylation. How this 23 might lead to the therapeutic response is unknown. Complete uncoup

ling cannot occur or the metabolic efficiency of the heart would he

impaired. Other smooth muscle types—bronchial, biliary, gastrointes

tinal, ureteral and uterine—are also relaxed by the nitrites.

Approximately twelve years ago, a new group of very powerful

inhibitors of excitation-contraction coupling was developed, with

some members in c u rre n t use in coronary th e ra p e u tic s (F lecken stein

e t a l . , I 967). Members of this group of compounds include verapamil,

compound C600 (a methoxy derivative of verapamil), nifedipine, prenyl-

amine, fendiline, perhexiline, and diltiazeme. These agents are specif

ic inhibitors of the transmembrane calcium influx through the slow

channel. There is no indication that these calcium antagonists have

any intracellular actions. These compounds are quite specific in that

doses required to produce calcium antagonism do not appear to produce

any other measureable pharmacological effects (Fleckenstein, 1977)•

Several lines of evidence support the specificity of these compounds

on calcium mobility. First, the results of studies on cardiac atrial

(Haas et a l., 1975) and ventricular muscle fibers (Kohlhardt et a l.,

1972), cardiac Purkinje fibers (Cranefield et al. , 197*0 an(l fibers in

the sinoatrial and atrioventricular nodes (Wit and Cranefield, 197*0

are consistent with the observation that these compounds depress the

slow calcium influx. Secondly, both verapamil and D600 block the slow

channel calcium entry in squid axon (Baker et al., 1973). Finally,

these agents have been shown to block excitation-contraction coupling

in smooth muscle (Haeusler, 1972) and skeletal muscle (Bondi et al.

1974), in addition to cardiac muscle (Fleckenstein et a l., 19&9) as

mentioned above. By virtue of their ability to block the transmembrane calcium supply to the contractile system, these drugs relax uterine, intestin al, and vascular smooth muscle as well as decrease myocardial contrac tility , Coronary smooth muscle seems to be particularly sensitive to these agents (Fleckenstein et a l,, 1975; Fleckenstein, 1 97?)• In creas ing the extracellular concentration of calcium can at least partially compensate for the decrease in transmembrane calcium conductivity.

These compounds have been proposed for use in treatment of hyperkinet ic heart function, angina pectoris, and other forms of coronary heart disease requiring restriction of cardiac metabolic activity.

Stimulus-secretion coupling is also affected by this group of compounds. High concentrations of verapamil and D600 specifically block release of oxytocin and vasopressin from the neurohypophysis

(Breifuss et a l., 1975)* Insulin release from beta cells of the islets of Langerhans (Bevis et a l., 1975) is also inhibited. Increasing cal cium concentration will restore secretory function in these systems.

The most extensively studied member of this group of compounds' is verapamil. Excessive doses (at least 1000 times the concentration necessary to produce myocardial failure) are needed to suppress calcium uptake in isolated mitochondria (Frey and Junke, 1975)• Similarly, in isolated sarcoplasmic reticulum preparations, verapamil has been shown capable of altering accumulation, binding or exchange of cal cium. Verapamil has the ability to ultimately deplete a membrane- bound calcium store found present on the inner aspect of the cell membrane (Nayler and Szeto, 1972). This i s achieved through in h ib itio n 25 of calcium influx through the slow channel which normally would re plenish the membrane-bound calcium store.

In addition to the clinical uses in ischemic heart disease, ver apamil has been studied for antiarrhythmic (Huang and Peng, 1977) and u te rin e re la x in g a c tiv ity (Gummerus, 1975» 1977)*

Prenylamine was originally investigated by Lindner (i 960). In addition to inhibition of calcium influx, prenylamine has central ner vous system sedative properties as well as antiadrenergic effects

(Charlier, 1971)• Prenylamine is known to deplete adrenergic storage granules in a manner similar to reserpine (Carlsson et a l., 19^3)•

However, unlike reserpine, prenylamine depletes only norepinephrine and dopamine but not serotonin (except at high doses). An alpha (Cloarec,

I 96I) and beta (Rahwan and Berger, 1970) adrenergic blocking effect has also been reported. Prenylamine has been investigated for its anti arrhythmic properties (Lindner and Kaiser, 1975)*

Nifedipine, probably the most potent of this series of compounds, has undergone extensive clinical trial and was the subject of two re cent symposia (Lichtlen, 1976; Lochner, 1975)* Fendiline has been much less studied (Fleckenstein et a l., 1977)* Perhexiline is presently an

investigational drug in the United States and has been studied in pa tients suffering from coronary heart disease, cardiac arrhythmias, and angina pectoris. Its particular pharmacology has been reported in de t a i l (Symposium, 1973)*

Antihypertensive Agents

The involvement of calcium ions in the maintenance of tone in vascular smooth muscle raises another potential therapeutic use for 26 calcium antagonists. Two drugs (sodium nitroprusside and diazoxide) currently useful in hypertensive crises affect calcium-dependent processes. Nitroprusside has a relaxant effect on various types of smooth muscle, being more pronounced on tonic type muscle (e.g. vascular and tracheal). Inhibition of excitation-contraction coupling by sodium nitroprusside is more dramatic on pharmacomechanieal coupling than on electromechanical coupling (Kreye and Luth, 1976). An antagonism be tween calcium and nitroprusside has been demonstrated in isolated prep arations of vascular smooth muscle (Kreye et. a l., 1975; Hausler and

Thorens, 1976). Conflicting reports have been obtained in studies concerning a possible verapamil-like effect of nitroprusside on cal cium influx into smooth muscle cells (Kreye et al., 1975; Hausler,

1975)* However, it is presently considered that inhibition of cal cium influx is not necessary for the action of nitroprusside (Kreye and Gross, 1977). Nitroprusside can relax potassium-depolarized rat aorta independently of extracellular calcium concentrations (Kreye and

Luth, 1976). These findings indicate that nitroprusside may interfere with intracellular calcium by inhibiting calcium uobilization from storage sites, facilitating extrusion of cellular calcium or enhancing sequestration of intracellular calcium (Kreye and Gross, 1977).

Due to the enhanced hypotensive potency of diazoxide in hyper tensive animals as compared to normal animals (Rubin et a l,, 1962;

Nohl et a l., 1968; Levy, 1975). a- concept of competitive blockade of calcium as a mechanism of antihypertensive action has been proposed.

Diazoxide has also been shown to competitively block the vasoconstrictor 27 action of ‘barium in vitro (Wohl et al. , 1967). Rhodes and Sutter

(1971) concluded from their studies using vascular smooth muscle that diazoxide inhibits smooth muscle contraction mainly by an effect at the cell membrane possibly relating to the role of calcium in regulating excitability of the cell membrane. This could occur through diazoxide binding to calcium sites on the outer surface of the membrane, stabil

izing the membrane, and preventing calcium release from the calcium pool on the inner aspect of the cell membrane, Although an effect on the cyclic nucleotide system has been proposed, it is now thought that diazoxide is not an inhibitor of cyclic nucleotide phosphodiesterase but may act either by depleting an intracellular calcium pool or inhib

iting the mobilization of calcium from such a pool (Gross, 1977) as mentioned above.

Miscellaneous Drugs

Antagonism of morphine analgesia by calcium and its potentiation by calcium chelators has been observed (Kakunaga et al, , I 966; Kaneto,

1971)* Harris et al, (1975» 1976) confirmed these findings and also

discovered that lanthanum, another calcium antagonist, has analgetic

properties. Lee and Berkowitz (1977) demonstrated that methadone,

1-pentazocine (at high doses), and 1-acetylmethadol behaved similarly

to verapamil, producing complete blockade of potassium-induced aortic

contraction and only partial antagonism of norepinephrine-induced con

tractures. Furthermore, the ability of these drugs to inhibit aortic

contractures could be reversed by increasing the calcium concentration

in the bath. An interesting finding of this studiy was the ability of

methadone to antagonize morphine-induced aortic contractions suggesting

that methadone and 1-acetylmethadol may produce opposite effects to 28 morphine at the molecular level. The calcium antagonistic actions of these compounds may possibly be of importance in mediating some of their pharmacological and/or toxicological effects (Lee and Berk- ow itz, 19 77)>

The aminoglycoside antibiotics, streptomycin and neomycin, can interfere with myocardial activity and autonomic ganglionic and neuro muscular transmission (Swain, 1956; Pittinger and Adamson, 1972). The neuromuscular blocking actions can be overcome by increasing calcium concentration, and it has been suggested that the aminoglycosides compete with calcium for specific-receptor sites (Pittinger and Adam son, 1972). Using fragmented sarcoplasmic reticulum of skeletal muscle as a model system, both aminoglycosides could inhibit the rate of cal cium uptake. Further, since calcium entry into cells triggers various physiological events, the calcium antagonistic properties exhibited by these compounds may be partially responsible for organ toxicities and other interference with physiological function produced by the aminoglycosides. (Fairhurst and Macri, 1975)•

SKF 525-A, an inducer of microsomal mixed function oxidase, also has calcium antagonistic properties (Kalsner et a l,, 1970)* Using isolated rabbit aortic strips, it was concluded that SKF 525~A selec tively blocks some step in the process by which potassium-induced depolarization promotes movement of extracellular and/or superficially bound intracellular calcium to the contractile element, but it does not interfere directly with mobilization of calcium from a separate firmly-bound (norepinephrine-sensitive) site, 29

The compound Org 6001 (3 a-am ino-2 a-hydroxy-5 P-androstan-17- one hydrochloride) has been found to exhibit antiarrhythmic activity

(Marshall and Parratt, 1975)* Org 6001 vfas more potent than procaine as a local anesthetic on desheathed frog nerve, did not block isopro terenol-induced atrial chronotropy, and produced a small negative ino tropic effect on atria which was overcome by increasing calcium con c e n tra tio n . I t was concluded th a t Org 6001 i s a calcium a n ta g o n istic of the local anesthetic type more related to lidocaine than quin idine (Salako et a l., 1976).

R337 H , a new potent calcium antagonist, inhibits entry of calcium into the beta cells of the pancreas thereby inhibiting insulin release.

This compound is 50 to 100 tim es more p o ten t than verapam il or D600 at inhibiting glucose-induced insulin release (Malaisse et al., 1976).

Flunarizine (E)-l-(bis(4-fluorophenyl)methyl)-4-(3-phenyl-2-pro- penyl)p iperazine d ih ydrochloride) i s a new compound being stu d ied fo r peripheral and central vascular insufficiency. In tests using contracted vascular smooth muscle strips and cat papillary muscle, it was found that flunarizine was about 150 times more selective for vascular smooth muscle than cardiac tissue, whereas, in the same experiments, verapam il showed no selectivity (Van Neuten et a l,, 1978)* These results in dicate that flunarizine is a selective antagonist of vasoconstrictor stimuli with little effect on myogenic tone.

There is a great deal of evidence that lanthanum inhibits the actions of calcium in a number of tissues. However, its site of action is still controversial. In 1964, Lettvin et al. suggested that 30 lanthanum, "because of the sim ilarity of its ionic radius to calcium and its higher valence, "binds at calcium binding sites on the outer surface of the membrane, thus s ta b iliz in g th e membrane by preventing calcium release from binding sites on the inner surface of the mem brane.

The effect of lanthanum on calcium-dependent secretory processes has also been studied. In isolated bovine adrenal glands, Borowitz

(1972) showed that lanthanum stimulates catecholamine release only on first exposure. Conversely, the acetylcholine- or potassium-in duced catecholamine secretion was inhibited by lanthanum. Similarly,

Foreman and Mongar (1972) reported that spontaneous histamine release from mast cells was increased but the calcium-dependent component of antigen-stimulated histamine release was inhibited by lanthanum. The ability of lanthanum to stimulate spontaneous secretion while inhibit ing induced release appears to parallel its actions on skeletal muscle.

Lanthanum stimulates spontaneous miniature endplate potential frequency

(MEPP) and inhibits the endplate potential (EPP) and calcium uptake in junctional transmission (Weiss, 197^)•

Van Breemen et al. (1972) proposed a method utilizing the prop erty of lanthanum as a calcium antagonist to measure changes in cellu lar calcium in smooth muscle, The "lanthanum method" assumes that a sufficiently high concentration of extracellular lanthanum will re place calcium a t i t s binding s i t e s on the o u ter membrane su rface, block calcium influx and consequently its efflux, and not itself enter the cell in appreciable quantities. Tissues can be exposed to a number of stimulatory agents in the presence of Ca and placed in a washout solution containing lanthanum to replace all extracellular calcium and prevent further calcium uptake or efflux. This method has "been criticized on the basis that lanthanum can indeed enter the cells be ing tested (Hodgson et a l., 1972).

Although lanthanum has received much recognition as an experimen tal tool to study calcium-dependent processes, several problems arise with its use. Different types of preparations vary considerably in their sensitivity to the inhibitory effects of lanthanum. Calcium and lanthanum may have different affinities for different membrane binding sites. High lanthanum concentrations may exhibit non-specific

(stabilizing) effects or actions detrimental to cellular integrity,

Finally, as mentioned above, there is a question of the ability of

lanthanum to enter the cell under physiological conditions.

A 1975 study of the interaction of lanthanum with mitochondria

indicated the complexity of actions exhibited by this ion (Villani

et al.). Lanthanum-inhibition of calcium influx into cells subsides with time, and the delayed calcium influx or exchange might overlap

with the action of lanthanum at the membrane. The existence of at

least two mitochondrial calcium receptor sites—one involved mainly

in storage (on the outer surface of the mitochondrial membrane) and

the other in calcium translocation processes (high affinity site on

the inner surface of the mitochondrial membrane) have been proposed

(Carafoli and Gazzotti, 1973)* Lanthanum may block mainly the first

type of receptor. This agrees with results obtained in sarcoplasmic

reticulum vesicles indicating that calcium transport is not affected

by lanthanum (Entman et a l., I 969). Recently, a study demonstrated that lanthanum may inhibit myo cardial contractility in the isolated perfused guinea pig heart.

This effect may be partially due to blockade of calcium influx into cardiac cells (Wong et a l., 1976). However, discontinuity of the dose-response curve suggests that lanthanum interacts at more than one site. The first site could be a high affinity site associated with the cell membrane. Lanthanum binding here could interfere with calcium influx and diminish intracellular calcium stores (Sanborn and Langer,

1970). This calcium antagonism is non-competitive in nature. The second site, a low affinity site, is not antagonized by high concen trations j lanthanum can penetrate the cell and interact with calcium binding sites in the sarcoplasmic reticulum presumably depleting intra cellular calcium stores.

Douglas and Rubin (I 963, 196*0 concluded th a t magnesium prevented acetylcholine-, potassium-, and barium-induced catecholamine release from the adrenal medulla due to blockade of entry of calcium or barium into the cell. However, a study by Rahwan et al. (1973b) showed that magnesium significantly diminished the secretory response of the adre nal medulla to caffeine, chlorpromazine, and amphetamine in calcium- fre e medium. Since these se c re to ry responses depend 0 1 1 the mobiliza tion of intracellular calcium, these findings suggested that magnesium interferes with the action of calcium at an intracellular receptor site.

This finding was confirmed by Lastowecka and Trifaro (197*0 using sodium deprivation of the bovine adrenal gland. Sodium deprivation normally causes a sharp increase in the output of catecholamines pre sumably through mobilization of intracellular calcium. Magnesium 33 prevented this catecholamine release "both in the presence and ab sence of extracellular calcium. The authors concluded that magnesium either prevented calcium binding to an intracellular receptor or blocked the release or translocation of intracellular calcium induced by so dium d ep riv atio n ,

Kanno e t a l. ( 1973) further demonstrated, by direct injection of magnesium into mast cells, the competition between magnesium and calcium for the intracellular calcium receptor site, Using rabbit vascular smooth muscle and atrial muscle from various species, Tur- lapaty and Carrier (1973) studied the role of magnesium in calcium-in duced responses. They concluded that magnesium competes with calcium a t the membrane and a t in tr a c e llu la r calcium -binding s ite s .

M iledi ( 1973)> using the squid stellate giant synapse, found that magnesium injected into the presynaptic terminal antagonized calcium- induced transmitter release. Krnjevic et al. ( 1976), using the cat spinal motor neuron, demonstrated magnesium-induced effects opposite to those of calcium and concluded that magnesium must compete principal ly with intracellular calcium, and that neuronal excitability can be regulated by the ratio of intracellular free ionic calcium/magnesium r a tio .

From the abundant evidence collected over the last five years, it can therefore be concluded that magnesium exhibits an intracellular calcium antagonistic action.

The io-(N,N-diethylamino)-alkyl-3,^,5_trimethoxybenzoate (TMB) series has been extensively studied for smooth muscle relaxant effects

(Sharma, i 960, 1962; Dell'Omodarme and Brunori, 1959; Lindner et a l., I 963). A structure-activity relationship study of the TMB-2 to TMB-6 compounds demonstrated that increasing the length of the intermediate alkyl chain (from two to six) increases drug potency (Robinson, 1971)*

Further, the dose range for antagonism of a wide spectrum of spasmogen ic agents in smooth muscle was narrowed. These findings suggest that the TMB compounds act at a step in the contractile process subsequent to membrane a c tiv a tio n ,

TMB-8 (8-(N,N-diethylamino)-octyl-3*^»5“trimethoxybenzoate) was synthesized in an attempt to produce a potent muscle relaxant which would act at a step in excitation-contraction coupling shared by both re c e p to r-sp e c ific and n o n -sp ecific agents. This compound was found capable of irreversibly blocking the spasmogenic effect of acetylcho line, norepinephrine, nicotine, potassium and dimethylphenylpiperazin- ium and reversibly inhibiting barium chloride in the guinea pig ileum

(Malagodi and Ghiou, 1974-) • TMB-8 inhibition of potassium-induced contractions of guinea pig ileum and vas deferens was reversed by in creasing calcium. These findings suggested that TMB-8 may interfere with calcium at an intracellular site. Further studies using rabbit aortic strips, guinea pig ileum, and rabbit skeletal muscle sarcoplas mic reticulum showed that TMB-8 stabilized calcium binding to cellular stores and inhibited release of calcium from such stores by contrac tile stimuli (Chiou and Malagodi, 1973)*

The TMB series was evaluated for antiarrhythmic activity. TMB-2 effectively inhibited acetylcholine-, aconitine-, and coronary liga tion-induced arrhythmias, but only at high doses (Sharma and Arora,

I 963). TMB-6 was comparable to lidocaine in converting digoxin-induced 35 arrhythmias (Chiou et a l., 1976). However, the magnitude of depression of atrial and ventricular rate normally seen with lidocaine was not evident with TMB-6. A negative inotropic effect was attributed either to decreased calcium availability to the contractile apparatus or in hibition of the calcium-dependent process of ectopic impulse generation.

TMB antagonism of contractile force in the myocardium could be reversed by elevating extracellular calcium levels. Chiou et al. (1976) con cluded that TMB-6 is a potent antiarrhythmic agent due to its ability to interfere with calcium movements in myocardial tissue.

In another study, TMB-8 demonstrated the ability to inhibit plate let secretion (Charo et a l., 1976). A dose-dependent inhibition of both thrombin-induced (mediated by extracellular calcium) and A23187

(a calcium ionophore)-induced (mediated by intracellular calcium) se cretion was noted. It was concluded that TMB-8 inhibits a calcium- dependent step in both thrombin- and ionophore-induced secretion, pos sibly by blocking release of calcium from an intracellular "trigger" pool. Similar results have been obtained with TMB-6 (LeBreton and

Dinerstein, 1977).

STATEMENT OF THE PROBLEM

A series of 2-substituted 3-d.imethylamino-5,6-methylenedioxyindene hydrochlorides (aminoindenes; fig. l) were synthesized by Witiak et al.

(197^) in the course of synthesis of the corresponding indanones, poten tial prostaglandin antagonists (Witiak, 1974). Preliminary investiga tions resulted in the serendipitous finding that, although the amino indenes lacked the requisite selectivity as prostaglandin antagonists, they possessed the attractive pharmacological property of calcium antagonism.

The aims of this research were three-fold;

1) To characterize the calcium antagonistic properties of the 2-sub-

stituted aminoindenes in isolated smooth muscle preparations (an

excitation-contraction coupling model).

2) To confirm the calcium antagonistic properties of the 2-substitu

ted aminoindenes and elucidate their probable site of action using

the isolated bovine adrenal medulla (a stimulus-secretion coupling

model).

3) To apply the calcium antagonistic properties of the 2-substituted

aminoindenes by investigating their potential usefulness as coro

nary vasodilators using isolated bovine coronary strips and iso

lated perfused rabbit hearts. 37

Figure 1. Chemical structures of the 2-substituted 3-dimethylamino-

5»6-methylenedioxyindene hydrochlorides. CH

CH CH

2-METHYL

CH CH.

2-n-PROPYL 2-n-BUT YL

Figure 1 CHAPTER II

MATERIALS AND METHODS

PHARMACOLOGICAL EVALUATION OF NEW CALCIUM ANTAGONISTS: 2-SUBSTITUTED 3 -DIMETHYLAMINO-5,6-METHYLENEDIOXYINDENES. EFFECTS ON SMOOTH MUSCLES (Rahwan, Faust, and Witiak, 1977).

Animals. Female albino rats (Cox Sprague-Dawley, Indianapolis,

Ind.) weighing 180~220g were fasted and treated with 0.1 mg/kg of

diethylstilbestrol 24 hours prior to sacrifice. Vaginal smears were

performed randomly to insure that the animals were in estrus at the

time of the experiments. Fasted male guinea pigs (Shirley Singer, N.

Lawrence, Ohio) weighing approximately 900g were used for certain

experiments.

Chemicals. Chemicals used in this study and their sources were:

prostaglandin F^a tromethamine salt (PGF^a) and prostaglandin E^

(PGEg) (gifts from Dr. J.E. Pike, The Upjohn Co., Kalamazoo, Michigan);

oxytocin (Pitocin, Parke Davis and Co., Detroit, Michigan); histamine

hydrochloride (Fisher Scientific Co., Fair Lawn, New Jersey); acetyl

choline chloride (Sigma Chemical Co,, St. Louis, Missouri); diphen

hydramine hydrochloride (Parke Davis and Co., Detroit, Michigan); atro

pine sulfate (Mallinkrodt Chemical Works, St. Louis, Missouri); ergo-

novine maleate (Ergotrate, Eli Lilly and Co., Indianapolis, Indiana);

barium chloride (BaCl^'EH^O, J.T, Baker Chemical Co., Phillipsburg,

New Jersey); calcium chloride (CaClg'SH^O, Allied Chemical Corp.,

39 40

Morristown, New Jersey); and diethylstilbestrol (Delestrogen, E.R.

Squibb and Sons, New York, New York). The concentrations of these chemicals as stated in the text refer to the salts given above, except where the drug is used as the base.

Prelim inary experim ents w ith PGEV,a: » PGE„ an<^ oxy'toc^Ln on isolated rat uterus. The rats were sacrificed by decapitation and uterine segments (approximately 2.5 cm in length) were prepared for

isotonic contraction recordings under 500 mg tension in 10 ml tis s u e baths containing a physiological solution (37° C) having the following composition (g/l): NaCl, 8.046; KC1, 0.20, CaCl^'SH^O, 0.132; MgC^*

6H20, 0.106; NaHGO^, 1.0; NaHgPO^'H^O, 0.065; dextrose, 1.0. Tyrode

solution was prepared fresh daily. Recordings were made with an iso

tonic MK II myograph transducer and a Physiograph 4 recorder (E & M In

strument company, Houston, Texas). The Tyrode solution was aerated with

% COg in oxygen for at least 20 minutes prior to use. A 30-rainute

equilibration time was allowed prior to all experiments to allow the

spontaneous activity of the tissue to subside.

All compounds were first tested for agonist activity by adding -5 -3 doses ranging from 10 M to 10 M to uterine strips not previously

treated with any spasmogen. No agonist activity was noted; therefore

the compounds were next tested for antagonist activity.

In each experiment on the isolated uterine strip, a control re-

sponse to FGI 22 (10 M bath concentration), PGF 2 q:(10 M bath concentra

tion), or oxytocin (10 ^ U/ml bath concentration) was obtained. The

bath was then washed three times prior to incubation of the tissue

with any of the aminoindene test compounds. The latter were always M l te s te d on d iffe re n t u te rin e s tr ip s . The aminoindene compound to be tested for antagonist activity was then added to the bath and left in contact with the tissue for 3 minutes. PGEg, PGF^a, or oxytocin (at the concentrations mentioned above) was added to the bath and the con tractions recorded. After 5 minutes, the bath was washed three times and the control response to the spasmogen regained. The volume of drug solution added to the 10 ml bath never exceeded 0.1 ml.

The results of these preliminary experiments (vide infra) showed that the 2-methyl and 2-ethyl aminoindenes (fig. l) had inferior an tagonistic properties as compared with the 2-n-propyl and 2-n-butyl derivatives. All subsequent experiments reported below, therefore, were performed with the 2-n-propyl and 2-n-butyl aminoindenes only.

Concentration-response experiments with PGF^rv and oxytocin on the isolated rat uterus. Rat uterine strips were incubated for 3 minutes with either the 2-n-propyl or the 2-n-butyl aminoindene antagonist -*5 at a concentration of 5 x 10 ^M. This concentration of the aminoindene antagonists was previously determined in preliminary experiments (vide supra) to almost completely block the oxytocic effect of 10 M and FGF2a without significantly (P<0,05) affecting the spasmogenic action of oxytocin (figs. 2 and 3). Concentration-response curves to

PGFgO; and oxytocin were constructed by varying the concentration of agonist added to the bath. After each addition of agonist, the bath was washed and the tissue reincubated with the same concentration of the antagonist.

Experiments with other smooth muscle spasmogens. In order to de termine the degree of selectivity of the 2-n-propyl and 2-n-butyl aminoindene antagonists, they were tested against various agonists

(acetylcholine, histamine, barium and ergonovine) on the isolated rat uterus (acetylcholine and ergonovine) and ileum (acetylcholine) and

on the guinea pig ileum (histamine). The same experimental protocol

described under "Preliminary experiments" was followed. The bathing

solution used for the ileum differed from that for the uterus only

in the following constituents (g/l): MgGl^’^HgO, 0.42; CaCl^^HgO,

0,52; NaHgPO^'H^O, 0,10, Diphenhydramine (10-^M) and atropine (10 ^M) were used as standard antagonists to the effects of histamine and acetylcholine, respectively.

Effect of increasing extracellular calcium in experiments on the

isolated rat uterus. The effect of increasing the concentration of calcium in the bath on the antagonistic action of the 2-n-propyl and

2-n-butyl aminoindenes against acetylcholine and barium were studied on the isolated rat uterus. In this set of experiments, the following protocol was adopted for each concentration of calcium tested. The tissue was incubated for 10 minutes with the chosen concentration of calcium to allow the spontaneous contractile activity to subside. The -6 agonist (acetylcholine 10 M or barium 0.05 mg/ml) was added to the bath and a control contraction recorded. The tissue was washed and reincubated for 10 minutes with the same concentration of bath calcium used to obtain the control response to the agonist. The antagonist

(loAl of the 2-n-propyl or 2-n-butyl aminoindene) was then added and left in contact with the tissue for 3 minutes before reintroduction of the agonist. The tissue was then washed and allowed to relax. The entire procedure was repeated at a higher concentration of bath calcium

(the concentration of agonists and antagonists being kept constant). Statistical analysis. Contractile responses produced by agonists in the presence of antagonists are reported as a percentage of the control response to the agonists obtained in the absence of antag onists. Comparison of 95^ confidence lim its of the mean was used whenever statistical evaluation of the data was deemed necessary

(Sokal and Rohlf, 1969). Statistical significance was set at P<0.05.

PHARMACOLOGICAL EVALUATION OF NEW CALCIUM ANTAGONISTS: 2-SUBSTITUTED 3-DIMETHYLAMINO-5,6-METHYLENEDIOXYINDENES. e f f e c t s o n adrenom edullary CATECHOLAMINE SECRETION (P ia sc ik , Rahwan, and W itiak, 1978).

Fresh bovine adrenal glands were obtained at a local slaughter house, transported on ice and used approximately one hour post mortem.

Prior to all experiments, the glands were perfused (retrograde) with aerated Locke's solution of the following composition (g/l): NaCl,

9.0; KC1, 0.418; CaCl2 *2H 0, 0.323; NaHPO^pH 0, 0.575; NaHgPO^-HgO,

0.117; glucose, 0.9 for 20 minutes or until a constant flow rate of

5 ml/min was achieved (Rahwan et al. , 19738), using individual channel metering pumps (Fluid Metering Inc., Oyster Bay, New Jersey). Flow rate during perfusion was maintained constant throughout the experiments.

Isotonicity was maintained throughout by adjusting the amount of sodium chloride in the buffered Locke's solution.

Experiments with carbachol. The purpose of these experiments was to determine the effect of the 2-substituted aminoindenes on the cal cium (extracellular)-dependent secretagogue action of carbachol (Schnei der, 1989) on the adrenal medulla. Cumulative concentration-effect curves were obtained as described by Rahwan et al. (19738, 1974) and

O'Neill and Rahwan (1975)- After adjusting the perfusion rate, the 44- control adrenal glands were perfused for 20 minutes with Locke's solu tion, followed by a 10-minute stimulation with increasing concentra tions of carbachol (0. 03 -3*3 mM; two minutes for each concentration).

The test glands were perfused with Locke's solution containing the . _8 . appropriate concentration (10 -10 M) of either the 2-n-propyl or the

2-n-butyl aminoindene for the 20 minutes prior to and the 10 m inutes of carbachol stimulation. Perfusates from the individual glands were collected in 2-minute fractions immediately prior to and during drug perfusion. Following carbachol stimulation, recovery of the secretory potential of the aminoindene (10 ^M)-treated test glands was deter mined by perfusing the glands with Locke's solution for an additional

30-60 minutes and restimulating with carbachol.

Total catecholamine content in the perfusate samples was assayed by the colorimetric method of von Euler and Hamberg (194-9)* Catechol amine content in perfusates from adrenal glands was determined in a 4- ml aliquot of the perfusate, To this aliquot was added 1 ml of 1 M a c e ta te b u ffer (pH 6. 5) and 0.2 ml of 0.1 N iodine with shaking. After

3 minutes, 0,2 ml of 0,2 N sodium thiosulfate was added, shaken until the solution was decolorized, and the optical density at 529 nm imme diately read in a Bausch and Lomb Spectronic 20 spectrophotometer

(Bausch and Lomb, Inc,, Rochester, Nevr York). Btrug-induced catechol amine release was corrected for resting secretion measured just prior to drug stimulation. None of the drugs used in these experiments in terfered with the assay for catecholamines. The aminoindenes did not alter resting secretion of the glands. Experiments with acetaldehyde. The purpose of these experiments was to determine the effect of the 2-substituted aminoindenes on the calcium-independent secretagogue action of acetaldehyde (O'Neill and

Rahwan, 1975) on the adrenal medulla. Cumulative c o n c e n tra tio n -e ffe c t curves with acetaldehyde in presence and in absence of the 2-substituted aminoindenes were obtained as described above for carbachol. The per fusate samples were analyzed immediately upon collection (Rahwan et a l,,

197*0 in order to avoid condensation of the released catecholamines with acetaldehyde in the collecting test tubes.

Experiments with barium. The purpose of these experiments was to determine the effect of the 2-substituted aminoindenes on the secre tagogue action of the presumed calcium substitute cation, barium

(Douglas and Rubin, 1964), on the adrenal medulla. After adjusting the perfusion rate, control adrenal glands were perfused for 20 minutes with normal Locke's solution, followed by a 30 -minute perfusion with isotonic calcium-free Locke's solution in order to wash out extracellular cal cium (Rahwan e t a l , , 1973L, 1974; O 'N eill and Rahwan, 1975)* The glands were then stimulated for 10 minutes with 1 mM barium chloride in cal cium -free medium. The p e rfu sate was c o lle c te d in 2-minute fra c tio n s .

The test glands were perfused in a similar manner except for the pres ence of either of the aminoindene compounds ( 10~%) or of magnesium

(5 x 10"^M) in the calcium-free perfusion medium 20 minutes prior to and during barium stimulation, Mg was used in these studies as an es tablished intracellular calcium antagonist (Rahwan et a l., 1973L; Las- towecka and Trifaro, 1974; Kanno et a l,, 1973; Turlapaty and Carrier,

1973; Miledi, 1973; and Krnjevic et a l., 1976). The removal of calcium 46 from or addition of magnesium to the perfusion medium did not alter the resting secretion rate of catecholamines.

4*5 Ca studies. Experiments with 45,-, Ca were performed in order to determine whether the aminoindene antagonists interfere with calcium uptake into the adrenal chromaffin cells. The procedure and its ra

tionale have been described by Rahwan et al. (1973b)* After adjusting the perfusion rate, control adrenal glands were perfused for 30 m inutes with calcium-free Locke's solution (to wash out extracellular calcium 45 and enhance the uptake of Ca into the chromaffin cells; Rahwan et a l.,

1973b), followed by a 2-minute infusion of ^Ca (l-jpi of ^CaGl^/ml,

specific activity 15 Ci/g of Ca) (iCN Pharmaceuticals, Inc., Irvine

California). The brief labeling period ensured the reintroduction of

a minimal amount of calcium to th e calcium -free p erfu sion medium. The 45 • extracellular Ca was washed out with calcium-free Locke's solution

for 60 minutes after the radioactive pulse. (Extracellular calcium is washed out rapidly from the glands, as indicated by the total loss of response of the adrenal medulla to acetylcholine after 30 minutes of

perfbsion w ith calcium -free medium). The procedure was the same fo r

the test glands except for the presence of either of the aminoindene

compounds (10 ^M) in the calcium-free perfusion medium for 20 m inutes 4 5 prior to and during the Ca pulse. The perfusate from the glands was 45 collected in 2-minute fractions during the Ca infusion and subsequent 45 60-minute washout of radioactivity. Ca in the perfusate samples

was counted by liquid scintillation as described by O'Neill and Rahwan

(1975)* Radioactivity in perfusates was determined by taking a 0.1 ml

aliquot of each of the 2-minute fractions and counting it in 10 ml o f a 47 counting cocktail consisting of 0,6% 2 i5-diphenyloxazole (PPO) and

0,01^ 1,4-bis(2-(5-phenyloxazolyl))benzene (POPOP) in a 1:1 mixture of toluene and 2-ethoxyethanol. Counting efficiency for perfusates was found to be 10k% by the internal standard method. Recovery of radi oactivity was essentially 100%. Liquid scintillation counting was per formed in a Packard Tri-Carb Scintillation Spectrometer Model 3375*

Statistical analysis. Statistical significance was set at

P<0.05 as determined by the Student's t test-(Sokal and Rohlf, 1969).

PHARMACOLOGICAL EVALUATION OF NEW CALCIUM ANTAGONISTS: 2-SUBSTITUTED 3-DIMETHYLAMIN0-5,6-METHYLENEDI0XYINDENES. CORONARY AND CARDIAC EFFECTS (P ia sc ik , Rahwan, and W itiak, J. Pharmacol. Exp. T h e r., subm itted).

Effect of aminoindenes on potassium-depolarized coronary strips.

Bovine extramural coronary arteries were obtained at a local slaught erhouse, transported on ice, and used within one hour post mortem. Cir cular or spiral strips, approximately 1.3 to 2 cm in length, were pre pared for isometric recording under 2 g tension in 10 ml jack eted t i s sue baths. The strips were bathed in aerated Tyrode solution ($%> CCt, in 0^) maintained at 37° C and having the following composition (g/l):

NaCl, 8.9; KC1, 0.3; CaCl 2*2H 20, 0.147; NaHCO^, 1.0; NaiyPO^HgO, 0.66j glucose, 1.0. The tissue was allowed to equilibrate for 3 to 4 hours prior to the start of each experiment to permit full relaxation of the coronary strip. Recordings were made with a model FT 03C Grass force displacement transducer and a model 7 Grass polygraph (Grass Instru ments, Quincy, Massachusetts).

To depolarize the tissue, the Tyrode solution was replaced with isotonic solution containing 43 mM KC1, and the strips were allowed to develop full contractile tone, The aminoindene hydrochlorides

(3 x 10 ^ to 10 ^M) or prenylamine gluconate (6 x 10 ^M; Hoechst Pharm aceutical, Somerville, New Jersey) was then added to the bath. The latter drug was included as a standard coronary relaxant. Once relaxa tion was complete, calcium chloride was added to the bath (final con centration of 7,2 mM) to demonstrate the calcium dependency and rever sibility of the relaxant effect of the aminoindenes and prenylamine.

Different coronary strips were used for each concentration of test compound.

Effect of aminoindenes on isolated perfused hearts. New Zealand white rabbits (Zane Bailey, Xenia, Ohio) of either sex, weighing 1 to

3 kg were pretreated with 1000 units of heparin (Abbott, N. Chicago,

Illinois) and sacrificed by cervical dislocation. Each heart was rap idly excised and the aorta attached to the glass cannula of an Anderson and Craver type perfusion apparatus (Anderson and Craver, 1948). The heart was constantly perfused with Ringer-Locke's solution (37° C; aerated with GO^) having the following composition (g/l)i Nad, 9«0»

KOI, 0,42; CaCl2*2H 20, 0.159; NaHCO^, 0.5; glucose, 1.0. The perfusion pressu re was 60 cm H^O, The apex of the h e a rt was connected to a type

B force displacement transducer (E & M Instrument Co., Houston, Texas).

Electrocardiogram was monitored via a Physiograph preamplifier (MK 111;

E & M Instrument Co.). Pin electrodes were inserted into the right atrial and left ventricular muscle. Coronary flow was measured with a

Physiograph drop counter. All recordings were made on a Physiograph IV reco rd er. Each heart was allowed to equilibrate for 20 minutes prior to introduction of drug solution. The drug dosages were calculated to re present the actual concentration of drug entering the coronary circu lation and were introduced immediately above the aortic cannula by means of a butterfly cannula (E-Z set with 21 guage needle) attached to a 5 ml glass syringe and syringe pump (Sage Instruments, Cambridge,

Massachusetts). Drug solutions were always infused at 1/10 the rate of perfusion of the Ringer-Locke's solution. Drug introduction was randomized, and each drug dose was infused for an 8-minute period.

A 20-minute washout period was allowed between doses. Doses of 2-n- propyl and 2-n-butyl aminoindene hydrochlorides ranged from 10 -9 to

J i o ^ 10 M. Theophylline (2.8 x 10 M) and prenylamine gluconate (6 x 10 M) were used as standard drugs to verify the functional responsiveness of each heart, both being coronary dilators but the former having positive chronotropic and inotropic effects while the latter having a negative inotropic action,

Effect of aminoindenes on ouabain-induced arrhythmias. In one preliminary experiment, a mongrel dog ( 7*5 kg) was anesthetized with

32 mg/kg pentobarbital (Abbott, N. Chicago, Illinois) administered in travenously. The electrocardiogram (Lead II) was monitored on a Physio graph IV recorder. Cardiac arrhythmias were precipitated by intravenous infusion (via the femoral vein) of ouabain (Eli Lilly and Co., Indiana polis, Indiana, total dose of 0.075 rng/^g body weight). The ability of intravenously-administered 2-n-propyl aminoindene hydrochloride (13 and

26 mg/kg; LD50 in mice was previously determined to be 185 mg/kg by intraperitoneal injection and ^0 mg/kg by intravenous injection (R.G. 50

Rahwan, T.L. Henry, and M.F. Piascik, unpublished observations)) to reverse the established arrhythmias was then determined.

Statistical analysis. Statistical significance was set at P<0.05 as determined by analysis of variance and Duncan's multiple range test as well as Student's t test (Sokal and Rohlf, i 960). CHAPTER III

RESULTS

PHARMACOLOGICAL EVALUATION OF NEW CALCIUM ANTAGONISTS: 2-SUBSTITUTED 3-DIMETHYLAMINO-5,6-METHYLENEDIOXYINDENES. EFFECTS ON SMOOTH MUSCLES (Rahwan, Faust, and Witiak, 1977).

Antagonism between aminoindenes and prostaglandins on the uterus.

The control contractile responses of the isolated rat uterus to pros taglandins were: 55*3 + 1*6 111111 f ° r an(^ 59 — ^'5 1111,1 ôr —*7 FGF^CÎO ' m). The 2-n-propyl and 2-n-butyl aminoindenes, at a concentration of 5 x 10 -5 M or greater, significantly blocked the oxytocic action of 10-^M KJE^ and PGF^C^figs. 2 and 3). A concentration-re sponse relationship was demonstrated for PGFô: in the presence of both aminoindene antagonists (fig. 4). Increasing the concentration of PGF^Ct progressively overcame the blockade induced by the aminoindenes. High er concentrations of PGF^Q! were not employed since the uterus responds with excessive spontaneous activity.

Antagonism between aminoindenes and oxytocin on the uterus. The _ O control contractile response for oxytocin (1 0 JU/ml bath concentration) in the isolated rat uterus was 56.4 + 1.9 mm« At a concentration of

5 x 10 (which significantly blocked the effects of prostaglandins on the uterus) the 2-n-propyl and 2 -n-butyl aminoindenes did not significantly alter the response of the uterus to oxytocin (10 U/ml). However, at -4 the higher concentration of 10 M the aminoindene test compounds 52

Figure 2. Antagonistic effect of 2-n-propyl 3“dimethylamino-5, 6- methylenedioxyindene hydrochloride on uterine contractions induced by prostaglandins and oxytocin. The control contractile responses of the isolated estrogenized rat uterus to PGE2 (10“?M), PGFgO: (10_?M) and oxytocin (10 ^ u/ml) were 55«3 + 1.6 mm, 59*8 + 1.5 mm and

56.4 + 1,9 mm, respectively. % CONTROL 100 60 60 20 40 0 5x 10~5 5 f --RPL CONCENTRATION2-n-PROPYL (M) Figure2 •—o .0 OXYTOCIN...0 A PGF2* -A

e g p 2 54

Figure 3. Antagonistic effect of 2-n-butyl 3-

(10 ?M) and oxytocin (10 ^ u/ml) were 55*3 + 1*6 mm, 59*8 + 1.5 mm and 56.4 + 1.9 mm, respectively. % CONTROL 100 20 40 60 80 10 H -5 --UY CONCENTRAT ION ( M) 2-n-BUTYL Figure 3 Figure x 10 x 5 -5 * - . OXYTOCIN ..B J PF « PGF -JL pge -4 2 2 55 56

Figure k-, Concentration-response curves for PGF^a -induced uterine contractions in presence of the 2-substituted 3 "dimethylamino- 5, 6- inethylenedioxyindene hydrochlorides. The isolated estrogenized rat u te ru s was incubated w ith the 2-substituted aminoindene antagonist

(5 x 10 ^M) for three minutes prior to each addition of PGF^cc . The control contractile response of the uterus to PGFgCHwas 57*3 + 1«8 mm.

Values represent the mean + S.E.M. of six to eight experiments. CONTROL 100 60 20 SO 10 -8 PGF2rf CONCENTRATION CM) CONCENTRATION PGF2rf Figure^ produced essentially complete blockade of oxytocin (figs. 2 and 3 )-

A concentration-response relationship was demonstrated for oxytocin in presence of both aminoindene antagonists (fig. 5)*

Antagonism between aminoindenes and selected spasmogens. Tables

1 and 2 show the inhibitory effect of different concentrations of the

2-n-propyl and 2-n-butyl aminoindenes on the spasmogenic action of various agonists in different smooth muscle preparations. It should be noted (see Discussion) that the data reported in these tables is derived from experiments designed solely for the purpose of investiga ting the selectivity (or lack thereof) of the aminoindene antagonists.

Consequently protocol consistency and systematism were sacrificed in favor of testing a wider variety of representative agonists and antag onist concentrations on different smooth muscle preparations. At a concentration of 5 x 10 (which blocked the oxytocic effects of pros taglandins but not oxytocin; see figs. 2 and 3 ) the 2-n-propyl and

2-n-butyl aminoindenes significantly blocked the spasmogenic action of agonists against which they were tested (10 acetylcholine in the -6 rat uterus and 10 M histamine in the guinea pig ileum). The antag onism of histamine was comparable to that obtained with 10 diphen hydramine (not shown in the tables). Furthermore, at the higher con- -4 centration of 10 M, the two aminoindene compounds significantly or completely blocked the spasmogenic action of 10 acetylcholine (rat ”6 Zi uterus and ileum), 10 M histamine (guinea pig ileum), 7*5 x 10~ M ergonovine (rat uterus), and 2,2 x 10 barium (rat uterus; see fig,

7). The antagonism of acetylcholine was comparable to that obtained -6 w ith 10 M atro p in e (not shown in ta b le s ). 59

Figure 5. Concentration-response curves for oxytocin-induced uterine contractions in presence of the 2-substituted 3 -d.iroethylam ino- 5, 6- raethylenedioxyindene hydrochlorides. The isolated estrogenized rat uterus was incubated with the 2-substituted aminoindene antagonist

(5 x 10 ^M) for three minutes prior to each addition of oxytocin. The control contractile response of the uterus to oxytocin was 58.9 + 2.2 mm. Values represent the mean + S.E.M. of six to eight experiments. CONTROL 100 0 4 0 6 20 0 8 0 2-n-BUTYl 5 OXYTOCIN CONCENTRATION CU/ML5 CONCENTRATION OXYTOCIN Figure 5 Figure -3 O O n TABLE 1 Effect of2-n-propyl-3-dimethylamino-5,6-methylenedioxyindene on the spasmogenic effect of several agonistsn

Control Con Contraction in Cone, of 2-n- traction in Ab Presence of 2-n- A g o n i s t T i s s u e 6 Propyl Am i sence of 2- % of Control Propyl Am inoin n o i n d e n e n-P r o p y l d e n e Aminoindene M mm mm

Acetylcholine chloride Rat uterus io-' 54.5 £ 7.7 57.7 £ 8.9 105.4 £ 1.8 ( 1 0 - s M ) • 5 x 1 0 - ' 65.5 - 9.8 49.5 £ 0.8 76.3 £ 7.9 IO*4 58.1 ± 4.0 9.3 £ 3.0 1 6 . 5 £ 5 . 0 Acetylcholine chloride Rat ileum i o - ' 33.7 ± 5.2 33.2 ± 8.8 95.6 £ 11.9

(10-B M) io-4 3 4 . 3 ± 7 . 4 8.2 £ 2.2 23.4 £ 2.8 10-3 40.0 ± 7.9 4.2 £ 4.2 4 . 2 £ 4 . 2 Histamine hydrochlo Guinea-pig ileum i o - s 32.8 * 6.3 30.3 £ 5.4 9 3 . 4 £ 4 . 7 ride (10'B M) 5 x 10-' 38.1 ± 6.0 10.4 £ 2.9 29.1 £ 8.4

io-4 42.4 ± 5.7 2 . 0 £ 1 . 2 4 . 2 £ 3 . 0

Ergonovine maleate Rat uterus i o - 4 5 9 . 3 ± 5 . 5 0 . 0 0 . 0 (7.5 x 10 4 M)

° For experimental conditions see text. 4 Number of experiments ranged from 3 to 15. TABLE 2 Effect of 2-n-butyl-3-dimethylamino-5,6-methylenedioxyindene on the spasmogenic effect of several“ agonists

Control Contrac Contraction in Cone, of 2-n- tion in Absence Presence of 2-n- A g o n i s t T i s s u e 6 B u t y l A m i o f C o n t r o l o f 2 -rc-B u ty l '•Butyl Am inoin n o i n d e n e Aminoindene d e n e M mm mm Acetylcholine chloride Rat uterus 10*5 49.5 ± 0.3 50.8 * '2.0 102.8 ± 4.4 (IO'* M) 5 x 10- 51.5 ± 0.8 12.0 ± 19.7 23.6 ± 23.8 1 0 - 59.5 ± 3.6 16.6 ± 4.0 24.7 ± 5.1 Acetylcholine chloride Rat ileum 10"5 43.3 ± 3.5 39.0 ± 1.7 88.9 t 2.0 (10- M) 10- 40.0 ± 2.4 9.2 ± 1.7 23.1 ± 4.7 Histamine hydrochlo Guinea-pig ileum 10- 26.0 i 3.2 20.4 ± 2.4 7S.4 ± 4.5 ride (10-fi M) 5 x 1 0 -’ 32.5 ± 5.4 5.0 - 2.6 26.9 ± 9.3 10 - 37.5 ± 2.9 1.0 ± 0.8 2.8 ± 1.8 Ergonovine maleate Rat uterus 10- 55.0 i 1.0 0.0 0.0 (7.5 x 10'" M) " For experimental conditions see text. “ Numbers of experiments ranged from 3 to 15. Effect of increasing extracellular calcium on aminoindene antag onism of acetylcholine and 'barium. In all experiments on the uterus previously described, the concentration of calcium (CaCl^^H^O) in the bath was 9 x 10-\l (0,132 g/l). In order to investigate the effect of increasing the extracellular calcium concentration on the spasmolytic action of the 2-substituted aminoindenes, two agonists (acetylcholine and barium) which utilize different calcium pools for their spasmogenic effects were selected. Figures 6 and 7 show the reversal of aminoin dene antagonism of acetylcholine (10 ^M) and barium ( 0.05 mg/ml), re spectively, when the concentration of extracellular calcium is increased -h. , from 9 x 10 M (at which concentration there is significant blockade of the agonists) to 7.2 x 10 ^M (at which concentration 80- 90^ of the oxytocic action of the agonists is regained), A further increase in the.extracellular calcium concentration resulted in excessive spontan eous uterine contractions.

PHARMACOLOGICAL EVALUATION OF NEW CALCIUM ANTAGONISTS: 2-SUBSTITUTED 3-DIMETHYLAMINO-5, 6-METHYLENEDIOXYINDENES. EFFECTS ON ADRENOMEDULLARY CATECHOLAMINE SECRETION (P ia sc ik , Rahwan, and W itiak, 1978).

Experiments with carbachol. The 2-n-propyl aminoindene produced a concentration-dependent blockade of carbachol-evoked catecholamine secretion from the adrenal medulla (fig. 8), At a concentration of

10 M, this aminoindene resulted in a complete but reversible (see below) blockade of carbachol-evoked adrenomedullary secretion. This corresponds well with the results obtained in smooth muscle using ace tylcholine as the spasmogen (Rahwan et a l., 1977)* Total or near total blockade of the agonist response was seen in both systems at a Figure 6. Effect of extracellular calcium on the antagonism of acetyl choline-induced uterine contractions by the 2-substituted 3 ~dimethylamino-5,6-methylenedioxyindene hydrochlorides. The tissue was incu bated for three minutes with the 2-substituted aminoindene antagonist

(10 ^M) at each higher concentration of extracellular calcium prior to -6 addition of acetylcholine chloride (10 M). For other experimental con ditions see text. The control response of the isolated es.trogenized rat uterus to acetylcholine at each concentration of extracellular calcium (GaGl^’SHgO) was: 60.8 + 2.7 mm a t 9 x 10 calcium; 58.1

+ 4 .8 ram a t 1.8 x 10 calcium; 57.9 + 3-^ mm at 3.6 x 10 ^ M calcium; and 62.5 +3*9 mm at 7.2 x 10 calcium. The values represent the mean + S.E.M. of 6-15 experim ents. ACETYLCHOLINE CONTROL RESPONSE 100 20 40 80 5* 10 4 5* ACU CONCENTRATIONCALCIUM CM} Figure 6 Figure 66

Figure 7. Effect of extracellular calcium on the antagonism of ■barium- induced uterine contractions "by the 2-substituted 3 ~dimethylamino- 5»6- methylenedioxyindene hydrochlorides. The tissue was incubated for three minutes with the 2-substituted arainoindene antagonist (10 Si) at each higher concentration of extracellular calcium prior to addition of barium (BaGl^^HgO, 2 .2 x 10 Si). For other experimental conditions see text. The control response of the isolated estrogenized rat uterus to barium at each concentration of extracellular calcium (CaClg^H^O)

_ h. was: 46.2 + 1.5 m at 9 x 10 M calcium; 48.7 + 2.2 mm at 1.8 x 10

_ o calcium; 48.6 +1.9 mm a t J .6 x 10 M calcium; and 46.2 + 2.5 mm at

7.2 x 10 M calcium. The values represent the mean + S.E.M. of six experiments. BARIUM CONTROL RESPONSE 100 0 6 40 80 20 0 2-n-BUTYl ACU CNETAIN CM5 CONCENTRATION CALCIUM Figure 7 Figure 68

Figure 8. Cumulative concentration-effect curves for carbachol- evoked catecholamine secretion from the isolated bovine adrenal gland in the presence and absence of 2-n-propyl aminoindene. Values on the right represent the molar concentration of the aminoindene. The recov ery curve for carbachol (in absence of aminoindene) was obtained one -4 hour after the 10 M aminoindene curve. Values are corrected for rest ing secretion. Each point is the mean + S.E.M. of 4-14 glands. All points in the figure, with the exception of those on the recovery -8 curve and on the 10 M aminoindene curve, are statistically different

3 00

> • RECOVERT c

vn < u< ac

Z 5 < z0 «-/

< u

0 0 3 0.1 0.3 1.0 3.3

CARBACHOl CONCENTRATION ImM)

Figure 8

o \o - h . concentration of 10 M of the aminoindenes. However, the adrenal med ulla proved to he a more sensitive tissue to the aminoindenes, since at —7 —6 concentrations of 10 M and 10 M o f these compounds (which did not

block acetylcholine-induced myometrial contractions; Rahwan et a l.,

1977), an approximately 60% blockade of carbachol-evoked adrenomedullary

secretion was observed.

The recovery of the carbachol response of the adrenal medulla _4 following the blockade instituted by 10 M 2-n-propyl aminoindene was

essentially complete (fig. 8).

Similar results were obtained with the 2-n-butyl aminoindene

(fig. 9). Thus, both aminoindene compounds were capable of reversibly

blocking the calcium-dependent carbachol-evoked catecholamine secretion

from the isolated bovine adrenal medulla.

Experiments with acetaldehyde. At the highest concentration of ~li 10 M, th e 2-n-propyl and 2-n-butyl aminoindenes did not interfere with

acetaldehyde-induced catecholamine secretion from the adrenal medulla

(fig . 10). These results indicate that the aminoindenes do not inter

fere with calcium-independent evoked adrenomedullary secretion.

A potentiation of acetaldehyde-induced catecholamine secretion

was noted with both aminoindenes at the highest concentration (lOOmM)

of the secretagogue (acetaldehyde). This paradoxical potentiation, for

which we currently have no explanation, was significant (P<0.05) only

fo r the 2-n-butyl aminoindene (fig. 10).

Experiments with barium. Over the 10-minute stimulation period

w ith 1 mM barium, catecholamine secretion from the adrenal medulla in

creased from an average of 4-5 + 6*5 M>s/2 min (resting secretion) to 71

Figure 9. Cumulative concentration-effect curves for carbachol- evoked catecholamine secretion from the isolated bovine adrenal gland in the presence and absence of 2-n-butyl aminoindene. Values on the right represent the molar concentration of the aminoindene. The re covery curve for carbachol (in absence of aminoindene) was obtained one hour after the 10 M aminoindene curve. Values are corrected for resting secretion. Each point is the mean + S.E.M. of 5-1^ glands.

All points in the figure, with the exception of those on the recovery —8 curve and on the 10 M aminoindene curve, are statistically differ en t (P<£.05) from the corresponding points on the control (no amino indene) carbachol curve. RECOVERY

CONTROL

300

® 200

T T /

TOO

0.03 0.1 0.3 1.0 3 3 CARBACHOL CONCENTRATION (mUl

Figure 9 73

Figure 10. Cumulative-concentration curves for acetaldehyde-induced catecholamine secretion from the isolated bovine adrenal gland in the -I/, presence and absence of 10 M 2-n-propyl or 2-n-butyl aminoindenes.

Values are corrected for resting secretion. Each point is the mean

+ S.E.M. of 6-? glands.

I CATECHOLAMINE RELEASE lHg /3m ln l 400 200 600 300 SOO too CTLEYE ON (mM) N IO T A R T N E C N O C ACETALDEHYDE 3 e d y h e d l a t e c a CONTROL ACETALDEHYDE Figure 10 Figure 10 BUTYL M)ACETALOEHYDE " O lO Y P O R P 33 100 75 187.5 1 54 tig/2 min (at the end of the stimulation period). The ef fect of barium was not altered by either of the aminoindenes (10 ^M) nor by magnesium (5 x 10 ^M). These results are in conflict with pre viously published data on barium and magnesium (Douglas and Rubin, 1964), and their interpretation is discussed below. 45 ^Ca uptake studies. In order to establish that the aminoindenes do not interfere with calcium uptake by the adrenal chromaffin cells, the washout curves (see Methods) for ^Ca in the presence and absence of the two aminoindenes were compared (fig. 11). The ^Ca washout curves for both aminoindenes were not statistically different from the control 45 curve at each time interval sampled. Total Ca washout for each aminoindene during the 60-minute washout period was also not statis tically different from the control value, These data should be com pared with results of an earlier study (Rahwan et a l., 1973b) which dem onstrated a more rap id washout of a rad io lab e led compound confined ,14 . b.5 to the extracellular space ( C-sorbitol) as compared to washout of Ca which is taken up by the chromaffin cells under the experimental con ditions described.

PHARMACOLOGICAL EVALUATION OP NEW CALCIUM ANTAGONISTS: 2-SUBSTITUTED 3-DIMETHYLAMINO-5,6-METHYLENEDIOXYINDENES. CORONARY AND CARDIAC EFFECTS (P iascik , Rahwan, and W itiak, J. Pharmacol. Exp. T h er., subm itted).

Effects of aminoindenes and prenylamine on potassium-depolarized

coronary strips. The average control contractile response of the bo

vine isolated extramural coronary strips to 43 mM potassium chloride

was 10 + 1.0 mm. This contraction was inhibited in a concentration-re

lated manner by both the 2-n-propyl and 2-n-butyl aminoindenes (fig. 12).

A concentration of 10 ^M of either of the aminoindenes or of 6 x 10 ^M 76

Figure 11. Effect of 2-substituted aminoindenes on washout of ^5ca from the isolated bovine adrenal gland. a was infused (2 mins) into the adrenals in presence or absence of 2-n-propyl or 2-n-butyl aminoindene in calcium-free medium, followed by washout of the radioactivity for 60 minutes (for details see Methods). Each point is the mean + S.E.M, of

3-4- glands. The ordinate represents counts per minute expressed as a percentage of the in itial counts per minute in the perfusate emanating froiji the glands during the 2-minute infusion of ^Ca. There is no statistically significant difference between any of the curves. 77

CONTROL

2-n PROPYL 10' M

2-n-BUTYl 10'4

1 10 20 30 40 50 60 MINUTES AFTER START OF W A S H O U T

Figure 11 78

Figure 12. The effects of 2-substituted 3-(iiIne'thylamino-.5»6-methyl-

enedioxyindenes on potassium-induced contractions of bovine coronary

strips. Different coronary strips were used for each concentration of

test compound. The control contractile response of the coronary strips

to 4-3 mM potassium chloride was 10 + 1 mm. Each point represents the mean + S.E.M. of four to six experiments. RELAXATION 100 0 6 80 20 40 -6 2-n- PROPYL2-n- 2-n- BUTYL OCNRTO CM2 CONCENTRATION g e 12 re igu F ,•4 5 80 prenylamine produced complete relaxation of the potassium-contracted coronary strips, although the relaxation induced by prenylamine devel oped much slower than that produced by the aminoindenes. Raising the calcium concentration in the bath to ?,2 mM rapidly and completely re versed the relaxing effect of the aminoindenes and prenylamine in all experiments. Control coronary strips exposed only to 43 mM potassium chloride remained contracted throughout the three to four hour dura tion of the experiments. Figure 13 illustrates a representative tracing of the ability of the aminoindenes and prenylamine to produce a rever sible relaxation of potassium-contracted coronary strips.

Effects of prenylamine and theophylline on isolated perfused hearts. Control values for the rabbit hearts used in these and sub sequent experiments were: coronary flow, 65+5 drops/minute; force of contraction, 17 + 2 mm; and heart rate, 112+5 beats/ minute.

Figure 14 summarizes the effects of the standard preparations, prenyl amine and theophylline, on the three parameters monitored. Saline pro duced no significant effect on coronary flow, force of cardiac con traction, or heart rate. Frenylamine produced a significant (K 0. 05) increase in coronary flow (fig. 14) at the same concentration (6 x 10 ^M) which produced complete relaxation of potassium-contracted coronary strips (fig. 13)• Heart rate was not affected by prenylamine, and force of cardiac contraction was depressed though not significantly so when compared to saline controls (calculated t = 1.61, table t value a t 0.05 probability = 1.86).

Theophylline significantly increased coronary flow (E~?0,05), force of cardiac contraction (F<0.0l), and heart rate (P<0.01) as compared to saline control values (fig. 14), 81

Figure 13. The reversible effects of 2-substituted 3-dimethylamino-5,6 methylenedioxyindenes and prenylamine on potassium-induced contractions of bovine coronary strips. Ca 7.2 mM KCI 43 mM 40 13070 190 200 210

2-n- Propyl 10‘4 M

KCI 43 mM Ca 7.2

20 50 6 0 70 85 95

KCI 43 mM 30 75 85 95130 160 TIME Cminl

F igu re 13 83

Figure 1^. Effects of prenylamine and theophylline on coronary flow, force of myocardial contraction, and heart rate in isolated perfused rabbit hearts. Values represent the mean + S.E.M. of five to ten experiments. CHANGE -40 -20 60 40 0 0 SALINE E R RATEHEART OOAY FLOW CORONARY FORCE Figure14 AMINE PRENYL PHYLLINE THEO • 85 Effect of 2-n-propyl aminoindene on isolated perfused hearts.

The 2-n-propyl aminoindene produced only a modest increase in coro nary flow at the concentrations tested (fig. 15)* At the concentration of 3 x of this aminoindene, the increase in coronary flow as compared to saline controls approached significance (calculated t =

1.8, table t value at 0.05 probability = 1.86), However, a dose-depen dent decrease in force of myocardial contraction was observed at 2-n- propyl aminoindene concentrations of 3 x 10 (P< 0«05) and 10

(P<0.0l), No effect on heart rate was observed at these concentrations.

An interesting, but as yet unexplained, increase ( 3 6.5%) in force of contraction was observed at the 10 concentration of the 2-n-propyl aminoindene (not shown in figure 15). This latter concentration is be low that necessary for calcium antagonistic activity in any system te s te d to d ate (Rahwan e t a l . , 1977; P iascik e t a l ., 1978).

Effect of 2-n-butyl aminoindene on isolated perfused hearts. The

2-n-butyl aminoindene produced a dose-dependent increase in coronary flow (fig. 16) which was significantly greater than saline control at both 3 x 10 (P<0.05) and 10 (BgO.Ol). The increase in coronary _Zt flow produced by 10 M 2-n-butyl aminoindene was also significantly -6 greater (P<0.05) than that produced by prenylamine (6 x 10 M) or

- 3 — L i theophylline (2.8 x 10 ^M). At the concentration of 10” M, the 2-n- butyl aminoindene produced a significant negative inotropic effect

(P<0,01); however, no chronotropic effects were observed at any con centration tested (fig. 16).

Effect of 2-n-propyl aminoindene on ouabain-Induced arrhythmias.

In the experiment depicted in figure 17, an intravenous infusion of 86

Figure 15. The effects of 2-n-propyl 3-dimethylamino-5,6-methylenedioxyindene on coronary flow, force of myocardial contraction, and heart rate in isolated perfused rabbit hearts. Values represent the mean +

S.E.M. of four to five experiments. CHANGE -20 -40 -60 40 20 0 ER RATE HEART OOAY FLOW CORONARY FORC E Figure15 PROPYL 88

Figure 16. The effects of 2-n-butyl 3“dimethylamino-5,6-methylenedioxyindene on coronary flow, force of myocardial contraction, and heart rate in isolated perfused rabbit hearts. Values represent the mean + S.E.M. of four experiments. CHANGE -60 -20 40 20 60 0 ER RATE HEART FLOW CORONARY FORCE-40 Figure 16Figure n BUTYL 2 - - X310‘5M 90

Figure 17. The effect of 2-n-propyl 3~diI>iethylamino-5,6-methylenedioxyindene on ouabain-induced arrhythmias in the dog. Panel A, normal EKG.

Panel B, arrhythmias induced by ouabain (0.075 mg/kg). Panel C transient reversal (duration 18,5 secs.) of the ouabain-induced arrhythmias by

2-n-propyl 3 - 6-methylenedioxyindene (13 mg/kg) followed by spontaneous resumption of abnormal rhythm, Panel D, complete reversal of ouabain-induced arrhythmias by the 2- rv-propyl aminoindene (26 mg/kg) with sinus rhythm persisting until termination of the experiment (30 m in.). ; A CONTROL

OUABAIN- NUUCED ARRHYTHMIA, 0.0 7.5 mfl / kfl

t 2*n*Propyl 13 mg/kg

2-n-Propyl 2 6 mg / kg

Figure 1? 92 ouabain (total dose of 0.075 mgAs) produced a classical arrhythmia characterized by left ventricular ectopia, periods of bigeminy, and ventricular tachycardia (fig. 17» panel B). Following approximately one hour of persistent arrhythmic activity, a dose of the 2-n-propyl aminoindene (13 mg/kg) was injected intravenously. This resulted in a transient reversal to sinus rhythm which lasted for 18.5 seconds and was followed by spontaneous resumption of the arrhythmia (fig. 17* panel C).

Following another 15 minutes of arrhythmic activity, a second dose of

26 mg/kg of 2-n-propyl aminoindene was administered intravenously.

A rapid reversal to sinus rhythm accompanied by a decrease in heart rate resulted (fig. 17* panel D). Sinus rhythm persisted thereafter until the experiment was terminated (30 m inutes). CHAPTER IV

DISCUSSION

PHARMACOLOGICAL EVALUATION OF NEW CALCIUM ANTAGONISTS: 2-SUBSTITUTED 3-DIMETHYLAMINO -5,6-METHYLENEDIOXYINDENES. EFFECTS ON SMOOTH MUSCLES (Rahwan, Faust, and Witiak, 1977)-

In preliminary experiments on the isolated rat uterus, it was found that the 2-methyl and 2-ethyl aminoindenes blocked the spasmogen ic actions of PGE^ (10 ^M) and PGF^a (10 ^M) at concentrations of

- h . -3 5 x 10 M and 10 M, respectively, whereas the 2-n-propyl and 2-n-butyl derivatives exhibited significant prostaglandin antagonism at a concen tration of 5 x 10 ^M each (figs. 2 and 3). Furthermore, the 2-methyl and

2-ethyl aminoindenes significantly blocked the action of oxytocin

(10 ^ U/ml bath concentration) in parallel with their effects on pros taglandins, whereas the 2-n-propyl and 2-n-butyl aminoindenes signif icantly blocked the action of oxytocin only at a concentration of the antagonists (10 %l) higher than that (5 x 10 ^M) which blocked the pros taglandins (figs. 2 and 3)- Based on these findings the 2-methyl and

2-ethyl aminoindenes were not studied further, and the apparent selec tive prostaglandin antagonism demonstrated by the 2-n-propyl and 2-n-bu- -5 tyl derivatives at a concentration of 5 x 10 M was pursued. -4 Although the blockade of oxytocin induced by the 10 M concentra tion of the 2-n-propyl and 2-n-butyl aminoindenes could be rationalized

93 on the basis of evidence implicating endogenous prostaglandins in the mediation of the uterine spasmogenic action of this hormone (Csapo and

Csapo, 19?4), it was essential to test the selectivity of the spasmo lytic aminoindenes against agonists whose spasmogenic action is not me diated by endogenous prostaglandins. Consequently, when tested against acetylcholine, histamine, and ergonovine at a variety of antagonist concentrations and on different tissues (tables 1 and 2), it was clear that the 2-n-propyl and 2-n-butyl aminoindenes were nonselective spas molytics since they blocked the spasmogenic action of all of the tested agonists. It was unlikely that the aminoindenes were causing a nonspe cific "poisoning" of smooth muscle since increasing the concentration of

PGFgO! or oxytocin significantly overcame the blockade by the aminoin denes (figs. 4 and 5# and see below).

It appeared likely that the 2-n-propyl and 2-n-butyl aminoindenes were acting at a step in excitation-contraction coupling common to the spasmogenic actions of the prostaglandins, oxytocin, ergonovine, his tamine, and acetylcholine. Since calcium and cyclic 3*»5'-adenosine monophosphate (cAMP) have received the greatest attention as intracellu lar messengers mediating smooth muscle contraction (Somlyo and Somlyo,

1970; Symposium, 1969 ) and re la x a tio n (Robison e t a l , , 1969; Robison and Sutherland, 1970; Andersson, 1972), respectively, we contemplated the possibilities that the 2-substituted aminoindenes could be blocking the spasmogenic action of the agonists listed above by increasing myometrial cAMP or by interfering'- with calcium influx into, or action within, the myometrial cell. In p relim in ary experim ents (P ia sc ik , P ia sc ik , and Rahwan, unpub lished observations) with 2-n-propyl aminoindene, it was found that th is compound, a t a concen tration of 10 produced a 5&% inhibition of the low Km form of cAMP phosphodiesterase (PDE). This inhibition of

PDE was approximately 1 greater than that produced by a similar con centration of papaverine (Piascik et a l., 1976). This finding, however, is of doubtful significance since Daniel and Janis (l975) have proposed that increases in uterine cAMP are probably coincidental and not causal for relaxation. Several lines of evidence support the latter contention.

For example, Vesin and Harbon (197*0 demonstrated that PGE^, PGE^, and epinephrine increased cAMP levels in estrogenized rat uteri probably by stimulating the same adenylate cyclase; however, the prostaglandins con tracted the uterus while epinephrine relaxed it. Similarly, Beatty et al.

(1973) found that oxytocin and epinephrine stimulated adenylate cyclase to the same extent in myometrium of pregnant rhesus monkeys, although oxytocin caused contraction and epinephrine caused relaxation in this tissue. Furthermore, Polacek et al. (1971) and Mitznegg et al. (197*0 have demonstrated that theophylline can suppress rat uterine contractions without increasing cAMP levels. Similar observations were made with papaverine (Polacek et a l., 1971)•

Daniel and Janis (1973) reviewed the complexities of calcium regu lation of myometrial contraction, and discussed the experimental diffi culties associated with studying the possible role of the different calcium pools (extracellular and intracellular) in excitation-eontraction coupling in this tissue. From the evidence reviewed by Daniel and Janis

(1975)i and from experimental data on other tissues (Rubin, 1970, 197*+; Symposium, 19^9)» i t would appear th a t acety lch o lin e owes i t s spasmo genic effects on certain smooth muscles at least in part to a depolar

izing action accompanied hy an influx of extracellular calcium into the

contractile cell. The extracellular calcium entering the cell may it

self serve to activate the contractile fibers, or may serve to trigger

the release of intracellular calcium and replenish such intracellular

calcium pools (Daniel and Janis, 1975)* On the other hand, although

no oxytocic agents have so far been established to act by releasing cal

cium from intracellular binding sites in the uterus (Daniel and Janis,

1975)—with the notable exception of the prostaglandins (Carsten, 1972,

1973)—there is considerable evidence from other tissues that barium

produces its spasmogenic effects primarily by releasing calcium from

intracellular storage pools (Antonio et a l., 1973; Caldwell and Walster,

I 963; D aniel, 196^; Karaki et a l., 1967; Saito et ad,, 1972). Because

acetylcholine and barium utilize different calcium pools for their spas

mogenic action, they were selected as pharmacological tools to investi

gate the mechanism of action of the 2-substituted aminoindene antag

onists. The action of both spasmogens was significantly blocked by the

aminoindene antagonists (tables 1 and 2, and fig. 7), and the blockade

could be reversed by increasing the concentration of extracellular cal

cium (figs. 6 and 7). That the 2-substituted aminoindenes did not in

terfere with calcium influx by stabilizing membranes in a manner similar

to high extracellular calcium (Tomita, 1970) is evidenced by the fact that

acetylcholine antagonism by the aminoindenes was reversed, rather than

potentiated, by increasing the extracellular calcium concentration.

This finding, and the demonstration of antagonism between the aminoindenes and barium, indicate that the aminoindenes may have inhib ited muscle responsiveness to spasmogens by interfering with the inter action between intracellular calcium and the contractile apparatus.

The possibility that the aminoindenes may be interfering with release of calcium from intracellular pools can only be contemplated if one assumes that the presumed increased influx of extracellular calcium induced by acetylcholine serves only to trigger the release of intra cellular calcium and replenish such intracellular calcium pools.

PHARMACOLOGICAL EVALUATION OF NEW CALCIUM ANTAGONISTS: 2-SUBSTITUTED 3-DIMETHYLAMINO - 5 ,6-METHYLENEDIOXYINDENES. EFFECTS ON ADRENOMEDULLARY CATECHOLAMINE SECRETION (P ia s c ik ,/ Rahwan, and W itiak, 1978).

In order to confirm the calcium antagonistic actions of the 2-sub stituted aminoindenes observed in smooth muscle studies, the bovine adrenal medulla was used as a model system to determine whether the aminoindenes would interfere with stimulus-secretion coupling in a manner similar to their interference with excitation-contraction coup ling in smooth muscle.

Carbachol-evoked catecholamine secretion from the isolated per fused bovine adrenal medulla is dependent upon the presence of extra cellular calcium (Schneider, 1969). On the other hand, acetaldehyde- induced adrenomedullary secretion is independent of both extracellular

(Rahwan e t a l , , 197^; Schneider, 1971) and in tr a c e llu la r (O 'N eill and

Rahwan, 1975) calcium . The 2-n-propyl and 2 -n-butyl aminoindenes were capable of blocking carbachol-evoked, but not acetaldehyde-induced, catecholamine secretion from the bovine adrenal medulla (figs. 8-10),

These findings support the earlier observation in smooth muscles 98 (Rahwan e t a l . , 1977) th a t the aminoindenes are calcium a n ta g o n ists, but do not elucidate the mechanism of the antagonism.

At least three possible mechanisms exist to explain the interfer

ence between the aminoindenes and calcium. First, the aminoindenes may interfere with the influx of extracellular calcium into the chro maffin cells, which is a prerequisite for carbachol-evoked adrenomed ullary secretion (Schneider, I 969). Second, the aminoindenes may inter

fere with the release of intracellular calcium, vrhich mediates the se

cretory action of certain adrenomedullary secretagogues (Rahwan et a l.,

1973h), although carbachol has not been shown to utilize this calcium

pool. Third, the aminoindenes may interfere with the action of calcium

by blocking the intracellular receptor site of this cation (as proposed

for smooth muscle by Rahwan et a l,, 197?) •

The possibility that the aminoindenes may be interfering with

calcium uptake by the chromaffin cells during stimulation can be re- 45 jected on the basis of data presented in figure 11. If ^Ca uptake into

the chromaffin cells was inhibited by the aminoindenes, the confinement

of this cation to the extracellular space should have resulted in a

significantly more rapid washout of radioactivity from the glands ex

posed to aminoindenes as compared to the washout from control glands / 14 not exposed to aminoindenes (as has been demonstrated for C-sorbitol

(Rahwan et a l., 19738)). However, the lack of statistical difference 45 in the Ca washout curves of glands exposed to aminoindenes and con

trols (fig. 11), as well as our previous finding that an increase in

extracellular calcium concentration can overcome the blockade in sti

tuted by the aminoindenes against smooth muscle spasmogens (Rahwan et a l., 99

1977)i argues against the possibility that the aminoindenes may be in terfering with calcium uptake by cells.

It is unlikely that carbachol-evoked catecholamine secretion from the bovine adrenal medulla is mediated by intracellular calcium since the action of this secretagogue is completely abolished by removal of extracellular calcium (Schneider, 1969). Furthermore, the secretory effect of acetylcholine on the bovine adrenal medulla is not accom panied by any changes in calcium content of mitochondria, lysosomes, microsomes, chromaffin granules, nuclei, or cytosol (Borowitz, 1969)* as has been demonstrated for other secretagogues which utilize intra cellular calcium (Rahwan et a l., 1973^). Consequently, it is unlikely that the aminoindenes interfere with the action of carbachol by inhib iting the release of calcium from intracellular pools in the chromaffin c e ll.

There is considerable evidence that barium stimulates muscle con traction primarily by releasing calcium from intracellular storage pools (Antonio et a l., 1973; Caldwell and Walster, 19&3; Daniel, 1964;

Karaki et al., 1967; Saito et a l., 1972). However, in the cat adrenal medulla, Douglas and Rubin (1964) proposed that barium induces catechol amine secretion either by increasing the permeability of the chromaffin c e ll membrane to e x tra c e llu la r calcium (when calcium i s p resen t in the perfusion medium) or by d ire c tly in te ra c tin g w ith the in tr a c e llu la r calcium receptor site (when extracellular calcium is absent), To elim inate the factor of extracellular calcium, the aminoindenes were tested against barium in a calcium-free environment (see Methods). It was reasoned that if the aminoindenes blocked the release or action of intracellular calcium, they should likewise block the secretory effect of barium on the bovine adrenal medulla regardless of whether barium acted by releasing intracellular calcium or by directly interacting with the intracellular calcium receptor site. However, the secretory effect of barium was not altered by the aminoindenes (see Results).

An attempt was then made to block the secretory effect of barium by magnesium. Magnesium has been shown to block th e in tr a c e llu la r calcium receptor site in the bovine adrenal medulla (Lastowecka and Trifaro,

1974; Rahwan et a l., 19731>) and other tissues (Kanno et a l., 1973;

Krnjevic et a l., 1976; Miledi, 1973; Turlapaty and Carrier, 1973)» and to block the secretory effect of barium on the in situ perfused adrenal medulla of the anesthetized cat (Douglas and Rubin, 1964). However, in the present investigation (see Results) with the isolated-bovine ad renal gland, magnesium did not block barium-induced catecholamine se cretion, It would appear, therefore, that in the bovine adrenal medulla, barium stimulates catecholamine secretion by a mechanism independent of intracellular calcium stores or the intracellular calcium receptor s i t e .

Prom the results discussed in this and the preceding section

(Rahwan e t a l . , 1977)» i t i s again concluded th a t the 2 -su b stitu te d .’ aminoindenes act as calcium antagonists by blocking an intracellular calcium receptor site. 101

PHARMACOLOGICAL EVALUATION OF NEW CALCIUM ANTAGONISTS: 2-SUBSTITUTED 3-DIMETHYLAMIN0-5» 6-METHYLENEDIOXYINDENES. CORONARY AND CARDIAC EFFECTS (P iascik , Rahwan, and W itiak, J. Pharmacol. Exp, T h er., submitted).

The coronary dilating nitrites and nitrates produce dubious and unpredictable effects in ischemic heart disease in presence of exten sive coronary atherosclerosis, and fail to produce a sustained increase in coronary flow during the limited period of their therapeutic useful ness (Melville et al,, 1965)* Dipyridamole, which produces coronary dilation without reducing myocardial oxygen consumption (but indeed ex hibits mild positive inotropic actions in animals and in man) (Boehring- er Ingelheim Product Information, 1978), fails to relieve anginal pain

(Kinseller et a l., 1962). In a brilliant series of articles published in the early 1960's, Raab ( 1962, I 963) emphasized the necessity for a reduction in myocardial oxygen consumption in conjunction with an in crease in myocardial oxygen supply as the rational basis for therapy of ischemic heart disease. The decrease in myocardial oxygen consumption can be achieved primarily through inhibition of adrenergic influences on the heart (Raab, 1962, 19&3) or through direct depression of myocar dial contractility (Fleckenstein et al., 1978; Fleckenstein, 1977)*

The effects of calcium antagonists on coronary and myocardial tis sue have been extensively reviewed (Fleckenstein et a l., 1976; Fleck e n ste in , 1977)* and a number of these agents are in clinical use as coronary dilators or antiarrhythmic agents. The results of the present investigation establish the 2-substituted aminoindenes as coronary di lators in vitro. The 2-substituted aminoindenes produced relaxation of potassium-

contracted strips of bovine extramural coronary vessels (figs. 12 and

13 ) and increased coronary flow in the non-stimulated isolated perfused rabbit heart preparation (figs. 15 and 16). The results obtained with

the 2-n-butyl aminoindene in the latter preparation (fig. 16) were more

impressive than those obtained with the 2-n-propyl derivative (fig. 15)•

This difference could possibly be attributed to the greater lipophil-

icity of the 2-n-butyl aminoindene, allowing greater cellular penetration

and tissue concentration of the compound. Although the use of extramu ral coronary vessels for the study of the coronary relaxant effects of

the 2-substituted aminoindenes is open to criticism, yet the results

obtained with these strips were in general agreement with those ob

tained with the perfused heart preparation. Furthermore, McGregor and

Fam (1966) proposed that the beneficial effects of coronary dilation in

ischemic heart disease would reside predominantly in dilation of the

large conducting coronary arteries which can direct more blood to ische

mic regions, rather than in any action on small resistance vessels which

would respond negligibly to coronary dilators due to their already max

imal dilation through autoregulatory mechanism in the ischemic heart

(Raab, 1962, 1963).

In the experiments with isolated perfused rabbit hearts, the 2-sub

stituted aminoindenes produced a significant decrease in myocardial

force of contraction without affecting the heart rate or rhythm (figs.

15 and 16). This negative inotropic effect should conceivably result

in decreased oxygen consumption by the myocardium. This has indeed

been shown to be the case with other calcium antagonists such as 103 prenylamine, nifedipine and verapamil (Fleckenstein et a l., 1 976),

The reduction of myocardial oxygen consumption resulting from the neg ative inotropic actions of calcium antagonists o n ly superficially re sembles that produced by the adrenergic beta receptor blockers, and offers the distinct advantage over the latter in that the negative inotropic effects of calcium antagonists are self-limited by a reflex activation of the sympathetic system should a disproportionate fall in arterial blood pressure occur (Fleckenstein et a l,, 1976).

The coronary smooth muscle relaxing effect of the 2-substituted aminoindenes closely resemble those produced by prenylamine and verap amil and their congeners, and differ from the effects produced by nitro glycerine and other nitrites in the following respects: Whereas the onset of relaxation of potassium-contracted coronary strips produced by nitroglycerine and other nitrites occurs within one minute even at very low drug concentrations (Fleckenstein et a l., 1976), the 2-substituted aminoindenes (fig. 13) and prenylamine (fig. 13 and Fleckenstein et a l,,

1976) produce a much slower onset of relaxation. Furthermore, nitro glycerine and other nitrites produce an incomplete and spontaneously reversible relaxation of potassium-contracted coronary strips even at high drug concentrations (Fleckenstein et a l., 1976), whereas the 2-sub stituted aminoindenes (fig. 12) and prenylamine and verapamil and their congeners (Fleckenstein et a l., 1976) produce a concentration-dependent and eventually complete relaxation of potassium-contracted coronary strips which is reversible only after elevating the concentration of calcium in the tissue bath (fig. 13)* Limited data is presented on the potential antiarrhythmic activity of the 2-substituted aminoindenes. The doses of the 2-n-propyl amino indene administered to the dog in the experiment depicted in figure 17 were based on a theoretical calculation to achieve, at the time of in travenous administration, a whole blood concentration of the drug of approximately 5 x 10 \ (hence the administered dose of 13 mg/kg body weight) or 10-^M (hence the administered dose of 26 mg/kg). Regardless of the accuracy or validity of these estimation, the administered doses were significantly lower than the previously determined LD50 of 2-n- propyl aminoindene in mice (R.G. Rahwan, T.L. Henry, and M.F. P ia sc ik , unpublished observations); the intraperitoneal and intravenous LD 50 of

2-n-propyl aminoindene in mice being I 85 mg/kg and 40 mg/kg, respectively.

As can be a n tic ip a te d from a compound w ith calcium a n ta g o n istic a c tiv ity , the 2-n-propyl aminoindene produced a dose-related antiarrhythmic effect in the ouabain-toxic dog. It is pertinent to note that other calcium antagonists with an intracellular site of action (the TMB compounds) have also been reported to exhibit antiarrhythmic properties (Chiou et a l., 1976).

Pharmacological Studies from other Laboratories. receptors located on the cardiac sarcolemma are thought to mediate the positive chrono tropic and inotropic responses of the heart to histamine. This stim ulation is thought to be due to histamine-induced rises in intracellular cAMP. A recent investigation demonstrated that the 2-substituted amino indenes antagonize histamine-induced increases in intracellular cAMP from cardiac sarcolemmal preparations. These compounds were more potent than the receptor blocking agent, cimetidine, indicating that the 105

2-substituted aminoindenes possess receptor blocking activity (C.L.

Johnson, personal communication).

Investigation of other calcium antagonists showed verapamil and both isomers of diltiazeme (one of which is practically inactive as a calcium antagonist) to be potent receptor blocking agents (more po tent than any of the 2-substituted aminoindenes). Nifedipine, one of the most potent calcium antagonists, had no receptor antagonist ac tivity. The only apparent structural similarity of all of these receptor blocking agents which also possess calcium antagonistic prop erties is an amine functionality. It seems most likely that the receptor blocking activity of the 2-substituted aminoindenes is unre lated to its calcium antagonistic properties.

The effect of 2-n-propyl aminoindene on platelet function has been studied by Murer and Davenport (1978), A concentration of 5 x 10 ^M produced 50$ loss of platelet-stored serotonin with only 17$ loss of cytoplasmic markers (^C-labeled nucleotides). At 10~^M, these results were reversed to inhibition of thrombin-induced secretion (E. Murer, personal communication). These results are very different from those

obtained with TMB-8, but the effect at low doses (below 10 ^M) resembled

that seen with A23187, an ionophore. Further studies are underway to verify or contradict the working hypothesis of these researchers. Con

trary to the results of the present study, Murer and Davenport (1978) believe that at low doses the 2-substituted aminoindenes release cal cium from intracellular stores (presumably membrane vesicles) while

at higher doses (10 ^M), the aminoindenes chelate the released calcium. 106

There is no justification for a chelating mechanism based on the structures of the aminoindenes. Furthermore, A. Schwartz and cowork ers (T. Wang, personal communication) indicate that the aminoindenes increase the binding (sequestration) of calcium to cardiac microsomes which would tend to confirm the conclusion of an intracellular calcium antagonistic action of the 2-substituted aminoindenes,

Proposed Future Studies with the Aminoindenes. Although the studies with the 2-substituted aminoindenes in excitation-contraction coupling and stimulus-secretion coupling systems indicate that the most likely mechanism of action of these compounds is intracellular calcium antag onism at a calcium receptor site, more conclusive evidence must be ob tained to confirm this hypothesis. Several different experiments may be proposed for this purpose. First, autoradiography using radiolabeled aminoindenes should demonstrate the site at which the aminoindenes con centrate. If this site is shown to be intracellular and if the resolu tion obtained is sufficiently high, a subcellular localization may also be possible.

A second study would involve the soluble calcium-binding protein isolated from the bovine adrenal medulla (Brooks and Siegel, 1973)*

This protein has a molecular weight of 11,900 and shows high-affinity calcium binding (K^ = 1.7 x 10 ^M) and binds one mole of calcium per mole of protein. An interaction between calcium, the calcium-binding protein (Brooks and Siegel, 1973)« tubulin and actomyosin (Redburn

et a l., 1972; Twomey and Poisner, 1972), and microtubules (Rahwan et a l.,

1973a)—all of which have been demonstrated in the adrenal medulla—may provide the elements required to substantiate a mechanism of hormone 107 secretion outlined by Rubin (197^)* The model proposes that the cal cium receptor protein may be associated with troponin, which is bound to the actin filaments. The ability of the 2-substituted aminoindenes to displace calcium from this calcium-binding protein would indeed in dicate that the aminoindenes exert their calcium antagonistic action via a direct blockade of an intracellular calcium receptor.

The coronary studies have demonstrated the ability of the 2-sub stituted aminoindenes to produce marked coronary vasodilation in vitro.

Evidence for the efficacy' of these compounds .in vivo can best be achieved by a series of experiments measuring increased survival in coronary li gated animals.

Finally, the promising findings of the preliminary experiment on the potential antiarrhythmic activity of the 2-substituted amino indenes definitely warrant a full-scale study of these compounds as possible antiarrhythmic agents. BIBLIOGRAPHY

Aceves, J. and Machne, X.: The action of calcium and local anesthet ics on nerve cells and their interaction during excitation. J. Phar macol, Exp. Ther. 140; 138-148, 19&3.

Anderson, F.F. and Craver, B.K.: A pyrex apparatus for the perfusion o f the coronary c irc u la tio n of mammalian h e a rts. J. Pharmacol. Exp. Ther. 22* 135-1^1, 1948. Andersson, R.G.G.; Cyclic AMP and calcium ions in mechanical and met abolic responses of smooth muscle; influence of some hormones and drugs. Acta Physiol. Scand. Suppl. 382, 1-59, 1972.

Antonio, A., Rocha E Silva, M. and Yashuda, Y.; The tachyphylactic ef fect of barium on intestinal smooth muscle. Arch. Int. Pharmacodyn, Ther. 204: 260-267, 1973.

Baker, P.F., Meves, H. and Ridgway, E.B.; Effects of manganese and other agents on the calcium uptake that follows depolarization of squid axons. J. Physiol. (London) 231: 511-326, 1973*

Beatty, C.H., Bocek, R.M. and Young, M.K.: Effect of oxytocin and epinephrine on the adenylate cyclase activity of myometrium from pregnant rhesus monkeys. Horm. Metab, Res. 3.: 213-215» 1973* Beeler, G.W. and Reuter, H.: Membrane calcium current in ventricular myocardial fibres. J. Physiol, (London) 207: 191-209, 1970.

Bianchi, C.P.: Pharmacology of excitation-contraction coupling in mus cle. Introduction: Statement of the Problem. Fed. Proc. 28: 1624- 1627, 1969. Blaustein, M.P.: Barbiturates block calcium uptake by stimulated and potassium-depolarized rat sympathetic ganglia. J. Pharmacol. Exp. Ther. 1^6: 80-86, 1976.

Blaustein, M.P. and Goldman, D.E,: Competitive action of calcium and procaine on lobster axon. J. Gen. Physiol. 42: 1043-1063, 1966.

Boehringer Ingelheim Product Information: Physician's Desk Reference, 32nd edition, p. 668, Medical Economics Co., Oradell, N.J., 1978. 108 109

Bondani, A. and Karler, It.: Interaction of calcium and local anes thetics with skeletal muscle microsomes. J. Cell Physiol. 199- 211, 1970. Bondi, A.Y., K irsten , E.B. and Hofmann, P.G.s E ffects of verapam il in frog skeletal muscle. Fed. Proc. 32 s 579 > 1974.

Borowitz, J.L.: Calcium binding by subcellular fractions of bovine' adrenal medulla. J, Cell Physiol. 6£: 3H “319» 19&7-

Borowitz, J.L.: Effects of acetylcholine on the subcellular distrib ution of ^5ca in the bovine adrenal medulla. Biochem, Pharmacol. 18; 715-723, 1969. Borowitz, J.L.; Effect of lanthanum on catecholamine release from adrenal medulla. Life Sciences. 1_1: 959-964, 1972.

Borowitz, J.L ., Fuwa, K. and Weiner, N.: Distribution of metals and catecholamines in bovine adrenal medulla subcellular fractions. Na ture (London) 205: 42-43, 1965.

Brooks, J.C. and Siegel, F.L.: Purification of a calcium-binding phosphoprotein from beef adrenal medulla. J. Biol. Chem, 248; 4189- 4193, 1973. Burn, J.H. and Gibbons, W.R.; The release of noradrenaline from sym pathetic fibres in relation to calcium concentration. J, Physiol. (London) 181; 214-223, 1965. Bustos, G. and Roth, R.H.: Release of monoamines from the striatum and hypothalamus: Effect of gamma-hydroxybutyrate. Brit. J. Pharmacol. 44; 817-820, 1972.

Caldwell, P.C. and Walster, G.; Studies on the microinjection of various substances into crab muscle fibers. J. Physiol. (London) 169: 353-372, 1963. Carafoli, E.: In vivo effect of uncoupling agents on the incorpora tion of calcium and strontium into mitochondria and other subcellu lar fractions of rat liver. J. Gen. Physiol. j)0: 1849-1864, 1967.

rj-1 j Carafoli, E, and Gazzotti, P.: The reaction of Ca with the inner and outer membrane of mitochondria. Experientia 22: 408-409, 1973.

Carlsson, A., Hillarp, N.A. and Waldeck, B.: Analysis of the Mg - ATP dependent storage mechanism in the amine granules of the adre nal medulla. Acta Physiol, Scand, 52s 474-477, 1963*

Carsten, M.E.: Prostaglandins' part in regulating uterine contractions by transport of calcium. In The Prostaglandins, ed. by E.M. Southern, PP. 59-66, Futura, Mount Kisco, N.Y., 1972. 110 Carsten, M.E.: Prostaglandins and cellular calcium transport in the pregnant human uterus. Amer. J. Obstet, Gynecol. 117: 824-832, 1973*

Charlier, R.: Antianginal Drugs. Handbook of Experimental Pharmacology vol. 31» Springer-Verlag, New York, 1971. Charo, I.F., Detwiler, T.C. and Feinman, R.D.: Inhibition of platelet secretion by an antagonist of intracellular calcium. Biochem. Bio- phys. Res. Comm. r]2 \ 1462-1467* 1976.

Chiou, C.Y. and Malagodi, M.H.s ’ Studies on the mechanism of action of a new Ca^+ antagonist, 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride in smooth and skeletal muscles. Br. J. Pharmacol. S h 279-285, 1975. Chiou, C.Y., Malagodi, M.H., Sastry, B.V.R. and Posner, P.: Effects of calcium antagonist, 6-(N,N-diethylamino)-hexyl-3,4,5-trimethoxybenzoate, on digitalis-induced arrhythmias and cardiac contractions. J, Pharmacol. Exp. Ther. 198; 444-449, 1976.

Cloarec, M.: Criteria of the efficacy of the treatment of coronary disease; "the coronary vasodilators," Sem. Ther. 2Z! 753-754, 1961.

Costantin, L,, Franzini-Armstrong, C. and Podolsky, R.J.: Localization of calcium-accumulating structures in striated muscle fibers. Science 147: 158-159, 1965. Cranefield, P.F., Aronson, R.S. and Wit, A.L.; Effect of verapamil on the normal action potential and on a calcium-dependent slow response of canine cardiac Purkinje fibers. Circulation Res. 204-213, 1974.

Csapo, A.I. and Csapo, E.E.: The "prostaglandin step," a bottleneck in the activation of the uterus. Life Sci. 14; 719-724, 1974.

Curry, D.L., Bennett, L.L. and Grodsky, G.M.: Requirement for calcium ion in insulin secretion by the perfused rat pancreas. Amer. J. Physiol. 214; 174-178, 1968.

Daniel, E.E.: Effects of drugs on contractions of vertebrate smooth muscle. Ann. Rev. Pharmacol. 4: 189-222, 1964.

Daniel, E.E.: In Muscle, ed. by W.M. Paul, E.E. Daniel, C.M. Kay and G. Monckton, p. 295, Pergamon, New York, 1965*

Daniel, E.E. and Janis, R.A.; Calcium regulation in the uterus. Pharmacol. Therap. IB; 695-729, 1975*

Dell'Omodarme, G. and Brunori, F .; Hypotensive and neurodepressive action of some trimethoxybenzoic acid derivatives. Rass. Med. Sper. 6s 279-288, 1959. DeLorenzo, R.J.: Antagonistic action of diphenylhydantoin and cal cium on the level of phosphorylation of particular rat and human brain proteins. Brain Res. 134: 125-138* 1977-

DeRobertis, E.D.P. and Vaz Ferreira, A.: Electron microscope study of the excretion of catechol-containing droplets in the adrenal medulla, Exptl. Cell Res. 12: 568, 1957.

Devis, G. Somers, G., Van Obberghen, E. and Malaisse, W.J.: Calcium antagonists and islet function. I. Inhibition of insulin release by verapamil. Diabetes 24: 547-551* 1975*

Douglas, W.W.: A possible mechanism of neurosecretion: Release of vasopressin by depolarization and its dependence on calcium. Na ture (London) 197: 81-82, 1963*

Douglas, W.W.: The mechanism of release of catecholamines from the adrenal medulla. Second International Symposium on Catecholamines Milan, Italy, July, 1965. Pharmacol. Rev. :L8; 471-480, 1966.

Douglas, W.W., Kanno, T. and Sampson, S.R.: Influence of the ionic environment on the membrane p o te n tia l of th e adrenal chrom affin cells and on the depolarizing effect of acetylcholine. J. Physiol, (London) 1 56: 515-522, 1967.

Douglas, W.W, and Poisner, A.M.: On the mode of acetylcholine in evoking adrenal medullary secretion: increased uptake of calcium during the secretory response. J. Physiol, (London) 162: 385“ 392, 1962.

Douglas, W.W. and Poisner, A.M.: Stimulus-secretion coupling in a neurosecretory organ: The role of calcium in the release of vaso pressin from the neurohypophysis. J. Physiol. (London) 172: 1-18, 1964a.

Douglas, W.W. and P oisner, A.M.; Calcium movement in the neurohypo physis of the rat and its relation to the release of vasopressin. J. Physiol. (London) 172: 19-30* 1964b.

Douglas, W.W. and Rubin, R.P.: The mechanism of catecholamine re lease from the adrenal medulla and the role of calcium stimulus- secretion coupling. J. Physiol. (London) 167: 288-310, 1963.

Douglas, W.W. and Rubin, R.P.: Stimulant action of barium on the ad renal medulla. Nature (London) 203: 305-30?, 1964.

Dreifuss, J.J., Grau, J.D. and Nordmann, J.J.: Calcium movements re lated to neurohypophysical hormone secretion. In Calcium Transport in Contraction and Secretion, ed. by E. Carafoli, F. Clementi, W. Drabikowski, and A, Margreth, pp. 271-279* North Holland/American Elsevier, New York, 1975* 112 Ebashi, S. and Endo, M.: Calcium ion and muscular contraction. Progr. Biophys. Mol. Biol. 18: 125-163, 1968.

Edman, K.A.P. and G rieve, D.W.: The decrem ental propagation of the ac tion potential and loss of mechanical response in frog sartorius muscle in the absence of calcium, Experientia 1£: 40-41, 1963*

Ellis, K.O. and Carpenter, J.F.: Studies on the mechanism of action of dantrolene sodium, a skeletal muscle relaxant. Naunyn-Schmiede- bergs Arch. Pharmac, 275: 83-94, 1972.

Ellis, K.O. and Carpenter, J.F .: Mechanism of control of skeletal muscle contraction by dantrolene sodium. Archs. Phys. Med. Rehab- il. 362-369, 1974.

E llis, K.O., Castellion, A.W. , Honkomp, L.J. , Wessels, F.L., Carpenter, J.F. and Halliday, R.P.: Dantrolene, a direct-acting skeletal muscle relaxant. J. Pharm. Sci. 62: 948-951, 1973.

Entman, M.L., Hansen, J.L. and Cook J.W .Jr,: Calcium metabolism in cardiac microsomes incubated with lanthanum ion. Biochem. Biophys. Res. Comm. 258-264, 1969.

Fairhufst, A.S. and Macri, J.: Aminoglycoside-Ca interactions in skeletal muscle preparations. Life Sci. L6: 1321-1330, 1975.

Feinstein, M.B.: Inhibition of contraction and calcium exchangeabil ity in rat uterus by local anesthetics. J. Pharmacol. Exp. Ther. 152: 516-524, 1966.

Feinstein, M.B. and Paimre, M.: Pharmacological action of local anes thetics on excitation-contraction coupling in striated and smooth muscle, Fed, Proc, 28; 1643-1648, 1969.

Fleckenstein, A.: Metabolic aspects of the excitation-contraction coupling. In The Cellular Functions of Membrane Transport, Symp. Soc. Gen. Physiol., ed. by J.F. Hoffman, pp. 71-93, Prentice-Hall, Englewood C liffs, N .J,, 1963*

Fleckenstein, A.: Specific pharmacology of calcium in myocardium, cardiac pacemakers, and vascular smooth muscle. Ann. Rev, Pharma col. Toxicol. 12: 149-166, 1977.

Fleckenstein, A., Byon, Y.K. and Fleckenstein-Grun, G.: Fundamental cardiovascular effects of Ca++-antagonistic coronary drug fendiline. Arzneimittel-Forschung. 27: 562-571, 1977.

Fleckenstein, A., Doring, H.J., Janke, J. and Byon, J.K.: Basic ac tions of ions and drugs on myocardial high-energy phosphate metab olism and contractility. In Handbook of Experimental Pharmacology, vol. 16/3, ed. by J. Schmier and 0. Eichler, pp. 3^5-405, Springer- Verlag, New York, 1975* 113

Fleckenstein, A., Nakayama, K,, Fleckenstein-Grun, G. and Byon Y.K.: Mechanism and sites of action of calcium-antagonistic coronary ther apeutics. In Coronary Angiography and Angina Pectoris, ed. by P.R. Lichtlen, pp. 297-315* Pub. Sciences Group, Inc., 1976.

Fleckenstein, A., Trirrhart, H,, Fleckenstein, B., Herbst, A. and Grun, G,: A new group of competitive Ca antagonists (iproveratril, D600, Prenylamine) with highly potent inhibitory effects on excitation- c o n tra ctio n coupling in mammalian myocardium. P flu g ers Arch. 307; 25, 1969. Foreman, J.C. and Mongar, J.L.: Dual effect of lanthanum on histamine release from mast cells. Nature 240: 255-256, 1972.

Frey, M. and Janke, J.: The effect of organic Ca-antagonists (verapam il, prenylamine) on the calcium transport system in isolated mito chondria of rat cardiac muscle. Pfluegers Arch. Gesamte Physiol Men- schen T iere. 252: R26, 1975.

Gross, F.: Drugs acting on arteriolar smooth muscle (Vasodilator Drugs), IV. Diazoxide, In Handbook of Experimental Pharmacology, vol. 39, ed. by F. Gross, pp. 430-443, 1977.

Gummerus, M.: Prevention of premature birth with nylidrin and verapam il. Zeitschrift fur geburtschulfe und perinatologie. 179; 261-268, 1975. Gummerus, M.: Treament of premature labor and antagonization of side- effects of tocolytic therapy with verapamil. Zeitschrift fur genurt- schulfe und perinatologie. 181: 33 ^ -339 , 1977.

Haas, H.G., Kern, R., Benninger, C. and Einwachter, H.M.: Effects of prenylamine on card iac membrane c u rre n ts and c o n tr a c tility . J. Phar macol. Exp. Ther. 192: 688-701, 1975-

Haeusler, G.: Differential effect of verapamil on excitation-contrac tion coupling in smooth muscle and on excitation-secretion coupling in adrenergic nerve terminals. J. Pharmacol. Exp„ Ther. 180: 672- 682, 1972. Hales, G.N. and Milner, R.D.G.: Cations and the secretion of insulin from rabbit pancreas in vitro. J. Physiol. (London) 199; 177-187, 1968.

H a rris, R.A., Loh, H.H. and Nay E.L.: E ffe c ts of d iv a le n t c a tio n s, c a t ion chelators and ionophore on morphine analgesia and tolerance, J. Pharmacol. Exp. Ther. 19£: 488-498, 1975.

Harris, R.A., Loh, H.H. and Way, E.L.: Antinociceptive effects of lanthanum and cerium in nontolerant and morphine tolerant-dependent animals. J. Pharmacol. Exp. Ther. I 96: 283-297, 1976. i i 4

Harvey, A.M. and Macintosh, F.C.: Calcium and synaptic transmission in a sympathetic ganglion. J. Physiol, (London) 27: *1-08-41 6, 1940.

Hasbani, M., Pincus, J.H. and Lee, S.H.: Diphcnylhydantoin and calcium movement in lo b s te r nerves, /arch. Neurol, 2 i .5 250-254, 1974.

Hasselbach, W. and Makinose, M.: Die Calciumpumpe der "Erschlaffungs- grana" des Muskels und ihre Abhangigkeit von der ATP-Spaltung. Bio- chera. Z. 222: 518-528, 1961. Hasselbach, W.s Relaxing factor and the relaxation of muscle. Progr. Biophys. Mol, Biol. 14: 169-222, 1964.

Hausler G.: The effects of sodium nitroprusside (SNP) on vascular smooth muscle. Expcrientia (Basel) 21: 729 > 1975*

Hausler G. and Thorens, S.: Pharmacology of vasoactive antihyperten sives. In Vascular Neuroeffector Mechanism, ed. by J.A, Bevan, G. Burnstock, B. Johansson, R.A. Maxwell and O.A. Nedergard-, pp. 232- 241, Karger, Basel, 1976.

Haugaard, N. Haugaard, E.S., Lee, N.H. and Horn, R.S.: Possible role of mitochondria in regulation of cardiac contractility. Fed. Proc. 28: 1657-1662, 1969.

Heilbrunn, L.V.: The action of calcium on muscle protoplasm. Physiol. Zool. 0 : 88-94, 1940.

Heilbrunn, L.V. and Wiercinski, F.J.: The action of various cations on muscle protoplasm. J. Cell Comp. Physiol. 2£: 15“32, 1947.

Hodgson, B.J., Kedwai, A.M. and Daniel, E.E.: Uptake of lanthanum by smooth muscle, Can. J. Physiol, Pharmacol. 52.: 730-733» 1972.

Huang, T.F. and Peng, Y.I.: Effect of verapamil on experimental ar rhythmias. Eur. J. Pharmacol. 42: 363-370, 1977.

Hurwitz, L, and Joiner, P.D.: Excitation-contraction coupling in smooth muscle. Fed. Proc. 28: I 629-I 633 , I 969.

Jaanus, S.D., Miele, E, and Rubin, R.P.: The analysis of the inhib itory effect of local anesthetics and propranolol on adrenomedullary secretion evoked by calcium or acetylcholine. Br, J. Pharmac. Che- mother. 21: 319-330, 1967.

Jaanus, S.D. and Rubin, R.P.: The effect of ACTH on calcium distribu tion in the perfused cat adrenal gland. J. Physiol. (London) 213: 581-598, 1971.

Jacobson, A. Schvrartz, M. and Rehm, W.S.: Effects of removal of calcium from bathing media on frog stomach. Amer. J. Physiol. 209: 134-140, 1965. 115

Jahn, T.L. and Bovee, E.C.: Protoplasmic movements within cells. Physiol. Rev. 42: 793-862, 1969.

Kakunaga, T., Kaneto, H, and Hano, K.L.: Pharmacologic studies on analgesics-VII. Significance of the calcium ion in morphine anal gesia. J. Pharmacol. Exp, Ther. 153; 134-141, 1966,

Kalsner, S., Nickerson, M. and Boyd, G.N.: Selective blockade of potassium-induced contraction of aortic strips by beta-diethylamino- ethyl-diphenylpropylacetate (SKF 525A). J. Pharmacol. Exp. Ther, 174-8 500-508, 1970.

Kaneto, H.: Inorganic ions: The role of calcium. In Narcotic Drugs- Biochemical Pharmacology, ed. by D.H. Clouet, pp. 300-309, Plenum Publishing Corp., New York, 1971.

Kanno, T., Cochrane, D.E, and Douglas, W.W.: Exocytosis (secretory granule extrusion) induced by injection of calcium into mast cells. Can. J. Physiol. Pharmacol, jjl: 1001-1004, 1973*

Karaki, H., Ikeda, M. and Urakawa, N.: Effects of external calcium and some metabolic inhibitors on barium-induced tension changes in guinea pig taenia coli. Jap. J. Pharmacol. 12.: 603-612, I 967.

Kinsellar, D., Troup, W. and McGregor, M.: Studies with a new coronary dilator drug: Persantin, Amer. Heart J. 63.: 14-6-150, 1962.

Kohlhardt, M., Bauer, B. Krause, H. and Fleckenstein, A.: Differen tia tio n of th e transmembrane Na and Ca channels in mammalian cardiac fibers by the use of specific inhibitors. Pflugers Arch. 335: 3°9- 322, 1972.

K ohlhardt, M., F ig u lla , H.R. and T rip a th i, 0: The slow membrane chan nel as the predominant mediator of the excitation process of the sino atrial pacemaker cell. Basic Res. Cardiol. 2L: 17-26, 1976.

Kraicer, J ., Milligan, J.V., Gosbee, J.L., Conrad, R.G. and Branson, C.M.: In vitro release of ACTH: Effects of potassium, calcium, and corticosterone. Endocrinology 8j>! 1144-1153> 1969*

Kreye, V.A.W., Baron, G.D., Luth, J.B. and Schmidt-Gayk, H.: Mode of a c tio n of sodium n itro p ru ssid e on v ascu lar smooth muscle. Naunyn Schmiedebergs Arch. Pharmacol. 288: 381-4-02, 1975*

Kreye, V.A.W. and Gross, F,: Drugs acting on arteriolar smooth muscle (V asodilator Drugs) I I I . N itroprusside. In Handbook of experim ental Pharmacology, vol. 39, ed. by F. Gross, pp. 4-18-4-30, Springer-Verlag, New York, 1977.

Kreye, V.A.W. and Luth, J.B.: Effect of sodium nitroprusside, temper ature, and calcium withdrawal on the relaxation speed of vascular smooth muscle. In Ionic Actions on V ascular Smooth Muscle, ed. by E. Betz, pp. 145-149, Springer-Verlag, New York, 1976. 116 2+ Kmjevic, K., Pull, E. and Herman, R.: ' Intracellular Mg increases neuronal excitability, Can J, Physiol, Pharmacol. £4: 73-77, 1976.

Kumagai, H,, Ebashi, S. and Takeda, F„: Essential relaxing factor in muscle other than myokinase and creatine phosphokinase. Nature (London) 126: 166-179, 1955. Lastowecka, A. and Trifaro, J.M.: The effect of sodium and calcium ions on the release of catecholamines from the adrenal medulla: So dium deprivation induces release by exocytosis in the absence of ex tracellular calcium. J. Physiol. (London) 236: 681-705, 1974.

LeBreton, G.C. and Dinerstein, R.J.: Effect of the calcium antagonist TMB-6 on intracellular calcium redistribution associated with plate let shape change. Thrombosis Res. L0: 521-523, 1977.

Lee, C, and Berkowitz, B.: Calcium antagonist activity of methadone, 1-acetylmethadol and 1-pentazocine in the rat aortic strip. J. Pharmacol. Exp. Ther. 202: 646-653, 1977.

Lettvin, J.Y., Pickard, W.F. and McCulloch, W.S.: A theory of passive ion flux through axon membranes. Nature (London) 202: 1338-1339, 1964.

Levy, J.V.: Verapamil and diazoxide antagonism of agonist-induced con tractions of aortic strips from normal and spontaneously hypertensive rats. Res. Comm. Chem, Path. Pharmacol. 11_: 387-404, 1975*

Lichtlen, P.R.; Coronary Angiography and Angina Pectoris, Symposium of the Eur. Soc. of Cardiology. Pub, Sciences Group, Inc., 1976,

Lindner, E.: N-(3-phenylisopropyl)-3,3“diphenylpropylamine, anew sub stance with a dilating action on the coronary vessels. Part 1. Action on the circulation. Arzneimittel-Forschung. 10: 569-573, I960.

Lindner, A., Claasen, R., Hendriksen, R.W.J. and Kralt, R.: Reserpine analogs: phenethylamine derivatives. J. Med. Chem. 6: 97-106, 1963.

Lindner, E, and Kaiser, J.: Anti-arrhythmic effect of Segontin (N-(3))- phenyl-propyl-(2))-l,1-diphenyl-propyl-( 3 )-amine-Further investiga tions with laboratory animals. Herz. Kreislauf. 2: 88-94, 1975.

Lochner, W. : New Therapy of Ischemic Heart Disease. 2nd Internation a l Adalat Symposium. Springer-V erlag, B e rlin , 1975*

Malagodi, M.H. and Chiou, C.Y.: Pharmacological evaluation of a new Ca 2+ antagonist, 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydro ch lo rid e (TMB-8): Studies in smooth m uscles. Eur, J. Pharmacol. 27: 25-33, 1974.

Halaisse, W.J., Sener, A., Devis, G. and Somers, G.: Calcium-antagonists and islet function. V. Effect of R33711# Horm. Metab, Res. 8: 434-438, 1976. 117 Marsh, B .B ,: The e ffe c ts of ATP on the fibre-volum e o f a muscle homog- enate. Biochim. Biophys. Acta £: 247-260, 1952.

Marshall, R.J. and Parratt, J.R.: Antiarrhythmic, haemodynamic and met abolic effects of 3 a-amino-5 a-androstan-2 (3-ol-17-one hydrochloride in greyhounds following acute coronary ligation. Br. J. Pharmacol. 52* 359-368, 1975. McGregor, M. and Fam, W.M.: Regulation of coronary blood flow. Bull. N.Y. Acad. Med. 42: 940-930, 1966.

Melville, K.I., G illis, R.A. and Sekelj, P.: Coronary flow, blood pressure, and heart rat dose-response changes after nitroglycerine administration. Can. J. Physiol. Pharmacol, 42.; 9-18, 196.5.

Miledi, R .T ransm itter release induced by injection of calcium ions into nerve terminals. Proc. Roy. Soc. London B Biol. Sci. 183: 421- 425, 1973. Milner, R.G.D. and Hales, C.N.: The role of calcium and magnesium in insulin secretion from rabbit pancreas studied in vitro. Diabetologia 2: 47-49, 1967. Mines, G.R.: On functional analysis by the action of electrolytes, J. Physiol. 46: 188-235, 1913.

Mitznegg, P., Schubert, E, and Heim, F.: Influence of low and high doses of theophylline on spontaneous m otility and cyclic 3 ',5 '-AMP’ content in isolated rat uterus. Life Sci, 14: 711-717, 1974.

Mommaerts, W.F.H.M.: E x citatio n and response in muscular tis s u e s . Ann. N.Y. Acad. Sci. 183: 425-432, 1971.

Mongar, J.L, and Schild, H.O.: The effect of calcium and pH on the anaphylactic reaction. J. Physiol. (London) 140: 272-284, 1958.

Moss, A.J. and Patton, R.D.: Antiarrhythmic agents, Charles C, Thomas, Pub,, Springfield, 111., 1973*

Murer, E.H. and Davenport, K.: Effect of calcium antagonists on plate let function and metabolism. Fed. Proc. 2Z! 1978.

Nayler, W.G. and Szeto, J .: Effect of verapamil on contractility, oxy gen u tiliz a tio n and calcium exch angeability in mammalian h e a rt mus cle. Cardiovasc. Res. 6: 120-128, 1972.

Needleman, P. and Hunter, F.E.: Effects of organic nitrates on mito chondrial respiration and swelling: possible correlation with the mechanism of pharmacological action. Mol. Pharmacol. 2: 134-143, 1966.

O 'N eill, P .J. and Rahwan, R.G.: Experim ental evidence f o r calcium -in dependent catecholamine secretion from the bovine adrenal medulla. J. Pharmacol. Exp. Ther. 193: 513-522, 1975- U 8 Otsuka, M., Iversen, L.L., Hall, Z.W. and Kravitz, E.A.: Release of gamma-aminobutyric acid from inhibitory nerves of lobster. Proc. Natl. Acad, Sci. 56: 1110-1115, 1966.

Papahadjopoulos, D.: Phospholipid model membranes. 3* Antagonistic effects of calcium and local anesthetics on the permeability of phosphatidyl serine vessicles. Biochim. Biophys. Acta 211: 467- 477, 1970. Parsons, J.A.: Calcium ion requirement for prolactin secretion by rat adenhypophyses in vitro. Amer. J. Physiol 217: 1599-1603, 1969*

Piascik, M. F., Rahwan, R.G. and Witiak, D.T.s Pharmacological evalua tion of new calcium antagonists: 2-substituted 3-d.iroethylamino-5,6- methylenedioxyindenes. Effects on adrenomedullary catecholamine se cretion. J, Pharmacol. Exp. Ther. 205 (in press), 1978.

Piascik, M.F., Rahwan, R.G. and Witiak, D.T.: Pharmacological eval uation of new calcium antagonists: 2-substituted 3-dimethylamino- 5,6-methylenedioxyindenes. Coronary and cardiac effects. J. Pharmacol. Exp.Ther., submitted.

Piascik, M.T., Miller, D.D. and Feller, D.R.: Phosphodiesterase inhib ition and lipolytic action of the stereoisomers of trimetoquinol. Res. Commun. Chem. Pathol. Pharmacol, ljh 203-215, 1976.

Pincus, J.H. and Lee S.H.: Diphenylhydantoin and norepinephrine re lease. Neurology 22: 410, 1973*

P ittin g e r , C. and Adamson, R.: A n tib io tic blockade of neurom uscular function, Ann. Rev. Pharmacol. 12: 169-184, 1972.

P oisner, A.M. and Hava, M.: The ro le of ATP and ATPase in the re le a s e of catecholamines from the adrenal medulla. IV. ATP-activated uptake of calcium by microsomes and mitochondria. Mol, Pharmacol. 6: 407- 415, 1970. P oisner, A.M. and T rifa ro , J.M .: The ro le of ATP and ATPase in the r e lease of catecholamines from the adrenal medulla. I. ATP-evoked re lease of catecholamines, ATP, and protein from isolated chromaffin granules. Mol. Pharmacol. 2 s 561-571, 1967.

Polacek, I., Bolan J. and Daniel, E.E.: Accumulation of adenosine 3 ',5 '-monophosphate and relaxation in the rat uterus in vitro. Can. J. Physiol, Pharmacol. 4£: 999-1004, 1971.

Putney, J.W., Jr, and Bianchi, C.P.: Site of action of dantrolene in frog sartorius muscle. J. Pharmacol. Exp. Ther. 189: 202-212, 1974.

Raab, W.: The sympathogenic biochemical trigger mechanism of angina pectoris. Its therapeutic suppression and long range prevention. Amer. J. C ardiol. 576-590, 1962, i 119 Raab, W.: The nonvascular metabolic myocardial vulnerability factor in "coronary heart disease." Fundamental pathogenesis, treatment, and prevention. Amer. Heart J. 66: 685- 706, 1963.

Rahwan, R.G. and Berger, J .E .: The e ffe c t o f prenylamine on ad ren alin e- induced hypercholesterolemia in mice. J, Pharm, Pharmacol. 22: 9&2- 964, 1970.

Rahwan, R.G. and Borowitz, J.L.: Mechanisms of stimulus-secretion coupling in adrenal medulla. J. Pharm, Sci. 62: 1911-1923. 1973*

Rahwan, R.G., Borowitz, J.L. and Hinsman, E.J.: Evidence for secretory tubules in the adrenal medulla. Arch, Int. Pharmacodyn, Ther. 206: 34-5-351. 1973a. Rahwan, R.G., Borowitz, J.L. and Miya, T.S.: The role of intracellular calcium catecholamine secretion from the bovine adrenal medulla. J. Pharmacol. Exp. Ther. 184: 106-118, 1973b*

Rahwan, R.G., Faust, M.M. and Witiak, D.T.: Pharmacological evaluation of new calcium antagonists; 2-substituted 3-dimethylamino-5,6-methylenedioxyindenes. J. Pharmacol, Exp. Ther. 201: 126-137, 1977*

Rahwan, R.G., O'Neill, P.J. and Miller D.D.: Differential secretion of catecholamines and tetrahydroisoquinolines from the bovine ad renal medulla. Life Sci. 14: 1927-1938. 1974.

Raines, A. and Standaert, F.G.: An effect of diphenylhydantoin on posttetanic hyperpolarization of intramedullary nerve terminals. J. Pharmacol. Exp. Ther. 156: 591-597, 1967.

Redburn, D.A., Poisner, A.M. and Samson, F.E. Jr.: Comparison of mi crotubule protein from adrenal medulla and brain. Brain Res. 44: 615-624-, 1972.

Reuter, H.: Localization of beta adrenergic receptors, and effects of noradrenaline and cyclic nucleotides on action potentials, ionic c u rre n ts and ten sio n in mammalian card iac muscle. J. P hysiol. (Lon don) 242: 429-4-51, 1973.

Rhodes, H.J. and Sutter, M.C.: The action of diazoxide on isolated vascular smooth muscle electrophysiology and contraction. Can. J. Physiol. Pharmacol. 49; 276-287, 1971.

Riddle, T.G., Mandel, L.J. and Goldner, M.M.: Dilantin-calcium inter action and active sodium transport in frog skin. Eur. J. Pharmacol. 22 : 189-192, 1975.

Ringer, S.: A further contribution regarding the influence of the dif ferent constituents of blood on the contraction of the heart. J. Physiol. 4: 29-47, I 883. 120 Robinson, C.: Pharmacometrics of «>-(N,N-diethylamino)-n-alkyl-3,4,5- trimethoxybenzoates patterned after the structure of reserpine on the nervous system. Doctoral Dissertation, Vanderbilt University, 1971. t Robison, G.A., Butcher R.W. and Sutherland, E.W.: On the relation of hormone receptors to adenyl cyclase. In Fundamental Concepts in Brug- Receptor Interactions, ed. by J.F. Danielle, J.F. Moran and D.J. Trig- gle, pp. 59-75, Academic Press, New York, 1969*

Robison, G.A. and Sutherland, E.W.: Sympathin E, sympathin I, and the intracellular level of cyclic AMP. Circ. Res. 27: suppl. 1, 147-161, 1970. Rubin, A.A,, Roth, F.E., Taylor, R.M. and Rosenkilde, H.: Pharmacol ogy of diazoxide, an antihypertensive non-diuretic benzothiadiazine. J. Pharmacol. Exp. Ther. 136; 344-352, 1962.

Rubin, R.P.: The role of calcium in the release of neurotransmitter substances and hormones. Pharmacol, Rev. 22: 3^9-428, 1970.

Rubin, R.P.: Calcium and the secretory process. Plenum Press, New York, 1974.

Rubin, R.P., Feinstein, M.B., Jaanus, S.D. and Paimre, M.: Inhibition of catecholamine secretion and calcium exchange in perfused cat adrenal glands by tetracaine and magnesium. J. Pharmacol. Exp. Ther. 164: 115-121, 1967.

Salako, L.A., Vaughan, E.M. and Wittig, J.H.: Investigations to charac terize a nevr anti-arrhythmic drug, Org 6001, including a simple test for calcium antagonism. Br. J. Pharmacol. 5Z! 251-262, 1976.

Saito, Y., Sakai, Y. and Urakawa, N.: Effect of cholinergic drugs and barium on oxygen consumption in guinea pig taenia coli. Jap, J. Pharmacol, 22: 653-661, 1972.

Samli, M.H. and Geschwind, I.I.: Some effects of energy-transfer in hibitors and of Ca^-free or I^-enhanced media on the release of lu teinizing hormone (LH) from the rat pituitary gland in vitro. Endocrin ology 82: 225- 231 , 1968.

Sanborn, W.G. and Danger, G.A.: Specific uncoupling of excitation and contraction in mammalian cardiac tissue by lanthanaum. Kinetic Studies. J. Gen. Physiol. j>6: 191-217, 1970.

Sandow, A.: Excitation-contraction coupling in skeletal muscle. Phar macol. Rev. 12: 265-3^0, 1965.

Schmitt, F.O.: Fibrous proteins—Neuronal organelles. Proc. Natl. Acad. Sci. 60: 1092-1101, I 968, 121 Schneider, F.H.: Drug-induced release of catecholamines, soluble protein and chromogranin A from the isolated bovine adrenal gland, Biochem. Pharmacol. l_8s 101-107, 19&9*

Schneider, F.H.: Acetaldehyde-induced catecholamine secretion from the cow adrenal medulla. J, Pharmacol. Exp, Ther. 177t 109-118, 1971.

Sharma, V.N.j Bronchodilator activity of twelve recently synthesized local anesthetics. Arch. Intern, Pharmacodyn. 125: 30^-310> i960.

Sharma, Y.N.: Antiacetylcholine antiaccelerator and local anesthetic activity of some quinidine-like drugs.Arch. Intern. Pharmacodyn. 137: 410-427, 1962. Sharma, V.N. and Arora R.B.: Antiarrhythmic activity of nine benzoic acid derivatives with special reference to their mode of action. Indian J. Med. Res. j51: 327-340, 1963*

Simpson, L.L.: The role of calcium in neurohumoral and neurohormonal extrusion processes, J. Pharm. Pharmacol. 20: 889-910, 1968.

Snyder, H.R., Davis C.S., Bickerton, R.K. and Halliday, R.P.: l-((5- arylfurfurylidene)amino)hydantoins. A new class of muscle relaxants. J, Mednl. Pharm, Chem. ID: 807-810, 19^7.

Sohn, R.S. and Ferrendelli, J.A.: Inhibition of Ca transport into r a t b rain synaptosomes by diphenylhydantoin (DPH). J. Pharmacol. Exp. Ther. 18£: 272-275. 1973.

Sohn R.S. and Ferrendelli, J.A.: Anticonvulsant drug mechanisms. Arch. Neurol, 626- 629, 1976.

Sokal, R.R. and Rohlf, F.J.: Biometry, pp. 145-149, Freeman, San Francisco, 1969.

Somlyo, A.P. and Somlyo, A.V.: Vascular smooth muscle II. Pharmacology of normal and hypertensive vessels. Pharmacol, Rev, 22: 249-353, 1970.

Steel, R.G.D. and Torrie, J.H.: Principles and Procedures of Statistics, McGraw-Hill Co,, New York, i 960.

Swain, A.W., Kiplinger, G.F. and Brody, T.M.: Actions of certain anti biotics on the isolated dog heart. J. Pharmacol. Exp. Ther. 117: 151- 159, 1956. Symposium: Biochemical asp ec ts o f prenylam ine, Biochim, A pplic, 14, Suppl. 1: 1-398, 1968.

Symposium: Pharmacology of e x c ita tio n -c o n tra c tio n coupling in muscle. Fed. Proc. 28: 1624-1669, 1969. 122

Symposium, P erh ex ilin e, Postgrad. Med. J. 4£ Suppl. 3* 1-132, 1973*

Taugner, G. and Hasselbach, W.s Uber den mechanismus der catecholamin- speicherung in den "chromaffinen granula" des nebennierenmarks. Naunyn-Schmiedeberg Arch. Pharmakol. Exp. Pathol, 255: 266-286, 1966,

Tomita, T .: E le c tric a l p ro p e rtie s of mammalian smooth muscle. In Smooth Muscle, ed. by E. Bulbring, A.F. Brading, A.W. Jones, and To Tomita, p. 197i The Williams and Wilkins Company, Baltimore, 1970.

Turlapaty, P.D.M.V. and Carrier, 0. Jr.: Influence of magnesium on calcium-induced responses of atrial and vascular muscle. J. Pharma col. Exp. Ther. 18£: 86-98, 1973.

Twomey, S.L. and Poisner, A.M.: An immunological comparison of brain microtubule protein (tubulin) with tubulin and actomyosin-like pro tein from the adrenal medulla. Brain Res. 46: 341-347i 1972.

Usherwood, P.N.R., Machili, P. and Leaf, G.: L-glutamate at insect excitatory nerve-muscle synapses. Nature 219: 1169-1172, 1968.

Vale, W., Burgus, R. and Guillemin, R.: Presence of calcium ions as a requisite for the in vitro stimulation of TSH-release by hypothal amic TRF. Experientia (Basel) 22: 853-855* 1967.

Van Breemen, C. and Daniel, E.E.: The influence of high potassium de polarization and acetylcholine on calcium exchange in the rat uterus. J. Gen. Physiol. 4£: 1299-1317, 1966.

Van Breemen, C„, Farinas, B.R. and Gerba, P.: Excitation-contraction coupling in rabbit aorta studied by the lanthanum method for mea suring cellular calcium influx. Circ, Res. 22.s 44-54, 1972.

Van Neuten, J.M., Tobia, A.J. and McGuire, J.L.: Antagonism of vascu lar smooth muscle contraction by flunarizine. Fed. Proc. 2Z5 793» 1978.

Vater, W., Herse, A., Kazda, S. and Stoepel, K.: Pharmacology of a new calcium antagonistic drug-nifedipine (Adalat). In Coronary An giography and Angina Pectoris, ed. by P.R. Lichtlen, p. 328-333* Pub. Sciences Group, Inc., 1976.

Vesin, M.F. and Harbon, S.: The effects of epinephrine, prostaglandins, and their antagonists on adenosine cyclic 3 '»5'-monophosphate concen trations and motility of the rat uterus. Mol. Pharmacol. 10^: 457- 473. 1974. Villani, F., Piccinini, F., Chiarra, A. and Brambilla, G.: Effect of lanthanum on calcium exchangeability in mitochondria. Biochem. Phar macol. 24: 1349-1351, 1975. 123 Von Euler, U.S. and Hamberg, U.: Colorimetric determination of adren aline and noradrenaline. Acta Physiol. Scand. 1£: 74-84, 1949.

Weber, A,, Herz, R. and Reiss, I.: On the mechanism of the relaxing effect of fragmented sarcoplasmic reticulum. J. Gen. Physiol. 46: 679-702, 1963.

Weiss, G.B.: Cellular pharmacology of lanthanum, Ann. Rev. of Pharmacol. 14: 3^3-364, 1974.

Winbury, M.M.: Problems in laboratory evaluation of antianginal agents, p. 1, North-Holland Pub, Co., Amsterdam, 1967*

Wit, A.L, and Cranefield, P.F.: Effect of verapamil on the sinoatrial and atrioventricular nodes of the rabbit and the mechanism by which it arrests reentrant atrioventricular nodal tachycardia. Circ. Res. 413-425, 1974.

Witiak, D.T.: The Vilsmeier-Haack cyclization reaction. Synthesis of potential prostaglandin antagonists. In Abstracts of the Fourteenth N ational M edicinal Chemistry Symposium, American Chemical Society, Durham, New Hampshire, 1974.

Witiak, D.T., Williams, D.R., Kakodkar, S.V., Hite, G. and Shen, M.S.: Vilsmeier-Haack cyclizations. Synthesis of 2-substituted 3-dimethylamino-5,6-methylenedioxyindenes and their corresponding indanones. J. Org. Chem. 2£: 1242-1247, 1974.

Wohl, A .J ., H aeusler, L.M. and Roth, F .E .: Studies on the mechanism of antihypertensive action of diazoxide in vitro vascular pharmacodynam ics. J. Pharmacol, Exp. Ther. 158: 531-539, 1967*

Wohl, A.J., Haeusler, L.M. and Roth, F.E.: Mechanism of the antihyper tensive effect of diazoxide: in vitro vascular studies in the hyper tensive rat, J, Pharmacol, Exp, Ther. 162: 109-114, I 968,

Wong, P.Y.D., Hwang, J.C. and Yeung, C.-H,: Interaction between lan thanum and calcium in isolated guinea-pig heart. Eur. J. Pharmacol, 36: 253-256, 1976.

Yahr, M.D.: Drugs in the treatment of convulsive disorders. In D rill's Pharmacology in Medicine, ed. by J.R. DiPalma, p. 305, McGraw-Hill, New York, 1971.