l M THE CABBERC EFFECTS OF COCAENE AND CERTAEN MTZEESTWERES: M3 RWBEFEESSPEHTS Tfimts §ozr Hm Dawn of M. 5. MICEEGMS Sum KIN-WEEKS!“ Roméd Site-1322mm Davis 3 97‘! n gum; lflzlllljlflfilll Lu: fll fl Mill" 1| 11qu Ill tun 1115-3" ABSTRACT THE CARDIAC EFFECTS OF COCAINE AND CERTAIN ANTIHISTAMINES AND ANTIDEPRESSANTS By Ronald Stephen Davis Cocaine and certain antihistamines and tricyclic antidepressants will potentiate the cardiovascular effects of norepinephrine. It is generally believed that at least part of the mechanism involved is a blockade of norepinephrine uptake. Recent evidence indicates that these drugs might have stimulating prOperties of their own as well and that this stimulation might be due to a release of norepinephrine from sympa- thetic nerve terminals. The present study was undertaken to further investigate this possibility. Chlorpheniramine, brompheniramine, tripelennamine, triprolidine, desipramine, imipramine, cocaine and tyramine were found to produce marked positive inotropic effects in isolated, Spontaneously beating guinea pig right atria. Promethazine, however, depressed the force of contraction. Propranolol was found to block the positive inotropic effects of these drugs. The reSponse was also absent in atria from animals treated with reserpine but could be restored by incubating these atria with nor- epinephrine. The increase in the force of contraction produced by tyramine and cocaine may have been due partially to an increase in rate. This wzs not true of the antihistamines and antidepressants as these drugs were found to decrease the spontaneous atrial rate. Ronald Stephen Davis Chlorpheniramine, tripelennamine, triprolidine, desipramine, cocaine and tyramine caused an increase in the efflux of H3-norepinephrine from isolated, perfused guinea pig hearts and from isolated right atria per- fused while in a tissue bath. In the latter tissue, this increase was associated with a positive inotropic effect for all drugs tested except desipramine. No increase in the efflux of Ola-urea was observed when these drugs were administered to atria previously labeled with the radio- isotope. Several of the drugs were tested for their ability to block H3-norepinephrine uptake at the doses which produced the greatest increase in force. The order of effectiveness from greatest to least was found to be desipramine, triprolidine, tripelennamine and promethazine. On the basis of these results, it was concluded that chlorphenir- amine, tripelennamine, triprolidine and cocaine exert their positive ino- trOpic effects via a tyramine-like mechanism, that is by releasing nor- epinephrine from sympathetic nerve endings. It is felt, however, that desipramine, at doses which are not depressant to the atria, is acting primarily by blocking the uptake of Spontaneously released norepinephrine. THE CARDIAC EFFECTS OF COCAINE AND CERTAIN ANTIHISTAMINES AND ANTIDEPRESSANTS By Ronald Stephen Davis A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pharmacology I971 ACKNOWLEDGMENTS Surely no thesis could ever be written without the assistance of many people. I am deeply indebted to all those who offered me their equipment, time and ideas during the course of this project. I eSpecially want to thank Dr. John H. McNeill for his interest, time, invaluable guid- ance and considerable patience. I wish to thank Dr. Theodore M. Brody, Dr. Kenneth E. Moore and Dr. Benedict R. Lucchesi for their time, assis- tance and evaluation in the preparation of this thesis. I would also like to thank the Michigan Heart Association for their financial support of the project. Finally, I wish to eXpress my utmost appreciation to my wife who aided me in countless ways in this work, gave her best and endured the worst. ii Chapter II III IV V TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES INTRODUCTION Norepinephrine as the Adrenergic Transmitter Release of Norepinephrine by Sympathomimetic Amines Other Drugs Which Alter the Norepinephrine Trans- mission Process METHODS Apparatus Preparation of Tissues Solutions and Drugs Calculations Reliability of Methods Statistical Methods RESULTS Positive InotrOpic Effect on Isolated Atria Effect of Reserpine and PrOpranolol Effect on Atrial Rate of Contraction Release of H3-norepinephrine in Isolated Hearts Release of H3-norepinephrine in Isolated Atria Effects on H3-norepinephrine Uptake Effects on Gin-urea Efflux DISCUSSION SUMMARY LIST OF REFERENCES iii Page iv :*H In IA 23 2A 27 28 29 3O 3O 39 1+6 '46 52 59 62 73 7h Table LIST OF TABLES Increase in force as a percent of maximum reSponse to norepinephrine: Comparison of chlorpheniramine, brom- pheniramine, tripelennamine, triprolidine, cocaine and tyramine Increase in force as a percent of maximum reSponse to norepinephrine: Comparison of imipramine and desi- pramine Drug-induced increases in H3-norepinephrine efflux in isolated atria Effect of drugs on uptake of H3-norepinephrine in iso- lated atria iv Page 31. 38 58 61 Figure 10 ll l2 l3 lh LIST OF FIGURES Possible sites of drug action at the adrenergic nerve ending Tissue bath for isolated atria Modified Langendorff heart perfusion apparatus Heart chamber Perfused tissue bath system Positive inotrOpic effects on isolated atria: Compari- son of tripelennamine, chlorpheniramine, bromphenir- amine, triprolidine and tyramine Positive inotrOpic effects on isolated atria: Compar- ison of desipramine, imipramine, cocaine and tyramine Effect of reserpine pretreatment and propranolol on the positive inotrOpic reSponse: Comparison of chlorphe- niramine, triprolidine, tripelennamine and tyramine Effect of prOpranolol on the reaponse to norepinephrine Effect of reserpine pretreatment and propranolol on the positive inotrOpic reSponse: Comparison of cocaine, imipramine and tyramine Effect of propranolol on the reSponse to norepinephrine Effect of norepinephrine incubation on drug reSponses in atria from reserpine pretreated guinea pigs Effect of drugs on atrial rate of contraction Effect of tyramine and tripelennamine on H3-norepine- phrine in isolated hearts Correlation between positive inotropic effect and increased H -norepinephrine efflux in isolated atria Effects of tripelennamine, chlorpheniramine, triprolidine and cocaine on Hj-norepinephrine and Clu-urea efflux in isolated atria Page 16 18 2o 22 32 36 Al A3 A5 #8 El 54 CHAPTER I INTRODUCTION Norepinephrine as the Adrenergic Transmitter Work during the early 1900's led to the development of the currently held view that certain drugs are capable of releasing nor- epinephrine from sympathetic nerve endings in the periphery, thereby mimicking the effects of electrical stimulation. The turn of the century marked the beginnings of the study of adrenergic neuro-transmission. Norepinephrine was first synthesized by Stolz in 1909 but received little attention at the time. During this same period, Elliot (1905) and Dixon and Hamill (1909) proposed the concept of chemical transmission at the synapse. It was thought that epinephrine might be the transmitter at the junction of sympathetic nerve endings and their effector organs. However, while studying the effects of various sympathomimetic drugs, Barger and Dale (I910) observed that not all the actions of nerve stimulation were mimicked by epinephrine as would be eXpected if this were indeed the transmitter. Then, in 1921, Cannon and Uridil in this country and Loewi in Germany provided the first experimental confirmation of the hypothesis that epinephrine was the transmitter by showing similarities between the actions of sympathetic nerve stimulation and administration of epineph- rine. It was subsequently demonstrated that extracts of adrenergic nerve fibers could produce many of the same effects as epinephrine both in mam- mals (Cannon and Lissak, i939) and in amphibians (Loewi, 1936; Lissak, 1939). That the adrenergic transmitter might be some substance other than epinephrine was not given much credence in view of the bulk of supportive evidence in favor of this amine. In retrOSpect, it can be seen how this misconception evolved. First, many of the effects of epinephrine and nor- epinephrine are quite similar, and early techniques might not have been able to distinguish between the two. For example, early assays lacked the sensitivity to detect the small amounts of norepinephrine present in rabbit adrenals (Euler, 1966). Species variation in the relative tissue content of the two amines resulted in a number of difficulties. In fact, epinephrine is now known to be the sympathetic transmitter in the frog heart (Euler, I966). Thus, extrapolation of this data to mammals was most unfortunate. With the exception of the early work of Barger and Dale (1910), significant challenges to the epinephrine hypothesis did not appear for many years. It was in 1933 that Cannon and Rosenblueth proposed their theory of two sympathins. Based on work which confirmed the findings of Barger and Dale (1910), they suggested that a common mediator was released from sympathetic nerve endings and combined with one of two substances at the receptor site. This combination resulted in the formation of either sympathin E (excitatory) or sympathin I (inhibitory). The following year, Bacq (I934) suggested that sympathin E might actually be norepinephrine while sympathin I could be epinephrine. However, Bacq (1935) later came to feel that sympathin E was instead a partially oxidized form of epineph- rine. Bacq's original view was supported by Stehle and Ellsworth (1937) who showed that the effects of sympathetic stimulation more closely resembled the actions of norepinephrine than they did epinephrine. Greer, at al. (1938) later modified Cannon and Rosenblueth's hypothesis by eliminating the intermediate transmitter substances. They postulated the release of either of two transmitters from sympathetic nerve endings. The excitatory transmitter, they felt, might be norepinephrine. The evidence implicating norepinephrine in the role of sympathetic transmitter was not well received due to the numerous eXperiments during this same period which tended to confirm the earlier hypothesis that epi- nephrine was in fact the transmitter. It was not until the late forties that Euler (l946a,b,c, I948, 1950) demonstrated that norepinephrine was the predominant catecholamine in peripheral adrenergic nerves and tissues. By more effectively purifying extracts of tissues and adrenergic fibers, he was able to show that the active substance diSplayed prOperties more closely resembling those of norepinephrine than epinephrine. In addition, he found that the norepinephrine present in these preparations was the Ievo-isomer and that amount was closely related to the density of adre- nergic fibers in the tissue or nerve. Since small quantities of epine- phrine were also detected in the extract, Euler did not rule out the possibility first suggested by Greer, at al, (I938) that there might be two transmitter substances. Bacq and Fischer (I947) confirmed Euler's findings that norepineph- rine was present in nerves and tissues. This amine was also detected in the urine by Holtz, et_al. (I947), in the liver by Gaddum and Goodwin (I947), and in the blood vessels of several Species by Schmiterléw (I948). This work was soon followed by direct evidence that norepinephrine was liberated on stimulation of postganglionic sympathetic nerves. Release of the amine was first shown by Peart (1949) who stimulated the Splenic nerve in the cat. Confirmation in other tissues and Species Was provided by West (1950, Mann and West (1950, 1951), Outschoorn (1952) and Outschoorn and Vogt (I952). Through the work of these and later inves- tigators, it has become generally accepted that norepinephrine is the only functionally important transmitter found in peripheral adrenergic terminals in mammals (Iversen, 1967). Release of Norepinephrine by Sympathomimetic Amines In their classical work in 1910, Barger and Dale showed that a number of amines structurally related to epinephrine and norepinephrine also possessed similar biological actions. The actions of two of these amines, tyramine and ephedrine, were later found to be antagonized by doses of cocaine which caused supersensitivity to the actions of epineph- rine (Fr6hlich and Loewi, I910; Tainter and Chang, 1927; Tainter, 1929). Thus originated the term ”cocaine paradox". Tyramine stimulates the heart as does epinephrine and cocaine lessens the action of tyramine, yet potentiates that of epinephrine. Similarly, it was noted by Burn and Tainter (I931) that when the nictitating membrane was denervated the actions of tyramine were abolished while those of epinephrine were greatly enhanced. In confirming these observations in the cat foreleg, Burn in 1932 suggested what now seems obvious--that these amines, rather than acting directly on the post synaptic receptor, might depend on the integ- rity of the adrenergic nerve ending for their effects. Several investigators in the l950's (Fleckenstein and Bass, I953; Fleckenstein and Burn, I953; Innes and Kosterlitz, I954; Fleckenstein and Stockle, I955) studied the actions of a large number of sympatho- mimetic amines and concluded that they could be classified into three groups: l) Direct-acting amines--those whose effects are potentiated by cocaine and denervation--e,g,, epinephrine, norepinephrine 2) Mixed-acting amines--those which are only partially affected by either procedure-~e,g,, ephedrine 3) Indirect-acting amines--those which are definitely less effective after denervation or cocaine--e,g,, tyramine Later, reserpine was also used to distinguish between these three classes. Carlsson, 3:.al- (1957) showed that tyramine activity was absent in the reserpine-pretreated cat. This was subsequently confirmed by Burn and Rand (I958, 1960) who also provided evidence for the possible mechanism when they demonstrated that the peripheral stores of norepi- nephrine were depleted in reserpine-treated animals and that infusion of norepinephrine temporarily restored the actions of indirect-acting amines. This resulted in their now generally accepted hypothesis that tyramine and other indirectly acting amines exert their sympathomimetic effect by releasing norepinephrine from adrenergic nerve terminals. The extensive substantiation of this work has been reviewed by Trendelenburg (1963) and Iversen (1967). Evidence that infused tyramine could cause an increase in the amount of norepinephrine in the effluent or venous outflow of an organ was first provided by Stjarne in 1961. In the same year, Burn and Burn showed that tyramine could diSplace radioactively labeled norepinephrine in the isolated cat atria. In order to diSplace norepinephrine from adrenergic neurons, it seems likely that sympathomimetic amines like tyramine must first gain access to the intraneural norepinephrine storage Site. It was postulated by Furchgott, 2: a1, (I963) that this process may be mediated by a cocaine-sensitive transfer mechanism, perhaps the same as that reSponsible for norepinephrine uptake. This was suggested by the fact that agents that block norepinephrine uptake also prevent the release of norepinephrine by tyramine (Swaine, I963). In addition, Trendelenburg (1962) has shown that tyramine can act as a potent inhibitor of norepinephrine uptake. Through the work of Axelrod, at al. (1962a), it was found that 014- tyramine is accumulated by the isolated rat heart. Since the tissue levels of tyramine were several times that of the normal endogenous norepinephrine content, only a fraction of which is available for release, it appeared that the uptake of tyramine was not accompanied by a stoichiometric release of norepinephrine. However, the hearts were removed and assayed immediately after infusion with Gin-tyramine. It is now felt that much of the tyramine initially taken up is non-Specifically bound (Lee, 3: al., 1967; McNeill and Brody, 1968), and, therefore, the tyramine levels mea- sured by Axelrod, 23.21' (I962a) may have little meaning in terms of elu- cidating norepinephrine release mechanism. In fact, Schfimann and Wegmann (1960) and Schumann and Philippu (1962) have shown that the tyramine-induced release of norepinephrine from isolated adrenal medullary storage granules is accompanied by a stoichiometric uptake of tyramine into the granule. The spontaneous release of norepinephrine from such granules as well as the release produced by reserpine is associated with a release of ATP in the molar ratio of 4 NE/ATP. 0n the other hand tyramine, along with ephedrine, amphetamine, phenylethylamine and methamphetamine, produces a release of norepinephrine without an equivalent release of ATP. This suggests that these amines may be acting simply by taking the place of norepinephrine molecules in their storage Sites. ‘3 Other Drugs Which Alter the Norepinephrine Transmission Process Shown in Figure l is a schematic representation of an adrenergic nerve ending and its receptor organ. Direct-acting amines, like epineph- rine and norepinephrine, exert their effects primarily by a direct action on the receptor, whereas indirect-acting amines, such as tyramine, pre- sumably release norepinephrine from the nerve terminal. While causing neither a direct nor an indirect stimulation of receptors, a number of compounds have been shown to potentiate the effects of norepinephrine by interfering with its deactivation or removal from the receptor site. Theoretically, this could be accomplished by inhibit- ing the enzymes which metabolize norepinephrine: catechol-O-methyltrans- ferase (COMT) and monoamine oxidase (MAO). However, it is now considered that the main method of inactivation of norepinephrine in the heart is by uptake of the amine and its subsequent storage in the adrenergic nerve endings. The importance of uptake in terminating the actions of norepinephrine can eXplain why the very small amount of norepinephrine release by tyr- amine and other indirect-acting amines have such potent effects. Since most of these amines block norepinephrine uptake, they will potentiate the effects of any norepinephrine released by interfering with the prime mechanism of norepinephrine inactivation. Among the drugs which have been shown to block uptake are cocaine, tyramine, guanethidine, bretylium, phenoxybenzamine, phentolamine and the tricyclic antidepressants, imipramine and desipramine (Iversen, I967). Certain members of another group of compounds, the antihistamines, have been known for more than twenty years to potentiate the cardiovas- cular effects of epinephrine and norepinephrine (Parrot, 1943; Yonkman, Figure 1. Possible sites of drug action at the adrenergic nerve ending. Norepinephrine (NE) exerts its effects by interacting directly with the receptor. The primary means of inactivation is by uptake into the neuron, although monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT) may participate to some degree. Drugs which alter these processes will modify the norepinephrine reSponse. The response may be enhanced by drugs which block neuronal uptake of norepinephrine, or it may be mimicked by drugs which release the amine. Depleting norepinephrine by releasing it intra- neuronally or by blocking its synthesis will deminish the reSponse to sympathetic stimulation or norepinephrine-releasing drugs. The response may also be blocked by preventing the release of norepineph- rine or by preventing interaction of the amine with the receptor. INTRANEURONAL /, .. RELEASE I N E ‘ ._ \ / 1--.:— \_ / /" I}E GRANULAR UPTAKE EXTRANEURONAL RELEASE ( N E: \\. // i1‘ Ey-E/ NEURONAL UPTAKE ACTION ON RECEPTOR C O M 'I' RECEPTOR Figure l 1O g £., 1946;. Sherrod, e_t a” 1947; Tislow, .62 31., 1949; Buchholz, e_t_ 21:: I951; Paasonen, I953; Kuriaki and Uchida, I955; Paasonen, et_al,, I956; Innes, I958; Altura and Zweifach, 1965). More recent work by several investigators indicates that the mechan- ism of this potentiation is by inhibition of norepinephrine uptake. Isaac and Goth (l965a,b, I967) showed that tripelennamine, chlorpheniramine, di- phenhydramine and phenindamine significantly prevented the uptake of H3- norepinephrine in the isolated rat atria and the in_viyo_rat heart. The degree to which this occurred was approximately the same as that found for cocaine. With the exception of phenindamine, these drugs also potentiated the effects of norepinephrine on blood pressure and heart rate. Pyrilamine and promethazine appeared to have no effect either on H3-norepinephrine up- take or the cardiovascular responses to norepinephrine. However, McNeill and Brody (I966) observed that promethazine could potentiate the norepineph- rine-induced activation of rat cardiac phOSphorylase though the degree was less than that obtained with chlorpheniramine, tripelennamine or phenindr- amine. They initially surmised that Isaac and Goth had failed to notice the activity of promethazine because assessing norepinephrine potentiation by measuring the effects of the drug on the rate reSponse might be a less sensi- tive method than measuring phosphorylase activation. This hypothesis was later modified when promethazine was found to have no significant effect on either norepinephrine (McNeill and Brody, 1968) or tyramine uptake (McNeill and Brody, I968; Commarato, at al., l969a); it was suggested that the actions of promethazine might be Specific for the enzyme system. A recent report (Muschek and McNeill, 1971) indicating that promethazine can inhibit phos- phodiesterase lends support to this suggestion. In an early report demonstrating the epinephrine-potentiating ll prOperties of the antihistamines Buchholz, et_al. (l95l) noted that tripelennamine could abolish the bovine carotid constrictor reSponse to tyramine. Later, investigators (Johnson, et_al3, l965; Johnson and Kahn, l966) reported that chlorpheniramine and tripelennamine inhibited the cardiovascular stimulating effects of tyramine and bretylium in open-chest dogs much the same as did cocaine, while triprolidine was without effect. Inhibition of the effects of tyramine and ephedrine by pyrilamine and chlorpheniramine have been observed in the isolated, Spontaneously beat- ing rabbit atria by Osterberg and Koppanyi (1969). They, along with Johnson and Kahn (1966) and later, McNeill and Brody (l966, l968), sup- ported the conclusions of Isaac and Goth, that those antihistamines which enhance the effects of norepinephrine do so by blocking the uptake of the amine. Such drugs will decrease the effect of tyramine by the Ssme mechanism, that is, by blocking amine uptake (Commarato, §t_al., l969a, b; McNeill and Commarato, l969). Similar effects on amine uptake have also been reported for certain tricyclic antidepreSSant compounds: imipramine and its derivative, desipramine (Axelrod, et_al., l96l; Dengler, et_al , 1961; Hertting, et al., 1961; Axelrod, et al., l962b; (D Titus and Spiegel, 1962; Carlsson, et_al., 1963; Shore, _t al., l96h; Brodie, §t_al.,l965; Iversen, l965; Titus, §£.§£:: l966; Brodie, at al., l968; McNeill and Brody, l968; Stjarne, et al., l968; Commarato, et al., l969a,b) and amitryptyline and its derivative, protryptyline (Carlsson and Waldeck, 1965; McNeill and Brody, 1968; Commarato, et al., l969a; McNeill and Commarato, l969). Since most norepinephrine-releasing drugs, such as tyramine, are known to prevent norepinephrine uptake as well (Iversen, 1967), it is possible that chlorpheniramine and the other antihistamines which block 12 uptake may also cause the release of norepinephrine. It was shown by McNeill and Brody (1966) that chlorpheniramine in addition to potentiating the effects of norepinephrine on cardiac phosphorylase, caused a slight increase in the amounts of the active form of this enzyme present. Similar results were noted with tripelennamine (McNeill and Brody, 1969). These effects could have been due to a release of endogenous norepinephrine since the effect was blocked by prOpranolol. On the other hand, the effect could also have been due to blockade of uptake if sympathetic discharge was causing the release of norepinephrine. None of the early investigators who studied the catecholamine- potentiating prOperties of the antihistamines reported any tyramine-like effects when the antihistamines were given alone. However, in pictures of tracings used by Buchholz, et a1. (1951) to describe the inhibition of the tyramine response, it appeared that the antihistamine synopen, which structurally resembles chlorpheniramine, also caused dose-related contractions of bovine isolated carotid strips. Other drugs which block uptake may also release norepinephrine. Furchgott, et_al, (1963) and Cervoni, et_al. (1966) have reported small to moderate positive inotropic reSponses to cocaine in the isolated guinea pig left atrium. However, the electrical stimulation used to drive the atria could have released norepinephrine. If so, its effect would have been potentiated by cocaine, due to the ability of this drug to block amine uptake. Recently, however, Trendelenburg (1968) reported positive chronotropic effects of cocaine in the isolated guinea pig right atrium due both to a release of endogenous norepinephrine and to an increase in sensitivity to this amine. Vohra (1969) confirmed the apparent ability of cocaine to release norepinephrine but stated that the amine 13 must have other actions as well. The ability to release norepinephrine from sympathetic nerve endings in the heart has also been described for desipramine. The initial work was that of Titus, et_al, (1966) who used rather high concentrations of the drug (2.5-4.1 x 10414) in the isolated rabbit heart. Nash, _e_t g. (1968) have shown that 10-6M desipramine caused an increase in the amount of radioactivity in the perfusate effluent of isolated rat hearts treated with H3-norepinephrine, whereas cocaine did not produce such an increase. A recent more SOphisticated study by Leitz and Stefano (1970) indicates that the norepinephrine released by desipramine in rat ventricle slices is primarily in the form of deaminated catechol compounds. This suggests that desipramine in some way causes the diSplacement of norepinephrine from the storage granules into the neuronal cytoplasm. On the basis of the work which has been cited, it appeared that chlorpheniramine and tripelennamine might be exerting their effects on cardiac phOSphorylase (McNeill and Brody, 1966, 1969) through a release of norepinephrine. The purpose of this study was to determine whether chlorpheniramine and other drugs known to block uptake could be causing such a release. The drugs studied were chlorpheniramine, brompheniramine, tripelennamine, triprolidine, promethazine and imipramine. As a means of comparison to previous work, 1-norepinephrine, tyramine, cocaine and desipramine were also used. CHAPTER II METHODS In these eXperiments, three different types of preparations were used: 1) a tissue bath for isolated atria, 2) a perfused whole heart system, and 3) a perfused tissue bath containing an isolated atria. Each of these will be described individually followed by a discussion of the methods used for preparation of the tissues, drugs and solutions. Apparatus A 180 milliliter (ml) tissue bath (Figure 2) was used for the initial experiments with isolated atria. Water was circulated through the outer jacket to maintain the temperature of solutions in the bath at 370C. A mixture of oxygen (95%) and carbon dioxide (5%) was bubbled through the solutions by means of a fritted glass disk at the bottom of the bath. A small (7 mm x 15 mm) Palmer heart clip or a curved suture needle attached to an L-shaped glass support rod was connected to the apex of the atria. The other end of the tissue was affixed with a similar clip or needle which in turn was connected with a fine silk thread to 2 Grass force diSplacement transducer (Model FTO3C). A Grass model 7 poly- graph was used in these and all other eXperiments for recording contrac- tions. Tension on the atria was adjusted by means of a Harvard isometric tension clamp. The isolated heart system (Figure 3) was essentially that described by Fallen, et_alf (1967). It consists of a modified Langendorff apparatus is 15 Figure 2. Tissue bath for isolated atria. A glass 180 ml tissue bath was used for determining the positive inotropic effects of drugs on isolated atria. Contractions were recorded by suSpending the atria between one clip attached to the glass support rod and another attached to the force transducer. Tension was adjusted by an isometric tension clamp. Heated water circulated through the outer jacket of the bath was used to maintain solutions and atria at 370C. Oxygenation was provided by passing a mixture of 95% oxygen and 5% carbon dioxide through a fritted glass disc at the base of the bath. 1 -- auntTMC «um»: “All" 4 ‘I r -“ 1 Inc: Tum-sou: . ‘ ' - _ ' ’ l7 .psasaaaa mammsmaom map mcflpomaaoo Mom pom: mm: pawoa map npwocmn amassm < .stao qoflmdmp oflaposomfi cw Eva: UmpmSmow mos :oflmdoB .Uopaoooa ohms Pawoz 639 Mo m:0flpomapsoo «mm: wasp QH .mhmaasm mo aflmm m afi> hoodomqwap ooaow map Op oopoocqoo was moaoflapco> map mo xomw ogp OP pogowppw msflapm < .pamog may mo madpwaomsop may campcflms Op oomaofl Omaw A©o>oan mafia ouo 39H? gzogmv aonsmgo mmwamfixoam < .oowm am on oasoz pamon may mafiaopcm mQOHPSHom asap ooadmmm mcflpsp wo nmeQH HmcflM map pudoam poxomm amps? < .mpwa 30am pumpmnoo w as pawon may opcfl pomadm oaoB maflo>aomoa 039 map go ono anm mcoHPSHOm powwog paw dopwqommxo .mpawon oaonz ompwaomfl pow pom: was Eopmmm mHQB .mdpmawmmm coamsmaom pawon wwaoosomqwq ooflwflooz .m madmflm 18 ‘ 1 a moaongt. momOu m assmam 19 Figure A. Heart chamber. This is a close-up view of the plexiglass chamber around the heart. The front side has been removed. Perfusion of the heart is retrograde via the aorta which has been secured to the tip of the glass cannula. A string from the force transducer is affixed to the apex of the ventricles with a small Palmer heart clip. 20 A TO FORCE TRANSDUCER PLCEMXIGLASS ‘ M ”In: {— .aiii 21 Figure 5. Perfused tissue bath system. This system was used for similtaneous recording of con- tractile activity and release of radiolabeled compounds. The appara- tus is identical to that shown in Figure 2 except that this bath has a smaller volumn (15 m1) and is perfused and drained at a constant rate. Oxygenated heated solutions were pumped into the bath through the glass cannula at a rate of 5 ml/min. A compression screwclamp was used to adjust the drainage from the bath to the same rate. 22 WLESS SUPPORT R05 v 'I‘ A V ‘7 \ ‘ Exf‘l 3‘ . . r 1; '41/r41 r1] Figure 5 23 in which the retrograde perfusion of the heart via the aorta was maintained at a constant flow rather than at a constant pressure. This was achieved by use of a Sigmamotor peristaltic pump. Oxygenated solutions were pumped into the heart from one of the two reservoirs. The water jacket surround- ing the final section of tubing and the plexiglass chamber (Figures 3 and A) helped to maintain the heart and solutions at 370C. A Grass force diSplacement transducer was connected to the heart via two small pulleys so that contractions could be monitored. A funnel underneath the plexi- glass chamber was used for collection of the perfusate effluent. The perfused tissue bath system (Figure 5) was designed for the isolated atria experiments in which radioisotopes were used. It differed from the tissue bath previously described only in that it had a smaller volume (15 ml) and was continuously perfused and drained at a constant rate (5 ml/min). Preparation of Tissues Guinea pigs ranging in weight from MOO to 700 grams were used in all eXperiments. They were pretreated with heparin (5OO USP units/kg, subcutaneously) at one to two hours prior to sacrifice. The animals were sacrificed by a blow on the head, and the chest cavity was rapidly Opened via an incision along the midline of the sternum. In order to wash the blood out of the heart, the vena cava was cut to eliminate venous return, and 1 ml of heparinized saline at 3700 was injected into the left ventricle. In those experiments using atria, the right atrial appendage and Parts of the right ventricle were dissected free from the heart and Placed in a tissue bath containing a modified Locke-Ringer solution (see 2h under Solutions and Drugs). Once in the bath any pieces of ventricle or connective tissue were trimmed away. The atrium was then prepared for recording of contractile activity by suSpending it vertically between two small clips or needles as previously described. Using a Harvard isometric tension clamp, tension on the atrium was adjusted to three- fourths of that which produced the maximum force. This was generally in the range of 0.75 to 1.25 grams. Once this was achieved, the atrium was washed several times by draining and quickly refilling the bath with fresh solution. With the perfused bath technique, perfusion was initiated following the washings. Prior to the addition of any drugs, the atria were allowed to stabilize for periods of thirty to ninety minutes. The preparations were deemed stable when there was no measurable change in the force of contraction for twenty minutes and when the Spontaneous rate was con- stant within ten beats per minute over a fifteen minute period. For the isolated whole heart preparations, the tissue was first dissected free from all blood vessels and connective tissue attachments. The heart was then quickly removed from the animal and the aorta secured to the perfusion cannula while fluid was being pumped through it at 5 ml/min. This resulted in a strong, viable preparation free of blood Clots. A clip or needle was attached to the apical portion of the ven- tricles for recording contractions, and the plexiglass chamber was placed around the heart. The stabilization criteria used were the same as those described for the isolated atria. §Qlutions and Drugs A modified Locke-Ringer solution described by Chenoweth and Koelle CL9A6) was used exclusively in all eXperimentS. The buffer was prepared 25 by dissolving the following amounts of reagents in one liter of distilled water (all weights are eXpressed as grams of the anhydrous compound): Glucose, 1.9; NaCl, 7.0; KCl, 0.h2; CaCl2, 0.29; MgC12, 0.20. A pH of '7.4 was obtained by adding NaHCO3 (2.0 g/liter) and bubbling a mixture of 95% oxygen and 5% carbon dioxide through the solution. All solutions in contact with cardiac tissue were maintained at 370C. The drugs used in this study were chlorpheniramine maleate (Mann Research Laboratories), brompheniramine maleate (A. H. Robins Co., Inc.), tripelennamine hydrochloride (Ciba Pharmaceutical Co.), triprolidine hydrochloride (Burroughs Wellcome & Co.), promethazine hydrochloride (Wyeth Laboratories, Inc.), imipramine and desipramine hydrochloride (Geigy Pharmaceuticals), cocaine hydrochloride (Merck & Co.), tyramine hydrochloride (Calbiochem), l-norepinephrine bitrartrate (Winthrop Laboratories), propranolol hydrochloride (Ayerst Laboratories, Inc.) and reserpine (Aldrich Chemical Co.). In the isolated atria eXperimentS, concentrated solutions of these drugs were pipetted into the tissue bath in amounts required to achieve the desired molar concentration. The cumulative method of determining dose reSponse curves was used. When the maximum response for a parti- cular dose of a drug was reached, sufficient drug was added to achieve the next highest molar concentration of that drug. In some experiments, prOpranolol (10'7M) was added to the bath twenty minutes prior to drug administration. Reserpine pretreatment consisted of a dose of 3 mg/kg intraperitoneally forty-eight hours prior to the experiment followed by 2 mg/kg twenty-four hours later. In the case of isolated whole hearts, drugs dissolved in Chenoweth- Koelle solution were added to one of the reservoirs and pumped into the 26 heart. A combination of the above two methods was utilized for the perfused tissue bath technique. When a drug was added to the tissue bath (by the method previously described), it was also added to the perfusate in the same concentration. Therefore, the bath could be instan- taneously brought up to a particular drug concentration, and this concen- tration would be maintained even though the bath was being continuously perfused. For experiments involving the use of radioisotOpes, dl-norepine- :phrine-Y-H3 (Specific activity, 5-13 curies/millimole) Efld urea-C11+ (Specific activity, h.7 millicuries/millimole) were obtained from New IEngland Nuclear Corp. The isolated hearts were perfused (at 5 ml/min) ‘with #0 ml of Chenoweth-Koelle solution containing 3 x lO‘aM (0.l5-0.39 Inicrocuries/ml) H3-norepinephrine. Ascorbic acid (0.1 mg/ml) was added as an antioxidant when H3-norepinephrine was used. The hearts were then 'washed for a period of ninety minutes with buffer prior to any drug treatments. The 15 ml perfused bath was employed for the radioisotope eXperi- Inents with isolated atria. In order to detect any release of radiolabeled c20mpounds, solutions were pumped into and drained from the bath at a con- Eitant rate. This technique was somewhat similar to superfusion of a 'tissue except that the atria remained immersed in a constant volume (l5 IIll) of solution. Atria were incubated in the bath in 3 x l0‘7M (1.5-3.9 Inicrocuries/ml) H3-norepinephrine for thirty minutes during which time ‘there was no perfusion. At the end of this period, the tissue was V"aid-shed several times and the bath refilled with Chenoweth-Koelle solution. P¥3rfusion was started and continued for ninety minutes (at a rate of 5 27 ml/min) before any drugs were given. Incubations in Clu-urea at a concentration of 6 x l0'4M (3 microcuries/ml) were carried out in the same way. The washout curves plotted for the radioisotOpe experiments were obtained by collecting samples of the perfusate effluent over a one minute period. One ml aliquots of these samples plus 1 ml of distilled water were pipetted into l0 ml of a modified Bray's solution (l00 g naphthalene plus 6 g of PPO in 1 liter of dioxane). The samples were stored overnight to reduce autoluminescence and then counted in a Beckman liquid scintillation counter (Model LSlOO). For determinations of H3-norepinephrine uptake, atria were incu- bated as described above. Following this, they were washed with 30 ml of fresh solution at fifteen minute intervals for forty-five minutes. At this time they were removed from the bath, blotted and weighed. They were then homogenized in 5 ml of 0.4N perchloric acid. A l ml aliquot of the homogenate plus l ml of water were added to l0 ml of Bray's solution and counted for radioactivity. diculations The reSponse of isolated atria to the various drugs is expressed £38 a percent of their maximal reSponse to norepinephrine. This was onm was Ewnoa wo msoflpwapcooqoo mayo an coosooam mooooaoeH .mpeoeflhomxo xflm pmwoa pm pom aoaao osmocmpm cum some one mason noamoa 05Hw> zoom .momoo mdowam> pm mmsao 0:9 uo comm pom co>flm ma oqflagmocfimoaou flpflz docfloppo ommoaoqfl Edeflxme one wo pqooaom m an commoamxo coapomapooo a0 ooaoy SH omwoaoqfl one .oQflEsazp pew oeflmooo «oqfiwflaoamwap «quEeroHomflsp «oGfiEmaHooQQEOHQ noQHEwaflsogmaoano a0 nomflameoo ”oqfiazmoqflmoao: OP omqomnos Edeflxms ao Pcooaom m mm ooaow ca omsoaoeH .H manta s-oaxm.a a.mn o.mn m.as o.mm 34 muoaxn.m m.¢Hm.Om m.mwm.mm o.mHo.oa o.mHo.mm JIOH m-oaxm.m n-0axo.a.w-oaxm eucaxe .mioaxm Ammoa m.mw o.na m.nn o.ma s.aa o.an m.ss m.sm s.sm m.oa m.@ 0.0 o.sHm.oa w.num.sm o.mHa.mm s.aum.oa m.nww.mm m.mua.ma m.+us.aa m.awa.s s s.mns.mm o.mam.ma o.mam.ma m.aum.s o m.mnm.aa m.awa.ma m.aam.m m.oam.s o m.eas.mm w.mum.mm s.mao.ma m.aHs.e o .mumwmw Iwnmmw. o-oaxm .mwmmw. _m apom :H msam mo AEV soapsapnoonoo osaaemoeamosoz bosomnom sesame: so aeooaom a no bosom ea ooooaoeH H mapmfi .E .o .m.H Pcooaom w mm omwoaoeHw oaHEwaha ocfloooo ocaoaaosaaaa oQflEmceoHomHaB oqfiemaficoQQanm oQHEmchoflmaoago msam 35 Figure 7. Positive inotrOpic effects on isolated atria: Comparison of desipramine, imipramine, cocaine and tyramine. The increase in force of contraction eXpressed as a per- cent of the maximum increase obtained with norepinephrine is plotted against the concentration (M) of drug in the bath. Drugs at the following concentrations produced increases that ere significantly greater than con rol (P<10.0l): desipramine, l0" 1 and above; imi- pramine, 3 x 10" A and above; cocaine, l0”7M and above; tyramine, 10' M and above. The bars above or below each point denote the standard error of the mean. INCREASE IN FORCE AS % OF MAXIMUM NE RESPONSE 40 30 10 36 f '\ Lo, .\ IYRAMINE .\. \ j ‘1 ossunnumt , \ I I / ,..-- / ya” I IMIPRQINE 3x100 10-7 3x104 10* 3x10 10‘ 3x10 10 CONC. OF DRUG (M) Figure 7 37 .Aao.ouvmv Hoapcoo doze aopmoam hapcwoflgflqmflm who? o>onm pew Ewnoa x m %o mQOHPmapdoodoo mowewamflefl pew o>onm paw Ewnoa wo meowpmapcooqoo ocfiaw namfimow mp doospoam momooaoQH .mpcoEHMoQXo o>H% pmmoa pm how aoaao pawodwpm dew some one menom loamoa odaw> zoom .momoo msoflam> Po mmdaw esp mo apop aoa so>fim ma onwanmoefimoaoe SPHS oodflmppo omwoaoefi EZEHXmE may go pnooaom w on oommohmxo dofipowapqoo %o ochom QH omwoaodfl ona .oQHEwamHmoo dew oQHEwamHEfl wo somflammEoo ”ooflagmosflmoaoc op omqommoa Edefixme wo Peooaom o no ooaom QH mommaocH .N oHQmB 38 o.mam.ma muoa .E o.mnw.mm w.sHm.cm s.sum.sm c.¢aa.om m.awm.aa H.mum.mm m.mws.am w.aam.ma s.aum.oa e.aHm.m c-0axm c-oa s-oasm s-oa w-oaxm npmm CH mdam wo AZV coflpmapeooqoo .o .n.H pcooaom mm omwoaoQHw o.aua.m m om.aam.m m w: OH m. onwanmoeflmoaoz op omqommom Edeflxmz wo Pcooaom w mm m oases moaom QH ommoaoQH oQHEwamHmoQ oGHEmamfieH mdam 39 The periods of drug administration utilized to obtain the cumula- tive dose-reSponse curves for the antihistamines, cocaine and tyramine were under twenty minutes, there being two to five minutes between doses. When the period between doses was greater than fifteen minutes, drug concentrations of l0-6M and above produced no positive inotropic effects and in most cases were depressant to the force of contraction. Periods of five to fifteen minutes were required for the antidepressants to achieve their maximum effect; in addition, they were less depressant than the aforementioned drugs. Effect of Reserpine and Propranolol Representative tracings of maximal responses to several of the drugs in the normal atrium are shown in Figures 8 and 9. Also shown are the effects of pretreatment with reserpine, which depletes norepineph- rine from the nerve endings, and prOpranolol, which blocks beta adrenergic receptors. As in the case of tyramine, the positive inotropic effects of chlorpheniramine, triprolidine, tripelennamine (Figure 8), cocaine and imipramine (Figure 9) were all abolished by pretreatment with reserpine (3 mg/kg at forty-eight hours before sacrifice; 2 mg/kg twenty-four hours later) or blocked by prOpranolol (lO'7M). Though sample tracings are not shown, the same results were obtained with desipramine and brompheniramine. The results were confirmed in three out of three experiments for each drug. The effectiveness of l0'7M propranolol in blocking the actions of the same dose of norepinephrine in this preparation is illustrated at the bottom of both Figure 8 and Figure 9. The maximum norepinephrine reSponse was not blocked by this concentration of propranolol. When the atria from reserpine pretreated animals were incubated in 5 mg/ml norepinephrine (approximately 3 x lO'5M), the positive inotropic A0 .Hoao:0amoam 9o :OH90a9:0o:oo 09:9 a: 00:0oa: 9o: 003 00:09009 0:9::m0:9m0:o: Edaflx0e 0:3 .Amzv 0:9: u:m0:9m0ao: mo 0000 0500 0:9 m:9:ooap :9 9090:0amoam Ewuoa mo 000:0>99o09m0 0:9 09 EO99o: 0:9 90 :30:m .9090:0:moag mo 0900990 0:9 «9:m9: 0:9 :0 0:0 «9:0E900a90am 0:9ma0m0a 9o 0900990 0:9 0:0 909:00 0:9 :H .mmda0 0:9 09 000:0900: 9089x0a 9o 00:90099 0>9909:000:m0a 0:0 :ESHOU 0:0: 9909 0:9 :H .:OH909909:HE00 w5a0 9o 09:9om 0:9 09o:00 msoaaw 0:9 0:0 «00:90099 :00390: 00mm090 0899 0:9 090090:9 090:0099 0:8 .Edfis90 0500 0:9 so: 00:om00: 00900a9 0 0:0 90:9:00 0 09:0009m0: 0m:900a9 Mo 9909 :00m .0:Ha:m0:9m0:o: 09 00:0m00a 0:9 :0 Hoao:0amoam mo 90099m .0:flE0am9 0:0 0:wE0::090m9:9 «0:90HHopmfla9 N0:080:9:0:mao9:o 9o :om9a0mEoo ”00:om00a 09moa9o:9 0>99900m 0:9 :o Hoao:0amoam 0:0 9:08900a909m 0:9m9000a Mo 900mmm .m 0a3w9m Ml m 0adwflm 1.....52 17¢..— 25%|...— 2. 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OnoJ 4..F 72:2. . __.._. 2222.....2_._:.._..2.:.22 o _ 80$ 1...... .. 2:22... 22.12... 2...; 2531302 3.... 02:23... .5552 46 reSponses to tyramine, chlorpheniramine, brompheniramine, tripelennamine, triprolidine, cocaine, desipramine, and imipramine were at least partially restored (n = 2). Representative tracings for several of the drugs are shown in Figure 10. In the left hand column are typical reSponses for normal atria; in the center, reSponses obtained with atria from reserpine pretreated guinea pigs; and on the right, reSponses in those same atria after incubation with norepinephrine. Effect on Atrial Rate of Contraction The increase in force of contraction produced by the antihistamines and antidepressants was not associated with any increase in rate. Cocaine and tyramine produced significant but highly variable increases in rate (10 to 80% above control at a concentration of 3 x lO'SM) concurrent with their positive inotropic effects. This variation appeared to be due primarily to differences in initial rate. However, in the experiments in which drug concentrations were maintained for fifteen minutes prior to the addition of the next highest dose, cocaine and the antihistamines produced a dose- and time-dependent depression of Spontaneous rate (Figure ll). The results obtained with cocaine were in agreement with those of Trendelenburg (l968). Release of H3-norepinephrine in Isolated Hearts Cocaine and the antihistamines and antidepressants under study were tested for their ability to release H3-norepinephrine from isolated perfused hearts which had been incubated with the radioisotOpe at a concentration of 3 x lO'SM for eight minutes. Following a washout period of ninety minutes, the drugs were administered in that concentration which produced the maximum positive isotropic effect in the atria. l+7 Figure ll. Effect of drugs on atrial rate of contraction. In the upper graph, the decrease from the control rate (in beats per minute) is plotted against the time (in minutes) after drug administration. The curves at the various concen rations represent the averzges for chlorpheniramine, tripelennamine and triprolidine (n = 2 for each drug). Stantard errors of the mean are denoted by the bars above or below ea " t‘ p , ”he decrease from the control rate in beats per ninu against he concentration (M) ' t t in the bath. The d creas ‘ . n o I I 1 I‘ g . ‘x 0 v 3‘! ‘9 l‘ ) "~ ““ ~ ‘3 1 ‘f§“*‘ “ y W .‘1‘ w'\n“1\.‘1.‘|\w .1- \K- L-.L hxu....LLL2D\-.‘1L2Q-1 \_I L25 .6. b2\.u.2 ,. ‘ c g . \ ~ - n o y. \‘\\‘ \‘u \'\-0 ‘ Q— .\ (a v_3~'i(: v; 3 ‘5‘; ‘ \ I‘bf‘.‘ 3 ,‘Qd vi 4-'1 fi'fi ffi‘p‘yx. fly 4 ysfi ‘ ‘— -y‘ -L . -L.\L..tb tue .1\'.-..5J.. L1;\.-.8.b€b . cal-.. ‘nl‘.-. L--l--.--t_1-- 3-22-9, p-1- .- .- .‘ \~ \‘\‘~\\.‘~‘V‘ V‘ ) (‘\\‘ +‘IV‘w‘j‘ \1. : “‘31“: F1‘ \vw~a“y‘r~—v1.y-\ ‘IQ-T‘r Cg'v“ed .. r“. Wagon. »-‘ k \' _'._L .L.-.‘~........L 42.1 L... .L\.. L2JLJ2--\. o g..-» L.--C~._.ine _L -'- u¢b C. LED-.. L221: .2 - ‘ - u u f \ \‘xo—“ 3\..‘ ‘\ ‘*‘ __3 [‘1-‘ '1 +1- \L b\\..\h-.igstb \.-\...\-\,§\.¢k.L2\a CALK.L\.L\.‘\O DECREASE FROM CONTROL RATE (Beats/Min.) DECREASE FROM CONTROL RATE (Beats/Min.) —4o -60 -20 -40 A8 a\~~§:“""”5‘ ““““ 5 ”'7“ \\ 5 310-6M ______ ____g_______—310-5M \ \110‘4M O 2 5 10 IS MINUTES AFTER DRUG ADMINISTRATION \\ Cocaine n=2 ........... I 3 \\\\I Antihistamine n=6 Promethazine ”:2 a". l o 10-7 10-6 10-5 10-4 CONC. (M) 0F DRUG IN BATH Figure ll 49 Results typical of those obtained with 3 x lO'5M tyramine and tripelenn- amine are shown in Figure l2. The peak increase in the efflux of the radioactive component was almost five times greater for tyramine than :for tripelennamine. Chlorpheniramine, triprolidine and cocaine caused an.elevation in radioactivity similar to that of tripelennamine, but a Inarked degree of variation for each of the drugs precluded precise analysis and quantitation. The lepe of the increased efflux seen with tyramine, cocaine and the antihistamines was quite steep. In contrast, the gradual rise observed with desipramine took as long as twenty minutes to reach a peak that was about half the tripelennamine peak. Other drugs were not tested in this system. The isolated whole heart appeared much more sensitive than the isolated atrium to the depressant effects of these drugs. In fact, no positive inotropic effects were observed with any of the drugs, and it was necessary to limit their perfusion time to four minutes to avoid severe myocardial depression. Chlorpheniramine was the most potent drug in this reapect. The failure to note stimulation with a drug such as tyramine wzs undoubtedly due in part to the method of measurement. The lack of a single axis of muscle fiber alignment in the heart makes a force trans- ducer connected to the apex of the ventricles an inadequate index of the force of contraction. Due to the inability to correlate release with increased force, the possibility of myocardial depression causing the release of H3-nor- epinephrine and the apparent failure to demonstrate maximum H3-norepin- ephrine releasing potential in the isolated heart, the search for a more suitable system was undertaken. 50 Figure l2: Effect of tyramine and tripelennamine on H3-norepinephrine efflux in isolated hearts. The amount of radioactivity in disintegrations per minute (DPM) per minute of effluent flow is plotted against the time in minutes after the end of H3-norepinephrine (Hj-NE) infusion. The heavy bar on the abscissa denotes the period during which the drug was admin- istered. The beginning of this period was taken as the zero level of radioactivity. DPM x 103/min. 80 60 4o- 20- 51 Tyramine 3 x To-5M Tripelennamine 3 x 10‘5M 20 4o 60 80 MINUTES AFTER H3-NE INFUSION Figure 12 Téo 52 Release of H3-norepinephrine in Isolated Atria The perfused tissue bath (Figure 5) offered the advantages of a system which utilized the tissue initially studied--the isolated atria-- as well as one in which any change in the efflux of radioactivity could be easily and accurately assessed. The administration of tripelennamine (3 x lO-SM) to isolated Spontaneously beating atria previously incubated in H3-norepinephrine (3 x lO'7M) produced a sharp rise in the amount of radioactivity in the perfusate effluent. This was accompanied by an increased force of con- traction characteristic of that seen in the initial series of eXperiments (Figure 13). Chlorpheniramine and triprolidine (3 x lO-SM) produced a similar increase in the efflux of tritiated compound (Figure lb, Table 3) which again correlated with the positive inotropic effect. These three drugs were significantly less effective (P<:0.0S) than tyramine (3 x lO‘5M). Desipramine was found to produce only a slight, gradual change in the efflux of H3-norepinephrine (Table 3) similar to that seen in the isolated heart. However, the marked inotrOpic effect previously observed with this compound was absent and, unexplainably, could no longer be demonstrated even using the original techniques. Cocaine appeared to be only about half as effective as the antihistamines in diSplacing H3-norepinephrine, although this difference was not statistically significant (Figure lb, Table 3). Effects on H3-norepinephrine gptake Since desipramine did not appear to be releasing H3-norepinephrine in the guinea pig heart or atrium, eXperiments were performed to deter- mine if the ability of the drug to block amine uptake was absent as well. 53 Figure l3. Correlation between positive inotropic effect and increased H -norepinephrine efflux in isolated atria. The amount of radioactivity in disintegrations per minute (DPM) per minute of effluent flow and the increase in force of con- traction in milligrams (mg) are plotted against the minutes following the termination of HJ-norepinephrine (Hj-NE) incubation. The arrow on the abscissa indicates the point of administration of 3 X lO'5M tripelennamine. 5A .‘PI. «- 300 :u I 50-' I I I l \ . Force-3.! . - 250 I \ I \ 4o.- H3-NE \\ I \ - 200 #4 . l \ § .5 ‘ ” E I \ 5:»: ‘ID 30-! I r“ x - I50 «1 z: I c: o- :0 c: I g} l a 3 zo-q | $1 I - Too 1 I I l 10-- l I - 50 -““-—____ o«---—---— ------ ?---- -o . . - . . . . . . 50 70 80 T 3 100 no MINUTES AFTER H3-NE INCUBATION Figure 13 55 .hpfi>fipom0fi©ma wo ao>ma oamm map can mEHp oamN mm dmxmp mm: soflpmapmfl uqflsdw mo pqflom one .QOwaQSoGH osflahmosflmoaosumm mo soapmsflsamp map ampww mopzsfla aposfic pm wmampmflsfiapc mamz mmdsa .ooMSIJHD mo mam>oa map «mocfia cognac map wow «osflsgmosflmoaosunm wo mao>oa map pqomoamoa mmcfla UHHOm mQB .nmpdsfie CH oEHp one Pmsflmmm Umppoam ma Boaw Pconammm mo mPSGHE Hog AEmQV opssfie hog mGOprawopsHmHU CH hpfl>flpomoflcmy Mo psdosm mQB .wflapw oopwaomfl Ga adawmm monoudao paw oqflanmosflmoaosnwm so msflmooo can ocfiwflaoamfiap «oQHEmaflsmsmaoago «mQflEmssoaomHap go mpoowwm .ja madmflm :H mpsmfim A.=,sv “zap o o o cm a. ow om m 3m am I» m m —T. _ . o IIIIIIIIIIWIIIIIIIIII... ........ .. I I I- 2m-op x m mammuou 2m-o. x m m=_st0Eswgh Emlop x m mcwsmgwcmsagopso ST} mega- m2um: IIIII U IIIII zmuop x m mewsmccmpmawgh fin" c op om mw N x (mm ,3 w. on m ow om 57 Table 3. Drug-induced increases in H3-norepinephrine efflux in isolated atria. The total increases in radioactivity elicited by tyrEmine, tripelennamine, chlorpheniramine, triprolidine, cocaine FHd desi- pramine were computed as disintegrations per minute (DPM) per gram of tissue. Each value represents the mean and standard error (s.e.m.) for A eXperiments. Tyramine was significantly more effective (P