AN ABSTRACT OF THE THESIS OF MASTER OF SCIENCE ROBERT EDWARD STUART for the degree of December 11, 1981 in FISHERIES presented on Title: PHYSIOLOGICAL AND PHARMACOLOGICAL INVESTIGATION OF THE VASCULARIZED AND NONVASCULARIZED TELEOST HEART WITH ADRENERGIC, CHOLINERGIC AND CORONARY DATING AGENTS Abstract approved: Redacted for Privacy Lavern J. Weber Teleost hearts can be classified into two types, vascularized (coronaries) and nonvascularized, based on the arrangement of the ventricular myocardium. Anatomical investigation of the buffalo sculpin (Enophrys bison) heart has revealed that it lacks coronary circulation. To see if the nonvascularized heart obeys Starling's law of the heart and to characterize it as to the type of adrenergic receptors present, isolated sculpin hearts were attached by the sinus venosus to a modified Langendorff perfusion apparatus. A pressure transducer was placed in series between the bulbus arteriosus and a vertical 15 cm length of glass tubing so as the heart pumped perfusate a resting pressure would be established for:the heart to work against. Heart rate, systolic pressure, and cardiac output were monitored. As atrial perfusion pressure was increased, the sculpin heart responded with a positive inotropy and an increase in cardiac output. Heart rate did not change. Epinephrine (0.64 pg /ml) accentuated the pulse pressure respone, causing an overall increase in pressure and heart rate. Acetylcholine (1.0 pg/ml) resulted in a negative chronotropy and a positive inotropy. The data indicate that the contractility increases with elevated filling pressure, demonstrating Starling's law. Isoproterenol results in positive chronotropy which could be blocked by propranolol. Methoxamine was without effect. The sculpin heart then appears to be under the control of beta adrenergic receptors. A further study was carried out to determine what effect vasoactive drugs would have on the fish heart. Chum salmon (Oncorhynchus keta) were used as a source of vascularized hearts. Hearts were mounted on the Langendorff perfusion apparatus as described previously with the exception that'the coronary artery was perfused in the salmon hearts via a cannula attached to a perfusion pump. The buffalo sculpin heart showed no responses when exposed to sodium nitrite (1 pg/ml), nitroglycerin (100 pg /mi), or perhexiline (1 pg/ml) while prenylamine (10 pg/ml) caused negative chronotropy and increased stroke volume. Nitroglycerin (100 pg/ml) and sodium nitrite (1 pg/ml) appeared to have no direct effect on the myocardial tissue of the chum salmon. Sodium nitrite, when administered through the coronary artery, produced a prolonged and gradual increase in coronary pressure which continued after their removal. results. Nitroglycerin produced no conclusive Acetylcholine (0.5 pg/ml) decreased heart rate, increased systolic pressure, stroke volume and vascular resistance by both atrial and coronary administration but these changes were greater when acetylcholine was administered through the atrium. Epinephrine (0.5 pg/ml) administered through the coronary artery had no significant effect on any of the parameter's measured. When per- fused through the chambers, stroke volume declined but none of the other parameters were changed. Physiological and Pharmacological Investigation of the Vascularized and Nonvascularized Teleost Heart with Adrenergic, Cholinergic and Coronary Dilating Agents by Robert Edward Stuart A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Master of Science Completed December 11, 1981 Commencement June 1982 APPROVED: Redacted for Privacy Professor of Fisheries in charge of major Redacted for Privacy Head of Department of Fisheries and Wildlife Redacted for Privacy Dean of Graduatk school Date thesis is presented U December 11, 1981 Typed by Anita deMercado Stuart for Robert Edward Stuart ACKNOWLEDGMENTS The completion of this thesis would not have been possible without the encouragement, guidance, and love of many people. those is my major professor, Dr. Lavern J. Weber. Foremost among I wish to thank him for always being available whenever I had questions or problems. The Thursday night groups, the Sunday dinners and the time spent spear fishing all contributed in some way to my education and will long be remembered. I would also like to thank the members of my committee, Dr. James L. Hedtke, Dr. John R. Smith, Dr. Robert E. Olson and Dr. Paul Kifer. Additionally, special thanks go to friend and fellow student Daniel B. Gant for helping me relieve my frustrations through the strangulationrelaxation technique. By rights 50% of this thesis belongs to my wife, Anita. She's the one who listened to my rantings and ravings when everything seemed to go wrong. She had faith in me when I had lost faith in myself and was always a source of understanding and love. Without her this thesis would never have been put on paper, especially since she typed it. To her go my love and thanks. I'm greatly indebted to my parents, Buren B. Stuart and Gertrude M. Stuart. Their love and encouragement was always given generously and was unrelenting. This work was supported by NIEHS ESO 1926. TABLE OF CONTENTS Page CHAPTER I. INTRODUCTION 1 MATERIALS AND METHODS 7 RESULTS 12 DISCUSSION 25 CHAPTER II. 29 INTRODUCTION 29 MATERIALS AND METHODS RESULTS Sculpin Salmon 31 34 34 45 DISCUSSION 61 BIBLIOGRAPHY 68 APPENDIX 75 LIST OF FIGURES Page Fig. 1 Fig. 2 Fig. 3 Schematic showing attachment of heart to perfusion manifold and pressure transducer 8 Electronmicrograph and light photomicrographs of the ventricular myocardium of the buffalo sculpin and chum salmon 13 Strip chart recordings of in vivo and in vitro heart rate and pulse pressure from the buffalo sculpin exposed to adrenergic and cholinergic agonists and antagonists 15 Fig. 4 Changes in heart rate, pulse pressure and stroke volume 17 over a two hour period for the control hearts Fig. 5 Effects on heart rate, stroke volume, pulse pressure and cardiac output of hearts exposed to varying atrial perfusion pressures in the absence and presence of epinephrine (0.64 pg/ml) 21 Effects on heart rate, stroke volume, pulse pressure and cardiac output of hearts exposed to varying atrial perfusion pressures in the absence and presence of acetylcholine (1.0 pg/ml) 23 Fig. 6 Fig. 7 Changes in heart rate, pulse pressure and stroke volume 35 over a 50 minute interval for the control hearts Fig. 8 Heart rate, pulse pressure and stroke volume changes in the isolated buffalo sculpin heart exposed to the vasodilators sodium nitrite (1.0 pg/m1) and nitro37 glycerin (100 pg/ml) Fig. 9 Strip chart recordings of heart rate and pulse pressure from the isolated buffalo sculpin heart exposed to the vasodilators prenylamine, perhexiline, nitroglycerin and sodium nitrite Fig. 10 Changes in heart rate, pulse pressure and stroke volume in the isolated buffalo sculpin heart administered the vasodilators perhexiline (1 pg/ml) and prenylamine (10 pg/m1) Fig. 11 39 41 Strip chart recordings of heart rate, pulse pressure and coronary pressure from isolated salmon hearts exposed to acetylcholine (0.5 pg/m1) and epinephrine (0.5 p9 /ml) 46 LIST OF FIGURES, Contd. Page Fig. Fig. Fig. Fig. Fig. 12 13 14 15 16 Graph of heart rate, pulse pressure, stroke volume and coronary pressure in the isolated salmon heart during administration of acetylcholine (1.0 pg/m1) first through the coronary artery and then through the sinus venosus 48 Effects of epinephrine (0.5 pg/ml) on heart rate, pulse pressure, stroke volume and coronary pressure in the Drug was added first through isolated salmon heart. the coronary and then through the sinus venosus 51 Strip chart recordings of heart rate, pulse pressure and coronary pressure from isolated salmon hearts exposed to the vasodilators sodium nitrite (1 pg/m1) and nitroglycerin (100 lig/m1) 53 Changes in heart rate, pulse pressure, stroke volume and coronary pressure in the isolated salmon heart exposed to sodium nitrite (1 pg/ml) first through the coronary and then through the sinus venosus 55 Changes in heart rate, pulse pressure, stroke volume and coronary pressure in the isolated salmon heart exposed to nitroglycerin (100 pg/m1) first through the coronary and then through the sinus venosus 57 LIST OF TABLES Page TABLE I. Summary of the Effects of Acetylcholine, Epinephrine and Vasodilators on the Isolated Buffalo Sculpin Heart TABLE II. Summary of the Effects of Acetylcholine, Epinephrine and Vasodilators on the Isolated Salmon Heart 44 60 PHYSIOLOGICAL AND PHARMACOLOGICAL INVESTIGATION OF THE VASCULARIZED AND NONVASCULARIZED TELEOST HEART WITH ADRENERGIC, CHOLINERGIC AND CORONARY DILATING AGENTS CHAPTER I INTRODUCTION Since Motti's review of the fish cardiovascular system in 1957, increasingly more work has been done on fish physiology and pharmacology.but there is still a relative paucity of information in this field. With over 20,000 species of fish, 96% of these represented by the infraclass Teleostei (Nelson, 1976), extending over a wide range of habitats, only a few species have been studied extensively. With such a diverse group it is difficult to make, generalizations based on the knowledge of only a few species. Anatomically, the cardiovascular system of the fish shows some conformity among the many species of fish, with the exception of the cyclostomes and lungfishes. The heart lies within a thin, compliant pericardium located just beneath and slightly posterior to the gills. Venous blood is pumped by the heart through the gills and systemic circulation and then returns to the heart. The teleost heart is a myogenic, branchial heart formed of typical vertebrate cardiac muscle fibers (Randall, 1970; Satchell, 1971). It consists of four chambers in series: the sinus venosus, atrium, ventricle and noncontractile but very elastic bulbus arteriosus, thus forming a single circuit pump (Hildebrand, 1974). 2 To meet the circulatory demands of the tissues the heart requires a regulatory mechanism. The two factors determining cardiac output are stroke volume and heart rate (Rushmer, 1961; Satchell, 1971). As in mammals, the teleost heart is controlled aneurally and neurally. A basic aneural control mechanism is demonstrated by Starling'd law of the heart which states that the strength of contraction is a function of the initial length of the muscle fiber (Rushmer, 1961; Randall, 1970). Bennion (1968) and Butler (1976), using the isolated hearts from trout and shark respectively, have confirmed that Starling's law is applicable to the hearts of these two species. It is believed that when the pacemaker tissue is extended in the heart it is more sensitive to stretch and depolarization (Jensen, 1961). Neural mechanisms for controlling the heart in fish share some similarities with those in mammals. In the mammal, neural control is mediated through the parasympathetic and sympathetic nervous systems. Hagfishes, Myxine and Eptatretus (Cyclostomata), appear to have aneural hearts receiving no vagal supply (Jensen, 1961; Falck, et al, 1961). Neural control of the other fish heart, however, is quite controversial. It was observed that the isolated trout heart could respond to catecholamines (Fange and Ostlund, 1954; Bennion, 1968). With fluorescence histochemistry, catecholamine-containing nerve fibers were found in the hearts of the cod, Gadus morhua (Holmgren, 1977), Salmo trutta and S. irideus (Gannon and Burnstock, 1969), Carassius auratus, and several other species (Cameron, 1979). Although it is commonly believed that acetylcholine is the major neurotransmitter in the fish vagus nerve, Euler and Fange (1961) also found norepinephrine in the vagus nerves of the cod (Gadus morhua). Randall (1970) reports that all fish hearts, with the exception of the hagfish, are innervated by a branch of the vagus. Stimulation of the vagus results in bradycardia (Randall, 1970) which can be blocked by atropine (Mott, 1957; Chan and Chow,- 1976; Gannon and Burnstock, 1969, Randall, 1966; Seibert, 1979). Many fish exhibit a reflex bradycardia (Cameron, 1979; Peyraud-Waitzenegger, et al., 1980) to visual, auditory and mechanical stimuli which suggests the slowing of the heart is due to an increasing level of vagal control (Randall, 1970). Saito (1973) observed both inhibitory and excitatory responses in the carp heart during or after vagal stimulation, which were abolished by atropine. It has been found by Stevens and Randall (1967a) and by Cameron (1978) that a resting vagal tone is present in many fish. Trout, (Stevens and Randall, 1967; Wood and Shelton, 1980) Asiatic eel (Anguilla japonica (Chan and Chow, 1976) and cod (Helgason, 1973) are reported to have little cardiac vagal tone while the eel Anguilla anguilla (Seibert, 1979) and the sea robin (Roberts, 1968) demonstrated considerable vagal tone to the heart. In Anguilla anguilla Seibert (1979) proposed that the degree of vagal tonus may depend upon temperature. No sympathetic fibers could be found in the heart of plaice, Pleuronectes platessa (Falck, et al., 1966) or in the eel, Anguilla japonica (Chow and Chan, 1975). Burnstock (1969) has reported sympathetic fibers running in the vagus nerve of fish. Sympathetic control of the fish heart may also be mediated through circulating 4 catecholamines released by chromaffin tissue (Nakano and Tomlinson, 1967; Holmgren, 1977; Wahlquist and Nilsson, 1977). It appears that there may be interspecific variation in the sympathetic innervation of the fish heart and that sympathetic control is exerted by one or both of these mechanisms. Adrenoreceptors have been demonstrated pharmacologically in the isolated and intact fish hearts. Falck, et al (1966) using the isolated hearts of Lampetra fluviatilis and Pleuronectes platessa, found adrenoreceptors of the beta type. Cardiac beta receptors were also demonstrated in the intact hearts of Salmo gairdneri (Wood and Shelton, 1980) and the isolated (Holmgren, 1977) and intact (Wahlquist and Nilsson, 1977) hearts of the cod, Gadus morhua. al Peyraud-Waitzenegger, et (1980), using Anguilla anguilla, demonstrated a seasonal variation in the type of cardiac adrenoreceptors. In summer, the fish exhibited beta adrenoreceptor mediated activity whereas in winter, alpha activity predominated. Anguilla anguilla is reported to have both alpha and beta adrenoreceptors (Forster, 1975) while Anguilla japonica demonstrated only alpha activity (Chan and Chow, 1976). In the isolated heart of the elasmobranch Squalus acanthias a negative chronotropic response was shown to be mediated by alpha receptors (Capra and Satchell, 1976a). Fish appear then to exhibit an interspecific, and perhaps, a seasonal variation in the type of cardiac adrenoreceptors. The myocardial catecholamines epinephrine and norepinephrine in fish are reported to be lower than in mammals with the predominant catecholamine being epinephrine (Euler and Frige, 1961; Burnstock, 1969; Lee, et al., 1970; Randall, 1970). In the isolated heart of Squalus acanthias epinephrine and norepinephrine produced positive inotropy but epinephrine resulted in positive chronotropy while norepinephrine exerted a negative chronotropic effect. The potency ratio was reported to be epinephrine (10):norepinephrine (1000) (Capra and Satchell, 1976a). Catecholamines produced no chronotropic effect in the eel Anguilla japonica (Chan and Chow, 1976), although a positive chronotropy in the cod, Gadus morhua was produced (Holmgren, 1977; Wahlquist and Nilsson, 1977). Flige and Ostlund (1954) demonstrated positive chronotropy and inotropy in the isolated fish heart exposed to epinephrine and norepinephrine while Helgason (1973) found a decrease in heart rate in the intact cod with these catecholamines. The isolated heart of the rainbow trout responded to epinephrine with positive inotropy and a decrease in heart rate (Bennion, 1968). The catecholamines affect the rest of the circulatory system as well, dilating the gill vasculature and constricting the general systemic vasculature (Ostlund and Fange, 1962). The general consensus until recently was that all teleostean hearts were vascularized. According to Randall (1970) the heart in all fishes, with the exception of the hagfish, had a coronary supply. However, Santer and Walker (1980) found that of 93 species of teleosts examined, only 19 possessed a coronary vasculature and in these 19 species plus the anguillids the ventricular myocardium was composed of an outer compact layer of muscle and an inner spongy layer. Of 14 elasmobranch species examined, all possessed 6 vascularized hearts. Physiological and pharmacological studies of the isolated fish heart have not taken this into account in the past. Dissection studies have revealed that the buffalo,sculpin (Enophrys bison), a marine teleost, contains no coronary vasculature. Light and electron micrographs (Bacon, unpublished data) revealed a ventricle composed of only a spongy, trabeculated myocardium, consistent with the nonvascularized hearts described by Santer and Walker (1980). A physiological and pharmacological study was carried out on both the intact and isolated heart of the-buffalo sculpin. 1. The object of this experiment was: To determine if the nonvascularized heart obeyed Starling's law. 2. To determine its response to acetylcholine and epinephrine. 3. To determine the type of adrenoreceptors present. MATERIALS AND METHODS Buffalo sculpin (Enophrys bison) were collected from Yaquina Bay, Newport, Oregon by otter trawl and maintained in aerated, running sea water at 12°C. weekly. A diet of chopped herring was fed once Animals weighed from 300 to 1100 gms. The animals were pithed and a midventral incision made to expose the heart. The heart was excised so as to include the sinus venosus and the bulbus arteriosus. Isolated hearts were immediately placed in cold marine teleost saline (hereafter referred to as saline) (NaCl: 7.25g/1; KC1: 0.38 g/1; CaC12.2H20: 0.23 g/1; NaHCO3: 1.0 g/1; MgSO47H20: 0.23 g/1; glucose: 1.00 g/1; Trizma Base: 0.80 g/1; Trizma HC1: 6.85 g/1), at pH 7.8 and at an osmolality of 350 mOs/kg, flushed with saline to remove residual blood or clots, and weighed. Hearts were then mounted on a modified Langendorff coronary perfusion apparatus by the sinus venosus and the bulbus arteriosus was cannulated with PE 90 tubing attached to a pressure transducer (Statham P23De). A 15cm length of glass tubing was mounted vertically to the exhaust port of the pressure transducer to establish a resting pressure (Fig. 1). The heart was perfused through the chambers with saline maintained at 12°C, (Neslab RTE-8 refrigerated circulating bath) pH 7.8 and also superfused with saline. All recordings were made with a Clevite Brush Mark 220 recorder. The modified Langendorff apparatus allowed the perfusion pressure to be varied. Hearts were allowed to equilibrate at the lowest perfusion pressure (0.8 kPa) for 30 min. before introducing drugs or altering perfusion pressures. After equilibration, the 8 Figure 1. Schematic showing attachment of heart to perfusion manifold and pressure transducer. The exhaust ports on the manifold allow atrial perfusion pressure to be varied. Small arrows indicate path of perfusate through heart, transducer and vertical length of glass tubing. Manifold 6 venosus 4Atrium Transducer <--Ventricle 4, Bulbus arteriosus Figure 1 10 perfusion pressure was raised to 1.1 kPa, 1.3 kPa, and 1.6 kPa at 5 min. intervals and finally returned to 0.8 kPa for an additional 5 min. to determine if the heart would recover its initial rate and pulse pressure. Saline with either L-epinephrine bitartrate (Sigma) or acetylcholine chloride (Sigma) was then perfUsed through the heart repeating the above sequence. Four hearts were used with each drug and each heart served as its own control. To characterize the autonomic receptors in the heart the following drugs were used: DL-isoproterenol HC1 (Sigma), DL- propranolol, methoxamine HC1 (Burroughs Wellcome Co.), acetylcholine Cl and atropine sulfate (J. L. Baker Chemical Co.). mounted and allowed to equilibrate as described. Hearts were During this time, heart rate, pulse pressure and cardiac output were recorded. At 30 min. a drug solution was perfused through the heart for 5 min. at the lowest perfusion pressure. After this 5 min. drug treatment, drug-free saline was reintroduced for 15 min. or until hearts regained pre-drug rates. Only one drug was used per heart. Histological examination of the hearts was performed by Dr. Robert Bacon at the Oregon Health Sciences University, Portland, Oregon (Department of Anatomy) using light microscopy, transmission electronmicroscopy and scanning electronmicroscopy. In vivo studies of the buffalo sculpin were performed by initially anesthetizing fish by alternate exposure of gills to oxygenated sea water with and without benzocaine (70 mg/1). A 24 inch length of PE 50 tubing filled with 500 I.U. heparinized saline was inserted ventrally into the branchial artery in the second or 11 third gill arch. The cannula was sutured into place and connected to a Statham P23De pressure transducer. Clevite Brush Mark 220 recorder. Recordings were made with a Animals were held in plexiglass restraining boxes placed in aerated running sea water aquaria. Recordings of heart rate and blood pressure were begun as soon as the animals were placed in water. A recovery period of two hours was allowed before any tests were performed. All drugs were prepared from concentrated stock solutions made with glass distilled, deionized water adjusted to a pH of 4.0 with HC1 and refrigerated. Drug solutions to be administered in vivo or in vitro were made just prior to administration with saline at the appropriate osmolality. Statistical analyses were by two way analysis of variance with a multiple range test. 12 RESULTS Figure 2A shows myocardial cells in the ventricle of the buffalo sculpin. myocardium. They appear to resemble the typical vertebrate Figure 2B pictures the ventricular wall of the sculpin demonstrating the arrangement of the myocardial fibers. No outer compact layer of muscle is present and vascularization is completely lacking in comparison to that of a salmonid (Fig. 2C). No vasculari- zation could be detected in this inner spongy layer of muscle of either sculpin or salmon. In vivo experiments with the buffalo sculpin demonstrated that a reflex bradycardia could be elicited with visual stimuli (Fig. 3A). Accompanying this reflex bradycardia was a sharp decrease in blood pressure. Administration of 0.25 mg/kg atropine abolished or reduced the bradycardia but resulted in a slight increase in pressure when the animal was exposed to visual stimuli (Fig. 3B). In Vitro Experiments Before the pharmacological tests were begun, hearts were mounted on the perfusion apparatus at the lowest pressure for two hrs to test their viability (Fig. 4). Heart rate decreased over the entire period, dropping 46% after two hrs. Sixty-five percent of this decrease occurred during the first 30 minutes. Pulse pressure and stroke volume increased over the two hr interval. Pulse pressure increased by 33% at the end of two hrs and had increased by 14% at 30 min. Stroke volume increased by 63% over the two hr test period and had increased by 36% at 30 min. Cardiac 13 Figure 2 Electronmicrograph (mag. 3400X) of the ventricular myocardium of the buffalo sculpin showing arrangement of muscle fibers. A. Photomicrograph of the ventricular wall of the sculpin heart (40X) showing trabecular arrangement of myocardial Arrow indicates luminal tissue and lack of vascularization. side of wall. B.. Photomicrograph of the ventricular wall of the salmon heart (40X) showing compact myocardial tissue with coronary Arrows indicate coronary vessels. vessels. C. 14 15 Figure 3. Strip,chart recordings of heart rate and pulse pressure from the buffalo sculpin. A and B are in vivo recordings; C through F are in the isolated heart. Horizontal and vertical arrows indicate direction of recording and point at which stimulus was administered, respectively. Pressure in kPa. In vivo recording demonstrating reflex bradycardia induced by visual stimulus. A. In vivo recording in same fish as ,A showing the inhibition of reflex bradycardia following atropine (0.25 mg/kg) (Atropine given at first arrow). administration. B. C. Acetylcholine (1.0 pg/ml) administration in the isolated heart. Acetylcholine. (1.0 pg /ml) following atropine (1.0 pg/ml) administration. D. E. Isoproterenol (1.0 pg/ml). F. Isoproterehol (1.0 pg /ml) following propranOlol (1.0 pg/ml) G. Epinephrine (0.64 pg/ml). 16 6.0 s 4.0 1 Min. 8.0 4.0 1 I !'ins. 4 10 sec. 2.0 1.0 2.0 E 1.0 10 sec. 1.5 1.0 10 sec. 2.0 C 0.5 25 sec. ricure 3 17 Figure 4. Changes in heart rate, pulse pressure and stroke volume over a two hour period for the control hearts (n = 4, x S.E.). O O STROKE VOLUME 0 PULSE PRESSURE(kPa) HEART RATE 19 output remained relatively stable over the entire test period with no significant changes occurring. The greatest rate of change for all parameters occurred within the first 30 mina Since the least amount of variation occurred between 30 and 90 min, all subsequent studies were confined to this interval, the initial 30 min being used as an equilibration period. Acetylcholine (1.0 pg/m1) resulted in negative chronotropy and positive inotropy (Figure 3C). At a dose of 1.0 pg/m1 the heart would be arrested for the entire duration of exposure or would stop for short periods and then continue beating again, resembling vagal escape. These effects of acetylcholine could be eliminated by washing the heart out with saline. Administration of atropine (1.0 pg/m1) for 5 min. followed by acetylcholine for 5 min. diminished or abolished the effects of acetylcholine (Fig. 3D). Epinephrine produced positive chronotropy and inotropy (Fig. 3G). The chronotropic effect of epinephrine persisted long after wash out with saline. Exposure to a high concentration of epineph- rine initially would mask the effects of a lower dose given later, therefore each heart was used only once for each dose. Isoproterenol increased heart rate, lowered pulse pressure and decreased stroke volume (Fig. 3E). As with epinephrine, the chronotropic effect of isoproterenol persisted after reintroduction of drug-free saline. nol (Fig. 3F). Propranolol abolished the effects of isoprotere- 20 Methoxamine was without effect. Starling's Law Increasing atrial perfusion pressure had no significant effect on heart rate but stroke volume and pulse pressure both increased. Cardiac output also increased with increasing atrial perfusion pressure (Fig. 5). Increasing atrial perfusion pressure in the presence of 0.64 pg/m1 epinephrine caused no change in heart rate between pressures. Stroke volume increased with atrial perfusion pressure when epinephrine was present but was less than in drug-free hearts. Pulse pressure was greater with epinephrine and continued to increase with rising atrial perfusion pressure. Cardiac output increased with increasing atrial perfusion pressure but was almost identical to that in the drug-free,hearts. The same tests repeated with acetylcholine (1 pg/m1) resulted in a negative chronotropic effect. However, there appeared to be little change in heart rate from one perfusion pressure to the next in the presence of acetylcholine. pressure were greater at all pressures. Stroke volume and pulse There was no significant difference in cardiac output between non-treated hearts and those administered acetylcholine (Fig. 6). 21 Figure 5. Effects on heart rate, stroke volume, pulse pressure and cardiac output of hearts (n = 4) exposed to varying atrial perfusion pressures in the absence (solid-bar) and presence (crosshatched bar) of epinephrine (0.64 pg/m1). S.E.). 22 bt) 40 20 0 MTS 0.16 MTS 8i Epi. 0.12 008 004 6 4 2 30 5 PI 45 70 P2 P3 Figure 5 P4 5075 MINS pi PRESSURE 23 Figure 6. Effects on heart rate, stroke volume, pulse pressure, and cardiac output of hearts (n = 4) exposed to varying atrial perfusion pressures in the absence (solid bar) and presence (crosshatched bar) of acetylcholine (1.0 pg/m1) [Perfusion P1 = 0.8 kPa; P2 = 1.1 kPa; P3 = 1.3 kPa; P4 = pressures: 30-50 minutes were in absence of drug and 55-75 1.6 kPa. (x ± S.E.)]. minutes were in presence of drug. m z C tn to z rn n \\\1\1.. HEART RATE ROM t \Mb, 111. Ilk,\11 0 a) STROKE VOLUME 111111L%\.\\B 0 0 1\121.\ PULSE PRESSURE(kPa) 1\11116, CARDIAC OUTPUT 25 DISCUSSION The ventricle of the sculpin heart is comprised entirely of trabeculated muscle surrounded by an epicardium and endoCardium. There is no outer compact layer of ventricular myocardium and coronary vasculature is completely lacking. The morphology of.the buffalo sculpin ventricle is similar to that described by Santer and Walker (1980) for nonvascularized teleost hearts which receive their oxygen supply from venous blood in the ventricular lumen. Santer and Walker (1980) hypothesized that those teleost species expending large amounts of metabolic energy throughout their lives would require a vascularized myocardium, whereas less active fish would not. The buffalo sculpin is a relatively inactive bottom fish remaining motionless most of the time except when feeding or disturbed. Based on its level of activity, one would expect that the buffalo sculpin heart would lack vascularization. The reflex bradycardia demonstrated by the buffalo sculpin in vivo and its reduction or abolishment by atropine is in agreement with the literature (Stevens, et al., 1972; Chan and Chow, 1976; Cameron, 1978). Randall (1970) suggests that this bradycardia is due to an increasing level of vagal tonus. There appears to be interspecific variation in the degree of vagal tonus to the teleost heart (Cameron, 1978). Wood and Shelton (1980) were unable to demonstrate any cholinergic vagal tone in intact rainbow trout while Priede (1974) was able to show an inhibitory vagal tonus in the same species. Seibert (1979) suggested the degree of vagal tonus depends on temperature. Roberts (1968) hypothesized that parasympathetic 26 activity was reduced in active species of teleosts but elevated in less active species. In Vitro Experiments The relatively rapid heart rate at the beginning of perfusion may have been due to catecholamines or other hormones released during dissection. As the effects of the catecholamines diminished during the first 30 min. of perfusion the heart rate declined and remained relatively stable thereafter. The rate decrease was accompan- ied by increased stroke volume and pulse pressure. explained by Starling's law. This may be As rate declines, more time is available for complete filling of the ventricle, which in turn stretches the myocardial muscle fibers more. The combination of increased filling and increased stretch results in greater stroke volume and pulse pressure. In the isolated sculpin heart acetylcholine consistently resulted in a positive inotropic and negative chronotropic response. Capra and Satchell (1977a) fpund a negative chronotropic and inotropic response in the isolated heart of Squalus acanthias, as one might expect to see in an isolated mammalian heart. In the intact animal a negative inotropic response might be expected with administration of acetylcholine due to vasodilation, but in the isolated heart one might expect to see just the opposite. As rate falls there will be more time for relaxation resulting in increased stretching of the myocardium. The greater stretching would then be expected to result in increased force of contraction. In the isolated heart of the buffalo sculpin, acetylcholine does not produce the expected negative 27 inotropy. Atropine produces an inhibition of the acetylcholine response as expected. The increase in rate and pressure associated with epinephrine administration is in agreement with the literature (Ffige and Ostlund, 1954; Bennion, 1968). Epinephrine administration caused a. persistent rate increase whereas the positive inotropy was transient. Administration of epinephrine to a failing or an arrhythmic heart improved cardiac function and restored rhythmicity. This phenomenon was also reported by Bennion (1968) in the isolated heart-of the rainbow trout. The persistent chronotropic response elicited by epinephrine may be due to its promotion of glycogenolysis. The presence of beta receptors within the buffalo sculpin heart is indicated by the positive chronotropy and negative inotropy produced by isoproterenol and the blockade of these effects by propranolol. Furthermore, the absence of any effect with methoxamine seems to indicate a lack of alpha receptors. The predominance of cardiac beta receptors concurs with evidence from other teleosts (Falck, et al, 1966; Bennion, 1968). However, Forster (1976b) found both alpha and beta adrenergic receptors in the heart of Anguilla anguilla. Peyraud-Waitzenegger, et al (1980), also working with Anguilla anguilla, reported seasonal variation in the predominant type of adrenergic receptor, alpha receptors predominating in winter and beta receptors in the summer. In this study, seasonal variation was not taken into account and whether such variation exists in the bUffalo sculpin is not known. 28 The positive inotropic effect produced by increasing atrial filling pressure demonstrates that this heart obeys Starling's law. Bennion (1968) reported similar results for the isolated heart of the trout. As the input pressure increased so did the end diastolic fiber length, and as a result the fibers contracted with greater force. Stroke volume and cardiac output increased as well. In Bennion's study, heart rate increased with increasing atrial perfusion pressure. However, in the sculpin heart, rising atrial perfusion pressure resulted in a slight decrease or no change at all in heart rate. Bennion postulated that the rate increase produced by increasing pressure was probably due to a direct effect of stretch on the pacemaker fibers causing a change in ionic conductance. Whether the differences in heart rate response between these two species are physiological in nature or due to differences in the mechanics of the preparation is not known. The two types of hearts may be different in more ways than just vascularization. 29 CHAPTER II INTRODUCTION The mammalian coronary vascular system has attracted much attention within the past three decades as a result of the high incidence of coronary artery disease in man. Much, if not the majority, of the information on the physiology and pharmacology of the coronary vasculature is clinical in nature. Very little literature is available describing the comparative aspects of the coronary system. Most research in this area has been conducted on mammals, ignoring the other vertebrate groups. By examining the coronary vasculature system of lower vertebrates, some insight may be gained in understanding the physiology and pathology of mammalian coronary systems. Until recently it was assumed that all vertebrate hearts were vascularized. However, Santer and Walker (1980), examining the ventricles of teleosts were able to describe two basically different arrangements of ventricular myocardium in fish. The first type of ventricular myocardium in fish is composed entirely of trabecular muscle enclosed by the endocardium and epicardium, deriving its oxygen supply from the venous blood in the lumen. The second arrangement of fish ventricular myocardium is made up of an outer compact myocardium surrounding the trabecular muscle. This cortical layer is supplied with a coronary vasculature system derived from epibranchial arteries (Watson and Cobb, 1979). Belaud and Peyraud (1971), working with the isolated heart of the Conger eel (Conger conger), cannulated and perfused what they 30 described as the coronary vasculature. They recorded a coronary blood flow between 0.1 and 0.3 ml/min at a pressure of 15 cm H20. They found that electrically induced tachycardia diminished coronary flow and that acetylcholine produced a coronary vasoconstriction which could be blocked by atropine. Physiological and pharmacological investigatiOns of the isolated fish heart have been done with little or no regard for perfusion Of the coronary vasculature in those species with vascularized hearts. Though the hearts of several species of fish have been described physiologically and pharmacologically the coronary vasculature has been neglected. Unlike the mammalian heart the fish heart consists of a single chambered atrium and a single chambered ventricle, which receives its oxygen supply either from luminal venous blood or from coronary vasculature derived from epibranchial arteries. It is possible to isolate the fish heart and perfuse the chambers and the coronary vasculature independently of one another. The present study compares the effects of several coronary vasodilators and neurotransmitters on the vascularized and non - vascularized fish heart. Additionally, it attempts to examine what differences, if any, exist when administering the vasodilators first through the coronary vasculature and then through the chambers of the heart. 31 MATERIALS AND METHODS Buffalo sculpin (Enophrys bison) were collected by otter trawl from Yaquina Bay, Newport, Oregon and held in running seawater aquaria at 12°C. Fish were fed a diet of chopped herring once weekly. Experimental animals were killed by a blow to the head, then weighed and bled by removing the tail. The heart was excised through a midventral incision so as to include the bulbus arteriosus and the sinus venosus, and immediately placed in cold saline. A cannula of PE90 tubing flared at the tip was inserted into the sinus venosus and tied in place. Cold saline was introduced to clear the lumen of re- maining blood and clots. Finally, a second cannula of PE 90 tubing was secured in the bulbus arteriosus. The isolated heart was then connected to a modified Langendorff coronary perfusion apparatus by the sinus venosus cannula. One port of a Statham P23De pressure transducer was attached to the bulbus arteriosus cannula while.the other port was connected to a 15 cm length of glass tubing so as to create a resting pressure of 1.5 kPa for the heart to work against. Returning adult male chum salmon (Oncorhynchus keta) were obtained from Oregon State University's salmon hatchery on Whiskey Creek, Netarts, Oregon. The salmon were held in aerated, freshwater tanks at 12°C without food and were kept no longer than a week before being used. The hearts of chum salmon were excised and prepared as described above with the exception that the coronary artery was cannulated. A cannula made of a short length of polyethylene tubing and a blunted hypodermiC needle was inserted into the coronary artery and sutured in 32 Cold saline was then injected to clear the coronary vasculature place. of blood and any possible clots. The coronary artery cannula was connected to an infusion pump (Harvard Apparatus Co., Inc.) via PE 90 tubing with an in-line Statham P23ID pressure transducer. The infusion pump was equipped with two 60 ml Monoject syringes, each with a Luer lock 3-way stopcock, so that it was possible to switch from one syringe to another. After completion of the cannulations the heart was placed in a temperature controlled (12°C) oxygenated organ bath. The heart was perfused with oxygenated teleost saline at 12°C and pH 7.8 at 1.1 kPa pressure. Coronaries were perfused at a rate of 0.4 ml/min. Coronary perfusion pressure varied between hearts. Isolated hearts were perfused for 30 minutewith drug-free teleost saline to allow equilibration. After equilibration, drugs were administered for a five minute interval followed by drug-free saline. Drugs were administered to the salmon hearts first through the coronary vasculature followed by a drug-free wash of 15 minutes and then the same drug was administered through the heart chambers. Only one drug was used per heart. Heart rate, pulse pressure and coronary pressure were recorded with a Clevite Brush Mark 220 recorder. Cardiac output was measured by collecting and weighing perfusate exiting the bulbus arteriosus cannula. Hearts of both species were sent to Oregon Health Sciences University for histological examination. Electron micrographs were done by Dr. Robert Bacon, Dept. of Anatomy. 33 Drugs used were nitroglycerine (10% in lactose, Key Pharmaceuticals, Inc.), perhexiline maleate (Merrell National Laboratories), prenylamine lactate (Hoechst-Roussell Pharmaceuticals, Inc.), Lepinephrine bitartrate (Sigma) and acetylcholine chloride (Sigma). All drugs were prepared in deionized double-distilled water at a concentration of 1 mg/ml. Drug solutions were made fresh for each experiment and kept refrigerated until used. A microliter pipet (Finnpipet) was used to withdraw the appropriate amount of concentrated stock solution (1 mg/ml) and add it to 500 ml of saline. The drugged saline solution was prepared 5 to 10 minutes prior to its use. A flow rate of 0.4 ml/min in the coronary vasculature was found to be the most optimum in terms of getting consistent recordings. This rate resulted in perfusion pressures in the range of those found in the dorsal aorta of salmon. 34 RESULTS Sculpin Controls To determine the viability of the isolated heart on the perfusion apparatus four hearts were used as controls. During a 45 min. interval the mean heart rate decreased 41.3% from the initial time. The greatest drop in rate occurred during the first 15 min., 26.4%. Pulse pressure and stroke volume did not change significantly during the 45 min. test interval (Fig. 7). Based on this information, all subsequent hearts were allowed to equilibrate for 15 min. before drugs were administered. Sodium Nitrite Sodium nitrite (1 pg/ml) was without significant effect on heart rate, pulse pressure or stroke volume when administered to the healthy heart (Fig. 8A and 9E). However, when administered to the arrhythmic failing heart sodium nitrite would occasionally restore rhythmicity (Fig. 9F). This was an inconsistent finding. Nitroglycerin Nitroglycerin (100 pg/ml) was without effect on the sculpin heart. No change could be seen in any of the parameters measured (Fig. 8B and 9D). Perhexiline Perhexiline (1 pg/ml ) was without effect (Fig. 9C and 10A). Prenylamine Prenylamine (10 pg/ml) resulted in a significant fall in heart rate accompanied by an insignificant rise in pulse pressure 35 Figure 7. Changes in heart rate, pulse pressure and stroke volume over S.E.). a 50 minute interval for the control hearts (n = 4, x -1 O 0' 0 O 0 9 I 0 I (MLS) STROKE VOLUME 1 (kPa) O PULSE PRESSURE HEART RATE 37 Figure 8. Heart rate, pulse pressure and stroke volume changes in the isolated buffalo sculpin heart exposed to the vasodilators sodium nitrite (1.0 pg/m1 - A) and nitroglycerin (100 pg /ml Drugs were introduced at 15 minutes (arrow) and perfused B). (n = 4; x ± S.E.). for 5 min. 38 RATE 20 PULSE PRESSURE ( k Pa) 0.4 STROKE VOLUME a- 50 HEART RATE B 40 T 4.0 PULSE PRESS RE (kPa) STROKE 0.4 VOLUME 0.3 I0 20 MINS. Figure 8 3 40 50 39 Figure 9. Strip chart recordings of heart rate and pulse pressure from Vertical arrows indicate the isolated buffalo sculpin heart. point of drug administration and horizontal arrows indicate direction of recording. Pressure in kPa. B. Prenylamine (10 pg/m1) Prenylamine (10 pg/m1) C. Perhexiline (1 pg /ml) D. Nitroglycerin (100 pg/ml) Sodium nitrite (1 pg/ml) in healthy heart Sodium nitrite (1 pg/ml) in arrhythmic heart A. E. F. 40 12 sec. 12 sc,c. 1.5 1-4 12 sec. 12 sec. E 2. [WiNlita HANNON ;774sec. 2.0 1.0 10 sec. Ficlure 9 41 Figure 10. Changes in heart rate, pulse pressure and stroke volume in the isolated buffalo sculpin heart administered the A) and prenylamine vasodilators perhexiline (1 pglml Drugs were introduced at 15 minutes (10 pg/ml - 13). (n = 4, (arrow) and perfused for 5 minutes. 42 5 0 A HEART RATE 30 PULSE PRESSURE (k Pa) 40 3.0 STROKE" VOLUME Q2 1 1 10 30 20 40 50 MINS. B 40 HEART RATE 20 PULSE 4 PRESSURE (kPa) 3 05 STROKE VOLUME 0.3 1 10 40 20 M INS. Figure 10 50 43 and stroke volume (Fig. 10B). perfusion. Heart rate fell 32% during prenylamine This negative chronotropy persisted even after prenylamine was discontinued. The transient increase in pulse pressure gave way to gradually decreasing pressure and a continuing decrease in heart rate 15 min. after discontinuation of prenylamine. dramatic effect however was on heart rate. effect of prenylamine on the sculpin heart. The most Fig. 9A shows the In this recording there was a noticeable increase in pulse pressure, however this was not a consistent finding as figure 9B shows. Stroke volume increased slightly during the 5 min. drug exposure but after removal of the prenylamine, stroke volume gradually decreased. When the hearts were perfused beyond the normal 45 min. test interval arrhythmias developed leading to failure,of the heart. After administration of prenylamine the hearts never returned to Pre-drug capacity. The data are summarized in Table I. 44 TABLE I. Summary of the Effects of Acetylcholine, Epinephrine and Vasodilators on the Isolated Buffalo Sculpin Heart Heart rate Systolic Pressure Acetylcholine (0.5 (pg/m1) +4, fi Epinpehrine (0.5 ug/m1) tt Stroke Volume 4, no change no change no change Nitroglycerin (100 pg/m1) no change no change no change Perhexiline (10 ug/m1) no change no change no change Sodium Nitrite (1 pg/m1) Prenylamine (10 ug/m1 t P < 0.05 4-4- = P < 0.01 no change 45 Salmon Figure 11A shows the simultaneous recording of heart rate and pulse presSure (top) and coronary pressure (bottom) in the isolated salmon heart perfused with saline solution. In a typical recording such as this, pulse pressure preceded peak coronary pressure indicating that ventricular contraction is probably producing the oscillations in coronary pressure. Acetylcholine Acetylcholine (0.5 pg /ml) administered through the coronary artery produced a slight, but insignificant, negative chronotropic and positive inotropic effect. significantly (22.6%). increase. Peak coronary pressure increased Stroke volume showed a slight but insignificant When the same dose was given through the sinus venosus there was a highly significant drop in mean heart rate (88%) (P < 0.01) and a highly significant increase (61%) in pulse pressure. Stroke volume and coronary pressure responded with significant increases as well. When drug-free saline was reintroduced the heart returned to the pre-drug conditions (Fig. 12). In Fig. 11B, the lower recording shows the increase in coronary pressure when 0.5 pg /ml of acetylcholine is perfused through the artery. There is only a minor effect on pulse pressure and virtually no change in rate. But the same dose administered through the chambers of the heart elicits a marked bradycardia (Fig. 11C). Epinephrine Epinephrine (0.5 pg/m1) administered through the coronary artery had no significant effect on any of the parameters measured 46 Figure 11. Strip chart recordings of heart rate, pulse pressure and coronary pressure from isolated salmon hearts. Upper recording is of heart rate and pulse pressure while Vertical the lower tracing is of coronary pressure. arrows indicate point of drug administration and horizontal arrows indicate direction of recording. Pressure in kPa. A. Control salmon heart Administration of 0.5 pg/ml acetylcholine through Spike is an injection artifact. the coronary artery. B. Same heart as in B but 15 minutes later but this time acetylcholine (0.5 11g/m1) was given through the sinus venosus. C. Epinephrine (0.5 pg /ml) administered through the coronary artery. D. Epinephrine (0.5 pg/ml) administered through the sinus venosus (same heart as D but 15 min. later). E. 47 3.0 A 2.5 0.5 3.5 1.5 12 sec. 12 sec. C 3.5 0_ 2.5 12 sec. E 2.5 0.5 12 sec. Figure 11 48 Figure 12. Graph of heart rate, pulse pressure, stroke volume and coronary pressure in the isolated salmon heart during administration of acetylcholine (1.0 pg /ml) first through the coronary artery (15-20 min) and then through the sinus venosus (35-40 min). The period of drug exposure-occurs between the dashed and solid lines. (n = 4, x ± S.E.). C n 0 0 to 0 0 0 1 V. (kPa) CORONARY PRESS. 9 cr, (MLS) STROKE VOL. ' (kPa) PULSE PRESSURE HEART RATE 50 during the 5 min. exposure time. But there was a gradual, but significant, increase in heart rate after epinephrine was replaced with drug-free saline. There was a slight, but insignificant rise in heart rate when epinephrine was perfused through the heart chambers. Pulse pressure and coronary pressure appeared to be unaffected by epinephrine through either route of administration. Stroke volume was not significantly altered by coronary administration of epinephrine but showed a significant decline (16.2%) when epinephrine was passed through the chambers of the heart (Fig. 11D, 11E and Fig. 13). Sodium Nitrite Sodium Nitrite (1 pg/m1) given through the coronary artery had no effect on heart rate, pulse pressure or stroke volume during the five minute perfusion time. Coronary arterial pressure showed a significant increase during this time and continued to increase after sodium nitrite was replaced with drug-free saline. in coronary pressure stabilized after 35 min. This rise Administration of sodium nitrite through the heart chambers showed no effect on any of the parameters measured (Fig. 1A, B and Fig. 15). Nitroglycerin The results of nitroglycerin (100 lig/m1) administration were inconsistent whether given through the coronary artery or the sinus venosus. During the 5 min. interval when nitroglycerin was perfused through the coronary vasculature no significant changes could be detected in any parameters (Fig. 14C). However, when nitroglycerin was discontinued, coronary pressure began to rise and pulse pressure showed a sharp decrease (Fig. 16). 51 Figure 13. Effects of epinephrine (0.5 lig/m1) on heart rate, pulse pressure, stroke volume and coronary pressure in the Drug was added first through the isolated salmon heart. coronary artery (15-20 min) and then through the sinus The period of drug exposure venosus (35-40 min). occurs between the dashed and solid lines (n = 4, + x - S.E.). 65 HEART RATE 45 PULSE 4 PRESS URE ( k Pa) 3 1 STROKE 0.4 VOLUME 0.3 CORONARY PRE SSURE (kPa) 2 H C 10 Figure 13 20 30 M INS. I 40 50 60 53 Figure 14. Strip chart recordings of heart rate, pulse pressure and coronary pressure from isolated salmon hearts. Upper recording is of heart rate and pulse pressure while the lower tracing is of coronary pressure. Vertical arrows indicate point of drug administration and horizontal Pressure in kPa. arrows indicate direction of recording. Sodium nitrite (1 pg/ml) administered through the coronary artery. A. Sodium nitrite (1 pg/ml) administered through the sinus venosus (same heart as in A,' 15 min later). B. Nitroglycerin (100 pg/ml) administered through the coronary artery. C. Nitroglycerin (100 pg/m1) administered through the sinus venosus (same heart as in C, 15 min later). D. 54 A 2.5 ['i.,1\ki'i'd'u'\,\,,I\Jklk,i'i\l"\i'dkillgjl\i'd\,1\ittill'i'ill'il,ildiliiilt;Vd 0.5 5.0 2.5 r-4 At` 0-- 12. sec. B C 2.5r 2.5 liklak* 1.0 3.0 4.0-- IteggiltIMAINNikl 1-1 1.57 12 sec. 0 12 sec. D 3.0 0. 5- 2.0 [e.*".~V,...JAAMMVAMMoMyanyowwwWhy,wo004", 1-4 12 sec. 0 Figure 14 55 Figure 15. Changes in heart rate, pulse pressure, stroke volume and coronary pressure in the isolated salmon heart The drug was exposed to sodium nitrite (1 pg/ml). administered first through the coronary artery (15-20 min) and then through the sinus venosus (35-40 min). The period of drug exposure occurs between the dashed and solid lines (n = 4, x ± S.E.). O O CORONARY PRESS. (kPa) (AILS) r) O (kPa) STROKE VOL. PULSE PRESSURE CT CT 4=. HEART RATE c^1 cn 57 Figure 16. Changes in heart rate, pulse pressure, stroke volume and coronary pressure in the isolated salmon heart exposed to nitroglycerin (100 pg /ml). The drug was administered first through the coronary artery (15-20 min) and then The period of through the sinus venosus (35-40 min). drug exposure occurs between the dashed and solid lines ± S.E.). (n = 4, x CORONARY PRESSURE(kPa) STROKE VOLUME PULSE PRESSURE(kPa) HEART RATE 59 Administration through the heart chambers in some cases resulted in a negative chronotropic and positive inotropic response (Fig. 14D) while increasing stroke volume and decreasing coronary pressure, (Fig. 16). However, in other cases, nitroglycerin was entirely without effect. The data for nitroglycerin are inconclusive and more work is needed. See Table II for a summary of the results. 60 Summary of the Effects of Acetylcholine, Epinephrine TABLE II. and Vasodilators on the Isolated Salmon Heart Systolic Pressure Stroke Volume H* C H C H C H 44 T ++ fi ++ + t -- + -- Heart Rate Acetylcholine (0.5 pg/m1) Epinephrine (0.5 pg/ml) Sodium Nitrite Coronary Pressure -- -- -- -- -- -- -- -- f -- (1 pg/ml) Nitroglycerin (100 pg/ml * C = Coronary; H = Heart chambers ** = no change + = P < 0.05 44 = P < 0.01 61 DISCUSSION The interpretation of the data obtained in this study will rely heavily on comparisons to mammalian studies since very little information exists on the pharmacology and physiology of the fish coronary system. Sculpin The negative results obtained with perhexiline, sodium nitrite, and nitroglycerin indicate that in the fish heart these drugs have no direct effect on the myocardial tissue itself. Winsor (1970), using dogs, demonstrated that the major pharmacological properties of perhexiline was dilation of the coronary and systemic vascular beds resulting in increased coronary flow and decreased heart rate. During exercise-induced tachycardia, Winsor also described a propranolol-like effect of perhexiline in lowering maximum heart rate during exercise. However, perhexiline did not lower the resting rate and it is not considered a beta blocker. Winsor describes a reflex mechanism for the action of perhexiline on the heart. As coronary flow is increased and systemic vascular resistence decreased the heart rate slows in response to a lower work load. Though Hudak, et al., (1970) concurs with Winsor on the dominant action of perhexiline on the systemic and coronary vascular beds, they suggest that perhexiline-induced bradycardia in dogs is due to a direct effect on cardiac membrane. If there is a direct effect of perhexiline on the cardiac membrane it is not demonstrated in the present study. This conflict with Hudak's findings could well be due to species differences. 62 The vasodilatory effect of the nitrates and nitrites in mammals is well documented in the literature. Their principal pharmacological action is dilation of vascular smooth muscle. Cardiac output, coronary blood flow, ventricular work and myocardial efficiency have been reported to be increased, decreased or unchanged by nitroglycerin. Using a modification of the Warburg.techniques, Honig, et al., (1960) found that the oxygen uptake of cardiac muscle was unaffected by direct exposure to nitroglycerin. They also found that cardiac efficiency was unchanged by nitroglycerin in the isolated, but metabolically supported, heart functioning at constant rate and volume. It seems unlikely that the nitrates and nitrites act by altering the oxygen consumption of resting heart muscle. Nitroglycerin administered in vivo and in vitro in the rat heart was reported by Ogawa, et al., (1967) to significantly inhibit the monoamine oxidase activity of the rat heart mitochondria and thereby increasing the norepinephrine content of the heart. If nitroglycerin has any MAO inhibiting activity in the fish heart it was not detectable in any of the physiological parameters measured in this study. The bradycardia elicited by prenylamine in the sculpin heart concurs with the results reported for mammalian hearts. Obianwu (1967) found that in various mammalian species prenylamine lowered mean arterial blood pressure and heart rate and potentiated the responses to norepinephrine and epinephrine in vivo. Using eats, Nielsen and Owman (1967) reported that prenylamine resulted in reduction in the catecholamine content of both the central nervous system and the sympatho-adrenal system thus preventing spontaneous 63 development of ventricular fibrillation during hypothermia by interfering with peripheral adrenergic mechanisms. In rats, 100 mg/kg of prenylamine administered subcutaneously reduced the serotonin level of the brain and the myocardium by 30% and the norepinephrine concentration by 50-60% (SchOne and. Lindner, 1960). The catecholamine- depleting action of prenylamine could possibly account for the bradycardia found in the buffalo sculpin. Salmon The vasodilatory action of acetylcholine in the mammalian cardiovascular system is well documented. In this study acetylcholine caused constriction of the coronary artery of the salmon heart. When considering the origin of the coronary vasculature of the fish heart this acetylcholine-induced vasoconstriction, might be expected. The coronary artery in fish originates from an epibranchial artery (Watson and Cobb, 1979) and acetylcholine produces vasoconstriction of the branchial vasculature (Randall, 1970). Belaud and Peyraud (1971), working with the isolated heart of the Conger eel (Conger conger L.), reported an acetylcholine-induced vasoconstriction in the cannulated coronary artery. blocked by atropine. This vasoconstriction could be However, Santer and Walker (1980), studying the morphology of the ventricles of teleost and elasmobranch hearts, were unable to detect a coronary vasculature supply in either Conger conger or Anguilla anguilla. What Belaud and Peyraud refer to as a coronary artery may have been an epicardial vascular system. Santer (1976), working with older (9-16 years) plaice (Pleuronectes platessa), found in the hearts of older fish only an extensive epicardial 64 vascH1,,,.r system, as revealed by arterial and venous profiles. This vascular system was confined to the epicardium and did not penetrate the myocardium to form a vascular bed, nor was it derived from extracardiac vessels. When administered through the coronary artery, acetylcholine resulted in a slight, but statistically insignificant, bradycardia. In the isolated mammalian heart mounted on a Langendorff perfusion apparatus administration of acetylcholine results in marked bradycardia (Broadley, 1979). The bradycardia was accompanied by slight but insignificant increases in pulse pressure and stroke volume. The acetylcholine-induced bradycardia resulting from administration through the sinus venosus of the salmon heart was significantly, greater, indicating that route of exposure is quite important. A possible explanation for this difference in response may involve the concentration of acetylcholine coming in contact with the sinoatrial (SA) node. Coronary blood flow in the fish empties into the coronary sinus at the base of the atrium after passing through the coronary vasculature of the sinus, atrium and ventricle. In this study coronary perfusion rate was 0.4 ml/min in contrast to an atrial perfusion rate of 8 ml/min so that the relative amount of perfusate entering the atrium through the coronary route was minimal. Therefore the SA node will be exposed to a greater quantity of drug per unit of time when the drug is perfused through the atrium than when the same concentration of drug is administered through the coronary vasculature. Furthermore, in this preparation, any drug entering the atrium through the coronary sinus was being diluted by 65 perfusate entering from the sinus venosus, thus further diminishing the quantity of drug reaching the SA node. In the classical Langendorff heart preparation, using the isolated guinea pig heart, 0.5 pg of acetylcholine administered through the coronary artery resulted in a significant bradycardia shortly after administration of the drug In the mammalian heart there appears to be better (Broadley, 1979). communication between the coronary vasculature and myocardial tissue, allowing neurohumoral agents access to receptors within the myocardium. Acetylcholine has no chronotropic or inotropic effects within the salmon heart until it reaches the atrium indicating that acetylcholine is not eliciting any effect on the myocardial tissue while it is within the coronary artery. This may be.due to impermeability of the coronary wall or to a lack of cholinergic receptors in the ventricular myocardium. Accompanying the bradycardia resulting from atrial perfusion of acetylcholine were increases in pulse pressure and stroke volume. As the heart slowed it had more time to relax, thus increasing the tension on the muscle fibers resulting in greater force of contraction and stroke volume. An increase in coronary pressure resulted from atrial perfusion of acetylcholine. This may have been an indirect effect due to extravascular compresSion from the increased force of contraction and greater filling of the heart. The initial reaction to the acetylcholine was transient cardiac arrest which caused the atrium to fill maximally, thus creating a back pressure at the coronary sinus. This could have also contributed to the increased coronary pressure. 66 Epinephrine (0.5 pg/ml) administered through the coronary artery had no significant effect on any of the parameters measured. When given through the chambers of the heart, rate was increased and stroke volume fell. In the fish cardiovascular system, epinephrine is reported to produce positive chronotropic and positive inotropic effects (Capra and Satchel], 1977). Broadly (1979) reported a biphasic response in the coronary artery of the isolated guinea pig to epinephrine. An initial short vasoconstriction was followed by a more prolonged and prominent fall in pressure. Epinephrine is reported to produce vasodilation in fish branchial vasculature. (Ostlund and Fnge, 1962) thus the coronary vasculature might be expected to respond in a similar manner. The absence of effects may be dose related, perhaps requiring a higher dose. Tachycardia produced by atrially perfused epinephrine appeared to have no indirect effects on coronary vasculature through extravascular compression. The rise in coronary pressure produced by NaNO2, indicative of vasoconstriction, is counter to the expected vasodilation normally seen with the nitrates and nitrites in mammals. continued even after discontinuing NaNO2. Vasoconstriction Accompanying this vasocon- striction was an insignificant rise. in rate and fall in pressure. Rubin, et al., (1963) reported a transient increase in heart rate and lowered systemic arterial pressure when NaNO2 was administered to the dog. The transient tachycardia in the dog may have been a feedback response to the lowered arterial pressure. It is difficult to speculate on the probable cause of this vasoconstriction. More 67 information is needed on the physiology and pharmacology of the fish coronary arterial system. Sodium nitrite administered through the atrium had no significant effects on any of the parameters being measured although there was a noticeable fall in heart rate. As in the sculpin it appears that the nitrites and nitrates do not exert any statistically significant direct effect on the myocardial tissue of the chum salmon. Their predominent effect appears to be on the vasculature with secondary effects on the heart itself. Nitroglycerin, as with NaNO2, appears to have no direct effect on the myocardial tissue of the chum salmon. 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A typical teleost heart performs the same basic function as the mammalian heart with the exception that it consists of one atrium and one ventricle pumping venous blood to the gills for oxygenation and from here the blood passes to the systemic circulation. To perform its task effectively the myocardial tissue itself must receive an oxygenated blood supply. In the mammalian heart, coronary arteries, which arise at the root of the aorta, supply the heart muscle with oxygenated blood. The right coronary artery supplies principally the right ventricle and atrium while the left coronary supplies the left ventricle and atrium. In the dog the left coronary artery supplies about 85% of the myocardium, whereas in humans the relative flow in the right and left coronary arteries is more variable, with the greatest percentage of individuals showing dominance of the right artery. The venous drainage of the coronary system can be divided into (Berne and Levy, 1977) a superficial drainage system consisting of the anterior cardiac veins and (Berne, 1974) the great cardiac veins, all of which drain into the coronary sinus (Berne, 1974). 76 Most of the coronary venous blood, after passing thorugh the myocardial capillary beds, returns to the right atrium through the coronary sinus while some reaches the right atrium via the anterior coronary veins. A second deep system of veins, the arteriosinusoidal, the arterioluminal and the thebesian vessels provide vascular communication between the myocardial vessels and the chambers of the heart. These deeper veins represent only a minor contribution to the total The arteriosinusoidal channels are small arteries which lose flow. their arterial structure as they penetrate the chamber walls and divide into irregular, endothelium-lined sinuses. These sinuses connect with other sinuses and with capillaries and communicate with the cardiac chambers. The arterioluminal vessels are small arteries or arterioles that open directly into the atria and ventricles. The thebesian vessels are small veins that connect capillary beds directly with the cardiac chambers and also communicate with cardiac veins and other thebesian veins. It appears that the minute vessels of the myocardium form an intercommunicating plexus of subendocardial vessels. It is postulated that myocardial tissue can derive nutrition from the cardiac chambers through these channels. In the mammal it has been found that blood in the cardiac chambers penetrates a short distance into the endocardium, but does not constitute a significant source of oxygen and nutrients to the myocardium (Berne and Levy, 1977). Factors Influencing Coronary Blood Flow Perfusion of the coronary vasculature is determined by the aortic pressure which is generated by the heart itself. Alterations 77 in aortic pressure influence the coronary blood flow, but other factors operate to maintain an adequate flow of blood to the myocardium. The ability to maintain a relatively stable coronary flow rate during changes in perfusion pressure is termed autoregulation of blood flOw and three mechanisms have been proposed to explain this; these are the tissue pressure, metabolic and myogenic hypotheses (Abel and McCutcheon, 1979). The tissue pressure hypothesis proposes that as perfusion pressure increases so does capillary hydrostatic pressure and this results in greater movement of fluid from the intravascular compartment to the interstitial space. interstitial pressure serves to compress increase vascular resistance. This elevated the thin-walled vessels and A reduction in perfusion pressure would therefore lead to reduced resistance. According to the metabolic hypothesis vascular resistance is closely linked to myocardial metabolic activity and that a chemical mediator produced in the tissue acts to change vascular resistance in accordance with the metabolic activity of the heart. (Increased myocardial metabolic activity results in lowered coronary resistance whereas reduced cardiac metabolism increases coronary resistance). The myogenic hypothesis maintains that stretch or increased tension of the vascular smooth muscle, produced by increased perfusion pressure, elicits an enhanced contractile response of the smooth muscle and thereby returns blood flow to the level existing before elevation. Extravascular compression produced by the contracting myocardium influences coronary blood flow by its squeezing effect on the coronary blood vessels. Depending on the diastolic pressure gradient and the 78 diastolic time, 70 to 90 percent of the coronary flow normally occurs during diastole. Flow in the coronary sinus tends to be opposite in phase to the arterial inflow pattern, venous flow is increased during systole because contraction forces blood out of capillary and venous beds (Berne and Levy, 1977). During tachycardia, when diastolic time is shortened, the heart spends more time in the contracted state, thus increasing extravascular resistance and restricting coronary blood flow. Because of increased myocardial metabolic activity during tachycardia the net effect on coronary resistance is a decrease due to the reduction in intravascular resistance by dilation of the arterioles. During bradycardia mechani- cal restriction of coronary blood flow is less as are the metabolic demands of the myocardium. Metabolic factors, which reduce intra- vascular resistance, predominate over mechanical factors, which increase extravascular resistance, in conditions such as tachycardia. Neural Regulation of the Coronary Vasculature The coronary vasculature receives both sympathetic and parasympathetic innervation (Berne, 1974; Berne and Levy, 1977; Abel and McCutcheon, 1979). Although circulating acetylcholine acts as a vasodilator in mammalian coronary arteries, the role of coronary cholinergic fibers is not clear. Vagus nerve stimulation has little direct effect on coronary arterial resistance. In the fibrillating heart with the coronary arteries perfused at a constant pressure, or in the beating, paced heart with flow measured in late diastole when there is essentially no extravascular compression, small increments in coronary blood flow can be observed with stimulation of the 79 peripheral ends of the vagus (Berne and Levy, 1977). It is likely that the vagi exert less effect on coronary resistance in the normal animal, because of the overriding influence of metabolic mechanisms (Berne and Levy, 1977). Acetylcholine-induced coronary artery vasodilation can be blocked by atropine. Coronary dilation can occur reflexly via the vagi by chemoreceptor activation. In order to evaluate the direct effects of sympathetic nerve stimulation on the coronary vasculature it has been necessary to eliminate the accompanying inotropic, chronotropic and metabolic factors which mask the effects on the coronary vessels. Both alpha and beta receptors have been demonstrated in the coronary vasculature. The larger surface vessels contain primarily alpha receptors while the deeper nutrient vessels are largely beta controlled. Additionally, betal receptors predominate in the larger vessels and beta2 receptors predominate in the smaller one (Berne, 1974). Using hearts in ventricular fibrillation it has been found that NE causes an initial coronary constriction followed quickly by vasodilation. The physio- logical importance of the coronary beta receptors has been questioned due to the interference of cardiac inotropic and chronotropic responses of sympathetic nerve stimulation. By using practolol, myocardial, but not vascular beta receptors can be blocked. Stimulation of the cardiac sympathetic nerves after practolol administration results in an increase in coronary resistance and this increased coronary resistance is attenuated if the alpha receptors are blocked with phentolamine (Abel and McCutcheon, 1979). 80 Coronary resistance can also be altered reflexly by the baroreceptors. In addition there seems to be some tonic activity in the sympathetic fibers that innervate the coronary resistance vessels, since acute cardiac denervation results in some decrease in coronary resistance (Abel and McCutcheon, 1979). Metabolic Factors Regulating Coronary Blood Flow The levels of myocardial metabolic activity and the magnitude of coronary blood flow are closely correlated. A direct relationship exists between the oxygen requirements of the myocardium and the degree of coronary blood flow. This relationship is seen in the perfused isolated heart as well as the intact heart. With the onset of ventricular fibrillation, there occurs an increase in coronary blood flow due to reduced myocardial metabolic demands and reduced extravascular compression. prefibrillation level. Flow then gradually returns toward the This increase in coronary resistance, which occurs despite the elimination of the extravascular compression, is a manifestation of the heart's ability to adjust its blood flow to meet its energy needs (Berne and Levy, 1977). The metabolic factors that control coronary blood flow appear to be significant (Berne, 1974). Numerous metabolic agents have been suggested as the mediator of coronary blood flow. Some of the substances implicated are CO2, 02, lactic acid, hydrogen ions, histamine, potassium ions, increased osmolarity, polypeptides, and adenine nucleotides. None of these agents has satisfied all the criteria for the physiological mediators, although adenosine seems to be the most acceptable (Berne and Levy, 1977). 81 Adenosine and its phosphorylated derivatives are all powerful vasodilators. When the myocardial metabolic rate increases, resulting in increased oxygen consumption, there is a decrease in myocardial partial pressure of oxygen. This leads to a decrease in ATP and an increase in inorganic phosphate, thereby increasing the amount of adenosine. As the extracellular concentration of adenosine increases, it produces a vasodilation of the coronaries and an increase in coronary blood flow to restore myocardial tissue oxygen to normal. The increase in blood flow washes out the additional adenosine, thus operating as a feedback mechanism to decrease adenosine and the resultant coronary vasodilation (Berne, 1974). Coronary Vasodilators The drugs employed as coronary vasodilators,,although used for conditions involving regional tissue ischemia, do not act specifically on the coronary vasculature but on the general systemic vasculature. A host of chemically dissimilar agents fall under the category of vasodilators. The earliest group of chemicals to be used as vasodi- lators were the nitrites and nitrates. Organic nitrates have been used for the relief of anginal pain since the 1800's. Despite all the research done on nitroglycerin and its widespread use, its mechanism of action remains controversial. The basic pharmacological action of the nitrates and nitrites is to relax smooth muscle, particularly vascular smooth muscle. The organic nitrates act as rapidly as the nitrites and do not liberate NO2 in the blood (Rath and Krantz, 1942; Krantz, et al., 1962; Nickerson, 1975), thus it appears that the nitrates have a direct 82 action on smooth muscle. The relief of angina pectoris by nitro- glycerin was attributed to vasodilation of occluded coronary vessels and thereby restores bloow flow to the ischemic myocardial tissue. However, it has been pointed out by Winbury (1969), that in an ischemic area tissue hypoxia probably already causes nearly maximal 'arteriolar dilation because of autoregulation (Winbury, 1969). The nitrates and nitrites produce both a local and systemic redistribution of blood flow (Mason and Braunwald, 1965; Fam and McGregor, 1968; Winbury, 1969; Winbury, et al., 1971; Becker, 1971; Horowitz, et al., 1971; Cohen, et al., 1973). Mason and Braunwald (1965) found that nitroglycerin reduced systemic arterial Pressure, elevated forearm blood flow, lowered forearm vascular resistance and decreased venous tone. By reducing the tone of the capacitance vessels, venous return to the heart is decreased thus diminishing the metabolic activity of the heart (Sharpey-Schafer, 1962; Ablad and Mellander, 1963; Robin, et al., 1967). At the local level the organic nitrates cause a redistribution of myocardial blood flow to the ischemic subendocardium by reducing extravascular compression on the deep lying capillaries and collaterals (Winbury, et al., 1971). By dilating the larger coronary conductance vessels blood is shunted to the passive collateral vessels leading to the ischemic area (Winbury, 1969; Winbury, et al, 1971; Fam and McGregor, 1968). Fam and McGregor (1964) suggest that nitroglycerin exerts its action only in the presence of well developed collateral circulation. Directly related to the reduction of coronary vascular resistance is oxygen delivery to the myocardial tissue. It is believed the 83 relief of ischemia results from enhancing oxygen delivery rather than by altering load (Honig, et al, 1960). However, they were able to find no effect of nitroglycerin on oxygen consumption in the resting heart muscle. Brachfeld, et al., (1959) found that following nitroglycerin administration, myocardial oxygen consumption increased, as did coronary flow, mediated by a lowered coronary vascular resistance. A reduction in 02 requirements, reported by Robin, et al., (1967) was attributed to decreased ventricular wall tension and velocity of myocardial fiber shortening after nitroglycerin.. Nitro- glycerin injected directly into the coronary artery selectively increased subendocardial oxygen tension due to a redistribution of coronary blood flow toward the subendocardium and a small direct reduction in cardiac metabolism (Weiss and Winbury, 1972). Another feasible therapeutic mechanism of the nitroglycerin type drugs is their action as functional antagonists of norepinephrine. Gillis and Melville (1970) found that nitroglycerin produced .only a transient increase in coronary flow and exerted significant blockade of the contractile force response induced by norepinephrine without affecting the contractile force response induced by Ca2+. No blockade of the heart rate response_ induced by norepinephrine was observed. Nitroglycerin also exerted significant blockade against the pressor effects of norepinephrine. Increasing doses of nitroglycerin produced a tolerance to both the coronary flow changes and the cardiac adrenergic blockade. effects. Very little tolerance developed to the hypotensive In addition to its coronary vasodilator action, nitroglycerin appears to exert a significant degree of cardiac blockade. The 84 .anti- epinephrine sympathetic effects of nitroglycerin regarding myocardial function suggest that the therapeutic action of this drug is not due to coronary dilation alone but also to a specific counteraction against the myocardial metabolic effects of an excess influx of adreno-sympathetic epinephrine in the heart (Raab and Humphreys, 1947; Darby and Aldinger, 1960). Systemic administration of nitroglyc- erin was found to cause a fall in systemic blood pressure, eliminated systolic bulging during coronary occlusion, and suppressed excitation of sympathetic afferent cardiac fibers (Bosnjak, et al., 1981). This nitroglycerin induced inhibition of cardiac afferents may have been due to reduction of substances which are released during myocardial ischemia, redistribution of epicardial to endocardial blood flow ratios, and elimination of systolic bulge. Related to its role as a functional antagonist of norepinephrine is its possible function as an MAO inhibitor. Ogawa, et al., (1967) demonstrated an increase in myocardial norepinephrine content after administration of nitroglycerin and found that rat heart mitochondrial MAO was inhibited. The increase in norepinephrine content after nitroglycerin administration may be explained by a reflex release of catecholamines (O'Rourke, et al., 1971). The nitrates, in particular the organic nitrates, have demonstrated strong inhibition of ATPase activity (Krantz, et al., 1951; Honig, et al., 1960). In summary the major therapeutic mechanisms of nitroglycerin are: 85 1. A reduction in capacitance vessel resistance leading to diminished venous return. This has a beneficial effect on myocardial oxygen requirements and coronary endocardial perfusion by reducing mechanical compression. 2. Dilation of large coronary vessels resulting in increased collateral and redistributed flow. 3. Central antagonism of the afferent limb of reflex vasoconstrictor innervation of coronary vasculature. Perhexiline Perhexiline maleate [2-(2,2-dicyclohexylethyl) piperidine] increases myocardial blood flow and decreases oxygen consumption in experimental animals and man. The dominant action of perhexiline is vasodilation of both coronary and systemic vascular beds (Hudak, et al., 1970; Winsor, 1970). Perhexiline has been reported to have a propranolol-like effect (lowering maximum heart rate during exercise); however, it does not lower the resting rate and is not considered a beta-blocker (Winsor, 1970). Perhexiline differs from nitroglycerin in that coronary flow is maintained above control values with perhexiline despite a 40% fall in mean arterial blood pressure and it acts preferentially on the arterioles rather than on postarteriolar vessels (Winsor, 1970; Hudak, 1970). To summarize the effects of perhexiline found by Hudak (1970) and Winsor (1970): 1. Increases coronary flow 2. Reduces heart rate (especially exercise-induced tachycardia) 3. Nonspecific inhibition of cardiac responsiveness to sympathetic stimulation which may involve inhibition of ionic transport 86 4. A diuretic effect, possibly leading to ventricular volume and tension reduction 5. Quinidine-like effect on cardiac cells which provides anti-dysrhythmia activity. Prenylamine Prenylamine IN(3'-phenyl-propyl-(2')-1,1-diphenyl-propyl-(3)amine; Segontin] is another agent employed in coronary insufficiency. Its principle action appears to be moderate depletion of peripheral and central catecholamines (Bohm, et al., 1960; Kochsiek, et al., 1960; Juorio and Vogt, 1965; Nielson and Oman, 1967). Obianwu (1967) found that in various animal species prenylamine lowered the mean arterial blood pressure and the heart rate, antagonized responses to tyramine and isoprenyline and potentiated those to norepinephrine and epinephrine. The potentiation of the actions of norepinephrine and epinephrine after prenylamine is due to inhibition of the uptake process of these amines at specific binding sites. Prenylamine exhibits alpha and beta blocking action in vitro but only moderate beta blockade in vivo (Obianwu, 1967). Prenylamine's beta blocking action may account for its beneficial effects in angina] patients. Juorio and Vogt (1965) have likened the action of prenylamine to the weaker analogs of reserpine. Granular amine storage mechanisms in adrenergic nerves are blocked by prenylamine (Nielsen, 1967). In vitro and in vivo tests with prenylamine have demonstrated good vasodilator effects on the coronary vasculature without having significant hemodynamic side effects such as decreased blood pressure or increased heart rate (Kochsiek, et al., 1960; Lindner, 1960). 87 Prenylamine appears also to have very little, if any, peripheral dilatory effect. In summary of prenylamine: 1. Increases coronary flow 2. Depletes cardiac catecholamines and inhibits calcium influx into cardiac cells thereby reducing basal tone and responsiveness to sympathetic discharges and consequent energy requirements. 3 Causes a slightly sustained decrease in blood pressure which lowers aortic resistance and ventricular compression, favoring reduced cardiac work and better coronary perfusion, respectively.