was—— Hf- EF‘FE‘CTS OF SEROTONI-N ('5 - HYDROXYTR‘YPTAMI'NE) ON MUSCLE VASCULAR RESISTANCE Thesis for the Degree of M. S. MICHIGAN STATE UNIVERSH’Y PAUL DANIEL MEIER 1969 ABSTRACT EFFECTS OF SEROTONIN (5-HYDROXYTRYPTAMINE) ON MUSCLE VASCULAR RESISTANCE BY Paul Daniel Meier Knowledge about the cardiovascular effects of serotonin in some of the vascular beds has grown in recent years, but the effects of serotonin (5-hydroxytryptamine) on vascular resistance in the muscle bed have not yet been established. Therefore, serotonin was infused at six different rates into isolated, collateral-free, innervated and denervated gracilis muscles of dogs. Vascular resistance was artificially raised in muscles with low initial resistance and artificially lowered in muscles with high initial resistance. Both natural flow and constant flow experiments were conducted. Serotonin was found to consistently lower the total vascular resistance in initially high and artificially high resistance muscles. Serotonin consistently raised total vascular resistance in initially low and artificially low resistance muscles. Paul Daniel Meier The decrease in resistance caused by serotonin in muscles with high initial resistance could be reversed to an increase in resistance when the resistance was subsequently lowered by metabolically induced vasodilation. This implies that the muscle's response to serotonin does not depend upon neurogenic tone, but rather upon the level of total muscle vascular resistance at the time serotonin is infused. EFFECTS OF SEROTONIN (5*HYDROXYTRYPTAMINE) ON MUSCLE VASCULAR RESISTANCE BY Paul Daniel Meier A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Physiology Department 1969 (7533791 /Cyfl227/@7' To my parents, Mr. and Mrs. Alexander Meier and to my wife, Jan ii ACKNOWLEDGEMENTS I would like to eXpress my deep appreciation to Dr. Thomas E. Emerson, Jr., for his invaluable advice and technical assistance. I am also grateful to Dr. Francis J. Haddy for his periodic advice throughout this project. iii TABLE OF CONTENTS INTRODUCTION REVIEW OF LITERATURE INTRODUCTION DISCOVERY AND EARLY DEVELOPMENT STRUCTURE AND METABOLISM OF SEROTONIN LOCATION OF SEROTONIN IN THE BODY CARDIOVASCULAR-RELATED ASPECTS OF SEROTONIN Variability of vascular responses to serotonin Transport of serotonin in the blood stream Effects of serotonin on the heart Effects of serotonin on systemic blood pressure Effects of serotonin in specific vascular beds ‘ Serotonin antagonists MATERIALS AND METHODS RESULTS DISCUSSION SUMMARY AND CONCLUSIONS BIBLIOGRAPHY iv LIST OF TABLES RESISTANCE CHANGES (mmHg/cc/min/IOOg); NATURAL FLOW EXPERIMENTS WITH LOW INITIAL RESISTANCE RESISTANCE CHANGES (mmHg/cc/min/lOOg); CONSTANT FLOW EXPERIMENTS WITH LOW INITIAL RESISTANCE RESISTANCE CHANGES (mmHg/cc/min/lOOg); NATURAL FLOW EXPERIMENTS WITH HIGH INITIAL RESISTANCE RESISTANCE CHANGES (mmHg/cc/min/lOOg); CONSTANT FLOW EXPERIMENTS WITH HIGH INITIAL RESISTANCE RESISTANCE CHANGES (mmHg/cc/min/lOOg); CONSTANT FLOW EXPERIMENTS IN MUSCLES WITH LOW INITIAL RESISTANCE AND WITH RESISTANCE SUBSEQUENTLY INCREASED BY SYMPATHETIC NERVE STIMULATION RESISTANCE CHANGES (mmHg/cc/min/lOOg); CONSTANT FLOW EXPERIMENTS IN MUSCLES WITH HIGH INITIAL RESISTANCE AND WITH RESISTANCE SUBSEQUENTLY LOWERED BY MOTOR NERVE STIMULATION LIST OF FIGURES Effects of I.A. serotonin infusion in gracilis muscles with low initial resistance. Flow = natural Effects of I.A. serotonin infusion in gracilis muscles with high initial resistance. Flow = natural Effects of I.A. serotonin infusion in gracilis muscles with low initial resistance. Flow = constant Effects of I.A. serotonin infusion in gracilis muscles with high initial resistance. Flow = constant Effects of I.A. serotonin infusion on total vascular resistance in muscles with low initial resistance and with resistance subsequently increased by nerve stimulation. Flow = constant Effects of I.A. serotonin infusion on total vascular resistance in muscles with high initial resistance and with resistance subsequently lowered by exercise dilation Effects of I.A. serotonin infusion on total vascular resistance in muscles with high initial resistance; in the same muscles after lowering resistance by exercise dilation; and in the same muscles again after subsequently raising resistance to a high level by sympathetic nerve stimulation. Flow = constant vi Effects of I.A. serotonin infusion on total muscle vascular resistance in 15 experiments with initial resistance levels near 12.3 units. Natural and constant flow experiments were both used in this group. Effects of I.A. serotonin infusion on total vascular resistance in all experiments combined. vii INTRODUCTION Serotonin is synthesized in the brain (White, Handler and Smith, 1964) and intestines (Humphrey and Toh, 1954) from tryptophan (Stacey, 1959; Gailitis and Sheiber, 1960; Enerback, 1965; Warner, 1967). It is distributed through- out the body, primarily by the platelets (Stacey, 1959; Whelan, 1959; Marshall, 1966). Serotonin's effects within the circulatory system are many and varied. It cannot be classified as either a pressor or depressor agent, since it may elevate, lower, or not affect blood pressure (Haddy, 1960). Many differences in vascular response to serotonin seem to be correlated with differences in pre-existing levels of neurogenic tone (Page, 1952, 1953; Page and McCubbin, 1956; Haddy, 1960; Garattini and Valzelli, 1965; Haddy and Scott, 1966; Emerson gt 31., 1968). Chemoreceptor stimulation also plays a role in vas- cular responses to serotonin (Page, 1952; McCubbin, Green and Salmoiraghi, 1956; Braun and Stern, 1961; Woolley and Shaw, 1962; Hamberger, Ritzen and Wersall, 1966). Serotonin causes a variety of responses in the different vascular beds (Takacs and Vajda, 1963; Garattini and Valzelli, 1965), including opposite effects in parallel skin and muscle beds (Daugherty gt a1.,1968; Emerson et al,, 1968). II. REVIEW OF LITERATURE A REVIEW OF SEROTONIN AND ITS EFFECTS IN THE CARDIOVASCULAR SYSTEM INTRODUCTION. A serum vasoconstrictor agent was known to exist over a hundred years ago (Heinzelman and Weisblat, 1951; Spies and Stone, 1952). But until 1948, Scien- tists were unable to isolate and study this vasoconstric- tor agent, which later became known as "serotonin" (Rapport, Page and Green, 1948; Rapport, 1949; Heinzel- man and Weisblat, 1951; Reid, 1952). DISCOVERY AND EARLY DEVELOPMENT. The existence of a potent vasoconstrictor agent in the sera of mammals was reported as early as 1868, by Ludwig and Schmidt, two German medical doctors (Heinzelman and Weisblat, 1951; Spies and Stone, 1952). Rapport, in 1948, described this agent as a "vasocon— strictor which is present in serum and defibrinated blood and which appears in connection with platelet destruction and the clotting process (Rapport, Page and Green, 1948)." Rapport was first to isolate this agent when he made a stable preparation of it in the form of a nitro-barbiturate complex. After further purification, it was shown on chemical grounds to be an indole derivative, 5—hydroxytryptamine (Rapport, 1949; Heinzelman and Weisblat, 1951). Rapport and his associates injected the substance intravenously into a dOg and found that it increased arterial pressure, and then they decided, "We should like provisionally to name it 'serotonin', which indi- cates that its source is serum and its activity is one of causing constriction (Rapport, Page and Green, 1948)." S-Hydroxytryptamine was first synthesized in August, 1951, by Hamlin and Fischer (1951). At about the same time Heinzelman and Weisblat (1951) also synthesized 5-hydroxytryptamine from S-benzyl- oxyindole and converted it to the creatinine sulfate salt. Shortly thereafter, Reid and Rand (1951) pre- pared serotonin from beef serum and ran a series of experiments with it in an assortment of animals and found that serotonin caused a rise in pulmonary and systemic arterial pressures in the cat; contraction of isolated arteries of sheep, dogs and oxen; con— striction of the guinea pig jejunum and uterus, rat uterus, nictitating membrane and pupillary sphincter of the cat; broncho-constriction of the vessels of the hind limb and kidney of the cat (Reid and Rand, 1951). In 1952, Stone and Spies (1952) injected 0.25—0.50 mg of serotonin into patients and noticed a sharp rise in systolic and diastolic blood pressures. III. In the ensuing years, interest and research in the area of serotonin mushroomed rapidly. STRUCTURE AND METABOLISM OF SEROTONIN. Serotonin (5—hydroxytryptamine) has the following structural formula (White, Handler and Smith, 1964): H0—— CHJ:—--CH,~-"'NHl 1 N 5-Hydroxytryptamine is synthesized in the brain (White, Handler and Smith, 1964) and in the intestines (Humphrey and Toh, 1954). In_yiyg, tryptophan is hydroxylated to 5-hydroxytryptophan, apparently by phenylalanine hydroxylase, and then decarboxylated by the enzyme S-hydroxytrytophan decarboxylase to yield 5-hydroxy- tryptamine, or serotonin (Stacey, 1959; Gailitis and Sheiber, 1960; Enerback, 1965; Warner, 1967). Sero- tonin is converted by mono-amine oxidase to an in- active compound called S-hydroxy—B-indolacetic acid, which is excreted in urine at 2-7 mg/day in man IV. (Borges and Bessman, 1957; Gailitis and Sheiber, 1960; White, Handler and Smith, 1964). It has been estimated that about three per cent of dietary tryptophan is metabolized via this pathway (White, Handler and Smith, 1964). Alcohol inhibits serotonin metabolism by depressing its biotransformation via oxidation and conjugation (Rosenfeld, 1960). Gursey and Olson (1960) have presented evidence which indi- cates that intravenous injection of 2 g/kg of ethanol results in decreased serotonin and norepinephrine levels in rabbit brain stems. Tryptophan—deficient rats also showed a significant decrease in serotonin content in all tissues analyzed (Gal and Drewes, 1962). Moran, Uvnas and Westerholm (1962) report that allicin, an SH group inhibitor, and ninhydrin, an NH2 group inhibitor, also intervene with serotonin metabolism. LOCATION OF SEROTONIN IN THE BODY. Vast amounts of serotonin are found throughout the brain and central nervous system (Woolley and Shaw, 1954; Borges and Bessman, 1957; Stacey, 1959; Woolley, 1960; Burton, 1966; Marchbanks, 1966), where it may play a role in mental processes and nervous transmis- sion (Woolley and Shaw, 1954; Whelan, 1959; Woolley, 1960). Serotonin has also been found throughout the gastrointestinal tract (Reid and Rand, 1951; Rosenberg, 1965; Davenport, 1966). Serotonin has been found in the uterus and placenta (Reid and Rand, 1951; Klinge, Penttila and Tissari, 1964), where it is reported to increase in concentration during pregnancy (Koren, Pfeifer and Sulman, 1965), and also in the spleen (Sanker 35 31., 1961), pancreas (Tobe, Fujiwara and Tanaka, 1966), kidney (Whelan, 1959), adrenal medulla (Stacey, 1959), and the pineal gland (Giarman and Freedman, 1960). Snyder, Zweig and Axelrod (1964) reported that a major portion of the serotonin in mammalian pineal glands is stored in sympathetic nerve endings. They also reported that a circadian rhythm in serotonin content of the rat pineal gland exists with its peak at noon and trough at about 10 PM, and that it is abolished by removal of the superior cervical ganglia. CARDIOVASCULAR-RELATED ASPECTS OF SEROTONIN. A. Variability of vascular reSponses to serotonin. The circulatory effects of serotonin are variable, depending not only on the species of animals and dose given, but also on variations in the same species under similar conditions (Schneider, 1954; Page and McCubbin, 1956; Kabins, Molina and Katz, 1959; Goodman, 1965). Haddy (1960) stated that "serotonin cannot be classified either as a pressor or depressor agent. Serotonin may lower, elevate or not affect blood pressure." Serotonin's effects upon blood pressure are so var— iable that Page and McCubbin (1956) created a new word, "amphibaric," to describe them. Haddy and Scott (1966) called the direct effects of serotonin on arterial and venous resistances “unusual." B. Transport of serotonin in the blood stream. DISTRIBUTION IN THE BLOOD Over half of the blood's supply of serotonin is contained in the platelets (Stacey, 1959; Whelan, 1959; Marshall, 1966). In rabbit platelets, serotonin appears to be bound to the granule fraction, and not to the platelet membrane (Wurzel, Marcus and Zweifach, 1965). Serotonin also is localized in mast cells, either through synthesis or specific uptake (Stacey, 1959; Moran, Uvnas and Westerholm, 1962; Enerback, 1963, 1965). Some serotonin also exists in the plasma (Robertson and Andrews, 1961) and serum (Davis, 1959). RELEASE FROM MAST CELLS The release of serotonin from mast cells is believed to be enzymatic and dependent upon pH and temperature (Moran, Uvnas and Westerholm, 1962). Allicin, an SH group inhibitor, and ninhydrin, an NH2 group inhibitor, block serotonin release from mast cells, but the injection of reserpine facilitates the release of serotonin until only 65% of the normal concentration remains (Moran, Uvnas and Westerholm, 1962). Cold exposure results in a marked increase in the number of mast cells in the abdominal skin, accompanied by the rapid and continuous excretion of serotonin in the urine (Leblanc, 1963). When mast cells are disrupted 13 21333, serotonin is released along with histamine, whose concentration in mast cells is about twenty times greater than that of serotonin (Archer, 1961). ABSORPTION OF SEROTONIN BY PLATELETS Zucker and Borrelli (1956) found that normal disc- shaped platelets readily absorbed serotonin. Weiss— bach and Redfield (1960) studied factors affecting the uptake of serotonin by human platelets in an inorganic medium and found that potassium and phOSphate are required for maximum uptake of serotonin at a pH of 5.7. At higher pH values, these ions had much less effect. Serotonin is taken up actively by platelets against a concentration gradient of several hundred to one, and its uptake is energy- and temperature-dependent (Burningham 33 31., 1966). An active transport system at the platelet surface is apparently responsible (Hughes and Brodie, 1959; Bridges and Baldini, 1966). Serotonin uptake, which is facilitated by phosphate and potassium ions (Weiss- bach and Redfield, 1960; Burningham 35 31., 1966), is inhibited by reserpine, fluoride, 2,4—dinitrophenol, chlor— promazine and tyramine (Burningham 3E 31., 1966). Platelets have a very high concentration of adenosine triphosphate (ATP), and the ATP content of platelets and their serotonin uptake are believed to be directly related (Burningham 3: 31., 1966). Platelets keep the concentration of free serotonin in plasma very low, and at low external concentrations the uptake is almost complete (Humphrey and Toh, 1954). Platelet serotonin is probably accumulated rather than synthesized in situ (Humphrey and Toh, 1954; Garattini and Valzelli, 1965). RELEASE OF SEROTONIN BY PLATELETS Serotonin is readily released from platelets under many circumstances (Reid, 1952), and its release apparently involves activation of enzymatic processes (Westerholm, 1966). Serotonin release is inhibited by heparin (Paasonen, 1965; Westerholm, 1966), and also by ninhydrin, and allicin (Westerholm, 1966). Honour and Mitchell (1963) discovered that when an artery is injured, white masses of platelets build up at the site of the injury and embolize. Swank and associates (1963) stated that serotonin greatly increases the tendency of the red blood Cells, leucocytes and platelets to aggregate. Differential centrifugation of homogenates of human platelets revealed that amino acids and potassium were largely free, but that serotonin was bound within granules 10 (Buckingham and Maynert, 1964). Platelets are not capable of synthesizing serotonin (Garattini and Valzelli, 1965), so most platelet serotonin probably originates in the enterochromaffin cells of the intestinal mucosa (Paasonen, 1965). C. Effects of serotonin on the heart. Intravenous infusion of serotonin usually results in tachycardia (Maxwell 3E_31., 1959; Noble and Nanson, 1959; Peskin and Miller, 1962), increased right ventricular work (Maxwell 33 31., 1959), and increased cardiac output (Takacs and Vajda, 1963). D. Effects of serotonin on systemic blood pressure. PRESSOR AND DEPRESSOR EFFECTS Hamilton (1966) calls serotonin "one of the most potent vasoconstrictors yet recognized." It must be remembered, however, that serotonin's effects vary, depending not only on the species of animals and doses given, but also on variations within the same species and under similar condi- tions (Schneider, 1954; Page and McCubbin, 1956; Kabins, Molina and Katz, 1959; Goodman, 1965). Injection of large doses of serotonin usually produces a pressor response in the systemic circuit (MacCanon and Howath, 1954; Braun and Stern, 1961). Page and McCubbin (1956) compared the effects of injection versus infusion of serotonin in the femoral veins of dogs. They found that a quick injection of serotonin 11 resulted in a pressor effect, whereas intravenous infusion at a slower rate, but with the same amount of serotonin, resulted in a sustained fall in arterial blood pressure. Other researchers have also found serotonin infusion to result in decreased arterial blood pressure and systemic vascular resistance (Maxwell 33 31., 1959; McGaff and Milnor, 1962). MacCanon and Howath (1954) found that in all their observations, both pulmonary and femoral arterial pressures began to rise within 3-6 seconds after the rapid injection of 500 ug of serotonin creatinine sulfate. The pressures reached maximum values in 12-20 seconds, and then decreased to control within 1-7 minutes. The mean recovery time was 2.75 minutes. BIPHASIC BLOOD PRESSURE RESPONSE TO SEROTONIN Gailitis and Sheiber (1960) injected 0.5-2.0 mg of serotonin into humans and reported a biphasic response: a brief fall in blood pressure followed by an overshoot for 1-5 minutes, accompanied by an increase in pulse rate. Reid (1952) also found that when he injected 36 ug of serotonin into dogs, there was an initial transient fall in blood pressure, succeeded by a pressor response which was followed in turn by a depression lasting for several minutes. The initial steep drop in systemic blood pressure was believed to be due primarily to vasoconstriction in the pulmonary circulation. When Reid intravenously injected 10-110 ug of 12 serotonin, the pressure in the pulmonary artery rose and, simultaneously, the pressure in the carotid artery fell. At the same time there was a fall in pulmonary venous pressure and a rise of pressure in the right atrium. Reid concluded that the rise of pressure in the pulmonary artery was due to an increase in pulmonary resistance and was not cardiac in origin. Serotonin liberates adrenaline from the suprarenal glands (Reid, 1952; Garattinin and Valzelli, 1965), and this may contribute to the pressor effect of large doses given intravenously (Reid, 1952; Reid and Rand, 1952; Garattini and Valzelli, 1965). But the drug also has an independent pressor action, since similar changes in blood pressure occur in adrenalectomized animals (Reid, 1952; Page and McCubbin, 1953). The secondary depressor phase may be due to decreases in either the peripheral resistance or the left ventricular output (Reid, 1952). The reduced peripheral resistance may be the result of a direct vasodilator action or an indirect action mediated through the nervous system, or it may be the result of the liberation of a vasodilator agent (Reid, 1952). Some of the depressor effects of serotonin may be the result of its ability to release histamine (Page and McCubbin, 1956; Moore, Normell and Eiseman, 1963; Goodman, 1965). 13 OPPOSITE RESPONSES IN LARGE AND SMALL VESSELS Haddy (1960) has stated, "It is clear that the calibers of arteries, veins and arterioles can actively change in opposite directions." Some evidence indicates that serotonin actively constricts large vessels at the same time that it actively dilates small vessels (Haddy, Fleishman and Emanuel, 1957; Haddy, 1960). Haddy (1960) found that when he ad- ministered serotonin into the brachial artery in amounts small enough to have no noticeable effect on systemic arterial and venous pressures, the small artery pressures fell while the pressures in the small veins rose in the dog forelimb, with no net change in blood flow through the limb. Histamine, which is released during the administration of serotonin (Page and McCubbin, 1956; Haddy, 1960; Moore, Normell and Eiseman, 1963; Goodman, 1965), might possibly be involved in the arteriolar dilation (Page and McCubbin, 1956; Haddy, 1960), since the administration of histamine causes the arterioles to dilate and the veins to constrict (Haddy, 1960). Haddy (1960) has also stated that "local (antagonism between serotonin and the adrenalines, whether (accurring at the molecular or physiologic level, might well account for the observed arteriolar dilation." Whelan (1959) and Chacalos (1963) have also reported small vessel dilation following the administration of serotonin. 14 FACTOR OF NEUROGENIC VASCULAR TONE Spinal cord transaction at the sixth cervical vertebra and removal of the cord in dogs resulted in a definitely augmented response to serotonin, marked by a large and prolonged initial fall in arterial pressure (Page, 1952). Serotonin has been observed to lower blood pressure in animals with neurogenic hypertension and elevate pressure in animals with neurogenic hypotension (Page and McCubbin, 1956; Haddy, 1960). After a series of eXperiments on the dog foreleg, Haddy and associates @1959) described this response by stating, Serotonin antagonizes extremes of vascular tone induced by neurogenic means. It produces net dilation when the bed is constricted and net constriction when the bed is dilated. This bi- directional response derives ultimately from the fact that changes in nervous activity change cal- ibers of small vessels without greatly altering calibers of large vessels, and that serotonin produces small vessel dilatation at the same time that it constricts large vessels. When small vessels are highly constricted, serotonin dilates small vessels more than it constricts large vessels. The net effect is "dilatation." 15 The most important mechanism controlling vascular reactivity to serotonin is probably the degree of autonomic nervous activity (Page, 1953; McCubbin, Kaneko and Page, 1962). McCubbin and associates (1962) found that when they stimulated the lumbar sympathetic trunk, both large and small arteries constricted; when serotonin was injected during stimulation, the formerly constricted small arteries relaxed, but the formerly constricted large arteries con- stricted even more. The net effect was a decrease in total vascular resistance, caused by the dilation of the small arteries and veins. ROLE OF CHEMORECEPTORS IN SEROTONIN RESPONSE Chemoreceptor stimulation appears to play a role in the rise of both pulmonary artery and pulmonary venous pressures after the administration of serotonin (Braun and Stern, 1961). Serotonin receptors apparently have a high degree of specificity since tryptamine and 5-hydroxy- tryptamine (serotonin) do not even activate the same recep- tors (Woolley and Shaw, 1962). Experiments performed with JDBMC (N—dimethylamide-N-benzyl-m—metoxycinnamamide) indicate ‘the existence of two types of serotonin receptors: DBMC— Sensitive and DBMC insensitive (Wurzel, 1966) . Serotonin ii; known to activate aortic and carotid chemoreceptors U?age, 1952; McCubbin, Green and Salmoiraghi, 1956; Braun anxi Stern, 1961). Serotonin causes a pronounced increase 16 in Chemoreceptor impulse traffic in the carotid sinus nerve when given in amounts of 12 ug into the common carotid artery (McCubbin, Green and Salmoiraghi, 1956). Serotonin is also said to activate chemoreceptors located in the heart and lung (Garattini and Valzelli, 1965). The presence of serotonin in the human carotid body was established by fluorescence microspectrophotometry in 1966 (Hamberger, Ritzen and Wersall, 1966). SEROTONIN AND SHOCK Anaphylactic reactions produce a release of serotonin from the platelets and a fall in blood serotonin concentra— tions (Rosenberg 33 31., 1959). Borges and Bessman (1957) report that serotonin is liberated from lung tissue, along with histamine, during anaphylaxis. Serum serotonin concentration falls immediately after administration of endotoxin and remains low throughout the shock state (Rosenberg 33 31., 1959). Total serotonin levels fall rapidly after the intravenous infusion of 3. 2211, as do the number of circulating platelets, along with marked changes in platelet morphology (Davis 3£_31., 1960). E. Effects of serotonin in specific vascular beds. CORONARY VESSELS Intravenous infusion of serotonin at 2.3 ug/kg/min in iflle rat results in a large increase in blood flow and (hacrease in coronary vascular resistance (Takacs and Vajda, 17 1963). Increases in coronary blood flow of 79% (Maxwell 33 31., 1959) and 134% (Hashimoto 33 31., 1964) have been reported following intravenous serotonin infusion. FORELIMB VE S SELS Haddy (1960) administered serotonin into the brachial artery of dog forelimbs in doses small enough to have no noticeable effect upon systemic arterial and venous pressures, and found that the pressures in the small arteries fell and those in small Veins rose, with no net change in blood flow through the limb. When serotonin (0.25—16 ug/min) was infused into the arteries of human forearms, the results were vasoconstriction, decreased blood flow, flushing and delayed cyanosis (Garattini and Valzelli, 1965). GASTROINTESTINAL VESSELS Intravenous infusion of serotonin (2.3 ug/kg/min) into the rat resulted in increased blood flow through the gut vessels (Takacs and Vajda, 1963). The topical effect of serotonin in the rat's mesoappendix is said to be about 200 times greater than that of general administration “Sarattini and Valzelli, 1965). HEPATIC VESSELS Intravenous serotonin infusion causes a slight increase iii the blood flow through the liver vessels of the rat Vlakacs and Vajda, 1963). In dogs, intravenous infusion of l8 serotonin increases portal pressure and resistance to hepa- tic blood flow, but it increases hepatic flow in man (Garattini and Valzelli, 1965). PULMONARY VESSELS Serotonin administration produces a marked pressor response in the pulmonary circuit (MacCanon, 1954; Shepherd 33 31., 1959; Rudolph and Auld, 1960; Vitolo 33 31., 1962; Rudolph and Scarpelli, 1964; Marshall, 1966) with a rapid increase in pulmonary arterial pressure (Reid and Rand, 1951; MacCanon and Howath, 1954; Maxwell 33 31., 1959; Aviado, 1960; Gailitis and Sheiber, 1960), along with pul— monary arteriolar and venous constriction (Kabins, Molina and Katz, 1959; Noble and Nanson, 1959) and a resultant increase in pulmonary vascular resistance (Noble and Nanson, 1959; Shepherd et a1., 1959; McGaff and Milnor, 1962). The degree of response of the pulmonary vasculature depends upon the state of vasomotor tone (Rudolph 33 31,, 1959). Haddy (1960) has stated, Serotonin is a potent constrictor of the pulmonary vascular bed. Dilator responses are not seen. The pulmonary vascular bed is low tone bed. The arterioles are widely dilated. Therefore, the predicted response would be constriction. 19 The principal site of increased resistance to flow through the lungs appears to be in the precapillary vessels (Shep- herd 33 31., 1959). McGaff and Milnor (1962) have stated that serotonin infusion reduces pulmonary blood volume by an average of about 26% below control values, which they say is an example of shifting of blood from pulmonic to systemic circuits "by reciprocal changes in the distensibility of these beds." Somlyo and Somlyo (1964) reported significant vasoconstriction in helically cut strips of canine main pulmonary artery. Marshall (1966) states that serotonin may be responsible for many of the effects of embolization of the lungs by blood clot or thrombus. Serotonin is a more effective pulmonary vasoconstrictor than adrenaline or noradrenaline, and the pulmonary hypertension it causes is not influenced by spinal section, vagotomy, adrenalectomy, ganglionic blockade, administration of antihistamines or the administration of reserpine (Garattini and Valzelli, 1965). RENAL VESSELS Renal blood flow in rats is decreased by intraperitoneal infusion of serotonin, but increased when serotonin is infused iJitravenously (Takacs and Vajda, 1963). The renal vessels O 2 5 IO 20 5O IOO 0 ml /min—+ O 0.02 0.05 DJ 0.2 0.5 Infusion Rate TABLE 23. MENTS WITH LOW INITIAL RESISTANCE: RESISTANCE CHANGES (mmHg/cc/min/IOOg); CONSTANT FLOW EXPERI- Exp. SEROTONIN: CC/MIN (lOOug/cc): No. Control 0.02 0.05 0.10 '0.20 ' 0.50 ‘1.00 ‘Rec. 1. 5.2 5.3 5.1 5.0 4.3 4.0 4.0 4.7 2. 9.2 9.8 10.1 9.9 9.9 11.6 12.9 10.3 3. 12.2 12.3 13.8 16.0 17.0 17.8 17.4 13.2 . 10.7 9.9 9.7 9.6 9.4 8.9 8.7 10.7 5. 12.1 14.7 14.2 14.7 14.7 15.0 13.2 12.1 6. 6.2 5.8 5.4 5.1 4.9 4.8 4.9 5.6 7. 7.6 8.1 8.5 8.4 8.1 8.1 8.0 7.8 8. 7.5 7.8 10.5 12.1 12.2 10.5 12.1 7.5 -9. 10.5 11.7 12.5 12.1 12.0 11.4 10.2 11.8 10. 6.5 9.6 11.2 11.3 11.5 11.5 11.3 6.7 11. 6.9 7.5 9.8 9.7 8.9 9.2 9.4 8.0 12. 6.3 6.4 8.4 8.4 8.2 7.7 7.4 5.3 13. 6.1 9.6 10.9 11.6 12.1 12.1 12.3 5.6 14. 12.2 13.8 13.6 14.1 14.4 14.5 14.1 11.6 15. 7.6 7.6 8.3 8.9 9.3 9.4 8.7 6.6 16. 5.7 7.2 7.6 7.8 7.8 7.6 7.2 6.6 17. 10.4 12.7 12.2 13.3 12.3 11.1 10.0 10.1 18. 12.0 12.8 12.8 13.2 12.4 12.5 11.1 12.2 19. 7.9 8.1 8.0 8.0 8.9 9.4 9.6 7.9 Ave. 8.6 9.5 10.1 10.5 10.4 10.4 10.1 8.6 :S.E. 0.56 0.64 0.61 0.70 0.74 0.78 0.75 0.62 TABLE 2b. 31 MENTS WITH LOW INITIAL RESISTANCE: RESISTANCE CHANGES (mmHg/cc/min/lOOg); CONSTANT FLOW EXPERI- Exp. SALINE, CC/MIN: No. Control 0.02 0.05 0.10 0.20 0.50 1.00 Rec. 1. 4.7 4.8 4.5 5.0 5.0 4.6 4.8 5.1 2. 10.3 10.5 10.5 10.7 10.4 10.5 10.3 11.0 3. 18.0 18.0 17.8 17.5 17.5 16.8 15.8 18.0 4. 10.3 10.5 10.5 10.5 10.5 10.2 9.5 10.6 5. 12.6 12.6 12.9 12.6 12.6 11.2 10.1 13.4 6. 6.4 6.6 6.7 6.7 6.7 6.6 6.4 6.7 7. 7.1 7.1 7.1 7.1 7.3 7.3 7.2 7.1 8. 7.2 ‘7.2 7.2 7.2 7.4 7.3 7.1 7.7 9. 13.9 13.6 12.7 12.3 11.8 10.9 10.1 12.3 10. 7.3 7.4 7.4 7.5 7.5 7.4 7.2 8.0. 11 6.6 6.6 6.6 6.3 6.1 6.1 6.6 7.0 12. 5.1 ‘ 5.1 5.2 5.4 5.5 6.1 5.5 6.2 13. 6.3 6.5 6.4 6.3 6.3 6.3 6.1 6.4 14. 12.4 12.5 13.0 13.2 13.0 12.5 11.5 12.9 15. 7.6 7.6 7.6 7.6 7.4 7.3 7.1 7.6 16. 7.4 7.4 7.4 7.4 6.7 6.4 6.0 6.7 17. 12.6 13.0 13.1 13.1 12.2 11.6 10.4 12.1 18. 12.4 12.4 12.4 12.4 12.4 11.9 11.4 12.5 9. 8.6 8.6 8.6 8.7 8.7 8.7 8.3 9.0 Ave. 9.3 9.4 9.3 9.3 9.2 8.9 8.5 9.5 EE;E 0.81 .0.81 0.80 0.78 0.76 0.70 0.62 0.76 Figure 2. 32 Effects of I.A. serotonin infusion in gracilis muscles with high initial resistance. Flow = natural. RT = total muscle vascular resistance; mean systemic arterial blood F= flow; PAS pressure; PLV muscle large vein pressure. R? (M ml xminxlOOg) ”Selling“o (N=I2) F (ml/min) I40 PA: (mmHg) '30 ----°-----°----‘0’----°-----o-----O-----o IZO IO PLv (mmHg) . . . I ,ug/min__.0 2 5 IO 20 50 I00 0 ml/m'n —-> O 0.02 0.05 DJ 0.2 0.5 LO 0 Infusion Role TABLE 3a. 33 RESISTANCE CHANGES (mmHg/Cc/min/lOOg)? NATURAL FLOW EXPERI- MENTS WITH HIGH INITIAL RESISTANCE: Exp. SEROTONIN: CC/MIN (lOOug/cc): No. Control 0.02 0.05 0.10 0.20 0.50 1.00 Rec. 1. 16.8 15.8 16.8 15.7 14.0 16.1 15.0 17.5 2. 12.6 11.6 11.7 11.7 11.4 11.5 10.3 14.4 3. 25.8 24.1 21.9 22.0 20.9 20.0 18.8 23.6 4. 19.2 14.7 12.8 11.9 11.2 9.0 8.7 15.2 5. 23.7 22.4‘ 20.3 21.5 20.4 20.3 16.1 20.7 6. 12.8 13.0 13.0 11.9 12.0 11.6 13.5 15.2 7. 15.6 14.8 13.8 12.6 10.8 10.0 11.2 11.5 8. 14.1 9.6 8.8 8.5 7.8 7.2 7.0 13.4 9. 17.9 15.7 14.6 12.8 11.9 11.5 11.6 17.9 10. 16.7 15.0 15.0 13.8 13.2 12.3 13.5 13.4 11. 13.3 12.6 12.6 11.4 10.3 10.0 9.4 14.3 12. 12.8 11.6 10.3 10.9 11.3 11.7 11.9 14.2 Ave. 16.8 15.1 14.3 13.7 12.9 12.6 12.3 16.1 :S.E. 1.25 1.23 1.10 1.18 1.13 1.18 0.96 0.99 TABLE 3b. RESISTANCE CHANGES (mmHg/cc/min/lOOgS; NATURAL FLOW EXPERI- MENTS WITH HIGH INITIAL RESISTANCE: Exp. SALINE: CC/MIN: No. Control 0.02 0.05 0.10 0.20 0.50 1.00 Rec. 1. 18.9 19.4 17.5 18.2 18.1 17.9 16.1 22.2 2. 13.4 15.2 16.2 15.8 15.7 14.8 12.6 16.0 3. 22.2 24.3 23.1 22.2 21.9 20.8 19.3 21.6 4. 15.2 15.5 16.3 13.7 13.1 12.8 10.8 15.1 5. 20.7 20.2 20.6 21.8 23.4 15.6 13.5 20.7 6. 17.0 16.1 16.0 15.2 14.4 13.0 12.0 16.0 7. 11.1 11.2 10.9 11.0 11.2 11.0 11.0 11.7 8. 13.5 12.9 12.9 11.8 11.8 11.4 11.0 12.8 9. 17.9 18.9 18.9 19.0 19.0 17.1 16.4 19.0 10. 12.5 12.6 14.0 12.7 12.9 11.7 10.5 13.6 11. 13.1 13.9 13.1 13.1 13.1 12.8 11.0 14.3 12. 14.2 15.0 14.4 13.4 11.3 11.4 10.7 14.6 Ave. 15.8 16.3 16.2 15.7 15.5 14.2 12.9 16.5 i§.E. 1.02 1.08 1.00 1.09 1.20 0.89 0.82 1.02 Figure 3. 35 Effects of I.A. serotonin infusion in gracilis muscles with low initial resistance. Flow = constant. RT = total muscle vascular resis- tance; F = flow; PAS = mean systemic arterial blood pressure. A33 In .0 r ”a. 3. -"8"--8'--'.0" "'O" \ " .3 _o \ x3. x 09 m @2281? 2....9 5‘ cumuomamno A219 _0 .u A3_\3§ _ m _u A IV :5 > 33 O O _NO . f . _ _ b _ 123510 N u 5 mo 00 .8 o 3. \ 33 lo CON 0.0m 0.. ON 00 _.O O .250: 23¢ 36 Figure 4 shows that in muscles with high initial resistance (RT:>12.3 units) and perfused at constant flow, serotonin infusion caused a progressive decrease in total muscle vascular resistance (p4:0.05). This decrease was again significantly greater than that caused by equivalent volumes of saline. The left hand portion of figure 5 shows that serotonin caused an average rise in muscle resistance when the initial resistance was low. However, when resistance was increased to a high level by sympathetic nerve stimulation, serotonin then caused a marked fall in total vascular resistance (p<(0.05). The left hand portion of figure 6 Shows that serotonin caused a marked drop in total muscle vascular resistance when the initial resistance was high. The right hand portion of figure 6, however, Shows that when resistance was decreased to a low steady-state level by exercise dilation, serotonin then caused a caused a rise in total vascular resistance in every case (p<0.05) . The left hand portion of figure 7 Shows that I.A. infusion of serotonin (50 ug/min) into 7 gracilis muscles with high initial resistance again resulted in a drop in resistance. The middle portion of figure 7 shows that when these same muscles were electrically caused to contract, thus lowering the resistance levels by exercise dilation, serotonin infusion resulted in a marked increase in total muscle vascular resistance, 37 which returned to near control level when infusion was stopped. The right hand portion of figure 7 Shows that when the resistance levels in these same muscles were subsequently raised by sympathetic nerve stimulation and serotonin was again infused at 50 ug/min, each muscle showed a marked drop in resistance equivalent to the drop caused by serotonin in the first portion of the experiment. Figure 8 represents the middle 15 experiments when all 52 are arranged according to their initial total resistances. The average effect of serotonin on total muscle vascular resistance is not significantly different in this group than the average effect of saline. Figure 9 represents the average results of all 52 ex- periments (25 with high and 27 with low initial resistances). Again, the average effects of serotonin and saline are very similar. Only when the experiments are divided into low and high initial resistance groups can one see significant differ- ences in the effects of serotonin as compared to the effects of saline. The average time allowed for total recovery from the effects of serotonin was approximately 15 minutes. The muscles recovered from the effects of saline in an average of approximately 5 minutes. TABLE 4a. 38 RESISTANCE CHANGES (mmHg/cc/min/lOOg); CONSTANT FLOW EXPERI- MENTS WITH HIGH INITIAL RESISTANCE: Exp. SEROTONIN: CC/MIN (lOOug/cc): No. Control 0.02 0.05 0.10 0.20 0.50 1.00 Rec. 1. 13.4 12.7 9.4 8.6 8.6 7.8 7.4 13.4 2. 12.5 9.8 9.4 9.5 9.3 8.9 8.9 12.5 _3. 12.5 13.0 13.4 13.4 13.1 9.0 6.4 15.8 4. 17.1 18.0 17.1 14.5 14.1 13.4 12.0 17.1 5. 12.4 12.1 12.3 12.2 10,4 10.2 10.4 12.3 6. 15.8 14.2 12.5 10.8 10.4 9.5 9.4 14.7 7. 12.5 12.4 12.4 12.4 11.8 11.6 12.5 12.6 8. 11.0 10.9 11.1 11.2 7.2 7.2 6.6 11.1 9. 13.3 10.9 11.1 11.3 11.3 11.3 12.4 13.3 10. 12.3 10.1 9.9 11.1 10.1 9.9 10.1 11.1 11. 25.0 20.9 20.1 19.0 17.8 14.9 12.9 23.2 12. 12.3 12.1 12.1 12.6 15.0 12.6 10.5 12.4 13. 12.3 11.8 12.3 12.6 12.6 12.3 12.1 11.5 Ave. 14.0 13.0 12.6 12.2 11.7 10.7 10.1 13.9 :S.E. 1.04 0.87 0.83 0.71 0.79 0.62 0.61 0.92 TABLE 4b. RESISTANCE CHANGES (mmHg/cc/minlOOg); CONSTANT FLOW EXPERI— MENTS WITH HIGH INITIAL RESISTANCE: Exp. SALINE, CC/MIN: No. Control 0.02 0.05 0.10 0.20 0.50 1.00 ReC. 1. 13.4 13.4 13.1 12.6 12.2 11.9 10.6 14.0 2. 12.1 12.0 12.1 12.0 12.0 11.8 11.4 12.3 3. 12.5 12.5 12.5 12.5 12.3 11.9 9.1 12.5 4. 19.8 18.9 19.1 19.2 17.5 17.1 15.0 20.6 5. 12.0 12.1 12.1 12.0 11.6 11.2 10.9 12.4 6. 16.6 16.7 16.6 16.3 16.1 15.8 15.4 16.3 7. 12.5 12.6 12.8 12.6 12.6 12.6 12.2 13.3 8. 10.9 10.9 10.9 10.9 10.9 10.9 10.6 11.3 9. 13.7 13.7 13.6 13.5 13.4 13.2 13.1 13.5 10. 10.4 10.9 10.7 11.7 11.5 11.5 11.2 10.7 11. 24.4 25.8 25.8 25.8 25.6 22.3 21.3 25.2 12. 15.9 15.9 15.7 15.5 15.3 13.1 11.4 14.4 13. 11.8 12.0 12.3 12.7 13.3 11.3 10.9 11.6 Ave. 14.3 14.4 14.4 14.4 14.2 13.4 12.6 14.5 :S.E. 1.13 1.15 1.15 1.14 1.09 0.89 0.87 0.80 Figure 4. 40 Effects of I.A. serotonin infusion in gracilis muscles with high initial resistance. Flow = constant. RT = total muscle vascular resis- tance; F = flow; PAS — mean systemic arterial blood pressure. 331a I... A 3. .w x 3.: x .00 a. E and. mess... .219 0" w v. .3335. m_ . bf 17 a . . . .upa .33 In. .mo _ . _ . _ _ to}? 0 N m .0 m0 mo _00 0 3 _ \35 0 0.0m 0.00 0.. 0M 0.0 _.0 0 3.6.40.0: mam 41 TABLE 5. RESISTANCE CHANGES (mmHgZCc/min/loog); CONSTANT FLOW EXPERI- MENTS IN MUSCLES WITH LOW INITIAL RESISTANCE AND WITH RESIS- TANCE SUBSEQUENTLY INCREASED BY SYMPATHETIC NERVE STIMULATION: Exp. SEROTONIN, CC/MIN (lOOug/cc): No. Pre—Stim. Control 0.5cc/min Recovery Post-Stim. 1. ---- 9.2 8.9 9.2 ---- 2. 9.6 17.7 13.5 16.8 9.8 3. 12.3 23.3 16.3 28.2 13.2 4. 6.9 13.8 12.4 13.3 7.3 5. ---- 25.7 19.9 22.2 -—-- 6. 11.1 18.7 16.6 19.2 12.4 7. 17.7 27.1 25.4 26.7 16.0 8. 11.9 25.7 22.3 23.5 11.9 9. 8.3 16.4 15.0 15.4 8.3 Ave. 11.1 19.7 16.7 19.4 11.3 +S.E 1.33 2.04 1.70 2.10 1.13 Figure 5. 42 Effects of I.A. serotonin infusion on total vascular resistance in muscles with low initial resistance and with resistance subsequently increased by nerve stimulation. Flow = constant. RT - total muscle vascular resis— tance. 33 In a... A 3. x3.:x_000. .c \35 3 9...: N0 .0 .m .b .N .0 2035. .20. J OOF' .35.? .66 9.38.320 292m 9.38.9.0: OO- TABLE 6. 43 RESISTANCE CHANGES (mmHg/cc/min/lOOg); CONSTANT FLOW EXPERI- MENTS IN MUSCLES WITH HIGH INITIAL RESISTANCE AND WITH RESIS— TANCE SUBSEQUENTLY LOWERED BY MOTOR NERVE STIMULATION: 33?. Pre-Stim. Control 0.50cc/min Recovery Post-Stim. 1. -—-- 5.24 7.15 7.15 ---- 2. ---- 5.09 4.72 5.00 ---- 3. ---- 9.70 12.00 10.10 ---- 4. ---- 4.03 5.50 4.07 ---- 5. -——- 3.42 4.92 3.72 ---- 6. ---- 9.26 10.50 8.82 ---- 7. -—-- 5.03 5.36 4.95 ---- 8. 9.67 6.50 10.80 6.18 9.67 9. 8.89 5.29 6.75 5.51 5.51 10. 8.00 4.07 6.30 3.93 6.94 11. 13.30 8.28 11.00 8.06 12.30 12. 12.00 10.10 23.60 9.28 13.30 13. 16.20 9.42 11.10 9.60 11.10 14 11.90 8.64 11.80 8.56 11.90 15. 8.33 3.53 6.07 3.53 8.33 16. ---- 7.42 9.58 7.77 --—- Ave. 11.00 6.56 9.20 6.64 9.88 :S.E. 0.95 0.59 1.22 0.57 0.65 Figure 6. 44 Effects of I.A. serotonin infusion on total vascular resistance in muscles with high ini- tial resistance and with resistance subsequently lowered by exercise dilation. Flow = constant. RT - total muscle vascular resistance. 3319 3a. A 3. .0: x 3.: x .00 a. >133... w 0 00 3_\3.: . 4 0 0.0 OOr SETS 39.6 .210 OC— Figure 7. 45 Effects of I.A. serotonin infusion on total vascular resistance in muscles with high ini— tial resistance; in the same muscles after low- ering resistance by exercise dilation; and in the same muscles again after subsequently raising resistance to a high level by sympathe— tic nerve stimulation. Flow - constant. RT = total muscle vascular resistance. Infusion rates were measured in ug/min (top numbers) and ml/min (bottom numbers). 03833.0 292m 2930. @5350 0:359:03 _ _ _ 3 4 N0 1 m. r .0 .. 0683:... End - ems .. 24. .4 1 A33 In . ad. :4- _w I .3... 1000. I .0 u . . u 0 r N .I. F t _ r . _ F . 0 00 0 0 00 0 0 00 0 00 0 0 0.0 0 0 0 0 Figure 8. 46 Effects of I.A. serotonin infusion on total muscle vascular resistance in 15 experiments with initial resistance levels near 12.3 units. Natural and constant flow experiments were both used in this group. RT = total muscle vascular resistance. .b I A33 In .0 u a... 3. . .m .. x 33 x .008 . . I _ _ _ . . L . . .cn\3.: III. 0 m 0 .0 NO 00 .00 0 3.\3.: |I|¢ 0 0.0m 0.00 0.. ON 0.0 ..0 0 53.5.8 no.8 063.83.: .2u.0. 02.3 .2u.0. 47 Figure 9. Effects of I.A. serotonin infusion on total vascular resistance in all experiments com— bined. RT = total muscle vascular resistance. a I o ..... o ..... o. 3310 nuaaouuuuuol em] ”a. A 3. / fl, 10’ 0 x3... 01000. .. _o .u r p t c . p . 1235 ll 0 m m _o mo 8 .oo 3.\3.: Ill 0 CON 0.00 0.. CM 0.0 ..O Eamssmam .mmqosoQP Azummv 0.3.81. .2 u 0N. 48 Since the serotonin was dissolved in normal saline, the effects of normal saline alone were of great importance. In every group of experiments, saline had very little, if any, effect on resistance at 0.02, 0.05, 0.1, and 0.2 ml/min levels of infusion, but at 0.5 and 1.0 ml/min there were small but consistent decreases in resistance. Since the average initial flow through the muscles was about 7.5 ml/min, an infusion of 1.0 ml/min of saline would make up approximately 13% of the total blood volume entering the muscle. The hematocrit would obviously be lower than normal, so it seems probable that this factor could be responsible for part of the decrease in resistance at the higher levels of infusion. 49 This study shows the direct effects of serotonin on the muscle vasculature, which may be different from the indirect effects. Serotonin was infused into the gracilis artery at rates that would not result in systemic effects, but would cause only local effects. The direct effect of serotonin on muscles with low initial resistance was an increase in resistance. Muscles with high initial resis- tances consistently showed drops in resistance when serotonin was infused. Takacs and Vajda (1963) reported an increase in blood flow in skeletal muscles following the intraperitoneal in— jection of 10 mg/kg of serotonin. Goodman (1965) has also stated that vasodilation is the usual response of the skeletal muscle vasculature to serotonin. Daugherty and colleagues (1968) recently made a comparison of the effects of intra- brachial and intravenous administration of serotonin on fore- limb blood flow in the dog. Almost no change in total flow from the limb was observed during intrabrachial or intravenous administration of serotonin. However, a shift in flow from the cephalic (skin) vein to the brachial (muscle) vein occurred with no net change in total outflow (during intrabrachial in- fusion at flow - K), suggesting that serotonin affects the two parallel beds in opposite directions, increasing vascular resistance in the skin and possibly decreasing it in the muscle (Daugherty et a1., 1968). From these earlier reports 50 one would think that infusion of serotonin and vasodilation of the muscle vasculature would go hand in hand. However, more recent research, including the author's study, indicate that this is not the case. Emerson and associates (1968) investigated the local effects of serotonin on resistance to blood flow in 20 isolated innervated gracilis muscles of dogs; six of the muscles were perfused at constant flow and 14 at natural flow. Intra-arterial infusion of serotonin at 24100 ug/min had variable effects on perfusion pressure and no effect on small vein pressure in the constant flow experiments. In the natural flow experiments, infusion of 2—100 ug/min of serotonin increased the blood flow in the ten experiments in which the initial total vascular resis- tance per 100 grams was above 10 mmHg/ml X min X 100g. In the four experiments with initial resistances below 10, blood flow fell in two and did not change in the other two. It was concluded that "the response of the innervated gracilis muscle was irregular and perhaps in part related to the initial level of resistance (Emerson et_al., 1968)." Others have related serotonin's effects, in part, to the initial level of resistance. Serotonin has been observed to lower blood pressure in animals with neurogenic hypertension and elevate pressure in animals with neurogenic hypotension (Page and McCubbin, 1956; Haddy, 1960; Garattini and Valzelli, 1965; Haddy and Scott, 1966). After completing a series of 51 experiments on the dog foreleg, Haddy described this response by stating, "Serotonin antagonizes extremes of vascular tone induced by neurogenic means. It produces net dilation when the bed is constricted and net constriction when the bed is dilated (Haddy, Gordon and Emanuel, 1959)." The author's own experiments Show quite clearly that the effect of serotonin upon total vascular resistance in the dog gracilis muscle is dependent upon the initial level of resis— tance in that muscle. In 50 out of 52 experiments (constant and natural flow), muscles with an initial total resistance (RT) greater than 12.3 mmHg/ml X min X 100g showed a drop in resistance and those with an initial RT lower than 12.3 showed a rise in resistance during serotonin infusion. Several muscles with initial resistances in the "twilight zone" (near 12.3) showed drops in resistance at some levels of serotonin infusion and rises at others. In two experiments serotonin initially dropped resistance, but later during the same ex- periments, resistance dropped spontaneously; serotonin now caused a marked increase in resistance. This information would seem to back previous statements calling the degree of autonomic nervous activity the most important mechanism controlling vascular reactivity to serotonin (Page, 1953; McCubbin, Kaneko and Page, 1962). However, the decrease in resistance caused by serotonin in 25 muscles with high initial resistance could be reversed to an increase in resistance when the resistance was 52 subsequently lowered by metabolically induced vasodilation. Thus the muscle's response to serotonin does not appear to depend upon neurogenic tone as such, but rather upon the level of resistance at the time serotonin is infused. It should be emphasized that the average effects of serotonin and saline in all 52 experiments combined are quite similar, i.e., if all experiments are simply evalu— ated together, serotonin appears to have no Significant effect on muscle resistance. In order to show the real effect of serotonin, the experiments must be divided into groups according to the initial level of vascular resis— tance based on a uniform muscle weight. 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