Functional Role of -Calcitonin Gene-Related Peptide in the Regulation of the Cardiovascular System

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1 /01/ $3.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 298, No. 2 Copyright 2001 by The American Society for Pharmacology and Experimental Therapeutics 3832/ JPET 298: , 2001 Printed in U.S.A. Functional Role of -Calcitonin Gene-Related Peptide in the Regulation of the Cardiovascular System YOU-TANG SHEN, TAMARA J. PITTMAN, PAMELA S. BUIE, DAVID L. BOLDUC, STEFANIE A. KANE, KENNETH S. KOBLAN, ROBERT J. GOULD, and JOSEPH J. LYNCH JR. Department of Pharmacology, Merck Research Laboratories, West Point, Pennsylvania Received February 2, 2001; accepted April 10, 2001 This paper is available online at ABSTRACT It remains unknown whether the extent of vasoactive response to exogenous calcitonin gene-related peptide (CGRP) varies among different regional vascular beds. It is also unclear whether endogenous CGRP plays a functional role in regulating basal vascular activity. To address these two issues, experiments were conducted in 27 anesthetized rats instrumented with a carotid flow probe and catheters in a jugular vein, left ventricle (LV), and femoral artery, and in 6 conscious dogs, chronically instrumented with LV pressure gauge, aortic and atrial catheters, and ascending aortic, coronary, carotid, and renal flow probes. In both species, administration of human -CGRP ( g/kg, i.v.) induced a dose-dependent peripheral vasodilation that was completely abolished by pretreatment with -CGRP[8-37] (30 g/kg/min, i.v.), a competitive antagonist of CGRP receptors. Regional blood flow measured by the radioactive microsphere technique in rats showed that the -CGRP (0.3 g/kg, i.v.)-induced increase in blood flow was greater (p 0.05) in the heart ( 53 16%) than in the brain ( 14 6%). In the presence of -adrenergic receptor blockade with propranolol, however, the increases in blood flow in these two vascular beds were identical. In conscious dogs, -CGRP (0.3 g/kg, i.v.) produced similar increases in coronary ( 24 6%), carotid ( 26 3%), and renal ( 26 6%) blood flow, which were different from the patterns induced by other vasodilators; at an equivalent level of reduction in mean arterial pressure and total peripheral resistance, -CGRP increased coronary and carotid blood flow significantly less (p 0.05) than adenosine or nitroprusside. Unlike -CGRP, adenosine and nitroprusside, as expected, induced pronounced differential blood flow changes in these vascular beds. Neither systemic hemodynamics nor regional blood flow distribution was altered by the administration of a pharmacological blocking dose of -CGRP[8-37] in the two species. Thus, we conclude that endogenous -CGRP does not play an important role in cardiovascular regulation under normal, resting conditions, although exogenous -CGRP induces a marked, comparable vasorelaxation in different regional vascular beds. Calcitonin gene-related peptide (CGRP), a 37-amino acid peptide, is generated by endocrine cells as well as by cells of the central and peripheral nervous systems. Immunohistochemical localization studies have found an abundance of CGRP-containing nerve fibers throughout many parts of the body, often associated with vascular smooth muscle (Rosenfeld et al., 1983). Although it has been generally accepted that CGRP induces a potent vasodilation, the extent to which this vasoactive response varies among different vascular beds remains unknown. In addition, previous studies of the vascular effects of CGRP[8-37], the peptide fragment antagonist of the CGRP receptor, were inconclusive. Some reports suggested that the administration of CGRP[8-37] induced vasoconstriction (Han et al., 1990; Yaoita et al., 1994), whereas other studies failed to find cardiovascular effects of CGRP[8-37] (Franco-Cereceda, 1991a; Gardiner et al., 1991; Sekiguchi et al., 1994). Recent studies have implicated CGRP in the pathogenesis of migraine (Goadsby et al., 1988, 1990; Goadsby and Edvinnson, 1993; Gallai et al., 1995) through a mechanism which probably involves dilation of the large intracranial and extracerebral arteries. Thus, CGRP receptor antagonists may represent a potential novel therapeutic approach to the treatment of migraine. Elucidating the vascular actions of CGRP and CGRP[8-37] has become more important for understanding the role of CGRP and the consequences of antagonizing CGRP receptors. Of particular interest is a comparison of the effects in the cerebral and coronary vasculature. Accordingly, the first goal of the present investigation was to establish a comprehensive pharmacodynamic profile of -CGRP in different vascular beds. To accomplish this goal, we not only continuously monitored systemic hemodynamic function in response to exogenous administration of CGRP, but we also used a radioactive microsphere technique to measure regional blood flow in two species, i.e., anesthetized rats and conscious dogs, allowing us to compare the relative effects of -CGRP in different vascular beds. Furthermore, ABBREVIATIONS: CGRP, calcitonin gene-related peptide; LV, left ventricular. 551

2 552 Shen et al. the effects of -CGRP were compared with those of the known vasodilators, i.e., nitroprusside and adenosine. To better understand the possible influence of reflex-mediated effects that could indirectly contribute to an increase in myocardial blood flow, some experiments in rats were conducted in the presence of -adrenergic receptor blockade. A second goal of the present study was to determine whether -CGRP[8-37], at a dose that completely blocked an exogenous -CGRP challenge, would affect resting systemic and regional vascular dynamics in the two species. Materials and Methods Animal Preparations. Sprague-Dawley rats, weighing 360 to 550 g, were anesthetized with pentobarbital sodium (30 40 mg/kg, i.p.). Following tracheal intubation and ventilation with room air using a rodent ventilator (Harvard Apparatus, South Natick, MA), a left lateral thoracotomy was performed. Polyethylene (PE 50) catheters (Norton Plastics, Akron, OH) were implanted in the left ventricular (LV) cavity through the apex for measurements of LV pressure and rate of change of LV pressure (LV dp/dt), and for injection of radiolabeled microspheres. The air in the chest was evacuated, and the thoracotomy was closed in layers. Through a midline cervical incision, a flow probe with a diameter of 1.5 mm (Transonic Systems Inc., Ithaca, NY) was implanted to measure carotid blood flow. A PE 50 catheter was placed in the jugular vein for infusion of drugs. The cervical incision was closed. Another PE 50 catheter was implanted in the abdominal aorta through an incision in the femoral artery to measure mean arterial pressure and to withdraw blood samples during microsphere injection. Mongrel dogs of either sex, weighing 10 to 15 kg, were anesthetized with pentothal (12 15 mg/kg, i.v.). Following tracheal intubation and ventilation, general anesthesia was maintained with isoflurane ( volume % in oxygen). A left thoracotomy was performed at the fourth intercostal space. Tygon catheters (Norton Plastics) were implanted in the descending aorta, right atrium, and left atrium for measurement of mean arterial pressure, administration of drugs, and administration of radiolabeled microspheres, respectively. A solid-state miniature pressure gauge (Konigsberg, Pasadena, CA) was implanted in the left ventricle for measurements of pressure and LV dp/dt. A flow probe (Transonic Systems Inc.) was placed around the ascending aorta to measure aortic blood flow, i.e., cardiac output. Another Transonic flow probe was implanted around the left circumflex coronary artery to measure coronary blood flow. The chest was closed in layers and evacuated of air. During the same surgical session, a midline laparotomy was performed. A third Transonic flow probe was placed around the right renal artery. In addition, a fourth Transonic flow probe was placed around the left carotid artery via a small neck incision. All catheters and electrical leads were tunneled and externalized to the back between the scapulae. The animals used in this study were maintained in accordance with the Guide for the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources (National Research Council, 1996), and the studies were approved by the Merck Research Laboratories (West Point, PA) Institutional Animal Care and Use Committee. Experimental Measurements. Hemodynamic recordings were made using a data tape recorder (TEAC, Montebello, CA) and a multiple-channel oscillograph (Gould, Cleveland, OH). Arterial pressure was measured using strain gauge manometers (Argon, Athens, TX), which were previously calibrated using a mercury manometer connected to the fluid-filled catheters. Carotid, renal, coronary, and aortic blood flows were measured using a volume flowmeter (Transonic Systems Inc.). Mean arterial pressure and blood flow were measured using an amplifier filter. LV dp/dt was calculated with an operational amplifier connected as a differentiator. A triangular wave signal was substituted for the pressure signal to directly calibrate the differentiator (Triton Inc., San Diego, CA). Total peripheral resistance was calculated as the quotient of mean arterial pressure and cardiac output. A cardiotachometer triggered by the pressure pulse provided instantaneous and continuous records of heart rate. Regional blood flow was measured by the radioactive microsphere technique. Microspheres (15 1 m) labeled with Nb 95,Ce 141,Sn 113, Ru 103,orSc 46 (New Life Science Products, Boston, MA) were suspended using an ultrasonic bath for 30 min. Each injection of microsphere suspension, which contained approximately 0.1 to 0.2 million spheres for the rats and 1 million spheres for the dogs, were administered through the left ventricular or left atrial catheter in the rats and dogs, respectively, and flushed with saline. An arterial blood reference sample was withdrawn at a rate of 0.5 ml/min for a total of 90 s from the rats and 7.75 ml/min for 120 s from the dogs. At the end of study, the animals received an overdose of pentobarbital, and regional tissue samples were collected and counted in a gamma counter (Packard BioScience, Meriden, CT) with appropriately selected energy windows. After a correction of the counts for background and crossover, the regional blood flow was calculated and expressed as milliliters per minute per gram of tissue. The value for each tissue type is an average from several samples. Experimental Protocols. The experiments in six conscious dogs were conducted 2 to 3 weeks after surgery. During this postoperative period, the dogs had been trained to lie quietly in the right lateral position. The experiments in 27 anesthetized rats were initiated when the hemodynamics were stable, i.e., at least 20 to 30 min after surgically implanting the instrumentation. The experimental protocols involved intravenous (i.v.) bolus injection of human -CGRP (Sigma, St. Louis, MO) at doses of 0.1, 0.2, 0.3, and 0.5 g/kg for rats and 0.1, 0.3, and 0.5 g/kg for dogs. To determine the dose of the -CGRP[8-37] required to block the effect of -CGRP challenge, the mean arterial pressure and carotid blood flow response to -CGRP at a dose of 0.3 g/kg, i.v. was tested in the presence of continuous i.v. infusion of human -CGRP[8-37] (Sigma) at doses of 10, 20, and 30 g/kg/min for 5 to 10 min. The results of the -CGRP and -CGRP[8-37] dose-response effects on systemic hemodynamics led us to use doses of 0.3 g/kg -CGRP and 30 g/kg/min -CGRP[8-37] to further examine the effects of -CGRP and -CGRP[8-37] on regional blood flow distribution in both species. In addition, nitroprusside at a dose of 1 to 10 g/kg, i.v. and adenosine at a dose of 0.04 to 0.32 mg/kg, i.v. were also tested in the dogs. The time interval between the doses was at least 10 min for rats and 15 min for dogs. To determine the direct effects of -CGRP on myocardial blood flow, -CGRP also was tested in the rat in the presence of -adrenergic receptor blockade with propranolol at a dose of 0.5 mg/kg, i.v., which completely blocked isoproterenol (0.1 g/kg, i.v.)-induced inotropic and chronotropic effects. Data Analysis. Data before and after administration of -CGRP or -CGRP[8-37] were compared using the Student s t test for paired data with a Bonferroni correction. The baseline values between the -CGRP and -CGRP[8-37] groups were compared using group t test. All values are expressed as the mean S.E. Statistical significance was accepted at the p 0.05 level. Results Effects of -CGRP in Anesthetized Rats and Conscious Dogs. In both anesthetized rats and conscious dogs, -CGRP i.v. induced a dose-dependent decrease in mean arterial pressure and increase in carotid blood flow, which lasted approximately 5 to 15 min. The peak effects on mean arterial pressure and carotid blood flow, which occurred about 1 to 2 min after injection in both species, are shown in Fig. 1. Note that the effects of the 0.3- g/kg dose of -CGRP in the anesthetized rats were near the plateau level of the dose-response curve. In conscious dogs, the increases in carotid blood flow by -CGRP at doses of 0.2 and 0.3 g/kg also

3 Fig. 1. Peak effects of -CGRP at doses of 0.1 to 0.5 g/kg, i.v. on mean arterial pressure and carotid blood flow in anesthetized rats and conscious dogs. Values are percentage changes from baseline levels. -CGRP induced a dose-dependent decrease in mean arterial pressure and increase in carotid blood flow. were similar. The baseline hemodynamic values and the peak responses to -CGRP at the dose of 0.3 g/kg for these two species are shown in Tables 1 and 2. At this dose, -CGRP significantly reduced (p 0.01) mean arterial pressure by 17 3 and 26 3% from baseline levels of and mm Hg, while the carotid blood flow was significantly increased (p 0.01) by 36 3 and 26 3% from baseline levels of and ml/min in the rats and dogs, respectively. In anesthetized rats, -CGRP at doses of 0.3, 0.5, and 0.8 g/kg slightly increased the heart rate by 2 1, 7 2, and 9 1%, respectively. In conscious dogs, Cardiovascular Effects of -CGRP 553 however, the heart rate was increased by 49 5% (p 0.01) at a dose of 0.3 g/kg of -CGRP. The time courses of the effects of -CGRP at a dose of 0.3 g/kg, i.v. on LV dp/dt, mean arterial pressure, heart rate, carotid, coronary, and renal blood flow in conscious dogs are shown in Fig. 2. Times to peak and the pattern of recovery in coronary, carotid, and renal blood flows following administration of -CGRP were almost identical. The peak increases in regional blood flow in the carotid ( 26 3%), coronary ( 24 6%), and renal ( 26 6%) vascular beds were similar. The time courses for -CGRP-induced increase in heart rate and decrease in mean arterial pressure were also similar to those of regional blood flow. A comparison of the effects of -CGRP, nitroprusside, and adenosine on systemic and regional blood flow in conscious dogs is shown in Fig. 3. At the doses that induced similar, approximately 40% reductions in total peripheral resistance, the increases in carotid and coronary blood flow induced by -CGRP were significantly less (p 0.05) than those produced by nitroprusside or adenosine. The nitroprusside-induced increase in renal blood flow ( 15 6%) was similar to that induced by -CGRP, but was significantly less (p 0.05) than the nitroprusside-induced increases in coronary ( 48 9%) and carotid ( 57 10%) blood flow. Adenosine induced the most intense increase in coronary blood flow ( %) and a decrease in renal blood flow. There were no differences in changes in heart rate among these three groups. Effects of -CGRP at a dose of 0.3 g/kg on regional blood flow in heart, brain, and kidney in anesthetized rats are shown in Fig. 4. After administration of -CGRP, regional blood flow was increased by 53 16, 14 6, and 13 10% from a baseline of , , and ml/min/g in heart, brain, and kidney, respectively. The increase in regional blood flow was significantly greater (p 0.05) in heart than in the brain or kidney. However, in the presence of -adrenergic receptor blockade with propranolol, as shown in the insert in Fig. 4, the increase in myocardial blood flow induced by -CGRP (0.3 g/kg) was attenuated compared with that observed in the absence of propranolol, such that regional blood flows in both heart ( 23 12%) and brain ( 21 12%) were increased similarly. The baseline blood flow in the heart ( ml/min/g), brain ( ml/min/ g), and kidney ( ml/min/g) was not significantly affected after the administration of propranolol. Effects of -CGRP[8-37] in Anesthetized Rats and Conscious Dogs. Baseline hemodynamic values, i.e., before administration of -CGRP[8-37], in the two species are TABLE 1 Cardiovascular effects of -CGRP (0.3 g/kg, i.v.) and -CGRP[8-37] (30 g/kg/min, i.v.) in anesthetized rats Baseline Peak Response % Change Mean arterial pressure (mm Hg) -CGRP (n 14) * -CGRP[8-37] (n 9) Carotid blood flow (ml/min) -CGRP (n 14) * -CGRP[8-37] (n 9) Heart rate (beat/min) -CGRP (n 14) * -CGRP[8-37] (n 9) * p 0.01, significant difference from baseline.

4 554 Shen et al. TABLE 2 Cardiovascular effects of -CGRP (0.3 g/kg, i.v.) and -CGRP[8-37] (30 g/kg/min, i.v.) in conscious dogs Baseline Peak Response % Change LV systolic pressure (mm Hg) -CGRP (n 5) * -CGRP[8-37] (n 5) LV dp/dt (mm Hg/s) -CGRP (n 5) ** -CGRP[8-37] (n 5) Mean arterial pressure (mm Hg) -CGRP (n 6) * -CGRP[8-37] (n 5) Cardiac output (l/min) -CGRP (n 3) * -CGRP[8-37] (n 3) Coronary blood flow (ml/min) -CGRP (n 6) * -CGRP[8-37] (n 5) Carotid blood flow (ml/min) -CGRP (n 6) * -CGRP[8-37] (n 5) Renal blood flow (ml/min) -CGRP (n 3) * -CGRP[8-37] (n 3) Heart rate (beat/min) -CGRP (n 6) * -CGRP[8-37] (n 5) * p 0.01, ** p 0.05, significant difference from baseline. shown in Tables 1 and 2. There were no differences compared with those obtained before administration of -CGRP. Before and during i.v. infusion of -CGRP[8-37] over a dose range of 10 to 30 g/kg/min, the effects of -CGRP challenge (0.3 g/kg, i.v.) on mean arterial pressure and carotid blood flow in anesthetized rats and conscious dogs were examined. In conscious dogs, -CGRP-induced increases in heart rate and carotid and coronary blood flows, and decrease in mean arterial pressure, were dose dependently attenuated by -CGRP[8-37], as shown in Fig. 5. In anesthetized rats, similar effects on carotid blood flow and mean arterial pressure were observed with -CGRP[8-37]. To confirm that the 30- g/kg/min dose of -CGRP[8-37] could completely block the exogenous -CGRP challenge, a higher dose of -CGRP (0.8 g/kg, i.v.) also was tested in the presence of -CGRP[8-37] in the anesthetized rats. The carotid blood flow increased by only 6 1% in the presence of -CGRP[8-37] at a dose of 30 g/kg/min, which was significantly less (p 0.01) than that observed in the absence of -CGRP[8-37] ( 47 5%). The hemodynamic effects of -CGRP[8-37] at a dose of 30 g/kg/min in anesthetized rats and conscious dogs are shown in Tables 1 and 2, respectively. Administration of -CGRP[8-37] at this pharmacological blocking dose did not alter any of these indices. Figure 6 shows the effects of -CGRP[8-37] (30 g/kg/min, i.v.) on regional blood flow distribution. Again, there were no significant differences in blood flow in any of the regional vascular beds, including muscle and skin, in either species. Discussion The potent coronary vasodilatory effects of CGRP have been studied extensively, both in animal preparations (Holman et al., 1986; Ezra et al., 1987; Franco-Cereceda, 1991a,b; Quebbeman et al., 1993; Sekiguchi et al., 1994; Yaoita et al., 1994), and in humans (McEwan et al., 1986; Ludman et al., 1991; Uren et al., 1993). A few studies also have reported that CGRP induces peripheral vascular relaxation (Gardiner et Fig. 2. Effects of -CGRP at a dose of 0.3 g/kg, i.v. on mean arterial pressure, LV dp/dt, heart rate (top), and carotid, coronary, and renal blood flows (bottom) in conscious dogs. Values are percentage changes from baseline levels. -CGRP decreased mean arterial pressure and increased heart rate and regional blood flow in all of the vascular beds studied. LV dp/dt was slightly changed. Note that the peak increases in regional blood flow induced by -CGRP were similar in the carotid, coronary, and renal beds.

5 Cardiovascular Effects of -CGRP 555 Fig. 3. Peak effects of -CGRP (0.1 g/kg, i.v.), nitroprusside (3 g/kg, i.v.), and adenosine (0.16 mg/kg, i.v.) on systemic and regional vascular dynamics in conscious dogs. Values are percentage changes from baseline levels. Each of the agents induced equivalent reductions in mean arterial pressure (MAP) and total peripheral resistance (TPR). -CGRP induced similar increases in the carotid, coronary, and renal blood flow (BF). In comparison with the other two vasodilators, the -CGRP-dependent increase in carotid and coronary blood flow was less than those produced by nitroprusside or adenosine. As expected, adenosine induced the most intense vasodilation in the coronary bed and vasoconstriction in the renal bed. HR, heart rate. Fig. 4. Effects of -CGRP at a dose of 0.3 g/kg, i.v. on regional blood flow distribution measured by radioactive microsphere technique in the absence and presence of -adrenergic receptor blockade (insert) in anesthetized rats. Values are percentage changes from baseline levels. Administration of the -CGRP resulted in a significantly greater (p 0.05) increase in blood flow in heart than in the brain or kidney. However, in the presence of -adrenergic receptor blockade, regional blood flows in both heart and brain were increased similarly. al., 1990, 1991), including in vessels supplying the skin (Brain et al., 1985, 1986; Hughes and Brain, 1991; Escott and Brain, 1993). However, it is not known whether CGRP induces a preferential vasodilation in the coronary circulation as compared with other vascular beds, particularly in the cerebrovascular system. An assessment of the relative functional effects of -CGRP and of CGRP antagonism on coronary versus cerebral vasculature would be particularly important, since CGRP has been implicated in the central mechanism of the pathogenesis of migraine, and as such constitutes a potential target for antimigraine therapy. The results from the present investigation show that administration of -CGRP induced a dose-dependent vasodilation both in anesthetized rats and in conscious dogs, consistent with results published previously. Furthermore, our data from the anesthetized rats indicated that exogenous -CGRP induced a significantly greater blood flow increase in the heart than in the brain. When the same experiment was performed in the presence of -adrenergic receptor blockade, however, the increases in regional blood flow in heart and brain were similar. This suggested that the greater increase in myocardial blood flow in intact rats possibly resulted from a reflex-mediated mechanism rather than from a direct effect via the CGRP receptors located in the coronary vasculature. Also, our results from conscious dogs using continuous measurements showed that both the time to peak and the duration of the increases in coronary and carotid blood flow, the latter mainly representing the cerebral blood flow, were almost identical. Notably, the heart rate response to -CGRP in rats was different from that observed in dogs.

6 556 Shen et al. Apparently, this was mainly due to the anesthetized versus conscious states, as it has previously been shown that the increase in heart rate was considerably greater in conscious rats than in anesthetized rats when a similar dose of CGRP was given (Fisher et al., 1983; Marshall et al., 1986). To better understand whether the vascular effects of -CGRP are similar to those of known vasodilators, we compared the effects of -CGRP, nitroprusside, and adenosine in conscious dogs at similarly reduced levels of mean arterial pressure and total peripheral resistance. -CGRP produced equivalent increases in carotid, coronary, and renal blood flow. Interestingly, -CGRP-induced increases in carotid and coronary blood flow were significantly less than the other two vasodilators. The -CGRP-induced increase in renal blood flow was similar to that induced by nitroprusside, where the least intense vasodilation was observed as compared with the other vascular beds. As expected, adenosine resulted in a preferential vasorelaxation in the coronary bed, but reduced renal blood flow, which is consistent with previous reports (Macias et al., 1983; Shen and Vatner, 1993). It has been suggested that CGRP plays a key role in migraine pathogenesis, presumably through a vasodilatory action on cerebral arteries, resulting in an activation of the trigeminovascular system (Hargreaves and Shepheard, 1999; May and Goadsby, 1999). Moreover, elevated CGRP levels have been observed in patients during migraine attacks (Goadsby et al., 1990; Goadsby and Edvinnson, 1993; Gallai et al., 1995). Determining whether or not systemic and regional vascular dynamics are altered by blocking CGRP receptors is critical to understand the role of endogenous CGRP in regulating peripheral vascular tone at rest. Obviously, this issue would also directly affect whether antagonism of CGRP receptors constitutes an alternative antimigraine therapy. Our current results from both species clearly showed that the Fig. 5. Effects of -CGRP at a dose of 0.3 g/kg, i.v. on mean arterial pressure, heart rate, and carotid and coronary blood flows in the absence and presence of -CGRP[8-37] over a dose range of 10 to 30 g/kg/min, i.v. in conscious dogs. Values are peak percentage changes from baseline levels. The -CGRP[8-37] dose dependently attenuated the -CGRP-induced cardiovascular effects. administration of a -CGRP receptor antagonist, -CGRP[8-37], at a dose that completely blocked the cardiovascular effects induced by exogenous -CGRP, did not alter systemic hemodynamics or regional blood flow in different vascular beds, including subcutaneous tissues. Although our study is the first comprehensive cardiovascular profile of -CGRP[8-37], a few prior studies using a single vascular bed also found that -CGRP[8-37] did not affect vascular tone (Franco-Cereceda, 1991a; Gardiner et al., 1991; Sekiguchi et al., 1994) nor mean arterial pressure and heart rate (Franco-Cereceda, 1991a). In contrast to these and the present study, other studies reported that administration of -CGRP[8-37] resulted in coronary or mesenteric vasoconstriction (Han et al., 1990; Yaoita et al., 1994). These different results could, at least in part, be attributable to the differences in measurements and types of experimental preparations used, i.e., using perfused flow or pressure measurements from isolated tissue versus direct measurements of systemic dynamics and regional blood flow from intact or conscious animal models. Additionally, it should be noted that the present investigation does not address the potential vascular effects of CGRP receptor antagonism in disease states, in which CGRP levels may be elevated as a potential compensatory mechanism. Several studies have reported that the plasma levels of CGRP were higher in patients with congestive heart failure (Ferrari et al., 1991) and acute myocardial infarction (Mair et al., 1990). Thus, further investigation using diseased animal models is warranted. In summary, the present investigation provides functional evidence that the exogenous administration of human -CGRP results in a dose-dependent vasodilation in two species. In anesthetized rats, -CGRP induced a preferential increase in myocardial blood flow that was abolished by -adrenergic receptor blockade, suggesting that a reflex-me-

7 Cardiovascular Effects of -CGRP 557 diated mechanism is also involved. Unlike other vasodilators, e.g., nitroprusside and adenosine, -CGRP produced a similar vasorelaxation in the carotid, coronary, and renal vascular beds in conscious dogs. Administration of -CGRP[8-37] at a dose that completely blocked the exogenous action of -CGRP did not alter systemic or regional hemodynamics, suggesting that endogenous -CGRP does not play an important functional role in regulating basal vascular tone under normal, resting conditions. Acknowledgments We gratefully acknowledge the support from the Laboratory Animal Resources, Merck Research Laboratories. We also thank Richard T. Wiedmann for editorial assistance. References Ashina M, Bendtsen L, Jensen R, Schifter S and Olesen J (2000) Evidence for increased plasma levels of calcitonin gene-related peptide migraine outside of attacks. Pain 86: Brain SD, Tippins JR, Morris HR, Macintyre I and Williams TJ (1986) Potent vasodilator activity of calcitonin gene-related peptide in human skin. J Invest Dermatol 87: Brain SD, Williams TJ, Tippins JR, Morris HR and MacIntyre I (1985) Calcitonin gene-related peptide is a potent vasodilator. Nature (Lond) 313: Fig. 6. Effects of -CGRP[8-37] at a dose of 30 g/kg/ min, i.v. on regional blood flow distribution in anesthetized rats (top) and conscious dogs (bottom). The regional blood flow in subcutaneous tissues is shown in the figure inserts. Values are the absolute change of blood flow (ml/min/g). Note that all of the regional blood flows were nearly identical before, i.e., baseline, and after administration of -CGRP[8-37]. Escott KJ and Brain SD (1993) Effects of calcitonin gene-related peptide antagonist (CGRP 8 37 ) on skin vasodilatation and edema induced by stimulation of the rat saphenous nerve. Br J Pharmacol 110: Ezra D, Laurindo FRM, Goldstein DS, Goldstein RE and Feuerstein G (1987) Calcitonin gene-related peptide: a potent modulator of coronary flow. Eur J Pharmacol 137: Ferrari R, Panzali AF, Poole-Wilson PA and Anand IS (1991) Plasma CGRP-like immunoreactivity in treated and untreated congestive heart failure. Lancet 338: Fisher LA, Kikkawa DO, Rivier JE, Amara SG, Evans RM, Rosenfeld MG, Vale WW and Brown MR (1983) Stimulation of noradrenergic sympathetic outflow by calcitonin gene-related peptide. Nature (Lond) 305: Franco-Cereceda A (1991a) Resiniferatoxin-, capsaicin- and CGRP-evoked porcine coronary vasodilatation is independent of EDRF mechanisms but antagonized by CGRP(8-37). Acta Physiol Scand 143: Franco-Cereceda A (1991b) Calcitonin gene-related peptide and human epicardial coronary arteries: presence, release and vasodilator effects. Br J Pharmacol 102: Gallai V, Sarchielli P, Floridi A, Franceschini M, Codini M, Glioti G, Trequattrini A and Palumbo R (1995) Vasoactive peptide levels in the plasma of young migraine patients with and without aura assessed both interictally and ictally. Cephalalgia 15: Gardiner SM, Compton AM, Kemp PA, Bennett T, Bose C, Foulkes R and Hughes B (1990) Antagonistic effect of human -CGRP [8-37] on the in vivo regional hemodynamic action of human -CGRP. Biochem Biophys Res Commun 171: Gardiner SM, Compton AM, Kemp PA, Bennett T, Bose C, Foulkes R and Hughes B (1991) Human -calcitonin gene-related peptide (CGRP)-(8-37), but not -(28-37), inhibits carotid vasodilator effects of human -CGRP in vivo. Eur J Pharmacol 199: Goadsby PJ and Edvinnson L (1993) The trigemino vascular system and migraine:

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