Introduction

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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, 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

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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.

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.

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 µg/kg for rats and 0.1, 0.3, and 0.5 µg/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 µg/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 µg/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 µg/kg -CGRP and 30 µg/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 µg/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 µg/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

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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-µg/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 µg/kg also were similar. The baseline hemodynamic values and the peak responses to -CGRP at the dose of 0.3 µg/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 103 ± 4 and 104 ± 6 mm Hg, while the carotid blood flow was significantly increased (p < 0.01) by 36 ± 3 and 26 ± 3% from baseline levels of 8.3 ± 0.5 and 131 ± 17 ml/min in the rats and dogs, respectively. In anesthetized rats, -CGRP at doses of 0.3, 0.5, and 0.8 µg/kg slightly increased the heart rate by 2 ± 1, 7 ± 2, and 9 ± 1%, respectively. In conscious dogs, however, the heart rate was increased by 49 ± 5% (p < 0.01) at a dose of 0.3 µg/kg of -CGRP.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Peak effects of -CGRP at doses of 0.1 to 0.5 µg/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.

View this table: [in this window] [in a new window]   TABLE 1 Cardiovascular effects of -CGRP (0.3 µg/kg, i.v.) and -CGRP[8-37] (30 µg/kg/min, i.v.) in anesthetized rats

View this table: [in this window] [in a new window]   TABLE 2 Cardiovascular effects of -CGRP (0.3 µg/kg, i.v.) and -CGRP[8-37] (30 µg/kg/min, i.v.) in conscious dogs

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Effects of -CGRP at a dose of 0.3 µg/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.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Peak effects of -CGRP (0.1 µg/kg, i.v.), nitroprusside (3 µg/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.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Effects of -CGRP at a dose of 0.3 µg/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.

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 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 µg/kg/min, the effects of -CGRP challenge (0.3 µg/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-µg/kg/min dose of -CGRP[8-37] could completely block the exogenous -CGRP challenge, a higher dose of -CGRP (0.8 µg/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 µg/kg/min, which was significantly less (p < 0.01) than that observed in the absence of -CGRP[8-37] (+47 ± 5%).

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Effects of -CGRP at a dose of 0.3 µg/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 µg/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.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Effects of -CGRP[8-37] at a dose of 30 µg/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].

	Discussion

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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 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. 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 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-mediated 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.