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CARDIOVASCULAR

Activation of Capsaicin-Sensitive Sensory Neurons by Carvedilol, a Nonselective -Blocker, in Spontaneous Hypertensive Rats

Department of Diagnostic Medicine, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan

Received for publication October 7, 2003
Accepted February 5, 2004.

	Abstract

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We performed a study in spontaneous hypertensive rats (SHR) to determine whether carvedilol, a nonselective -adrenoceptor antagonist, activates capsaicin-sensitive sensory neurons (CSSNs), thereby promoting the release of calcitonin gene-related peptide (CGRP), a neuropeptide with an important role in maintenance of cardiovascular homeostasis. Carvedilol given intravenously at a dose of 0.3 mg/kg transiently decreased the mean arterial blood pressure (MABP) and increased renal tissue blood flow with increases in CGRP levels in plasma and kidney. These effects induced by carvedilol were not seen in animals pretreated with capsazepine, an antagonist of capsaicin. Although 1.0 mg/kg cavedilol markedly decreased MABP, it neither increased renal tissue blood flow nor CGRP levels in plasma and kidney. Prazosin, a selective ₁-adrenoceptor antagonist, and bisoprolol, a selective ₁-adrenoceptor antagonist, decreased MABP with capsazepine, showing no antagonistic action in either cases, and these agents increased neither renal tissue blood flow nor levels of CGRP in plasma and kidney. Both ICI 118,551 [(±)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol], a selective ₂-adrenoceptor antagonist, at a dose of 0.25 mg/kg and capsaicin mimicked effects induced by 0.3 mg/kg carvedilol. Administration of 1.0 mg/kg ICI 118,551 produced effects similar to those induced by 1.0 mg/kg carvedilol. These observations strongly suggested that the low dose of carvedilol might activate CSSNs in SHR to increase the release of CGRP, thereby decreasing blood pressure with an increase in renal tissue blood flow. The effects induced by carvedilol seemed to be mediated by its ₂-adrenoceptor blockade activity.

Carvedilol, a nonselective -adrenoceptor antagonist with ₁-adrenoceptor blockade activity, has been used as a therapeutic agent for hypertension and CHF (Nichols et al., 1991; Cleland, 1997). Carvedilol has been shown to improve the outcome of patients with severe CHF in a randomized, double-blind, placebo-controlled trial (Packer et al., 2001). Furthermore, a multicenter, double-blind, randomized parallel group trial demonstrated that mortality in patients with mild to severe CHF was significantly lower with carvedilol than with metoprolol, a selective ₁-adrenoceptor anatagonist (Pole-Wilson et al., 2003). Although the antioxidant activity of carvedilol is considered to be one explanation for its superior effect in the treatment of CHF (Cleland and Swedberg, 1996), other unknown effects might underlie the therapeutic usefulness.

Bowles et al. (2003) recently demonstrated that ₂-adrenoceptor stimulation resulted in inhibition of CGRP release from sensory neurons in vitro, suggesting that carvedilol might increase CGRP release via inhibition of ₂-adrenoceptor activation when the sympathetic nervous system is activated. Thus, we hypothesized that carvedilol might increase the release of CGRP from CSSN, thereby exerting beneficial effects in the treatment of hypertension and CHF. To examine this hypothesis, we attempted to determine in the present study whether carvedilol affects hemodynamic conditions in spontaneous hypertensive rats (SHR) by stimulating CSSN. Effects of carvedilol were compared with those of prazosin, a selective ₁-adrenoceptor antagonist; bisoprolol, a selective ₁-adrenoceptor antagonist; and ICI 118,551, a selective ₂-adrenoceptor antagonist, to determine whether the ₂-adrenoceptor blockade activity of carvedilol might be important for enhancement of CGRP release in SHR.

	Materials and Methods

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Reagents. Carvedilol was kindly provided by Daiichi Pharmaceutical Co. (Tokyo, Japan). Bisoprolol was kindly provided by Tanabe Seiyaku Co. (Tokyo, Japan). Capsaicin, capsazepine (CPZ) (a vanilloid receptor-1 antagonist), and prazosin hydrochloride (a selective ₁-adrenoceptor antagonist) were purchased from Sigma-Aldrich (St. Louis, MO). ICI 118,551 (a selective ₂-adrenoceptor antagonist) was purchased from Tocris Cookson Inc. (Bristol, UK). All other reagents were of analytical grade.

Administration of Various Agents. Carvedilol, capsazepine, and capsaicin were dissolved in 10% Tween 20/10% ethanol with saline. Carvedilol (0.3 and 1.0 mg/kg) was injected i.v. as described previously (Hashimoto et al., 1991). Bisoprolol (0.3 mg/kg), prazosin (0.3 mg/kg), and ICI 118,551 (0.25 and 1.0 mg/kg) were dissolved in saline and injected i.v. as described previously (Smith et al., 1992, Aidonidis et al., 1994; Quevedo et al., 1999). Capsazepine (15 mg/kg) was injected s.c. 30 min before administration of various agents as described previously (Perkins and Campbell, 1992). Capsaicin (1.0 mg/kg) was injected s.c. as described previously (Erin et al., 2000). Solutions were prepared immediately before the experiments. Each control animal received the vehicle in these experiments. However, because results in control experiments using the vehicle of each solution were not significantly different from those obtained by using saline (data not shown), we used, as a representative control, the data obtained by using saline in the present study.

Measurement of Arterial Blood Pressure. Male SHR (280–320 g) aged 10 weeks were purchased from Nihon SLC (Hamamatsu, Japan). The care and handling of the animals were in accordance with the National Institutes of Health guidelines. All experimental procedures described below were approved by the Kumamoto University Animal Care and Use Committee. Animals were anesthetized with intraperitoneal injection of sodium pentobarbital (50 mg/kg). The right femoral artery was cannulated with 22-gauge angiocatheter and connected with a strain-gauge transducer (Viggo-Spectramed, Singapore, Singapore) for measuring the arterial blood pressure continuously as described previously (Uchiba et al., 1996). The data were recorded using a MacLab data acquisition system (ADI Diagnostics Inc., Rexdale, ON, Canada) in conjunction with a Macintosh 7200 computer (Apple Computers, Cupertino, CA). Anesthesia was maintained by intraperitoneal administration of sodium pentobarbital (10 mg/kg) during the experiment.

Determination of Plasma Calcitonin Gene-Related Peptide Level. Plasma levels of CGRP were determined in animals by modification of the methods as described previously (Gangula et al., 2000). Plasma samples were acidified with 10% of trifluoroacetic acid (TFA) (450 µl plasma with 50 µl of TFA) and incubated on ice for 20 min. Acidified samples were centrifuged at 6000g for 15 min at 4°C, and the supernatant was retained. The pellets were then treated with 300 µl of 10% TFA, resuspended, and recentrifuged as described above. CGRP was extracted from the supernatant by using reverse phase C18 columns (Amersham Biosciences UK, Ltd., Buckinghamshire, Little Chalfont, UK). Columns were prepared by washing with 5 ml of methanol, followed by 10 ml of water before use. The supernatant was applied onto the column, followed by washing with 20 ml of 0.1% TFA. CGRP was eluted with 3 ml of 60% acetonitrile in 0.1% TFA, and the solvent was evaporated under a stream of nitrogen gas. The concentration of CGRP was assayed by using a specific enzyme immunoassay kit (SPI-BIO, Massey Cedex, France). The sensitivity of the CGRP assay was 10 pg/ml. The antiserum cross-reacted 100% of rat - and -CGRP according to the manufacturer's data sheet.

Measurement of Renal Tissue Blood Flow. Renal tissue blood flow was measured by laser-Doppler flowmeter (ALF21N; Advance, Tokyo, Japan), as described previously (Mizutani et al., 2000). After anesthesia with intraperitoneal administration of sodium pentobarbital (50 mg/kg), the right jugular veins of these animals were cannulated with a PE-10 catheter for continuous infusion of normal saline. The Doppler flowmeter probe was placed on the renal cortex. Renal tissue blood flow was measured from 30 min before drug administration. The results are expressed as percentage of initial levels.

Determination of Renal Calcitonin Gene-Related Peptide Level. Renal levels of CGRP were determined in animals by modification of the methods as described previously (Harada et al., 2002). In brief, the kidney was weighed and then homogenized in 3 ml of 2 N acetic acid. The homogenates were bathed in 90°C water for 20 min and then centrifuged at 4500g for 10 min (4°C). CGRP was extracted from the supernatant by using reverse phase C18 columns (Amersham Biosciences UK, Ltd.). Columns were prepared by washing with 5 ml of methanol, followed by 10 ml of water before use. The supernatant was applied onto the column, followed by washing with 20 ml of 0.1% trifluoroacetic acid. CGRP was eluted with 3 ml of 60% acetonitrile in 0.1% trifluoroacetic acid, and the solvent was evaporated under a stream of nitrogen gas. The concentration of CGRP was assayed by using a specific enzyme immunoassay kit (SPI-BIO). The sensitivity of the CGRP assay was 10 pg/ml. The antiserum cross-reacted 100% of rat - and -CGRP according to the manufacturer's data sheet. Results are expressed as micrograms of CGRP per gram of tissue.

Statistical Analysis. Data are expressed as the mean ± S.D. The results were compared using either analysis of variance followed by Scheffé post hoc test or an unpaired t test. A level of p < 0.05 was considered statistically significant.

	Results

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Effects of Carvedilol and/or Capsazepine on Mean Arterial Blood Pressure (MABP) and Plasma Levels of CGRP in SHR. When administered intravenously, carvedilol at a dose of 0.3 mg/kg transiently decreased the MABP by about 40 mm Hg at 15 min after administration (Fig. 1A). Administration of capsazepine, a vanilloid receptor-1 antagonist, slightly, but significantly, increased MABP from 30 to 60 min after administration (Fig. 1A). Carvedilol-induced decrease in MABP was completely antagonized by pretreatment with capsazepine (Fig. 1A). Although carvedilol at a higher dose (1.0 mg/kg) markedly decreased MABP, pretreatment with capsazepine did not antagonize this decrease (Fig. 1B). Plasma levels of CGRP were significantly increased at 15 min after administration of 0.3 mg/kg carvedilol, but these levels were not increased by administration of a higher dose of cravedilol (1.0 mg/kg) (Fig. 2). Plasma levels of CGRP were slightly, but significantly, lower in animals given capsazepine at 45 min after administration than those given saline alone (control animals) (Fig. 2). Increases in plasma levels of CGRP seen in animals given 0.3 mg/kg carvedilol were almost completely inhibited by pretreatment with capsazepine (Fig. 2). Plasma levels of CGRP in animals given 0.3 and 1.0 mg/kg carvedilol, but pretreated with capsazepine, were significantly lower than those of control animals (Fig. 2).

View larger version (34K): [in this window] [in a new window]   Fig. 1. Effects of carvedilol and/or capsazepine on mean arterial blood pressure in SHR. Mean arterial blood pressure was continuously measured by pressure transducer after intravenous administration of 0.3 mg/kg (A) or 1.0 mg/kg (B) carvedilol in SHR. CPZ (15 mg/kg) was injected subcutaneously 30 min before the administration of carvedilol. Control animals received saline instead of capsazepine or carvedilol. Each value represents the mean ± S.D. derived from five animal experiments. A, open circles, saline + saline; closed circles, CPZ + saline; open squares, saline + carvedilol (0.3 mg/kg i.v.); and closed squares, CPZ + carvedilol (0.3 mg/kg i.v.). B, open circles, saline + saline; closed circles, CPZ + saline; open squares, saline + carvedilol (1.0 mg/kg i.v.); and closed squares, CPZ + carvedilol (1.0 mg/kg i.v.). **, p < 0.01 versus saline; , p < 0.01 versus carvedilol.

View larger version (21K): [in this window] [in a new window]   Fig. 2. Effects of carvedilol and/or capsazepine on plasma levels of CGRP in SHR. Plasma levels of CGRP in SHR were determined at 15 min after intravenous injection of 0.3 or 1.0 mg/kg carvedilol. CPZ (15 mg/kg) was subcutaneously injected 30 min before the administration of carvedilol. Control animals received saline instead of capsazepine or carvedilol. Each value represents mean ± S.D. derived from six animal experiments. **, p < 0.01 versus saline; , p < 0.01 versus carvedilol (0.3 mg/kg i.v.); , p < 0.01 versus carvedilol (1.0 mg/kg i.v.).

Effects of Prazosin, Bisoprolol, ICI 118,551, Capsaicin, and/or Capsazepine on Mean Arterial Blood Pressure and Plasma Levels of CGRP in SHR. Intravenous administration of prazosin (0.3 mg/kg), a selective ₁-adrenoceptor antagonist, and bisoprolol (0.3 mg/kg), a selective ₁-adrenoceptor antagonist, decreased MABP by about 30 and 25 mm Hg at 15 min after administration, respectively (Fig. 3, A and B). However, decreases in MABP induced by prazosin and bisoprolol were not reversed by capsazepine pretreatment (Fig. 3, A and B). Administration of ICI 118,551, a selective ₂-adrenoceptor antagonist, at doses of 0.25 and 1.0 mg/kg decreased MABP by about 30 and 80 mm Hg, at 15 min after administration, respectively (Fig. 3, C and D). Although the decrease in MABP induced by 0.25 mg/kg ICI 118,551 was antagonized by capsazepine, that induced by 1.0 mg/kg ICI 118,551 was not (Fig. 3, C and D). Subcutaneous administration of capsaicin (1.0 mg/kg) decreased MABP by about 40 mm Hg at 15 min after administration and this decrease in MABP was completely antagonized by pretreatment with capsazepine (Fig. 3E). MABP of animals given prazosin, bisoprolol, or ICI 118,551 at doses of 0.25 and 1.0 mg/kg, and capsaicin, but pretreated with capsazepine, were significantly higher than those of control animals (Fig. 3, A–E).

View larger version (25K): [in this window] [in a new window]   Fig. 3. Effects of prazosin, bisoprolol, ICI 118,551, capsaicin, and/or capsazepine on mean arterial blood pressure in SHR. Mean arterial blood pressure was continuously measured by pressure transducer after intravenous administration of prazosin (A), bisoprolol (B), 0.25 mg/kg ICI 118,551 (C), 1.0 mg/kg ICI 118,551 (D), and capsaicin (E) in SHR. CPZ (15 mg/kg) was injected s.c. 30 min before the administration of these agents. Control animals received saline instead of capsazepine, prazosin, or -adrenoceptor antagonists and capsaicin. Each value represents the mean ± S.D. derived from five animal experiments. A, open circles, saline + saline; closed circles, CPZ + saline; open squares, saline + prazosin (0.3 mg/kg i.v.); and closed squares, CPZ + prazosin (0.3 mg/kg i.v.). B, open circles, saline + saline; closed circles, CPZ + saline; open squares, saline + bisoprolol (0.3 mg/kg i.v.); and closed squares, CPZ + bisoprolol (0.3 mg/kg i.v.). C, open circles, saline + saline; closed circles, CPZ + saline; open squares, saline + ICI 118,551 (0.25 mg/kg i.v.); and closed squares, CPZ + ICI 118,551 (0.25 mg/kg i.v.). D, open circles, saline + saline; closed circles, CPZ + saline; open squares, saline + ICI 118,551 (1.0 mg/kg i.v.); and closed squares, CPZ + ICI 118,551 (1.0 mg/kg i.v.). E, open circles, saline + saline; closed circles, CPZ + saline; open squares, saline + capsaicin (1.0 mg/kg s.c.); and closed squares, CPZ + capsaicin (1.0 mg/kg s.c.). **, p < 0.01 versus saline; , p < 0.01 versus ICI 118,551 (C) or capsaicin (E).

Plasma levels of CGRP were not changed at 15 min after administration of prazosin and bisoprolol (Fig. 4). ICI 118,551 at a dose of 0.25 mg/kg significantly increased plasma levels of CGRP at 15 min after administration, whereas this agent at a dose of 1.0 mg/kg did not (Fig. 4). Subcutaneous administration of capsaicin increased plasma levels of CGRP at 15 min after administration (Fig. 4). Increases in plasma levels of CGRP seen in animals given ICI 118,551 at doses of 0.25 and 1.0 mg/kg as well as capsaicin, but pretreated with capsazepine, were significantly lower than those of control animals (Fig. 4).

View larger version (20K): [in this window] [in a new window]   Fig. 4. Effects of prazosin, bisoprolol, ICI 118,551, capsaicin, and/or capsazepine on plasma levels of CGRP in SHR. Plasma levels of CGRP in SHR were determined at 15 min after intravenous injection of prazosin (0.3 mg/kg), bisoprolol (0.3 mg/kg), and 0.3 or 1 mg/kg ICI 118,551 (ICI), 30 min after subcutaneous injection of capsaicin (1 mg/kg) and/or capsazepine. CPZ (15 mg/kg) was injected subcutaneously 30 min before the administration of these agents. Control animals received saline instead of capsazepine, prazosin, or -adrenoceptor antagonists and capsaicin. Each value represents mean ± S.D. derived from six animal experiments. **, p < 0.01 versus saline; , p < 0.01 versus ICI (0.25 mg/kg i.v.); , p < 0.01 versus ICI (1.0 mg/kg i.v.); ¶¶, p < 0.01 versus capsaicin.

Effects of Carvedilol and/or Capsazepine on Renal Tissue Blood Flow and Renal Tissue Levels of CGRP in SHR. Intravenous administration of carvedilol at a dose of 0.3 mg/kg significantly increased renal tissue blood flow in SHR, whereas that of carvedilol at a higher dose (1.0 mg/kg) did not (Fig. 5). Renal tissue blood flow was significantly decreased transiently after administration of capsazepine (Fig. 5). Increases in renal tissue blood flow in animals given carvedilol at a dose of 0.3 mg/kg were completely inhibited by pretreatment with capsazepine (Fig. 5). Renal tissue levels of CGRP were significantly increased at 30 min after administration of 0.3 mg/kg carvedilol, whereas they were not increased by administration of 1.0 mg/kg carvedilol (Fig. 6). Increases in renal tissue levels of CGRP in animals given carvedilol at a dose of 0.3 mg/kg were completely inhibited by pretreatment with capsazepine, and renal tissue levels of CGRP in animals given carvedilol at doses of 0.3 and 1.0 mg/kg, but pretreated with capsazepine, were significantly lower than those of control animals (Fig. 6).

View larger version (32K): [in this window] [in a new window]   Fig. 5. Effects of carvedilol and/or capsazepine on renal tissue blood flow in SHR. Renal tissue blood flow was continuously measured by laser-Doppler flowmeter after intravenous administration of 0.3 mg/kg carvedilol (A), 1.0 mg/kg carvedilol (B), and/or capsazepine in SHR. CPZ (15 mg/kg) was injected subcutaneously 30 min before the administration of carvedilol. Control animals received saline instead of capsazepine or carvedilol. Each value represents the mean ± S.D. derived from five animal experiments. A, open circles, saline + saline; closed circles, CPZ + saline; open squares, saline + carvedilol (0.3 mg/kg i.v.); and closed squares, CPZ + carvedilol (0.3 mg/kg i.v.). B, open circles, saline + saline; closed circles, CPZ + saline; open squares, saline + carvedilol (1.0 mg/kg i.v.); and closed squares, CPZ + carvedilol (1.0 mg/kg i.v.). **, p < 0.01 versus saline; , p < 0.01 versus carvedilol.

View larger version (21K): [in this window] [in a new window]   Fig. 6. Effects of carvedilol and/or capsazepine on renal tissue levels of CGRP in SHR. Renal tissue levels of CGRP in SHR were determined at 30 min after intravenous injection of 0.3 and 1.0 mg/kg carvedilol and/or capsazepine. CPZ (15 mg/kg) was injected subcutaneously 30 min before the administration of carvedilol. Control animals received saline instead of capsazepine or carvedilol. Each value represents mean ± S.D. derived from six animal experiments. **, p < 0.01 versus saline; , p < 0.01 versus carvedilol (0.3 mg/kg i.v.); , p < 0.01 versus carvedilol (1.0 mg/kg i.v.).

Effects of Prazosin, Bisoprolol, ICI 118,551, Capsaicin, and/or Capsazepine on Renal Tissue Blood Flow and Renal Tissue Levels of CGRP in SHR. Neither prazosin nor bisoprolol given intravenously affected the renal tissue blood flow (data not shown). Although intravenous administration of ICI 118,551 at a dose of 0.25 mg/kg significantly increased renal tissue blood flow, that of a higher dose of ICI 118,551 (1.0 mg/kg) did not (Fig. 7, A and B). Subcutaneous administration of capsaicin significantly increased renal tissue blood flow (Fig. 7C). Administration of capsazepine significantly decreased renal tissue blood flow (Fig. 7, A–C) and almost completely inhibited increases in renal tissue blood flow induced by 0.25 mg/kg ICI 118,551 and capsaicin (Fig. 7, A and C). Renal tissue blood flow in animals given ICI 118,551 at doses of 0.25 and 1.0 mg/kg and capsaicin, but pretreated with capsazepine, was significantly lower than that of control animals (Fig. 7, A–C).

View larger version (29K): [in this window] [in a new window]   Fig. 7. Effects of prazosin, bisoprolol, ICI 118,551, capsaicin, and/or capsazepine on renal tissue blood flow in SHR. Renal tissue blood flow was continuously measured by laser-Doppler flowmeter after intravenous administration of 0.25 mg/kg ICI 118,551 (A), 1.0 mg/kg ICI 118,551 (B), and capsaicin (C) and/or capsazepine in SHR. CPZ (15 mg/kg) was injected s.c. 30 min before the administration of these agents. Control animals received saline instead of capsazepine, prazosin, or -adrenoceptor antagonists and capsaicin. Each value represents the mean ± S.D. derived from five animal experiments. A, open circles, saline + saline; closed circles, CPZ + saline; open squares, saline + ICI 118,551 (0.25 mg/kg i.v.); and closed squares, CPZ + ICI 118,551 (0.25 mg/kg i.v.). B, open circles, saline + saline; closed circles, CPZ + saline; open squares, saline + ICI 118,551 (1.0 mg/kg i.v.); and closed squares, CPZ + ICI 118,551 (1.0 mg/kg i.v.). C, open circles, saline + saline; closed circles, CPZ + saline; open squares, saline + capsaicin (1.0 mg/kg s.c.); and closed squares, CPZ + capsaicin (1.0 mg/kg s.c.). **, p < 0.01 versus saline; , p < 0.01 versus ICI 118,551 (A and B) or capsaicin (C).

Renal tissue levels of CGRP in animals given prazosin and bisoprolol were not changed (data not shown). Although renal tissue levels of CGRP in animals given ICI 118,551 at a dose of 0.25 mg/kg were significantly increased, they were not changed in animals given ICI 118, 551 at a dose of 1.0 mg/kg (Fig. 8). Subcutaneous administration of capsaicin increased renal tissue levels of CGRP (Fig. 8). Pretreatment of animals with capsazepine significantly decreased renal tissue levels of CGRP compared with those of control animals (Fig. 8). Renal tissue levels of CGRP in animals given ICI 118,551 at doses of 0.25 and 1.0 mg/kg and capsaicin were significantly lower than those of control animals (Fig. 8).

View larger version (20K): [in this window] [in a new window]   Fig. 8. Effects of prazosin, bisoprolol, ICI 118,551, capsaicin, and/or capsazepine on renal tissue levels of CGRP in SHR. Renal tissue levels of CGRP in SHR were determined at 30 min after intravenous injection of 0.3 and 1.0 mg/kg ICI 118,551 (ICI), or 45 min after s.c. injection of capsaicin (1.0 mg/kg) and/or capsazepine. CPZ (15 mg/kg) was injected subcutaneously 30 min before the administration of ICI 118,551 and capsaicin. Control animals received saline instead of capsazepine, prazosin, or -adrenoceptor antagonists and capsaicin. Each value represents mean ± S.D. derived from six animal experiments. **, p < 0.01 versus saline; , p < 0.01 versus ICI (0.25 mg/kg i.v.); , p < 0.01 versus ICI (1.0 mg/kg i.v.); ¶¶, p < 0.01 versus capsaicin.

	Discussion

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As shown in the present study, intravenous administration of carvedilol transiently, but significantly, decreased MABP in SHR in a dose-dependent manner. Pretreatment with capsazepine completely reversed the decrease in MABP induced by 0.3 mg/kg carvedilol, whereas it did not reverse the decrease in MABP induced by 1.0 mg/kg carvedilol. Although plasma levels of CGRP were increased after administration of 0.3 mg/kg carvedilol, they were not changed by administration of 1.0 mg/kg carvedilol. Pretreatment with capsazepine completely reversed the MABP decrease induced by 0.3 mg/kg carvedilol, whereas it did not reverse the decrease in MABP induced by 1.0 mg/kg carvedilol. These observations strongly suggested that the low dose of carvedilol might increase CGRP release, thereby decreasing MABP in SHR. Because MABP was slightly, but significantly, increased with decreases in plasma levels of CGRP after administration of capsazapine in SHR, CGRP might play a critical role in regulation of MABP in SHR. Consistent with this assumption is a previous report demonstrating that the sensitivity of the vasculature to vasodilation by exogenous CGRP was significantly increased in SHR compared with that in corresponding normotensive Wistar-Kyoto rats (Kawasaki et al., 1990).

Although both prazosin, a selective ₁-adrenoceptor antagonist, and bisoprolol, a selective ₁-adrenoceptor antagonist, decreased MABP in SHR, decreases in MABP induced by these agents were not antagonized by pretreatment with capsazepine. Furthermore, neither of these agents increased plasma levels of CGRP in SHR. ICI 118.551, a selective ₂-adrenoceptor antagonist at doses of 0.25 and 1.0 mg/kg, decreased MABP in SHR, whereas pretreatment with capsazepine only antagonized the decrease in MABP in animals given 0.25 mg/kg ICI 118,551. ICI 118,551 at a dose of 0.25 mg/kg increased plasma levels of CGRP, whereas this agent at a dose of 1.0 mg/kg did not. Administration of capsaicin mimicked the effects induced by low doses of carvedilol and ICI 118,551 in SHR. These observations strongly suggested that the low dose of carvedilol might decrease MABP by increasing CGRP release in SHR and this effect might depend mainly on its ₂-adrenoceptor blockade activity. These observations are consistent with the previous reports demonstrating that ₂-adrenoceptor activation inhibited CGRP release from sensory neurons in in vitro superfusion of bovine dental pulp (Bowles et al., 2003).

Both blood flow and tissue levels of CGRP in the kidney of SHR were increased by administration of 0.3 mg/kg carvedilol, whereas they were not increased by administration of 1.0 mg/kg carvedilol as shown in the present study. Both increases in renal tissue blood flow and renal tissue levels of CGRP in animals given 0.3 mg/kg carvedilol were completely antagonized by pretreatment with capsazepine, suggesting that the low dose of carvedilol might increase renal tissue blood flow by increasing CGRP release in SHR. Pretreatment with capsazepine significantly decreased both renal tissue blood flow and renal tissue levels of CGRP, suggesting that CGRP might play an important role in regulation of renal tissue blood flow in SHR. Neither prazosin nor bisoprolol increased renal tissue blood flow or the renal tissue levels of CGRP as shown in the present study. In contrast, ICI 118551, only at the dose of 0.25 mg/kg, increased renal tissue blood flow and renal tissue levels of CGRP. Increases in both blood flow and tissue levels of CGRP in the kidney of animals given 0.25 mg/kg ICI 118,551 were completely antagonized by pretreatment with capsazepine. Administration of capsaicin produced effects similar to those induced by low doses of carvedilol and ICI 118,551. These observations strongly suggested that the low dose of carvedilol might increase the renal tissue blood flow by increasing CGRP release from sensory neurons via inhibition of ₂-adrenoceptor activation in SHR. Systemic infusion of CGRP in rats has been shown to decrease blood pressure with increases in both renal tissue blood flow and the glomerular filtration rate (Amuchasteguiet al., 1994), supporting the assumption described above. However, the reason why inhibition of ₂-adrenoceptor activation by high doses of carvedilol and ICI 118,551 did not increase CGRP release in SHR is not known.

In the present study, higher dose of capsazepine (15 mg/kg) was administered intravenously to SHR compared with that used in another study using SHR (Li et al., 2003). Because capsazepine has been shown to block the voltage-activated calcium current in sensory neurons (Docherty et al., 1997), it increases MABP and decreases renal tissue blood flow in SHR by inhibiting CGRP release not only via its competitive antagonism with vanilloid receptor-1 but also by inhibition of calcium influx into sensory neurons.

Treatment of CHF with -adrenoceptor antagonists is considered advantageous, because these drugs attenuate reflex sympathetic activation and improve cardiac function (Cleland et al., 1996). Consistent with this assumption, the mean increase in ejection fraction in patients with CHF was about 5% with administration of several -adrenoceptor antagonists other than carvedilol (Packer et al., 2001). However, Cleland (1997) reported that the increase in ejection fraction was more than 9% in patients with CHF treated with carvedilol. CGRP has been shown to possess positive inotropic and chronotropic effects (Franco-Cereceda et al., 1987), and it was demonstrated to inhibit sympathetic nervous activity (Oh-hashi et al., 2001). Plasma levels of CGRP were markedly increased in patients with untreated CHF and these levels were decreased after treatment (Ferrari et al., 1991), suggesting that activation of CSSNs might be a compensatory response that maintains cardiovascular homeostasis in patients with CHF. Consistent with this hypothesis is the observation that infusion of CGRP in patients with CHF significantly improved their hemodynamic condition (Nichols et al., 1991). These observations raise the possibility that therapeutic effects of carvedilol in patients with CHF can at least partly be mediated by CGRP released from CSSN through inhibition of ₂-adrenoceptor activation.

Although additional activities of carvedilol such as -adrenoceptor blockade activity (van Zweiten, 1993), antioxidant activity (Feuerstein et al., 1993), and antiendothelin effects (Massart et al., 1999) may be particularly important for reduction of mortality in patients with severe CHF, the precise mechanisms of the therapeutic effects remain unclear. In addition, Cheng et al. (1999) demonstrated that carvedilol blocked K⁺ and Ca²⁺ currents concomitantly in rabbit ventricular myocytes, suggesting that carvedilol might be beneficial in the treatment of ventricular tachyarrythmias by prolongation of action potential duration with minimal reverse frequency dependence. This activity of carvedilol might also contribute to the beneficial effects in the treatment of CHF. Based on these observations in the present study, it is possible that enhanced activation of CSSN by carvedilol, leading to the release of CGRP, might contribute to ameliorate the pathological conditions of CHF by improving the hemodynamic conditions of CHF. Because this effect of carvedilol might depend on its ₂-adrenodceptor blockade activity as shown in the present study, the difference in the therapeutic effects in CHF between carvedilol and metoprolol, a selective ₁-adrenoceptor antagonist (Pole-Wilson et al., 2003), might be at least partly explained by the ₂-adrenoceptor blockade activity of carvedilol.

	Footnotes

 
This study was supported, in part, by departmental funds from Kumamoto University School of Medicine.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

DOI: 10.1124/jpet.103.061150.

ABBREVIATIONS: CSSN, capsaicin-sensitive sensory neuron(s); CGRP, calcitonin gene-related peptide; CHF, congestive heart failure; SHR; spontaneous hypertensive rats; CPZ, capsazepine; TFA, trifluoroacetic acid; MABP, mean arterial blood pressure; ICI 118,551, (±)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol.

Address correspondence to: Dr. Kenji Okajima, Department of Diagnostic Medicine, Graduate School of Medical Sciences, Kumamoto University, Honjo, 1-1-1 Kumamoto 860-0811, Japan. E-mail: whynot{at}kaiju.medic.kumamoto-u.ac.jp

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