Pharmacological characterization of CGRP receptors mediating relaxation of the rat pulmonary artery and inhibition of twitch responses of the rat vas deferens

Abstract

1. CGRP receptors mediating vasorelaxation of the rat isolated pulmonary artery and inhibition of contractions of the rat isolated prostatic vas deferens were investigated using CGRP agonists, homologues and the antagonist CGRP_8-37.

2. In the pulmonary artery, human (h)α-CGRP-induced relaxation of phenylephrine-evoked tone was abolished either by removal of the endothelium or by N^G-nitro-L-arginine (10⁻⁵ M). The inhibitory effect of N^G-nitro-L-arginine was stereoselectively reversed by L- but not by D-arginine (10⁻⁴ M). Thus, CGRP acts via nitric oxide released from the endothelium.

3. In the endothelium-intact artery, hα-CGRP, hβ-CGRP and human adrenomedullin (10⁻¹⁰–3×10⁻⁷ M), dose-dependently relaxed the phenylephrine-induced tone with similar potency. Compared with hα-CGRP, rat amylin was around 50 fold less potent, while [Cys(ACM^2,7)] hα-CGRP (10⁻⁷–10⁻⁴ M) was at least 3000 fold less potent. Salmon calcitonin was inactive (up to 10⁻⁴ M).

4. Human α-CGRP_8-37 (3×10⁻⁷–3×10⁻⁶ M) antagonized hα-CGRP (pA₂ 6.9, Schild plot slope 1.2±0.1) and hβ-CGRP (apparent pK_B of 7.1±0.1 for hα-CGRP_8-37 10⁻⁶ M) in the pulmonary artery. Human β-CGRP_8-37 (10⁻⁶ M) antagonized hα-CGRP responses with a similar affinity (apparent pK_B 7.1±0.1). Human adrenomedullin responses were not inhibited by hα-CGRP_8-37 (10⁻⁶ M).

5. In the prostatic vas deferens, hα-CGRP, hβ-CGRP and rat β-CGRP (10⁻¹⁰–3×10⁻⁷ M) concentration-dependently inhibited twitch responses with about equal potency, while rat amylin (10⁻⁸–10⁻⁵ M) was around 10 fold less potent and the linear analogue [Cys(ACM^2,7)] hα-CGRP was at least 3000 fold weaker. Salmon calcitonin was inactive (up to 10⁻⁴ M).

6. The antagonist effect of hα-CGRP_8-37 (10⁻⁵–3×10⁻⁵) in the vas deferens was independent of the agonist, with pA₂ values against hα-CGRP of 6.0 (slope 0.9±0.1), against hβ-CGRP of 5.8 (slope 1.1±0.1), and an apparent pK_B value of 5.8±0.1 against both rat β-CGRP and rat amylin. Human β-CGRP_8-37 (3×10⁻⁵–10⁻⁴ M) competitively antagonized hα-CGRP responses (pA₂ 5.6, slope 1.1±0.2). The inhibitory effect of hα-CGRP on noradrenaline-induced contractions in both the prostatic and epididymal vas deferens was antagonized by hα-CGRP_8-37 (pA₂ 5.8 and 5.8, slope 1.0±0.2 and 1.0±0.3, respectively).

7. The effects of hα-CGRP and hα-CGRP_8-37 in both rat pulmonary artery and vas deferens were not significantly altered by pretreatment with peptidase inhibitors (amastatin, bestatin, captopril, phosphoramidon and thiorphan, all at 10⁻⁶ M). The weak agonist activity of [Cys(ACM^2,7)] hα-CGRP in the vas deferens was not increased by peptidase inhibitors.

8. These data demonstrate that two different CGRP receptors may exist in the rat pulmonary artery and vas deferens, a CGRP₁ receptor subtype in the rat pulmonary artery (CGRP_8-37 pA₂ 6.9), while the lower affinity for CGRP_8-37 (pA₂ 6.0) in the vas deferens is consistent with a CGRP₂ receptor.

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