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 CGRP8-37. 2. In the pulmonary artery, human (h)alpha-CGRP-induced relaxation of phenylephrine-evoked tone was abolished either by removal of the endothelium or by NG-nitro-L-arginine (10(-5) M). The inhibitory effect of NG-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, halpha-CGRP, hbeta-CGRP and human adrenomedullin (10(-10) - 3 x 10(-7) M), dose-dependently relaxed the phenylephrine-induced tone with similar potency. Compared with halpha-CGRP, rat amylin was around 50 fold less potent, while [Cys(ACM2,7)] halpha-CGRP (10(-7) - 10(-4) M) was at least 3000 fold less potent. Salmon calcitonin was inactive (up to 10(-4) M). 4 Human alpha-CGRP8-37 (3 x 10(-7) - 3 x 10(-6) M) antagonized halpha-CGRP (pA2 6.9, Schild plot slope 1.2+/-0.1) and hbeta-CGRP (apparent pKB of 7.1+/-0.1 for halpha-CGRP8-37 10(-6) M) in the pulmonary artery. Human beta-CGRP8-37 (10(-6) M) antagonized halpha-CGRP responses with a similar affinity (apparent pKB 7.1+/-0.1). Human adrenomedullin responses were not inhibited by halpha-CGRP8-37 (10(-6) M). 5. In the prostatic vas deferens, halpha-CGRP, hbeta-CGRP and rat beta-CGRP (10(-10) - 3 x 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(ACM2,7)] halpha-CGRP was at least 3000 fold weaker. Salmon calcitonin was inactive (up to 10(-4) M). 6 The antagonist effect of halpha-CGRP8-37 (10(-5) 3 x 10(-5)) in the vas deferens was independent of the agonist, with pA2 values against halpha-CGRP of 6.0 (slope 0.9+/-0.1), against hbeta-CGRP of 5.8 (slope 1.1+/-0.1), and an apparent pKB value of 5.8+/-0.1 against both rat beta-CGRP and rat amylin. Human beta-CGRP8-37 (3 x 10(-5) - 10(-4) M) competitively antagonized halpha-CGRP responses (pA2 5.6, slope 1.1+/-0.2). The inhibitory effect of halpha-CGRP on noradrenaline-induced contractions in both the prostatic and epididymal vas deferens was antagonized by halpha-CGRP8-37 (pA2 5.8 and 5.8, slope 1.0+/-0.2 and 1.0+/-0.3, respectively). 7 The effects of halpha-CGRP and halpha-CGRP8-37 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(ACM2,7)] halpha-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 (CGRP8-37 pA2 6.9), while the lower affinity for CGRP8-37 (pA2 6.0) in the vas deferens is consistent with a CGRP2 receptor.