Calcitonin gene-related peptide (CGRP) in the circular muscle of guinea-pig colon: role as inhibitory transmitter and mechanisms of relaxation

Abstract

In the presence of 1 microM tetrodotoxin (TTX), human alpha calcitonin gene-related peptide (CGRP) produced a concentration-dependent relaxation (EC50 1.1 nM; Emax 86% of the relaxation to 1 microM isoprenaline) of mucosa-free circular muscle strips from the guinea-pig proximal colon. In the presence of TTX, the C-terminal fragment CGRP(8-37) produced a concentration (0.3-3 microM)-dependent rightward shift of the curve to CGRP. The TTX-resistant, receptor-mediated, CGRP-induced relaxation was unaffected by apamin (0.3 microM) and L-nitroarginine (L-NOARG, 100 microM), alone or in combination, as well as by glibenclamide (3 microM) or (S)-ketoprofen (10 microM). Tetraethylammonium (TEA, 1-10 mM) and cyclopiazonic acid (CPA, 3-10 microM) produced a concentration-dependent partial inhibition of the relaxant response to CGRP. The inhibitory effect of TEA on the maximal relaxation produced by CGRP was prevented by nifedipine (1 microM) which did not affect the CGRP-relaxation of its own. In the presence of atropine (1 microM), guanethidine (3 microM), SR 140,333 (0.3 microM), MEN 10,627 (1 microM), apamin (0.3 microM) and L-NOARG (100 microM), the application of 1 microM capsaicin produced a transient relaxation of the strips. This response was not reproduced upon a second application of capsaicin, 60 min later, indicating complete desensitization. CGRP(8-37) (0.3-1.0 microM) produced a partial inhibitory effect (about 50% inhibition) of the relaxant response to capsaicin. In the presence of atropine (1 microM), guanethidine (3 microM), SR 140,333 (0.3 microM), MEN 10,627 (1 microM), apamin (0.3 microM), L-NOARG (100 microM) and after capsaicin in vitro pretreatment (10 microM for 15 min), electrical field stimulation (EFS, 10 Hz for 5 s) produced a transient relaxation which was unchanged by CGRP(8-37) (1 microM) while being abolished by TTX. In sucrose gap, brief superfusion with 0.3 microM CGRP produced a TTX (1 microM)- resistant membrane hyperpolarization and relaxation: the hyperpolarization produced by CGRP was inhibited by about 50% by either TEA (10 mM) or CPA (10 microM), while being unaffected by glibenclamide (3 microM). The combined application of TEA and CPA was not more effective (65% inhibition) in inhibiting the CGRP-induced hyperpolarization than each drug alone. We conclude that CGRP produces a direct relaxation of the circular muscle of the guinea-pig proximal colon by activating receptors sensitive to blockade by CGRP(8-37). Activation of Ca-dependent potassium channels and Ca release/reuptake from internal store(s) appear both to be involved in the action of CGRP. Endogenous CGRP mediates part of the relaxant response evoked by stimulation of capsaicin-sensitive primary afferent nerves in the circular muscle of guinea-pig colon, while it is not involved in the apamin and L-NOARG-resistant nonadrenergic noncholinergic (NANC) relaxation produced by electrical field stimulation of intrinsic inhibitory nerves.