Limitations in using peptide drugs to characterize calcitonin gene-relatedpeptide receptors

Calcitonin gene-related peptide (CGRP) is an endogenous vasodilator peptide that produces its effects by activation of CGRP(1) and CGRP(2) receptor su btypes, These receptor subtypes are characterized in functional studies usi ng the agonist Cys(Acm)(2,7)-human-alpha-calcitonin gene-related peptide (C ys(ACM)(2,7)-h-alpha-CGRP), which activates CGRP(2) receptors, and the anta gonist h-alpha CGRP(8-37) which has a high affinity for CGRP, receptors and a low affinity for CGRP(2) receptors. Our aim was to identify factors that may limit the use of these drugs to characterize CGRP receptor subtypes. W e studied CGRP receptors using isolated ring segments of pig coronary and b asilar arteries studied in vitro. The affinity of the antagonist h-alpha CG RP(8-37) for inhibiting h-alpha CGRP-induced relaxation of coronary arterie s (log(10) of the antagonist equilibrium dissociation constant = -5.33) was determined from Schild plots that had steep slopes. Therefore, we used cap saicin to investigate the role of endogenous CGRP in confounding affinity m easurements for h-alpha CGRP(8-37). After capsaicin treatment, the slopes o f the Schild plots were not different from one, and a higher affinity of h- CGRP(8-37) in blocking relaxation was obtained (log(10) of the antagonist e quilibrium dissociation constant = -6.01). We also investigated the agonist activity of the putative CGRP, receptor selective agonist Cys(Acm)(2,7)-h- alpha-CGRP. We found that maximal relaxation of coronary arteries caused by Cys(Acm)(2,7)-h-alpha CGRP was dependent upon the level of contractile ton e induced by KCI. We also determined the K-A for Cys(Acm)(2,7)-h-alpha CGRP and found that the K-A (817 nM) was not significantly different from the E C50 (503 nM) for this drug in causing relaxation, indicating that Cys(Acm)( 2,7)-h-alpha CGRP is a partial agonist. Because experimental conditions aff ect the actions of h-CGRP(8-37) and Cys(Acm)(2,7)-h-alpha CGRP, the conditi ons must be carefully controlled to reliably identify CGRP receptor subtype s.