CGRP(2) receptor in the internal anal sphincter of the rat: implications for CGRP receptor classification

Abstract

The CGRP receptor mediating relaxation of the rat internal anal sphincter (IAS) has been characterized using CGRP analogues, homologues, the antagonist CGRP(8 - 37) and its analogues. In isolated IAS strips, the spontaneously developed tone was concentration-dependently relaxed by halpha CGRP, hbeta CGRP and rat beta CGRP (pEC(50) 8.1+/-0.2, 8.3+/-0.1 and 8.4+/-0.2, respectively; 100% maximum response). Vasoactive intestinal polypeptide (VIP) was around 7 fold more potent than halpha CGRP (pEC(50) 9.0+/-0.1; 100% maximum relaxation). [Cys(ACM(2.7))] halpha CGRP and salmon calcitonin were inactive (up to 10(-5) M). Halpha CGRP(8 - 37) (10(-5) M) antagonized responses to halpha CGRP (apparent pK(B) 5.7+/-0.3) and rat beta CGRP (apparent pK(B) 5.8+/-0.2), but not to VIP. Hbeta CGRP(8 - 37) (10(-5) M) was an antagonist against halpha CGRP (apparent pK(B) 6.1+/-0.1). Halpha CGRP(8 - 37) analogues (10(-5) M), with substitutions at the N-terminus by either glycine(8) or des-NH(2) valine(8) or proline(8), antagonized halpha CGRP responses with similar affinities (apparent pK(B) 5.8+/-0.1, 5.8+/-0.1 and 5.5+/-0.1, respectively). Peptidase inhibitors (amastatin, bestatin, captopril, phosphoramidon and thiorphan, 10(-6) M each) did not increase the agonist potency of either halpha CGRP or [Cys(ACM(2,7))] halpha CGRP, or the antagonist affinity of halpha CGRP(8 - 37) against halpha CGRP or rat beta CGRP. These data demonstrate for the first time a CGRP receptor in the rat IAS for which halpha CGRP (8 - 37) and its analogues have an affinity that is consistent with a CGRP(2) receptor. However, there is a marked species difference as the antagonist has a 100 fold lower affinity in the rat than in the same tissue of the opossum (Chakder & Rattan, 1991).

Figure 1

Relaxation to hα CGRP of spontaneous tone in rat internal anal sphincter (IAS). Traces showing (a) development and plateau of spontaneous contractions, and (b) concentration-dependent relaxation to cumulatively administered hα CGRP on spontaneous tone, added in half-log molar increments. Numbers represent log molar concentrations of hα CGRP.

Figure 2

Agonist activities of CGRP analogues, homologues and VIP in rat IAS. Concentration response curves to hα CGRP, hβ CGRP, rat β CGRP, [Cys(ACM^2,7)] hα CGRP, salmon calcitonin, and vasoactive intestinal polypeptide (VIP) on spontaneous tone. Results are expressed as percentage relaxation of the spontaneous tone. Points and error bars represent the mean±s.e.mean of four or five separate experiments.

Figure 3

The effect of hα CGRP_8–37 on hα CGRP, rat β CGRP and VIP responses and the effect of hβ CGRP_8–37 on hα CGRP responses in the rat IAS. Concentration response curves to (a,d) hα CGRP, (b) rat β CGRP and (c) VIP on spontaneous tone, and in the presence of 10⁻⁵ M of (a–c) hα CGRP_8–37 and (d) hβ CGRP_8–37. Results are expressed as percentage relaxation of the spontaneous tone. Points and error bars represent the mean±s.e.mean of four or five individual experiments.

Figure 4

The effect of hα CGRP_8–37 analogues substituted at the N-terminus on hα CGRP responses in rat IAS. Concentration response curves to hα CGRP on spontaneous tone, and in the presence of 10⁻⁵ M of (a) hα CGRP_8–37Gly⁸, (b) hα CGRP_8–37 des-NH₂ Val⁸, and (c) hα CGRP_8–37Pro⁸. Results are expressed as percentage relaxation of the spontaneous tone. Points and error bars represent the mean±s.e.mean of four individual experiments.

Figure 5

Effect of peptidase inhibitors (amastatin, bestatin, captopril, phosphoramidon, thiorphan; 10⁻⁶ each) on hα CGRP and [Cys(ACM^2,7)] hα CGRP responses and on antagonism by hα CGRP_8–37 against hα CGRP in rat IAS. Concentration response curves to (a) hα CGRP, [Cys(ACM^2,7)] hα CGRP, and to (b) hα CGRP in the absence and presence of hα CGRP_8–37 (10⁻⁵ M) on spontaneous tone, before and after treatment with peptidase inhibitors. Results are expressed as percentage relaxation of the spontaneous tone. Points and error bars represent the mean±s.e.mean of four individual experiments.