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

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).

Bioactive beta-bend structures for the antagonist halpha CGRP(8 - 37) at the CGRP(1) receptor of the rat pulmonary artery.

The aim of this study was to determine beta-bend structures and the role of the N- and C-terminus in the antagonist halpha CGRP(8 - 37) at the rat pulmonary artery CGRP receptor mediating halpha CGRP relaxation. Halpha CGRP(8 - 37) Pro(16) (10(-6) M), with a bend-biasing residue (proline) at position 16, did not antagonize halpha CGRP responses, while a structure-conserving amino acid (alanine(16)) at the same position retained antagonist activity (apparent pK(B) 6.6+/-0.1; 10(-6) M). Halpha CGRP(8 - 37) Pro(19) (10(-6) M), with proline at position 19 was an antagonist (apparent pK(B) 6.9+/-0.1). Incorporation of a beta-bend forcing residue, BTD (beta-turn dipeptide), at positions 19 and 20 in halpha CGRP(8 - 37) (10(-6) M) antagonized halpha CGRP responses (apparent pK(B) 7.2+/-0.2); and BTD at positions 19,20 and 33,34 within halpha CGRP(8 - 37) was a competitive antagonist (pA(2) 7.2; Schild plot slope 1.0+/-0.1). Halpha CGRP(8 - 37) analogues, substituted at the N-terminus by either glycine(8) or des-NH(2) valine(8) or proline(8) were all antagonists (apparent pK(B) 6.9+/-0.1; (10(-6) M), 7.0+/-0.1 (10(-6) M), and pA(2) 7.0 (slope 1.0+/-0.2), respectively); while replacements by proline(8) together with glutamic acid(10,14) in halpha CGRP(8 - 37) (10(-6) M) or alanine amide(37) at the C-terminus of halpha CGRP(8 - 37) (10(-5) M) were both inactive compounds. In conclusion, possible bioactive structures of halpha CGRP(8 - 37) include two beta-bends (at 18 - 21 and 32 - 35), which were mimicked by BTD incorporation. Within halpha CGRP(8 - 37), the N-terminus is not essential for antagonism while the C-terminus may interact directly with CGRP(1) receptors in the rat pulmonary artery.

Receptors mediating CGRP-induced relaxation in the rat isolated thoracic aorta and porcine isolated coronary artery differentiated by h(alpha) CGRP(8-37).

1 Receptors mediating CGRP-induced vasorelaxation were investigated in rat thoracic aorta and porcine left anterior descending (LAD) coronary artery and anterior interventricular artery (AIA), using CGRP agonists, homologues and the antagonist h(alpha) CGRP(8-37). 2 In the endothelium-intact rat aorta, h(alpha) CGRP, h(beta) CGRP, rat beta CGRP and human adrenomedullin caused relaxation with similar potencies. Compared with h(alpha) CGRP, rat amylin was about 25 fold less potent, while [Cys(ACM2,7)] h(alpha) CGRP and salmon calcitonin were at least 1000 fold weaker. 3 H(alpha) CGRP(8-37) (up to 10(-5) M) did not antagonize responses to h(alpha) CGRP, h(beta) CGRP or rat beta CGRP (apparent pKB <5). Peptidase inhibitors did not increase either the effect of h(alpha) CGRP or [Cys(ACM,2,7)] h(alpha) CGRP, while h(alpha) CGRP(8-37) remained inactive. Endothelium-dependent relaxation produced by h(alpha) CGRP was accompanied by increases in cyclic AMP and cyclic GMP, that were not inhibited by h(alpha) CGRP(8-37) (10(-5) M). 4 In porcine LAD and AIA, h(alpha) CGRP produced relaxation in an endothelium-independent manner. H(alpha) CGRP(8-37) competitively antagonized h(alpha) CGRP responses (pA2 6.3 and 6.7 (Schild slope 0.9+/-0.1, each), in LAD and AIA, respectively). In LAD artery, h(alpha) CGRP-induced relaxation was accompanied by increases in cyclic AMP that were inhibited by h(alpha) CGRP(8-37) (10(-7)-10(5 )). 5 In conclusion, the antagonist affinity for h(alpha) CGRP(8-37) in porcine coronary artery is consistent with a CGRP1 receptor, while the lack of h(alpha) CGRP(8-37) antagonism in rat aorta could suggest either a CGRP receptor different from CGRP1 and CGRP2 type, or a non-CGRP receptor.

Conformational restraints revealing bioactive beta-bend structures for halpha CGRP8-37 at the CGRP2 receptor of the rat prostatic vas deferens.

1. The main aim of this study was to identify putative beta-bends and the role of the N- and C-terminus in the CGRP receptor antagonist halpha CGRP8-37, which was measured against halpha CGRP inhibition of twitch responses in the rat prostatic vas deferens. 2. With a bend-biasing residue (proline) at position 16 in halpha CGRP8-37 (10(-5) M) an inactive compound was produced, while alanine at the same position retained antagonist activity (apparent pKB 5.6+/-0.1 at 10(-5) M). Proline at position 19 within halpha CGRP8-37 (10(-5) M) was an antagonist (apparent pKB 5.8+/-0.1). 3. Incorporation of a bend-forcing structure (beta-turn dipeptide or BTD) at either positions 19,20 or 33,34 in halpha CGRP8-37 (10(-5) M) antagonized halpha CGRP responses (apparent pKB 6.0+/-0.1 and 6.1+/-0.1, respectively). Replacement by BTD at both positions 19,20 and 33,34 within halpha CGRP8-37 competitively antagonized responses to halpha CGRP (pA2 6.2; Schild plot slope 1.0+/-0.1). 4. Halpha CGRP8-37 analogues (10(-5) M), substituted at the N-terminus by either glycine8, or des-NH2 valine8 or proline8 were all antagonists against halpha CGRP (apparent pKB 6.1+/-0.1, 6.5+/-0.1 and 6.1+/-0.1, respectively), while halpha CGRP8-37 (10(-5) M) substituted in three places by proline8 and glutamic acid10,14 was inactive. 5. Replacement of the C-terminus by alanine amide37 in halpha CGRP8-37 (10(-5) M) failed to antagonize halpha CGRP responses. 6. Peptidase inhibitors did not alter either the agonist potency of halpha CGRP or the antagonist affinities of halpha CGRP8-37 BTD19,20 and 33,34 and halpha CGRP8-37 Gly8 (against halpha CGRP responses). 7. In conclusion, two beta-bends at positions 18-21 and 32-35 are compatible with high affinity by BTD and is the first approach of modelling the bioactive structure of halpha CGRP8-37. Further, the N-terminus of halpha CGRP8-37 is not essential for antagonism, while the C-terminus interacts directly with CGRP receptor binding sites of the rat vas deferens.

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

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.