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 CGRP . 2 In the pulmonary artery, human (h)a-CGRP-induced relaxation of phenylephrine-evoked tone was abolished either by removal of the endothelium or by N G -nitro-L-arginine (10 75 M). The inhibitory e ect of N G -nitro-L-arginine was stereoselectively reversed by L-but not by D-arginine (10 74 M). Thus, CGRP acts via nitric oxide released from the endothelium. 3 In the endothelium-intact artery, ha-CGRP, hb-CGRP and human adrenomedullin (10 710 ± 3610 77 M), dose-dependently relaxed the phenylephrine-induced tone with similar potency. Compared with ha-CGRP, rat amylin was around 50 fold less potent, while [Cys(ACM 2,7 )] ha-CGRP (10 77 ± 10 74 M) was at least 3000 fold less potent. Salmon calcitonin was inactive (up to 10 74 M). 4 Human a-CGRP 8-37 (3610 77 ± 3610 76 M) antagonized ha-CGRP (pA 2 6.9, Schild plot slope 1.2+0.1) and hb-CGRP (apparent pK B of 7.1+0.1 for ha-CGRP 8-37 10 76 M) in the pulmonary artery. Human b-CGRP 8-37 (10 76 M) antagonized ha-CGRP responses with a similar a nity (apparent pK B 7.1+0.1). Human adrenomedullin responses were not inhibited by ha-CGRP 8-37 (10 76 M). 5 In the prostatic vas deferens, ha-CGRP, hb-CGRP and rat b-CGRP (10 710 ± 3610 77 M) concentration-dependently inhibited twitch responses with about equal potency, while rat amylin (10 78 ± 10 75 M) was around 10 fold less potent and the linear analogue [Cys(ACM 2,7 )] ha-CGRP was at least 3000 fold weaker. Salmon calcitonin was inactive (up to 10 74 M). 6 The antagonist e ect of ha-CGRP 8-37 (10 75 ± 3610 75 ) in the vas deferens was independent of the agonist, with pA 2 values against ha-CGRP of 6.0 (slope 0.9+0.1), against hb-CGRP of 5.8 (slope 1.1+0.1), and an apparent pK B value of 5.8+0.1 against both rat b-CGRP and rat amylin. Human b-CGRP 8-37 (3610 75 ± 10 74 M) competitively antagonized ha-CGRP responses (pA 2 5.6, slope 1.1+0.2).The inhibitory e ect of ha-CGRP on noradrenaline-induced contractions in both the prostatic and epididymal vas deferens was antagonized by ha-CGRP 8-37 (pA 2 5.8 and 5.8, slope 1.0+0.2 and 1.0+0.3, respectively). 7 The e ects of ha-CGRP and ha-CGRP 8-37 in both rat pulmonary artery and vas deferens were not signi®cantly altered by pretreatment with peptidase inhibitors (amastatin, bestatin, captopril, phosphoramidon and thiorphan, all at 10 76 M). The weak agonist activity of [Cys(ACM 2,7 )] ha-CGRP in the vas deferens was not increased by peptidase inhibitors. 8 These data demonstrate that two di erent CGRP receptors may exist in the rat pulmonary artery and vas deferens, a CGRP 1 receptor subtype in the rat pulmonary artery (CGRP 8-37 pA 2 6.9), while the lower a nity for CGRP 8-37 (pA 2 6.0) in the vas deferens is consistent with a CGRP 2 receptor.

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 ha CGRP 8 ± 37 . 2 In the endothelium-intact rat aorta, ha CGRP, hb CGRP, rat b CGRP and human adrenomedullin caused relaxation with similar potencies. Compared with ha CGRP, rat amylin was about 25 fold less potent, while [Cys(ACM 2,7 )] ha CGRP and salmon calcitonin were at least 1000 fold weaker. )] ha CGRP, while ha CGRP 8 ± 37 remained inactive. Endothelium-dependent relaxation produced by ha CGRP was accompanied by increases in cyclic AMP and cyclic GMP, that were not inhibited by ha CGRP 8 ± 37 (10 75 M).4 In porcine LAD and AIA, ha CGRP produced relaxation in an endothelium-independent manner. Ha CGRP 8-37 competitively antagonized ha CGRP responses (pA 2 6.3 and 6.7 (Schild slope 0.9+0.1, each), in LAD and AIA, respectively). In LAD artery, ha CGRP-induced relaxation was accompanied by increases in cyclic AMP that were inhibited by ha CGRP 8 ± 37 (10 77 ± 10 75 M).5 In conclusion, the antagonist anity for ha CGRP 8 ± 37 in porcine coronary artery is consistent with a CGRP 1 receptor, while the lack of ha CGRP 8 ± 37 antagonism in rat aorta could suggest either a CGRP receptor dierent from CGRP 1 and CGRP 2 type, or a non-CGRP receptor.

1 The main aim of this study was to identify putative b-bends and the role of the N-and Cterminus in the CGRP receptor antagonist ha CGRP 8 ± 37 , which was measured against ha CGRP inhibition of twitch responses in the rat prostatic vas deferens. 2 With a bend-biasing residue (proline) at position 16 in ha CGRP 8 ± 37 (10 75 M) an inactive compound was produced, while alanine at the same position retained antagonist activity (apparent pK B 5.6+0.1 at 10 75 M). Proline at position 19 within ha CGRP 8 ± 37 (10 75 M) was an antagonist (apparent pK B 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 ha CGRP 8 ± 37 (10 75 M) antagonized ha CGRP responses (apparent pK B 6.0+0.1 and 6.1+0.1, respectively). Replacement by BTD at both positions 19,20 and 33,34 within ha CGRP 8 ± 37 competitively antagonized responses to ha CGRP (pA 2 6.2; Schild plot slope 1.0+0.1). 4 Ha CGRP 8 ± 37 analogues (10 75 M), substituted at the N-terminus by either glycine 8 , or des-NH 2 valine 8 or proline 8 were all antagonists against ha CGRP (apparent pK B 6.1+0.1, 6.5+0.1 and 6.1+0.1, respectively), while ha CGRP 8 ± 37 (10 75 M) substituted in three places by proline 8 and glutamic acid 10,14 was inactive. 5 Replacement of the C-terminus by alanine amide 37 in ha CGRP 8 ± 37 (10 75 M) failed to antagonize ha CGRP responses. 6 Peptidase inhibitors did not alter either the agonist potency of ha CGRP or the antagonist a nities of ha CGRP 8 ± 37 BTD 19,20 and 33,34 and ha CGRP 8 ± 37 Gly 8 (against ha CGRP responses). 7 In conclusion, two b-bends at positions 18 ± 21 and 32 ± 35 are compatible with high a nity by BTD and is the ®rst approach of modelling the bioactive structure of ha CGRP 8 ± 37 . Further, the Nterminus of ha CGRP 8 ± 37 is not essential for antagonism, while the C-terminus interacts directly with CGRP receptor binding sites of the rat vas deferens.

The aim of this study was to determine β‐bend structures and the role of the N‐ and C‐terminus in the antagonist hα CGRP8–37 at the rat pulmonary artery CGRP receptor mediating hα CGRP relaxation. Hα CGRP8–37 Pro16 (10−6 M), with a bend‐biasing residue (proline) at position 16, did not antagonize hα CGRP responses, while a structure‐conserving amino acid (alanine16) at the same position retained antagonist activity (apparent pKB 6.6±0.1; 10−6 M). Hα CGRP8–37 Pro19 (10−6 M), with proline at position 19 was an antagonist (apparent pKB 6.9±0.1). Incorporation of a β‐bend forcing residue, BTD (beta‐turn dipeptide), at positions 19 and 20 in hα CGRP8–37 (10−6 M) antagonized hα CGRP responses (apparent pKB 7.2±0.2); and BTD at positions 19,20 and 33,34 within hα CGRP8–37 was a competitive antagonist (pA2 7.2; Schild plot slope 1.0±0.1). Hα CGRP8–37 analogues, substituted at the N‐terminus by either glycine8 or des‐NH2 valine8 or proline8 were all antagonists (apparent pKB 6.9±0.1; (10−6 M), 7.0±0.1 (10−6 M), and pA2 7.0 (slope 1.0±0.2), respectively); while replacements by proline8 together with glutamic acid10,14 in hα CGRP8–37 (10−6 M) or alanine amide37 at the C‐terminus of hα CGRP8–37 (10−5 M) were both inactive compounds. In conclusion, possible bioactive structures of hα CGRP8–37 include two β‐bends (at 18–21 and 32–35), which were mimicked by BTD incorporation. Within hα CGRP8–37, the N‐terminus is not essential for antagonism while the C‐terminus may interact directly with CGRP1 receptors in the rat pulmonary artery. British Journal of Pharmacology (2000) 129, 1049–1055; doi:

1 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. 2 In isolated IAS strips, the spontaneously developed tone was concentration-dependently relaxed by ha CGRP, hb CGRP and rat b 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 ha CGRP (pEC 50 9.0+0.1; 100% maximum relaxation). [Cys(ACM 2.7 )] ha CGRP and salmon calcitonin were inactive (up to 10 75 M). 3 Ha CGRP 8 ± 37 (10 75 M) antagonized responses to ha CGRP (apparent pK B 5.7+0.3) and rat b CGRP (apparent pK B 5.8+0.2), but not to VIP. Hb CGRP 8 ± 37 (10 75 M) was an antagonist against ha CGRP (apparent pK B 6.1+0.1). Ha CGRP 8 ± 37 analogues (10 75 M), with substitutions at the Nterminus by either glycine 8 or des-NH 2 valine 8 or proline 8 , antagonized ha CGRP responses with similar a nities (apparent pK B 5.8+0.1, 5.8+0.1 and 5.5+0.1, respectively). 4 Peptidase inhibitors (amastatin, bestatin, captopril, phosphoramidon and thiorphan, 10 76 M each) did not increase the agonist potency of either ha CGRP or [Cys(ACM 2,7 )] ha CGRP, or the antagonist a nity of ha CGRP 8 ± 37 against ha CGRP or rat b CGRP. 5 These data demonstrate for the ®rst time a CGRP receptor in the rat IAS for which ha CGRP 8 ± 37 and its analogues have an a nity that is consistent with a CGRP 2 receptor. However, there is a marked species di erence as the antagonist has a 100 fold lower a nity in the rat than in the same tissue of the opossum (Chakder & Rattan, 1991).