European Journal of Pharmacology, 219 (1992) 415-425 415 ~ 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00 EJP 52621 Regional distribution and regulation of [ 25I]calcitonin gene-related peptide binding sites in coronary arteries Gregory A. Knock, John Wharton, Jullien A.R. Gaer a, Magdi H. Yacoub b, Kenneth M. Taylor ~ and Julia M. Polak Departments of Histochemistry and ~ Cardiothoracic SurgeD', Royal Postgraduate Medical School, Hammersmith Hospital, London W12 ONA and h The Cardiothoracic Unit, Harefield llospital, Harefield, Middlessex UB9 611t, UK Received 30 March 1992, revised MS received 3 June 1992, accepted 9 June 1992 UK Quantitative in vitro autoradiographic techniques were used to localize and characterize J25I-labcllcd human calcitonin gcnc-related peptide ([~25I]hCGRP) binding sites in sections of bovine left anterior descending coronary artery (LAD). Specific high affinity (Kd 0.4 nM) [i25I]hCGRP binding sites were localized to the media of both cpicardial and myocardial coronary arteries. Binding site density was greater in distal cpicardial and myocardial arteries than in proximal epicardial regions of the LAD. Binding sites exhibited a significantly higher affinity for a-hCGRP (K, 1.1 nM) than for hCGRP-(8-37) (K, 7.0 nM) and [Cys(ACM)2'7]hCGRP (K i 27.4 nM). Guanosinc-5'-O-(3-thiotriphosphatc) inhibited [125I]hCGRP binding in a concentration-de- pendent manner. Extrinsic denervation of the bovine heart resulted in a depletion of CGRP-Iike immunoreactive perivascular nerve fibres and an increase in the density of coronary artery [~-~51]hCGRPbinding sites (P = 0.0092). The regional distribution of binding sites in human coronary arteries differed from that observed in bovine and porcine vessels. It is concluded that selective, G protein-coupled, CGRP receptors are present in the media of bovine coronary arteries; there are both regional and species differences in the distribution of CGRP binding sites in coronary arteries and endogenous CGRP may exert a tonic influence on coronary vasomotor tone. CGRP (calcitonin gene-related peptide); CGRP receptors; Autoradiography; Coronary arteries; Neuropeptides 1. Introduction Calcitonin gene-related peptide (CGRP) is a 37 amino acid peptide produced by alternative processing of calcitonin gene transcripts (Amara et al., 1982; Rosenfeld et al., 1983). CGRP-Iike immunoreactivity occurs, together with the tachykinin substance P, in sensory nerve fibres in the mammalian cardiovascular system (Wharton and Gulbenkian, 1987; Wharton et al., 1988) and is a potent inhibitor of vasomotor tone in bovine (Greenberg et al., 1987), porcine (Foulkes et al., 1991) and human epicardial coronary arteries, both in vivo (McEwan et al., 1986; Ludman et al., 1991) and in vitro (Franco-Cereceda and Rudehill, 1989; Franco- Cereceda, 1991). Two molecular forms of human CGRP have been identified, which differ by three amino acids (Steenbergh et al., 1985), but it is uncertain which form Correspondence to: J. Wharton, Department of llistochemistry, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK. Tel. 44 81 743 2030 ext. 2150, fax 44 81 743 5362. is present in cardiac nerves. Endogenous CGRP is thought to mediate the coronary vasodilation induced by the activation of eapsaicin-sensitive sensory nerve terminals in the heart and the action of CGRP on human (Franco-Cereceda and Rudehill, 1989; Franco- Cereceda, 1991), porcine (Foulkes et al., 1991) and bovine coronary arteries (Greenberg et al., 1987) is an endothelium-independent effect, which does not in- volve the release of other vasoactive agents such as endothelium-derived relaxing factor or NO. In rat coronary arteries (Prieto et al., 1991) and some other mammalian blood vessels (Samuelson and Jernbeck, 1991), however, CGRP induces relaxation by endothe- lium-dependent as well as endothelium-independent mechanisms. Regional variation in CGRP vasodilator activity and CGRP-tachyphylaxis has been found in porcine (Foulkes et al., 1991) and human coronary vessels (Ludman et al., 1991) and may reflect differ- ences in receptor density or the distribution of CGRP receptor subtypes. More than one molecular form of CGRP binding site has been identified in affinity cross-linking studies on porcine coronary artery mem- branes (Sano et al., 1989) and at least two CGRP receptor subtypes have been distinguished in rat and