Characterisation of CGRP receptors in human and porcine isolated coronary arteries: Evidence for CGRP receptor heterogeneity
Introduction
The human calcitonin gene-related peptide (h-αCGRP), a 37-amino acid peptide, is one of the most potent endogenous vasodilators known. It exists in two forms, h-αCGRP and h-βCGRP, which differ from each other by three amino acids. In humans, these forms have almost similar biological actions (Quirion et al., 1992, Van Rossum et al., 1997), but are encoded by separate genes. Although CGRP was first described in 1982 (Amara et al., 1982) the classification of CGRP receptors has been painstakingly slow because of the lack of selective and potent ligands.
Presently, CGRP receptors are functionally classified into CGRP1 and CGRP2 types. The CGRP1 receptor has been cloned and consists of at least three main entities, namely, the calcitonin receptor like receptor (CLR), receptor activity modifying protein-1 (RAMP-1) and receptor component protein (RCP) (Luebke et al., 1996, Poyner et al., 2002); in contrast, the CGRP2 receptor, described in some animal tissues (Dumont et al., 1997, Quirion et al., 1992), has not yet been reported in humans or deciphered molecularly. Therefore, the classification and characterisation of CGRP receptors are primarily based on different functional pharmacological responses. In this respect, the C-terminal fragment h-αCGRP8-37 has a higher antagonist potency at the prototypic CGRP1 receptor described in guinea-pig atrium (pKb: 7–8) than at the CGRP2 receptor described in rat vas deferens (pKb: 5.5–6.5) (Dennis et al., 1989, Quirion et al., 1992). However, the range of antagonist affinities reported for h-αCGRP8-37 within and between different species (Poyner et al., 2002) is too wide to be explained by the existence of just two receptors. Further, the CGRP2 receptor seems to be more sensitive to the linear agonists [ethylamide-Cys2,7]-h-αCGRP ([Cys(Et)2,7]-h-αCGRP) and [acetimidomethyl-Cys2,7]-h-αCGRP ([Cys(Acm)2,7]-h-αCGRP) (pEC50: ≈7) (Dumont et al., 1997) than the CGRP1 receptor (Dennis et al., 1990, Mimeault et al., 1991). It should be noted that the selectivity of these linear agonists is ambiguous; in porcine large coronary artery [Cys(Acm)2,7]-h-αCGRP acts like a partial agonist (Waugh et al., 1999), while [Cys(Et)2,7]-h-αCGRP is known to activate the CGRP1 receptor in cell lines (Choksi et al., 2002). However, these linear agonists still are important for the study of CGRP receptors in the absence of more selective CGRP receptor agonists and antagonists.
Interestingly, the responses to CGRP receptor agonists vary depending on the location and the size of blood vessels (Foulkes et al., 1991, Sheykhzade and Nyborg, 1998). Moreover, the relaxation induced by CGRP is endothelium-dependent in the rat aorta (Wisskirchen et al., 1999), while it is endothelium-independent in human and porcine arteries (Luu et al., 1995, Wisskirchen et al., 1999). Therefore, the extrapolation of affinities from one species to another to classify CGRP receptors should be done with caution.
BIBN4096BS, a lys-tyr dipeptide derivative, (1-piperidinecarboxamide, N-[2-[[5-amino-1-[[4-(4-pyridinyl)-1-piperazinyl] carbonyl] pentyl] amino]-1-[(3,5-dibromo-4-hydroxyphenyl) methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl), displays a high antagonist potency and selectivity for human CGRP receptors (Doods et al., 2000, Durham, 2004). Moreover, this antagonist is reported to have a 10-fold higher affinity for the CGRP1 receptor (Wu et al., 2000) as compared to the CGRP2 receptor. These properties of BIBN4096BS provide the possibility for an in-depth characterisation of CGRP receptors. Although the antagonist potency of BIBN4096BS in the human coronary artery has been studied earlier (Edvinsson et al., 2002), a detailed investigation of CGRP receptors in human coronary arteries has not yet been performed. The fact that BIBN4096BS is effective in the acute treatment of migraine (Olesen et al., 2004) underlines the need for a detailed investigation of CGRP receptors in human blood vessels. Therefore, we used BIBN4096BS and other available conventional CGRP receptor ligands to characterise CGRP receptors in human and porcine isolated coronary arteries. A part of this study has been published as an abstract (Gupta et al., 2004).
Section snippets
Functional studies
Human hearts were obtained from ‘heart-beating’ donors (22 male, 28 female; 48 ± 2 years, range 3–66 years) who died due to non-cardiac causes. The hearts were provided by the Heart Valve Bank, Rotterdam after donor mediation by Bio Implant Services Foundation/Euro Transplant Foundation (Leiden, The Netherlands). Porcine hearts (pigs of either sex; 6–12 months of age) were obtained from a local slaughterhouse. In both cases, proximal (internal diameter: 2–3 mm) and distal (internal diameter:
Human coronary arteries
The contraction induced by 100 mM KCl was significantly higher in the proximal (56 ± 3 mN) than in the distal (8 ± 1 mN) coronary arteries. In contrast, the endothelium-dependent relaxant response to substance P (10 nM) was less in the proximal than in the distal coronary segments (26 ± 13 and 76 ± 4%, respectively).
h-αCGRP induced a concentration-dependent relaxation in precontracted human coronary arteries (Fig. 1). The maximal response to h-αCGRP was significantly less in the proximal (Emax: 43 ± 7%
Functional interaction between CGRP receptor agonists and antagonists
In both human and porcine proximal (internal diameter: 2–3 mm) as well as distal (internal diameter: 250–600 μm) segments of the right coronary artery, h-αCGRP elicited a concentration-dependent relaxation that was antagonised by BIBN4096BS, which proved more potent in the humans than in the pig. Moreover, our results demonstrate that these relaxations were endothelium-independent in both human and porcine distal coronary artery. Our findings, showing that the responses to h-αCGRP in all four
Acknowledgements
We would like to acknowledge Dr. H. Doods (Boehringer Ingelheim Pharma, Biberach, Germany) for the generous gift of BIBN4096BS and other CGRP receptor-ligands and Dr. P.G.H. Mulder (Department of Epidemiology and Biostatistics, Erasmus MC, Rotterdam, The Netherlands) for his kind help in the statistical analyses. This study was partly supported from funds obtained from Boehringer Ingelheim Pharma KG (Biberach, Germany) for a contract research project with Erasmus Pharma B.V.
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