Characterisation of CGRP receptors in the human isolated middle meningeal artery
Introduction
Migraine is a neurovascular syndrome that is characterised by vasodilatation of meningeal blood vessels; this vasodilatation is associated with activation of perivascular trigeminal sensory nerves and release of calcitonin gene-related peptide (CGRP) (Hargreaves and Shepheard, 1999, Villalón et al., 2002). Indeed, plasma concentrations of CGRP, but not of other neuropeptides, are elevated during migraine attacks (Goadsby et al., 1990) and these levels are normalized by triptans in parallel with alleviation of headache (Goadsby and Edvinsson, 1993). Further, the antimigraine action of triptans has been attributed to vasoconstriction of cranial arteries, including the middle meningeal artery (Henkes et al., 1996, Tfelt-Hansen et al., 2000).
On the basis of pharmacological criteria, it is known that CGRP may act mainly on CGRP1 and CGRP2 receptors (Arulmani et al., 2004), with h-αCGRP8–37 being a 10-fold more potent antagonist at CGRP1 receptors than at CGRP2 receptors (Quirion et al., 1992). While CGRP1 receptors are widely distributed, CGRP2 receptors have only been described in rat vas deferens, and are 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) (Dumont et al., 1997). Using these pharmacological tools, CGRP receptors have been characterised in human cerebral (Sams et al., 2000) and middle meningeal (Jansen-Olesen et al., 2003) arteries.
BIBN4096BS (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)-, [R-(R⁎,S⁎)]-) has been developed as a very potent and selective human CGRP receptor antagonist (Doods et al., 2000, Durham, 2004) which: (i) displays a 10-fold higher affinity for CGRP1 than for CGRP2 receptors (Wu et al., 2000); and (ii) is effective in the acute treatment of migraine (Olesen et al., 2004). Hence, the clinical potential of CGRP receptor antagonists makes it of pivotal importance to characterise CGRP receptors using such novel antagonists in human blood vessels implicated in the pathophysiology of migraine. Using this antagonist, Edvinsson et al. (2002) showed the presence of CGRP1 receptors in human cerebral arteries. While a non-competitive behaviour of BIBN4096BS has been reported in human subcutaneous arteries (Sheykhzade et al., 2004), we have observed CGRP receptor heterogeneity in the human coronary artery using BIBN4096BS (Gupta et al., 2006) and others have described a diversity of CGRP receptors in human temporal (Verheggen et al., 2002) and meningeal arteries (Moreno et al., 2002). Regarding the latter blood vessel, it is noteworthy that the conclusions drawn by Moreno et al. were based on the reversal of already established relaxations to CGRP by BIBN4096BS, and no experiments were performed with linear agonists such as [Cys(Et)2,7]h-αCGRP.
Molecularly, CGRP1 receptors consist of at least three main different entities, namely, the calcitonin receptor like receptor (CLR), receptor activity modifying protein-1 (RAMP-1) (McLatchie et al., 1998) and receptor component protein (RCP) (Luebke et al., 1996, Poyner et al., 2002), whereas CGRP2 receptors have not yet been molecularly characterised. The molecular components of CGRP1 receptors that have been demonstrated in the human cerebral and meningeal artery include CLR and the RAMPs 1, 2 and 3 (Oliver et al., 2002, Jansen-Olesen et al., 2003); nevertheless, there is no report about RCP, which is required for the formation of a high-affinity G-protein-coupled receptor, thereby ensuring the signal transduction of CLR (Evans et al., 2000).
In view of: (i) the relevance of the meningeal artery in the pathophysiology of migraine (Henkes et al., 1996, Hargreaves and Shepheard, 1999); (ii) the fact that homo- or heterogeneity of the CGRP receptor population in this blood vessel has not yet been assessed using a classical pharmacological approach; and (iii) the CGRP2 receptor selective agonist [Cys(Et)2,7]h-αCGRP has not yet been studied in this blood vessel, we performed an analysis, using the Schild plot approach with the antagonists BIBN4096BS and h-αCGRP8–37 as well as the agonists h-αCGRP and [Cys(Et)2,7]h-αCGRP in this artery. This would allow a comparison of results obtained in other human blood vessels with BIBN4069BS (Edvinsson et al., 2002, Gupta et al., 2006, Verheggen et al., 2005). Since not all essential components required for a functional CGRP1 receptor have yet been demonstrated in the human meningeal artery we tried to confirm the presence of mRNA of all these components.
Section snippets
Tissue preparation
Human meningeal arteries were obtained from 22 individuals (10 male, 12 females; mean age: 50 ± 3 years), either undergoing neurosurgical procedures (n = 17) or from autopsy within 24 h of death (n = 5). During the surgical procedure, the dura mater together with a small piece of the meningeal artery was collected in ice-cold (0–4 °C) saline and was immediately transported to the laboratory. Subsequently, the artery was placed in a cold oxygenated Krebs bicarbonate solution (composition mM: NaCl 119,
Functional experiments
The contraction induced by 100 mM KCl in human middle meningeal arteries was 7.59 ± 0.86 mN (n = 19). Substance P (10 nM) elicited endothelium-dependent relaxant response that was 47 ± 6% of the precontraction to U46619 (10–100 nM; n = 19).
h-αCGRP concentration-dependently relaxed arterial segments with no significant difference in relaxation between the vessel segments obtained perioperatively or post mortem, as illustrated by the similar pEC50 (8.50 ± 0.19 and 8.56 ± 0.28; n = 15 and 4, respectively) and E
Discussion
Since meningeal vasodilatation induced by trigeminal release of CGRP is likely to play a prominent role in the pathophysiology of migraine (Hargreaves and Shepheard, 1999, Villalón et al., 2002, Arulmani et al., 2004), our study set out to characterise the CGRP receptors mediating vasodilatation in the human isolated middle meningeal artery. Apart from the implications discussed below, our results clearly demonstrate that BIBN4096BS is a potent competitive antagonist of h-αCGRP-induced
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