Evidence for a functional calcium-sensing receptor in the rat mesenteric artery

Annie Geraghty, Mais Absi, Arthur Weston, Gillian Edwards
University of Manchester, Manchester, United Kingdom

The G-protein coupled calcium-sensing receptor (CaR) plays an important role in systemic calcium homeostasis (Brown et al., 2001). Agonists at the CaR, including Ca2+, Mg2+ and spermine stimulate both isolated vascular cells and blood vessels (Bukoski et al., 1997, Wonneberger et al., 2000, Ohanian et al., 2005, Weston et al. 2005, Ziegelstein et al., 2006), and Western blotting and immunohistochemical studies have shown the presence of the CaR in porcine and rat blood vessels (Weston et al., 2005). The aim of the present study was to investigate the possible role of this receptor in the rat mesenteric artery using specific CaR modulators.

Mesenteric artery branches (200-300μm) were dissected from male, Wistar rats (250-300g) previously killed by CO2 asphyxiation and cervical dislocation in compliance with the Scientific Procedures Act 1986. These were either fixed and stained for CaR immunofluoresence or used for sharp microelectrode recordings and pressure myography. All values are given as mean ± s.e.mean. Statistical analysis was carried out using an unpaired Student’s t-test, one-way or two-way repeated measures ANOVA with post hoc Dunnett’s or Bonferroni tests as appropriate. A value of p<0.05 was considered significant.

Immunohistochemical studies confirmed the presence of the CaR in both endothelial cells and the adventitia. Calindol (0.3-1μM), a CaR positive allosteric modulator, induced endothelium-dependent myocyte hyperpolarizations (1μM: Δ -10.8±1.3 mV; n=4). These responses were sensitive to TRAM-34 (10μM), an intermediate conductance, Ca2+-sensitive K+ channel (IKCa) inhibitor. In separate pressure myograph experiments, corresponding calindol-induced vasodilation (3μM: 22% of maximal dilation; n=4) of pre-contracted arteries (U46619, a TXA2 agonist) was seen. Ca2+, a CaR agonist, induced a biphasic response on pre-contracted (U44619), isolated rat mesenteric arteries (n=4). Increasing [Ca2+]o from 0.5-10mM produced an initial vasoconstriction (maximal at 1mM: 48±7.2% of constricted vessel diameter). With increasing [Ca2+]o a vasodilation was seen which was statistically significant at 6-9mM (maximal at 7mM: 29±4.8% of constricted vessel diameter). Calhex 231, a negative allosteric modulator at the CaR which alone had essentially no effect on vascular tone, significantly inhibited the dilator component of this response (from 4-10mM Ca2+o; n=4).

This study presents the first specific evidence that the vasodilator effects of Ca2+ in the low millimolar range are mediated via the CaR. Together with the results of earlier studies (Weston et al., 2005, Absi et al., 2007), the data suggest that the CaR is coupled specifically to intermediate conductance, Ca2+-sensitive K+ channels and indicate that this endothelial cell receptor-channel complex could function in the modulation of vascular tone.

Absi et al. (2007) Br. J. Pharmacol. 151: 332-340, Brown et al. (2001) Phys. Rev. 81: 239-297, Bukoski et al. (1997) Hypertension 30: 1431-1439, Ohanian et al. (2005) Am. J. Physiol. 288: H1756-H1762, Weston et al. (2005) Circ. Res. 97: 391-398, Wonneberger et al. (2000) J. Memb. Biol. V175: 203-212, Ziegelstein et al. (2006) Biochem. Biophy. Res. Commun. 342: 153-163.

Supported by the British Heart Foundation (FS/06/067/21444)