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Role of the calcium-sensing receptor (CaR) in vascular endothelial cell microdomains The extracellular calcium-sensing receptor (CaR) is a G protein-coupled receptor which responds to physiologically-relevant changes in [Ca2+]o, and plays an important role in systemic Ca2+ homeostasis. The CaR is also expressed in blood vessels although its exact physiological function in the vasculature is unknown1. Calindol (a positive allosteric modulator of the CaR) induces myocyte hyperpolarization following activation of endothelial cell intermediated-conductance Ca2+-sensitive K+ (IKCa) channels and would thus be expected to induce vasorelaxation. However, calindol-induced relaxations of precontracted vessels are difficult to demonstrate and are only routinely revealed in the presence of a cocktail of drugs (L-NOARG, indomethacin and iberiotoxin)2. In the present study, pressure myography was used to investigate the role of the CaR in the control of tone in rat isolated mesenteric arteries. Following precontraction with U46619 (10-100nM), no relaxation was seen on exposure to calindol (300nM-3μM; n = 7). However, when arteries were incubated with even low concentrations of calindol (300nM; 30 min), U46619 (0.1-300nM) failed to constrict the vessels (300nM U46619; calindol; 2±1% maximal contraction (n = 4) : ethanol solvent control: 117±12.3% maximal contraction; n = 4) in the continuing presence of calindol. Similarly, phenylephrine (1nM–3μM) was also a relatively ineffective vasoconstrictor (3μM: 20±10% maximal contraction; n = 6: ethanol solvent control: 88±1.9% maximal contraction; n = 4). When the endothelium was destroyed, the anti-constrictor effects of pre-incubation with calindol were lost (300nM U46619: 94±3.7% maximal contraction; n = 4). Sharp microelectrodes (3M KCl) were used to measure vascular myocyte membrane potential. Pre-exposure to calindol (300 nM) elicited a 4.7±0.2mV (ethanol solvent control: 0.2±0.1mV) maintained hyperpolarization and inhibited U46619-induced depolarizations by 85±4.6% (100nM U46619: calindol; area under depolarization curve (AUDC), 191±45.8mV.s; n = 3: ethanol solvent control: AUDC, 1331±356.9mV.s; n = 3). In 3 experiments, levcromakalim (500nM) elicited hyperpolarizations of 5.6±0.8mV with subsequent inhibition of U46619 depolarizations (100nM U46619: 82±31.2mV.s; n = 3). Using confocal microscopy, 3-dimensional immunofluorescence images of the rat superior mesenteric arterial endothelium were labelled with an anti-CaR antibody. Staining was largely limited to the foot-like projections of the endothelium that passed through holes in the internal elastic lamina and that probably constitute microdomains involved in myoendothelial signalling3. Collectively these findings suggest that the CaR-IKCa complex may act in a feedback system that senses local [Ca2 = ]0 changes. Further experiments are underway to determine why prior activation of the CaR system is able to exert such a powerful anti-spasmogenic effect.
1. Weston et al. (2005) Circ. Res. 97: 391-398.
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