Evidence for a calcium-sensing receptor in the vascular smooth muscle cells of the spiral modiolar artery

The vascular diameter of the gerbilline spiral modiolar artery has been shown to depend on the presence of extracellular Ca2+ but it remained unknown whether the smooth muscle cells of this arteriole contain a Ca2+ sensing receptor (CaSR). The cytosolic Ca2+ concentration ([Ca2+](i)) was monitored as fluo 3 fluorescence and the vascular diameter was measured by videomicroscopy in isolated in vitro superfused spiral modiolar arteries. RT-PCR was used to probe for the presence of CaSR transcripts. Increasing the extracellular Ca2+ concentration ([Ca2+](o)) from 1 to 10 mM caused a biphasic increase in [Ca2+](i) that was paralleled by a vasoconstriction. The initial rate of this vasoconstriction, 2.01 +/- 0.07 mu m/sec (n = 131), was inhibited when cytosolic Ca2+ stores were presumably depleted with thapsigargin (IC50 = 3 x 10(-9) M, n = 26) or ryanodine (IC50 = 4 x 10(-8) M, n = 25) or when PLC was inhibited by 10(-6) M U73122 (n = 8). The initial rate of this constriction was not affected by the L-type Ca2+ channel blocker 10(-6) M nifedipine (n = 5), by 10(-6) M U73343 (n = 6), which is the inactive analogue of U73122, by the T-type Ca2+ channel blocker 10(-6) Gd3+ (n = 6) or the Na+/Ca2+ exchanger blocker 10(-4) M Ni2+ (n = 5). The agonist rank potency order was Gd3+ > Ni2+ > Ca2+ >> neomycin = Mg2+. Analysis of RNA isolated from the SMA revealed a RT-PCR product of the appropriate size for the CaSR (448 bp). Sequence analysis of the amplified cDNA fragment revealed a 94-96% amino acid identity compared to other CaSRs. These results demonstrate that the spiral modiolar artery contains a CaSR, which is most likely located in the vascular smooth muscle cells.