In this study, the presence of GPRC6A receptors in rat mesenteric artery was investigated. In artery homogenates, GPRC6A mRNA was detected and Western blotting showed the presence of GPRC6A protein. Immunohistochemical studies revealed GPRC6A in both endothelial cells and myocytes. In whole vessel segments, the GPRC6A activators, 300 microM l-ornithine and 100 microM Al(3+), induced endothelium-dependent myocyte hyperpolarizations sensitive to 10 microM TRAM-34, a blocker of intermediate conductance, Ca(2+)-sensitive K(+) channels (IK(Ca)). Activation of IK(Ca) with calindol (300 nM; a positive allosteric Ca(2+)-sensing receptor - CaR - modulator) was inhibited by 500 nM ouabain (inhibition of rat type 2 and type 3 Na(+)/K(+)-ATPases) but unaffected by 30 microM Ba(2+) (blockade of inwardly rectifying K(+) channels). Neither l-ornithine nor Al(3+) activated CaRs heterologously expressed in CHO or HEK293 cells. In the presence of 300 microM l-ornithine or 100 microM Al(3+), myocyte hyperpolarizations to calindol were potentiated whereas this potentiation and hyperpolarizations to l-ornithine were lost following incubation with an anti-GPRC6A antibody. It is concluded that GPRC6A receptors are present on mesenteric artery endothelial cells and myocytes and that their activation selectively opens IK(Ca) channels. This triggers a ouabain-sensitive myocyte hyperpolarization suggesting a close functional relationship between GPRC6A, the IK(Ca) channel and type 2 and/or type 3 Na(+)/K(+)-ATPases.