Expression of extracellular calcium-sensing receptor in human osteoblasticMG-63 cell line

We have previously shown the expression of the extracellular calcium (Ca-o( 2+))-sensing receptor (CaR) in osteoblast-like cell lines, and others have documented its expression in sections of murine, bovine, and rat bone. The existence of the CaR in osteoblasts remains controversial, however, since s ome studies have failed to document its expression in the same osteoblast-l ike cell lines. The goals of the present study were twofold. 1) We sought t o determine whether the CaR is expressed in the human osteoblast-like cell line, MG-63, which has recently been reported by others not to express this receptor. 2) We investigated whether the CaR, if present in MG-63 cells, i s functionally active, since most previous studies have not proven the role of the CaR in mediating known actions of Ca-o(2+) on osteoblast-like cells . We used immunocytochemistry and Western blotting with the specific, affin ity-purified anti-CaR antiserum 4637 as well as Northern blot analysis and RT-PCR using a riboprobe and PCR primers specific for the human CaR, respec tively, to show readily detectable CaR protein and mRNA expression in MG-63 cells. Finally, we employed the patch-clamp technique to show that an elev ation in Ca-o(2+) as well as the specific, allosteric CaR activator NPS R-4 67 (0.5 muM), but not its less active stereoisomer NPS S-467 (0.5 muM), act ivate an outward K+ channel in MG-63 cells, strongly suggesting that the Ca R in MG-63 cells is not only expressed but is functionally active.