Am J Physiol Cell Physiol AJP: Endocrinology and Metabolism
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Am J Physiol Cell Physiol 280: C382-C393, 2001;
0363-6143/01 $5.00
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Vol. 280, Issue 2, C382-C393, February 2001

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

Toru Yamaguchi, Naibedya Chattopadhyay, Olga Kifor, Chianping Ye, Peter M. Vassilev, Jennifer L. Sanders, and Edward M. Brown

Endocrine-Hypertension Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115

We have previously shown the expression of the extracellular calcium (Cao2+)-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 some studies have failed to document its expression in the same osteoblast-like cell lines. The goals of the present study were twofold. 1) We sought to 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, is functionally active, since most previous studies have not proven the role of the CaR in mediating known actions of Cao2+ on osteoblast-like cells. We used immunocytochemistry and Western blotting with the specific, affinity-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, respectively, to show readily detectable CaR protein and mRNA expression in MG-63 cells. Finally, we employed the patch-clamp technique to show that an elevation in Cao2+ as well as the specific, allosteric CaR activator NPS R-467 (0.5 µM), but not its less active stereoisomer NPS S-467 (0.5 µM), activate an outward K+ channel in MG-63 cells, strongly suggesting that the CaR in MG-63 cells is not only expressed but is functionally active.

G protein-coupled receptor; potassium channel; Northern analysis; reverse transcriptase-polymerase chain reaction; Western analysis; immunocytochemistry


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