Store-operated calcium entry induced by activation of Gq-coupled alpha1B adrenergic receptor in human osteoblast

Abstract

Recent studies have revealed that the sympathetic nervous system is involved in bone metabolism. We previously reported that noradrenaline (NA) suppressed K(+) currents via Gi/o protein-coupled alpha1B-adrenergic receptor (α1B-AR) in human osteoblast SaM-1 cells. Additionally, it has been demonstrated that the intracellular Ca(2+) level ([Ca(2+)]i) was increased by NA via α1B-AR. In this study, we investigated the signal pathway of NA-induced [Ca(2+)]i elevation by using Ca(2+) fluorescence imaging in SaM-1 cells. NA-induced [Ca(2+)]i elevation was suppressed by pretreatment with a PLC inhibitor, U73122. This suggested that the [Ca(2+)]i elevation was mediated by Gq protein-coupled α1B-AR. On the other hand, NA-induced [Ca(2+)]i elevation was completely abolished in Ca(2+)-free solution, which suggested that Ca(2+) influx is the predominant pathway of NA-induced [Ca(2+)]i elevation. Although the inhibition of K(+) channel by NA caused membrane depolarization, the [Ca(2+)]i elevation was not affected by voltage-dependent Ca(2+) channel blockers, nifedipine and mibefradil. Meanwhile, NA-induced [Ca(2+)]i elevation was abolished following activation of store-operated Ca(2+) channel by thapsigargin. Additionally, the [Ca(2+)]i elevation was suppressed by store-operated channel inhibitors, 2-APB, flufenamate, GdCl3 and LaCl3. These results suggest that Ca(2+) influx through store-operated Ca(2+) channels plays a critical role in the signal transduction pathway of Gq protein-coupled α1B-AR in human osteoblasts.

Keywords: 2-APB; 2-aminoethyl diphenylborate; AR; Gq protein-coupled α(1)-adrenergic receptor; MG-63; NA; Osteoblast; PDL; PI-PLC; PLC; SaM-1; Store-operated Ca(2+) channel; Sympathetic nervous system; [Ca(2+)](i); adrenergic receptor; human osteosarcoma-derived osteoblast-like cell line; human periosteum-derived osteoblastic cells; intracellular Ca(2+) concentration; noradrenaline; phosphoinositide-phospholipase C; phospholipase C; population doubling level.