Elevation of Extracellular Ca(2+) Induces Store-Operated Calcium Entry via Calcium-Sensing Receptors: A Pathway Contributes to the Proliferation of Osteoblasts

AIMS: The local concentration of extracellular Ca(2+) ([Ca(2+)](o)) in bone microenvironment is accumulated during bone remodeling. In the present study we investigated whether elevating [Ca(2+)](o) induced store-operated calcium entry (SOCE) in primary rat calvarial osteoblasts and further examined the contribution of elevating [Ca(2+)](o) to osteoblastic proliferation. METHODS: Cytosolic Ca(2+) concentration ([Ca(2+)](c)) of primary cultured rat osteoblasts was detected by fluorescence imaging using calcium-sensitive probe fura-2/AM. Osteoblastic proliferation was estimated by cell counting, MTS assay and ATP assay. Agonists and antagonists of calcium-sensing receptors (CaSR) as well as inhibitors of phospholipase C (PLC), SOCE and voltage-gated calcium (Cav) channels were applied to study the mechanism in detail. RESULTS: Our data showed that elevating [Ca(2+)](o) evoked a sustained increase of [Ca(2+)](c) in a dose-dependent manner. This [Ca(2+)](c) increase was blocked by TMB-8 (Ca(2+) release inhibitor), 2-APB and BTP-2 (both SOCE blockers), respectively, whereas not affected by Cav channels blockers nifedipine and verapamil. Furthermore, NPS2143 (a CaSR antagonist) or U73122 (a PLC inhibitor) strongly reduced the [Ca(2+)](o)-induced [Ca(2+)](c) increase. The similar responses were observed when cells were stimulated with CaSR agonist spermine. These data indicated that elevating [Ca(2+)](o) resulted in SOCE depending on the activation of CaSR and PLC in osteoblasts. In addition, high [Ca(2+)](o) significantly promoted osteoblastic proliferation, which was notably reversed by BAPTA-AM (an intracellular calcium chelator), 2-APB, BTP-2, TMB-8, NPS2143 and U73122, respectively, but not affected by Cav channels antagonists. CONCLUSIONS: Elevating [Ca(2+)](o) induced SOCE by triggering the activation of CaSR and PLC. This process was involved in osteoblastic proliferation induced by high level of extracellular Ca(2+) concentration.