title: Elevation of extracellular Ca2+ induces store-operated calcium entry via calcium-sensing receptors: a pathway contributes to the proliferation of osteoblasts.
abstract: OBJECTIVE
  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.
authors.affiliation: The Key Laboratory of Weak-Light Nonlinear Photonics, Ministry of Education, TEDA Applied Physics Institute and School of Physics, Nankai University, Tianjin, China.
authors.forename: Fen
authors.initials: F
authors.lastname: Hu
authors.affiliation: The Key Laboratory of Weak-Light Nonlinear Photonics, Ministry of Education, TEDA Applied Physics Institute and School of Physics, Nankai University, Tianjin, China.
authors.forename: Leiting
authors.initials: L
authors.lastname: Pan
authors.affiliation: The Key Laboratory of Weak-Light Nonlinear Photonics, Ministry of Education, TEDA Applied Physics Institute and School of Physics, Nankai University, Tianjin, China.
authors.forename: Kai
authors.initials: K
authors.lastname: Zhang
authors.affiliation: The Key Laboratory of Weak-Light Nonlinear Photonics, Ministry of Education, TEDA Applied Physics Institute and School of Physics, Nankai University, Tianjin, China.
authors.forename: Fulin
authors.initials: F
authors.lastname: Xing
authors.affiliation: The Key Laboratory of Weak-Light Nonlinear Photonics, Ministry of Education, TEDA Applied Physics Institute and School of Physics, Nankai University, Tianjin, China.
authors.forename: Xinyu
authors.initials: X
authors.lastname: Wang
authors.affiliation: The Key Laboratory of Weak-Light Nonlinear Photonics, Ministry of Education, TEDA Applied Physics Institute and School of Physics, Nankai University, Tianjin, China.
authors.forename: Imshik
authors.initials: I
authors.lastname: Lee
authors.affiliation: The Key Laboratory of Weak-Light Nonlinear Photonics, Ministry of Education, TEDA Applied Physics Institute and School of Physics, Nankai University, Tianjin, China.
authors.forename: Xinzheng
authors.initials: X
authors.lastname: Zhang
authors.affiliation: The Key Laboratory of Weak-Light Nonlinear Photonics, Ministry of Education, TEDA Applied Physics Institute and School of Physics, Nankai University, Tianjin, China.
authors.forename: Jingjun
authors.initials: J
authors.lastname: Xu
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism*; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D044169:Receptors, Calcium-Sensing / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D019284:Thapsigargin / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism
chemical_list: D015220:Calcium Channels; D044169:Receptors, Calcium-Sensing; D019284:Thapsigargin; D015544:Inositol 1,4,5-Trisphosphate; D010738:Type C Phospholipases; D002118:Calcium