We investigated the direct effects of changes in free ionized extracellular calcium concentrations ([Ca 2؉ ]o) on osteoblast function and the involvement of the calcium-sensing receptor (CaR) in mediating these responses. CaR mRNA and protein were detected in osteoblast models, freshly isolated fetal rat calvarial cells and murine clonal osteoblastic 2T3 cells, and in freshly frozen, undecalcified preparations of human mandible and rat femur. In fetal rat calvarial cells, elevating [Ca 2؉ ]o and treatment with gadolinium, a nonpermeant CaR agonist, resulted in phosphorylation of the extracellular signal-regulated kinases 1 and 2, Akt, and glycogensynthase kinase 3␤, consistent with signals of cell survival and proliferation. In agreement, cell number was increased under these conditions. Expression of the osteoblast differentiation markers core binding factor ␣1, osteocalcin, osteopontin, and collagen I mRNAs was increased by high [ (2, 3) and alter the levels of expression of some differentiation markers (4, 5). During mineralization, decreases in [Ca 2ϩ ] o are also likely to occur (6), but the effect of lowering [Ca 2ϩ ] o in bone cells has not been extensively addressed.The mechanism of [Ca 2ϩ ] o -sensing by osteoblasts is unclear. The parathyroid extracellular calcium-sensing receptor (CaR) is a key player in the maintenance of a constant systemic [Ca 2ϩ ] o , predominantly through regulation of parathyroid hormone (PTH) secretion and urinary calcium excretion (7,8). CaR is also present in osteoblasts (9 and references therein), where a functional role is currently debated. Recently two studies have shown that CaRdeficient mice exhibit an essentially normal skeletal phenotype when the hyperparathyroidism resulting from the lack of the parathyroid CaR is prevented (10, 11). Thus, it remains unclear whether the osteoblast CaR is a true regulator of bone function or whether its expression is vestigial (12).In this study, we investigated the effects of both decreasing and increasing [Ca 2ϩ ] o on osteoblast proliferation and intracellular signaling events, the expression of several osteoblast differentiation markers [core binding factor ␣1 (Cbfa1, also termed Runx2 and Osf2), osteocalcin (OC), osteopontin (OP), and type I collagen (collaI)], the activity of alkaline phosphatase (AlP), and mineralized nodule formation in the absence of systemic calciotropic factors, namely PTH and vitamin D. We further investigated the role played by the CaR in these events using an alternative, nonpermeant CaR agonist, gadolinium (Gd 3ϩ ) and a CaR inhibitor, NPS 89636 (a ''calcilytic''). We used well characterized osteoblast models, freshly isolated fetal rat calvarial cells (FRC) (13) and the clonal murine osteoblast cell line, 2T3 cells (14). The expression of CaR in freshly frozen sections of rat and human bone was also determined. Materials and MethodsAnimals. Sprague-Dawley rats (Charles River Breeding Laboratories) were killed by cervical dislocation and used in accordance to the U.K. Animals Scientific Procedures...

Higher plants share with animals a responsiveness to the Ca 2؉ mobilizing agents inositol 1,4,5-trisphosphate (InsP3) and cyclic ADP-ribose (cADPR). In this study, by using a vesicular 45 Ca 2؉ flux assay, we demonstrate that microsomal vesicles from red beet and cauliflower also respond to nicotinic acid adenine dinucleotide phosphate (NAADP), a Ca 2؉ -releasing molecule recently described in marine invertebrates. NAADP potently mobilizes Ca 2؉ with a K 1/2 ‫؍‬ 96 nM from microsomes of nonvacuolar origin in red beet. Analysis of sucrose gradient-separated cauliflower microsomes revealed that the NAADP-sensitive Ca 2؉ pool was derived from the endoplasmic reticulum. This exclusively nonvacuolar location of the NAADP-sensitive Ca 2؉ pathway distinguishes it from the InsP3-and cADPR-gated pathways. Desensitization experiments revealed that homogenates derived from cauliflower tissue contained low levels of NAADP (125 pmol͞mg) and were competent in NAADP synthesis when provided with the substrates NADP and nicotinic acid. NAADP-induced Ca 2؉ release is insensitive to heparin and 8-NH2-cADPR, specific inhibitors of the InsP3-and cADPR-controlled mechanisms, respectively. However, NAADP-induced Ca 2؉ release could be blocked by pretreatment with a subthreshold dose of NAADP, as previously observed in sea urchin eggs. Furthermore, the NAADP-gated Ca 2؉ release pathway is independent of cytosolic free Ca 2؉ and therefore incapable of operating Ca 2؉ -induced Ca 2؉ release. In contrast to the sea urchin system, the NAADPgated Ca 2؉ release pathway in plants is not blocked by L-type channel antagonists. The existence of multiple Ca 2؉ mobilization pathways and Ca 2؉ release sites might contribute to the generation of stimulus-specific Ca 2؉ signals in plant cells.

Platelet activation is associated with an increase of cytosolic Ca++ levels. The 1,4,5IP3receptors [1,4,5IP3R] are known to mediate Ca++ release from intracellular stores of many cell types. Currently there are at least 3 distinct subtypes of1,4,5IP3R—type I, type II, and type III—with suggestions of distinct roles in Ca++ elevation. Specific receptors for 1,3,4,5IP4 belonging to the GAP1 family have also been described though their involvement with Ca++ regulation is controversial. In this study we report that platelets contain all 3 subtypes of1,4,5IP3R but in different amounts. Type I and type II receptors are predominant. In studies using highly purified platelet plasma (PM) and intracellular membranes (IM) we report a distinct localization of these receptors. The PM fractions were found to contain the type III 1,4,5IP3R and GAP1IP4BP in contrast to IM, which contained type I1,4,5IP3R. The type II receptor exhibited a dual distribution. In studies examining the labeling of surface proteins with biotin in intact platelets only the type III1,4,5IP3R was significantly labeled. Immunogold studies of ultracryosections of human platelets showed significantly more labeling of the PM with the type III receptor antibodies than with type I receptor antibodies. Ca++ flux studies were carried out with the PM to demonstrate in vitro function of inositol phosphate receptors. Ca++ release activities were present with both 1,4,5IP3 and1,3,4,5IP4 (EC50 = 1.3 and 0.8 μmol/L, respectively). Discrimination of the Ca++-releasing activities was demonstrated with cyclic adenosine monophosphate (cAMP)-dependent protein kinase (cAMP-PK) specifically inhibiting 1,4,5IP3 but not1,3,4,5IP4-induced Ca++ flux. In experiments with both PM and intact platelets, the1,4,5IP3Rs but not GAP1IP4BP were found to be substrates of cAMP-PK and cGMP-PK. Thus the Ca++ flux property of1,3,4,5IP4 is insensitive to cAMP-PK. These studies suggest distinct roles for the1,4,5IP3R subtypes in Ca++movements, with the type III receptor and GAP1IP4BPassociated with cation entry in human platelets and the type I receptor involved with Ca++ release from intracellular stores.