The Ca2+-sensing receptor (CaR) activates phospholipases C, A(2), and D in bovine parathyroid and CaR-transfected, human embryonic kidney (HEK293) cells

The extracellular Ca2+ (Ca-o(2+))-sensing receptor (CaR) is a G protein-coupled receptor that activates phospholipase C (PLC). In the present studies, we assessed Ca-o(2+)-dependent changes in the generation of inositol phosphates (IP), free arachidonic acid (AA), and phosphatidylbutanol (PtdBtOH) by PLC, phospholipase A(2) (PLA(2)), and phospholipase D (PLD), respectively, in bovine parathyroid cells as well as in wild-type or CaR-transfected human embryonic kidney (HEK293) cells (HEK-WT and HEK-CaR respectively). Elevated Ca-o(2+) increased the formation of IPs in parathyroid cells as well in HEK-CaR but not in HEK-WT cells. High Ca-o(2+) also elicited time- and dose-dependent increases in PtdBtOH in parathyroid cells and HEK-CaR but not in HEK-WT cells. Brief treatment of parathyroid and HEK-CaR cells with an activator of protein kinase C (PKC), phorbol 12-myristate, 13-acetate (PMA), stimulated PLD activity at both low and high Ca-o(2+). Moreover, high Ca-o(2+)-stimulated PLD activity was abolished following down-regulation of PKC by overnight phorbol myristate acetate (PMA) pretreatment, suggesting that CaR-mediated activation of PLD depends largely upon stimulation of PKC. High Ca-o(2+) likewise increased the release of free AA in parathyroid and HEK-CaR but not in HEK-WT cells. Mepacrine, a general PLA(2) inhibitor, and AACOCF(3), an inhibitor of cytosolic PLA(2), reduced AA release in parathyroid cells at high Ca-o(2+), suggesting a major role for PLA(2) in high Ca-o(2+)-elicited AA release. Pretreatment of parathyroid cells with PMA stimulated release of AA at low and high Ca-o(2+), while a PKC inhibitor, chelerythrine, reduced AA release at high Ca-o(2+) to the level observed with low Ca-o(2+) alone. Thus, PKC contributes importantly to the high Ca-o(2+)-evoked, CaR-mediated activation of not only PLD but also PLA(2). Finally, high Ca-o(2+)-stimulated production of IP, PtdBtOH, and AA all decreased substantially in parathyroid cells cultured for 4 days, in which expression of the CaR decreases by 80% or more, consistent with mediation of these effects by the receptor. Thus, the CaR activates, directly or indirectly, at least three phospholipases in bovine parathyroid and CaR-transfected HEK293 cells, providing for coordinate, receptor-mediated regulation of multiple signal transduction pathways in parathyroid and presumably other CaR-expressing cells.