The Ca2+-Sensing Receptor (CaR) Activates Phospholipases C, A2, and D in Bovine Parathyroid and CaR-Transfected, Human Embryonic Kidney (HEK293) Cells

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Abstract
The extracellular Ca2+ (Cao2+)-sensing receptor (CaR) is a G protein–coupled receptor that activates phospholipase C (PLC). In the present studies, we assessed Cao2+-dependent changes in the generation of inositol phosphates (IP), free arachidonic acid (AA), and phosphatidylbutanol (PtdBtOH) by PLC, phospholipase A2 (PLA2), 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 Cao2+ increased the formation of IPs in parathyroid cells as well in HEK-CaR but not in HEK-WT cells. High Cao2+ 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 Cao2+. Moreover, high Cao2+-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 Cao2+ likewise increased the release of free AA in parathyroid and HEK-CaR but not in HEK-WT cells. Mepacrine, a general PLA2 inhibitor, and AACOCF3, an inhibitor of cytosolic PLA2, reduced AA release in parathyroid cells at high Cao2+, suggesting a major role for PLA2 in high Cao2+-elicited AA release. Pretreatment of parathyroid cells with PMA stimulated release of AA at low and high Cao2+, while a PKC inhibitor, chelerythrine, reduced AA release at high Cao2+ to the level observed with low Cao2+ alone. Thus, PKC contributes importantly to the high Cao2+-evoked, CaR-mediated activation of not only PLD but also PLA2. Finally, high Cao2+-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.