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The Ca 2+ ‐Sensing Receptor (CaR) Activates Phospholipases C, A 2 , and D in Bovine Parathyroid and CaR‐Transfected, Human Embryonic Kidney (HEK293) Cells
Author(s) -
Kifor Olga,
Diaz Ruben,
Butters Robert,
Brown Edward M.
Publication year - 1997
Publication title -
journal of bone and mineral research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.882
H-Index - 241
eISSN - 1523-4681
pISSN - 0884-0431
DOI - 10.1359/jbmr.1997.12.5.715
Subject(s) - hek 293 cells , transfection , phospholipase c , parathyroid chief cell , protein kinase c , phorbol , medicine , receptor , arachidonic acid , chemistry , phospholipase , endocrinology , microbiology and biotechnology , parathyroid hormone , calcium , biology , biochemistry , signal transduction , enzyme , gene
The extracellular Ca 2+ (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.