The Ca2+-sensing receptor (CaR) activates phospholipases C, A2, and D in bovine parathyroid and CaR-transfected, human embryonic kidney (HEK293) cells.
The extracellular Ca2+ (Ca2+(o))-sensing receptor (CaR) is a G protein-coupled receptor that activates phospholipase C (PLC). In the present studies, we assessed Ca2+(o)-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 Ca2+(o) increased the formation of IPs in parathyroid cells as well in HEK-CaR but not in HEK-WT cells. High Ca2+(o) 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 Ca2+(o). Moreover, high Ca2+(o)-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 Ca2+(o) 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 Ca2+(o), suggesting a major role for PLA2 in high Ca2+(o)-elicited AA release. Pretreatment of parathyroid cells with PMA stimulated release of AA at low and high Ca2+(o), while a PKC inhibitor, chelerythrine, reduced AA release at high Ca2+(o) to the level observed with low Ca2+(o) alone. Thus, PKC contributes importantly to the high Ca2+(o)-evoked, CaR-mediated activation of not only PLD but also PLA2. Finally, high Ca2+(o)-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.
Kifor
O
O
Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115, U.S.A.
Diaz
R
R
Butters
R
R
Brown
E M
EM
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D002118:Calcium / Q000502:physiology; D002135:Calcium-Binding Proteins / Q000502:physiology*; D002417:Cattle; D002460:Cell Line; D004789:Enzyme Activation; D020404:Glycerophospholipids; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000196:embryology / Q000378:metabolism*; D010280:Parathyroid Glands / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010712:Phosphatidic Acids / Q000494:pharmacology; D010739:Phospholipase D / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D020404:Glycerophospholipids; D007295:Inositol Phosphates; D010712:Phosphatidic Acids; C068564:phosphatidylbutanol; D016718:Arachidonic Acid; D010741:Phospholipases A; D054467:Phospholipases A2; D010738:Type C Phospholipases; D010739:Phospholipase D; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium