Differential effects of phorbol esters on PGE2 and bradykinin-induced elevation of [Cai2+] in MDCK cells.
Prostaglandin (PG) activation of the phosphoinositol transduction pathway in MDCK cells and modulation of this process by phorbol esters was studied by monitoring changes in cytosolic free Ca2+ concentration, [Cai2+], with the Ca2+-sensitive fluorescent probe, fura-2 and measurement of stimulation of inositol phosphates by anion-exchange chromatography. Cells challenged with PGE1 or PGE2 responded with a prompt and transient increase in [Cai2+] that was independent of extracellular Ca2+. The K0.5 for PGE2 for the process was 6.1 X 10(-7) M. PGE1 and PGE2 appeared to be recognized by a common receptor. PGF2 alpha was without effect. 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) but not verapamil, a Ca2+ channel inhibitor, blocked the PGE2-evoked Ca2+ transient. Under identical conditions PGE2 increased inositol phosphate accumulation by 54 +/- 8% (inositol-1-monophosphate), 23 +/- 6% (inositol-1,4-bisphosphate), and 49 +/- 3% (total inositol trisphosphate), above control values. Brief (30-60 s) exposure of cells to phorbol-12,13-myristate (PMA) or phorbol-12,13-dibutyrate (PDB) completely blocked the PGE2-induced Ca2+ transient. The K0.5 for the process for PMA and PDB was 0.3 +/- 0.1 and 4.5 +/- 2.2 nM, respectively. Neither 4 alpha-nor 4 beta-phorbol, which lack the ability to activate protein kinase C, were effective in this regard. In contrast to complete blockade by 10(-8) PMA of the PGE2 (10(-5) M)-elicited Ca2+ transient, this concentration of PMA inhibited the Ca2+ transient evoked by 10(-9) M bradykinin (BK) by 50%. In fact 10(-4) M PMA only partially blocked the BK-elicited Ca2+ transient. In summary, in MDCK cells, the PG receptor is coupled both to the adenylate cyclase system and inositol phospholipid transduction pathway. The PG receptor appears to be regulated by protein kinase C. In addition to protein kinase C other factors regulate the BK receptor.
Aboolian
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Department of Medicine, School of Medicine, State University of New York, Stony Brook 11794.
Vander Molen
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Nord
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D000818:Animals; D001572:Benzofurans; D001920:Bradykinin / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D015232:Dinoprostone / Q000494:pharmacology*; D005456:Fluorescent Dyes; D016257:Fura-2; D007668:Kidney; D007700:Kinetics; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D011453:Prostaglandins / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D001572:Benzofurans; D005456:Fluorescent Dyes; D011453:Prostaglandins; D015240:Phorbol 12,13-Dibutyrate; D015232:Dinoprostone; D013755:Tetradecanoylphorbol Acetate; D001920:Bradykinin; D002118:Calcium; D016257:Fura-2