Phorbol esters inhibit ionomycin-induced hydrolysis of phosphoinositides and phosphatidylcholine in bovine corneal epithelial cells.
The effects of phorbol esters on phospholipase C (PLC) activity towards phosphoinositides and phosphatidylcholine (PC) in bovine corneal epithelial cells were examined. The cells were labeled with 32Pi, myo[3H]inositol or methyl[14C]choline, and PLC stimulated by incubation of the cells with Ca2+ ionophore, ionomycin. The PLC activity was assessed by monitoring the loss of radioactivity from the labeled phospholipids or the accumulation of their radioactive metabolites. The data from this study can be summarized as follows: Addition of 20 microM ionomycin to the prelabeled cells resulted in a rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) and somewhat slower hydrolysis of phosphatidylinositol (PI) and phosphatidylcholine (PC) with concomitant several-fold increase in phosphatidic acid (PA). The effects of the ionophore were time- and dose-dependent. Incubation of the cells with phorbol 12,13-dibutyrate (PDBu) or phorbol 12-myristate 13-acetate (PMA) caused increased radioactivity in PC and PA, whereas the radioactivity in PI and PIP2 remained unchanged. The effects of PDBu were inhibited by staurosporine and H-7, and inactive derivatives of phorbol esters failed to exert any effect on phospholipid metabolism. Pretreatment of the corneal epithelial cells with PDBu or PMA abolished the ionomycin-induced hydrolysis of phosphoinositides and PC. The data suggest that activation of protein kinase C by phorbol esters in corneal epithelial cells results in inhibition of PLC activity towards phosphoinositides and PC through a mechanism probably involving phosphorylation of the enzyme.
Akhtar
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Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta 30912-2100.
Wilmoth
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D000818:Animals; D002417:Cattle; D002852:Chromatography, Ion Exchange; D002855:Chromatography, Thin Layer; D003315:Cornea / Q000187:drug effects* / Q000201:enzymology; D004789:Enzyme Activation; D004848:Epithelium / Q000187:drug effects / Q000201:enzymology; D006868:Hydrolysis; D015759:Ionomycin / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D010713:Phosphatidylcholines / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D010738:Type C Phospholipases / Q000378:metabolism
D010713:Phosphatidylcholines; D010716:Phosphatidylinositols; D015240:Phorbol 12,13-Dibutyrate; D015759:Ionomycin; D011493:Protein Kinase C; D010738:Type C Phospholipases; D013755:Tetradecanoylphorbol Acetate