Cholinergic activation of phospholipase D in lacrimal gland acini is independent of protein kinase C and calcium.
To determine if rat lacrimal gland acini contain phospholipase D (PLD) activity, we took advantage of PLD's unique ability, in the presence of ethanol, to catalyze a transphosphatidylation reaction to produce phosphatidylethanol (PEth). Lacrimal gland acini were labeled for 3 h with [14C]stearic acid, preincubated for 20 min in the presence of 2% ethanol, and incubated for 20 min with or without agonists. Total cellular lipids were then extracted and analyzed by thin-layer chromatography, and the radioactivity was determined by liquid scintillation counting. Carbachol (1 mM), a cholinergic agonist, stimulated the production of both [14C]PEth and [14C]phosphatidic acid ([14C]PA) twofold. This effect was completely blocked by the muscarinic antagonist atropine (10 microM). [14C]PEth accumulation was also stimulated twofold by the active phorbol esters, 4 beta-phorbol 12-myristate 13-acetate and 4 beta-phorbol 12,13-dibutyrate at 1 microM. Ionomycin (1 microM), a Ca2+ ionophore, also stimulated the production of [14C]PEth twofold. In contrast to carbachol, neither phorbol esters nor ionomycin stimulated [14C]PA production. Neither [14C]PEth nor [14C]PA production was altered by epinephrine (1 mM), a nonselective adrenergic agonist, or phenylephrine (0.1 and 1 mM), a specific alpha 1-adrenergic agonist. We concluded that PLD activity, modulated by muscarinic receptors, protein kinase C, and Ca2+, but not by adrenergic receptors, is present in rat lacrimal gland acini. We also concluded that cholinergic activation of PLD appears to be independent of PKC and Ca2+.
Zoukhri
D
D
Schepens Eye Research Institute, Boston, Massachusetts.
Dartt
D A
DA
D000818:Animals; D001285:Atropine / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology*; D002217:Carbachol / Q000494:pharmacology; D019852:Diacylglycerol Kinase; D004789:Enzyme Activation; D004837:Epinephrine / Q000494:pharmacology; D000431:Ethanol / Q000494:pharmacology; D007765:Lacrimal Apparatus / Q000201:enzymology*; D008297:Male; D010656:Phenylephrine / Q000494:pharmacology; D010712:Phosphatidic Acids / Q000096:biosynthesis; D010714:Phosphatidylethanolamines / Q000096:biosynthesis; D010739:Phospholipase D / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D011950:Receptors, Cholinergic / Q000502:physiology*; D013229:Stearic Acids / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D010712:Phosphatidic Acids; D010714:Phosphatidylethanolamines; D011950:Receptors, Cholinergic; D013229:Stearic Acids; D010656:Phenylephrine; D000431:Ethanol; C031183:stearic acid; D001285:Atropine; D002217:Carbachol; D017853:Phosphotransferases (Alcohol Group Acceptor); D019852:Diacylglycerol Kinase; D011493:Protein Kinase C; D010739:Phospholipase D; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D004837:Epinephrine