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 p hosphatidylethanol (PEth). Lacrimal gland acini were labeled for 3 h w ith [C-14]stearic acid, preincubated for 20 min in the presence of 2% ethanol, and incubated for 20 min with or without agonists. Total cell ular lipids were then extracted and analyzed by thin-layer chromatogra phy, and the radioactivity was determined by liquid scintillation coun ting. Carbachol (1 mM), a cholinergic agonist, stimulated the producti on of both [C-14]PEth and [C-14]phosphatidic acid ([C-14]pA) twofold. This effect was completely blocked by the muscarinic antagonist atropi ne (10 mu M). [C-14]PEth accumulation was also stimulated twofold by t he active phorbol esters, 4 beta-phorbol 12-myristate 13-acetate and 4 beta-phorbol 12,13-dibutyrate at 1 mu M. Ionomycin (1 mu M), a Ca2+ i onophore, also stimulated the production of [C-14]PEth twofold. In con trast to carbachol, neither phorbol esters nor ionomycin stimulated [C -14]PA production. Neither [C-14]PEth nor [C-14]PA production was alte red by epinephrine (1 mM), a nonselective adrenergic agonist, or pheny lephrine (0.1 and 1 mM), a specific al-adrenergic agonist. We conclude d that PLD activity, modulated by muscarinic receptors, protein kinase C, and Ca2+, but not by adrenergic receptors, is present in rat lacri mal gland acini. We also concluded that cholinergic activation of PLD appears to be independent of PKC and Ca2+.