Production of phosphatidylethanol by phospholipase D phosphatidyl transferase in intact or dispersed pancreatic islets: evidence for the in situ metabolism of phosphatidylethanol.
To determine if phospholipase D is present in intact adult islets, we took advantage of the fact that, in the presence of ethanol, this enzyme generates phosphatidylethanol via transphosphatidylation. Extracts of cells prelabeled with [14C]arachidonate, [14C]myristate, or [14C]stearate were analyzed via three TLC systems; the identify of phosphatidylethanol was further confirmed via incorporation of [14C]ethanol into the same phospholipid bands. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate stimulated phosphatidylethanol (to 603% of basal by 60 min) both in intact adult islets and in dispersed neonatal islet cells. A nonphorbol activator of protein kinase C (mezerein) also stimulated phospholipase D, whereas a phorbol which does not activate protein kinase C (4 alpha-phorbol-12,13-didecanoate) was virtually inactive. The effects of the active phorbol ester or of mezerein were reduced by the protein kinase C inhibitor H-7 and were virtually eliminated by prior down-regulation of that enzyme. In addition, a calcium-selective ionophore (ionomycin) or fluoroaluminate also activated the islet phospholipase D. When accumulation of phosphatidylethanol (labeled with any of three fatty acids) was induced by a preincubation in the presence of ethanol plus agonist, which then were removed, phosphatidylethanol declined by 34-47% over a subsequent 60-min incubation. Thus, while phosphatidylethanol is relatively stable metabolically, it is detectably degraded (a variable overlooked in previous studies). In the absence of ethanol, stimulated islet cells generated phosphatidic acid, although such hydrolysis was less evident than transphosphatidylation. Ethanol provision distinguished phosphatidate formed via phospholipase D (inhibition, via phosphatidylethanol formation) from that due predominantly to phospholipase C (phosphatidate not inhibited). In view of our recent findings that phosphatidic acid (or exogenous phospholipase D) has potent insulinotropic effects, this pathway could play a role in stimulus-secretion coupling; conversely, stimulation of transphosphatidylation at the expense of hydrolysis could contribute to the inhibition of secretion caused by ethanol.
Metz
S A
SA
Research and Medical Services, Denver VA Medical Center, Colorado 80220.
Dunlop
M
M
D000375:Aging; D000818:Animals; D000831:Animals, Newborn; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism; D066298:In Vitro Techniques; D015759:Ionomycin / Q000494:pharmacology; D007515:Islets of Langerhans / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D007700:Kinetics; D008297:Male; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*; D010743:Phospholipids / Q000096:biosynthesis* / Q000302:isolation & purification; D051381:Rats; D011919:Rats, Inbred Strains; D013229:Stearic Acids / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D001095:Arachidonic Acids; D009227:Myristic Acids; D010743:Phospholipids; D013229:Stearic Acids; D019814:Myristic Acid; D016718:Arachidonic Acid; C031183:stearic acid; D015759:Ionomycin; D010739:Phospholipase D; D013755:Tetradecanoylphorbol Acetate