Alpha 1-adrenergic receptor-mediated activation of phospholipase D in rat cerebral cortex.
We have investigated phospholipase D activity in rat brain cortical slices prelabeled with [32P]orthophosphoric acid. In the presence of ethanol (170 mM), norepinephrine stimulated, in a dose-dependent manner (EC50 = 2.2 microM), the accumulation of [32P]phosphatidylethanol as a result of phospholipase D activity. Norepinephrine-stimulated phospholipase D activity was completely inhibited by prazosin, a specific alpha 1-adrenergic antagonist (Ki = 2.8 nM). However, no accumulation of phosphatidylethanol was observed in the presence of the muscarinic agonist carbachol. The Ca2+ ionophore ionomycin and the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) also stimulated [32P]phosphatidylethanol accumulation in cortical slices, in a dose- and time-dependent manner, whereas the inactive phorbol, 4 alpha-phorbol 12,13-didecanoate, did not stimulate phospholipase D activity. Staurosporine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, two potent inhibitors of protein kinase C, inhibited PMA and ionomycin stimulation of phospholipase D activity, but did not affect the response to norepinephrine. Furthermore, the effects of PMA and norepinephrine were additive. Differences between PMA and norepinephrine stimulation of phospholipase D activity were also found with regard to the extracellular Ca2+ requirement and time course of phosphatidylethanol accumulation. No stimulation of phospholipase D activity by norepinephrine was observed in slices from cerebellum, a brain area with a low density of alpha 1-adrenergic receptors, while the effect of PMA was greater in the cerebellum than in cortical or hippocampal slices. These results strongly suggest that activation of phospholipase D in cortical slices by norepinephrine and PMA involve different mechanisms.
Llahi
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Department of Biochemistry, University of Tennessee, Memphis 38163.
Fain
J N
JN
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D002540:Cerebral Cortex / Q000187:drug effects / Q000201:enzymology*; D002855:Chromatography, Thin Layer; D004336:Drug Antagonism; D004789:Enzyme Activation; D020404:Glycerophospholipids; D015759:Ionomycin / Q000494:pharmacology; D008297:Male; D009638:Norepinephrine / Q000494:pharmacology; D010712:Phosphatidic Acids / Q000096:biosynthesis; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010739:Phospholipase D / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011224:Prazosin / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D051381:Rats; D011919:Rats, Inbred Strains; D011942:Receptors, Adrenergic, alpha / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D020404:Glycerophospholipids; D010712:Phosphatidic Acids; D011942:Receptors, Adrenergic, alpha; C051521:phosphatidylethanol; D015759:Ionomycin; D002217:Carbachol; D011493:Protein Kinase C; D010727:Phosphoric Diester Hydrolases; D010739:Phospholipase D; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D009638:Norepinephrine; D011224:Prazosin