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To investigate membrane lipid metabolism during smooth-muscle activation, the role of phospholipase D (PLD) in the production of phosphatidate (PA) was studied in rat small arteries stimulated with noradrenaline. Incubation with [3H]myristate preferentially labelled phosphatidylcholine (PtdCho), and in the presence of 0.5% ethanol [3H]phosphatidylethanol ([3H]PEt) was formed, demonstrating PLD activity. Noradrenaline (NA) stimulation resulted in an increase in PtdCho derived [3H]PA and [3H]PEt formation, indicating PLD activation. Stimulation of [14C]choline release confirmed PLD-mediated hydrolysis of PtdCho. Propranolol, an inhibitor of PA phosphohydrolase, increased [3H]PA levels in non-stimulated tissue and decreased the rate of degradation of both [3H]PA and [3H]PEt, implying that this is an active route for PA metabolism in small arteries. However, [3H]diacylglycerol levels were not increased during NA stimulation. Fluoroaluminate increased [3H]PEt formation and [14C]choline release, whereas high K+ in the presence of alpha 1-adrenoceptor blockade did not. Pervanadate increased phosphotyrosine levels in small arteries, and markedly stimulated [3H]PEt formation and [14C]choline release. The combination of pervanadate and NA stimulation resulted in a dramatic increase in [3H]PEt formation, which was greater than the sum of the individual responses to the two agonists. Pervanadate and fluoroaluminate in combination appeared to give an additive response, whereas high K+ did not alter the pervanadate-induced formation of [3H]PEt. Phosphotyrosine levels were increased by NA in the presence of tyrosine phosphatase inhibitors. This effect was blocked by genistein, a tyrosine kinase inhibitor. These data demonstrate that in NA-stimulated small arteries PLD-induced PtdCho hydrolysis contributes to accumulation of PA, but not of diacylglycerol. Furthermore, regulation of PLD activity appears to require G-protein and tyrosine-phosphorylation-linked pathways.

biochemical journal

Phospholipase D-induced phosphatidate production in intact small arteries during noradrenaline stimulation: involvement of both G-protein and tyrosine-phosphorylation-linked pathways