PHOSPHOLIPASE D-INDUCED PHOSPHATIDATE PRODUCTION IN INTACT SMALL ARTERIES DURING NORADRENALINE STIMULATION - INVOLVEMENT OF BOTH G-PROTEIN AND TYROSINE-PHOSPHORYLATION-LINKED PATHWAYS

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 [H-3]myristate preferentially labelled phosphatidylcholine (PtdCho), and in the presence of 0.5% ethanol [H-3]phosphatidylethanol ([H-3]PEt) was formed, demonstrating PLD activity, Noradrenaline (NA) stimulation resulted in an increase in PtdCho derived [H-3]PA and [H-3]PEt formation, indicating PLD activation. Stimulation of [C-14]choline release confirmed PLD-mediated hydrolysis of PtdCho. Propranolol, an inhibitor of PA phosphohydrolase, increased [H-3]PA levels in non-stimulated tissue and decreased the rate of degradation of both [H-3]PA and [H-3]PEt, implying that this is an active route for PA metabolism in small arteries, However, [H-3]diacylglycerol levels were not increased during NA stimulation. Fluoroaluminate increased [H-3]PEt formation and [C-14]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 [H-3]PEt formation and [C-14]choline release. The combination of pervanadate and NA stimulation resulted in a dramatic increase in [H-3]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 [H-3]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.