STIMULATION OF PHOSPHOLIPASE-D IN RABBIT PLATELET MEMBRANES BY NUCLEOSIDE TRIPHOSPHATES AND BY PHOSPHOCREATINE - ROLES OF MEMBRANE-BOUND GDP, NUCLEOSIDE DIPHOSPHATE KINASE AND CREATINE-KINASE

Previous work has shown that guanosine 5'-[gamma-thio]triphosphate (GT P[S]) and GTP stimulate phospholipase D (PLD) in rabbit platelet membr anes and that these effects are greatly enhanced by pretreatment of pl atelets with phorbol esters that activate protein kinase C [Van der Me ulen and Haslam (1990), Biochem. J. 271, 693-700]. In the present stud y, the effects of Mg2+, various nucleoside triphosphates and phosphocr eatine (PCr) were investigated. Platelet membranes containing phosphol ipids labelled with [H-3]glycerol were assayed for PLD in the presence of an optimal Mg2+ concentration (10 mM) by measuring [H-3]phosphatid ylethanol formation in incubations that included 300 mM ethanol. In me mbranes from phorbolester-treated platelets, the same maximal increase s in PLD activity (5-fold) were seen with 1 mu M GTP[S] and 100 mu M G TP. Addition of adenosine 5'-[gamma-thio]triphosphate (ATP[S]), ITP, X TP, UTP and CTP had similar stimulatory effects, but only at greater t han or equal to 1 mM. In contrast, ATP had a biphasic action, causing a maximal (2-fold) stimulation at 10 mu M and smaller effects at highe r concentrations; the inhibitory component of the action of ATP was bl ocked by 2 mu M staurosporine. Guanosine 5'-[beta-thio]diphosphate dec reased the stimulatory effects of ATP and ATP[S]. UDP, which can inhib it nucleoside diphosphate kinase (NDPK), decreased the activation of P LD by ATP[S], ATP, XTP, CTP and to a lesser extent ITP, but had no eff ect on the actions of GTP[S] and GTP. Rabbit platelet membranes contai ned NDPK and addition of [gamma-P-32]ATP led to the formation of [P-32 ]GTP in amounts sufficient to explain most or all of the activation of PLD; UDP prevented GTP formation. PCr (0.04-1 mM) also stimulated mem brane PLD activity, an effect that was dependent on endogenous membran e-bound creatine kinase (CK). UDP and guanosine 5'-[beta-thio]diphosph ate each inhibited this effect of PCr. The results show that in rabbit platelet membranes, CK, NDPK and the GTP-binding protein that activat es PLD can be functionally coupled. However, assay of membrane prepara tions at increasing dilutions showed that stimulation of PLD by the co mpounds studied, with the partial exception of ATP[S], involved diffus ible rather than protein-bound intermediates.