Abstract
G protein regulation of human platelet membrane phospholipase A2 activity was investigated at pH 8.0 and 9.0 by studying the effects of the nonhydrolyzable GTP analogue, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), and of F-/Al3+ ions on arachidonic acid (AA) release. The membrane acted as the source of the enzyme, the substrate, and the G protein. At pH 8.0, 10 and 100 microM GTP gamma S stimulated AA mobilization at least 6-fold. Optimum AA release conditions required 1 mM Ca2+ and 5 mM Mg2+. Nonspecific nucleotide effect was excluded since similar stimulatory effects on AA release were not observed by ATP, GTP, ADP, and NADP. Although at pH 9.0 the GTP gamma S-stimulated AA release was greater than at pH 8.0, it constituted only 26% of the total. At both pH values the effect of F- (10 mM) in the presence of Al3+ (2 microM) was similar to that of GTP gamma S. The G protein inhibitor, guanosine 5'-O-(2-thiodiphosphate), inhibited the GTP gamma S-stimulated AA release by about 80% at pH 8.0 and by 100% at pH 9.0. To determine a possible contribution to AA mobilization by the phospholipase C and diacylglycerol lipase pathway, the effects of neomycin, a phospholipase C inhibitor, were investigated. 100 microM neomycin did not inhibit the GTP gamma S-stimulated AA release at pH 8.0 and only slightly so (17%) at pH 9.0. At pH 8.0 in the presence of Ca2+ the released fatty acids consisted mainly of arachidonic and docosahexaenoic acids (80 and 8%, respectively). GTP gamma S had no effect on the fatty acid profile but only on their quantity. These results provide evidence of G protein regulation of phospholipase A2 activity in isolated platelet membranes.
Highlights
From the $Department of Biochemistry, New York College of Osteopathic Medicine, Old Westbury, New York 11568 preference for arachidonic acid (AA) [6]
Optimum AA release conditions required 1 mM Ca2+ and5 mM Mg2+.Nonspecific nucleotide effect was excluded since similar stimulatory effects on AA releasewere not observed by ATP, GTP, ADP, and and Lapetina [17] reported that the adenylatecyclase inhibitory G protein
The previously demonstratedPLA2 activity in isolated platelet membranes required high pH and high Ca2+ concentrationfor optimum activity [18].In view of such nonphysiological activating conditions and the recently emerging evidence of G protein regulation of a number of PLA2s [10,11,12,13,14,15,16], we investigated the existence of a similarregulating mechanisminplatelet membranes
Summary
Ificity for AA by the soluble PLA2sposed a serious objection to thePLAZ pathway [5]. Thisobjection, seemed to (Received for publication, May 1, 1989) have been alleviated with the isolation of a PLA, from mac-. Some evidence of G protein regulation of PLA2 activity phospholipase Az activity was investigated at pH 8.0 has been reported in platelets[10, 11]as well as in other and 9.0 by studying the effectsof the nonhydrolyzable cells [12,13,14,15,16]. At both pH values the effect of F- (10 to AA mobilizationin intact cells by the findings that the mM) in the presence of Ai3+(2 )IM) was similar to that release ofAA in isolated membranes and in thrombin- and of GTPyS. GTPyS had noeffect on the fatty acid profile but only on their quantity These results provide evidence of G protein regulation of phospholipase A2 activity in isolated platelet membranes.
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