Abstract
Two inhibitors of thrombin-stimulated arachidonic acid release from platelets, p-bromophenacyl bromide and mepacrine, were examined for their ability to inhibit the phospholipase C-diglyceride lipase pathway. This pathway involves hydrolysis of phosphatidylinositol to diglyceride, followed by release of arachidonate from diglyceride, and has been proposed as an alternative or addition to phospholipase A 2 as a mechanism for arachidonate release. p-Bromophenacyl bromide, a potent alkylating agent, was shown to cause a time-dependent inhibition of phosphatidylinositol-specific phospholipase C activity in crude platelet extracts; the inhibition was >90% after 15 min incubation with 100 μ m p-bromophenacyl bromide. However, p-bromophenacyl bromide was also shown to destroy about one-half of the titratable sulfhydryl groups in whole platelets under similar conditions. The lack of specificity of p-bromophenacyl bromide was further demonstrated by our finding that thrombin-stimulated serotonin release was also inhibited by conditions inhibiting arachidonate release and that diglyceride lipase activity was decreased by higher levels of p-bromophenacyl bromide. Mepacrine was found to inhibit the activity of phosphatidylinositol-specific phospholipase C and had a greater effect at low substrate concentrations. The loss of [ 14C]arachidonate from both endogenous phosphatidylinositol and phosphatidylcholine in intact platelets was also inhibited. Thrombin-stimulated serotonin release was impaired by mepacrine also but only at a concentration 10-fold greater than that required to prevent arachidonate release. Thus we have shown that these two agents which inhibit arachidonate release are inhibitors of the phosphatidylinositol-specific phospholipase C-diglyceride lipase pathway. The multiple effects produced by both compounds limit their utility as agents to examine the source and mechanism of arachidonate release.
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