Interleukin 1-stimulated prostacyclin synthesis in endothelium: Lack of phospholipase C, phospholipase D, or protein kinase C involvement in early signal transduction

The cascade of transmembrane signaling events that follow the occupancy of the interleukin 1 receptor remain poorly defined. We examined potential postreceptor transduction systems involved in human recombinant interleukin 1-β-stimulated prostacylin synthesis in human umbilical vein endothelium. Challenge of human umbilical vein endothelium monolayers with recombinant interleukin 1-β resulted in dose- and time-dependent tritiated arachidonate release and prostacyclin synthesis consistent with phospholipase A₂ activation. Prostacyclin synthesis after interleukin 1-β (10 ng/ml) was detected 4 hours after stimulation and peaked at 16 to 24 hours. To examine whether interleukin 1-β produced early activation of a phosphoinositide-specific phospholipase C, human umbilical vein endothelium monolayers were labeled with tritiated-2-myoinositol and inositol polyphosphates recovered after interleukin 1-β stimulation. In contrast to the potent agonist, α-thrombin, interleukin 1-β failed to significantly increase inositol phosphate production when examined for up to 4 hours. The absence of a significant increase in the Ca_I⁺⁺ secretagogue, IP₃, was confirmed in human umbilical vein endothelium monolayers loaded with the Ca⁺⁺ photoprotein probe aequorin. Basal aequorin luminescence was unaltered after interleukin 1-β (0 to 2 hours), whereas both α-thrombin and Ca⁺⁺ ionophore A23187 produced rapid rises in Ca_I⁺⁺. The intracellular Ca⁺⁺ antagonist BAPTA and the extracellular Ca⁺⁺ chelator EGTA produced significant inhibition of interleukin 1-β-stimulated prostacylin generation at 4 to 8 hours, suggesting either an indirect inhibitory effect of these agents on phospholipase A₂ activity or that an increase in Ca⁺⁺ may be a late event in the transduction scheme after interleukin 1 stimulation. Interleukin 1-β-stimulated protein kinase C, phospholipase D, and adenylyl cyclase activities (0 to 4 hours) were unchanged from controls. Despite the absence of increased plasma membrane protein kinase C activity up to 4 hours after interleukin 1, pretreatment of human umbilical vein endothelium monolayers with staurosporine or phorbol myristate acetate (18 hours) to reduce protein kinase C activities, significantly attenuated the interleukin 1-stimulated prostanoid responses at 16 hours but not at 4 hours. Furthermore, short (5 minute) pretreatment with phorbol myristate acetate dramatically augmented interleukin 1-mediated prostacylin responses in synergistic fashion, suggesting that protein kinase C may modulate interleukin 1 signal transducing pathways. In summary, these studies suggest that interleukin 1-β-mediated endothelial cell phospholipase A₂ activity and prostacyclin synthesis occur via a novel transducing pathway that does not involve early activation of phospholipase C, phospholipase D, or adenylate cyclase. As a result, IP₃, DAG, Ca⁺⁺, phosphatidic acid, and cyclic adenosine monophosphate are unlikely second messengers for interleukin 1-induced endothelial cell activation that results in prostacyclin synthesis. Protein kinase C activity and possibly increased Ca_I⁺⁺ appear to be involved late (>4 hours) in the signal transduction scheme. The exact site of protein kinase C modulatory effects as well as the mechanism of interleukin 1-induced protein kinase C activation, however, requires further investigation.