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

J Lab Clin Med. 1992 Dec;120(6):929-40.

Abstract

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-beta-stimulated prostacyclin synthesis in human umbilical vein endothelium. Challenge of human umbilical vein endothelium monolayers with recombinant interleukin 1-beta resulted in dose- and time-dependent tritiated arachidonate release and prostacyclin synthesis consistent with phospholipase A2 activation. Prostacyclin synthesis after interleukin 1-beta (10 ng/ml) was detected 4 hours after stimulation and peaked at 16 to 24 hours. To examine whether interleukin 1-beta 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-beta stimulation. In contrast to the potent agonist, alpha-thrombin, interleukin 1-beta failed to significantly increase inositol phosphate production when examined for up to 4 hours. The absence of a significant increase in the Cai++ 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-beta (0 to 2 hours), whereas both alpha-thrombin and Ca++ ionophore A23187 produced rapid rises in Cai++. The intracellular Ca++ antagonist BAPTA and the extracellular Ca++ chelator EGTA produced significant inhibition of interleukin 1-beta-stimulated prostacyclin 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-beta-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 prostacyclin responses in synergistic fashion, suggesting that protein kinase C may modulate interleukin 1 signal transducing pathways. In summary, these studies suggest that interleukin 1-beta-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.(ABSTRACT TRUNCATED AT 400 WORDS)

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Arachidonic Acid / metabolism
  • Cells, Cultured
  • Cyclic AMP / biosynthesis
  • Egtazic Acid / analogs & derivatives
  • Egtazic Acid / pharmacology
  • Endothelium, Vascular / metabolism*
  • Epoprostenol / biosynthesis*
  • Humans
  • Interleukin-1 / pharmacology*
  • Phospholipase D / physiology*
  • Protein Kinase C / physiology*
  • Signal Transduction*
  • Tetradecanoylphorbol Acetate / pharmacology
  • Type C Phospholipases / physiology*

Substances

  • Interleukin-1
  • Arachidonic Acid
  • Egtazic Acid
  • Epoprostenol
  • Cyclic AMP
  • Protein Kinase C
  • Type C Phospholipases
  • Phospholipase D
  • 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
  • Tetradecanoylphorbol Acetate