EFFECT OF DEXAMETHASONE ON PROSTAGLANDIN SYNTHESIS AND ON LIPOCORTIN STATUS IN HUMAN-ENDOTHELIAL CELLS - INHIBITION OF PROSTAGLANDIN I-2 SYNTHESIS OCCURRING WITHOUT ALTERATION OF ARACHIDONIC-ACID LIBERATION AND OF LIPOCORTIN SYNTHESIS

  • 25 February 1989
    • journal article
    • research article
    • Vol. 264  (6) , 3506-3513
Abstract
Glucocorticoids have been shown to decrease prostaglandin I2 synthesis in human endothelial cells, suggesting the possible involvement of lipocortin in the inhibition of arachidonic acid liberation achieved by phospholipase A2 (De Caterina, R., and Weksler, B. B. (1986) Thromb. Haemostasis 55, 369-374). To test this hypothesis, human endothelial cells labeled with [14C]arachidonic acid were stimulated with thrombin (2 units/ml, 10 min), resulting in the secretion of free arachidonic acid togehter with various 14C-labeled metabolites, mainly 6-keto-prostaglandin F1.alpha., the stable derivative of prostaglandin I2. Under conditions where prior incubation of cells with dexamethasone reduced by 51% 6-keto-prostaglandin F1.alpha. production, phospholipid hydrolysis induced by thrombin remained unaffected. Using three rabbit polyclonal antibodies directed against endonexin I, lipocortin I, and lipocortin II, evidence was obtained for the presence in human endothelial cells of equivalent amounts of lipocortin I and an immunologically unrelated 33-kDa protein, together with lower quantities of 67-kDa calelectrin/calcimedin. These Ca2+- and phospholipid-binding proteins were selectively extracted with [ethylenebis(oxyethylene-nitrilo)]tetraacetic acid (EGTA) from cell membranes precipitated in the presence of Ca2+, and they displayed an inhibitory activity against pig pancreas phospholipase A2. However, the amounts of the three proteins were not changed by cell treatment with 2.5 .mu.M dexamethasone, as detected upon polyacrylamide gel electrophoresis by silver staining, immunoblotting, or autoradiography following [35S]methionine in vivo labeling. Since the antiphospholipase A2 activity of EGTA extracts was hardly modified, it was concluded that an increased synthesis of lipocortin cannot account for the inhibition of prostaglandin synthesis brought about by dexamethasone, suggesting other biological functions for these proteins.

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