Effect of dexamethasone on prostaglandin synthesis and on lipocortin status in human endothelial cells. Inhibition of prostaglandin I2 synthesis occurring without alteration of arachidonic acid liberation and of lipocortin synthesis

• Published in: The Journal of biological chemistry 1989-02, Vol.264 (6), p.3506-3513
• Main Authors: HULLIN, F, RAYNAL, P, RAGAB-THOMAS, J. M. F, FAUVEL, J, CHAP, H
• Format: Article
• Language: English
• Subjects: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 6-Ketoprostaglandin F1 alpha - biosynthesis; Annexins; Arachidonic Acid; Arachidonic Acids - metabolism; Biological and medical sciences; Calcium-Binding Proteins - analysis; Cell physiology; Dexamethasone - pharmacology; Dinoprost - biosynthesis; Dinoprostone - biosynthesis; Egtazic Acid - pharmacology; Endothelium, Vascular - drug effects; Endothelium, Vascular - metabolism; Epoprostenol - biosynthesis; Fundamental and applied biological sciences. Psychology; Glycoproteins - biosynthesis; Humans; Hydroxyeicosatetraenoic Acids - biosynthesis; Immunoassay; Kinetics; Molecular and cellular biology; Phospholipases A - antagonists & inhibitors; Phospholipases A2; Phospholipids - metabolism; Secretion. Exocytosis; Thrombin - pharmacology
• ISSN: 0021-9258; 1083-351X

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 together 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 [ethylene-bis(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 microM 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.