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Stimulation of Human Amnion Prostaglandin E₂ Production by Activators of Protein Kinase-C^*

TAMAS ZAKAR and DAVID M. OLSON

Departments of Pediatrics and Physiology, The Lawson Research Institute, St. Joseph’s Health Centre, The University of Western Ontario London, Ontario, Canada N6A 4V2

Address all correspondence and requests for reprints to: Dr. David M. Olson, The Lawson Research Institute, St. Joseph’s Health Centre, 268 Grosvenor Street, London, Ontario, Canada N6A 4V2.

We tested the possibility that the activation of protein kinase-C by the tumor-promoter phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) or diacylglycerol stimulates the production of prostaglandin E₂ (PGE₂) by the amnion. Confluent primary cultures of human amnion epithelial cells were adapted to serum-free medium and treated with the agonists for up to 8 h. Cumulative PGE₂ output in the medium was measured by RIA. TPA, a potent activator of protein kinase-C, stimulated basal PGE₂ output from less than 50 pg/well·5 h to 3 ng/well·5 h (P < 0.01) in a time- and dose-dependent manner. 4-Methoxy-TPA, a weak tumor promoter derivative of TPA, was ineffective when tested in the same concentration range as TPA (1 nmol/L to 1 µmol/L). Neither calcium ionophore A23187 (20 nmol/L) nor arachidonate (1 µmol/L) stimulated PGE₂ output alone, but each agonist potentiated the effect of TPA as much as 5-fold (P < 0.01). 1,2-Dioctanoyl-sn-glycerol stimulated PGE₂ output 4- to 7-fold (P < 0.05), and this effect was potentiated by Ca ionophore and arachidonate. Studies involving actinomycin-D and cycloheximide indicated that the stimulatory effect of TPA was dependent on RNA synthesis during the first 60 min and on protein synthesis during the entire length of the phorbol ester treatment period (300 min). TPA was also able to stimulate PGE₂ production after irreversible inactivation of PG endoperoxide synthase activity with acetylsalicylic acid. These results suggest that activation of protein kinase-C in amnion cells increases the de novo synthesis of the PG endoperoxide synthase enzyme in a RNA synthesis-dependent manner. Elevated intracellular calcium levels contribute to the stimulation apparently by increasing the availability of endogenous arachidonate for subsequent conversion to PGE₂.

^* This work was supported by a Basil O’Connor Starter Research Grant (no. 5-530) from the March of Dimes Birth Defects Foundation, the Department of Pediatrics, University of Western Ontario; and The Lawson Research Institute.

Visiting Professor of Pediatrics to University of Western Ontario, Permanent address: First Institute of Biochemistry, Semmelweis University Medical School, Budapest, Hungary.

Scholar of the Medical Research Council of Canada.

Received February 19, 1988.

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