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Mechanism of 12-O-Tetradecanoylphorbol-13-acetate Enhanced Metabolism of Arachidonic Acid in Dog Urothelial Cells¹

Geriatric Research, Education, and Clinical Center, Veterans Administration Medical Center, and Departments of Biochemistry and Internal Medicine, St. Louis University School of Medicine, St. Louis, Missouri [T. V. Z., Y-H. H. W., B. B. D.], and Laboratory of Molecular Biophysics, National Institute of Environmental Health Science, Research Triangle Park, North Carolina [T. E. E., Z. M. D.]

The mechanism of 12-O-tetradecanoylphorbol-13-acetate (TPA) enhanced arachidonic acid metabolism was investigated in dog urothelial cells. Primary cultures of dog urothelial cells were grown to confluency and evaluated in the presence or absence of overnight prelabeling with [³H]arachidonic acid. High-performance liquid chromatography analysis of media from TPA stimulated cells indicated that prostaglandin E₂ (PGE₂) was the major eicosanoid produced. Lipoxygenase products were not detected. Control cell media contained only arachidonic acid. Effects of selected inhibitors on TPA and exogenous arachidonic acid mediated increases in radioimmunoassayable PGE₂ were assessed. Prostaglandin H synthase inhibitors (indomethacin and aspirin) prevented both TPA and arachidonic acid increases in PGE₂. By contrast, inhibitors of phospholipases (quinacrine, W-7, and trifluoropromazine), protein synthesis (cycloheximide), and protein kinase C (staurosporine) prevented TPA but not arachidonic acid increases in PGE₂. The latter agents also reduced TPA mediated increases in the release of total radioactivity from cells labeled with [³H]arachidonic acid. However, aspirin reduced the amount of ³H-prostaglandins formed with TPA. A calcium requirement was demonstrated when increases in radioimmunoassayable PGE₂ elicited by TPA and the calcium ionophore A23187 were reduced with calcium depleted media. When epidermal growth factor in combination with either TPA or bradykinin was used, at least additive effects were observed with respect to release of [³H]arachidonic acid, ³H-prostaglandins, and radioimmunoassayable PGE₂. These experiments suggest that separate pathways may be involved in enhanced arachidonic acid metabolism demonstrated with different agonists. For TPA, increased arachidonic acid release occurs by a calcium dependent process involving phospholipase(s), protein synthesis, and protein kinase C.

¹ This work was supported by the Department of Veterans Affairs and by USPHS Grant CA-28015 from the National Cancer Institute through the National Bladder Cancer Project.

² To whom requests for reprints should be addressed, at Geriatric Center (111G-JB), Veterans Administration Medical Center, St. Louis, MO 63125.

Received 3/16/90.

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