Relative Activities of Retrovirally Expressed Murine Prostaglandin Synthase-1 and -2 Depend on Source of Arachidonic Acid

• Published in: Archives of biochemistry and biophysics 1996-06, Vol.330 (2), p.301-313
• Main Authors: Chulada, Patricia C., Loftin, Charles D., Winn, Virginia D., Young, Donald A., Tiano, Howard F., Eling, Thomas E., Langenbach, Robert
• Format: Article
• Language: English
• Subjects: Animals; Arachidonic Acid - metabolism; Cell Line; Chinese Hamster Ovary cells; CHO Cells; constitutive prostaglandin synthase; Cricetinae; DNA, Complementary - genetics; endogenous arachidonic acid; Enzyme Stability; exogenous arachidonic acid; Gene Expression; inducible prostaglandin synthase; Isoenzymes - genetics; Isoenzymes - metabolism; Mice; murine embryo fibroblasts; Prostaglandin-Endoperoxide Synthases - genetics; Prostaglandin-Endoperoxide Synthases - metabolism; retroviral expression; Retroviridae - genetics
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1006/abbi.1996.0257

We have developed derivatives of mouse embryonic fibroblasts (10T1/2) and Chinese hamster ovary (AS52) cells that stably express high levels of murine prostaglandin synthase-1 or -2 (PGHS-1 or -2). The cDNAs were transferred using retroviral vectors and the resulting G418-resistant clones were analyzed for prostaglandin E2(PGE2) production. Specific expression was confirmed by Western and Northern analyses. Enzyme activities, protein, and message levels peaked 1 (10T1/2) or 2 (AS52) days after seeding but decreased as cells became density arrested. Upon subculturing, enzyme activities returned to their initial high levels. With 10 μmexogenous arachidonic acid (AA) as the substrate, PGHS-1 activities were approximately 3- to 5-fold higher than PGHS-2 activities. Conversely, when exogenous AA was left out of the medium and only endogenous AA was available as substrate, enzyme activities were lower; but PGHS-2 activities were 5-fold (10T1/2) or 1.5-fold (AS52) higher than PGHS-1 activities. Following phorbol ester treatment to stimulate endogenous AA release, PGHS-2 activities increased over time and by 6 hours, were 4-fold (10T1/2) or 2-fold (AS52) higher than PGHS-1 activities. However, when calcium ionophore A23187 was used to stimulate endogenous AA release, maximum PGHS activities occurred within 30 min of treatment; PGHS-1 activities were equal to (10T1/2) or 2-fold higher (AS52) than PGHS-2 activities. Because these cell lines allow us to measure specific PGHS activity in intact cells, we were able to demonstrate that the relative activities of the two PGHS isozymes depend on the source of AA (exogenous versus endogenous) or biochemical stimulus used to mobilize endogenous AA (A23187 versus phorbol ester). These data suggest that PGHS-1 and PGHS-2 preferentially utilize different pools of AA and may be modulated through different stimulus-initiated pathways.