Abstract:
: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 microM exogenous 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.
journal_name
Arch Biochem Biophysjournal_title
Archives of biochemistry and biophysicsauthors
Chulada PC,Loftin CD,Winn VD,Young DA,Tiano HF,Eling TE,Langenbach Rdoi
10.1006/abbi.1996.0257subject
Has Abstractpub_date
1996-06-15 00:00:00pages
301-13issue
2eissn
0003-9861issn
1096-0384pii
S0003-9861(96)90257-Xjournal_volume
330pub_type
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更新日期:2009-04-01 00:00:00
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更新日期:2015-03-15 00:00:00
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更新日期:1997-08-01 00:00:00
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journal_title:Archives of biochemistry and biophysics
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更新日期:1996-09-01 00:00:00