Cellular mechanism of synergistic stimulation of PGE2 production by phorbol diester and Ca2+ ionophore A23187 in cultured Madin-Darby canine kidney cells.
The synergistic stimulation of arachidonic acid release and prostaglandin (PG) E2 production was observed when quiescent Madin-Darby canine kidney (MDCK) cells were exposed to phorbol 12-myristate 13-acetate (PMA) and Ca2+ ionophore, A23187. Addition of PMA or A23187 by itself was not effective. When cells were treated with 50 nM PMA and 0.1 microM A23187 at a suboptimal concentration, there was a marked increase in the production of PGE2. In the presence of higher concentrations of A23187 (5-10 microM), 50 nM PMA was only synergistic in potentiating the liberation of free arachidonic acid, but failed to stimulate the PGE2 production. The amount of free arachidonic acid liberated by 50 nM PMA and 10 microM A23187 reached a maximum level within several hours, whereas PGE2 synthesis induced by 50 nM PMA and 0.1 microM A23187 proceeded with a slower process requiring more than 24 h to reach a maximum. The stimulated PGE2 synthesis was blocked by transcription and translation inhibitors. The addition of 50 nM PMA alone or the mixture of 50 nM PMA and 0.1 microM A23187 was found similarly to increase the cellular PG endoperoxide synthase activity, suggesting that PMA was responsible for the increased enzyme activity. However, these agents failed to enhance the activities of phospholipases and PGE2 synthase from PGH2. Northern blot analysis confirmed the increased level of PG endoperoxide synthase mRNA in the cells treated with PMA. The effect of PMA was mimicked by other protein kinase C activators. The pretreatment with PMA caused a down-regulation in the PGE2 production. The stimulated PGE2 production was abolished in the presence of selective protein kinase C inhibitors such as staurosporine and H-7. In addition, sphingosine, dihyrosphingosine, and psychosine, recently found to be protein kinase C inhibitors, blocked the effect of PMA in intact MDCK cells. Thus, the results indicate that the synergistically stimulated PGE2 production with phorbol diesters and 0.1 microM A23187 occurred principally through the de novo synthesis of PG endoperoxide synthase, also implying a role for protein kinase C.
Yokota
K
K
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
D000818:Animals; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism; D000001:Calcimycin / Q000008:administration & dosage*; D002460:Cell Line; D015232:Dinoprostone / Q000096:biosynthesis*; D004285:Dogs; D004357:Drug Synergism; D007668:Kidney / Q000187:drug effects / Q000378:metabolism; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000008:administration & dosage*
D001095:Arachidonic Acids; D016718:Arachidonic Acid; D000001:Calcimycin; D011451:Prostaglandin-Endoperoxide Synthases; D011493:Protein Kinase C; D015232:Dinoprostone; D013755:Tetradecanoylphorbol Acetate