Bioscience, Biotechnology, and Biochemistry
Online ISSN : 1347-6947
Print ISSN : 0916-8451
Activation Mechanism of Phospholipase D Involved in the Generation of Lipid Mediators in Cultured Madin-Darby Canine Kidney Cells
Kazushige YokotaJun-Ichi TakeuchiMitsuo JisakaKoichi Takinami
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JOURNAL FREE ACCESS

1995 Volume 59 Issue 7 Pages 1291-1299

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Abstract

Addition of 100 nM phorbol 12-myristate 13-acetate (PMA), an active phorbol diester, to quiescent cultured Madin-Darby canine kidney (MDCK) cells caused a maximal stimulation of phosphatidylethanol formation within 1-2 h in the presence of 1% ethanol, indicating the activation of phospholipase D (PLD). The specificity of phorbol diesters for the activation of PLD activation was confirmed by the fact that phorbol 12, 13-dibutyrate (PDBu) was effective, whereas 4α-phorbol 12, 13-didecanoate (4α-PDD) was without effect. Down-regulation caused by the long-term pretreatment of the cells with active phorbol diesters significantly decreased the production of phosphatidylethanol. Staurosporine, a well known protein kinase (PK)C inhibitor at 1μM, decreased the activation of PLD. Taken together, these observations suggested the involvement of PKC in the activation of PLD. The cellular PLD activity was found to be selectively localized in the particulate fraction by centrifugation at 12, 000×g. The particulate PLD showed the selective substrate specificity for phosphatidylcholine rather than phosphatidyl-ethanolamine. In response to the addition of 100 nM PMA, 1, 2-diacylglycerol (DG) increased in a biphasic fashion. In view of the time course of the activation of PLD, the second increase in the 1, 2-DG around 20 min was contributed by the activation of PLD. In reponse to the simultaneous addition of 100 nM PMA and 100 nM A23187, the cultured MDCK cells activated the arachidonate cascades to form prostaglandin (PG)E2 and PGF as major products, requiring slower 24h to reach maximal levels. The pretreatment of the cells with 1% ethanol caused a significant drop in the synthesis of PGE2 rather than PGF. The results indicated that either phosphatidic acid (PA) or 1, 2-DG from the hydrolysis of PA served as an activator of cellular response or a direct source of free arachidonic acid as substrates for phospholipases.

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