Regulation of prostaglandin synthesis in Madin Darby canine kidney cells: role of prostaglandin G/H synthase and secreted phospholipase A 2

Abstract

The renal epithelial cell line MDCK (Madin Darby canine kidney) was used as a model system to investigate the contribution of the secreted phospholipase A 2 type II (sPLA 2) and cyclooxygenases to prostaglandin E 2 (PGE 2) synthesis. Activation of protein kinase C by the phorbol ester TPA led to an enhanced PGE 2 synthesis within 1 hour, which continued for more than 20 hours. Treatment of the cells with TPA increased the activities of sPLA 2 and cyclooxygenase. Activation of cyclooxygenase was reflected by an increase in cyclooxygenase-2 mRNA. Coincubation of the cells with TPA and a specific sPLA 2 inhibitor (BM 16.2224) almost completely inhibited sPLA 2 activity in the cell culture supernatants. TPA-induced PGE 2 synthesis was reduced by the inhibitor to about 50%. The inhibitor had no effect on cyclooxygenase activity or expression, indicating an involvement of sPLA 2 in PGE 2 synthesis. These experiments show that in resting cells, even in the presence of exogenous arachidonic acid, PGE 2 synthesis was limited by the low abundance of cyclooxygenase. Enhanced expression and activity of cyclooxygenase, however, was not sufficient for increased prostaglandin synthesis. Availability of the precursor arachidonic acid seemed to be rate limiting in prostaglandin synthesis in stimulated MDCK cells.