Key role for constitutive cyclooxygenase-2 of MDCK cells in basal signaling and response to released ATP

Madin-Darby canine kidney (MDCK) cells release ATP upon mechanical or bioch emical activation, initiating P2Y receptor signaling that regulates basal l evels of multiple second messengers, including cAMP (J Biol Chem 275: 11735 -11739, 2000). Data shown here document inhibition of cAMP formation by Gd3 + and niflumic acid, channel inhibitors that block ATP release. cAMP produc tion is stimulated via Ca2+-dependent activation of cytosolic phospholipase A(2), release of arachidonic acid (AA), and cyclooxygenase (COX)-dependent production of prostaglandins, which activate prostanoid receptors coupled to G(s) and adenylyl cyclase. In the current investigation, we assessed the expression and functional role of the two known isoforms of COX, COX-1 and COX-2. Treatment of cells with either a COX-1-selective inhibitor, SC-560, or COX-2- selective inhibitors, SC-58125 or NS-398, inhibited basal and UT P-stimulated cAMP levels. COX inhibitors also decreased forskolin-stimulate d cAMP formation, implying this response is in part attributable to an acti on of AA metabolites. These findings imply an important role for the induci ble form of COX, COX-2, under basal conditions. Indeed, COX-2 expression wa s readily detectable by immunoblot, and treatments that induce or reduce CO X-2 expression in other cells (interleukin-1 beta, tumor necrosis factor-al pha, phorbol ester, or dexamethasone) had minimal or no effect on the level s of COX-2 immunoreactivity. RT-PCR using isoform-specific primers detected COX-2 mRNA. We conclude that COX-2 is constitutively expressed in MDCK-D-1 cells and participates in basal and P2Y(2)-mediated signaling, implying a key role for COX-2 in regulation of epithelial cell function.