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 biochemical activation, initiating P2Y receptor signaling that regulates basal levels 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 production is stimulated via Ca²⁺-dependent activation of cytosolic phospholipase A₂, 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 UTP-stimulated cAMP levels. COX inhibitors also decreased forskolin-stimulated cAMP formation, implying this response is in part attributable to an action of AA metabolites. These findings imply an important role for the inducible form of COX, COX-2, under basal conditions. Indeed, COX-2 expression was readily detectable by immunoblot, and treatments that induce or reduce COX-2 expression in other cells (interleukin-1β, tumor necrosis factor-α, phorbol ester, or dexamethasone) had minimal or no effect on the levels of COX-2 immunoreactivity. RT-PCR using isoform-specific primers detected COX-2 mRNA. We conclude that COX-2 is constitutively expressed in MDCK-D₁ cells and participates in basal and P2Y₂-mediated signaling, implying a key role for COX-2 in regulation of epithelial cell function.