ATP activates cAMP production via multiple purinergic receptors in MDCK-D1 epithelial cells - Blockade of an autocrine/paracrine pathway to define receptor preference of an agonist

Extracellular nucleotides regulate function in many cell types via activation of multiple P-2-purinergic receptor subtypes, However, it has been difficult to define which individual subtypes mediate responses to the physiological agonist ATP, We report a novel means to determine this by exploiting the differential activation of an autocrine/paracrine signaling pathway. We used Madin-Darby canine kidney epithelial cells (MDCK-D1) and assessed the regulation of cAMP formation by nucleotides, We found that ATP, 2-methylthio-ATP (MT-ATP) and UTP increase cAMP production. The cyclooxygenase inhibitor indomethacin completely inhibited UTP-stimulated, did not inhibit MT-ATP-stimulated, and only partially blocked ATP-stimulated cAMP formation. In parallel studies, ATP and UTP but not MT-ATP stimulated prostaglandin production. By pretreating cells with indomethacin to eliminate the P-2Y2/prostaglandin component of cAMP formation, we could assess the indomethacin-insensitive P-2 receptor component. Under these conditions, ATP displayed a ten-fold lower potency for stimulation of cAMP formation compared with untreated cells. These data indicate that ATP preferentially activates P-2Y2 relative to other P-2 receptors in MDCK-D1 cells (P-2Y1 and P-2Y11, as shown by reverse transcriptase polymerase chain reaction) and that P-2Y2 receptor activation is the principal means by which ATP increases cAMP formation in these cells. Blockade of autocrine/paracrine signaling can aid in dissecting the contribution of multiple receptor subtypes activated by an agonist.