ATP ACTIVATES CAMP PRODUCTION VIA MULTIPLE PURINERGIC RECEPTORS IN MDCK-D1 EPITHELIAL-CELLS - BLOCKADE OF AN AUTOCRINE PARACRINE PATHWAY TODEFINE RECEPTOR PREFERENCE OF AN AGONIST/

Extracellular nucleotides regulate function in many cell types via act ivation of multiple P-2-purinergic receptor subtypes, However, it has been difficult to define which individual subtypes mediate responses t o the physiological agonist ATP, We report a novel means to determine this by exploiting the differential activation of an autocrine/paracri ne signaling pathway. We used Madin-Darby canine kidney epithelial cel ls (MDCK-D1) and assessed the regulation of cAMP formation by nucleoti des, We found that ATP, 2-methylthio-ATP (MT-ATP) and UTP increase cAM P production. The cyclooxygenase inhibitor indomethacin completely inh ibited UTP-stimulated, did not inhibit MT-ATP-stimulated, and only par tially blocked ATP-stimulated cAMP formation. In parallel studies, ATP and UTP but not MT-ATP stimulated prostaglandin production. By pretre ating cells with indomethacin to eliminate the P-2Y2/prostaglandin com ponent of cAMP formation, we could assess the indomethacin-insensitive P-2 receptor component. Under these conditions, ATP displayed a ten-f old lower potency for stimulation of cAMP formation compared with untr eated cells. These data indicate that ATP preferentially activates P-2 Y2 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 increas es cAMP formation in these cells. Blockade of autocrine/paracrine sign aling can aid in dissecting the contribution of multiple receptor subt ypes activated by an agonist.