P-2-PURINOCEPTORS UTILIZE MULTIPLE SIGNALING PATHWAYS IN MDCK-D-1 CELLS

1 Madin-Darby canine kidney (MDCK) cells are a widely used model syste m for the study of epithelial cells. We have utilized a clonal variant , MDCK-D-1, to examine signalling by P-2-purinoceptors. 2 Several line s of evidence that lead us to conclude that MDCK-D-1 cells co-express p(2u-) and P-2y-purinoceptors and that both subtypes are linked to the release of arachidonic acid and metabolites (AA) include: (a) relativ e potency of nucleotide analogues in promoting AA release; (b) blockad e by the antagonist suramin of response to the P-2y-selective agonist, 2-methylthio ATP (2-MT-ATP), but not to the P-2u-selective agonist, U TP; and (c) additivity of response to 2-MT-ATP and UTP. AA release is a consequence of activation of phospholipase A(2) (PLA(2)), most likel y the 85 kDa cytosolic PLA(2). 3 Treatment of MDCK-D-1 cells with ATP, but not UTP, increases inositol 1,4,5-trisphosphate formation while b oth UTP and ATP increase phosphatidylcholine hydrolysis. ATP, UTP, and 2-MT-ATP can also stimulate phospholipase D activity. 4 Purine nucleo tides increase cellular cAMP levels in MDCK-D-1 cells in a manner that depends, at least in part, on activation of cyclooxygenase, since cAM P generation stimulated by ATP or UTP is inhibited by treatment of cel ls with indomethacin. Because cyclooxygenase-derived PGE(2) can bind t o prostaglandin receptors and stimulate synthesis of cAMP, nucleotides may raise cAMP in an autocrine or paracrine fashion. 5 Taken together , these results indicate that MDCK-D-1 cells co-express P-2u and P-2y- purinoceptors and that these receptors utilize several mechanisms to r egulate cell function, including activation of multiple phospholipases and autocrine/paracrine action of products.