ATP-induced inhibition of Na+, K+, Cl- cotransport in Madin-Darby canine kidney cells: Lack of involvement of known purinoceptor-coupled signaling pathways

P-2U/2Y-receptors elicit multiple signaling in Madin-Darby canine kidney (M DCK) cells, including a transient increase of [Ca2+](i), activation of phos pholipases C (PLC) and A(2) (PLA(2)), protein kinase C (PKC) and mitogen-ac tivated protein kinase (MAPK). This study examines the involvement of these signaling pathways in the inhibition of Na+,K+,Cl- cotransport in MDCK cel ls by ATP. The level of ATP-induced inhibition of this carrier (similar to 50% of control values) was insensitive to cholera and pertussis toxins, to the PKC inhibitor calphostin C, to the cyclic nucleotide-dependent protein kinase inhibitors, H-89 and H-8 as well as to the inhibitor of serine-threo nine type 1 and 2A phosphoprotein phosphatases okadaic acid. ATP led to a t ransient increase of [Ca2+](i) that was abolished by a chelator of Ca-i(2+) , BAPTA. However, neither BAPTA nor the Ca2+ ionophore A231287, or an inhib itor of endoplasmic reticulum Ca2+-pump, thapsigargin, modified ATP-induced inhibition of Na+,K+,Cl- cotransport. An inhibitor of PLC, U73122, and an inhibitor of MAPK kinase (MEK), PD98059, blocked ATP-induced inositol-1,4,5 -triphosphate production and MAPK phosphorylation, respectively. However, t hese compounds did not modify the effect of ATP on Na+,K+,Cl- cotransport a ctivity. Inhibitors of PLA(2) (AACOCF(3)), cycloxygenase (indomethacin) and lypoxygenase (NDGA) as well as exogenous arachidonic acid also did not aff ect ATP-induced inhibition of Na+,K+,Cl- cotransport. Inhibition of the car rier by ATP persisted in the presence of inhibitors of epithelial Na+ chann els (amiloride), Cl- channels (NPPB) and Na+/H+ exchanger (ETPA) and was in sensitive to cell volume modulation in anisosmotic media and to depletion o f cells with monovalent ions, thus ruling out the role of other ion transpo rters in purinoceptor-induced inhibition of Na+,K+,Cl- cotransport. Our dat a demonstrate that none of the known purinoceptor-stimulated signaling path ways mediate ATP-induced inhibition of Na+,K+,Cl- cotransport and suggest t he pres ence of a novel P-2-receptor-coupled signaling mechanism.