PURINERGIC-MEDIATED INHIBITION OF NA-K+-ATPASE IN PROXIMAL TUBULE CELLS - ELEVATED CYTOSOLIC CA2+ IS NOT REQUIRED()

The involvement of cytosolic Ca2+ concentration ([Ca2+](i)) as messeng er for the regulation of Na+-K+-ATPase activity was investigated in a renal cell line recently developed by immortalization of early proxima l tubule primary cultures from the Wistar-Kyoto rat strain. Na+-K+-ATP ase was measured as short-circuit current (I-sc) in intact monolayers after permeabilization of the apical plasma membrane with amphotericin B. With symmetrical solutions, I-sc quantitatively reflects Na+-K+-AT Pase activity as judged by ouabain inhibition and dependence on Na+ an d K+. Extracellular ATP (50%) effective concentration = 0.32 mM) on th e apical side produced acute inhibition of Na+-K+-ATPase-generated I-s c of up to 50%. The inhibition peaked within 1 min and lasted similar to 5 min. The potency order was ATP > ADP >> beta,gamma-methyleneadeno sine 5'-triphosphate UTP, consistent with a P-2y receptor. Extracellul ar ATP also stimulated a transient increase in [Ca2+](i). This increas e had a similar time course as the inhibition of ATPase and reached a peak change of similar to 120 nM. However, the elevation of [Ca2+](i) is not required in the purinergic inhibition of the Na+-K+-ATPase, sin ce, first, increases in [Ca2+](i) produced with a Ca2+ ionophore (iono mycin) failed to mimic the purinergic inhibition and, second, ,2-bis(2 -aminophenoxy)ethane=N,N,N',N'-tetraacetic acid, which abolished the [ Ca2+](i) elevation, failed to block the purinergic inhibition.