Sch-28080 depletes intracellular ATP selectively in mIMCD-3 cells

Two H⁺-K⁺-ATPase isoforms are presentin kidney: the gastric, highly sensitive to Sch-28080, and the colonic,partially sensitive to ouabain. Upregulation of Sch-28080-sensitiveH⁺-K⁺-ATPase, or "gastric"H⁺-K⁺-ATPase, has been demonstrated inhypokalemic rat inner medullary collecting duct cells (IMCDs).Nevertheless, only colonic H⁺-K⁺-ATPase mRNAand protein abundance increase in this condition. This study wasdesigned to determine whether Sch-28080 inhibits transporters otherthan the gastric H⁺-K⁺-ATPase. In the presenceof bumetanide, Sch-28080 (200 µM) and ouabain (2 mM) inhibited⁸⁶Rb⁺ uptake (>90%). That⁸⁶Rb⁺ uptake was almost completely abolished bySch-28080 indicates an effect of this agent on theNa⁺-K⁺-ATPase. ATPase assays in membranes, orlysed cells, demonstrated sensitivity to ouabain but not Sch-28080.Thus the inhibitory effect of Sch-28080 was dependent on cellintegrity. ⁸⁶Rb⁺-uptake studies withoutbumetanide demonstrated that ouabain inhibited activity by only50%. Addition of Sch-28080 (200 µM) blocked all residualactivity. Intracellular ATP declined after Sch-28080 (200 µM) butrecovered after removal of this agent. In conclusion, highconcentrations of Sch-28080 inhibit K⁺-ATPase activity inmouse IMCD-3 (mIMCD-3) cells as a result of ATP depletion.