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

Abstract

Two H+-K+-ATPase isoforms are present in kidney: the gastric, highly sensitive to Sch-28080, and the colonic, partially sensitive to ouabain. Upregulation of Sch-28080-sensitive H+-K+-ATPase, or “gastric” H+-K+-ATPase, has been demonstrated in hypokalemic rat inner medullary collecting duct cells (IMCDs). Nevertheless, only colonic H+-K+-ATPase mRNA and protein abundance increase in this condition. This study was designed to determine whether Sch-28080 inhibits transporters other than the gastric H+-K+-ATPase. In the presence of bumetanide, Sch-28080 (200 μM) and ouabain (2 mM) inhibited86Rb+ uptake (>90%). That86Rb+ uptake was almost completely abolished by Sch-28080 indicates an effect of this agent on the Na+-K+-ATPase. ATPase assays in membranes, or lysed cells, demonstrated sensitivity to ouabain but not Sch-28080. Thus the inhibitory effect of Sch-28080 was dependent on cell integrity. 86Rb+-uptake studies without bumetanide demonstrated that ouabain inhibited activity by only 50%. Addition of Sch-28080 (200 μM) blocked all residual activity. Intracellular ATP declined after Sch-28080 (200 μM) but recovered after removal of this agent. In conclusion, high concentrations of Sch-28080 inhibit K+-ATPase activity in mouse IMCD-3 (mIMCD-3) cells as a result of ATP depletion.