Pharmacological profiles of the murine gastric and colonic H,K-ATPases

BackgroundThe H,K-ATPase, consisting of α and β subunits, belongs to the P-type ATPase family. There are two isoforms of the α subunit, HKα1 and HKα2 encoded by different genes. The ouabain-resistant gastric HKα1-H,K-ATPase is Sch28080-sensitive. However, the colonic HKα2-H,K-ATPase from different species shows poor primary structure conservation of the HKα2 subunit between species and diverse pharmacological sensitivity to ouabain and Sch28080. This study sought to determine the contribution of each gene to functional activity and its pharmacological profile using mouse models with targeted disruption of HKα1, HKα2, or HKβ genes.MethodsMembrane vesicles from gastric mucosa and distal colon in wild-type (WT), HKα1, HKα2, or HKβ knockout (KO) mice were extracted. K-ATPase activity and pharmacological profiles were examined.ResultsThe colonic H,K-ATPase demonstrated slightly greater affinity for K+ than the gastric H,K-ATPase. This K-ATPase activity was not detected in the colon of HKα2 KO but was observed in HKβ KO with properties indistinguishable from WT. Neither ouabain nor Sch28080 had a significant effect on the WT colonic K-ATPase activity, but orthovanadate abolished this activity. Amiloride and its analogs benzamil and 5-N-ethyl-N-isopropylamiloride inhibited K-ATPase activity of HKα1-containing H,K-ATPase; the dose dependence of inhibition was similar for all three inhibitors. In contrast, the colonic HKα2-H,K-ATPase was not inhibited by these compounds.ConclusionsThese data demonstrate that the mouse colonic H,K-ATPase exhibits a ouabain- and Sch28080-insensitive, orthovanadate-sensitive K-ATPase activity. Interestingly, pharmacological studies suggested that the mouse gastric H,K-ATPase is sensitive to amiloride.General SignificanceCharacterization of the pharmacological profiles of the H,K-ATPases is important for understanding the relevant knockout animals and for considering the specificity of the inhibitors.