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

Abstract

Background The H,K-ATPase, consisting of α and β subunits, belongs to the P-type ATPase family. There are two isoforms of the α subunit, HKα 1 and HKα 2 encoded by different genes. The ouabain-resistant gastric HKα 1-H,K-ATPase is Sch28080-sensitive. However, the colonic HKα 2-H,K-ATPase from different species shows poor primary structure conservation of the HKα 2 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 HKα 1, HKα 2, or HKβ genes. Methods Membrane vesicles from gastric mucosa and distal colon in wild-type (WT), HKα 1, HKα 2, or HKβ knockout (KO) mice were extracted. K-ATPase activity and pharmacological profiles were examined. Results The 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 HKα 2 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 HKα 1-containing H,K-ATPase; the dose dependence of inhibition was similar for all three inhibitors. In contrast, the colonic HKα 2-H,K-ATPase was not inhibited by these compounds. Conclusions These 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 Significance Characterization 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.