OMEPRAZOLE DECREASES H+-K+-ATPASE PROTEIN AND INCREASES PERMEABILITY OF OXYNTIC SECRETORY MEMBRANES IN RABBITS

Amounts and fractional distributions of gastric H+-K+-adenosinetriphosphatase (ATPase) activity and H+-K+-ATPase protein as well as properties of H+-K+-ATPase-containing membranes were studied in rabbits injected with omeprazole (OM; 1 mg/kg sc twice daily for 5 days). Total H+-K+-ATPase activity decreased to 22 +/- 2% of control (n = 4). Densitometry of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blots showed H+-K+-ATPase protein was decreased to 60-70% of control. In vitro reduction of the enzyme-OM disulfide bond with 0.1 M 2-mercaptoethanol increased microsomal H+-K+-ATPase activity to 56 +/- 7% of control (n = 3), consistent with a substantial decrease in enzyme protein. Incorporation of S-35-labeled methionine for 30 min before death resulted in 2.2-fold more label per unit of microsomal a-subunit protein (5 days OM vs. control). Thus the decrease in enzyme protein resulted from increased breakdown rather than decreased synthesis. A striking shift in distribution of H+-K+-ATPase-containing microsomes (tubulovesicles) on sucrose gradients reflected slow equilibration of most control vesicles with the gradient medium and faster equilibration after 5 days OM, indicating increased permeability. After 5 days OM, microsomal vesicle acidification (by acridine orange uptake assay) was negligible, even with 2-mercaptoethanol treatment, and H+ leakage on sudden DELTApH was faster than control. We conclude that extended OM treatment not only inhibits H+-K+-ATPase but accelerates its breakdown and renders H+-K+-ATPase-containing membranes more permeable. It is thus possible that increased backward H+ flux contributes to profound inhibition of acid secretion during extended omeprazole treatment. In parallel experiments, H+-K+-ATPase activity and density gradient sedimentation of tubulovesicles returned to near normal 3 days after OM withdrawal.