Abstract
In the resting state of gastric parietal cells, most of H+,K+-ATPase is located in intracellular tubulovesicles. Upon stimulation, the tubulovesicles fuse with the apical surface membrane. Here, two types of gastric vesicles (intracellular tubulovesicles (TV) and stimulation-associated vesicles (SA) derived from apical surface membrane) were prepared from hog gastric mucosa. In the TV sample, 60% of H+,K+-ATPase were found in the detergent (1% CHAPS)-resistant membrane microdomains (DRM) and 40% of H+,K+-ATPase were in non-DRM. In the SA sample, 30% of H+,K+-ATPase were in the DRM and 70% of H+,K+-ATPase were in the non-DRM. Phospholipids were abundant in the DRM of the TV, while those were present both in the DRM and non-DRM of SA. Cholesterol was abundantly present in the DRM of TV and SA. Expression level of caveolin-1, a caveolae marker, in TV was much higher than that in SA. In contrast, expression level of flotillin-2, a raft marker, in SA was higher than that in TV. The H+,K+-ATPase activity in TV was significantly reduced by treatment of the vesicles with methyl-β-cyclodextrin (MβCD), and the MβCD-decreased activity was significantly restored by exogenous addition of cholesterol. These results suggest that H+,K+-ATPase is present in intracellular caveosome (TV) and in the raft- and non-raft-domains in the apical surface membrane (SA) of parietal cells, and that cholesterol is necessary to maintain the pump activity. [J Physiol Sci. 2008;58 Suppl:S54]
