Role of Na+/H+ exchanger 3 in acidification and morphological remodeling of tubulovesicles in gastric parietal cells.

Gastric parietal cells undergo dramatic morphological changes upon stimulation, during which intracellular tubulovesicles (TV) fuse with the apical canalicular membrane to support massive H+ secretion by the gastric proton pump (H+,K+-ATPase). Although the activation process has been extensively characterized, the mechanisms underlying the return to the resting state remain largely unknown. To elucidate this mechanism, we performed proteomic analysis and Western blotting of tubulovesicles isolated from hog stomach. Interestingly, these analyses revealed high expression of the Na+/H+ exchanger 3 (NHE3) in these tubulovsicles. Immunocytochemical studies further demonstrated colocalization of NHE3 with H+,K+-ATPase in hog and rat gastric parietal cells. Acridine orange-based measurements of vesicular acidification (H+ uptake) in hog tubulovesicles showed that the selective NHE3 inhibitors S3226 and tenapanor significantly enhanced H+,K+-ATPase-mediated acidification. These inhibitors did not affect the ATPase activity of H+,K+-ATPase in freeze-dried hog tubulovesicles. This excessive acidification was abolished under Na+-free conditions. In addition, ATP-dependent 22Na+ uptake into hog tubulovesicles, which was inhibited by NHE3 inhibitors, was also suppressed by the H+,K+-ATPase inhibitor SCH28080. In rat primary cultured parietal cells, apical canalicular structures were visualized using an extracellularly applied fluorescent antibody against H+,K+-ATPase to monitor morphological recovery from the stimulated state (acid-secreting). This analysis revealed that NHE3 inhibitors prevented the transition from the stimulated to the resting state. These findings suggest that NHE3 is functionally coupled with H+,K+-ATPase in tubulovesicles and may play a critical role in preventing excessive vesicular acidification and facilitating membrane remodeling during the recovery phase.NEW & NOTEWORTHY This study identifies Na+/H+ exchanger 3 (NHE3) as a key regulator of the transition of gastric parietal cells from the acid-secreting state to the resting state. Using purified gastric tubulovesicles and primary cultured rat parietal cells, we demonstrate for the first time that NHE3 prevents excessive tubulovesicular acidification and promotes membrane remodeling. These findings provide new insight into the homeostasis of parietal cells in the regulation of gastric acid secretion.