Functional studies of acid transporter in cultured rat epididymal cell

ObjectiveTo explore the functional role of vacuolar H+-ATPase in the pH regulation of epididymal fluid and its effect on sperm motility.DesignExperimental study.SettingPhysiology laboratory in a university.Animal(s)Immature male Sprague-Dawley rats.Intervention(s)The H+-ATPase inhibitor was applied to the primary culture of epididymal cells.Main Outcome Measure(s)The intracellular luminal fluid pH and sperm percent motility were recorded.Result(s)Double immunofluorescence of H+-ATPase and carbonic anhydrase II in primary culture of cauda epididymal epithelial cells showed that the system was a suitable model for investigation of acid secretion by clear cells. Clear cells were pharmacologically distinct from principal cells in acid/base transportation. The intracellular pH recovery from cellular acidification was suppressed by the H+-ATPase inhibitor bafilomycin A1(100 nM) and the Na+/H+ exchanger inhibitor amiloride (1 mM) by 85% and 54%, respectively. These results suggest that, in addition to Na+/H+ exchanger, clear cells actively pump proton from cytoplasm into extracellular space through H+-ATPase. In addition, inhibition of H+-ATPase by bafilomycin A1 blocked the acidification of luminal fluid with IC50 values of 12 nM, which supports that H+-ATPase acidifies the luminal fluid. We also confirm that the acid fluid regulates rat cauda sperm motility.Conclusion(s)The present work shows that clear cells, the minority cell type of epididymal cell population, play an important role in the pH regulation of epididymal fluid by H+-ATPase.