Functional studies of acid transporter in cultured rat epididymal cell.

OBJECTIVE

To explore the functional role of vacuolar H(+)-ATPase in the pH regulation of epididymal fluid and its effect on sperm motility.

DESIGN

Experimental study.

SETTING

Physiology 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 IC(50) 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.