V-type H+-ATPase in the human eccrine sweat duct: immunolocalization and functional demonstration

Abstract

We investigated for the presence of a vacuolar-type H+-ATPase (V-ATPase) in the human eccrine sweat duct (SD). With the use of immunocytochemistry, an anti-V- ATPase antibody showed a strong staining at the apical membrane and a weaker one in the cytoplasm. Cold preservation followed by rewarming did not alter this staining pattern. With the use of the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein on isolated and perfused straight SD under HCO-free conditions and in the absence of Na+, proton extrusion was determined from the recovery rate of intracellular pH (dpH(i)/dt) following an acid load. Oligomycin (25 microM), an inhibitor of F-type ATPases, decreased dpH(i)/dt by 88 +/- 6%, suggesting a role for an ATP-dependent process involved in pH(i) recovery. Moreover, dpH(i)/dt was inhibited at 95 +/- 3% by 100 nM luminal concanamycin A, a specific inhibitor of V-ATPases, whereas 10 microM bafilomycin A1, another specific inhibitor of V-ATPases, was required to decrease dpH(i)/dt by 73%. These results strongly suggest that a V-ATPase is involved in proton secretion in the human eccrine SD.