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

1 Groupe de Recherche en Transport Membranaire, Université de Montréal, Montreal, Quebec, Canada H3C 3J7; and 2 Unilever Research, Port Sunlight, UK CH63 3JW 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 /d t ) following an acid load. Oligomycin (25 µM), an inhibitor of F-type ATPases, decreased dpH i /d t by 88 ± 6%, suggesting a role for an ATP-dependent process involved in pH i recovery. Moreover, dpH i /d t was inhibited at 95 ± 3% by 100 nM luminal concanamycin A, a specific inhibitor of V-ATPases, whereas 10 µM bafilomycin A 1 , another specific inhibitor of V-ATPases, was required to decrease dpH i /d t by 73%. These results strongly suggest that a V-ATPase is involved in proton secretion in the human eccrine SD. proton pump; intracellular pH measurement; concanamycin A; bafilomycin A 1 ; oligomycin