Effect of sulfhydryl compounds on ATP-stimulated H+ transport and Cl− uptake in rabbit renal cortical endosomes

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Abstract
The vacuolar H+ ATPase is inhibited by N-ethylmaleimide (NEM), a sulfhydryl compound, suggesting the involvement of a sulfhydryl group in this transport process. We have examined the effects of several sulfhydryl-containing compounds on the vacuolar H+ ATPase of rabbit renal cortical endosomes. A number of such compounds were effective inhibitors of endosomal H+ transport at 10−5–10−6 m, including NEM, mersalyl, aldrithiol, 5,5′ dithiobis (2-nitrobenzoic acid),p-chloromercuribenzoic acid (PCMB) andp-chloromercuriphenyl sulfonic acid (PCMBS). NEM, mersalyl, aldrithiol and PCMBS had no effect on pH-gradient dissipation, whereas PCMB decreased the pH gradient faster than control. In the absence of ATP, PCMB (10−4 m) stimulated endosomal36Cl− uptake, particularly in the presence of an inside-alkaline pH gradient (pHin=7.6/pHout=5.5.). This result was not an effect of PCMB on the Cl−-conductive pathway. The less permeable PCMBS did not stimulate36Cl− uptake. The effects of PCMB were concentration dependent and were prevented by dithioerithritol,. ATP-dependent36Cl− uptake was decreased by addition of PCMB. Finally, PCMB had no effect on45Ca2+ uptake. These results support the presence of two functionally important sulfhydryl groups in this endosomal preparation. One such group is involved with ATP-driven H+ transport and must be located on the cytoplasmic surface of the endosomal membrane. The second sulfhydryl group must reside on the internal surface of the endosomal membrane and relates to a PCMB-activated Cl−/OH− exchanger that is functional both in the presence and absence of ATP. This endosomal transporter is similar to the PCMB-activated Cl−/OH− exchanger recently described in rabbit renal brush-border membranes.