2 DISTINCT TYPES OF SH-GROUPS ARE NECESSARY FOR BUMETANIDE AND BILE-ACID UPTAKE INTO ISOLATED RAT HEPATOCYTES

Substances that block SH-groups were studied in respect to their effects on the uptake of the loop diuretic bumetanide and the bile acids cholate and taurocholate into isolated rat hepatocytes. SH-blockers, e.g., p-chloromercuribenzenesulfonate (PCMBS), N-ethylmaleimide (NEM), dithiobis-nitropyridine (DTNP) and dithiobis-2-nitrobenzoic acid (DTNB) reduced bumetanide transport in a concentration-dependent manner. Inhibition of the organic mercurial PCMBS was reversed by the addition of 500 muM dithiothreitol (DTT), indicating an interaction of this substance with free SH-groups. NEM irreversibly blocked SH-groups by covalent binding and was the most effective inhibitor of bumetanide and cholate uptake. In contrast, PCMBS was the most effective inhibitor of taurocholate uptake. Photoaffinity studies with [H-3]bumetanide and [H-3]7,7-azotaurocholate were performed with isolated rat hepatocytes in the presence of PCMBS and DTNP. Binding of the photolabels was not reduced by SH-group blockers. Newly synthesized sulfhydryl-modifying reagents such as dithio-sulfonate-ethyl-nitrobenzoic acid (DTSNB) and dithio-octyl-nitrobenzoic acid (DTONB), are derivatives of the alkylating agent DTNB. DTSNB is regarded as a selective blocker for SH-groups in a hydrophilic environment, while DTONB is more lipophilic and interacts with SH-groups in the transmembrane domain of transport proteins. The IC50-values of these blockers for bumetanide uptake (DTSNB 250 muM, DTONB 141 muM) and for cholate uptake (DTSNB 250 muM, DTONB 115 muM) were almost identical. These findings support the concept of a common uptake mechanism for cholate and bumetanide and indicate that two distinct moieties of SH-groups are required for the uptake of both organic anions. One of these is probably located on the outer surface and the other within the membrane of hepatocytes.