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  • Title: Membrane transport of conjugated and unconjugated bile acids into hepatocytes is susceptible to SH-blocking reagents.
    Author: Blumrich M, Petzinger E.
    Journal: Biochim Biophys Acta; 1990 Nov 02; 1029(1):1-12. PubMed ID: 2171648.
    Abstract:
    The present study indicates that SH-groups are essential for the uptake of [3H]taurocholate and [14C]cholate into isolated rat hepatocytes. Several sulfhydryl-modifying reagents viz. p-chloromercuribenzenesulfonate (PCMBS), N-ethylmaleimide (NEM), dithio-bis(5-nitropyridine) (DTNP), bromosuccinimide and HgCl2 inhibited uptake of bile acids in a concentration-dependent manner. PCMBS was the most effective inhibitor in the uptake of taurocholate, while NEM is preferentially blocking the cholate uptake. PCMBS inhibited both the sodium- dependent and the sodium-independent bile acid uptake. Two different moieties of SH-groups seemed to be important for bile acid transport. One group was susceptible to DTNP and NEM, whereas PCMBS was able to block another type of SH-groups in addition. Cell viability was altered by SH-blockers, except by PCMBS. Efflux studies with 86Rb+ demonstrated that the transmembrane potential of hepatocytes was less effected by 100 microM PCMBS in contrast to 100 microM HgCl2. Efflux of tetra[3H]phenylphosphonium and of [3H]aflatoxin in PCMBS-treated hepatocytes documented membrane integrity during at least 10 min. PCMBS did not reduce cellular ATP levels significantly (minus 7%) nor did it markedly increase the amount of the Trypan-blue stained hepatocytes (plus 8.5%). The blocking effect of PCMBS was immediate and was completely reversed by the addition of 500 microM dithiothreitol (DTT), indicating a specific interaction with sulfhydryl-groups. This antagonizing effect of DTT depends on the concentration and exposure time of PCMBS. Six other thiols viz. 2-mercaptoethanol, 1,2-dimercaptoethane, 1,4-dimercaptobutane, 1,6-dimercaptohexane, L-cysteine and L-glutathione were less effective. The results suggest that free SH-groups on the outer surface of hepatocytes play an important role in the uptake process for conjugated and unconjugated bile acids.
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