Inhibition of rat sinusoidal GSH transporter by thioethers: specificity, sidedness, and kinetics

In isolated hepatocytes, cystathionine, methionine, and thioether analogues of methionine, cysteine, and homocysteine, including S-adenosyl derivatives, inhibited reduced glutathione (GSH) efflux. The potency of inhibition by thioethers with different S-alkyl moieties was methyl < ethyl < butyryl < aminoethyl < alpha-aminopropionyl. Inhibition of GSH efflux by cystathionine from hepatocytes that were allowed to resynthesize GSH resulted in greater repletion (30-40%) of GSH levels compared with absence of cystathionine. To address unequivocally the sidedness of inhibition, i.e., cis vs. trans, we examined the effect of cystathionine on the activity of GSH transport in Xenopus oocytes expressing the cRNA of cloned rat liver sinusoidal (RsGshT) and canalicular (RcGshT) GSH transporters. Cystathionine trans inhibited efflux of GSH and cis inhibited uptake of GSH by oocytes expressing RsGshT. Conversely, when oocytes expressing RsGshT were loaded with cystathionine, no inhibition of uptake or efflux was observed. The same structural requirement of a thioether bond to exert an inhibitory effect on GSH transport was observed in oocytes expressing RsGshT. Oocytes expressing RsGshT do not transport methionine, whereas oocytes expressing total rat liver mRNA express methionine transport. Inhibition of both GSH efflux from and uptake by oocytes expressing RsGshT exhibited a competitive type of kinetics: cystathionine increased the Michaelis constant for GSH transport (4.5 +/- 0.9 vs. 10 +/- 2.5 mM and 7.5 +/- 0.6 vs. 12.9 +/- 1.5 mM for uptake and efflux, respectively) without affecting the maximal velocity for transport. Thus thioethers such as methionine and cystathionine inhibit the transport of GSH by interacting in a competitive and specific fashion with the sinusoidal GSH transporter without themselves being transported by this carrier.