INHIBITION OF RAT SINUSOIDAL GSH TRANSPORTER BY THIOETHERS - SPECIFICITY, SIDEDNESS, AND KINETICS

In isolated hepatocytes, cystathionine, methionine, and thioether anal ogues of methionine, cysteine, and homocysteine, including S-adenosyl derivatives, inhibited reduced glutathione (GSH) efflux. The potency o f inhibition by thioethers with different S-alkyl moieties was methyl < ethyl < butyryl < aminoethyl < alpha-aminopropionyl. Inhibition of G SH efflux by cystathionine from hepatocytes that were allowed to resyn thesize GSH resulted in greater repletion (30-40%) of GSH levels compa red with absence of cystathionine. To address unequivocally the sided ness of inhibition, i.e., cis vs. trans, we examined the effect of cys tathionine on the activity of GSH transport in Xenopus oocytes express ing 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. Converse ly, when oocytes expressing RsGshT were loaded with cystathionine, no inhibition of uptake or efflux was observed. The same structural requi rement of a thioether bond to exert an inhibitory effect on GSH transp ort was observed in oocytes expressing RsGshT. Oocytes expressing RsGs hT do not transport methionine, whereas oocytes expressing total rat l iver mRNA express methionine transport. Inhibition of both GSH efflux from and uptake by oocytes expressing RsGshT exhibited a competitive t ype of kinetics: cystathionine increased the Michaelis constant for GS H 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 ma ximal velocity for transport. Thus thioethers such as methionine and c ystathionine inhibit the transport of GSH by interacting in a competit ive and specific fashion with the sinusoidal GSH transporter without t hemselves being transported by this carrier.