SPECIFICITY AND DIRECTIONALITY OF THIOL EFFECTS ON SINUSOIDAL GLUTATHIONE TRANSPORT IN RAT-LIVER

In rats the sinusoidal glutathione (GSH) carrier transports GSH bidire ctionally, and its activity is influenced by the thiol-disulfide statu s; the V-max of sinusoidal GSH efflux was increased by dithiothreitol (DTT) and decreased by cystine. In the present work we examined the sp ecificity and directionality of the thiol effect. Using in situ perfus ed livers, we found that 1 mM DTT and other dithiols, including 1,2-et hanedithiol, 1,3-propanedithiol, and 1,4-butanedithiol, stimulated sin usoidal GSH efflux by 200-500% but dihydrothioctic acid, which is nega tively charged, had no effect. Uncharged or positively charged monothi ols (2 mM), such as dimercaprol, monothioglycerol, 2-mercaptoethanol, 3-mercapto-2-butanol, 1-mercapto-2-propanoI, and cysteamine, also exer ted a stimulatory effect on sinusoidal GSH efflux. In contrast, monoth iols containing a negatively charged substituent, such as penicillamin e, captopril, N-acetylcysteine, mercaptopropionylglycine, mercaptoetha nesulfonic acid, mercaptoacetic acid, and mercaptopropionic acid, had no effect. The thiol moiety was essential for activity, inasmuch as et hanol, propanol, propanediol, and glycerol had no effect on sinusoidal GSH efflux. The effect of DTT or cystine pretreatment (2 mM or 0.5 mM , respectively, for 30 min) on GSH uptake was then examined using cult ured rat hepatocytes. The linear rate of [S-35]GSH uptake and the conc entration dependence were measured after cells were pretreated with ac ivicin (0.5 mM, for 15 min) and buthionine sulfoximine (10 mM, 15 min) , to prevent breakdown and resynthesis of GSH from precursors, respect ively. Uptake buffer also contained 20 mM alpha-(methylamino)isobutyri c acid and 20 mM threonine (inhibitors of amino acid transport systems A and ASC, respectively), to prevent uptake of cysteine. Pretreatment with DTT decreased the V-max of GSH uptake by similar to 50% (control V-max value, 24 nmol/10(6) cells/30 min), whereas the K-m remained un affected (similar to 8 mM). Cystine pretreatment had no influence on G SH uptake but inhibited efflux. In conclusion, the presence of at leas t one thiol group and the absence of negative charge are required to s timulate sinusoidal GSH efflux. The direction of GSH transport is modu lated by the thiol-disulfide status, so that thiol reduction changes t he GSH transporter from a bidirectional GSH transporter into a prefere ntially unidirectional (outward) transporter by inhibiting uptake whil e stimulating efflux and thiol oxidation favors inward transport by in hibiting only efflux.