Inhibition of glutathione efflux in the perfused rat liver and isolated hepatocytes by organic anions and bilirubin. Kinetics, sidedness, and molecular forms.

Abstract

Using isolated, in situ, single-pass perfused rat livers, incubations of freshly isolated hepatocytes, and sinusoidal membrane-enriched vesicles, we and others have shown the saturability of transport (efflux) of hepatic glutathione (GSH). These observations have implicated a carrier mechanism. Our present studies were designed to provide further evidence in support of a carrier mechanism for hepatic GSH efflux by demonstrating competition by liver-specific ligands which are taken up by hepatocytes. Perfusing livers with different substances, we found that: (a) sulfobromophthalein-GSH (BSP-GSH) had a dose-dependent and fully reversible inhibitory effect on GSH efflux, while GSH alone did not have any effect; (b) taurocholate had no inhibitory effect; (c) all of the organic anions studied, i.e., BSP, rose bengal, indocyanine green, and unconjugated bilirubin (UCB), manifested potent, dose-dependent inhibitory effects, with absence of toxic effects and complete reversibility of inhibition in the case of UCB. The inhibitory effects of UCB could be overcome partially by raising (CoC12-induced) hepatic GSH concentration.Because of the physiological importance of UCB, we conducted a detailed study of its inhibitory kinetics in the isolated hepatocyte model in the range of circulating concentrations of UCB. Studies with Cl1-free media, to inhibit the uptake of UCB by hepatocytes, showed that the inhibition of GSH efflux by UCB is apparently from inside the cell. This point was confirmed by showing that the inhibition is overcome only when bilirubin-loaded cells are cleared of bilirubin (incubation with 5% bovine serum albumin). Using Gunn rat hepatocytes and purified bilirubin mono-and diglucuronides, we found that both UCB and glucuronide forms of bilirubin inhibit GSH efflux in a dose-dependent manner. We conclude that the organic anions, although taken up by a mechanism independent of GSH, may competitively inhibit the carrier for GSH efflux from inside the hepatocyte.

Thiol-disulfide effects on hepatic glutathione transport. Studies in cultured rat hepatocytes and perfused livers.

“…As shown in Fig. 2, when cultured hepatocytes were incubated with BSP-GSH (40 ,4M), total GSH efflux was inhibited by -40%, similar to that observed previously (7). Cystine (0.5 mM) coincubation did not result in further inhibition of GSH efflux.…”

“…Our laboratory has studied GSH transport extensively using different experimental models. The effilux ofGSH from the perfused liver and isolated hepatocytes is a saturable process (5,6) that can be competitively inhibited by organic anions, bilirubin (7), and methionine (8). Furthermore, we have shown that membrane potential is a driving force for the transport of GSH, so that hyperpolarization increases and depolarization decreases the release of GSH (9).…”

Role of two recently cloned rat liver GSH transporters in the ubiquitous transport of GSH in mammalian cells.

“…To the best of our knowledge, no information exists on GSH efflux from the other four cell lines. We examined basal GSH efflux and the response to DTT and BSP-GSH, which is know to cis-inhibit sinusoidal GSH transport selectively (3,6,24). Fig.…”

“…The sinusoidal GSH transporter mRNA was only expressed in liver whereas the canalicular transporter mRNA was found in all organs examined (4,5). The functional distinction between these two transporters relies on the selective cis-inhibitory effect of BSP-GSH on the sinusoidal GSH transporter but not the low affinity canalicular GSH transporter (3)(4)(5)(6). Another feature of the sinusoidal GSH transporter is its sensitivity to thiol modifying agents such that thiol reduction with dithiothreitol (DTT) led to increased GSH efflux from hepatocytes while inhibiting uptake, favoring outward movement of GSH while the opposite was observed with thiol oxidation (7,8).…”

The Thiol Sensitivity of Glutathione Transport in Sidedness-Sorted Basolateral Liver Plasma Membrane and in Oatp1-Expressing HeLa Cell Membrane

“…Sinusoidal GSH efflux is trans-inhibited by methionine and cystathionine (Aw et al, 1986;Ghibelli et al, 1998), cis-inhibited by unconjugated and mono-and diglucuronide forms of bilirubin, and sulfobromophthalein-glutathione conjugate (Aw et al, 1986;Ookhtens et al, 1988;FernandezCheca et al, 1993). Certain disulfides (cystine and oxidized glutathione disulfide) and uncharged thiols such as dithiothreitol (DTT) have opposite effects on low-affinity, high-capacity sinusoidal GSH transport (Lu et al, 1993(Lu et al, , 1994.…”