Expression cloning of the cDNA for a polypeptide associated with rat hepatic sinusoidal reduced glutathione transport: characteristics and comparison with the canalicular transporter.

Abstract

Using the Xenopus oocyte expression system, we previously identified an -4-kb fraction of mRNA from rat liver that expresses sulfobromophthalein reduced glutathione S-conjugate (BSP-GSH)-insensitive and an -2.5-kb fraction expressing BSP-GSH-sensitive reduced glutathione (GSH) transport. From the former, a 4.05-kb cDNA was cloned and characterized as the putative rat canalicular GSH transporter. Starting with a cDNA library constructed from the -2.5-kb fraction, we have now isolated a single clone that leads to expression of a BSP-GSH-and cystathionine- bases. An '40-kDa protein is generated in in vitro translation of cRNA for RsGshT. Northern blot analysis revealed a single -2.8-kb transcript in rat and human liver with negligible hybridization signal in other organs. The abundance of mRNA for RsGshT did not increase with phenobarbital treatment. Cis-inhibition by BSP-GSH and trans-inhibition by cystathionine and lack of induction by phenobarbital are characteristic of sinusoidal GSH secretion and thus indicate that RsGshT either encodes the sinusoidal GSH transporter itself or a regulatory subunit of the transporter that determines its liver-specific activity.

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

“…RNA was isolated from various cell lines according to the method of Chomczynski and Sacchi (19). RNA (20 g) was separated on a 1% agarose gel with 6.7% formaldehyde, blotted onto a nylon membrane by capillary transfer, then hybridized to a 1.5-kb cDNA derived from a subcloned insert corresponding to nucleotide 1132-2623 of the rat canalicular GSH transporter (RcGshT) and to a full length 2.7-kb cDNA for the rat sinusoidal GSH transporter (RsGshT) as described previously (4,5). The cDNA probes were labeled with 32 P-dCTP by random priming (Primer-It II Kit; Stratagene, La Jolla, CA).…”

“…Both transporters exhibit low affinity (high K m ), bidirectional, Na-independent GSH transport, and they show no sequence homology and represent two unique genes. 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).…”

“…Our laboratory has successfully cloned two of these transports from rat liver cDNA libraries (4,5). Both transporters exhibit low affinity (high K m ), bidirectional, Na-independent GSH transport, and they show no sequence homology and represent two unique genes.…”

“…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).…”

Nonclonal growth of preneoplastic cells positive for glutathione S‐transferase P‐form in the rat liver

“…(7,11,12) Furthermore, Devi and Devaraj (13) reported that the acrolein-generating cyclophosphamide (36,37) induced both weak and strong GST-P expression in all rat hepatocytes, and indicated that the strongly GST-P + single hepatocytes and minifoci were distributed along the hepatocyte strands. Since acrolein is excreted through the GS-X pump as a GSH conjugate, (38,39) this finding indicated that the induction of GST-P expression is mediated through injury of the transporter by the toxic action of acrolein. Thus, the induction of GST-P expression serves to inactivate acrolein toxicity, resulting in the formation of GST-P + single hepatocytes, as reported previously.…”