A membrane transporter mediates access of uridine 5′-diphosphoglucuronic acid from the cytosol into the endoplasmic reticulum of rat hepatocytes: implications for glucuronidation reactions

Abstract

Hepatic glucuronidation of a wide variety of substrates is catalyzed by the membrane-bound UDP-glucuronosyltransferases. Uridine 5′-diphosphoglucuronic acid (UDP-GlcUA) is the essential cosubstrate for all UDP-glucuronosyltransferase-mediated reactions. The mechanism by which this bulky, hydrophilic nucleotide-sugar is transported from the cytosol (where it is synthesized) to its binding sites(s) on the enzyme is unknown. To determine whether a membrane carrier mediates the access of UDP-GlcUA into the endoplasmic reticulum, the transport of uridine 5′-diphospho- D-[U- 14C]glucuronic acid into vesicles of rough and smooth endoplasmic reticulum isolated from rat liver was investigated at 38°C using a rapid filtration technique. Uptake of UDP-GlcUA by both rough and smooth vesicles was extremely rapid (linear for only 10–20 s) and temperature-dependent (negligible at 4°C). UDP-GlcUA uptake was saturable, and similar kinetic parameters were obtained for rough and smooth vesicles ( K m 1.9 μM, V max 443 pmol/mg protein per min, and K m 1.3 μM, V max 503 mol/mg protein per min, respectively). The uptake of UDP-GlcUA also exhibited a high degree of specificity, since many related compounds, including UMP, UDP and UDP-Glc, did not influence uptake. In addition, the non-penetrating inhibitors of anion transport, 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS), 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), and probenecid, markedly inhibited UDP-GlcUA uptake. Finally, osmotic modulation of the intravesicular volume did not affect total uptake of UDP-GlcUA by membrane vesicles at equilibrium, indicating that this nucleotide-sugar is transported into the membrane rather than the intravesicular space. Collectively, these data provide direct evidence for a specific, carrier-mediated uptake process, which transports UDP-GlcUA from the cytosol into the endoplasmic reticulum of hepatocytes. This UDP-GlcUA transporter may be involved in the regulation of hepatic glucuronidation reactions.