Molecular and functional characterization of microsomal UDP-glucuronic acid uptake by members of the nucleotide sugar transporter (NST) family.

Transport of the co-substrate UDPGA (UDP-glucuronic acid) into the lumen of the endoplasmic reticulum is an essential step in glucuronidation reactions due to the intraluminal location of the catalytic site of the enzyme UGT (UDP-glucuronosyltransferase). In the present study, we have characterized the function of several NSTs (nucleotide sugar transporters) and UGTs as potential carriers of UDPGA for glucuronidation reactions. UDPGlcNAc (UDP-N-acetylglucosamine)-dependent UDPGA uptake was found both in rat liver microsomes and in microsomes prepared from the rat hepatoma cell line H4IIE. The latency of UGT activity in microsomes derived from rat liver and V79 cells expressing UGT1A6 correlated well with mannose-6-phosphatase latency, confirming the UGT in the recombinant cells retained a physiology similar to rat liver microsomes. In the present study, four cDNAs coding for NSTs were obtained; two were previously reported (UGTrel1 and UGTrel7) and two newly identified (huYEA4 and huYEA4S). Localization of NSTs within the human genome sequence revealed that huYEA4S is an alternatively spliced form of huYEA4. All the cloned NSTs were stably expressed in V79 (Chinese hamster fibroblast) cells, and were able to transport UDPGA after preloading of isolated microsomal vesicles with UDPGlcNAc. The highest uptake was seen with UGTrel7, which displayed a V(max) approx. 1% of rat liver microsomes. Treatment of H4IIE cells with beta-naphthoflavone induced UGT protein expression but did not affect the rate of UDPGA uptake. Furthermore, microsomes from UGT1-deficient Gunn rat liver showed UDPGA uptake similar to those from control rats. These data show that NSTs can act as UDPGA transporters for glucuronidation reactions, and indicate that UGTs of the 1A family do not function as UDPGA carriers in microsomes. The cell line H4IIE is a useful model for the study of UDPGA transporters for glucuronidation reactions.

Molecular characterization of human UDP-glucuronic acid/UDP-N-acetylgalactosamine transporter, a novel nucleotide sugar transporter with dual substrate specificity.

A novel human nucleotide sugar transporter (NST) which transports both UDP-glucuronic acid (UDP-GlcA) and UDP-N-acetylgalactosamine (UDP-GalNAc) has been identified, cloned and characterized. The strategy for the identification of the novel NST involved a search of the expressed sequence tags database for genes related to the human UDP-galactose transporter-related isozyme 1, followed by heterologous expression of a candidate gene (hUGTrel7) in Saccharomyces cerevisiae and biochemical analyses. Significantly more UDP-GlcA and UDP-GalNAc were translocated from the reaction medium into the lumen of microsomes prepared from the hUGTrel7-expressing yeast cells than into the control microsomes from cells not expressing hUGTrel7. The possibility that this transporter participates in glucuronidation and/or chondroitin sulfate biosynthesis is discussed.

Evidence for an UDP-glucuronic acid/phenol glucuronide antiport in rat liver microsomal vesicles.

The transport of glucuronides synthesized in the luminal compartment of the endoplasmic reticulum by UDP-glucuronosyltransferase isoenzymes was studied in rat liver microsomal vesicles. Microsomal vesicles were loaded with p-nitrophenol glucuronide (5 mM), phenolphthalein glucuronide or UDP-glucuronic acid, by a freeze-thawing method. In was shown that: (i) the loading procedure resulted in millimolar intravesicular concentrations of the different loading compounds; (ii) addition of UDP-glucuronic acid (5 mM) to the vesicles released both intravesicular glucuronides within 1 min; (iii) glucuronides stimulated the release of UDP-glucuronic acid from UDP acid-loaded microsomal vesicles; (iv) trans-stimulation of UDP-glucuronic acid entry by loading of microsomal vesicles with p-nitrophenol glucuronide, phenolphthalein glucuronide, UDP-glucuronic acid and UDP-N-acetyl-glucosamine almost completely abolished the latency of UDP-glucuronosyltransferase, although mannose 6-phosphatase latency remained unaltered; (v) the loading compounds by themselves did not stimulate UDP-glucuronosyltransferase activity. This study indicates that glucuronides synthesized in the lumen of endoplasmic reticulum can leave by an antiport, which concurrently transports USP-glucuronic acid into the lumen of the endoplasmic reticulum.

Uridine diphosphoxylose enhances hepatic microsomal UDP-glucuronosyltransferase activity by stimulating transport of UDP-glucuronic acid across the endoplasmic reticulum membrane.

The UDP-glucuronosyltransferase (UGT) system fulfils a pivotal role in the biotransformation of potentially toxic endogenous and exogenous compounds. Here we report that the activity of UGT in rat liver is stimulated by UDP-xylose. This stimulation was found in native microsomal vesicles as well as in the intact endoplasmic reticulum (ER) membrane, as studied in permeabilized hepatocytes, indicating the potential physiological importance of UDP-xylose in the regulation of UGT. We present evidence that UDP-xylose enhances UGT activity by stimulation of (i) the uptake of UDP-glucuronic acid across the ER membrane and (ii) the elimination of the UDP and/or UMP reaction product out of the ER lumen. UDP-xyloe produced a marked trans-stimulation of microsomal UDP-glucuronic acid uptake when it was present within the lumen of the ER. When UDP-xylose was presented at the cytosolic side of the ER, it acted as a weak inhibitor of UDP-glucuronic acid uptake. Likewise, cytosolic UDP-glucuronic acid strongly trans-stimulated efflux of intravesicular UDP-xylose, whereas cytosolic UDP-xylose was inefficient in trans-stimulating efflux of UDP-glucuronic acid. Microsomal UDP-xylose influx was markedly stimulated by UMP and UDP. Such stimulation was only apparent when microsomes had been preincubated and thereby preloaded with UMP or UDP, indicating that UMP and UDP exeted their effect on UDP-xylose uptake by trans-stimulation from the luminal side of the ER membrane.

Mechanism of stimulation of microsomal UDP-glucuronosyltransferase by UDP-N-acetylglucosamine.

We propose the existence in rat liver endoplasmic reticulum (ER) of two asymmetric carrier systems. One system couples UDP-N-acetylglucosamine (UDPGlcNAc) transport to UDP-glucuronic acid (UDPGlcA) transport. When UDPGlcNAc was presented at the cytosolic side of the ER, it then acted as a weak inhibitor of UDPGlcA uptake. By contrast, UDPGlcNAc produced a forceful trans-stimulation of microsomal UDPGlcA uptake when it was present within the lumen of the ER. Likewise, cytosolic UDPGlcA strongly trans-stimulated efflux of intravesicular UDPGlcNAc, whereas cytosolic UDPGlcNAc was ineffective in trans-stimulating efflux of UDPGlcA. A second asymmetric carrier system couples UDPGlcNAc transport to UMP transport. Microsomal UDPGlcNAc influx was markedly stimulated by UMP present inside the microsomes. Such stimulation was only apparent when microsomes had been preincubated and thereby preloaded with UMP, indicating that UMP exerted its effect on UDPGlcNAc uptake by trans-stimulation from the lumenal side of the ER membrane. Contrariwise, extravesicular UMP only minimally trans-stimulated efflux of intramicrosomal UDPGlcNAc. It is widely accepted that UDPGlcNAc acts as a physiological activator of hepatic glucuronidation, but the mechanism of this effect has remained elusive. Based on our findings, we propose a model in which the interaction of two asymmetric transport pathways, i.e. UDPGlcA influx coupled to UDPGlcNAc efflux and UDPGlcNAc influx coupled to UMP efflux, combined with intravesicular metabolism of UDPGlcA, forms a mechanism that leads to stimulation of glucuronidation by UDPGlcNAc.

Membrane translocation and regulation of uridine diphosphate-glucuronic acid uptake in rat liver microsomal vesicles.

BACKGROUND/AIMS: Hepatic glucuronidation is quantitatively the most important conjugation reaction by which an array of endogenous compounds and xenobiotics undergo biotransformation and detoxification. The active site of the uridine diphosphate (UDP) glucuronosyltransferases, which catalyze glucuronidation reactions, has been postulated to reside in the lumen of the endoplasmic reticulum. The aim of this study was to characterize the process whereby UDP glucuronic acid (UDP-GlcUA), the cosubstrate for all glucuronidation reactions, is transported into microsomal vesicles. METHODS: The uptake process was analyzed using rapid filtration techniques, radiolabeled UDP-GlcUA, and rat liver microsomes. RESULTS: Uptake was saturable with respect to time and concentration, inhibited by 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid and 4-acetamido-4'-isothio-cyanatostilbene-2-2'-disulfonic acid, and was osmotically sensitive. Transport was stimulated by Mg2+ and guanosine triphosphate (50 mumol/L) but not guanosine 5'-O-(3-thiotriphosphate) or adenosine triphosphate. Luminal UDP-N-acetylglucosamine (1 mmol/L) produced enhanced uptake of UDP-GlcUA (trans stimulation). In contrast to nucleotide sugar transport in the Golgi apparatus, trans uridine monophosphate and UDP did not alter UDP-GlcUA transport in microsomes, indicating distinct processes. CONCLUSIONS: These data provide unambiguous evidence for the existence of a unique, substrate-specific, regulated, carrier-mediated process that transports UDP-GlcUA into the lumen of hepatocyte microsomes. This transporter may regulate glucuronidation in vivo.

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

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 site(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 degrees 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 degrees C). UDP-GlcUA uptake was saturable, and similar kinetic parameters were obtained for rough and smooth vesicles (Km 1.9 microM, Vmax 443 pmol/mg protein per min, and Km 1.3 microM, Vmax 503 pmol/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.