Identification of a new urate and high affinity nicotinate transporter, hOAT10 (SLC22A13)

The orphan transporter hORCTL3 (human organic cation transporter like 3; SLC22A13) is highly expressed in kidneys and to a weaker extent in brain, heart, and intestine. hORCTL3-expressing Xenopus laevis oocytes showed uptake of [(3)H]nicotinate, [(3)H]p-aminohippurate, and [(14)C]urate. Hence, hORCTL3 is an organic anion transporter, and we renamed it hOAT10. [(3)H]Nicotinate transport by hOAT10 into X. laevis oocytes and into Caco-2 cells was saturable with Michaelis constants (K(m)) of 22 and 44 microm, respectively, suggesting that hOAT10 may be the molecular equivalent of the postulated high affinity nicotinate transporter in kidneys and intestine. The pH dependence of hOAT10 suggests p-aminohippurate(-)/OH(-), urate(-)/OH(-), and nicotinate(-)/OH(-) exchange as possible transport modes. Urate inhibited [(3)H]nicotinate transport by hOAT10 with an IC(50) value of 759 microm, assuming that hOAT10 represents a low affinity urate transporter. hOAT10-mediated [(14)C]urate uptake was elevated by an exchange with l -lactate, pyrazinoate, and nicotinate. Surprisingly, we have detected urate(-)/glutathione exchange by hOAT10, consistent with an involvement of hOAT10 in the renal glutathione cycle. Uricosurics, diuretics, and cyclosporine A showed substantial interactions with hOAT10, of which cyclosporine A enhanced [(14)C]urate uptake, providing the first molecular evidence for cyclosporine A-induced hyperuricemia.     

Polarized expression of different monocarboxylate transporters in rat medullary thick limbs of Henle.

Extracellular lactic acid is a major fuel for the mammalian medullary thick ascending limb (MTAL), whereas under anoxic conditions, this nephron segment generates a large amount of lactic acid, which needs to be excreted. We therefore evaluated, at both the functional and molecular levels, the possible presence of monocarboxylate transporters in basolateral (BLMVs) and luminal (LMVs) membrane vesicles isolated from rat MTALs. Imposing an inward H(+) gradient induced the transient uphill accumulation of L-[(14)C]lactate in both types of vesicles. However, whereas the pH gradient-stimulated uptake of L-[(14)C]lactate in BLMVs was inhibited by anion transport blockers such as alpha-cyano-4-hydroxycinnamate, 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS), and furosemide, it was unaffected by these agents in LMVs, indicating the presence of a L-lactate/H(+) cotransporter in BLMVs, but not in LMVs. Under non-pH gradient conditions, however, the uptake of L-[(14)C]lactate in LMVs was transstimulated 100% by L-lactate, but by only 30% by D-lactate. Furthermore, this L-lactate self-exchange was markedly inhibited by alpha-cyano-4-hydroxycinnamate and DIDS and almost completely by 1 mM furosemide, findings consistent with the existence of a stereospecific carrier-mediated lactate transport system in LMVs. Using immunofluorescence confocal microscopy and immunoblotting, the monocarboxylate transporter (MCT)-2 isoform was shown to be specifically expressed on the basolateral domain of the rat MTAL, whereas the MCT1 isoform could not be detected in this nephron segment. This study thus demonstrates the presence of different monocarboxylate transporters in rat MTALs; the basolateral H(+)/L-lactate cotransporter (MCT2) and the luminal H(+)-independent organic anion exchanger are adapted to play distinct roles in the transport of monocarboxylates in MTALs.     

Involvement of rBAT in Na(+)-dependent and -independent transport of the neurotransmitter candidate L-DOPA in Xenopus laevis oocytes injected with rabbit small intestinal epithelium poly A(+) RNA

Although L-3,4-dihydroxyphenylalanine (L-DOPA) is claimed to be a neurotransmitter in the central nervous system (CNS), receptor or transporter molecules for L-DOPA have not been determined. In an attempt to identify a transporter for L-DOPA, we examined whether or not an active and high affinity L-DOPA transport system is expressed in Xenopus laevis oocytes injected with poly A(+) RNA prepared from several tissues. Among the poly A(+) RNAs tested, rabbit intestinal epithelium poly A(+) RNA gave the highest transport activity for L-[(14)C]DOPA in the oocytes. The uptake was approximately five times higher than that of water-injected oocytes, and was partially Na(+)-dependent. L-Tyrosine, L-phenylalanine, L-leucine and L-lysine inhibited this transport activity, whereas D-DOPA, dopamine, glutamate and L-DOPA cyclohexylester, an L-DOPA antagonist did not affect this transport. Coinjection of an antisense cRNA, as well as oligonucleotide complementary to rabbit rBAT (NBAT) cDNA almost completely inhibited the uptake of L-[(14)C]DOPA in the oocytes. On the other hand, an antisense cRNA of rabbit 4F2hc barely affected this L-[(14)C]DOPA uptake activity. rBAT was thus responsible for the L-[(14)C]DOPA uptake activity expressed in X. laevis oocytes injected with poly A(+) RNA from rabbit intestinal epithelium. As rBAT is localized at the target regions of L-DOPA in the CNS, rBAT might be one of the components involved in L-DOPAergic neurotransmission.     

Functional analysis of ABCA8, a new drug transporter

We examined the transport capacity in Xenopus laevis oocytes of human EST KIAA0822/ABCA8, a member of the ABC superfamily. Substrates of ABCC2/MRP-2 such as [14C]estradiol-beta-glucuronide, taurocholate, and LTC4, and of organic anion transporter (OAT), such as para-aminohippuric acid, ochratoxin-A, were significantly accumulated while tetraethylammonium and doxorubicin were not. The transport of [14C]estradiol-beta-glucuronide was ATP-dependent and K(m) and V(max) values of 30.4microM and 66.9pmol/h/egg, respectively, were estimated. The transport of [14C]estradiol-beta-glucuronide was inhibited by substrates/inhibitors of ABCC2/MRP-2, but not by those of the organic cation transporter and multidrug resistance protein (MDR)-1. KIAA0822/ABCA8 possesses two ATP-binding sites and fourteen transmembrane domains. Northern blot analysis revealed expression in most organs, especially in heart, skeletal muscle, and liver. Thus, ABCA8 is a new member of the xenobiotic transporter ABC-subfamily.     

Endogenous lactate transport in Xenopus laevis oocyte: dependence on cytoskeleton and regulation by protein kinases

Carbon flux in Xenopus laevis oocyte is glycogenic and an endogenous monocarboxylate transporter is responsible for intracellular lactate uptake. The aim of the present study was to determine if direct activation of protein kinases C and A modulates the activity of lactate transporter, as well as to investigate the possible role of cytoskeleton in these regulatory phenomena. The modulation was studied in isolated Xenopus oocytes of stage V–VI by measuring ¹⁴C-lactate uptake, both in the absence and in the presence of cytoskeletal-perturbing toxins. We found that the basal lactate transporter activity depends on the integrity of the cytoskeleton since it is partially inhibited by cytoskeleton disorganisation. Both PKA and PKC activation caused a significant decrease in transport activity and this decrease could be blocked by specific protein kinase inhibitors. The evidenced effects were not additive. Transport inhibition was annulled by agents that destabilize actin filaments or microtubules. We conclude that both protein kinases A and C, whose effects are mediated by cytoskeleton, negatively regulate the endogenous lactate transporter of Xenopus oocyte, suggesting that these kinases may have a role in the control of cytosolic pyruvate/lactate pool in the oocyte. All the experiments in this study comply with the current laws of Italy.     

The organic cation transporters rOCT1 and hOCT2 are inhibited by cGMP

The electrogenic cation transporters OCT1 and OCT2 in the basolateral membrane of renal proximal tubules mediate the first step during secretion of organic cations. Previously we demonstrated stimulation and change of selectivity for rat OCT1 (rOCT1) by protein kinase C. Here we investigated the effect of cGMP on cation transport by rOCT1 or human OCT2 (hOCT2) after expression in human embryonic kidney cells (HEK293) or oocytes of Xenopus laevis. In HEK293 cells, uptake was measured by microfluorimetry using the fluorescent cation 4-(4-(dimethyl-amino)styryl)-N-methylpyridinium iodide (ASP + ) as substrate, whereas uptake into Xenopus laevis oocytes was measured with radioactively labelled cations. In addition, ASP +-induced depolarizations of membrane voltages (Vm) were measured in HEK293 cells using the slow whole-cell patch-clamp method. Incubation of rOCT1-expressing HEK293 cells for 10 min with 100 mM 8-Br-cGMP reduced initial ASP + uptake by maximally 78% with an IC50 value of 24 +/- 16 mM. This effect was not abolished by the specific PKG inhibitor KT5823, indicating that a cGMP-dependent kinase is not involved. An inhibition of ASP + uptake by rOCT1 in HEK293 cells was also obtained when the cells were incubated for 10 min with 100 mM cGMP, whereas no effect was obtained when cGMP was given together with ASP +. ASP + (100 mM)-induced depolarizations of Vm were reduced in the presence of 8-Br-cGMP (100 mM) by 44 +/- 11% (n = 6). Since it could be demonstrated that [3H]cGMP is taken up by an endogeneous cyanine863-inhibitable transporter, the effect of cGMP is probably mediated from inside the cell. Uptake measurements with [14C]tetraethylammonium and [3H]2-methyl-4-phenylpyridinium in Xenopus laevis oocytes expressing rOCT1 performed in the absence and presence of 8-Br-cGMP showed that cGMP does not interact directly with the transporter. The data suggest that the inhibition mediated by cGMP observed in HEK293 cells occurs most likely via a mammalian cGMP-binding protein that interacts with OCT1-2 transporters.     

γ-Aminobutyric acid transporter 2 mediates the hepatic uptake of guanidinoacetate, the creatine biosynthetic precursor, in rats

Guanidinoacetic acid (GAA) is the biosynthetic precursor of creatine which is involved in storage and transmission of phosphate-bound energy. Hepatocytes readily convert GAA to creatine, raising the possibility that the active uptake of GAA by hepatocytes is a regulatory factor. The purpose of this study is to investigate and identify the transporter responsible for GAA uptake by hepatocytes. The characteristics of [(14)C]GAA uptake by hepatocytes were elucidated using the in vivo liver uptake method, freshly isolated rat hepatocytes, an expression system of Xenopus laevis oocytes, gene knockdown, and an immunohistochemical technique. In vivo injection of [(14)C]GAA into the rat femoral vein and portal vein results in the rapid uptake of [(14)C]GAA by the liver. The uptake was markedly inhibited by γ-aminobutyric acid (GABA) and nipecotinic acid, an inhibitor of GABA transporters (GATs). The characteristics of Na(+)- and Cl(-)-dependent [(14)C]GAA uptake by freshly isolated rat hepatocytes were consistent with those of GAT2. The Km value of the GAA uptake (134 μM) was close to that of GAT2-mediated GAA transport (78.9 μM). GABA caused a marked inhibition with an IC(50) value of 8.81 μM. The [(14)C]GAA uptake exhibited a significant reduction corresponding to the reduction in GAT2 protein expression. GAT2 was localized on the sinusoidal membrane of the hepatocytes predominantly in the periportal region. This distribution pattern was consistent with that of the creatine biosynthetic enzyme, S-adenosylmethionine:guanidinoacetate N-methyltransferase. GAT2 makes a major contribution to the sinusoidal GAA uptake by periportal hepatocytes, thus regulating creatine biosynthesis in the liver.     

Pyruvate transport in isolated cardiac mitochondria from two species of amphibian exhibiting dissimilar aerobic scope: Bufo marinus and Rana catesbeiana

Cardiac mitochondria were isolated from Bufo marinus and Rana catesbeiana, two species of amphibian whose cardiovascular systems are adapted to either predominantly aerobic or glycolytic modes of locomotion. Mitochondrial oxidative capacity was compared using VO2 max and respiratory control ratios in the presence of a variety of substrates including pyruvate, lactate, oxaloacetate, beta-hydroxybutyrate, and octanoyl-carnitine. B. marinus cardiac mitochondria exhibited VO2 max values twice that of R. catesbeiana cardiac mitochondria when oxidizing carbohydrate substrates. Pyruvate transport was measured via a radiolabeled-tracer assay in isolated B. marinus and R. catesbeiana cardiac mitochondria. Time-course experiments described both alpha-cyano-4-hydroxycinnamate-sensitive (MCT-like) and phenylsuccinate-sensitive pyruvate uptake mechanisms in both species. Pyruvate uptake by the MCT-like transporter was enhanced in the presence of a pH gradient, whereas the phenylsuccinate-sensitive transporter was inhibited. Notably, anuran cardiac mitochondria exhibited activities of lactate dehydrogenase and pyruvate carboxylase. The presence of both transporters on the inner mitochondrial membrane affords the net uptake of monocarboxylates including pyruvate, beta-hydroxybutyrate, and lactate; the latter potentially indicating the presence of a lactate/pyruvate shuttle allowing oxidation of extramitochondrial NADH. Intramitochondrial lactate dehydrogenase and pyruvate carboxylase enables lactate to be oxidized to pyruvate or converted to anaplerotic oxaloacetate. Kinetics of the MCT-like transporter differed significantly between the two species, suggesting differences in aerobic scope may be in part attributable to differences in mitochondrial carbohydrate utilization.     

The highly toxic oxyanion tellurite (TeO<Stack><Subscript>3</Subscript><Superscript>2−</Superscript></Stack>) enters the phototrophic bacterium <Emphasis Type="Italic">Rhodobacter capsulatus</Emphasis> via an as yet uncharacterized monocarboxylate transport system

The facultative phototroph Rhodobacter capsulatus takes up the highly toxic oxyanion tellurite when grown under both photosynthetic and respiratory growth conditions. Previous works on Escherichia coli and R. capsulatus suggested that tellurite uptake occurred through a phosphate transporter. Here we present evidences indicating that tellurite enters R. capsulatus cells via a monocarboxylate transport system. Indeed, intracellular accumulation of tellurite was inhibited by the addition of monocarboxylates such as pyruvate, lactate and acetate, but not by dicarboxylates like malate or succinate. Acetate was the strongest tellurite uptake antagonist and this effect was concentration dependent, being already evident at 1 μM acetate. Conversely, tellurite at 100 μM was able to restrict the acetate entry into the cells. Both tellurite and acetate uptakes were energy dependent processes, since they were abolished by the protonophore FCCP and by the respiratory electron transport inhibitor KCN. Interestingly, cells grown on acetate, lactate or pyruvate showed a high level resistance to tellurite, whereas cells grown on malate or succinate proved to be very sensitive to the oxyanion. Taking these data together, we propose that: (a) tellurite enters R. capsulatus cells via an as yet uncharacterized monocarboxylate(s) transporter, (b) competition between acetate and tellurite results in a much higher level of tolerance against the oxyanion and (c) the toxic action of tellurite at the cytosolic level is significantly restricted by preventing tellurite uptake.     

Monocarboxylate transporters in the central nervous system: distribution, regulation and function

Monocarboxylate transporters (MCTs) are proton-linked membrane carriers involved in the transport of monocarboxylates such as lactate, pyruvate, as well as ketone bodies. They belong to a larger family of transporters composed of 14 members in mammals based on sequence homologies. MCTs are found in various tissues including the brain where three isoforms, MCT1, MCT2 and MCT4, have been described. Each of these isoforms exhibits a distinct regional and cellular distribution in rodent brain. At the cellular level, MCT1 is expressed by endothelial cells of microvessels, by ependymocytes as well as by astrocytes. MCT4 expression appears to be specific for astrocytes. By contrast, the predominant neuronal monocarboxylate transporter is MCT2. Interestingly, part of MCT2 immunoreactivity is located at postsynaptic sites, suggesting a particular role of monocarboxylates and their transporters in synaptic transmission. In addition to variation in expression during development and upon nutritional modifications, new data indicate that MCT expression is regulated at the translational level by neurotransmitters. Understanding how transport of monocarboxylates is regulated could be of particular importance not only for neuroenergetics but also for areas such as functional brain imaging, regulation of food intake and glucose homeostasis, or for central nervous system disorders such as ischaemia and neurodegenerative diseases.     

Lactate transport by skeletal muscle sarcolemmal vesicles

Recent studies have indicated that lactate traversal of the sarcolemmal membrane of skeletal muscle could be a carrier mediated process. In the present study, the initial rates of L(+)-lactate flux (J_lact) were measured in highly purified rat hindlimb skeletal muscle sarcolemmal vesicles. Fluxes were determined by the vesicle uptake of L(+)-[U-¹⁴C] lactate from the extra-vesicular medium. J_lact was saturable with respect to increasing concentrations of L(+)-lactate. Regression of these data to the Michaelis-Menten equation yielded a Km of 12.5 mM. J_lact was inhibited 81% by 10 mM pyruvate and 83% by 5mM alpha-cyano 4 hydroxycinnamate (p<0.05), but not by D-lactate indicating the presence of a stereoselective monocarboxylate transporter in the sarcolemmal membrane. Preincubation of the vesicles with the protein modifier, N-ethylmaleimide (20mM), inhibited J_lact by 86% (p<0.05). An inhibitor of the inorganic anion exchanger, SITS (1mM), had no effect on J_lact. However, J_lact was markedly sensitive to an inwardly directed proton gradient (p<0.05), and the flux was more closely related to the concentration of external ionic L(+)-lactate than to the protonated (HLa) form. These studies suggest that skeletal muscle sarcolemmal membranes possess a specific transport system for L-lactate and other monocarboxylates, which has similar properties to the lactate carrier described for several other tissues.     

Identification of monocarboxylate transporter 8 as a specific thyroid hormone transporter

Transport of thyroid hormone across the cell membrane is required for its action and metabolism. Recently, a T-type amino acid transporter was cloned which transports aromatic amino acids but not iodothyronines. This transporter belongs to the monocarboxylate transporter (MCT) family and is most homologous with MCT8 (SLC16A2). Therefore, we cloned rat MCT8 and tested it for thyroid hormone transport in Xenopus laevis oocytes. Oocytes were injected with rat MCT8 cRNA, and after 3 days immunofluorescence microscopy demonstrated expression of the protein at the plasma membrane. MCT8 cRNA induced an approximately 10-fold increase in uptake of 10 nM 125I-labeled thyroxine (T4), 3,3',5-triiodothyronine (T3), 3,3',5'-triiodothyronine (rT3) and 3,3'-diiodothyronine. Because of the rapid uptake of the ligands, transport was only linear with time for <4 min. MCT8 did not transport Leu, Phe, Trp, or Tyr. [125I]T4 transport was strongly inhibited by L-T4, D-T4, L-T3, D-T3, 3,3',5-triiodothyroacetic acid, N-bromoacetyl-T3, and bromosulfophthalein. T3 transport was less affected by these inhibitors. Iodothyronine uptake in uninjected oocytes was reduced by albumin, but the stimulation induced by MCT8 was markedly increased. Saturation analysis provided apparent Km values of 2-5 microM for T4, T3, and rT3. Immunohistochemistry showed high expression in liver, kidney, brain, and heart. In conclusion, we have identified MCT8 as a very active and specific thyroid hormone transporter.     

Functional expression of pig renal organic anion transporter 3 (pOAT3)

With the cloning of pig renal organic anion transporter 1 (pOAT1) (Biochimie 84 (2002) 1219) we set up a model system for comparative studies of cloned and natively isolated membrane located transport proteins. Meanwhile, another transport protein involved in p-aminohippurate (PAH) uptake on the basolateral side of the proximal tubule cells was identified, designated organic anion transporter 3 (OAT3). To explore the contribution of pOAT1 to the PAH clearance in comparison to OAT3, it was the aim of this study to extend our model by cloning of the pig ortholog of OAT3. Sequence comparisons of human organic anion transporter 3 (hOAT3) with the expressed sequence tag (EST) database revealed a clone and partial sequence of the pig renal organic anion transporter 3 (pOAT3) ortholog. Sequencing of the entire open reading frame resulted in a protein of 543 amino acid residues encoded by 1632 base pairs (EMBL Acc. No. AJ587003). It showed high homologies of 81%, 80%, 76%, and 77% to the human, rabbit, rat, and mouse OAT3, respectively. A functional characterization of pOAT3 in Xenopus laevis oocytes yielded an apparent Km (Kt) for [3H]estrone sulfate of 7.8 +/- 1.3 microM. Moreover, pOAT3 mediated [3H]estrone sulfate uptake was almost abolished by 0.5 mM of glutarate, dehydroepiandosterone sulfate, or probenecid consistent with the hallmarks of OAT3 function.     

The Neuroplastin Adhesion Molecules Are Accessory Proteins That Chaperone the Monocarboxylate Transporter MCT2 to the Neuronal Cell Surface

Background

The neuroplastins np65 and np55 are two synapse-enriched immunoglobulin (Ig) superfamily adhesion molecules that contain 3 and 2 Ig domains respectively. Np65 is implicated in long term, activity dependent synaptic plasticity, including LTP. Np65 regulates the surface expression of GluR1 receptor subunits and the localisation of GABA_A receptor subtypes in hippocampal neurones. The brain is dependent not only on glucose but on monocarboxylates as sources of energy. The. monocarboxylate transporters (MCTs) 1–4 are responsible for the rapid proton-linked translocation of monocarboxylates including pyruvate and lactate across the plasma membrane and require association with either embigin or basigin, proteins closely related to neuroplastin, for plasma membrane expression and activity. MCT2 plays a key role in providing lactate as an energy source to neurons.

Methodology/Findings

Here we use co-transfection of neuroplastins and monocarboxylate transporters into COS-7 cells to demonstrate that neuroplastins can act as ancillary proteins for MCT2. We also show that Xenopus laevis oocytes contain endogenous neuroplastin and its knockdown with antisense RNA reduces the surface expression of MCT2 and associated lactate transport. Immunocytochemical studies show that MCT2 and the neuroplastins are co-localised in rat cerebellum. Strikingly neuroplastin and MCT2 are enriched in the same parasagittal zebrin II-negative stripes.

Conclusions

These data strongly suggest that neuroplastins act as key ancillary proteins for MCT2 cell surface localisation and activity in some neuronal populations, thus playing an important role in facilitating the uptake of lactate for use as a respiratory fuel.     

Functional identification of SLC5A8, a tumor suppressor down-regulated in colon cancer, as a Na(+)-coupled transporter for short-chain fatty acids

SLC5A8, a tumor suppressor gene down-regulated in human colon cancer, codes for a transporter in the Na(+)/glucose cotransporter gene family, but the definitive functional identity of the transporter protein is not known. Since this gene is expressed abundantly in the colon where short-chain fatty acids are generated by bacterial fermentation, we tested the hypothesis that it codes for a Na(+)-coupled transporter for these fatty acids. The coding region of SLC5A8 mRNA was amplified from human intestine and expressed heterologously in Xenopus laevis oocytes. Transport function was monitored by uptake of radiolabeled substrates and by substrate-induced currents under voltage-clamp conditions. Uptake of short-chain fatty acids (lactate, pyruvate, acetate, propionate, and butyrate) in oocytes expressing SLC5A8 was severalfold higher than in uninjected oocytes. Exposure of SLC5A8-expressing oocytes to these fatty acids induced inward currents under voltage-clamp conditions in a Na(+)-dependent manner. These currents were saturable and the substrate concentrations needed for half-maximal induction of the current were in the range of 0.08-2.5 mm. The substrate-induced currents decreased as the carbon chain length of the substrates increased. The Na(+)-activation kinetics indicated involvement of more than one Na(+) ion in the activation process. Direct measurements of substrate (propionate) and charge transfer showed that three positive charges are transferred into oocytes per substrate molecule. These studies establish the functional identity of SLC5A8 as a Na(+)-coupled transporter for short-chain fatty acids.     

Regulation of butyrate uptake in Caco-2 cells by phorbol 12-myristate 13-acetate

Butyrate and the other short-chain fatty acids (SCFAs) are the most abundant anions in the colonic lumen. Also, butyrate is the preferred energy source for colonocytes and has been shown to regulate colonic electrolyte and fluid absorption. Previous studies from our group have demonstrated that the HCO(3)(-)/SCFA(-) anion exchange process is one of the major mechanisms of butyrate transport across the purified human colonic apical membrane vesicles and the apical membrane of human colonic adenocarcinoma cell line Caco-2 and have suggested that it is mainly mediated via monocarboxylate transporter-1 (MCT-1) isoform. However, little is known regarding the regulation of SCFA transport by various hormones and signal transduction pathways. Therefore, the present studies were undertaken to examine whether hydrocortisone and phorbol 12-myristate 13-acetate (PMA) are involved in a possible regulation of the butyrate/anion exchange process in Caco-2 cells. The butyrate/anion exchange process was assessed by measuring a pH-driven [(14)C]butyrate uptake in Caco-2 cells. Our results demonstrated that 24-h incubation with PMA (1 microM) significantly increased [(14)C]butyrate uptake compared with incubation with 4alphaPMA (inactive form). In contrast, incubation with hydrocortisone had no significant effect on butyrate uptake in Caco-2 cells compared with vehicle (ethanol) alone. Induction of butyrate uptake by PMA appeared to be via an increase in the maximum velocity (V(max)) of the transport process with no significant changes in the K(m) of the transporter for butyrate. Parallel to the increase in the V(max) of [(14)C]butyrate uptake, the MCT-1 protein level was also increased in response to PMA incubation. Our studies demonstrated that the butyrate/anion exchange was increased in response to PMA treatment along with the induction in the level of MCT-1 expression in Caco-2 cells.     

Co-expression of a mammalian accessory trafficking protein enables functional expression of the rat MCT1 monocarboxylate transporter in Saccharomyces cerevisiae

We have developed a new heterologous expression system for monocarboxylate transporters. The system is based on a Saccharomyces cerevisiae pyk1 mae1 jen1 triple-deletion strain that is auxotrophic for pyruvate and deficient in monocarboxylate uptake. Growth of the yeast cells on ethanol medium supplemented with pyruvate or lactate was dependent on the expression of a suitable monocarboxylate transporter. We have used the system to characterize the functional significance of interactions between the rat MCT1 transporter and its ancillary protein CD147. CD147 was shown to improve trafficking of MCT1 to the plasma membrane and its uptake activity. Our results demonstrate a new strategy for the production of properly folded and correctly targeted membrane proteins in a microbial expression system by co-expression of appropriate accessory proteins.     

Functional expression of the rabbit intestinal Na+/L-proline cotransporter (IMINO system) in Xenopus laevis oocytes

Proline absorption across small intestine takes place mainly through a Na+-dependent cotransporter localized at the brush border membrane of the enterocyte named IMINO system. It transports L-proline and 4-OH-proline but not L-alanine, neither cationic nor anionic amino acids. The present work demonstrates the functional expression of this transporter in Xenopus laevis oocytes by mRNA microinjection and radiotracer uptake techniques. Poly (A)+-RNA was isolated from rabbit jejunal mucosa and injected into oocytes. Five days after the injection, results showed 1.5 fold stimulation of 50 microM 3H-proline uptake by the injected oocytes when compared to the non injected oocytes uptake. Poly (A)+-RNA was sized fractionated and fractions were injected again. Increase on Na+-dependent L-proline uptake was obtained with a mRNA fraction between 2,4 and 4,4 kb, which was used to construct a cDNA library. The library was sequentially divided and cRNAs injected into oocytes in order to screen for an increment on the signal. A subdivision containing around 2,000 colonies was found to augment L-proline uptake 25 fold over the non injected oocytes uptake. This cRNA pool was used to further characterize the transporter. Results showed that in the absence of Na+ there was no L-proline uptake, 2 mM 4-OH-L-proline completely inhibited 50 microM proline uptake and there was no 50 microM alanine uptake. In summary, these results demonstrate the expression of the rabbit small intestine IMINO transporter in Xenopus laevis oocytes and support the next steps in the isolation of the clone.     

Voltage dependence of H+ buffering mediated by sodium bicarbonate cotransport expressed in Xenopus oocytes

The electrogenic sodium bicarbonate cotransporter (NBCe1) is expressed in many epithelial cells and, in the brain, in glial cells. Little is known about the physiological significance of the NBCe1 for proton homeostasis and for other acid/base-coupled transporters in these cells. We have measured the voltage-dependent transport activity of an NBC from human kidney, type hkNBCe1, expressed in oocytes of the frog Xenopus laevis, by recording membrane current and the changes in intracellular pH and sodium at different membrane potentials between -20 and -100 mV. The apparent intracellular buffer capacity was increased and became dependent upon membrane voltage when the NBCe1 was expressed; the measured buffer capacity increased by up to 7 mm/10 mV of membrane depolarization. Lactate transport by the electroneutral monocarboxylate transporter became enhanced and dependent upon membrane potential, when the monocarboxylate transporter (isoform 1) was co-expressed with NBCe1 in oocytes. Our results indicate that the electrogenic NBCe1 renders the cell membrane potential an effective regulator of intracellular H(+) buffering and acid/base-coupled metabolite transport.