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The cytosolic sulfotransferases (SULTs) catalyze the sulfate conjugation of nucleophilic substrates, and the cofactor for sulfonation, 3′-phosphoadenosine-5′-phosphosulfate (PAPS), is biosynthesized from sulfate and ATP. The phenotype of male knockout mice for the NaS1 sodium sulfate cotransporter includes hyposulfatemia and increased hepatic expression of mouse cytoplasmic sulfotransferase Sult2a and Sult3a1. Here we report that in 8-week-old female NaS1-null mice, hepatic Sult2a1 mRNA levels were ∼51-fold higher than they were in a wild-type liver but expression of no other Sult was affected. To address whether hyposulfatemia-inducible Sult2a1 expression might be due to reduced PAPS levels, we stably knocked down PAPS synthases 1 and 2 in HepG2 cells (shPAPSS1/2 cells). When a reporter plasmid containing at least 233 nucleotides (nt) of Sult2a1 5′-flanking sequence was transfected into shPAPSS1/2 cells, reporter activity was significantly increased relative to the activity that was seen for reporters containing 179 or fewer nucleotides. Mutation of an IR0 (inverted repeat of AGGTCA, with 0 intervening bases) nuclear receptor motif at nt −191 to 180 significantly attenuated the PAPSS1/2 knockdown-mediated increase. PAPSS1/2 knockdown significantly activated farnesoid X receptor (FXR), retinoid-related orphan receptor, and pregnane X receptor responsive reporters, and treatment with the FXR agonist GW4064 [3-(2,6-dichlorophenyl)-4-(3′-carboxy-2-chlorostilben-4-yl)oxymethyl-5-isopropylisoxazole] increased Sult2a1 promoter activity when the IR0 was intact. Transfection of shPAPSS1/2 cells with FXR small interfering RNA (siRNA) significantly reduced the Sult2a1 promoter activity. The impact of PAPSS1/2 knockdown on Sult2a1 promoter activity was recapitulated by knocking down endogenous SULT2A1 expression in HepG2 cells. We propose that hyposulfatemia leads to hepatic PAPS depletion, which causes loss of SULT2A1 activity and results in accumulation of nonsulfated bile acids and FXR activation.

OBJECTIVE: Sulfate (SO(4)(2-)) is an abundant component of intestinal mucins and its content is decreased in certain gastrointestinal diseases, including inflammatory bowel disease. In this study, the hyposulfataemic NaS1 sulfate transporter null (Nas1(-/-)) mice were used to investigate the physiological consequences of disturbed sulfate homeostasis on (1) intestinal sulfomucin content and mRNA expression; (2) intestinal permeability and proliferation; (3) dextran sulfate sodium (DSS)-induced colitis; and (4) intestinal barrier function against the bacterial pathogen, Campylobacter jejuni. METHODS: Intestinal sulfomucins and sialomucins were detected by high iron diamine staining, permeability was assessed by fluorescein isothiocyanate (FITC)-dextran uptake, and proliferation was assessed by 5-bromodeoxyuridine (BrdU) incorporation. Nas1(-/-) and wild-type (Nas1(+/+)) mice received DSS in drinking water, and intestinal damage was assessed by histological, clinical and haematological measurements. Mice were orally inoculated with C jejuni, and intestinal and systemic infection was assessed. Ileal mRNA expression profiles of Nas1(-/-) and Nas1(+/+) mice were determined by cDNA microarrays and validated by quantitative real-time PCR. RESULTS: Nas1(-/-) mice exhibited reduced intestinal sulfomucin content, enhanced intestinal permeability and DSS-induced colitis, and developed systemic infections when challenged orally with C jejuni. The transcriptional profile of 41 genes was altered in Nas1(-/-) mice, with the most upregulated gene being pancreatic lipase-related protein 2 and the most downregulated gene being carbonic anhydrase 1 (Car1). CONCLUSION: Sulfate homeostasis is essential for maintaining a normal intestinal metabolic state, and hyposulfataemia leads to reduced intestinal sulfomucin content, enhanced susceptibility to toxin-induced colitis and impaired intestinal barrier to bacterial infection.

This protocol describes the method of expression cloning of heterologous proteins using Xenopus laevis oocytes and the functional characterization of membrane proteins using radiotracer assays. It can be used to isolate proteins for which sequence data is unavailable and to characterize the functions of proteins. A cDNA library is generated, that is, translated into proteins in Xenopus oocytes, and the function of these proteins is assessed by a radiotracer assay. Their large size, high degree of expression and ease of handling makes Xenopus oocytes an optimal tool for the expression and characterization of protein function when compared with traditional expression systems, such as Escherichia coli, yeast or eukaryotic cell lines. The expected results of this technique include the following: functional identification of novel proteins; molecular (kinetic) characterization of protein function; and determination of functionally relevant residues and domains of membrane proteins, including transporters, ion channels and receptors. The identification of novel proteins can take several months, whereas functional characterization in Xenopus oocytes can take 1 week.

PDZ-binding motifs are found in the C-terminal tails of numerous integral membrane proteins where they mediate specific protein-protein interactions by binding to PDZ-containing proteins. Conventional yeast two-hybrid screens have been used to probe protein-protein interactions of these soluble C termini. However, to date no in vivo technology has been available to study interactions between the full-length integral membrane proteins and their cognate PDZ-interacting partners. We previously developed a split-ubiquitin membrane yeast two-hybrid (MYTH) system to test interactions between such integral membrane proteins by using a transcriptional output based on cleavage of a transcription factor from the C terminus of membrane-inserted baits. Here we modified MYTH to permit detection of C-terminal PDZ domain interactions by redirecting the transcription factor moiety from the C to the N terminus of a given integral membrane protein thus liberating their native C termini. We successfully applied this “MYTH 2.0” system to five different mammalian full-length renal transporters and identified novel PDZ domain-containing partners of the phosphate (NaPi-IIa) and sulfate (NaS1) transporters that would have otherwise not been detectable. Furthermore this assay was applied to locate the PDZ-binding domain on the NaS1 protein. We showed that the PDZ-binding domain for PDZK1 on NaS1 is upstream of its C terminus, whereas the two interacting proteins, NHERF-1 and NHERF-2, bind at a location closer to the N terminus of NaS1. Moreover NHERF-1 and NHERF-2 increased functional sulfate uptake in Xenopus oocytes when co-expressed with NaS1. Finally we used MYTH 2.0 to demonstrate that the NaPi-IIa transporter homodimerizes via protein-protein interactions within the lipid bilayer. In summary, our study establishes the MYTH 2.0 system as a novel tool for interactive proteomics studies of membrane protein complexes.

An HPLC-MS/MS method has been developed for the selective quantitative analysis of paracetamol and its two major metabolites. The use of tandem MS enabled the detection and quantitation of metabolites in small sample sizes with high sensitivity and selectivity. Isocratic elution using acetonitrile and water containing formic acid combined with electrospray-tandem MS enabled the separation and accurate quantitation of each analyte and the internal standard 3-acetamidophenol. The on-column limits of detection for paracetamol, paracetamol sulfate, and paracetamol glucuronide were 2.4, 1.2, and 1.2 pmol, respectively. The method was applied to quantitate paracetamol and its metabolites in mouse urine. It is highly specific, sensitive, and easily adaptable to measure these analytes in biological fluids of other animals.

Sulfate (SO ) is an abundant nutrient in the blood and is essential for normal growth and development. SO is conjugated (sulfonated) to many compounds in the body, including glycosaminoglycans, steroid hormones and bile acids. Sulfonation also plays an important role in the metabolism of xenobiotics and certain drugs, such as acetaminophen. SO enters and exits cells: via plasma membrane SO transporters. To date, ten human SO transporters belonging to the Solute Linked Carrier 13 (SLC13) or 26 (SLC26) gene families have been identified. Clinical interest has focused on two SLC26 SO transporters which are associated with recessive human disorders: SLC26A2 is defective in four different chondrodysplasias (MED, DTD, AO2 and ACGIB) and SLC26A3 is associated with congenital chloride diarrhea (CLD). Ongoing studies arc focused on the physiological significance of the other eight SO transporters (SLCI3Al, A4, and SLC26Al, A6, A7, A8, A9, A11), yet to be characterized in human diseases. Our Slcl3al null (Nas) mouse studies, have revealed several pathophysiological features associated with this transporter: hyposulfatemia, hypersulfaturia, reduced growth, seizures, behavioural abnormalities, hypercholesterolemia, fatty liver, reduced fertility and enhanced acetaminophen-induced hepatotoxicity. These findings can be relevant to single nucleotide polymorphisms (SNPs) in human SLC13Al, which lead to changes in SO transport function. This paper summarises non-synonymous SNPs (nsSNPs) found in the sulflite transporters SLC13A1, A4, and SLC26A1, A6, A7, A8, A9, A11, with a special focus on SLC13A1, which we have identified to be responsible for maintaining blood SO concentrations.

NaSi-1 encodes a Na(+)-sulfate cotransporter expressed on the apical membrane of renal proximal tubular cells, which is responsible for body sulfate homeostasis. Limited information is available on NaSi-1 protein structure and the mechanisms controlling its apical membrane sorting. The aims of this study were to biochemically determine the quaternary structure of the rat NaSi-1 protein and to characterize its expression in renal epithelial cell lines. Hexahistidyl-tagged NaSi-1 (NaSi-1-His) proteins expressed in Xenopus oocytes, appeared as two bands of about 60 and 75 kDa. PNGase F treatment shifted both bands to 57 kDa while endoglycosidase H treatment led to a downward shift of the lower molecular mass band only. Mutagenesis of a putative N-glycosylation site (N591S) produced a single band that was not shifted by endoglycosidase H or PNGase F, confirming a single glycosylation site at residue 591. Blue native-PAGE and cross-linking experiments revealed dimeric complexes, suggesting the native form of NaSi-1 to be a dimer. Transient transfection of EGFP/NaSi-1 in renal epithelial cells (OK, LLC-PK1 and MDCK) demonstrated apical membrane sorting, which was insensitive to tunicamycin. Transfection of the EGFP/NaSi-1 N591S glycosylation mutant also showed apical expression, suggesting N591 is not essential for apical sorting. Treatment with cholesterol depleting compounds did not disrupt apical sorting, but brefeldin A led to misrouting to the basolateral membrane, suggesting that NaSi-1 sorting is through the ER to Golgi pathway. Our data demonstrates that NaSi-1 forms a dimeric protein which is glycosylated at N591, whose sorting to the apical membrane in renal epithelial cells is brefeldin A-sensitive and independent of lipid rafts or glycosylation.

Sulfate (SO(4)2-) is an important anion regulating many metabolic and cellular processes. Maintenance of SO4(2-) homeostasis occurs in the renal proximal tubule via membrane transport proteins. Two SO(4)2- transporters that have been characterized and implicated in regulating serum SO4(2-) levels are: NaSi-1, a Na+-SO(4)2- cotransporter located at the brush border membrane and Sat-1, a SO4(2-)-anion exchanger located on the basolateral membranes of proximal tubular cells. Unlike Sat-1, for which very few studies have looked at regulation of its expression, NaSi-1 has been shown to be regulated by various hormones and dietary conditions in vivo. To study this further, NaSi-1 (SLC13A1) and Sat-1 (SLC26A1) gene structures were determined and recent studies have characterized their respective gene promoters. This review presents the current understanding of the transcriptional regulation of NaSi-1 and Sat-1, and describes possible pathogenetic implications which arise as a consequence of altered SO(4)2- homeostasis.

Sulfate plays an essential role in human growth and development. Here, we characterized the functional properties of the human Na(+)-sulfate cotransporter (hNaS2), determined its tissue distribution, and identified its gene (SLC13A4) structure. Expression of hNaS2 protein in Xenopus oocytes led to a Na(+)-dependent transport of sulfate that was inhibited by thiosulfate, phosphate, molybdate, selenate and tungstate, but not by oxalate, citrate, succinate, phenol red or DIDS. Transport kinetics of hNaS2 determined a K(m) for sulfate of 0.38mM, suggestive of a high affinity sulfate transporter. Na(+) kinetics determined a Hill coefficient of n=1.6+/-0.6, suggesting a Na:SO(4)(2-) stoichiometry of 2:1. hNaS2 mRNA was highly expressed in placenta and testis, with intermediate levels in brain and lower levels found in the heart, thymus, and liver. The SLC13A4 gene contains 16 exons, spanning over 47kb in length. Its 5'-flanking region contains CAAT- and GC-box motifs, and a number of putative transcription factor binding sites, including GATA-1, AP-1, and AP-2 consensus sequences. This is the first study to characterize hNaS2 transport kinetics, define its tissue distribution, and resolve its gene (SLC13A4) structure and 5' flanking region.

Inorganic sulfate is required for numerous functions in mammalian physiology, and its circulating levels are proposed to be maintained by the Na + -SO 4 2- cotransporter, (NaS i -1). To determine the role of NaS i -1 in sulfate homeostasis and the physiological consequences in its absence, we have generated a mouse lacking a functional NaS i -1 gene, Nas1 . Serum sulfate concentration was reduced by >75% in Nas1 -/- mice when compared with Nas1 +/+ mice. Nas1 -/- mice exhibit increased urinary sulfate excretion, reduced renal and intestinal Na + -SO 4 2- cotransport, and a general growth retardation. Nas1 -/- mouse body weight was reduced by >20% when compared with Nas1 +/+ and Nas1 +/- littermates at 2 weeks of age and remained so throughout adulthood. Nas1 -/- females had a lowered fertility, with a 60% reduction in litter size. Spontaneous clonic seizures were observed in Nas1 -/- mice from 8 months of age. These data demonstrate NaS i -1 is essential for maintaining sulfate homeostasis, and its expression is necessary for a wide range of physiological functions.

Both stimulation of purinergic receptors by ATP and activation of the cystic fibrosis transmembrane conductance regulator (CFTR) inhibit amiloride-sensitive Na+ transport and activate Cl- secretion. These changes in ion transport may well affect cell volume. We therefore examined whether cell shrinkage or cell swelling do affect amiloride-sensitive Na+ transport in epithelial tissues or Xenopus oocytes and whether osmotic stress interferes with regulation of Na+ transport by ATP or CFTR. Stimulation of purinergic receptors by ATP/UTP or activation of CFTR by IBMX and forskolin inhibited amiloride-sensitive transport in mouse trachea and colon, respectively, by a mechanism that was Cl- dependent. When exposed to a hypertonic but not hypotonic bath solution, amiloride-sensitive Na+ transport was inhibited in mouse trachea and colon, independent of the extracellular Cl- concentration. Both inhibition of Na+ transport by hypertonic bath solution and ATP were additive. When coexpressed in Xenopus oocytes, activation of CFTR by IBMX and forskolin inhibited the epithelial Na+ channel (ENaC) in a Cl- dependent fashion. However, both hypertonic and hypotonic bath solutions showed only minor effects on amiloride-sensitive conductance, independent of the bath Cl- concentration. Moreover, CFTR-induced inhibition of ENaC could be detected in oocytes even after exposure to hypertonic or hypotonic bath solutions. We conclude that amiloride-sensitive Na+ absorption in mouse airways and colon is inhibited by cell shrinkage by a mechanism that does not interfere with purinergic and CFTR-mediated inhibition of ENaC.

Contains fulltext : 142011.pdf (Publisher’s version ) (Open Access) In renal collecting ducts, a vasopressin-induced cAMP increase results in the phosphorylation of aquaporin-2 (AQP2) water channels at Ser-256 and its redistribution from intracellular vesicles to the apical membrane. Hormones that activate protein kinase C (PKC) proteins counteract this process. To determine the role of the putative kinase sites in the trafficking and hormonal regulation of human AQP2, three putative casein kinase II (Ser-148, Ser-229, Thr-244), one PKC (Ser-231), and one protein kinase A (Ser-256) site were altered to mimic a constitutively non-phosphorylated/phosphorylated state and were expressed in Madin-Darby canine kidney cells. Except for Ser-256 mutants, seven correctly folded AQP2 kinase mutants trafficked as wild-type AQP2 to the apical membrane via forskolin-sensitive intracellular vesicles. With or without forskolin, AQP2-Ser-256A was localized in intracellular vesicles, whereas AQP2-S256D was localized in the apical membrane. Phorbol 12-myristate 13-acetate-induced PKC activation following forskolin treatment resulted in vesicular distribution of all AQP2 kinase mutants, while all were still phosphorylated at Ser-256. Our data indicate that in collecting duct cells, AQP2 trafficking to vasopressin-sensitive vesicles is phosphorylation-independent, that phosphorylation of Ser-256 is necessary and sufficient for expression of AQP2 in the apical membrane, and that PMA-induced PKC-mediated endocytosis of AQP2 is independent of the AQP2 phosphorylation state.

Inorganic sulfate is one of the most abundant anions in mammalian plasma and is essential for proper cell growth and development, as well as detoxification and activation of many biological compounds. To date, little is understood how physiological levels of sulfate are maintained in the body. Our studies, and of others, have identified the NAS(i)-1 protein to be a functional sulfate transporter in the kidney and intestine, and due to this localization, constitutes a strong candidate gene for maintaining body sulfate homeostasis. Several factors, including hormones and metabolic conditions, have been shown to alter NAS(i)-1 mRNA and protein levels in vivo. In this study, we describe the transcriptional regulation of NaSi-1, with a focus on the mouse NaSi-1 gene (Nas1) that was recently cloned in our laboratory. Vitamin D (1,25-(OH)(2)D-3) and thyroid hormone (T-3) led to an increase in Nas1 promoter activity in OK cells. Mutational analysis of the Nas1 promoter resulted in identification of a direct repeat 6-type vitamin-D-responsive element (DR6 VDRE) at -525 to -508 and an imperfect inverted repeat 0-type T-3 responsive element (IRO T3RE) at -426 to -425 which conferred 1,25-(OH)(2)D-3 and T-3 responsiveness respectively. These findings suggest for vitamin D and thyroid hormone regulation of NaSi-1, may provide important clues to the physiological control of sulfate homeostasis.

Sulfate (SO42−) plays an important role in mammalian growth and development. In this study, hyposulfatemic NaS1 null (Nas1−/−) mice were used to investigate the consequences of perturbed SO42− homeostasis on longevity. Median life spans were increased (by ≈ 25%) in male and female Nas1−/− mice when compared with Nas1+/+ mice. At 1 yr of age, serum SO42− levels remained low in Nas1−/− mice (≈ 0.16 mM) when compared to Nas1+/+ mice (≈ 0.96 mM). RT-PCR revealed increased hepatic mRNA levels of Sirt1 (by ≈60%), Cat (by ≈48%), Hdac3 (by ≈22%), Trp53 and Cd55 (by ≈36%) in Nas1−/− mice, genes linked to ageing. Histological analyses of livers from 2 yr old mice revealed neoplasms in > 50% of Nas1+/+ mice but not in Nas1−/− mice. This is the first study to report increased lifespan, decreased hepatic tumours and increased hepatic expression of genes linked to ageing in hyposulfatemic Nas1−/− mice, implicating a potential role of SO42− in mammalian longevity and cancer.

D-Glucose entry into erythrocytes from adult grey-headed flying fox fruit bats (Pteropus poliocephalus) was rapid and showed saturation at high substrate concentrations. Kinetic parameters were estimated from the concentration dependence of initial rates of zero-trans D-glucose entry at 5.5 degrees C as Michaelis constant (K-m) 1.64 +/- 0.56 mM, and maximal velocity (V-max) 1162 +/- 152 mu mol . l cell water(-1).min(-1). D-Glucose entry was inhibited by cytochalasin B; mass law analysis of D-glucose-displaceable cytochalasin B binding gave Values of K-d 37.1 +/- 5.0 nM and B-max 361.2 +/- 9.1 pmol/mg membrane protein. Entry of 2-deoxy-D-glucose, and 3-O-methyl-D-glucose, into P. poliocephalus red cells was rapid, entry of D-fructose was very slow. Glucose transporter polypeptides were identified on immunoblots as a band M-r 47 000-54 000 and their identity confirmed by D-glucose-sensitive photolabeling of membranes with [H-3]-cytochalasin B. Peptide-N-glycanase F digestion of both human and bat erythrocyte membranes generated GLUT-1-derived bands M-r 37 000. Trypsin digestion of human and fruit bat erythrocyte membranes generated fragmentation patterns consistent with similar GLUT-1 polypeptide structures in both species. Erythrocytes from adult Australian ghost bats (Macroderma gigas), a carnivorous microchiropteran bat, also expressed high levels of GLUT-1. (C) 2000 Elsevier Science Inc. All rights reserved.

NaSi-1 is a Na+-sulfate cotransporter expressed on the apical membrane of the renal proximal tubule and plays an important role in sulfate reabsorption. To understand the molecular mechanisms that mediate the regulation of NaSi-1, we have isolated and characterized the mouse NaSi-1 cDNA (mNaSi-1), gene (Nas1), and promoter region and determinedNas1 chromosomal localization. The mNaSi-1 cDNA encodes a protein of 594 amino acids with 13 putative transmembrane segments, inducing high affinity Na+-dependent transport of sulfate in Xenopus oocytes. Three different mNaSi-1 transcripts derived from alternative polyadenylation and splicing were identified in kidney and intestine. The Nas1 gene is a single copy gene comprising 15 exons spread over 75 kilobase pairs that maps to mouse chromosome 6. Transcription initiation occurs from a single site, 29 base pairs downstream to a TATA box-like sequence. The promoter is AT-rich (61%), contains a number of well characterizedcis-acting elements, and can drive basal transcriptional activity in opossum kidney cells but not in COS-1 or NIH3T3 cells. We demonstrated that 1,25-dihydroxyvitamin D3 stimulated the transcriptional activity of the Nas1 promoter in transiently transfected opossum kidney cells. This study represents the first characterization of the genomic organization of a Na+-sulfate cotransporter gene. It also provides the basis for a detailed analysis of Nas1 gene regulation and the tools required for assessing Nas1 role in sulfate homeostasis using targeted gene manipulation in mice.

Sulfate is an essential anion involved in many biosynthetic and pharmacological reactions. Sulfate is an important constituent of myelin membranes in the brain; however, very little is known as to how sulfate enters brain cells. In this study, our aim was to determine whether the mammalian brain possesses a sulfate transporter. Injection of rat brain poly A(+) RNA into Xenopus oocytes led to an induction of Na(+)-independent sulfate transport, which was inhibited by oxalate, probenecid, phenol red, thiosulfate and DIDS. Hybrid depletion using sat-1 antisense oligodeoxyribonucleotides led to a complete inhibition of brain mRNA-induced sulfate transport in Xenopus oocytes, suggesting the presence of a functional sat-1 transcript in the brain. By RT-PCR, sat-1 mRNA was detected throughout the rat brain and in situ hybridisation showed highest sat-1 expression in the hippocampus and cerebellum. This is the first study to identify and characterise a functional mammalian brain sulfate transporter.

Recently, we cloned a cDNA (NaSi-1) localized to rat renal proximal tubules and encoding the brush-border membrane (BBM) Na gradient-dependent inorganic sulfate (Si) transport protein (Na-Sicotransporter). The purpose of the present study was to determine the effect of metabolic acidosis (MA) on Na-Sicotransport activity and NaSi-1 protein and mRNA expression. In rats with MA for 24 h (but not 6 or 12 h), there was a significant increase in the fractional excretion of Si, which was associated with a 2.4-fold decrease in BBM Na-Sicotransport activity. The decrease in Na-Sicotransport correlated with a 2.8-fold decrease in BBM NaSi-1 protein abundance and a 2.2-fold decrease in cortical NaSi-1 mRNA abundance. The inhibitory effect of MA on BBM Na-Sicotransport was also sustained in rats with chronic (10 days) MA. In addition, in Xenopus laevis oocytes injected with mRNA from kidney cortex, there was a significant reduction in the induced Na-Sicotransport in rats with MA compared with control rats, suggesting that MA causes a decrease in the abundance of functional mRNA encoding the NaSi-1 cotransporter. These findings indicate that MA reduces Sireabsorption by causing decreases in BBM Na-Sicotransport activity and that decreases in the expression of NaSi-1 protein and mRNA abundance, at least in part, play an important role in the inhibition of Na-Sicotransport activity during MA.

Activated macrophages require L-arginine uptake to sustain NO synthesis. Several transport systems could mediate this L-arginine influx. Using competition analysis and gene-expression studies, amino acid transport system y+ was identified as the major carrier responsible for this activity. To identify which of the four known y+ transport-system genes is involved in macrophage-induced L-arginine uptake, we used a hybrid-depletion study in Xenopus oocytes. Cationic amino acid transporter (CAT) 2 antisense oligodeoxyribonucleotides abolished the activated-macrophage-mRNA-induced L-arginine transport. Together with expression studies documenting that CAT2 mRNA and protein levels are elevated with increased L-arginine uptake, our data demonstrate that CAT2 mediates the L-arginine transport that is required for the raised NO production in activated J774 macrophages.

Dietary inorganic sulfate (Si) intake is an important factor in the regulation of renal proximal tubular sodium-dependent Si transport (Na/Si cotransport). The purpose of the present study was to determine whether modulation of Na/Si cotransport activity by dietary Si is mediated through regulation of the renal expression of the recently cloned NaSi-1 protein located in the apical brush border membrane (BBM) of the proximal tubule. It was found that rats fed a high Si diet had a marked increase in the renal excretion of Si and a concomitant decrease in BBM Na/Si cotransport activity when compared with rats on a control Si diet. The 43% decrease in BBM Na/Si cotransport activity was associated with a 33% decrease in BBM NaSi-1 protein abundance, as determined by Western blotting, and a 2.7-fold decrease in cortical NaSi-1 mRNA abundance, as determined by Northern blotting. Furthermore, cortical mRNA from rats fed a high Si diet when injected into Xenopus laevis oocytes led to a 2.2-fold decrease in Na/Si cotransport activity compared with mRNA isolated from control Si diet rats. This study indicates that adaptation to a high Si diet is accompanied by a decrease in renal cortical NaSi-1 mRNA abundance, which results in reduced expression of the NaSi-1 protein at the level of the proximal tubular BBM.

The Na + /dicarboxylate cotransporter, NaDC-1, and the Na + /sulfate cotransporter, NaSi-1, share 43% sequence identity, but they exhibit no overlap in substrate specificity. A functional chimera, SiDC-4, was prepared from NaDC-1 and NaSi-1 by homologous recombination and expressed in Xenopus oocytes. SiDC-4 contains putative transmembrane domains 1–4 of NaSi-1 (amino acids 1–139) and putative transmembrane domains 5–11 of NaDC-1 (amino acids 141–593). SiDC-4 retains the substrate specificity of NaDC-1, which suggests that the substrate recognition domain is found in the carboxy-terminal portion of the protein, past amino acid 141. However, residues that affect substrate affinity and inhibition by furosemide and flufenamate are found in the amino terminal third of the protein. The cation binding properties of SiDC-4, including a stimulation of transport by lithium, differed from both parental transporters, suggesting that cation binding is determined by interactions between the amino- and carboxy-terminal portions of the protein. We conclude that the substrate recognition site of NaDC-1 and NaSi-1 is found in the carboxy-terminal portion of the protein, past amino acid 141, but residues in the amino terminus can affect substrate affinity, inhibitor sensitivity, and cation selectivity.

Heavy metal intoxication leads to a number of reabsorptive and secretory defects in renal transport systems. We have studied the effects of several heavy metals on the expression of the renal Na-Sicotransporter NaSi-1. NaSi-1 cRNA was injected into Xenopusoocytes, and Na-Sicotransport activity was measured in the presence of mercury, lead, cadmium, or chromium. Mercury strongly inhibited NaSi-1 transport irreversibly by reducing both maximal velocity ( Vmax) and Michaelis constant ( Km) for inorganic sulfate (Si). Lead inhibited NaSi-1 transport reversibly by decreasing Vmaxbut not Kmfor Si. Cadmium showed weak reversible inhibition of NaSi-1 transport by decreasing only NaSi-1 Vmax. Chromium strongly inhibited NaSi-1 cotransport reversibly by reducing Kmfor Siby sevenfold, most probably by binding to the Sisite, due to the strong structural similarity between the C[Formula: see text] and[Formula: see text] substrates. In conclusion, this study presents an initial report demonstrating heavy metals inhibit renal brush border Na-Sicotransport via the NaSi-1 protein through various mechanisms and that this blockade may be responsible for sulfaturia following heavy metal intoxication.

In a previous report we documented an increased Na+-dependent transport of inorganic phosphate (P i ) in Xenopus laevis oocytes injected with mRNA isolated from rabbit duodenum (Yagci et al., Pfluegers Arch. 422:211–216, 1992; ref 24). In the present study we have used expression cloning in oocytes to search for the cDNA/mRNA involved in this effect. The identified cDNA (provisionally named PiUS; for P i -uptake stimulator) lead to a 3-4-fold stimulation of Na+-dependent P i -uptake (10ng cRNA injected, 3–5 days of expression). Na+-independent uptake of P i was also affected but transport of sulphate and l-arginine (in the presence or absence of sodium) remained unchanged. The apparent K m -values for the induced Na+-dependent uptake were 0.26 ± 0.04 mm for P i and 14.8 ± 3.0 mm for Na+. The 1796 bp cDNA codes for a protein of 425 amino acids. Hydropathy analysis suggests a lack of transmembrane segments. In vitro translation resulted in a protein of 60 kDa and provided no evidence of glycosylation. In Northern blots a mRNA of ∼2 kb was recognized in various tissues including different intestinal segments, kidney cortex, kidney medulla, liver and heart. Homology searches showed no similarity to proteins involved in membrane transport and its control. In conclusion, we have cloned from a rabbit small intestinal cDNA library a novel cDNA encoding a protein stimulating P i -uptake into Xenopus laevis oocytes, but which is not a P i -transporter itself.

Sulfate is essential for normal physiology. The kidney plays a major role in sulfate homeostasis. Sulfate is freely filtered and strongly reabsorbed in the proximal tubule. The apical membrane Na(+)-sulfate cotransporter NaS1 (SLC13A1) mediates sulfate (re)absorption across renal proximal tubule and small intestinal epithelia. NaS1 encodes a 595-amino acid (≈ 66 kDa) protein with 13 putative transmembrane domains. Its substrate preferences are sodium and sulfate, thiosulfate, and selenate, and its activity is inhibited by molybdate, selenate, tungstate, thiosulfate, succinate, and citrate. NaS1 is primarily expressed in the kidney (proximal tubule) and intestine (duodenum to colon). NaS1 expression is down-regulated in the renal cortex by high sulfate diet, hypothyroidism, vitamin D depletion, glucocorticoids, hypokalemia, metabolic acidosis, and NSAIDs and up-regulated by low sulfate diet, thyroid hormone, vitamin D supplementation, growth hormone, chronic renal failure, and during post-natal growth. Disruption of murine NaS1 gene leads to hyposulfatemia and hypersulfaturia, as well as changes in metabolism, growth, fecundity, behavior, gut physiology, and liver detoxification. This suggests that NaS1 is an important sulfate transporter and its disruption leads to perturbed sulfate homeostasis, which contributes to numerous pathophysiological conditions.

A complementary deoxyribonucleic acid (cDNA) corresponding to a murine renal cortical Na/phosphate-(Na/Pi-) cotransporter was isolated and its transport properties characterized by electrophysiological techniques after expression in Xenopus laevis oocytes. A Na-dependent inward movement of positive charges (“short-circuit current”) was observed upon superfusion with Pi (and with arsenate). Increasing the Na concentration led to a sigmoidal elevation in Pi-induced short-circuit current; the apparent Michaelis constant, K m, (around 40 mM Na) was increased by lowering the pH of the superfusate but was not influenced by altering the Pi concentration. Increasing the Pi (and arsenate) concentration led to a hyperbolic elevation in Na-dependent short-circuit current (apparent Km for Pi at 100 mM Na was around 0.1 mM; apparent Km for arsenate was around 1 mM); lowering the Na concentration decreased the apparent affinity for Pi. The Pi-induced short-circuit current was lower at more acidic pH values (at pH 6.3 it was about 50% of the value at pH 7.8); this pH dependence was similar if the Pi concentration was calculated in total, or if distinction was made between its mono- and divalent forms. Thus, the pH dependence of Na-dependent Pi transport (total Pi) may not be related primarily to a pH-dependent alteration in the availability of divalent Pi, but includes also a competitive interaction of Na with protons. The effect of Pi and Na concentration on the apparent Km values for Na or Pi, respectively, provides evidence for an ordered interaction of “cosubstrate” (Na first) and “substrate” (Pi or arsenate second).

Heavy metal mediated inhibition of rBAT-induced amino acid transport. rBAT, a protein which is located in the brush border membranes of intestine and renal proximal tubule cells, was recently shown to induce electrogenic countertransport of neutral and dibasic amino acids after its expression in Xenopus oocytes [1]. Here, we studied the effects of heavy metals on rBAT induced amino acid transport in Xenopus oocytes to clarify a possible involvement of rBAT in heavy metal-induced aminoaciduria. The heavy metals Hg 2+ and Pb 2+ inhibited rBAT-induced amino acid transport with a different profile of action. The Pb 2+ mediated inhibition occurred rapidly upon superfusion and was readily reversible upon washout. The maximal inhibition caused by Pb 2+ was about 50% of the amino acid-induced currents at an apparent affinity (K m ) of about 10 µM. In contrast, the Hg 2+ -mediated inhibition occurred rather slowly, depending on its concentration, and was not reversible during washout with control solution. However, the Hg 2+ -mediated amino acid transport inhibition could be reversed with Hg 2+ chelating agents and reducing compounds. Other oxidative agents, such as the membrane permeable 2,2′-Dithio-bis(5-Nitropyridine) (DTNP), but not the membrane impermeable 5,5′-Dithio-bis (2-Nitrobenzoic acid) (DTNB), mimicked the effect of Hg 2+ , and their effect could similarly be reversed with 2,3-Dihydroxybutane-l,4-dithiol (DTE). In conclusion, Pb 2+ and Hg 2+ inhibit rBAT-induced amino acid transport in a noncompetitive, allosteric fashion. Blockade of rBAT-induced amino acid transport may be involved in aminoaciduria following mercury or lead intoxication.

The SLC13 gene family is comprised of five sequence related proteins that are found in animals, plants, yeast and bacteria. Proteins encoded by the SLC13 genes are divided into the following two groups of transporters with distinct anion specificities: the Na(+)-sulfate (NaS) cotransporters and the Na(+)-carboxylate (NaC) cotransporters. Members of this gene family (in ascending order) are: SLC13A1 (NaS1), SLC13A2 (NaC1), SLC13A3 (NaC3), SLC13A4 (NaS2) and SLC13A5 (NaC2). SLC13 proteins encode plasma membrane polypeptides with 8-13 putative transmembrane domains, and are expressed in a variety of tissues. They are all Na(+)-coupled symporters with strong cation preference for Na(+), and insensitive to the stilbene 4, 4'-diisothiocyanatostilbene-2, 2'-disulphonic acid (DIDS). Their Na(+):anion coupling ratio is 3:1, indicative of electrogenic properties. They have a substrate preference for divalent anions, which include tetra-oxyanions for the NaS cotransporters or Krebs cycle intermediates (including mono-, di- and tricarboxylates) for the NaC cotransporters. This review will describe the molecular and cellular mechanisms underlying the biochemical, physiological and structural properties of the SLC13 gene family.

The SLC13 family comprises five genes (SLC13A1, SLC13A2, SLC13A3, SLC13A4, and SLC13A5) encoding structurally related multi-spanning transporters (8-13 transmembrane domains) with orthologues found in prokaryotes and eukaryotes. Mammalian SLC13 members mediate the electrogenic Na(+)-coupled anion cotransport at the plasma membrane of epithelial cells (mainly kidney, small intestine, placenta and liver) or cells of the central nervous system. While the two SLC13 cotransporters NaS1 (SLC13A1) and NaS2 (SLC13A4) transport anions such sulfate, selenate and thiosulfate, the three other SLC13 members, NaDC1 (SLC13A2), NaCT (SLC13A5) and NaDC3 (SLC13A3), transport di- and tri-carboxylate Krebs cycle intermediates such as succinate, citrate and α-ketoglutarate. All these transporters play a variety of physiological and pathophysiological roles in the different organs. Thus, the purpose of this review is to summarize the roles of SLC13 members in human physiology and pathophysiology and what the therapeutic perspectives are. We have also described the most recent advances on the structure, expression, function and regulation of SLC13 transporters.

Mice deficient for the apical membrane oxalate transporter SLC26A6 develop hyperoxalemia, hyperoxaluria, and calcium oxalate stones due to a defect in intestinal oxalate secretion. However, the nature of the basolateral membrane oxalate transport process that operates in series with SLC26A6 to mediate active oxalate secretion in the intestine remains unknown. Sulfate anion transporter-1 (Sat1 or SLC26A1) is a basolateral membrane anion exchanger that mediates intestinal oxalate transport. Moreover, Sat1-deficient mice also have a phenotype of hyperoxalemia, hyperoxaluria, and calcium oxalate stones. We, therefore, tested the role of Sat1 in mouse duodenum, a tissue with Sat1 expression and SLC26A6-dependent oxalate secretion. Although the active secretory flux of oxalate across mouse duodenum was strongly inhibited (>90%) by addition of the disulfonic stilbene DIDS to the basolateral solution, secretion was unaffected by changes in medium concentrations of sulfate and bicarbonate, key substrates for Sat1-mediated anion exchange. Inhibition of intracellular bicarbonate production by acetazolamide and complete removal of bicarbonate from the buffer also produced no change in oxalate secretion. Finally, active oxalate secretion was not reduced in Sat1-null mice. We conclude that a DIDS-sensitive basolateral transporter is involved in mediating oxalate secretion across mouse duodenum, but Sat1 itself is dispensable for this process.

Anion transporters NaS1 (SLC13A1) and Sat1 (SLC26A1) mediate sulfate (re)absorption across renal proximal tubule and small intestinal epithelia, thereby regulating blood sulfate levels. Disruption of murine NaS1 and Sat1 genes leads to hyposulfatemia and hypersulfaturia. Sat1-null mice also exhibit hyperoxalemia, hyperoxaluria, and calcium oxalate urolithiasis. This review will highlight the current pathophysiological features of NaS1- and Sat1-null mice resulting from alterations in circulating sulfate and oxalate anion levels.

We have isolated a cDNA (ileal NaSi-1) from rat small intestine by homology screening with a cDNA (renal NaSi-1) encoding rat kidney cortex Na(+)-SO4(2-) cotransport. Ileal NaSi-1 cRNA specifically stimulates Na(+)-dependent SO4(2-) uptake in a time- and dose-dependent manner in Xenopus laevis oocytes, with kinetic parameters almost identical to those of the renal NaSi-1. Ileal NaSi-1 cDNA contains 2722 base pairs (bp), almost 500 bp more than the renal NaSi-1 cDNA; however, it encodes a protein of 595 amino acids identical to the renal NaSi-1 protein. Northern blot analysis shows strong signals in rat lower small intestine and kidney cortex (2.9 x 10(3) and 2.3 x 10(3) bases), with the ileal NaSi-1 corresponding to the longer transcript. We conclude that we have identified a rat ileal cDNA that encodes a membrane protein most likely involved in brush-border Na(+)-SO4(2-) cotransport. It differs to the renal NaSi-1 only in the length of the 3' untranslated region, suggesting that the major difference lies in the differential use of polyadenylation signals.

Renal proximal tubular sulfate reabsorption is mediated by brush border membrane Na+/sulfate-cotransport and basolateral Na(+)-independent sulfate transport. Injection of rat kidney cortex mRNA into Xenopus laevis oocytes induced Na(+)-dependent as well as Na(+)-independent sulfate transport. The inhibition pattern of Na(+)-dependent uptake coincided with that known for the brush border membrane; the inhibition pattern of Na(+)-independent uptake suggested that this activity could be related to the basolateral cell surface. By Northern blot hybridization of size-fractionated mRNA, we provide evidence that the Na(+)-dependent uptake is induced by an mRNA species related to a recently cloned cDNA encoding rat renal cortex Na+/SO4 cotransport (NaSi-1; Markovich, D., Forgo, J., Stange, G., Biber, J., and Murer, H. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 8073-8077); the Na(+)-independent sulfate transport activity seems to be related to an mRNA species encoding a rat liver Na(+)-independent sulfate transporter (Bissig, M., Hagenbuch, B., Stieger, B., Koller, T., and Meier, P. J. (1994) J. Biol. Chem. 269, 3017-3021). Hybrid depletion experiments using antisense oligonucleotides provided further evidence for the association of the expressed transport activities to NaSi-1 and sat-1, respectively.

This review will briefly summarize current knowledge on the renal anion transporters sodium-sulfate cotransporter-1 (NaS1; Slc13a1) and sulfate-anion transporter-1 (Sat1; Slc26a1). NaS1 and Sat1 mediate renal proximal tubular sulfate reabsorption and thereby regulate blood sulfate levels. Sat1 also mediates renal oxalate transport and controls blood oxalate levels. Targeted disruption of murine NaS1 and Sat1 leads to hyposulfatemia and hypersulfaturia. Sat1 null mice also exhibit hyperoxalemia, hyperoxaluria, and calcium oxalate urolithiasis. NaS1 and Sat1 null mice also have other phenotypes that result due to changes in blood sulfate and oxalate levels. Experimental data indicate that NaS1 is essential for maintaining sulfate homeostasis, whereas Sat1 controls both sulfate and oxalate homeostasis in vivo.

Sulfate is important for growth and development, and is supplied from mother to fetus throughout pregnancy. We used NaS1 sulfate transporter null (Nas1(-/-)) mice to investigate the role of NaS1 in maintaining sulfate homeostasis during pregnancy and to determine the physiological consequences of maternal hyposulfataemia on fetal, placental and postnatal growth. We show that maternal serum (≤0.5 mM), fetal serum (0.1 mM) and amniotic fluid (≤0.5 mM) sulfate levels were significantly lower in pregnant Nas1(-/-) mice when compared with maternal serum (≍2.0 mM), fetal serum (≍1.5 mM) and amniotic fluid (≍1.7 mM) sulfate levels in pregnant Nas1(+/+) mice. After 12 days of pregnancy, fetal reabsorptions led to markedly reduced (by ≥50%) fetal numbers in Nas1(-/-) mice. Placental labyrinth and spongiotrophoblast layers were increased (by ≍140%) in pregnant Nas1(-/-) mice when compared to pregnant Nas1(+/+) mice. Birth weights of progeny from female Nas1(-/-) mice were increased (by ≍7%) when compared to progeny of Nas1(+/+) mice. These findings show that NaS1 is essential to maintain high maternal and fetal sulfate levels, which is important for maintaining pregnancy, placental development and normal birth weight.

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Urolithiasis, a condition in which stones are present in the urinary system, including the kidneys and bladder, is a poorly understood yet common disorder worldwide that leads to significant health care costs, morbidity, and work loss. Acetaminophen-induced liver damage is a major cause of death in patients with acute liver failure. Kidney and urinary stones and liver toxicity are disturbances linked to alterations in oxalate and sulfate homeostasis, respectively. The sulfate anion transporter-1 (Sat1; also known as Slc26a1) mediates epithelial transport of oxalate and sulfate, and its localization in the kidney, liver, and intestine suggests that it may play a role in oxalate and sulfate homeostasis. To determine the physiological roles of Sat1, we created Sat1-/- mice by gene disruption. These mice exhibited hyperoxaluria with hyperoxalemia, nephrocalcinosis, and calcium oxalate stones in their renal tubules and bladder. Sat1-/- mice also displayed hypersulfaturia, hyposulfatemia, and enhanced acetaminophen-induced liver toxicity. These data suggest that Sat1 regulates both oxalate and sulfate homeostasis and may be critical to the development of calcium oxalate urolithiasis and hepatotoxicity.

A kidney cortex cDNA clone (rBAT) has recently been isolated, which upon in vitro transcription and capping complementary RNA (cRNA) and injection into Xenopus laevis oocytes induces a system b0,(+)-like amino acid transport activity. This cDNA encodes a type II membrane glycoprotein that shows significant homology to another type II membrane glycoprotein, the heavy chain of the human and mouse 4F2 surface antigen (4F2hc). Here we demonstrate that injection of human 4F2hc cRNA into oocytes results in the activation of a cation-preferring amino acid transport system that appears to be identical to the y(+)-like transport already present in the oocyte. This is based on the following results: (i) Injection of in vitro transcripts from 4F2hc cDNA (4F2hc cRNA) into oocytes stimulates up to 10-fold the sodium-independent uptake of L-arginine and up to 4.1-fold the sodium-dependent uptake of L-leucine. In contrast, 4F2hc cRNA does not increase the basal sodium-independent uptake of L-leucine. (ii) Basal and 4F2hc cRNA-stimulated sodium-independent uptake of L-arginine is completely inhibited by L-leucine in the presence of sodium. Similarly, the basal and 4F2hc cRNA-stimulated sodium-dependent uptake of L-leucine is entirely inhibited by L-arginine. (iii) The stimulation of sodium-independent uptake of L-arginine and the stimulation of sodium-dependent uptake of L-leucine induced by injection of 4F2hc cRNA are both completely inhibited by dibasic L amino acids and to a lesser extent by D-ornithine. (iv) Both basal and 4F2hc cRNA-stimulated sodium-independent uptake of L-arginine show two additional characteristics of the system y+ transport activity: inhibition of L-arginine uptake by L-homoserine only in the presence of sodium and an increase in the inhibition exerted by L-histidine as the extracellular pH decreased. Our results allow us to propose that an additional family of type II membrane glycoproteins (composed by rBAT and 4F2hc) is involved in amino acid transport, either as specific activators or as components of amino acid transport systems.

Sulfate plays an important role in maintaining normal structure and function of tissues, and its content is decreased in certain cancers including lung carcinoma. In this study, we investigated tumor growth in a mouse model of hyposulfatemia (Nas1(-/-)) and compared it to wild-type (Nas1(+/+)) mice. Lung epithelial tumor cells (TC-1 cell line) were injected subcutaneously into male Nas1(-/-) and Nas1(+/+) mice on a mixed 129Sv and C57BL/6 genetic background. Tumor sections were stained with anti-glycosaminoglycan antibodies to assess the distribution of proteoglycans and Gomori's trichrome to detect collagen. After 14 days, tumor weights were markedly increased (by approximately 12-fold) in Nas1(-/-) mice when compared with Nas1(+/+) mice. Histological analyses of tumors revealed increased (by approximately 2.4-fold) vessel content, as well as markedly reduced collagen and immunoreactivity against glycosaminoglycan structural epitopes in the tumors from Nas1(-/-) mice. No significant differences were found for the growth of cultured TC-1 cells supplemented with Nas1(-/-) or Nas1(+/+) serum, as determined by (3)H-thymidine incorporation, implying that the cell culture conditions may not reflect the in vivo situation of enhanced tumor growth. This study has revealed increased tumor growth and an altered extracellular tumor matrix in hyposulfatemic Nas1(-/-) mice. These findings highlight the importance of blood sulfate levels as a possible modulator of tumor growth, and could lead to future cancer studies in humans with altered sulfate homeostasis.

This chapter will summarize the most recent data available for sulfate and phosphate transport in mammalian renal and gastrointestinal systems. Dietary derived sulfate and phosphate are absorbed in the intestines and their circulating levels are controlled by renal tubular mechanisms. Such processes are facilitated by sulfate and phosphate transporters that exist in the epithelial cells of the kidneys and intestines. Sulfate transporters belong to two gene families, the Na+-coupled sulfate transporters (SLC13) and the sulfate anion exchangers (SLC26). Phosphate transporters belong to three gene families, Type I (SLC17), Type II (SLC34), and Type III (SLC20) of which all members are Na+-coupled phosphate transporters. Tissue distribution for these transporters is diverse, with some being restricted to either renal or intestinal tissues and with others showing more broad tissue distribution. Various modes of regulation affect the expression of these genes and the proteins they encode in their respective tissues. Lessons from knock-out mice reveal the physiological (and pathophysiological) roles these proteins play in the body and their contributions to sulfate and phosphate homeostasis.