Sulfate Efflux Research Articles

Sulfation disposition of liquiritigenin in SULT1A3 overexpressing HEK293 cells: The role of breast cancer resistance protein (BCRP) and multidrug resistance-associated protein 4 (MRP4) in sulfate efflux of liquiritigenin

This study aimed to investigate the cellular disposition of liquiritigenin via the sulfonation pathway and the role of efflux transporters in liquiritigenin sulfate excretion. The sulfonation disposition of liquiritigenin was investigated using SULT1A3 overexpressed HEK293 cells (HEK-SULT1A3 cells). Liquiritigenin generated one mono-sulfate metabolite (7-O-sulfate) in HEK-SULT1A3 cell lysate. And the sulfonation followed the Michaelis-Menten kinetic (Vmax = 0.84 nmol/min/mg and Km = 7.12 μM). Expectedly, recombinant SULT1A3 (hSULT1A3) showed a highly similar kinetic profile with cell lysate. Furthermore, 7-O-sulfate was rapidly generated and excreted in HEK-SULT1A3 cells. Ko143 (a BCRP-selective inhibitor) at 20 μM significantly decreased the excretion rate of liquiritigenin sulfate (>42.5%, p < 0.001). Moreover, the pan-MRPs inhibitor MK-571 at 20 μM essentially abolished the liquiritigenin sulfate effluxion, resulting in the marked reduction of excretion rate (>97.4%, p < 0.001). Furthermore, knockdown of BCRP led to moderate reduction in sulfate excretion (15.9%–16.9%, p < 0.05). Silencing of MRP4 caused significant decreased in sulfate excretion (20.2%–32.5%, p < 0.01). In conclusion, one sulfate metabolite was generated from liquiritigenin in HEK-SULT1A3 cells. BCRP and MRP4 should be the key factors for the cellular excretion of liquiritigenin sulfate.

Loss of the anion exchanger DRA (Slc26a3), or PAT1 (Slc26a6), alters sulfate transport by the distal ileum and overall sulfate homeostasis.

The ileum is considered the primary site of inorganic sulfate ([Formula: see text]) absorption. In the present study, we explored the contributions of the apical chloride/bicarbonate (Cl-/[Formula: see text]) exchangers downregulated in adenoma (DRA; Slc26a3), and putative anion transporter 1 (PAT1; Slc26a6), to the underlying transport mechanism. Transepithelial 35[Formula: see text] and 36Cl- fluxes were determined across isolated, short-circuited segments of the distal ileum from wild-type (WT), DRA-knockout (KO), and PAT1-KO mice, together with measurements of urine and plasma sulfate. The WT distal ileum supported net sulfate absorption [197.37 ± 13.61 (SE) nmol·cm-2·h-1], but neither DRA nor PAT1 directly contributed to the unidirectional mucosal-to-serosal flux ([Formula: see text]), which was sensitive to serosal (but not mucosal) DIDS, dependent on Cl-, and regulated by cAMP. However, the absence of DRA significantly enhanced net sulfate absorption by one-third via a simultaneous rise in [Formula: see text] and a 30% reduction to the secretory serosal-to-mucosal flux ([Formula: see text]). We propose that DRA, together with PAT1, contributes to [Formula: see text] by mediating sulfate efflux across the apical membrane. Associated with increased ileal sulfate absorption in vitro, plasma sulfate was 61% greater, and urinary sulfate excretion (USO4) 2.2-fold higher, in DRA-KO mice compared with WT controls, whereas USO4 was increased 1.8-fold in PAT1-KO mice. These alterations to sulfate homeostasis could not be accounted for by any changes to renal sulfate handling suggesting that the source of this additional sulfate was intestinal. In summary, we characterized transepithelial sulfate fluxes across the mouse distal ileum demonstrating that DRA (and to a lesser extent, PAT1) secretes sulfate with significant implications for intestinal sulfate absorption and overall homeostasis.NEW & NOTEWORTHY Sulfate is an essential anion that is actively absorbed from the small intestine involving members of the Slc26 gene family. Here, we show that the main intestinal chloride transporter Slc26a3, known as downregulated in adenoma (DRA), also handles sulfate and contributes to its secretion into the lumen. In the absence of functional DRA (as in the disease congenital chloride diarrhea), net intestinal sulfate absorption was significantly enhanced resulting in substantial alterations to overall sulfate homeostasis.

Chronic exposure to short-chain fatty acids modulates transport and metabolism of microbiome-derived phenolics in human intestinal cells

Dietary fiber-derived short-chain fatty acids (SCFA) and phenolics produced by the gut microbiome have multiple effects on health. We have tested the hypothesis that long-term exposure to physiological concentrations of SCFA can affect the transport and metabolism of (poly)phenols by the intestinal epithelium using the Caco-2 cell model. Metabolites and conjugates of hesperetin (HT) and ferulic acid (FA), gut-derived from dietary hesperidin and chlorogenic acid, respectively, were quantified by LC-MS with authentic standards following transport across differentiated cell monolayers. Changes in metabolite levels were correlated with effects on mRNA and protein expression of key enzymes and transporters. Propionate and butyrate increased both FA transport and rate of appearance of FA glucuronide apically and basolaterally, linked to an induction of MCT1. Propionate was the only SCFA that augmented the rate of formation of basolateral FA sulfate conjugates, possibly via basolateral transporter up-regulation. In addition, propionate enhanced the formation of HT glucuronide conjugates and increased HT sulfate efflux toward the basolateral compartment. Acetate treatment amplified transepithelial transport of FA in the apical to basolateral direction, associated with lower levels of MCT1 protein expression. Metabolism and transport of both HT and FA were curtailed by the organic acid lactate owing to a reduction of UGT1A1 protein levels. Our data indicate a direct interaction between microbiota-derived metabolites of (poly)phenols and SCFA through modulation of transporters and conjugating enzymes and increase our understanding of how dietary fiber, via the microbiome, may affect and enhance uptake of bioactive molecules.

Chloride and sulfate salinity differently affect biomass, mineral nutrient composition and expression of sulfate transport and assimilation genes in Brassica rapa

Background and aimsIt remains uncertain whether a higher toxicity of either NaCl or Na2SO4 in plants is due to an altered toxicity of sodium or a different toxicity of the anions. The aim of this study was to determine the contributions of sodium and the two anions to the different toxicities of chloride and sulfate salinity. The effects of the different salts on physiological parameters, mineral nutrient composition and expression of genes of sulfate transport and assimilation were studied.MethodsSeedlings of Brassica rapa L. have been exposed to NaCl, Na2SO4, KCl and K2SO4 to assess the potential synergistic effect of the anions with the toxic cation sodium, as well as their separate toxicities if accompanied by the non-toxic cation potassium. Biomass production, stomatal resistance and Fv/fm were measured to determine differences in ionic and osmotic stress caused by the salts. Anion content (HPLC), mineral nutrient composition (ICP-AES) and gene expression of sulfate transporters and sulfur assimilatory enzymes (real-time qPCR) were analyzed.ResultsNa2SO4 impeded growth to a higher extent than NaCl and was the only salt to decrease Fv/fm. K2SO4 reduced plant growth more than NaCl. Analysis of mineral nutrient contents of plant tissue revealed that differences in sodium accumulation could not explain the increased toxicity of sulfate over chloride salts. Shoot contents of calcium, manganese and phosphorus were decreased more strongly by exposure to Na2SO4 than by NaCl. The expression levels of genes encoding proteins for sulfate transport and assimilation were differently affected by the different salts. While gene expression of primary sulfate uptake at roots was down-regulated upon exposure to sulfate salts, presumably to prevent an excessive uptake, genes encoding for the vacuolar sulfate transporter Sultr4;1 were upregulated. Gene expression of ATP sulfurylase was hardly affected by salinity in shoot and roots, the transcript level of 5′-adenylylsulfate reductase (APR) was decreased upon exposure to sulfate salts in roots. Sulfite reductase was decreased in the shoot by all salts similarly and remained unaffected in roots.ConclusionsThe higher toxicity of Na2SO4 over NaCl in B. rapa seemed to be due to an increased toxicity of sulfate over chloride, as indicated by the higher toxicity of K2SO4 over KCl. Thus, toxicity of sodium was not promoted by sulfate. The observed stronger negative effect on the tissue contents of calcium, manganese and phosphorus could contribute to the increased toxicity of sulfate over chloride. The upregulation of Sultr4;1 and 4;2 under sulfate salinity might lead to a detrimental efflux of stored sulfate from the vacuole into the cytosol and the chloroplasts. It remains unclear why expression of Sultr4;1 and 4;2 was upregulated. A possible explanation is a control of the gene expression of these transporters by the sulfate gradient across the tonoplast.

Interplay of metabolizing enzymes and transporter of xenobiotics

1. Xenobiotics are metabolized and eliminated through the coordinated interplay of their metabolizing enzymes and transporters. However, these two activities in vitro are measured separately, with the addition of ATP as a pre-requisite.2. We propose a human renal cell-line model which integrates the sulfate and glutathione conjugation of xenobiotics with the efflux of their respective conjugates. Sulfation and glutathionylation represent two major Phase II detoxification of xenobiotics in man. The reactions are catalyzed, respectively, by phenolsulfotransferase and glutathione-S-transferase followed by extrusion of their respective conjugates.3. Using Ko-143, a specific inhibitor of breast cancer resistance protein (BCRP), an ATP-binding cassette (ABC) transporter, we identified this transporter to be responsible for the efflux of p-cresol sulfate, harmol sulfate and the glutathione conjugate of 1-chloro-2,4-dinitrobenzene.4. The conjugation-cum-efflux was inhibited by oligomycin and uncouplers, which highlights the role of cellular mitochondria in providing ATP for the biosynthesis of their conjugating agents, 3′-phosphoadenosine-5′-phosphosulfate (PAPS) and reduced glutathione as well as for the transport function of BCRP.5. The human 786-O renal cell-line provides a “3-in-1” system linking ATP biosynthesis to metabolism of xenobiotics and their ultimate transport and elimination by BCRP; this integrated system was not apparent in other human cell-lines examined.

Legume adaptation to sulfur deficiency revealed by comparing nutrient allocation and seed traits in Medicago truncatula

Reductions in sulfur dioxide emissions and the use of sulfur-free mineral fertilizers are decreasing soil sulfur levels and threaten the adequate fertilization of most crops. To provide knowledge regarding legume adaptation to sulfur restriction, we subjected Medicago truncatula, a model legume species, to sulfur deficiency at various developmental stages, and compared the yield, nutrient allocation and seed traits. This comparative analysis revealed that sulfur deficiency at the mid-vegetative stage decreased yield and altered the allocation of nitrogen and carbon to seeds, leading to reduced levels of major oligosaccharides in mature seeds, whose germination was dramatically affected. In contrast, during the reproductive period, sulfur deficiency had little influence on yield and nutrient allocation, but the seeds germinated slowly and were characterized by low levels of a biotinylated protein, a putative indicator of germination vigor that has not been previously related to sulfur nutrition. Significantly, plants deprived of sulfur at an intermediary stage (flowering) adapted well by remobilizing nutrients from source organs to seeds, ensuring adequate quantities of carbon and nitrogen in seeds. This efficient remobilization of photosynthates may be explained by vacuolar sulfate efflux to maintain leaf metabolism throughout reproductive growth, as suggested by transcript and metabolite profiling. The seeds from these plants, deprived of sulfur at the floral transition, contained normal levels of major oligosaccharides but their germination was delayed, consistent with low levels of sucrose and the glycolytic enzymes required to restart seed metabolism during imbibition. Overall, our findings provide an integrative view of the legume response to sulfur deficiency.

Glyoxylate is a substrate of the sulfate-oxalate exchanger, sat-1, and increases its expression in HepG2 cells

Hyperoxaluria is a major problem causing nephrolithiasis. Little is known about the regulation of oxalate transport from the liver, the main organ for oxalate synthesis, into the circulation. Since the sulfate anion transporter-1(sat-1) is present in the sinusoidal membrane of hepatocytes and translocates oxalate, its impact on increased oxalate synthesis was studied. Sat-1 expressing oocytes were used for cis-inhibition, trans-stimulation, and efflux experiments with labelled sulfate and oxalate to demonstrate the interactions of oxalate, glyoxylate, and glycolate with sat-1. HepG2 cells were incubated with oxalate and its precursors (glycine, hydroxyproline, glyoxylate, and glycolate). Changes in endogenous sat-1 mRNA-expression were examined using real-time PCR. After incubation of HepG2 cells in glyoxylate, sat-1 protein-expression was analysed by Western blotting, and sulfate uptake into HepG2 cells was measured. RT-PCR was used to screen for mRNA of other transporters. While oxalate and glyoxylate inhibited sulfate uptake, glycolate did not. Sulfate and oxalate uptake were trans-stimulated by glyoxylate but not by glycolate. Glyoxylate enhanced sulfate efflux. Glyoxylate was the only oxalate precursor stimulating sat-1 mRNA-expression. After incubation of HepG2 cells in glyoxylate, both sat-1 protein-expression and sulfate uptake into the cells increased. mRNA-expression of other transporters in HepG2 cells was not affected by glyoxylate treatment. The oxalate precursor glyoxylate was identified as a substrate of sat-1. Upregulated expression of sat-1 mRNA and of a functional sat-1 protein indicates that glyoxylate may be responsible for the elevated oxalate release from hepatocytes observed in hyperoxaluria.

Purification of functional human [formula omitted] exchanger, AE1, over-expressed in Saccharomyces cerevisiae

There is no high-resolution structure for the membrane domain of the human erythrocyte anion exchanger, AE1 (Band 3). In this report, we have developed an expression and purification strategy for AE1 to be used in crystallization trials. Saccharomyces cerevisiae strain BJ5457 was transformed with an expression vector encoding the AE1 membrane domain (AE1MD, amino acids 388–911), fused C-terminally to an epitope tag, corresponding to the nine C-terminal amino acids of rhodopsin. The fusion protein, AE1MD-Rho, was expressed at a concentration of 0.3 mg/l of culture. Confocal immunofluorescence microscopy and sucrose gradient ultracentrifugation revealed that AE1MD-Rho did not process to the plasma membrane of S. cerevisiae, but was retained in an intracellular membrane fraction. Treatment with the endoglycosidase, PNGase F, showed that AE1MD-Rho is not N-glycosylated. AE1MD-Rho solubilized from yeast membranes, with Fos-choline detergent, was purified to 93% homogeneity in a single-step, using a 1D4 antibody affinity resin, in amounts up to 2.5 mg from 18 l of culture. The ability of purified AE1MD-Rho to transport sulfate was examined in reconstituted vesicles. The rate of sulfate efflux mediated by vesicles reconstituted with AE1MD-Rho was indistinguishable from vesicles with purified erythrocyte-source AE1. Using this purification strategy, sufficient amounts of functional, homogeneous AE1MD-Rho can be purified to enable crystallization trials.

Sultr4;1 mutant seeds of Arabidopsis have an enhanced sulphate content and modified proteome suggesting metabolic adaptations to altered sulphate compartmentalization

BackgroundSulphur is an essential macronutrient needed for the synthesis of many cellular components. Sulphur containing amino acids and stress response-related compounds, such as glutathione, are derived from reduction of root-absorbed sulphate. Sulphate distribution in cell compartments necessitates specific transport systems. The low-affinity sulphate transporters SULTR4;1 and SULTR4;2 have been localized to the vacuolar membrane, where they may facilitate sulphate efflux from the vacuole.ResultsIn the present study, we demonstrated that the Sultr4;1 gene is expressed in developing Arabidopsis seeds to a level over 10-fold higher than the Sultr4;2 gene. A characterization of dry mature seeds from a Sultr4;1 T-DNA mutant revealed a higher sulphate content, implying a function for this transporter in developing seeds. A fine dissection of the Sultr4;1 seed proteome identified 29 spots whose abundance varied compared to wild-type. Specific metabolic features characteristic of an adaptive response were revealed, such as an up-accumulation of various proteins involved in sugar metabolism and in detoxification processes.ConclusionsThis study revealed a role for SULTR4;1 in determining sulphate content of mature Arabidopsis seeds. Moreover, the adaptive response of sultr4;1 mutant seeds as revealed by proteomics suggests a function of SULTR4;1 in redox homeostasis, a mechanism that has to be tightly controlled during development of orthodox seeds.

Remobilization of leaf S compounds and senescence in response to restricted sulphate supply during the vegetative stage of oilseed rape are affected by mineral N availability.

The impact of sulphur limitation on the remobilization of endogenous S compounds during the rosette stage of oilseed rape, and the interactions with N availability on these processes, were examined using a long-term 34SO42− labelling method combined with a study of leaf senescence progression (using SAG12/Cab as a molecular indicator) and gene expression of the transporters, BnSultr4;1 and BnSultr4;2, involved in vacuolar sulphate efflux. After 51 d on hydroponic culture at 0.3 mM 34SO42− (1 atom% excess), the labelling was stopped and plants were subject for 28 d to High S-High N (HS-HN, control), Low S-High N (LS-HN) or Low S-Low N (LS-LN) conditions. Compared with the control, LS-HN plants showed delayed leaf senescence and, whilst the shoot growth and the foliar soluble protein amounts were not affected, S, 34S, and SO42− amounts in the old leaves declined rapidly and were associated with the up-regulation of BnSultr4;1. In LS-LN plants, shoot growth was reduced, leaf senescence was accelerated, and the rapid S mobilization in old leaves was accompanied by decreased 34S and SO42−, higher protein mobilization, and up-regulation of BnSultr4;2, but without any change of expression of BnSultr4;1. The data suggest that to sustain the S demand for growth under S restriction (i) vacuolar SO42− is specifically remobilized in LS-HN conditions without any acceleration of leaf senescence, (ii) SO42− mobilization is related to an up-regulation of BnSultr4;1 and/or BnSultr4;2 expression, and (iii) the relationship between sulphate mobilization and up-regulation of expression of BnSultr4 genes is specifically dependent on the N availability.

Molecular characterization of Slc26a3 and Slc26a6 anion transporters in guinea pig pancreatic duct

HCO3 in pancreatic juice arises from the pancreatic duct cells. Secretin stimulates HCO3 secretion via a mechanism that involves activation of the adenylate cyclase pathway, activation of both basolateral K channels and the apical CFTR Cl channel, and stimulation of an apical Slc26mediated Cl/HCO3 exchanger (1). The Slc26 anion exchangers, Slc26a3 and Slc26a6, have both been localized to the apical membrane of human pancreatic duct and mouse pancreatic duct although their functional roles in pancreatic HCO3 secretion remain uncertain. The high rates of pancreatic bicarbonate secretion shared by guinea pig (gp) and human, in contrast to the low rates in mouse, prompted us to study the functional characteristics of gp Slc26 anion transporters. Furthermore, recent data from isolated guinea pig pancreatic duct experiments demonstrated that during stimulated HCO3 secretion an apical Slc26a6-like Cl/HCO3 exchanger is functionally coupled with CFTR (2). This study prompted us to clone cDNAs encoding Slc26a3 and Slc26a6 polypeptides from the gp pancreas and to compare their functional characteristics in Xenopus oocytes with those of their mouse and human orthologs (3-5), and with those of luminal Cl/HCO3 exchange measured in isolated gp pancreatic interlobular ducts (2). Whereas gp Slc26a6 mediated Cl/Cl exchange and bidirectional exchange of Cl for oxalate and HCO3, gp Slc26a6 mediated only sulfate influx but not sulfate efflux in preliminary experiments. Gp Slc26a3 mediated robust Cl/Cl exchange and minimal oxalate efflux in exchange for Cl, but did not mediate detectable influx of oxalate or sulfate. Measurement of intracellular pH by BCECF ratio fluorimetry in HEK cells transfected with Slc26a6 or in Slc26a3-expressing Xenopus oocytes during removal and restoration of Clo in the presence of HCO3 indicated that both gp Slc26a6 and Slc26a3 function as Cl/HCO3 exchangers. These substrate specificity experiments reveal that whereas Slc26a3 functions predominantly as a Cl/HCO3 exchanger, Slc26a6 can exchange a wider range of substrates, functioning as a Cl/HCO3 exchanger or a Cl/anion exchanger. This finding allows for the possibility that Slc26a6 may have alternative roles in transepithelial solute transport in the pancreatic duct. The apparent affinity of gp Slc26a6 for extracellular Cl (K1/2) was 2 mM during Cli/Clo exchange and 30 mM during C-oxalatei/Clo exchange. These experiments reveal that binding and/ or translocation rates of extracellular Cl with respect to gp slc26a6 is/are dependent on the internal transanion substrate and show species-specificity (5). The high affinity for extracellular Clo suggests that even when duct luminal Cl is low, such as might occur with passage of pancreatic juice along the duct lumen, Slc26a6-mediated Cl/HCO3 exchange would still be active. Whereas gp Slc26a6-mediated isotopic flux was moderately sensitive to DNDS (500 μM), niflumic acid (100 μM) and DIDS (500 μM), gp Slc26a3 was minimally sensitive to inhibition by DIDS and DNDS but strongly sensitive to niflumic acid. These pharmacological data, combined with EXPANDED ABSTRACT

Enteric oxalate secretion is not directly mediated by the human CFTR chloride channel

The secretion of the oxalate anion by intestinal epithelia is a functionally significant component of oxalate homeostasis and hence a relevant factor in the etiology and management of calcium oxalate urolithiasis. To test the hypothesis that human cystic fibrosis transmembrane conductance regulator (hCFTR) can directly mediate the efflux of the oxalate anion, we compared cAMP-stimulated 36Cl-, 14C-oxalate, and 35SO(4)2- efflux from Xenopus oocytes expressing hCFTR with water-injected control oocytes. hCFTR-expressing oocytes exhibited a large, reversible cAMP-dependent increase in whole cell conductance measured using a two-electrode voltage clamp and a 13-fold increase in rate of cAMP-stimulated 36Cl- efflux. In contrast, the rate constants of oxalate and sulfate efflux were low and unaffected by cAMP in either control or hCFTR-expressing oocytes. We conclude that the human CFTR gene product does not directly mediate oxalate efflux in secretory epithelia and hence is not directly involved in oxalate homeostasis in humans.

Mouse Ae1 E699Q mediates SO42−i/aniono exchange with [SO42−]i-dependent reversal of wild-type pHo sensitivity

The SLC4A1/AE1 gene encodes the electroneutral Cl(-)/HCO(3)(-) exchanger of erythrocytes and renal type A intercalated cells. AE1 mutations cause familial spherocytic and stomatocytic anemias, ovalocytosis, and distal renal tubular acidosis. The mutant mouse Ae1 polypeptide E699Q expressed in Xenopus oocytes cannot mediate Cl(-)/HCO(3)(-) exchange or (36)Cl(-) efflux but exhibits enhanced dual sulfate efflux mechanisms: electroneutral exchange of intracellular sulfate for extracellular sulfate (SO(4)(2-)(i)/SO(4)(2-)(o) exchange), and electrogenic exchange of intracellular sulfate for extracellular chloride (SO(4)(2-)(i)/Cl(-)(o) exchange). Whereas wild-type AE1 mediates 1:1 H(+)/SO(4)(2-) cotransport in exchange for either Cl(-) or for the H(+)/SO(4)(2-) ion pair, mutant Ae1 E699Q transports sulfate without cotransport of protons, similar to human erythrocyte AE1 in which the corresponding E681 carboxylate has been chemically converted to the alcohol (hAE1 E681OH). We now show that in contrast to the normal cis-stimulation by protons of wild-type AE1-mediated SO(4)(2-) transport, both SO(4)(2-)(i)/Cl(-)(o) exchange and SO(4)(2-)(i)/SO(4)(2-)(o) exchange mediated by mutant Ae1 E699Q are inhibited by acidic pH(o) and activated by alkaline pH(o). hAE1 E681OH displays a similarly altered pH(o) dependence of SO(4)(2-)(i)/Cl(-)(o) exchange. Elevated [SO(4)(2-)](i) increases the K(1/2) of Ae1 E699Q for both extracellular Cl(-) and SO(4)(2-), while reducing inhibition of both exchange mechanisms by acid pH(o). The E699Q mutation also leads to increased potency of self-inhibition by extracellular SO(4)(2-). Study of the Ae1 E699Q mutation has revealed the existence of a novel pH-regulatory site of the Ae1 polypeptide and should continue to provide valuable paths toward understanding substrate selectivity and self-inhibition in SLC4 anion transporters.

The impacts of future climate change and sulphur emission reductions on acidification recovery at Plastic Lake, Ontario

Abstract. Climate-induced drought events have a significant influence on sulphate export from forested catchments in central Ontario, subsequently delaying the recovery of surface waters from acidification. In the current study, a model chain that employed a statistical downscaling model, a hydrological model and two hydrochemical models was used to forecast the chemical recovery of Plastic Lake sub-catchment 1 (PC1) from acidification under proposed deposition reductions and the A2 emission scenario of the Intergovernmental Panel on Climate Change. Any predicted recovery in stream acid neutralising capacity and pH owing to deposition reductions were clearly offset by large acid effluxes from climate-induced drought events. By 2100, ANC is predicted to show large variations ranging between 10 and −30 μmolc L−1. Similarly, predicted pH in 2100 is lower (&gt;0.05 of a pH unit) than the value simulated for 2000 (pH 4.35). Despite emission reductions, the future scenario paints a bleak picture of reacidification at PC1 to levels commensurate with those of the late 1970s. The principal process behind this reacidification is the oxidation of previously stored (reduced) sulphur compounds in wetlands during periods of low-flow (or drought), with subsequent efflux of sulphate upon re-wetting. Simulated catchment runoff under the A2 emissions scenario predictes increased intensity and frequency of low-flow events from approximately 2030 onwards. The Integrated Catchments model for Carbon indicated that stream DOC concentrations at PC1 will also increase under the future climate scenario, with temperature being the principal driver. Despite the predicted (significant) increase in DOC, pH is not predicted to further decline (beyond the climate-induced oxidation scenario), instead pH shows greater variability throughout the simulation. As echoed by many recent studies, hydrochemical models and model frameworks need to incorporate the drivers and mechanisms (at appropriate time-scales) that affect the key biogeochemical processes to reliably predict the impacts of climate change.

Leaf Developmental Stage Affects Sulfate Depletion and Specific Sulfate Transporter Expression During Sulfur Deprivation in Brassica napus L.

BRASSICA NAPUS was grown under hydroponic conditions and responses to the removal of the external supply of sulfur (S) were analysed in roots and in leaves of different developmental age. The concentrations of sulfate and nitrate were greatest in the older leaves and least in younger leaves, whilst phosphate was greatest in roots and youngest leaves and least in old leaves. S-deprivation resulted in decreases in tissue sulfate concentrations at variable rates in the order: roots and young leaves > middle-aged leaves > oldest leaves. Phosphate concentrations were unaffected and nitrate concentrations were only depleted in the oldest leaves. Expression of representative members of the sulfate transporter gene family was assessed by Northern blotting in the respective tissues. Group 1 transporters (high affinity type) were induced in response to S-deprivation in all tissues except old leaves, where no expression was detected, and to the greatest extent in roots. Groups 2 and 5 (a BRASSICA Group 5 sulfate transporter is reported here, accession number: AJ311389) transporters showed either no or only a small induction by S-deprivation. Group 4 transporters (localised in the tonoplast membrane and thought to be involved in vacuolar sulfate efflux) were induced by S-deprivation with a complex pattern: 4;1 was expressed in root and mature leaves, was strongly induced by sulfur-deprivation in roots, and was also induced in the middle-aged leaves alone; 4;2 was only expressed under S-deprivation in parallel with the observed pattern of tissue sulfate concentrations. Expression patterns indicated that both differences in intracellular sulfate pools and localised aspects of the signal transduction pathway link tissue sulfate-status and sulfur-nutrition regulated gene expression.

Evaluation of the Role of Breast Cancer Resistance Protein (BCRP/ABCG2) and Multidrug Resistance-Associated Protein 4 (MRP4/ABCC4) in The Urinary Excretion of Sulfate and Glucuronide Metabolites of Edaravone (MCI-186; 3-Methyl-1-phenyl-2-pyrazolin-5-one)

Edaravone (MCI-186; 3-methyl-1-phenyl-2-pyrazolin-5-one), a free radical scavenger, is used for the treatment of acute cerebral infarction. Edaravone is mainly excreted into the urine after conjugation to glucuronide or sulfate. Previous studies have demonstrated that edaravone sulfate is a good substrate of human organic anion transporter (OAT) 1 (SLC22A6) and human OAT3 (SLC22A8). In this study, we examined the involvement of breast cancer resistance protein [BCRP (ABCG2)] and [multidrug resistance-associated protein 4 MRP4 (ABCC4)] in the luminal efflux in the kidney. Increased ATP-dependent uptake of edaravone sulfate but not edaravone glucuronide was observed in BCRP-expressing membrane vesicles compared with control vesicles (Km = 16.5 microM). In contrast, edaravone glucuronide, but not edaravone sulfate, exhibited greater ATP-dependent uptake in MRP4-expressing membrane vesicles than that in control vesicles (Km = 9.85 microM). Unlike taurocholate uptake, S-methylglutathione had no effect on the ATP-dependent uptake of edaravone glucuronide by MRP4. The functional importance of BCRP and MRP4 in the urinary excretion of edaravone sulfate and edaravone glucuronide, respectively, was investigated using Bcrp and Mrp4 knockout mice. The renal clearance with respect to the kidney concentration of edaravone sulfate was reduced significantly but not abolished in Bcrp knockout mice compared with wild-type mice (3.62 versus 4.85 ml/min/kg b.wt.). The renal clearance of edaravone glucuronide was lower in Mrp4 knockout mice than wild-type mice (2.01 versus 5.06 ml/min/kg BW). Our results suggest that Bcrp and Mrp4 are partly involved in the luminal efflux of edaravone sulfate and edaravone glucuronide, respectively.

Regulation of the Sodium/Sulfate Co-transporter by Farnesoid X Receptor α

Fxralpha is known to regulate a variety of metabolic processes, including bile acid, cholesterol, and carbohydrate metabolism. In this study, we show direct evidence that Fxralpha is a key player in maintaining sulfate homeostasis. We identified and characterized the sodium/sulfate co-transporter (NaS-1; Slc13a1) as an Fxralpha target gene expressed in the kidney and intestine. Electromobility shift assays, chromatin immunoprecipitation, and promoter reporter studies identified a single functional Fxralpha response element in the second intron of the mouse Slc13a1 gene. Treatment of wild-type mice with GW4064, a synthetic Fxralpha agonist, induced Slc13a1 mRNA in the intestine and kidney. Slc13a1 mRNA was also induced in the kidney and intestine of wild-type, but not Fxralpha-/- mice, after treatment with the hepatotoxin alpha-naphthylisothiocyanate, which is known to result in elevated blood bile acid levels. Finally, we observed a decrease in Slc13a1 mRNA in the kidney and intestine of Fxralpha-/- mice and a corresponding increase in urinary excretion of free sulfates as compared with wild-type mice. These results demonstrate that mouse Slc13a1 is a novel Fxralpha target gene expressed in the kidney and intestine and that in the absence of Fxralpha, mice waste sulfate into the urine. Thus, Fxralpha is necessary for normal sulfate homeostasis in vivo.

Transport and metabolism of MitoQ10, a mitochondria-targeted antioxidant, in Caco-2 cell monolayers

Mitoquinone (MitoQ(10) mesylate) is a mitochondria-targeted antioxidant formulated for oral administration in the treatment of neurodegenerative diseases. We have investigated the absorption and metabolism of MitoQ(10) in Caco-2 cell monolayers. The intracellular accumulation of MitoQ(10) was 18-41% of the total amount of MitoQ(10) added. Some of the intracellular MitoQ(10) was reduced to mitoquinol and subsequently metabolized to glucuronide and sulfate conjugates. Transport of MitoQ(10) was polarized with the apparent permeability (P(app)) from basolateral (BL) to apical (AP) (P(appBL-->AP)) being >2.5-fold the P(app) from apical to basolateral (P(appAP-->BL)). In the presence of 4% bovine serum albumin on the basolateral side, the P(appAP-->BL) value increased 7-fold compared with control. The P(appBL-->AP) value decreased by 26, 31 and 61% in the presence of verapamil 100 microM, ciclosporin 10 and 30 microM, respectively, whereas the P(appAP-->BL) value increased 71% in the presence of ciclosporin 30 microM. Apical efflux of mitoquinol sulfate and mitoquinol glucuronide conjugates was significantly decreased by ciclosporin 30 microM and the breast cancer receptor protein (BCRP) inhibitor, reserpine 25 microM, respectively. These results suggested that the bioavailability of MitoQ(10) may be limited by intracellular metabolism and the action of P-glycoprotein and BCRP. However, the dramatic increase in absorptive P(app) in the presence of bovine serum albumin on the receiver side suggests these barrier functions may be less significant in-vivo.

Involvement of Breast Cancer Resistance Protein (BCRP/ABCG2) in the Biliary Excretion and Intestinal Efflux of Troglitazone Sulfate, the Major Metabolite of Troglitazone with a Cholestatic Effect

Troglitazone sulfate (TGZS) is the major metabolite of troglitazone (TGZ), an antidiabetic agent, and thought to be a cause of the cholestasis induced by TGZ. The aim of the present study is to elucidate the involvement of breast cancer resistance protein (BCRP/ABCG2) in the hepatic disposition of TGZS. The basal-to-apical transport of TGZS was enhanced in organic anion transporting polypeptide 1B1-expressing Madin-Darby canine kidney II cells by infection of recombinant adenovirus harboring human BCRP and mouse Bcrp cDNA. TGZS was given to wild-type and Bcrp (-/-) mice by constant infusion. Biliary excretion is the predominant elimination pathway of TGZS in wild-type mice, and the biliary excretion clearance of TGZS with regard to the hepatic concentration was reduced to 30% of the control in Bcrp (-/-) mice. However, plasma and hepatic concentrations were unchanged, suggesting induction of compensatory mechanisms in Bcrp (-/-) mice for the elimination of TGZS. Involvement of BCRP in the intestinal efflux transport of TGZS was examined using everted sacs. The mucosal efflux clearance of TGZS showed only a slight reduction (15% reduction) in Bcrp (-/-) mice. Our results suggest that BCRP plays a major role in the biliary excretion but a minor role in the intestinal transport of TGZS.

Localization of the sulfate/anion exchanger in the rat liver

Although the sulfate/anion transporter (sat-1; SLC26A1) was isolated from a rat liver cDNA library by expression cloning, localization of sat-1 within the liver and its contribution to the transport of sulfate and organo sulfates have remained unresolved. In situ hybridization and immunohistochemical studies were undertaken to demonstrate the localization of sat-1 in liver tissue. RT-PCR studies on isolated hepatocytes and liver endothelial and stellate cells in culture were performed to test for the presence of sat-1 in these cells. In sulfate uptake and efflux experiments, the substrate specificity of sat-1 was evaluated. Sat-1 mRNA was found in hepatocytes and endothelial cells. Sat-1 protein was localized in sinusoidal membranes and along the borders of hepatocytes. The canalicular region and bile capillaries were not stained. Sulfate uptake was only slightly affected by sulfamoyl diuretics or organo sulfates. Sulfate efflux from sat-1-expressing oocytes was enhanced in the presence of bicarbonate, indicating sulfate/bicarbonate exchange. Estrone sulfate was not transported by sat-1. Sat-1 may be responsible for the uptake of inorganic sulfate from the blood into hepatocytes to enable sulfation reactions. In hepatocytes and endothelial cells, sat-1 may also supply sulfate for proteoglycan synthesis.