Localization of electrogenic Na/bicarbonate cotransporter NBCe1 variants in rat brain.

The activity of HCO(3)(-) transporters contributes to the acid-base environment of the nervous system. In the present study, we used in situ hybridization, immunoblotting, immunohistochemistry, and immunogold electron microscopy to localize electrogenic Na/bicarbonate cotransporter NBCe1 splice variants (-A, -B, and -C) in rat brain. The in situ hybridization data are consistent with NBCe1-B and -C, but not -A, being the predominant NBCe1 variants in brain, particularly in the cerebellum, hippocampus, piriform cortex, and olfactory bulb. An antisense probe to the B and C variants strongly labeled granule neurons in the dentate gyrus of the hippocampus, and cells in the granule layer and Purkinje layer (e.g. Bergmann glia) of the cerebellum. Weaker labeling was observed in the pyramidal layer of the hippocampus and in astrocytes throughout the brain. Similar, but weaker labeling was obtained with an antisense probe to the A and B variants. In immunoblot studies, antibodies to the A and B variants (alphaA/B) and C variant (alphaC) labeled approximately 130-kDa proteins in various brain regions. From immunohistochemistry data, both alphaA/B and alphaC exhibited diffuse labeling throughout brain, but alphaA/B labeling was more intracellular and punctate. Based on co-localization studies with antibodies to neuronal or astrocytic markers, alphaA/B labeled neurons in the pyramidal layer and dentate gyrus of the hippocampus, as well as cortex. alphaC labeled glia surrounding neurons (and possibly neurons) in the neuropil of the Purkinje cell layer of the cerebellum, the pyramidal cell layer and dentate gyrus of the hippocampus, and the cortex. According to electron microscopy data from the cerebellum, alphaA/B primarily labeled neurons intracellularly and alphaC labeled astrocytes at the plasma membrane. In summary, the B and C variants are the predominant NBCe1 variants in rat brain and exhibit different localization profiles.

Behavioral and anatomical abnormalities in Mecp2 mutant mice: a model for Rett syndrome.

Over 90% of Rett syndrome (RTT) cases have a mutation in the X-linked gene encoding methyl CpG binding-protein 2 (MeCP2). A mouse model that reprises clinical manifestations of the disease would be valuable for examining disease mechanisms. Here, we characterize physical and behavioral measures, as well as brain region volumes in young adult mice that have mutations in mouse methyl CpG binding-protein 2 gene (Mecp2) to serve as a baseline for other studies. Hemizygous males, which produce no functional protein, exhibit hypoactivity and abnormalities in locomotion, stereotypies, and anxiety reminiscent of the clinical condition. The mutant males also exhibit cognitive deficits in fear conditioning and object recognition relative to wildtypes. Volumetric analyses of male brains revealed a 25% reduction in whole brain volume in mutants relative to wildtypes; regional differences were also apparent. Mutants had decreased volumes in three specific brain regions: the amygdala (39%), hippocampus (21%), and striatum (29%). Heterozygous females, which produce varying amounts of functional protein, displayed a less severe behavioral phenotype. The mutant females exhibit abnormalities in locomotion, anxiety measures, and cognitive deficits in object recognition in an open field. This study provides the first evidence that the abnormal motor and cognitive behavioral phenotype in Mecp2 mice is consistent with specific volume reductions in brain regions associated with these behaviors, and shows how these data parallel the human condition. The Mecp2 mutant mice provide a very good model in which to examine molecular and behavioral mechanisms, as well as potential therapeutic interventions in RTT.

cAMP-mediated regulation of murine intestinal/pancreatic Na+/HCO3- cotransporter subtype pNBC1.

Basolateral Na(+)-HCO(3)(-) cotransport is essential for intestinal anion secretion, and indirect evidence suggests that it may be stimulated by a rise of intracellular cAMP. We therefore investigated the expression, activity, and regulation by cAMP of the Na(+)-HCO(3)(-) cotransporter isoforms NBC1 and NBCn1 in isolated murine colonic crypts. Na(+)-HCO(3)(-) transport rates were measured fluorometrically in BCECF-loaded crypts, and mRNA expression levels and localization were determined by semiquantitative PCR and in situ hybridization. Acid-activated Na(+)-HCO(3)(-) cotransport rates were 5.07 +/- 0.7 mM/min and increased by 62% after forskolin stimulation. NBC1 mRNA was more abundant in colonic crypts than in surface cells, and crypts expressed far more NBC1 than NBCn1. To investigate whether the cAMP-induced Na(+)-HCO(3)(-) cotransport activation was secondary to secretion-associated changes in HCO(3)(-) or cell volume, we measured potential forskolin-induced changes in intracellular pH and assessed Na(+)-HCO(3)(-) transport activity in CFTR -/- crypts (in which no forskolin-induced cell shrinkage occurs). We found 30% reduced Na(+)-HCO(3)(-) transport rates in CFTR -/- compared with CFTR +/+ crypts but similar Na(+)-HCO(3)(-) cotransport activation by forskolin. These studies establish the existence of an intracellular HCO(3)(-) concentration- and cell volume-independent activation of colonic NBC by an increase in intracellular cAMP.

Claudin-2 is selectively expressed in proximal nephron in mouse kidney.

The proximal nephron possesses a leaky epithelium with unique paracellular permeability properties that underlie its high rate of passive NaCl and water reabsorption, but the molecular basis is unknown. The claudins are a large family of transmembrane proteins that are part of the tight junction complex and likely form structural components of a paracellular pore. To localize claudin-2 in the mouse kidney, we performed in situ hybridization using an isoform-specific riboprobe and immunohistochemistry using a polyclonal antibody directed against a COOH-terminal peptide. Claudin-2 mRNA and protein were found throughout the proximal tubule and in the contiguous early segment of the thin descending limb of long-looped nephrons. The level of expression demonstrated an axial increase from proximal to distal segments. In confocal images, the subcellular localization of claudin-2 protein coincided with that of the tight junction protein ZO-1. Our findings suggest that claudin-2 is a component of the paracellular pathway of the most proximal segments of the nephron and that it may be responsible for their uniquely leaky permeability properties.

CaT1 expression correlates with tumor grade in prostate cancer.

Ca(2+) signaling is important for growth and survival of prostatic carcinoma (PCa) cells. Here we report that the gene for CaT1, a channel protein highly selective for Ca(2+), is expressed at high levels in human PCa and in the LNCaP PCa cell line. CaT1 mRNA levels were elevated in PCa specimens in comparison to benign prostatic hyperplasia (BPH) specimens and positively correlated with Gleason grade in a PCa series. CaT1 mRNA was suppressed by androgen and was induced by a specific androgen receptor antagonist in LNCaP cells, suggesting that the gene is negatively regulated by androgen. These findings are the first to implicate a Ca(2+) channel in PCa progression and suggest that CaT1 may be a novel target for therapy.

Differential distribution of the glutamate transporters GLT-1 and GLAST in tanycytes of the third ventricle.

The ventral one-third of the ventricular lining in the hypothalamus is formed by specialized ependymal cells called the tanycytes. These cells may serve a neuroendocrine transport function because of their structural specializations, which include apical microvili on the ventricular surface and long basal processes that terminate on blood vessels or on the glia limitans. Here, we describe the expression of mRNA and protein for the glutamate transporters GLT-1 and GLAST in unique tanycyte populations of the third ventricle in rat brain. Using nonisotopic in situ hybridization, we demonstrate GLAST mRNA labeling in tanycytes of the ventral floor and lateral walls in the tuberal and mammillary recess portions of the third ventricle. This GLAST mRNA labeling had a higher intensity than the labeling intensity observed in regular ependymal cells throughout the ventricular system. Furthermore, we have identified strong GLT-1 mRNA labeling in a population of tanycytes situated in the dorsolateral walls of caudal tuberal and mammillary recess portions. Immunocytochemical staining indicates that both GLT-1 and GLAST protein are expressed in the tanycyte populations as well. These data corroborate previous findings that third ventricle tanycytes are functionally heterogeneous. Furthermore, the GLT-1-expressing tanycytes represent a population of tanycytes that, to date, has not been recognized as functionally distinct. The strong GLAST expression by the ventral tanycytes in the hypophysiotropic area suggests a role of tanycyte-mediated glutamate transport in neuroendocrine activity. The functional role of GLT-1 in dorsal wall tanycytes remains to be explored.

Molecular characterization of a novel urea transporter from kidney inner medullary collecting ducts.

In the terminal part of the kidney collecting duct, rapid urea reabsorption is essential to maintaining medullary hypertonicity, allowing maximal urinary concentration to occur. This process is mediated by facilitated urea transporters on both apical and basolateral membranes. Our previous studies have identified three rat urea transporters involved in the urinary concentrating mechanism, UT1, UT2 and UT3, herein renamed UrT1-A, UrT1-B, and UrT2, which exhibit distinct spatial distribution in the kidney. Here we report the molecular characterization of an additional urea transporter isoform, UrT1-C, from rat kidney that encodes a 460-amino acid residue protein. UrT1-C has 70 and 62% amino acid identity to rat UrT1-B and UrT2 (UT3), respectively, and 99% identity to a recently reported rat isoform (UT-A3; Karakashian A, Timmer RT, Klein JD, Gunn RB, Sands JM, and Bagnasco SM. J Am Soc Nephrol 10: 230-237, 1999). We report the anatomic distribution of UrT1-C in the rat kidney tubule system as well as a detailed functional characterization. UrT1-C m RNA is primarily expressed in the deep part of the inner medulla. When expressed in Xenopus laevis oocytes, UrT1-C induced a 15-fold stimulation of urea uptake, which was inhibited almost completely by phloretin (0.7 mM) and 60-95% by thiourea analogs (150 mM). The characteristics are consistent with those described in perfusion studies with inner medullary collecting duct (IMCD) segments, but, contrary to UrT1-A, UrT1-C-mediated urea uptake was not stimulated by activation of protein kinase A. Our data show that UrT1-C is a phloretin-inhibitable urea transporter expressed in the terminal collecting duct that likely serves as an exit mechanism for urea at the basolateral membrane of IMCD cells.

Diurnal rhythmicity in intestinal SGLT-1 function, V(max), and mRNA expression topography.

Mechanisms underlying the circadian rhythmicity in intestinal sugar absorption remain unclear. To test whether this rhythmicity is caused by changes in Na(+)-glucose cotransporter 1 (SGLT-1) function, we measured phloridzin-inhibitable sugar fluxes as an index of SGLT-1 activity. Jejunum obtained from rats killed at 6-h intervals during a 12-h light-dark cycle (CT0 is circadian time 0 h, time of light onset) were mounted in Ussing chambers, and 3-O-methylglucose (3-OMG) fluxes were calculated before and after addition of phloridzin. 3-OMG-induced change in short-circuit current and absorptive flux were significantly greater at CT9 than at CT3. This increase was phloridzin inhibitable. Kinetic studies indicated a significant increase in SGLT-1 maximal velocity (V(max)) at CT9. Food intake between CT3 and CT9 was <10% of the daily total, indicating that the increased SGLT-1 activity was anticipatory. Diurnicity of SGLT-1 mRNA was confirmed by Northern blotting. Expression topography analyzed by in situ hybridization revealed more intense labeling along the entire villus axis at CT9 and CT15 compared with CT3 and CT21. We conclude that diurnicity in intestinal sugar absorption is caused by periodicity in SGLT-1 V(max).

Amyotrophic lateral sclerosis-linked glutamate transporter mutant has impaired glutamate clearance capacity.

We have investigated the functional impact of a naturally occurring mutation of the human glutamate transporter GLT1 (EAAT2), which had been detected in a patient with sporadic amyotrophic lateral sclerosis. The mutation involves a substitution of the putative N-linked glycosylation site asparagine 206 by a serine residue (N206S) and results in reduced glycosylation of the transporter and decreased uptake activity. Electrophysiological analysis of N206S revealed a pronounced reduction in transport rate compared with wild-type, but there was no alteration in the apparent affinities for glutamate and sodium. In addition, no change in the sensitivity for the specific transport inhibitor dihydrokainate was observed. However, the decreased rate of transport was associated with a reduction of the N206S transporter in the plasma membrane. Under ionic conditions, which favor the reverse operation mode of the transporter, N206S exhibited an increased reverse transport capacity. Furthermore, if coexpressed in the same cell, N206S manifested a dominant negative effect on the wild-type GLT1 activity, whereas it did not affect wild-type EAAC1. These findings provide evidence for a role of the N-linked glycosylation in both cellular trafficking and transport function. The resulting alteration in glutamate clearance capacity likely contributes to excitotoxicity that participates in motor neuron degeneration in amyotrophic lateral sclerosis.

Human calcium transport protein CaT1.

Transcellular calcium transport occurs in many epithelial tissues including intestine, kidney, and placenta. We identified the human ortholog (hCaT1) of a recently cloned rat calcium transport protein, CaT1, that mediates intestinal calcium uptake. hCaT1 messenger RNA is present in the gastrointestinal tract, including esophagus, stomach, duodenum, jejunum, ileum, and colon. High levels of hCaT1 transcripts are also present in pancreas, placenta, prostate, and salivary gland, while moderate levels are present in liver, kidney, and testis. hCaT1 mRNA is also expressed in the colorectal cancer cell line, SW480, and the chronic myelogenous leukemia cell line, K-562. The hCaT1 gene was assigned to the long arm of chromosome 7, bands q33-34, by fluorescence in situ hybridization. When expressed in Xenopus laevis oocytes, hCaT1 promotes saturable Ca(2+) uptake with a Michaelis constant of 0.25 mM. Our studies suggest a role for hCaT1 in cellular calcium uptake in a variety of tissues, including the transcellular calcium transport pathway in intestine.

Na/HCO3 cotransporters in rat brain: expression in glia, neurons, and choroid plexus.

We studied the expression and distribution of Na/HCO(3) cotransporters in rat brain using polynucleotide probes and polyclonal antibodies derived from the electrogenic rat kidney Na/HCO(3) cotransporter (rkNBC). In whole brain, we observed a single mRNA ( approximately 7.5 kb) by Northern hybridization and a major approximately 130 kDa protein by immunoblotting with a polyclonal antiserum directed against the C terminus of rkNBC. NBC mRNA and protein were present in cortex, brainstem-diencephalon, and cerebellum. In situ hybridization revealed NBC mRNA expression throughout the CNS, with particularly high levels in olfactory bulb, hippocampal dentate gyrus, and cerebellum. NBC mRNA was present in glial cells (e.g., Bergmann glia of cerebellum and hippocampal astrocytes) and neurons (e.g., granule cells of dentate gyrus and neurons of cortex or striatum). Double hybridization of mRNA encoding NBC and glutamate transporter 1 (glial marker) confirmed that both glia and neurons express NBC. Indirect immunofluorescence microscopy demonstrated NBC protein throughout the CNS, particularly in hippocampus and cerebellum. Although NBC mRNA was restricted to cell bodies, NBC protein was distributed diffusely, compatible with a localization in cell processes and perhaps cell bodies. Double labeling with glial fibrillary acidic protein (astrocytic marker), microtubule-associated protein 2 (neuronal marker), or 2',3'-cyclic mononucleotide 3'-phosphodiesterase (oligodendrocytic marker) demonstrated expression of NBC protein in specific subpopulations of both glia and neurons. Moreover, NBC protein was present in both cultured hippocampal astrocytes and cortical neurons. NBC mRNA and protein were also present in epithelial cells of choroid plexus, ependyma, and meninges. Our results are thus consistent with multiple novel roles for Na/HCO(3) cotransport in CNS physiology.

A rat kidney-specific calcium transporter in the distal nephron.

Active absorption of calcium from the intestine and reabsorption of calcium from the kidney are major determinants of whole body calcium homeostasis. Two recently cloned proteins, CaT1 and ECaC, have been postulated to mediate apical calcium uptake by rat intestine and rabbit kidney, respectively. By screening a rat kidney cortex library with a CaT1 probe, we isolated a cDNA encoding a protein (CaT2) with 84.2 and 73.4% amino acid identities to ECaC and CaT1, respectively. Unlike ECaC, CaT2 is kidney-specific in the rat and was not detected in intestine, brain, adrenal gland, heart, skeletal muscle, liver, lung, spleen, thymus, and testis by Northern analysis or reverse transcription polymerase chain reaction. The expression pattern of CaT2 in kidney was similar to that of calbindin D(28K) and the sodium calcium exchanger 1, NCX1, by in situ hybridization of adjacent sections. Furthermore, the mRNAs for CaT2 and calbindin D(28K) were colocalized in the same cells. CaT2 mediated saturable calcium uptake with a Michaelis constant (K(m)) of 0.66 mm when expressed in Xenopus laevis oocytes. Under voltage clamp condition, CaT2 promoted inward currents in X. laevis oocytes upon external application of Ca(2+). Sr(2+) and Ba(2+) but not Mg(2+) also evoked inward currents in CaT2-expressing oocytes. Similar to the alkaline earth metal ions, application of Cd(2+) elicited inward current in CaT2-expressing oocytes with a K(m) of 1.3 mm. Cd(2+), however, also potently inhibited CaT2-mediated Ca(2+) uptake with an IC(50) of 5.4 micrometer. Ca(2+) evoked currents were reduced at low pH and increased at high pH and were only slightly affected by the L-type voltage-dependent calcium channel antagonists, nifedipine, verapamil, diltiazem, and the agonist, Bay K 8644, even at relatively high concentrations. In conclusion, CaT2 may participate in calcium entry into the cells of the distal convoluted tubule and connecting segment of the nephron, where active reabsorption of calcium takes place via the transcellular route. The high sensitivity of CaT2 to Cd(2+) also provides a potential explanation for Cd(2+)-induced hypercalciuria and resultant renal stone formation.

The vitamin C transporter SVCT2 is expressed by astrocytes in culture but not in situ.

Ascorbic acid (vitamin C) is known to be selectively accumulated by brain cells through sodium-dependent vitamin C transporters. It is unclear however, whether this uptake occurs in neurons, astrocytes or both. Using Northern analysis we demonstrate that the recently cloned ascorbate transporter isoform SVCT2 is expressed by cultured astrocytes. In contrast, in situ hybridization experiments reveal that SVCT2 mRNA is expressed only in neurons and not in normal astrocytes or astrocytes stimulated by an intrastriatal injection of the neurotoxin quinolinic acid. We conclude that SVCT2 is neuron specific and that the majority of ascorbate storage occurs in neurons. Furthermore, we propose that the observed sodium-dependent ascorbate transport in cultured astrocytes may be due to artificial upregulation of SVCT2 during cell culturing.

Distribution of the glutamate transporters GLAST and GLT-1 in rat circumventricular organs, meninges, and dorsal root ganglia.

The glial glutamate transporters GLAST and GLT-1 are primarily responsible for the removal of glutamate from brain extracellular fluid. This study compares the distribution of GLAST and GLT-1 expression in the circumventricular organs of the brain, in the meninges, and in the dorsal root ganglion. By using a highly sensitive nonisotopic in situ hybridization method and immunostaining, we demonstrate marked differences in the expression patterns for the two transporters. In the three sensory circumventricular organs that contain neuronal elements, i.e., the subfornical organ, the vascular organ of the lamina terminalis, and the area postrema, GLAST is strongly expressed, whereas GLT-1 is faintly expressed or absent. Both transporters are absent from the choroid plexus, and only GLAST mRNA is found in the subcommisural organ. In the pineal gland, GLAST is expressed by astrocytic cells near the pineal stalk, whereas GLT-1 is expressed by pinealocytes throughout the gland. In the pituitary gland, GLAST is likely expressed by folliculo-stellate cells in the anterior lobe, by a group of astrocyte-like cells and by marginal cells in the intermediate lobe, and by pituicytes in the posterior lobe, whereas GLT-1 is expressed only by the astrocyte-like cells in the intermediate lobe. Finally, GLAST, but not GLT-1, is expressed by specific layers of the meninges, and by satellite cells in the dorsal root ganglion. These results show that GLAST is the primary glutamate transporter in the circumventricular organs. The data provide further evidence that these two glutamate transporters fulfill markedly different functions in the nervous system.

Glutamate carboxypeptidase II is expressed by astrocytes in the adult rat nervous system.

The enzyme glutamate carboxypeptidase II (GCP II) has been cloned from rat brain and human prostate. This enzyme, which catabolizes the neuropeptide N-acetylaspartylglutamate, has also been known as N-acetylated alpha-linked acidic dipeptidase (NAALADase), and is identical to the prostate-specific membrane antigen and to the jejunal folylpoly-gamma-glutamate carboxypeptidase. The goals of the present study were to elucidate the cell specificity and regional pattern of GCP II expression in the rat nervous system by using Northern blots and enzymatic assays of brain and subfractionated primary neuronal and glial cultures together with in situ hybridization histochemistry (ISHH) in sections of adult rat tissue. GCP II activity was assayed in astrocyte cultures (4.4 pmol/mg protein per minute), neuronal-glial cocultures (2.5 pmol/mg protein per minute) and neuron-enriched cultures (0.38 pmol/mg protein per minute), with the activity in each preparation correlating to its astrocytic content (r = 0.99). No activity was detected in cultured oligodendrocytes or microglia. Northern blots probed with a GCP II cDNA detected mRNAs exclusively in activity-positive cell preparations. ISHH results show that GCP II is expressed by virtually all astrocytes, by Bergmann glial cells in cerebellum, by Müller cells in retina and by the satellite cells in dorsal root ganglia. Astrocytes in select groups of nuclei (e.g., habenula, supraoptic nucleus, pontine nucleus) contained pronounced levels of GCP II message. The data of the present study suggest that GCP II is expressed in the adult rat nervous system exclusively in astrocytic glial cells.

Molecular cloning and characterization of a channel-like transporter mediating intestinal calcium absorption.

Calcium is a major component of the mineral phase of bone and serves as a key intracellular second messenger. Postnatally, all bodily calcium must be absorbed from the diet through the intestine. Here we report the properties of a calcium transport protein (CaT1) cloned from rat duodenum using an expression cloning strategy in Xenopus laevis oocytes, which likely plays a key role in the intestinal uptake of calcium. CaT1 shows homology (75% amino acid sequence identity) to the apical calcium channel ECaC recently cloned from vitamin D-responsive cells of rabbit kidney and is structurally related to the capsaicin receptor and the TRP family of ion channels. Based on Northern analysis of rat tissues, a 3-kilobase CaT1 transcript is present in rat duodenum, proximal jejunum, cecum, and colon, and a 6.5-kilobase transcript is present in brain, thymus, and adrenal gland. In situ hybridization revealed strong CaT1 mRNA expression in enterocytes of duodenum, proximal jejunum, and cecum. No signals were detected in kidney, heart, liver, lung, spleen, and skeletal muscle. When expressed in Xenopus oocytes, CaT1 mediates saturable Ca(2+) uptake with a Michaelis constant of 0.44 mM. Transport of Ca(2+) by CaT1 is electrogenic, voltage-dependent, and exhibits a charge/Ca(2+) uptake ratio close to 2:1, indicating that CaT1-mediated Ca(2+) influx is not coupled to other ions. CaT1 activity is pH-sensitive, exhibiting significant inhibition by low pH. CaT1 is also permeant to Sr(2+) and Ba(2+) (but not Mg(2+)), although the currents evoked by Sr(2+) and Ba(2+) are much smaller than those evoked by Ca(2+). The trivalent cations Gd(3+) and La(3+) and the divalent cations Cu(2+), Pb(2+), Cd(2+), Co(2+), and Ni(2+) (each at 100 microM) do not evoke currents themselves, but inhibit CaT1-mediated Ca(2+) transport. Fe(3+), Fe(2+), Mn(2+), and Zn(2+) have no significant effects at 100 microM on CaT1-mediated Ca(2+) transport. CaT1 mRNA levels are not responsive to 1,25-dihydroxyvitamin D(3) administration or to calcium deficiency. Our studies strongly suggest that CaT1 provides the principal mechanism for Ca(2+) entry into enterocytes as part of the transcellular pathway of calcium absorption in the intestine.

A family of mammalian Na+-dependent L-ascorbic acid transporters.

Vitamin C (L-ascorbic acid) is essential for many enzymatic reactions, in which it serves to maintain prosthetic metal ions in their reduced forms (for example, Fe2+, Cu+), and for scavenging free radicals in order to protect tissues from oxidative damage. The facilitative sugar transporters of the GLUT type can transport the oxidized form of the vitamin, dehydroascorbic acid, but these transporters are unlikely to allow significant physiological amounts of vitamin C to be taken up in the presence of normal glucose concentrations, because the vitamin is present in plasma essentially only in its reduced form. Here we describe the isolation of two L-ascorbic acid transporters, SVCT1 and SVCT2, from rat complementary DNA libraries, as the first step in investigating the importance of L-ascorbic acid transport in regulating the supply and metabolism of vitamin C. We find that SVCT1 and SVCT2 each mediate concentrative, high-affinity L-ascorbic acid transport that is stereospecific and is driven by the Na+ electrochemical gradient. Despite their close sequence homology and similar functions, the two isoforms of the transporter are discretely distributed: SVCT1 is mainly confined to epithelial systems (intestine, kidney, liver), whereas SVCT2 serves a host of metabolically active cells and specialized tissues in the brain, eye and other organs.

Molecular and functional analysis of SDCT2, a novel rat sodium-dependent dicarboxylate transporter.

Kidney proximal tubule cells take up Krebs cycle intermediates for metabolic purposes and for secretion of organic anions through dicarboxylate/organic anion exchange. Alteration in reabsorption of citrate is closely related to renal stone formation. The presence of distinct types of sodium-coupled dicarboxylate transporters has been postulated on either side of the polarized epithelial membrane in the kidney proximal tubule. Using a PCR-based approach, we isolated a novel member of the sodium-dependent dicarboxylate/sulfate transporter called SDCT2. SDCT2 is a 600-amino acid residue protein that has 47-48% amino acid identity to SDCT1 and NaDC-1, previously identified in kidney and intestine. Northern analysis gave a single band of 3.3 kb for SDCT2 in kidney, liver, and brain. In situ hybridization revealed that SDCT2 is prominently expressed in kidney proximal tubule S3 segments and in perivenous hepatocytes, consistent with the sites of high-affinity dicarboxylate transport identified based on vesicle studies. A signal was also detected in the meningeal layers of the brain. SDCT2 expressed in Xenopus oocytes mediated sodium-dependent transport of di- and tricarboxylates with substrate preference for succinate rather than citrate, but excluding monocarboxylates. SDCT2, unlike SDCT1, displayed a unique pH dependence for succinate transport (optimal pH 7.5-8.5) and showed a high affinity for dimethylsuccinate, two features characteristic of basolateral transport. These data help to interpret the mechanisms of renal citrate transport, their alteration in pathophysiological conditions, and their role in the elimination of organic anions and therapeutic drugs.

Distribution of peptide transporter PEPT2 mRNA in the rat nervous system.

The signaling action of neuropeptides in the brain is terminated by breakdown through extracellular peptidases and subsequent removal of the peptide fragments from the extracellular fluid via specific transporter proteins. Here we describe the anatomical distribution in the rat nervous system of the recently isolated high affinity peptide transporter PEPT2. Using nonisotopic in situ hybridization we demonstrate that PEPT2 mRNA is expressed in brain by astrocytes, subependymal cells, ependymal cells and epithelial cells of choroid plexus. Furthermore, PEPT2 is expressed in retina by Müller cells and in dorsal root ganglia by satellite cells. The mRNA levels of PEPT2 in astrocytes are moderate and relatively homogenous throughout the brain except for an area in ventral forebrain where PEPT2 levels are below average. PEPT2 mRNA expression is weakly upregulated in reactive astrocytes that were stimulated through an injection of the glutamatergic neurotoxin quinolinic acid. These data suggest that removal of neuropeptide fragments from brain extracellular fluid occurs via PEPT2 expressed in astrocytes, ependymal cells and choroid plexus epithelial cells.

Localization of sodium bicarbonate cotransporter (NBC) protein and messenger ribonucleic acid in rat epididymis.

An acidic environment is important for sperm maturation in the epididymis and also helps to maintain mature sperm in an immotile state during storage in this organ. Both an Na+/H+ exchanger and an H+ATPase have been implicated in this process. The H+ATPase is concentrated in specialized apical (and/or narrow) and clear cells of the epididymis, while the Na+/H+ exchanger has not yet been localized in situ. As in other proton-secreting epithelia, bicarbonate transport occurs in the epididymis, where it is implicated in luminal acidification. In this study we used an antibody raised against a fusion protein (maltose-binding protein: MBP-NBC-5) from the C-terminus of the recently cloned rat kidney Na+/HCO3- cotransporter (NBC) to localize this protein in the epididymis and vas deferens of the rat. The distribution of the respective mRNA was mapped by in situ hybridization. NBC message was strongly expressed in the initial segment and the intermediate zone of the epididymis, and the NBC-5 antibody gave a strong basolateral staining in both principal cells and apical/narrow cells in this region. Western blotting revealed a single band at about 160 kDa in the epididymis. The intensity of staining as well as mRNA levels decreased in the cauda epididymidis and in the vas deferens, where only weak staining was seen. Basolateral NBC may function in parallel with apical proton secretion to regulate luminal acidification and/or bicarbonate reabsorption in the excurrent duct system.