Sodium-dependent Uptake Research Articles

Regulation of Glutamate Transport in Developing Rat Oligodendrocytes

Glutamate released from synaptic vesicles mediates excitatory neurotransmission by stimulating glutamate receptors. Glutamate transporters maintain low synaptic glutamate levels critical for this process, a role primarily attributed to astrocytes. Recently, vesicular release of glutamate from unmyelinated axons in the rat corpus callosum has been shown to elicit AMPA receptor-mediated currents in glial progenitor cells. Glutamate transporters are the only mechanism of glutamate clearance, yet very little is known about the role of glutamate transporters in normal development of oligodendrocytes (OLs) or in excitotoxic injury to OLs. We found that OLs in culture are capable of sodium-dependent glutamate uptake with a K(m) of 10 +/- 2 microm and a V(max) of 2.6, 5.0, and 3.8 nmol x min(-1) x mg(-1) for preoligodendrocytes, immature, and mature OLs, respectively. Surprisingly, EAAC1, thought to be exclusively a neuronal transporter, contributes more to [(3)H]l-glutamate uptake in OLs than GLT1 or GLAST. These data suggest that glutamate transporters on oligodendrocytes may serve a critical role in maintaining glutamate homeostasis at a time when unmyelinated callosal axons are engaging in glutamatergic signaling with glial progenitors. Furthermore, GLT1 was significantly increased in cultured mature OLs contrary to in vivo data in which we have shown that, although GLT1 is present on developing OLs when unmyelinated axons are prevalent in the developing rat corpus callosum, after myelination, GLT1 is not expressed on mature OLs. The absence of GLT1 in mature OLs in the rat corpus callosum and its presence in mature rat cultured OLs may indicate that a signaling process in vivo is not activated in vitro.

Identification of a novel sodium-dependent fructose transport activity in the hepatopancreas of the Atlantic lobsterHomarus americanus

[(3)H]Fructose and [(3)H]glucose transport were determined in brush-border membrane vesicles (BBMV), basolateral membrane vesicles (BLMV) and isolated cells (E, R, F, B) of H. americanus (Atlantic lobster) hepatopancreas. Glucose transport in BBMV was equilibrative in the absence of sodium and concentrative in the presence of sodium. Sodium-dependent glucose transport by BBMV was not inhibited by a tenfold molar excess of fructose. Glucose transport by BLMV was equilibrative and sodium independent. Fructose uptake by BBMV and BLMV was equilibrative in the absence of sodium and concentrative in the presence of sodium. This enhancement was not affected by a tenfold molar excess of glucose in the presence of sodium. E-, F- and B-cells showed sodium-dependent uptake of fructose, while R-cells did not. Sodium-dependent fructose uptake by E-cells was not inhibited by a tenfold molar excess of glucose or mannose. Western blot analysis of BBMV, BLMV and E-, R-, F- and B-cells using rabbit polyclonal antibodies directed against epitopes of mammalian GLUT2, GLUT5, SGLT1 and SGLT4 indicated the presence of cross-reacting lobster proteins. Sequence alignment of the mammalian proteins with translated, lobster expressed sequence tags also indicated significant identity between species. Comparison of fructose and glucose uptake in the absence and presence of sodium by BBMV, BLMV and isolated cells indicated the presence of a distinct sodium-dependent transport activity for each sugar in the Atlantic lobster.

The Drug of Abuse γ-Hydroxybutyrate Is a Substrate for Sodium-Coupled Monocarboxylate Transporter (SMCT) 1 (SLC5A8): Characterization of SMCT-Mediated Uptake and Inhibition

Gamma-hydroxybutyric acid (GHB), a drug of abuse, is a substrate of monocarboxylate transporters (MCTs). Sodium-coupled monocarboxylate transporter 1 (SMCT1; SLC5A8) is expressed in kidney, thyroid gland, neurons, and intestinal tract and exhibits substrate specificity similar to that of the proton-dependent MCT (SLC16A) family. The role of SMCT1 in GHB disposition has not been determined. In this study we characterized the driving force, transport kinetics, and inhibitors of GHB uptake, as well as expression of SMCT and MCT isoforms, in rat thyroid follicular (FRTL-5) cells. GHB, as well as the monocarboxylates butyrate and d-lactate, exhibited sodium-dependent uptake at pH 7.4, which could be described with a simple Michaelis-Menten equation plus a diffusional component [K(m) 0.68 +/- 0.30 mM, V(max) 3.50 +/- 1.58 nmol . mg(-1) . min(-1), and diffusional clearance (P) 0.25 +/- 0.08 microl . mg(-1) . min(-1)]. In the absence of sodium, GHB uptake was significantly increased at lower pH, suggesting proton-gradient dependent transport. Reverse transcriptase-polymerase chain reaction and Western analyses demonstrated the expression of SMCT1, MCT1, and MCT2 in FRTL-5 cells, supporting the activity results. Sodium-dependent GHB uptake in FRTL-5 cells was inhibited by MCT substrates (d-lactate, l-lactate, pyruvate, and butyrate), nonsteroidal anti-inflammatory drugs (ibuprofen, ketoprofen, and naproxen), and probenecid. IC(50) values for l-lactate, ibuprofen, ketoprofen, and probenecid were 101, 31.6, 64.4, and 380 muM, respectively. All four inhibitors also significantly inhibited GHB uptake in rat MCT1 gene-transfected MDA/MB231 cells, suggesting they are not specific for SMCT1. Luteolin and alpha-cyano-4-hydroxycinnimate represent specific proton-dependent MCT inhibitors. Our findings indicate that GHB is a substrate for both sodium- and proton-dependent MCTs and identified specific inhibitors of MCTs.

The membrane-bound bile acid receptor TGR5 is localized in the epithelium of human gallbladders #

TGR5 (Gpbar-1) is a plasma membrane-bound, G protein-coupled receptor for bile acids. TGR5 messenger RNA (mRNA) has been detected in many tissues, including rat cholangiocytes and mouse gallbladder. A role for TGR5 in gallstone formation has been suggested, because TGR5 knockout mice did not develop gallstones when fed a lithogenic diet. In this study, expression and localization of TGR5 was studied in human gallbladders. TGR5 mRNA and protein were detected in all 19 gallbladders. Although TGR5 mRNA was significantly elevated in the presence of gallstones, no such relation was found for TGR5 protein levels. In order to study the localization of TGR5 in human gallbladders, a novel antibody was generated. The receptor was localized in the apical membrane and the rab11-positive recycling endosome of gallbladder epithelial cells. Furthermore, the TGR5 staining colocalized with the cyclic adenosine monophosphate-regulated chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) and the apical sodium-dependent bile salt uptake transporter, suggesting a functional coupling of TGR5 to bile acid uptake and chloride secretion. Stimulation with bile acids significantly increased cyclic adenosine monophosphate concentration in human gallbladder tissue. Incubation of gallbladder epithelial cells with a TGR5 agonist led to a rise of N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE)-fluorescence, suggestive of a decrease in intracellular chloride concentration. The TGR5 agonist-dependent increase in MQAE-fluorescence was absent in TGR5 knockout mice or in the presence of a CFTR inhibitor, indicating that TGR5 mediates chloride secretion via activation of CFTR. The presence of the receptor in both the plasma membrane and the recycling endosome indicate that TGR5 can be regulated by translocation. The data suggest a role for TGR5 in bile acid-induced fluid secretion in biliary epithelial cells.

Protein kinase C–dependent trafficking of glutamate transporters excitatory amino acid carrier 1 and glutamate transporter 1b in cultured cerebellar granule cells

Previous data showed that cell surface expression of the glutamate transporters GLT1a and excitatory amino acid carrier 1 (EAAC1), localized in glia and neurons of the CNS, can be regulated by protein kinase C (PKC). Regulation and physiological importance of GLT1b, a splice variant of GLT1a, is not understood. In the present study we used cultured cerebellar granule cells (CGCs) from mice to investigate PKC dependent trafficking of GLT1b in comparison to GLT1a and EAAC1 using immunohistochemistry and subcellular fractionation followed by Western blotting. In neurites of CGCs, GLT1b and EAAC1 were localized to different aggregates of vesicles that were different from vesicle aggregates containing vesicular glutamate transporters. In CGCs cultured with low-potassium medium, stimulation of PKC by phorbol ester enhanced the formation of large varicosities in neurites that exhibited immunoreactivity for GLT1a, GLT1b, and EAAC1. Stimulation of PKC leads to a significant increase of GLT1b and EAAC1 in the plasma membrane whereas GLT1a in the plasma membrane was decreased. Following PKC stimulation, also a significant increase of transporter-mediated glutamate uptake representing sodium dependent glutamate uptake, was observed. Similarly, the fraction of glutamate uptake, that was sensitive to the inhibitor WAY-213613 and represents uptake by GLT1a and GLT1b, was increased after stimulation by PKC. The findings suggest that PKC is similarly involved in regulation of surface trafficking of GLT1b and EAAC1 and that PKC stimulated increase in surface location of GLT1b and EAAC1 in glutamatergic CGCs.

Substrate specificity of rat Na+/taurocholate cotransporting polypeptide

Human Na+/taurocholate cotransporting polypeptide (NTCP) has recently been proposed to be a multispecific transporter that mediates uptake of xenobiotics including some statins in addition to its prototypical substrate (bile acids). In rat basolateral plasma membrane vesicles Na+‐dependent taurocholate uptake was competitively inhibited by several glitazones, suggesting that these drugs could be substrates of rat Ntcp. Therefore we used CHO cells that stably express rat Ntcp to screen for drugs and other xenobiotics that inhibit sodium dependent taurocholate uptake and therefore might be substrates of Ntcp. We tested selected inhibitors as substrates using LC‐MS/MS analysis. We confirmed that troglitazone and rosiglitazone are strong inhibitors of rat Ntcp (IC50 = 13 μM for troglitazone, 8 μM for rosiglitazone). Furthermore, we determined that several additional drugs including losartan, ritonavir, saquinavir, fluvastatin, ketoconazole, diclofenac and carbenoxolone inhibited Ntcp mediated taurocholate transport in a concentration dependent pattern. The so far tested troglitazone, rosiglitazone, ritonavir, saquinavir and pravastatin however are not substrates for rat Ntcp although pravastatin is transported by human NTCP. In conclusion, rat Ntcp is inhibited by numerous drugs but its substrate specificity seems to be more restricted than the substrate specificity of human NTCP.

Membrane targeting and intracellular trafficking of the human sodium-dependent multivitamin transporter in polarized epithelial cells.

The human sodium-dependent multivitamin transporter (hSMVT) mediates sodium-dependent uptake of biotin in renal and intestinal epithelia. To date, however, there is nothing known about the structure-function relationship or targeting sequences in the hSMVT polypeptide that control its polarized expression within epithelia. Here, we focused on the role of the COOH-terminal tail of hSMVT in the targeting and functionality of this transporter. A full-length hSMVT-green fluorescent protein (GFP) fusion protein was functional and expressed at the apical membrane in renal and intestinal cell lines. Microtubule disrupting agents disrupted the mobility of trafficking vesicles and impaired cell surface delivery of hSMVT, which was also prevented in cells treated with dynamitin (p50), brefeldin, or monensin. Progressive truncation of the COOH-terminal tail impaired the functionality and targeting of the transporter. First, biotin transport decreased by approximately 20-30% on deletion of up to 15 COOH-terminal amino acids of hSMVT, a decrease mimicked solely by deletion of the terminal PDZ motif (TSL). Second, deletions into the COOH-terminal tail (between residues 584-612, containing a region of predicted high surface accessibility) resulted in a further drop in hSMVT transport (to approximately 40% of wild-type). Third, apical targeting was lost on deletion of a helical-prone region between amino acids 570-584. We conclude that the COOH tail of hSMVT contains several determinants important for polarized targeting and biotin transport.

The Four Major N- and C-Terminal Splice Variants of the Excitatory Amino Acid Transporter GLT-1 Form Cell Surface Homomeric and Heteromeric Assemblies

The L-glutamate transporter GLT-1 is an abundant central nervous system (CNS) membrane protein of the excitatory amino acid transporter (EAAT) family that controls extracellular L-glutamate levels and is important in limiting excitotoxic neuronal death. Using reverse transcription-polymerase chain reaction, we have determined that four mRNAs encoding GLT-1 exist in mouse brain, with the potential to encode four GLT-1 isoforms that differ in their N and C termini. We expressed all four isoforms (termed MAST-KREK, MPK-KREK, MAST-DIETCI, and MPK-DIETCI according to amino acid sequence) in a range of cell lines and primary astrocytes and show that each isoform can reach the cell surface. In transfected human embryonic kidney (HEK) 293 or COS-7 cells, all four isoforms support high-affinity sodium-dependent L-glutamate uptake with identical pharmacological and kinetic properties. Inserting a viral epitope (tagged with V5, hemagglutinin, or FLAG) into the second extracellular domain of each isoform allowed coimmunoprecipitation and time-resolved Förster resonance energy transfer (tr-FRET) studies using transfected HEK-293 cells. Here we show for the first time that each of the four isoforms is able to combine to form homomeric and heteromeric assemblies, each of which is expressed at the cell surface of primary astrocytes. After activation of protein kinase C by phorbol ester, V5-tagged GLT-1 is rapidly removed from the cell surface of HEK-293 cells and degraded. This study provides direct biochemical evidence for oligomeric assembly of GLT-1 and reports the development of novel tools to provide insight into the trafficking of GLT-1.

Lactaturia and Loss of Sodium-dependent Lactate Uptake in the Colon of SLC5A8-deficient Mice

SLC5A8 is a member of the sodium/glucose cotransporter family. It has been proposed that SLC5A8 might act as an apical iodide transporter in the thyroid follicular cells or as a transporter of short chain monocarboxylates. We have directly addressed the functional role of SLC5A8 in vivo by generation of SLC5A8 mutant mice. We found that SLC5A8 is responsible for the re-absorption of lactate at the apical membrane of the kidney proximal tubules and of serous salivary gland ducts. In addition, SLC5A8 mediated the uptake of lactate into colonocytes under physiological conditions. We did not find any evidence of SLC5A8 being essential for the apical iodide transport in the thyroid gland, even if the ion-cotransporter SLC26A4, causing the human Pendred syndrome, is missing. Because SLC5A8 is transcriptionally silenced in many tumors, it has been proposed that SLC5A8-mediated transport of butyrate suppresses tumor formation. Treatment of Slc5a8(-/-) mice with carcinogens and breeding to the Apc(min) mouse line did not reveal a higher incidence of tumor formation. We conclude that SLC5A8 is instrumental in preventing lactaturia and loss of sodium-dependent uptake of lactate in the colon but does not have any apparent role in the prevention of tumor formation and growth.

Ubiquitination-mediated internalization and degradation of the astroglial glutamate transporter, GLT-1

Sodium-dependent glutamate uptake is essential for limiting excitotoxicity, and dysregulation of this process has been implicated in a wide array of neurological disorders. The majority of forebrain glutamate uptake is mediated by the astroglial glutamate transporter, GLT-1. We and others have shown that this transporter undergoes endocytosis and degradation in response to activation of protein kinase C (PKC), however, the mechanisms involved remain unclear. In the current study, transfected C6 glioma cells or primary cortical cultures were used to show that PKC activation results in incorporation of ubiquitin into GLT-1 immunoprecipitates. Mutation of all 11 lysine residues in the amino and carboxyl-terminal domains to arginine (11R) abolished this signal. Selective mutation of the seven lysine residues in the carboxyl terminus (C7K–R) did not eliminate ubiquitination, but it completely blocked PKC-dependent internalization and degradation. Two families of variants of GLT-1 were prepared with various lysine residues mutated to arginine. Analyses of these constructs indicated that redundant lysine residues in the carboxyl terminus were sufficient for the appearance of ubiquitinated product and degradation of GLT-1. Together these data define a novel mechanism by which the predominant forebrain glutamate transporter can be rapidly targeted for degradation.

HTERT alone immortalizes epithelial cells of renal proximal tubules without changing their functional characteristics

Telomere-dependent replicative senescence is one of the mechanisms that limit the number of population doublings of normal human cells. By overexpression of telomerase, cells of various origins have been successfully immortalized without changing the phenotype. While a limited number of telomerase-immortalized cells of epithelial origin are available, none of renal origin has been reported so far. Here we have established simple and safe conditions that allow serial passaging of renal proximal tubule epithelial cells (RPTECs) until entry into telomere-dependent replicative senescence. As reported for other cells, senescence of RPTECs is characterized by arrest in G1 phase, shortened telomeres, staining for senescence-associated beta-galactosidase, and accumulation of gamma-H2AX foci. Furthermore, ectopic expression of the catalytic subunit of telomerase (TERT) was sufficient to immortalize these cells. Characterization of immortalized RPTEC/TERT1 cells shows characteristic morphological and functional properties like formation of tight junctions and domes, expression of aminopeptidase N, cAMP induction by parathyroid hormone, sodium-dependent phosphate uptake, and the megalin/cubilin transport system. No genomic instability within up to 90 population doublings has been observed. Therefore, these cells are proposed as a valuable model system not only for cell biology but also for toxicology, drug screening, biogerontology, as well as tissue engineering approaches.

Hormonal Regulation of Sodium/Sulfate Co-Transport in Renal Epithelial Cells

Abstract. Serum sulfate concentrations are elevated in infants, young children, and pregnant women due, at least in part, to increased renal sulfate reabsorption. Little is known about the effects of hormones, particularly those involved in growth, development, and pregnancy, on renal sulfate reabsorption. The objective of this investigation was to examine the effects of growth hormone (GH), insulin-like growth factor 1 (IGF-1), progesterone (PG), and 17β-estradiol (EST) on renal sodium/sulfate co-transport. 35S-sulfate uptake was determined in Madin-Darby canine kidney (MDCK)/NaSi-1 cells (MDCK cells that have been stably transfected with rat sodium/sulfate co-transporter (NaSi-1) cDNA) and in opossum kidney (OK) cells. NaSi-1 mRNA was determined by RT-PCR and protein levels by ELISA. GH (0.1 nM) significantly increased the sodium/sulfate co-transport in MDCK/NaSi-1 cells up to 35%. IGF-1 induced a concentration-related stimulation of the sodium/sulfate co-transport with a maximal response observed at 1000 nM (59% increase). Sodium-dependent sulfate uptake was significantly increased when cells were preincubated with 10 nM PG, 10 nM EST, or 10 nM PG/10 nM EST up to 41%, 46%, or 39%, respectively. OK cells exhibited endogenous sodium-dependent sulfate transport; significantly increased sodium/sulfate co-transport was also observed in OK cells that were preincubated with GH, IGF-1, and PG/EST, although not with EST alone. The NaSi-1 mRNA and NaSi-1 protein levels were significantly increased in MDCK/NaSi-1 cells treated with 0.1 nM GH, 100 nM IGF-1, 10 nM PG, and/or 10 nM EST compared with control. These results suggest that the increased renal sulfate reabsorption that occurs in neonates, young and pregnant humans, and animals could be mediated by the increased steady-state levels of NaSi-1 mRNA produced by the higher plasma concentrations of GH, IGF-1, or PG/EST.

Study of enteral nutrients transport in intestinal hypoperfused rat model

To study the transport of glutamine and glucose, expression of their transporters and tissue morphology in intestinal hypoperfusion. Sprague-Dawley rats were randomized to receive 60 min of intestinal hypoperfusion (superior mesenteric artery clamp) or serve as normoxic controls (celiotomy only). At the same time, jejunal loops were randomized to receive in situ perfusion of mannitol,glucose,or glutamine.Intestinal brush border membrane vesicles (BBMV) were prepared by calcium precipitation. Sodium-dependent uptake of glucose and glutamine into BBMV were quantitated by rapid mixing and filtration. Histologic examination and immunohistochemistry were performed by pathologists blinded to the groups. When compared with the control group, tissue lactate concentration of the hypoperfused group increased significantly (4.9+/-0.3 vs 3.1+/-0.2), especially in the glucose perfused groups (P<0.01). Transport and transporters of glucose in brush border, but not glutamine, decreased during hypoperfusion [(76+/-10) pmol d mg(-1) d 10 s(-1) vs (290+/-13)pmol d mg(-1) d 10 s(-1)]. Tissue structural damage was most severe in glucose perfused groups during hypoperfusion. Transport and expression of transporters of glucose and glutamine in enteral nutrition are differently regulated under conditions of trauma and stress.

Novel NaPi-2c mutations that cause mistargeting of NaPi-2c protein and uncoupling of Na-Picotransport cause HHRH

hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is an autosomal recessive inherited disorder of mineral and bone metabolism. It is characterized by hypophosphatemia, rickets, and increased serum 1,25-dihydroxyvitamin D concentration, resulting in secondary absortive hypercalciuria and

Sodium signals in cerebellar Purkinje neurons and Bergmann glial cells evoked by glutamatergic synaptic transmission

Glial cells express specific high-affinity transporters for glutamate that play a central role in glutamate clearance at excitatory synapses in the brain. These transporters are electrogenic and are mainly energized by the electrochemical gradient for sodium. In the present study, we combined somatic whole-cell patch-clamp recordings with quantitative Na+ imaging in fine cellular branches of cerebellar Bergmann glial cells and in dendrites of Purkinje neurons to analyze intracellular Na+ signals close to activated synapses. We demonstrate that pressure application of glutamate and glutamate agonists causes local Na+ signals in the mM range. Furthermore, we analyzed the pharmacological profile, as well as the time course and spatial distribution of Na+ signals following short synaptic burst stimulation of parallel or climbing fibers. While parallel fibers stimulation resulted in local sodium transients that were largest in processes close to the stimulation pipette, climbing fibers stimulation elicited global sodium transients throughout the entire cell. Glial sodium signals amounted to several mM, were mainly caused by sodium influx following inward transport of glutamate and persisted for tens of seconds. Sodium transients in dendrites of Purkinje neurons, in contrast, were mainly caused by activation of AMPA receptors and had much faster kinetics. By reducing the driving force for sodium-dependent glutamate uptake, intracellular sodium accumulation in glial cells upon repetitive activity might provide a negative feedback mechanism, promoting the diffusion of glutamate and the activation of extrasynaptic glutamate receptors at active synapses in the cerebellum.

Influence of starch and casein administered postruminally on small intestinal sodium-glucose cotransport activity and expression

Twenty-four Holstein steer calves (88 ± 3kg) with abomasal catheters were randomly assigned within blocks to one of four treatments. The treatments consisted of four abomasal infusions: water (control), 4 g/kg BW.d-1 of partially hydrolyzed starch (SH), 0.6 g/kg BW.d-1 of casein, and a mix of SH + casein. The small intestine was collected and five equidistan, 1m sites were identified (duodenum, jejunum 1, jejunum 2 jejunum 3 and ileum). Maltase specific activity in mucosal homogenate and brush border membrane vesicles, SGLT1 protein abundance, and sodium-dependent glucose uptake in brush border membrane vesicles did not differ between the calves receiving different abomasal infusion treatments. However, maltase specific activity in homogenates and brush border membrane vesicles increased four-fold from the duodenum to the first jejunal site before declining steadily towards the ileum (p=0.0145 p=0.0020, respectively). The SGLT1 abundance differed by intestinal sampling site (p=0.0162). These data indicated that cattle might not have the ability to alter the capacity for glucose uptake when challenged with different substrates and that the regulation of SGLT1 expression could differ between bovine and other species.

Impact of Inorganic Carbon Availability on Microcystin Production by Microcystis aeruginosa PCC 7806

Batch culture experiments with the cyanobacterium Microcystis aeruginosa PCC 7806 were performed in order to test the hypothesis that microcystins (MCYSTs) are produced in response to a relative deficiency of intracellular inorganic carbon (C(i,i)). In the first experiment, MCYST production was studied under increased C(i,i) deficiency conditions, achieved by restricting sodium-dependent bicarbonate uptake through replacement of sodium bicarbonate in the medium with its potassium analog. The same experimental approach was used in a second experiment to compare the response of the wild-type strain M. aeruginosa PCC 7806 with its mcyB mutant, which lacks the ability to produce MCYSTs. In a third experiment, the impact of varying the C(i,i) status on MCYST production was examined without suppressing the sodium-dependent bicarbonate transporter; instead, a detailed investigation of a dark-light cycle was performed. In all experiments, a relative C(i,i) deficiency was indicated by an elevated variable fluorescence signal and led to enhanced phycocyanin cell quotas. Higher MCYST cell quotas (in the first and third experiments) and increased total (intracellular plus extracellular) MCYST production (in the first experiment) were detected with increased C(i,i) deficiency. Furthermore, the MCYST-producing wild-type strain and its mcyB mutant showed basically the same response to restrained inorganic carbon uptake, with elevated variable fluorescence and phycocyanin cell quotas with increased C(i,i) deficiency. The response of the wild type, however, was distinctly stronger and also included elevated chlorophyll a cell quotas. These differences indicate the limited ability of the mutant to adapt to low-C(i,i) conditions. We concluded that MCYSTs may be involved in enhancing the efficiency of the adaptation of the photosynthetic apparatus to fluctuating inorganic carbon conditions in cyanobacterial cells.

NCX3 is a major functional isoform of the sodium–calcium exchanger in osteoblasts

The calcium phosphate-based skeleton of vertebrates serves as the major reservoir for metabolically available calcium ions. The skeleton is formed by osteoblasts which first secrete a proteinaceous matrix and then provide Ca++ for the calcification process. The two calcium efflux ports found in most cells are the plasma membrane Ca-ATPase (PMCA) and the sodium-calcium exchanger (NCX). In osteoblasts, PMCA and NCX are located on opposing sides of the cell with NCX facing the mineralizing bone surface. Two isoforms of NCX have been identified in osteoblasts NCX1, and NCX3. The purpose of this study was to determine the extent to which each of the two NCX isoforms support delivery of Ca++ into sites of calcification and to discern if one could compensate for the other. SiRNA technology was used to knockdown each isoform separately in MC3T3-E1 osteoblasts. Osteoblasts in which either NCX1 or NCX3 was impaired were tested for Ca++ efflux using the Ca++ specific fluorophore, fluo-4, in a sodium-dependent calcium uptake assay adapted for image analysis. NCX3 was found to serve as a major contributor of Ca++ translocation out of osteoblasts into calcifying bone matrix. NCX1 had little to no involvement.

Nutritional, physiological, and histological responses in Atlantic salmon, Salmo salar L. fed diets with genetically modified maize

The objective of this study was to evaluate whether standard fish meal diets prepared with increasing levels of genetically modified (GM; 150 and 300 g kg−1) maize (event MON810®) as a starch source, showed any nutritional or physiological adverse effects on Atlantic salmon, Salmo salar L. postsmolt. The diets with low or high inclusions of GM maize and its near-isogenic parental line (nongenetically modified; nGM maize), were balanced with Suprex maize (Reference) to obtain compositional equivalency of diet starch, sugars and all other nutrients. Total starch level in all diets was 160 g kg−1. After 82 days of feeding, fish growth was high in all groups, however fish fed the GM maize showed slight but significant lower feed intake, which was followed by slight but significant lower specific growth rate and final body weights, compared with fish fed nGM maize, none of the groups varied significantly from fish fed the Reference diet. There was no variation in feed conversion ratios (FCR), protein and lipid efficiency ratios (PER and LER), or protein- and lipid-productive values (PPV and LPV) in this study. No significant effect of maize type was detected on apparent digestibility coefficients (ADC) of dry matter, protein or lipid. Hematological analysis and plasma nutrients varied within normal ranges for Atlantic salmon in all diet groups, except for somewhat elevated aspartate aminotransferase (ASAT) values in all groups. Hepatosomatic index (HSI) with values ranging from 1.37 to 1.60, was significantly higher for the high GM maize group compared with the high nGM maize group but not when compared with the Reference diet group. Lowered spleen (SSI) and head-kidney somatic indices (H-KSI) were registered when fed GM compared with nGM maize, the Reference treatment was however, equal to both. Distal intestine somatic index (DISI) was significantly higher for GM maize-fed fish compared with nGM maize-fed fish, but not significantly different from the Reference diet group. Histological evaluation of the mid- and distal intestine, liver, spleen and head-kidney did not reveal any diet-related morphological changes. Maltase activities in the mid- and distal intestinal tissue homogenates were affected by diet, the fish fed high GM maize having higher activities compared with high nGM maize-fed fish. Leucine aminopeptidase (LAP) and alkaline phosphatase (AP) activities were not affected by diet. Sodium-dependent d-glucose uptake in brush border membrane vesicles (BBMV) isolated from pyloric caeca of fish fed high GM maize was significantly higher than that found in fish fed the analogous diet with high nGM maize. Based on the present findings, the conclusions made are: Atlantic salmon smolts fed GM maize (event MON810®), its near-isogenic parental line and suprex maize (Reference diet), all resulted in high growth rates, ADC and feed utilization. Health, when evaluated by means of mortality (low), normal ranges of blood and plasma parameters, except somewhat elevated ASAT values and minor variations in organ sizes, were considered good in all diet groups. The changes in the glucose transport mechanism and intestinal maltase enzyme activity in the gastrointestinal tract warrant further studies.

Na+-d-glucose cotransporter in the kidney of Leucoraja erinacea: molecular identification and intrarenal distribution

Studies on membrane vesicles from the kidney of Leucoraja erinacea suggested the sole presence of a sodium-D-glucose cotransporter type 1 involved in renal D-glucose reabsorption. For molecular characterization of this transport system, an mRNA library was screened with primers directed against conserved regions of human sglt1. A cDNA was cloned whose nucleotide and derived amino acid sequence revealed high homology to sodium glucose cotransporter 1 (SGLT1). Xenopus laevis oocytes injected with the respective cRNA showed sodium-dependent high-affinity uptake of D-glucose. Many positions considered functionally essential for sodium glucose cotransporter 1 (SGLT1) are also found in the skate protein. High conservation preferentially in transmembrane helices and small linking loops suggests early appearance and continued preservation of these regions. Larger loops, especially loop 13, which is associated with phlorizin binding, were more variable, as is the interaction with the specific inhibitor in various species. To study the intrarenal distribution of the transporter, a skate SGLT1-specific antibody was generated. In cryosections of skate kidney, various nephron segments could be differentiated by lectin staining. Immunoreaction with the antibody was observed in the proximal tubule segments PIa and PIIa, the early distal tubule, and the collecting tubule. Thus Leucoraja, in contrast to the mammalian kidney, employs only SGLT1 to reabsorb d-glucose in the early, as well as in the late segments of the proximal tubule and probably also in the late distal tubule (LDT). Thereby, it differs also partly from the kidney of the close relative Squalus acanthias, which uses SGLT2 in more distal proximal tubule segments but shows also expression in the later nephron parts.