Biomedical subjects

E M Wright

Publications and source records attributed to E M Wright.

At least 91 records · Page 5Linked to original sources

PubMed1997

Water transport by the Na+/glucose cotransporter under isotonic conditions.

Solute cotransport in the Na+/glucose cotransporter is directly coupled to significant water fluxes. The water fluxes are energized by the downhill fluxes of the other substrates by a mechanism within the protein itself. In the present paper we investigate the Na+/glucose cotransporter expressed in Xenopus oocytes. We present a method which allows short-term exposures to sugar under voltage clamp conditions. We demonstrate that water is cotransported with the solutes despite no osmotic differences between the external and intracellular solutions. There is a fixed ratio of 195:1 between the number of water molecules and the number of Na+ ions transported, equivalent to 390 water molecules per glucose molecule. Unstirred layer effects are ruled out on the basis of experiments on native oocytes incubated with the ionophores gramicidin D or nystatin.

Animals↗

PubMed1997

Na/D-glucose cotransport and SGLT1 expression in hen colon correlates with dietary Na+.

We have tested whether separately varying the content of either Na or Cl in diets causes earlier observed increase in Na-coupled sugar and amino acid transport induced by high NaCl diets in hen colon. A comparison was also made between the dependence of the Na-coupled transport on a pure wheat/barley/soya diet against a diet with supplements of essential amino acids, fatty acids, vitamins, and trace elements, as a test for possible elimination of the cotransporters due to a deficient diet. Na/nutrient-coupled transport was measured as changes in short circuit current. The level of expressed Na/glucose cotransporters, SGLT1, due to dietary alterations was followed by quantitative Western blot and immunodetection of SGLT1 in colon, and the dietary effects on plasma aldosterone were assessed as well. An observed switch in transport from amiloride-sensitive electrodiffusive Na transport to phlorizin-sensitive Na/D-glucose cotransport and Na/amino acid-coupled transport is caused solely by increasing Na+ in the diet. Thus, neither dietary Cl- nor the dietary supplements altered the expression of Na(+)-coupled nutrient transport processes. Corroborating these findings, only Na+ in the diet increased the expression of SGLT1 in colon epithelium and suppressed aldosterone level in plasma.

Animals↗

PubMed1997

Caffeine overrides the S-phase cell cycle block in sea urchin embryos.

During the early mitotic cell cycles of the sea urchin embryo, the cell oscillates between S-phase and M-phase. In the presence of aphidicolin, a DNA synthesis inhibitor, a checkpoint control blocks the activation of the p34cdc2 protein kinase, by keeping it in the inactive, tyrosine phosphorylated form, and the embryos do not enter mitosis. Caffeine has been shown to bypass the G2/M-phase checkpoint in mammalian cells and in cycling Xenopus extracts and to induce mitosis despite the presence of damaged or unreplicated DNA. In this study we show that caffeine also induces mitosis and cell division in sea urchin embryos, in the presence of unreplicated DNA, by stimulating the tyrosine dephosphorylation of p34cdc2 and switching on its protein kinase activity. We also show that the caffeine-induced activation of the p34cdc2 protein kinase is not mediated by either of the two second messengers, calcium and cAMP, or by inhibition of the p34cdc2 tyrosine kinase. Thus, none of the mechanisms proposed for caffeine's action can explain how it overrides the S-phase checkpoint in the early cell cycles of the sea urchin embryo.

Animals↗

PubMed1997

Enhancing motivation to reduce the risk of HIV infection for economically disadvantaged urban women.

This research evaluated a motivation-based HIV risk reduction intervention for economically disadvantaged urban women. Participants completed a survey that assessed HIV-related knowledge, risk perceptions, behavioral intentions, sexual communication, substance use, and risk behavior. A total of 102 at-risk women (76% African American) were randomly assigned to either the risk reduction intervention or to a waiting list. Women were reassessed at 3 and 12 weeks. Results indicated that treated women increased their knowledge and risk awareness, strengthened their intentions to adopt safer sexual practices, communicated their intentions with partners, reduced substance use proximal to sexual activities, and engaged in fewer acts of unprotected vaginal intercourse. These effects were observed immediately, and most were maintained at follow-up.

Adolescent↗

PubMed1997

Regulation of Na+/glucose cotransporters.

Na+/glucose cotransporters (SGLTs) are expressed in the small intestine and the proximal renal tubule, where they play a central role in the absorption of glucose and galactose from food and the reabsorption of glucose from the glomerular filtrate. The regulation of intestinal sugar absorption occurs over two distinct time scales, one over days and the other over minutes. This review focuses on the mechanisms involved in the shorter-term regulation. Recent studies of the mouse intestine in vitro demonstrated that Na+/glucose cotransport is increased two- to eightfold within minutes by the application of forskolin, an agent that increases intracellular cyclic AMP levels. Here we explore how cyclic AMP may upregulate Na+/glucose cotransport. Our strategy was to express cloned SGLT1s in Xenopus laevis oocytes and then use electrophysiological methods to measure (i) the kinetics of Na+/glucose cotransport, (ii) the number of cotransporters in the plasma membrane, and (iii) the net rate of exo- and endocytosis before and after activation of protein kinases. To evaluate the role of cotransporter phosphorylation, we have examined the effect of protein kinase activation on various SGLT1 isoforms and other cotransporters. In oocytes expressing rabbit SGLT1, the activation of protein kinase A (PKA) increased the maximum rate of Na+/glucose cotransport by 30%, and the activation of protein kinase C (PKC) decreased the maximum rate of transport by 60%. Changes in maximum transport rates were accompanied by proportional changes in the number of cotransporters in the plasma membrane and by changes in the area of the membrane. We conclude that PKA and PKC regulate rabbit SGLT1 activity by modulating the number of cotransporters in the plasma membrane and that this occurs through regulation of exocytosis and endocytosis. Given the size of intracellular transport vesicles containing SGLT1, 100-120 nm in diameter, and the density of cotransporters in these vesicles, 10-20 per vesicle, we estimate that the net rate of SGLT1 vesicle exocytosis is about 10,000 s-1 and that this rate increases 100-fold after activation of PKA. The effect of PKA is independent of the presence or absence of consensus sites for phosphorylation on SGLT1. Surprisingly, the effects of PKA or PKC depend critically on the sequence of the contransporter being expressed in the oocyte, e.g. activation of PKC inhibited rabbit and rat SGLT1, but stimulated human SGLT1. This dependency suggests that the regulation of vesicle trafficking by protein kinases depends upon the structure of the cotransporter expressed in the oocyte. Similar considerations apply to other classes of cotransporters, such as the neurotransmitter and dipeptide cotransporters. Our working hypothesis is that the regulation of cotransporter expression by protein kinases occurs largely by regulated exo- and endocytosis, and that the effect of the protein kinases is indirect and determined by critical domains in the cotransporter.

Animals↗

PubMed1996

Biophysical characteristics of the pig kidney Na+/glucose cotransporter SGLT2 reveal a common mechanism for SGLT1 and SGLT2.

The Na+-dependent, low affinity glucose transporter SGLT2 cloned from pig kidney is 76% identical (at the amino acid level) to its high affinity homologue SGLT1. Using two-microelectrode voltage clamp, we have characterized the presteady-state and steady-state kinetics of SGLT2 expressed in Xenopus oocytes. The kinetic properties of the steady-state sugar-evoked currents as a function of external Na+ and alpha-methyl-D-glucopyranoside (alphaMG) concentrations were consistent with an ordered, simultaneous transport model in which Na+ binds first. Na+ binding was voltage-dependent and saturated with hyperpolarizing voltages. Phlorizin was a potent inhibitor of the sugar-evoked currents (KiPz approximately 10 microM) and blocked an inward Na+ current in the absence of sugar. SGLT2 exhibited Na+-dependent presteady-state currents with time constants 3-7 ms. Charge movements were described by Boltzmann relations with apparent valence approximately 1 and maximal charge transfer approximately 11 nC, and were reduced by the addition of sugar or phlorizin. The differences between SGLT1 and SGLT2 were that (i) the apparent affinity constant (K0.5) for alphaMG (approximately 3 mM) was an order of magnitude higher for SGLT2; (ii) SGLT2 excluded galactose, suggesting discrete sugar binding; (iii) K0.5 for Na+ was lower in SGLT2; and (iv) the Hill coefficient for Na+ was 1 for SGLT2 but 2 for SGLT1. Simulations of the six-state kinetic model previously proposed for SGLT1 indicated that many of the kinetic properties observed in SGLT2 are expected by simply reducing the Na+/glucose coupling from 2 to 1.

Animals↗

PubMed1996

Cotransport of water by the Na+/glucose cotransporter.

Water is transported across epithelial membranes in the absence of any hydrostatic or osmotic gradients. A prime example is the small intestine, where 10 liters of water are absorbed each day. Although water absorption is secondary to active solute transport, the coupling mechanism between solute and water flow is not understood. We have tested the hypothesis that water transport is directly linked to solute transport by cotransport proteins such as the brush border Na+/glucose cotransporter. The Na+/glucose cotransporter was expressed in Xenopus oocytes, and the changes in cell volume were measured under sugar-transporting and nontransporting conditions. We demonstrate that 260 water molecules are directly coupled to each sugar molecule transported and estimate that in the human intestine this accounts for 5 liters of water absorption per day. Other animal and plant cotransporters such as the Na+/CI-/gamma-aminobutyric acid, Na+/iodide and H+/amino acid transporters are also able to transport water and this suggests that cotransporters play an important role in water homeostasis.

Animals↗

PubMed1996

Transport mechanism of the cloned potato H+/sucrose cotransporter StSUT1.

The transport mechanism of the potato StSUT1 H+/sucrose cotransporter expressed in Xenopus oocytes was investigated using the 2-electrode voltage clamp and radiotracer flux methods. Sucrose induced inward currents through the transporter that were dependent on the extracellular sucrose and H+ concentrations and the membrane voltage. The activation of StSUT1 by H+ and sucrose displayed Michaelis-Menten-type kinetics suggestive of a 1:1 H+:sucrose stoichiometry. This was confirmed by simultaneously measuring inward currents and sucrose flux in voltage-clamped oocytes. The apparent affinities K0.5 for H+ and sucrose were voltage-dependent. At -150 mV Ksuc0.5 was 0.5 +/- 0.07 mM at 10 microM H+o, and KH0.5 was 0.1 +/- 0.05 microM at 20 mM sucroseo. StSUT1 exhibited presteady-state transient currents, which relaxed with time constants between <1 and 4 ms and fitted to the Boltzmann equation: maximum charge transfer Qmax approximately 1.8 nanocoulombs; apparent valence z approximately 1; potential for 50% charge transfer V0.5 approximately -15 mV at 0.032 microM H+o and -45 mV at 10 microM H+o. The steady-state data were used to formulate a kinetic model for sucrose transport, and computer simulations were performed to obtain rate constants for the partial reaction steps. Our model is consistent with protons binding to StSUT1 before sucrose with both ligands transported simultaneously across the membrane.

Animals↗

PubMed1996

Regulation of the mouse retinal taurine transporter (TAUT) by protein kinases in Xenopus oocytes.

The goal was to investigate the role of protein kinases in modulating taurine transporter activity in Xenopus laevis oocytes expressing the mouse retinal Na+/C-/taurine transporter. The currents generated by the taurine transporter were studied with a two-electrode voltage clamp and we recorded the maximal current (Imax), presteady-state charge transfer Q, and membrane capacitance Cm. 8-Br-cAMP, a membrane-permeable activator of the cAMP-dependent protein kinase (PKA), decreased Imax (41%), Q (41%) and Cm (10%). Similarly, 1 microM sn-1,2-dioctanoylglycerol (DOG), an activator of the Ca2+/diacylglycerol-dependent protein kinase (PKC), decreased Imax (56%), Q (37%), and Cm (9%). Calyculin A, a specific inhibitor of protein phosphatases 1 and 2A, also produced effects similar to those of 8-Br-cAMP and DOG, and decreased Imax (64 %), Q (38%), and Cm (10%). We conclude that the taurine transporter is regulated by activators of PKA and PKC, and regulation occurs largely by changes in the number of transporters in the plasma membrane.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1996

Regulation of Na+/glucose cotransporter expression by protein kinases in Xenopus laevis oocytes.

Cotransporters are proteins responsible for the accumulation of nutrients, neurotransmitters, and drugs in cells. As forskolin has been shown to stimulate intestinal Na+/glucose cotransport, we have used electrophysiological techniques to examine the role of protein kinases in regulating Na+/glucose cotransporters, SGLT1, expressed in Xenopus laevis oocytes. We monitored SGLT1 kinetics, the number of SGLT1 cotransporters in the plasma membrane, and plasma membrane area before and after activation of protein kinases. 8-Bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) and sn-1, 2-dioctanoylglycerol (DOG) were used as membrane permeable activators of protein kinases A (PKA) and C (PKC), respectively. In oocytes expressing rabbit SGLT1 8-Br-cAMP increased by 28 +/- 4% (n = 10), and DOG decreased by 51 +/- 5% (n = 13) the maximum rate of Na+/glucose cotransport. These reversible changes in the maximum transport rate occurred within minutes, and were accompanied by proportional changes in the number of cotransporters in the membrane and area of the plasma membrane. This suggests that protein kinases regulate rabbit SGLT1 activity by controlling the distribution of transporters between intracellular compartments and the plasma membrane, and that this occurs by exo- and endocytosis. Similar increases in maximum transport were obtained with activation of PKA in oocytes expressing rabbit, human, and rat SGLT1 isoforms, but with activation of PKC the response was isoform-dependent. PKC activation decreased the maximum rate of transport by rabbit and rat SGLT1, but increased transport by human SGLT1. We conclude that: (i) the regulation of SGLT1 expression in oocytes by protein kinases occurs mainly by regulated endo- and exocytosis; (ii) it is independent of consensus phosphorylation sites in the transporter; and (iii) the effect of a given kinase depends upon the actual sequence of the cotransporter expressed. These considerations may also apply to the regulation of other cotransporters by protein kinases in oocytes, cells, and tissues.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1996

Sugar binding to Na+/glucose cotransporters is determined by the carboxyl-terminal half of the protein.

d-Glucose is absorbed across the proximal tubule of the kidney by two Na+/glucose cotransporters (SGLT1 and SGLT2). The low affinity SGLT2 is expressed in the S1 and S2 segments, has a Na+:glucose coupling ratio of 1, a K0.5 for sugar of approximately 2 mM, and a K0.5 for Na+ of approximately 1 mM. The high affinity SGLT1, found in the S3 segment, has a coupling ratio of 2, and K0.5 for sugar and Na+ of approximately 0.2 and 5 mM, respectively. We have constructed a chimeric protein consisting of amino acids 1-380 of porcine SGLT2 and amino acids 381-662 of porcine SGLT1. The chimera was expressed in Xenopus oocytes, and steady-state kinetics were characterized by a two-electrode voltage-clamp. The K0.5 for alpha-methyl-d-glucopyranoside (0.2 mM) was similar to that for SGLT1, and like SGLT1 the chimera transported D-galactose and 3-O-methylglucose. In contrast, SGLT2 transports poorly D-galactose and excludes 3-O-methylglucose. The apparent K0.5Na was 3.5 mM (at -150 mV), and the Hill coefficient ranged between 0.8 and 1.5. We conclude that recognition/transport of organic substrate is mediated by interactions distal to amino acid 380, while cation binding is determined by interactions arising from the amino- and carboxyl-terminal halves of the transporters. Surprisingly, the chimera transported alpha-phenyl derivatives of D-glucose as well as the inhibitors of sugar transport: phlorizin, deoxyphlorizin, and beta-D-glucopyranosylphenyl isothiocyanate are transported with high affinity (K0.5 for phlorizin was 5 microM). Thus, the pocket for organic substrate binding is increased from 10 x 5 x 5 (A) for SGLT1 to 11 x 18 x 5 (A) for the chimera.

Amino Acid Sequence↗

PubMed1996

Mechanisms of the human intestinal H+-coupled oligopeptide transporter hPEPT1.

The hPEPT1 cDNA cloned from human intestine (Liang, R., Fei, Y.-J., Prasad, P. D., Ramamoorthy, S., Han, H., Yang-Feng, T. L., Hediger, M. A., Ganapathy, V., and Leibach, F. H. (1995) J. Biol. Chem. 270, 6456-6463) encodes a H+/oligopeptide cotransporter. Using two-microelectrode voltage-clamp in Xenopus oocytes expressing hPEPT1, we have investigated the transport mechanisms of hPEPT1 with regard to voltage dependence, steady-state kinetics, and transient charge movements. The currents evoked by 20 mM glycyl-sarcosine (Gly-Sar) at pH 5.0 were dependent upon membrane potential (Vm) between -150 mV and +50 mV. Gly-Sar-evoked currents increased hyperbolically with increasing extracellular [H+], with Hill coefficient approximately 1, and the apparent affinity constant (K0.5H) for H+ was in the range of 0.05 1 microM. K0.5 for Gly-Sar (K0.5GS) was dependent upon Vm and pH; at -50 mV, K0.5H was minimal (approximately 0.7 mM) at pH 6.0. Following step-changes in Vm, in the absence of Gly-Sar, hPEPT1 exhibited H+-dependent transient currents with characteristics similar to those of Na+-coupled transporters. These charge movements (which relaxed with time constants of 2-10 ms) were fitted to Boltzmann relations with maximal charge (Qmax) of up to 12 nC; the apparent valence was determined to be approximately 1. Qmax is an index of the level of transporter expression which for hPEPT1 was in the order of 1011/oocyte. In general our data are consistent with an ordered, simultaneous transport model for hPEPT1 in which H+ binds first.

Animals↗

PubMed1996

Membrane topology of the human Na+/glucose cotransporter SGLT1.

The membrane topology of the human Na+/glucose cotransporter SGLT1 has been probed using N-glycosylation scanning mutants and nested truncations. Functional analysis proved essential for establishment of signal-anchor topology. The resultant model diverges significantly from previously held suppositions of structure based primarily on hydropathy analysis. SGLT1 incorporates 14 membrane spans. The N terminus resides extracellularly, and two hydrophobic regions form newly recognized membrane spans 4 and 12; the large charged domain near the C terminus is cytoplasmic. This model was evaluated further using two advanced empirically-based algorithms predictive of transmembrane helices. Helix ends were predicted using thermo-dynamically-based algorithms known to predict x-ray crystallographically determined transmembrane helix ends. Several considerations suggest the hydrophobic C terminus forms a 14th transmembrane helix, differentiating the eukaryotic members of the SGLT1 family from bacterial homologues. Our data inferentially indicate that these bacterial homologues incorporate 13 spans, with an extracellular N terminus. The model of SGLT1 secondary structure and the predicted helix ends signify information prerequisite for the rational design of further experiments on structure/function relationships.

Amino Acid Sequence↗

PubMed1996

Kinetics and specificity of a H+/amino acid transporter from Arabidopsis thaliana.

The amino acid transporter AAP1/NAT2 recently cloned from Arabidopsis thaliana was expressed in Xenopus oocytes, and we used electrophysiological, radiotracer flux, and electron microscopic methods to characterize the biophysical properties, kinetics, and specificity of the transporter. Uptake of alanine was H(+)-dependent increasing from 14 pmol/oocyte/h at 0.032 microM H+ to 370 pmol/oocyte/h at 10 microM H+. AAP1 was electrogenic; there was an amino acid-induced depolarization of the oocyte plasma membrane and net inward currents through the transporter due to the transport of amino acids favoring neutral amino acids with shortside chains. The maximal current (imax) for alanine, proline, glutamine, histidine, and glutamate was voltage and [H+]o-dependent. Similarly, the imaxH was voltage and [amino acid]o-dependent. The imax for both H+ and amino acid were dependent on the concentrations of their respective cosubstrates, suggesting that both ligands bind randomly to the transporter. The K0.5 of the transporter for amino acids decreased as [H+]o increased and was lower at negative membrane potentials. The K0.5 for H+ was relatively voltage-independent and decreased as [amino acid]o increased. This positive cooperativity suggests that the transporter operates via a simultaneous mechanism. The Hill coefficients n for amino acids and H+ were > 1, suggesting that the transporter has more than one binding site for both H+ and amino acid. Freeze-fracture electron microscopy was used to estimate the number of transporters expressed in the plasma membrane of oocytes. The density of particles on the protoplasmic face of the plasma membrane of oocytes expressing AAP1 increased approximately 5-fold above water-injected controls and corresponded to a turnover number 350 to 800 s-1.

Amino Acid Transport Systems↗

PubMed1996

Prenatal identification of a heterozygous status in two fetuses at risk for glucose-galactose malabsorption.

Glucose-galactose malabsorption (GGM) is an autosomal recessive disorder which presents with severe osmotic diarrhoea shortly after birth. Two proband siblings with GGM were previously demonstrated to contain a missense mutation (D28N) in the Na(+)-dependent glucose/galactose cotransporter (SGLT1) that accounts for the defect in sugar absorption. Prenatal screening for GGM was performed in two subsequent pregnancies in this large consanguineous family. The first exon of the SGLT1 gene was PCR-amplified from genomic DNA and screened for the presence of the D28N mutation by EcoRV restriction digestion. The proband's sibling was heterozygous and a cousin was not a carrier of the D28N mutation. Both children at 2-years of age remain healthy and have had no diarrhoeal symptoms. Molecular biology techniques will allow a prospective determination of the presence of an abnormal SGLT1 allete and potentially decrease the postnatal morbidity.

Amniocentesis↗

PubMed1996

Sodium cotransporters.

Recent studies of cloned mammalian sodium cotransporters in heterologous systems have revealed that these integral membrane proteins serve multiple functions as cotransporters, uniporters, channels and water transporters. Some progress has been gained in understanding their secondary structure, but information on helical bundling and tertiary structure is lacking. Site-directed mutagenesis and the construction of chimeras have resulted in the identification of residues and domains involved in ligand binding, and natural mutations have also been found that are responsible for human genetic diseases. Major factors in the short-term regulations of cotransporter function by protein kinases are exocytosis and endocytosis.

Biological Transport↗

PubMed1996

Defects in Na+/glucose cotransporter (SGLT1) trafficking and function cause glucose-galactose malabsorption.

Cotransporters harness ion gradients to drive 'active' transport of substrates into cells, for example, the Na+/glucose cotransporter (SGLT1) couples sugar transport to Na+ gradients across the intestinal brush border. Glucose-Galactose Malabsorption (GGM) is caused by a defect in SGLT1. The phenotype is neonatal onset of diarrhea that results in death unless these sugars are removed from the diet. Previously we showed that two sisters with GGM had a missense mutation in the SGLT1 gene. The gene has now been screened in 30 new patients, and a heterologous expression system has been used to link the mutations to the phenotype.

Amino Acid Sequence↗

PubMed1996

Local perinuclear calcium signals associated with mitosis-entry in early sea urchin embryos.

Using calcium-sensitive dyes together with their dextran conjugates and confocal microscopy, we have looked for evidence of localized calcium signaling in the region of the nucleus before entry into mitosis, using the sea urchin egg first mitotic cell cycle as a model. Global calcium transients that appear to originate from the nuclear area are often observed just before nuclear envelope breakdown (NEB). In the absence of global increases in calcium, confocal microscopy using Calcium Green-1 dextran indicator dye revealed localized calcium transients in the perinuclear region. We have also used a photoinactivatable calcium chelator, nitrophenyl EGTA (NP-EGTA), to test whether the chelator-induced block of mitosis entry can be reversed after inactivation of the chelator. Cells arrested before NEB by injection of NP-EGTA resume the cell cycle after flash photolysis of the chelator. Photolysis of chelator triggers calcium release. TreatmenT with caFfeine to enhance calcium-induced calcium release increases the amplitude of NEB-associated calcium transients. These results indicate that calcium increases local to the nucleus are required to trigger entry into mitosis. Local calcium transients arise in the perinuclear region and can spread from this region into the cytoplasm. Thus, cell cycle calcium signals are generated by the perinuclear mitotic machinery in early sea urchin embryos.

Animals↗