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Biomedical subjects

E M Wright

Publications and source records attributed to E M Wright.

At least 127 records · Page 7Linked to original sources
PubMed1993

Regulation of mouse choroid plexus apical Cl- and K+ channels by serotonin.

Using patch-clamp techniques, we have characterized ion channels in the apical membrane of the mouse choroid plexus epithelium and have examined the effect of serotonin on these channels. When the pipette contained 140 mM KCl and the bath contained NaCl Ringer solution, cell-attached patches revealed both Cl- and K+ channels. The Cl- channel was activated by hyperpolarizing membrane potentials, and 70% were also activated by large depolarizing potentials (pipette potential, Vp, more negative than -40 mV). The channel exhibited linear current-voltage (I-V) relations with a conductance of 4 +/- 1 pS (n = 30), and a reversal potential at Vp = -14 +/- 1 mV (n = 30). The majority of the K+ channels (84%) were activated by depolarizing membrane potentials. These exhibited linear I-V relations with a conductance of 18 +/- 1 pS (n = 10) and a reversal potential at Vp = -51 +/- 8 mV (n = 10). Serotonin (10(-6) M) increased the open probability (Po) of active Cl- channels (n = 20) by an order of magnitude at the resting potential (Vp = 0 mV) as well as activating previously silent Cl- channels. In contrast, complete inhibition of K+ channel activity was observed in the majority of experiments. There was a 30 s delay after exposure of the tissue to serotonin, thereafter the K+ channel was rapidly inhibited (within 1 min) prior to the stimulation of the Cl- channel. Stimulation of the Cl- channel by serotonin was abolished by mianserin (10(-3) M). We conclude that serotonin exerts its effect on apical Cl- channels via the 5-HT1c receptor. The modulation of these channels by serotonin may be important to CSF secretion and its regulation.

Animals↗
PubMed1993

Relaxation kinetics of the Na+/glucose cotransporter.

An important class of integral membrane proteins, cotransporters, couple solute transport to electrochemical potential gradients; e.g., the Na+/glucose cotransporter uses the Na+ electrochemical potential gradient to accumulate sugar in cells. So far, kinetic analysis of cotransporters has mostly been limited to steady-state parameters. In this study, we have examined pre-steady-state kinetics of Na+/glucose cotransport. The cloned human transporter (hSGLT1) was expressed in Xenopus oocytes, and voltage-clamp techniques were used to monitor current transients after step changes in membrane potential. Transients exhibited a voltage-dependent time constant (tau) ranging between 2 and 10 ms. The charge movement Q was fitted to a Boltzmann relation with maximal charge Qmax of approximately 20 nC, apparent valence z of 1, and potential V0.5 of -39 mV for 50% Qmax. Lowering external Na+ from 100 to 10 mM reduced Qmax 40%, shifted V0.5 from -39 to -70 mV, had no effect on z, and reduced the voltage dependence of tau. Qmax was independent of, but tau was dependent on, temperature (a 10 degrees C increase increased tau by a factor of approximately 2.5 at -50 mV). Addition of sugar or phlorizin reduced Qmax. Analyses of hSGLT1 pre-steady-state kinetics indicate that transfer upon a step of membrane potential in the absence of sugar is due to two steps in the reaction cycle: Na+ binding/dissociation (30%) and reorientation of the protein in the membrane field (70%). The rate-limiting step appears to be Na+ binding/dissociation. Qmax provides a measure of transporter density (approximately 10(4)/microns 2). Charge transfer measurements give insight into the partial reactions of the Na+/glucose cotransporter, and, combined with genetic engineering of the protein, provide a powerful tool for studying transport mechanisms.

Animals↗
PubMed1993

Regulation of the ovine intestinal Na+/glucose co-transporter (SGLT1) is dissociated from mRNA abundance.

We have investigated the mechanisms of regulation of the Na+/glucose co-transporter (SGLT1) in a ruminant animal, which is an exceptional model system for studying intestinal glucose transport. Pre-ruminant lambs absorb glucose, produced by hydrolysis of the milk sugar lactose, in the intestine via apical SGLT1 and basolateral facilitative glucose transporters (GLUT2). Weaning coincides with the development of the rumen, and consequently the amount of hexoses reaching the small intestine of the ruminant sheep is undetectable. During development, SGLT1 activity and abundance in intestinal brush-border membranes decreased by over 200-fold, and either maintaining lambs on a milk replacer diet or infusing sheep intestine with D-glucose restored co-transporter activity and expression. We have measured ovine intestinal SGLT1 mRNA levels during development, with changes in diet and after direct infusion of D-glucose or methyl alpha-D-glucopyranoside into the intestinal lumen, in order to determine the level of regulation. During development, mRNA levels decreased only 4-fold. Lambs maintained on a milk replacer diet showed no change in mRNA levels relative to age-matched controls. Finally, upon infusion of the intestine of the ruminant sheep with sugars, D-glucose infusion increased SGLT1 mRNA, but only by 2-fold, compared with a 60-90-fold increase in co-transporter number and activity. Since the change in Na(+)-dependent glucose transport activity is correlated with SGLT1 protein abundance, and since changes in mRNA levels do not account for the dramatic changes in protein abundance, we conclude that the principal level of SGLT1 regulation by luminal sugar is translational or post-translational.

Animals↗
PubMed1993

Assignment of the human Na+/glucose cotransporter gene SGLT1 to chromosome 22q13.1.

The Na+/glucose cotransporter gene SGLT1 encodes the primary carrier protein responsible for the uptake of the dietary sugars glucose and galactose from the intestinal lumen. SGLT1 transport activity is currently exploited in oral rehydration therapy. The 75-kDa glycoprotein is localized in the brush border of the intestinal epithelium and is predicted to comprise 12 membrane spans. In two patients with the autosomal recessive disease glucose/galactose malabsorption, the underlying cause was found to be a missense mutation in SGLT1, and the Asp28-->Asn change was demonstrated in vitro to eliminate SGLT1 transport activity. The SGLT1 gene was previously shown to reside on the distal q arm of chromosome 22 (11.2-->qter). We have used a cosmid probe for fluorescence in situ hybridization, which refines the localization to 22q13.1, and provide an example of the utility of the SGLT1 probe as a diagnostic for genetic diseases associated with translocations of chromosome 22.

Carrier Proteins↗
PubMed1993

[30 years' work on congenital glucose and galactose malabsorption: from phenotype to genotype].

Intestinal absorption of glucose plays a key role in water economy as attested by the congenital and selective glucose and galactose malabsorption which is expressed as severe watery diarrhea just after birth, leading to life-threatening dehydration. This syndrome, transmitted on an autosomal recessive mode, is the consequence of a functional defect of the glucose-sodium cotransporter at the luminal membrane of the enterocyte of the small intestine. In one family, this defect was associated with a misense mutation at position 92 of the SGLT1 gene coding for the cotransporter. The mutant RNA reproduced the transport defect after injection in xenopus oocytes. These results confirm the genetic origin of the congenital defect; in addition they indicate that the study of the relationship between phenotype and genotype of congenital defects of intestinal transport may help in the understanding of basic intestinal functions in relation with human nutrition.

Galactose↗
PubMed1993

[Thirty years of research on congenital glucose and galactose malabsorption: from phenotype to genotype].

Intestinal absorption of glucose plays a key role in water economy as attested by the congenital and selective glucose and galactose malabsorption which is expressed as severe watery diarrhea just after birth, leading to life-threatening dehydration. This syndrome, transmitted on an autosomal recessive mode, is the consequence of a functional defect of the glucose-sodium cotransporter at the luminal membrane of the enterocyte of the small intestine. In one family, this defect was associated with a missense mutation at position 92 of the SGLT1 gene coding for the cotransporter. The mutant RNA reproduced the transport defect after injection in xenopus oocytes. These results confirm the genetic origin of the congenital defect; in addition they indicate that the study of the relationship between phenotype and genotype of congenital defects of intestinal transport may help in the understanding of basic intestinal functions in relation with human nutrition.

Dietary Carbohydrates↗
PubMed1992

Molecular evidence for two renal Na+/glucose cotransporters.

Previous studies have shown that two kinetically and genetically distinct Na+/glucose cotransporters exist in mammalian kidney. We have recently cloned and sequenced one of the rabbit renal Na+/glucose cotransporters (SGLT1) and have found that it is identical in sequence to the intestinal Na+/glucose cotransporter. Northern blots showed that SGLT1 mRNA was found predominantly in the outer medulla of rabbit kidney. Injection of mRNA from outer medulla and outer cortex into Xenopus oocytes resulted in equal expression of Na(+)-dependent sugar uptake, indicating that the outer cortex sample contained mRNA encoding both SGLT1 and a second Na+/glucose cotransporter. Western blots using antipeptide antibodies against SGLT1 showed that the SGLT1 protein is more abundant in outer medulla than outer cortex. However, brush border membrane vesicles prepared from outer cortex had a greater capacity for Na(+)-dependent glucose transport, indicating the presence of a second transporter in the vesicles from outer cortex. It appears that the cloned renal Na+/glucose cotransporter, SGLT1, is the 'high affinity, low capacity' transporter found predominantly in outer medulla. There is evidence that a second transporter, the 'low affinity, high capacity' transporter, is in outer cortex. Finally, the cDNA and protein sequences of the two renal Na+/glucose cotransporters are predicted to differ by more than 20%.

Animals↗
PubMed1992

Cloning and functional expression of a mammalian Na+/nucleoside cotransporter. A member of the SGLT family.

Complementary DNAs encoding seven different proteins related to the rabbit intestinal Na+/glucose cotransporter, SGLT1, were isolated from a rabbit renal cDNA library at relatively high stringency. The messages for RK-B to RK-F were single mRNA species at 2.3 kilobases (kb) in heart and kidney. The message for RK-A was 4 kb and was found in brain, lung, intestine, liver, and kidney. RK-I mRNA was approximately 3 kb and was found in all tissues tested. The most abundant clone, RK-C, constituted nucleotides 66-2150 of the sodium-nucleoside cotransporter, SNST1. The 672-amino acid protein encoded by SNST1 is 61% identical and 80% similar in sequence to SGLT1. Expression of SNST1c in Xenopus oocytes resulted in nucleoside-stimulated 22Na uptake and sodium-dependent [3H]uridine uptake. The uptake of [3H]uridine was inhibited by a range of nucleosides, including the anti-human immunodeficiency virus drug, dideoxycytidine. The results of this study show that there is a family of SGLT1-related proteins found in a wide variety of tissues and that one of these is a Na+/nucleoside cotransporter.

Amino Acid Sequence↗
PubMed1992

Baculovirus-mediated expression of the Na+/glucose cotransporter in Sf9 cells.

We have used baculovirus (AcNPV) to express the Na+/glucose cotransporter protein in cultured Sf9 cells. We constructed a baculovirus transfer vector containing the cDNA for the rabbit intestinal Na+/glucose cotransporter (SGLT1) under the control of the polyhedrin gene promoter. Recombinant baculovirus was obtained by cotransfection of SF9 cells with wild-type AcNPV DNA and the transfer vector. Recombinant virus was identified by Southern blotting and then purified. Recombinant infected Sf9 cells expressed a protein which was recognized by anti-peptide antibodies raised to sequences of the cloned Na+/glucose cotransporter. This protein migrated with a molecular mass of 55 kD by SDS-PAGE, similar to the in vitro translation product of SGLT1. An identical protein was metabolically labeled with [35S]methionine. Cells which synthesized the transport protein showed Na(+)-dependent alpha MeGlc transport. Micromolar phlorizin inhibited transport. Uninfected and wild-type virus infected Sf9 cells did not have Na(+)-dependent glucose transport. All transport protein migrated at 45% sucrose (w/w) by density gradient sedimentation, suggesting that the expressed transporter is membrane associated. We conclude that we have functionally expressed the rabbit intestinal Na+/glucose cotransporter in Sf9 cells. The transporter is not heavily glycosylated, and this is consistent with previous work showing that glycosylation is not necessary for function. We are poised to purify and characterize this protein from a structure-function perspective.

Amino Acid Sequence↗
PubMed1992

Glycosylation of the rabbit intestinal brush border Na+/glucose cotransporter.

The glycosylation of the mature form of the rabbit intestinal Na+/glucose cotransporter was investigated by using both glycosidases and chemical treatment. The protein was identified on Western blots using polyclonal antibodies directed against peptide sequences from the cloned transporter as a Mr 68,000 polypeptide. The effect of these treatments on the size of the transporter is consistent with the major post-translational processing being a single N-linked glycosylation of either the tri- or tetra-antennary complex type. Either method of deglycosylation reduced the SDS-PAGE size by 11,000 to Mr 57,000. These results also suggest that O-linked glycosylation, if present, contributes little to the apparent size of the transporter. The relative size of the deglycosylated mature protein appears to be greater than that of the in vitro primary transcript (Mr 45,000), suggesting either a difference in a stable conformational state insensitive to reduction and denaturation by SDS or an additional post-translational modification. In addition, deglycosylation of the native transporter does not affect transport activity in brush border membrane vesicles. The transporter, an integral membrane protein having several membrane-spanning regions, has an anomalous mobility in SDS-PAGE as shown by Ferguson analysis. We estimate that the actual size of the mature Na+/glucose cotransporter is 86,000, and that N-linked glycosylation contributes about 15,000 to the mass.

Animals↗
PubMed1992

Electrogenic properties of the cloned Na+/glucose cotransporter: I. Voltage-clamp studies.

The cloned rabbit intestinal Na+/glucose cotransporter was expressed in Xenopus laevis oocytes. Presteady-state and steady-state currents associated with cotransporter activity were measured with the two-electrode voltage-clamp technique. Steady-state sugar-dependent currents were measured between -150 and +90 mV as a function of external Na+ ([Na]o) and alpha-methyl-D-glucopyranoside concentrations ([alpha MDG]o). K alpha MDG0.5 was found to be dependent upon [Na]o and the membrane potential. At Vm = -50 mV, increasing [Na]o from 10 to 100 mM decreased K alpha MDG0.5 from 1.5 mM to 180 microM. Increasing membrane potential toward negative values decreased K alpha MDG0.5 at nonsaturating [Na]o. For instance, at 10 mM [Na]o, K alpha MDG0.5 decreased from 1.5 mM to 360 microM on increasing the membrane potential from -50 to -150 mV. The i alpha MDGmax was relatively insensitive to [Na]o between 10 and 100 mM and weakly voltage dependent (e-fold increase per 140 mV). KNa0.5 and iNamax were found to be dependent upon membrane potential and [sugar]o. In the presence of 1 mM [alpha MDG]o, KNa0.5 decreased from 50 to 5 mM between 0 and -150 mV and iNamax increased twofold between -30 and -200 mV. The voltage dependence of KNa0.5 is consistent with an effect of potential on Na+ binding (Na(+)-well effect), whereas the voltage dependence of iNamax is compatible with the translocation step being voltage dependent. It is concluded that voltage influences both Na+ binding and translocation. Presteady-state currents were observed for depolarization pulses in the presence of 100 mM [Na]o. The transient current relaxed with a half time of approximately 10 msec, and both the half time and magnitude of the transient varied with the holding potential and the size of depolarization pulse. Presteady-state currents were not observed after the addition of phlorizin or alpha MDG to the external Na+ solution and were not observed for water-injected control oocytes. We conclude that presteady-state currents are due to the activity of the carrier and that they may give a novel insight to the transport mechanism of the Na+/glucose cotransporter.

Animals↗
PubMed1992

Electrogenic properties of the cloned Na+/glucose cotransporter: II. A transport model under nonrapid equilibrium conditions.

The results of the accompanying electrophysiological study of the cloned Na+/glucose cotransporter from small intestine (Parent, L., Supplisson, S., Loo, D.D.F., Wright, E.M. (1992) J. Mémbrane Biol. 125:49-62) were evaluated in terms of a kinetic model. The steady-state and presteady-state cotransporter properties are described by a 6-state ordered kinetic model ("mirror" symmetry) with a Na+:alpha MDG stoichiometry of 2. Carrier translocation in the membrane as well as Na+ and sugar binding and dissociation are treated as a function of their individual rate constants. Empty carrier translocation and Na+ binding/dissociation are the only steps considered to be voltage dependent. Currents were associated with the translocation of the negatively charged carrier in the membrane. Negative membrane potential facilitates sugar transport. One numerical solution was found for the 14 rate constants that account quantitatively for our experiment observations: i.e., (i) sigmoidal shape of the sugar-specific current-voltage curves (absence of outward currents and inward current saturation at high negative potentials), (ii) Na+ and voltage dependence of Ksugar0.5 and isugarmax, (iii) sugar and voltage dependence of KNa0.5 and iNamax, (iv) presteady-state currents and their dependence on external Na+, alpha MDG and membrane potential, and (v) and carrier Na+ leak current. We conclude that the main voltage effect is on carrier translocation. Na+ ions that migrate from the extracellular medium to their binding sites sense 25 to 35% of the transmembrane voltage, whereas charges associated with the carrier translocation experiences 60 to 75% of the membrane electrical field. Internal Na+ ion binding is not voltage dependent. In our nonrapid equilibrium model, the rate-limiting step for sugar transport is a function of the membrane potential, [Na]o and [alpha MDG]o. At 0 mV and at saturating [Na]o and [alpha MDG]o, the rate-limiting step for sugar transport is the empty carrier translocation (5 sec-1). As the membrane potential is made more negative, the empty carrier translocation gets faster and the internal Na+ dissociation becomes increasingly rate limiting. However, as [Na]o is decreased to less than 10 mM, the rate-limiting step is the external Na+ ions binding in the 0 to -150 mV potential range. At 0 mV, the external Na+ dissociation constant KNa' is 80 mM and decreases to 24 mM at -150 mV. The external sugar dissociation constant KNaS' is estimated to be 200 microM and voltage independent. Finally, the internal leak pathway (CNa2 translocation) is insignificant.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding Sites↗
PubMed1992

Neurotrophic factors promote the maturation of developing sensory neurons before they become dependent on these factors for survival.

We have studied the early development of chicken embryo sensory neurons in culture before they become dependent on neurotrophic factors for survival. During this period, they undergo a distinct change in morphology:initially they have small, spindle-shaped, phase-dark cell bodies, which become spherical and phase bright and extend long neurites. Although this maturational change occurs in isolated cells grown in chemically defined medium, it is accelerated by brain-derived neurotrophic factor (BDNF) or neurotrophin-3 and is retarded by antisense oligonucleotides that inhibit expression of the common, low affinity neurotrophic factor receptor (gp75NGFR) and by antisense BDNF oligonucleotides. We conclude that neurotrophic factors play a role in the earliest stages of sensory neuron development and suggest that they operate by an autocrine mechanism at this time.

Animals↗
PubMed1992

Sodium cotransport proteins.

Significant advances have been made in elucidating the structure of Na+ cotransport proteins. Some fifteen of these low-abundance proteins have been cloned, sequenced and functionally expressed. They are members of the 12 membrane-spanning superfamily and they segregate into two groups, the Na+/glucose (SGLT1) and Na+/Cl-/GABA (GAT-1) families. SGLT1 transporters are expressed in bacteria and animal cells, while GAT-1 transporters are mostly expressed in the brain. None have yet been found in plants.

Amino Acid Sequence↗