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E M Wright

Publications and source records attributed to E M Wright.

At least 163 records · Page 9Linked to original sources
PubMed1990

Expression and characterization of the intestinal Na+/glucose cotransporter in COS-7 cells.

Cells derived from the simian kidney, COS-7 cells, were transfected with a eucaryotic expression vector (pEUK-C1) containing the clone for the rabbit intestinal Na+/glucose cotransporter. Expression was monitored after transfection with lipofectin by measuring the initial rate of alpha-methylglucopyranoside (MeGlc) uptake. Cells transfected with vector containing the cDNA for the Na+/glucose cotransporter expressed Na(+)-dependent MeGlc transport. Neither control cells nor cells transfected with vector lacking cloned cDNA expressed the cotransporter. Na(+)-dependent MeGlc uptake into transfected cells was saturable (Km 150 microM), phlorizin-sensitive (Ki 11 microM), and inhibited by sugar analogs (D-glucose greater than MeGlc greater than D-galactose greater than 3-O-methyl-D-glucoside greater than D-allose much greater than L-glucose). Europium was able to mimic Na+ in driving MeGIC uptake. Finally, tunicamycin, an inhibitor of asparagine-linked glycosylation, inhibited the expression of Na(+)-dependent MeGlc transport 80%. We conclude that the rabbit intestinal Na+/glucose cotransporter expressed in COS-7 cell exhibits very similar kinetic properties to that in the native brush border and to that expressed in Xenopus oocytes. In addition, N-linked glycosylation appears to be important for functional expression of this membrane protein.

Animals↗
PubMed1990

Intestinal Na+/glucose cotransporter expressed in Xenopus oocytes is electrogenic.

The cloned rabbit intestinal Na+/glucose cotransporter was expressed in Xenopus oocytes, and transmembrane currents associated with this transporter were monitored using a two-electrode voltage clamp. Addition of D-glucose to a Na(+)-containing solution bathing these oocytes generated a current which was blocked by phlorizin. Water-injected control oocytes did not exhibit any currents under these conditions. The magnitude and shape of the currents were dependent on the extracellular glucose and Na+ concentrations and the membrane potential. At Vhold = -50 mV, the Km values for glucose and Na+ were 14 +/- 2 (N = 4) microM and 17 +/- 1 (N = 3) mM, respectively. These Km values and imax exhibited voltage dependence: increasing the membrane potential from -30 to -150 mV increased KGlcm and imax threefold and decreased KNam eightfold. The reversal potential (VR) of the phlorizin-sensitive, glucose-dependent current varied with log Nao+ (slope 46 +/- 6 [N = 9] mV). In the absence of sugar, a Na(+)-dependent, phlorizin-sensitive (Ki = 3 +/- 0.5 microM) current was detected only in RNA-injected oocytes. The amplitude of this current at -50 mV was 6 +/- 1% (N = 13) of the maximum current measured in the presence of D-glucose. The VR of this sugar-independent current varied with log Nao+ (slope 63 +/- 1 [N = 4] mV), indicating that the cotransporter may carry Na+ in the absence of sugar. We conclude that the Na+/glucose cotransporter is electrogenic and that investigations of currents associated with its operation can yield valuable insights into the mechanisms of solute translocation.

Animals↗
PubMed1990

Intestinal brush border membrane Na+/glucose cotransporter functions in situ as a homotetramer.

The functional unit molecular size of the intestinal brush border membrane-bound Na+/glucose cotransporter was determined by radiation inactivation. Purified brush border membrane vesicles preserved in cryoprotectant buffer were irradiated (-135 degrees C) with high-energy electrons from a 13-MeV (1 eV = 1.602 x 10(-19) J) linear accelerator at doses from 0 to 70 Mrad (1 rad = 0.01 Gy). After each dose, the cotransporter was investigated with respect to (i) Na(+)-dependent transport activity and (ii) immunologic blot analysis with antibodies against the cloned rabbit intestinal cotransporter. Increasing radiation decreased the maximal Na(+)-dependent cotransporter activity Jmax without affecting apparent Km. The size of the transporting functional unit was 290 +/- 5 kDa. Immunologic blot analysis of brush border membranes gave a single band of Mr 70,000, which decreased in intensity with increased radiation dose and gave a target size of 66 +/- 11 kDa. We conclude that activity of the intestinal Na+/glucose cotransporter in situ in the brush border membrane requires the simultaneous presence of four intact, independent, identical subunits arranged as a homotetramer.

Animals↗
PubMed1990

Pharmacokinetic interaction of propofol and fentanyl: single bolus injection study.

The effect of pretreatment with fentanyl on the pharmacokinetics of a single bolus of propofol was studied in 17 female patients (mean age 35 yr), ASA grade I. Eight patients received fentanyl 1.5 micrograms kg-1 5 min before induction of anaesthesia. In all patients anaesthesia was induced with propofol 2.5 mg kg-1 and maintained with halothane and nitrous oxide in oxygen. Pretreatment with fentanyl resulted in prolonged apnoea in all eight patients compared with three of nine patients in the control group. The pharmacokinetic values for propofol were described by a three-compartment mammillary model with rapid distribution phases (T1/2 alpha mean (SEM) 3.1 (2.0) min and T1/2 beta 44 (9.1) min) and a slower final phase of T1/2 gamma 520 (96) min. The clearance of propofol was rapid (mean 1.6 (0.24) litre min-1). Propofol was distributed initially into a relatively large central compartment (mean 23.7 (6.6) litre) and was extensively redistributed (mean Vss 593 (157) litre). There was no difference in the pharmacokinetic profile of propofol between the two groups.

Adolescent↗
PubMed1990

Sequence homologies among intestinal and renal Na+/glucose cotransporters.

Sodium-dependent glucose transport occurs in the intestine and kidney of most animal species. The cDNA encoding the Na+/glucose cotransporter from rabbit jejunum was used to examine the distribution of homologous mRNA in other rabbit tissues and in the intestines of other species. Northern blots of mRNA extracted from various tissues were probed with radiolabeled cDNA of the cloned rabbit transporter. The probe hybridized with mRNA of approximately 2.2 kb from rabbit jejunum, renal cortex, and renal medulla, indicating that related mRNA of the same size is found in these tissues. With the use of the same cDNA probe, a 1.6-kb partial-length clone encoding 484 amino acids was isolated from a rabbit renal cortex cDNA library. There was greater than 99% identity between the cDNA sequences, and 100% identity between the amino acid sequences, of the renal clone and the rabbit intestinal Na+/glucose cotransporter. The 2.2-kb transcript was seen in mRNA from duodenum, jejunum, and ileum, with a distribution that matched the Na+/glucose transport capacity in these tissues. A faint signal at 2.2 kb was also seen in colon mRNA. There was no detectable hybridization to blots of stomach and heart mRNA. The rabbit probe also hybridized to intestinal mRNA from a number of species from trout to humans. We conclude that a Na+/glucose cotransporter of rabbit renal cortex is very similar to that of the intestine and that the intestinal transporter has been conserved during evolution.

Amino Acid Sequence↗
PubMed1989

Molecular genetics of the human Na+/glucose cotransporter.

Recent success in expression cloning has revealed the primary structure of the Na+/glucose cotransporter from rabbit small intestine, and this has subsequently led to the cloning of the Na+/glucose cotransporters from human small intestine and human kidney. Close homology is evident between the rabbit and human intestinal Na+/glucose cotransporters at the DNA level, and the predicted amino acid and secondary structure levels. The Na+/glucose cotransporter amino acid sequence from human kidney is 57% identical with that from human small intestine. Significant homology also exists between these Na+/glucose cotransporters and the E. coli Na+/proline cotransporter (putP). The rabbit intestinal Na+/glucose cotransporter has 11 potential membrane spanning regions and 2 hydrophilic regions containing highly charged residues. The amino acid sequence shows two potential N-glycosylation sites (N-X-T/S). Using an in vitro translation approach we were able to determine that only one of these (Asn 248) is glycosylated. Expression experiments with Xenopus oocytes using the N-glycosylation inhibitor tunicamycin indicate that glycosylation of Asn 248 is required for functional expression of the transporter. The N-X-T/S sequence at Asn 248 is conserved in the human intestinal and the human renal Na+/glucose cotransporter. Chromosomal localization studies map the human intestinal Na+/glucose cotransporter gene (SGLT1) to the q11.2----qter region of chromosome 22 and the human renal Na+/glucose cotransporter gene (SGLT2) to the q-arm of chromosome 16.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Characterization of a Na+/glucose cotransporter cloned from rabbit small intestine.

The Na+/glucose cotransporter from rabbit intestinal brush border membranes has been cloned, sequenced, and expressed in Xenopus oocytes. Injection of cloned RNA into oocytes increased Na+/sugar cotransport by three orders of magnitude. In this study, we have compared and contrasted the transport properties of this cloned protein expressed in Xenopus oocytes with the native transporter present in rabbit intestinal brush borders. Initial rates of 14C-alpha-methyl-D-glucopyranoside uptake into brush border membrane vesicles and Xenopus oocytes were measured as a function of the external sodium, sugar, and phlorizin concentrations. Sugar uptake into oocytes and brush borders was Na+-dependent (Hill coefficient 1.5 and 1.7), phlorizin inhibitable (Ki 6 and 9 microM), and saturable (alpha-methyl-D-glucopyranoside Km 110 and 570 microM). The sugar specificity was examined by competition experiments, and in both cases the selectivity was D-glucose greater than alpha-methyl-D-glucopyranoside greater than D-galactose greater than 3-O-methyl-D-glucoside. In view of the close similarity between the properties of the cloned protein expressed in oocytes and the native brush border transporter, we conclude that we have cloned the classical Na+/glucose cotransporter.

Animals↗
PubMed1989

Assignment of the human intestinal Na+/glucose cotransporter gene (SGLT1) to the q11.2----qter region of chromosome 22.

The chromosomal location of the human intestinal Na+/glucose cotransporter gene (SGLT1) was determined using human cDNA and genomic probes for this transporter gene. Southern blot analysis of genomic DNA from 15 mouse-human somatic cell hybrids showed that the human gene for this transporter resides on chromosome 22. Analysis of hamster-human hybrids selectively retaining chromosome 22 or a portion of it allowed specific assignment of the locus to the q11.2----qter region of chromosome 22. A restriction fragment length polymorphism was identified with EcoRI.

Animals↗
PubMed1989

Homology of the human intestinal Na+/glucose and Escherichia coli Na+/proline cotransporters.

Cotransport proteins are responsible for the active accumulation of organic substrates in cells. Na+ gradients provide the driving force for uptake of most substrates into eukaryotes and for a few substrates in some prokaryotes. We report here the cloning and sequencing of the human intestinal Na+/glucose cotransporter (SGLT1) and compare its structure with other cloned transporters. At the DNA level and the predicted amino acid and secondary structure levels, close homology is evident between the human and rabbit intestinal Na+/glucose cotransporters, and a significant homology is found between these and the Escherichia coli Na+/proline cotransporter (putP). No homology is detectible with other known proteins. We infer from these results that the mammalian Na+/glucose and prokaryote Na+/proline cotransporters share a common ancestral gene.

Amino Acid Sequence↗
PubMed1988

Ca2+-activated K+ channels in the apical membrane of Necturus choroid plexus.

The properties of Ca2+-activated K+ channels in the apical membrane of the Necturus choroid plexus were studied using single-channel recording techniques in the cell-attached and excised-patch configurations. Channels with large unitary conductances clustered around 150 and 220 pS were most commonly observed. These channels exhibited a high selectivity for K+ over Na+ and K+ over Cs+. They were blocked by high cytoplasmic Na+ concentrations (110 mM). Channel activity increased with depolarizing membrane potentials, and with increasing cytoplasmic Ca2+ concentrations. Increasing Ca2+ from 5 to 500 nM, increased open probability by an order of magnitude, without changing single-channel conductance. Open probability increased up to 10-fold with a 20-mV depolarization when Ca2+ was 500 nM. Lowering intracellular pH one unit, decreased open probability by more than two orders of magnitude, but pH did not affect single-channel conductance. Cytoplasmic Ba2+ reduced both channel-open probability and conductance. The sites for the action of Ba2+ are located at a distance more than halfway through the applied electric field from the inside of the membrane. Values of 0.013 and 117 mM were calculated as the apparent Ba2+ dissociation constants (KD(0 mV] for the effects on probability and conductance, respectively. TEA+ (tetraethylammonium) reduced single-channel current. Applied to the cytoplasmic side, it acted on a site 20% of the distance through the membrane, with a KD(0 mV) = 5.6 mM. A second site, with a higher affinity, KD(0 mV) = 0.23 mM, may account for the near total block of channel conductance by 2 mM TEA+ applied to the outside of the membrane. It is concluded that the channels in Necturus choroid plexus exhibit many of the properties of "maxi" Ca2+-activated K+ channels found in other tissues.

Animals↗
PubMed1988

Ca2+-activated K+ currents in Necturus choroid plexus.

The tight-seal whole-cell recording method has been used to study Necturus choroid plexus epithelium. A cell potential of -59 +/- 2 mV and a whole cell resistance of 56 +/- 6 M omega were measured using this technique. Application of depolarizing step potentials activated voltage-dependent outward currents that developed with time. For example, when the cell was bathed in 110 mM NaCl Ringer solution and the interior of the cell contained a solution of 110 mM KCl and 5 nM Ca2+, stepping the membrane potential from a holding value of -50 to -10 mV evoked outward currents which, after a delay of greater than 50 msec, increased to a steady state in 500 msec. The voltage dependence of the delayed currents suggests that they may be currents through Ca2+-activated K+ channels. Based on the voltage dependence of the activation of Ca2+-activated K+ channels, we have devised a general method to isolate the delayed currents. The delayed currents were highly selective for K+ as their reversal potential at different K+ concentration gradients followed the Nernst potential for K+. These currents were reduced by the addition of TEA+ to the bath solution and were eliminated when Cs+ or Na+ replaced intracellular K+. Increasing the membrane potential to more positive values decreased both the delay and the half-times (t1/2) to the steady value. Increasing the pipette Ca2+ also decreased the delay and decreased t1/2. For instance, when pipette Ca2+ was increased from 5 to 500 nM, the delay and t1/2 decreased from values greater than 50 and 150 msec to values less than 10 and 50 msec. We conclude that the delayed currents are K+ currents through Ca2+-activated K+ channels. At the resting membrane potential of -60 mV, Ca2+-activated K+ channels contribute between 13 to 25% of the total conductance of the cell. The contribution of these channels to cell conductance nearly doubles with membrane depolarization of 20-30 mV. Such depolarizations have been observed when cerebrospinal fluid (CSF) secretion is stimulated by cAMP and with intracellular Ca2+. Thus the Ca2+-activated K+ channels may play a specific role in maintaining intracellular K+ concentrations during CSF secretion.

Animals↗
PubMed1987

Regulation of intracellular chloride in bullfrog choroid plexus.

Intracellular Cl- activity (AiCl) of the bullfrog choroidal epithelium has been studied using double-barreled Cl(-)-selective microelectrodes. In bicarbonate-buffered saline, the brush-border membrane potential (VVC) was -43 mV, and AiCl was 24 mM which was twice the predicted equilibrium activity. The uphill Cl- accumulation required the presence of external Na+ and was inhibited by furosemide added to the basolateral side. Removal of HCO3- from the bath solution slightly increased AiCl. On addition of 3-isobutyl-1-methylxanthine (IBMX), VVC depolarized, and AiCl approached the equilibrium activity. It is concluded that net Cl- secretion by the choroidal epithelium is mediated by a furosemide-sensitive, Na+-coupled Cl- uptake mechanism at the basolateral border and a Cl- conductive pathway at the brush border membrane. The results suggest that intracellular cAMP either increases the Cl- conductance of the epithelial membranes and/or inhibits the NaCl co-transport mechanism.

1-Methyl-3-isobutylxanthine↗
PubMed1987

Examination of the Na+-induced conformational change of the intestinal brush border sodium/glucose symporter using fluorescent probes.

The Na+-induced change in conformation of the intestinal brush border glucose carrier has been examined by three procedures. In the first, we have measured the effect of Na+ on the binding of fluorescein isothiocyanate (FITC) to the glucose site; 100 mM Na increased the specific [blocked by D-glucose, p-(chloromercuri)benzenesulfonic acid, and N-acetylimidazole] FITC binding to a 75-kilodalton polypeptide 3-fold. In the second series, we have examined the effect of Na+ on the susceptibility of the fluorescently labeled glucose site [pyrene isothiocyanate (PYTC) labeled] to a hydrophilic quencher (Tl+); 100 mM NaCl increased the fraction of PYTC sites available to Tl+ from 32% to 92% and decreased the apparent quenching constant from 94 to 44 M-1. Finally, in the third series, we probed the distribution of tryptophan residues 15-30 A from the glucose site using a "distant reporter group method", where tryptophan was used an an energy donor to anthracene isothiocyanate bound to the glucose site. Tryptophan quench reagents (I-, Cs+, and acrylamide) were then employed to probe the accessibility of the glucose site tryptophans in the presence and absence of sodium. In the absence of Na+, there were two major classes of glucose tryptophans--exterior surface residues and residues buried in the hydrophobic protein matrix. Na+ caused a redistribution of the donor tryptophans such that a higher percentage were accessible to I- (51% vs. 25%) and fewer were accessible to Cs+ (13% vs. 25%) and acrylamide (27% vs. 57%).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Kinetics of the intestinal brush border proline (Imino) carrier.

The kinetics of L-proline transport across intestinal brush borders via the Imino carrier were studied using membrane vesicles. The Imino carrier is defined as the agent responsible for L-alanine insensitive. Na+-dependent uptake of L-proline. Initial rate measurements were made under voltage clamped conditions (pD = 0) to investigate L-proline transport as a function of cis and trans Na+ and proline concentrations. Under zero-trans conditions, increasing cis Na+ activated proline uptake with a Hill coefficient of 1.7 and decreased the apparent Kt with no change in Jimax. The Jimax was approximately 60 pmol mg-1 s-1 and the apparent Kt ranged from 0.25 mM at cis Na = 100 to 1.0 mM at cis Na+ = 30 mM. Trans Na inhibited proline uptake via a reduction in Jimax. Trans proline had no significant effect in the absence of trans Na+, but it relieved the trans Na+ inhibition. Under equilibrium exchange conditions, the Jimax was twice that observed under zero-trans conditions. These kinetics of L-proline transport suggest a model in which uptake occurs by a rapid equilibrium iso-ordered ter ter system. Two Na+ ions bind first to the carrier on the cis face of the membrane to increase the affinity of the carrier for proline. The fully loaded complex then isomerizes to release the substrates to the trans side. The partially loaded Na+-only forms are unable to translocate across the membrane. A rate-limiting step appears to be the isomerization of unloaded carrier from the trans to the cis side of the membrane.

Amino Acid Transport Systems, Neutral↗
PubMed1987

Effects of lidocaine on transport properties of renal brush-border membranes.

The effect of lidocaine was examined in membrane vesicles from rabbit renal brush borders. Changes in the ionic permeability and the kinetics of Na+-dependent metabolite transport were observed at different concentrations of anesthetic. Lidocaine was found to alter the membrane permeability of all inorganic cations examined (Li+, Rb+, K+, and Na+). At low lidocaine concentrations, there was a saturable decrease in permeability, whereas at higher concentrations (greater than 0.2 mM) there was a non-saturable general increase in cation permeability. Lidocaine (1.0 mM) inhibited Na+-coupled transport of all ten substrates examined (sugars, amino acids and Krebs cycle intermediates). The affinity for the substrate decreased in the presence of the anesthetic.

Animals↗
PubMed1987

Expression of size-selected mRNA encoding the intestinal Na/glucose cotransporter in Xenopus laevis oocytes.

The expression of the rabbit intestinal brushborder Na/glucose cotransporter has been studied in Xenopus oocytes. Poly(A)+ RNA isolated from the intestinal mucosa was injected into oocytes, and the expression of the transporter in the oocyte plasma membrane was assayed by measuring the Na-dependent phlorizin-sensitive uptake of methyl alpha-D-[14C]glucopyranoside (MeGlc). Expression of the glucose carrier was detected 3-7 days after mRNA injection, and the rate of glucose transport was proportional to the amount of mRNA injected. mRNA (50 ng) increased the maximum velocity (Vmax) of MeGlc uptake by as much as 10-fold over background. The total mRNA was fractionated by preparative agarose gel electrophoresis and each fraction was assayed for its ability to induce transport activity. The mRNA encoding the Na/glucose cotransporter was found in a single fraction of approximately 2.3 kilobases (kb), which contained 3% of the total mRNA. A similar mRNA fraction (2.0-2.6 kb) isolated from colon did not induce expression of this transporter. In vitro translation of the fractionated intestinal mRNA showed enhanced synthesis of two protein bands at 57 and 63 kDa. The mRNA encoding the cotransporter is smaller (2.3 kb) than that (2.6-2.9 kb) encoding the 55-kDa facilitated glucose carrier in human hepatoma cells and rat brain.

Animals↗