Role of -SH groups in rat sugar intestinal transport in vivo.

The effect of p-chloromercuribenzoic acid (pCMB), either alone or in the presence of 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), on the 1 mM galactose absorption by in vivo perfused rat intestine has been studied. At 0.25 mM concentration, pCMB inhibits galactose absorption in about 32% but it does not modify the absorption of this sugar when the transport is blocked by 0.5 mM phlorizin, or that of the non-transportable monosaccharide derivative 2-deoxy-D-glucose. This shows that only the active transport component of galactose absorption is inhibited. A 2 min preexposure period is required for the inhibition to appear. The inhibition was not reversed by washing with saline solution even when it contained 0.5 mM dithioerythritol, 10 mM cysteine or 5 and 10 mM EDTA. The simultaneous exposure to 0.25 pCMB and 0.25 mM DTNB inhibits the total galactose entry in about 50%, an effect higher than the one exerted by each reagent separately and close to the one obtained with 0.5 mM phlorizin. Our results, in vivo, confirm the importance of the thiol groups in the cotransport of Na+ and sugar. As DTNB is an SH-reagent of lesser liposolubility than pCMB, the existence of two populations of sulfhydryl groups related to sugar transport which differ in their location within the brushborder membrane and in accessibility from the intestinal lumen, is suggested.     

Inhibition of sugar active transport across rat intestine in vivo by cadmium

Galactose absorption by rat jejunum perfused in vivo is inhibited by 0.5 mM Cd2+. This effect is explained by impairment of the phlorizin-sensitive sugar transport system, as Cd does not modify the absorption of L-sorbose or that of galactose in the presence of 0.5 mM phlorizin. Cd inhibition is observed as early as in the 1st minute, does not increase by previous exposure of the mucosa to the metal and does not decrease after washing with saline solution, but it is entirely reversed by EDTA or dithioerythritol. Results agree with a Cd2+ binding to HS- groups of membrane proteins at the brush border, appertaining or functionally related to the phlorizin-sensitive and Na+ dependent transport system for sugars.     

D-galactose uptake by snail intestine: competitive inhibition by phlorizin and sodium dependence

The net entry of galactose into the tissue of snail everted intestinal rings with 2 or 15 minute long incubation periods has been measured. With 10(-4) M phlorizin, the mediated transport is completely blocked while only the passive entry of sugar is produced. Lower concentrations of the glycoside partially inhibit transport according to competitive inhibition kinetics (K1 = 10(-7) M). The transport of galactose is Na+ dependent. In the absence of Na+, transport ceases and the sugar entry can be explained through simple diffusion. With 15 mM Na+ (control 71,4 mM) transport diminishes and a marked increase in the apparent Km with no changes in the Vmax is observed. One mM harmaline completely blocks galactose (0.5 mM) transport. One mM ouabain also makes transport null, but only after tissue preincubation with the inhibitor on the serosal side.     

Accumulation of cadmium in pancreatic beta cells is similar to that of calcium in being stimulated by both glucose and high potassium

The transport of Cd2+ and the effects of this ion on secretory activity and metabolism were investigated in beta cell-rich pancreatic islets isolated from obese-hyperglycemic mice. The endogenous cadmium content was 2.5 mumol/kg dry wt. After 60 min of incubation in a Ca2+-deficient medium containing 2.5 microM Cd2+ the islet cadmium content increased to 0.18 mmol/kg dry wt. This uptake was reduced by approx. 50% in the presence of 1.28 mM Ca2+. The incorporation of Cd2+ was stimulated either by raising the concentration of glucose to 20 mM or K+ to 30.9 mM. Whereas D-600 suppressed the stimulatory effect of glucose by 75%, it completely abolished that obtained with high K+. Only about 40% of the incorporated cadmium was mobilized during 60 min of incubation in a Cd2+-free medium containing 0.5 mM EGTA. It was possible to demonstrate a glucose-induced suppression of Cd2+ efflux into a Ca2+-deficient medium. Concentrations of Cd2+ up to 2.5 microM did not affect glucose oxidation, whereas, there was a progressive inhibition when the Cd2+ concentration was above 10 microM. Basal insulin release was stimulated by 5 microM Cd2+. At a concentration of 160 microM, Cd2+ did not affect basal insulin release but significantly inhibited the secretory response to glucose. It is concluded that the beta cell uptake of Cd2+ is facilitated by the activation of voltage-dependent Ca2+ channels. Apparently, the accumulation of Cd2+ mimics that of Ca2+ also involving a component of intracellular sequestration promoted by glucose.     

Inhibition of sugar transport across rat jejunum, in vivo, by cupric ions

Cupric ions inhibit galactose absorption by in vivo perfused rat jejunum. It takes some delay for the inhibitory action to display its maximal levels, and previous exposure of the mucosa to Cu markedly increases inhibition. Copper effects were only scarcely reversed by saline solution washing, more effectively by EDTA and more so by dithioerythritol, in no case reaching control values. Absorption of L-sorbose, or that of galactose in the presence of 0.5 mM phlorizin, are not modified by 0.5 mM cupric ions. Cu action may be understood as a selective impairment of the phlorizin-sensitive sugar transport system by binding of the metal to prevailing thiol chemical groups of proteins at the brush border, located at different depth within the thickness of the membrane.     

Inhibition of intestinal absorption of 5-methyltetrahydrofolate by fluoxetine

Thein vitro uptake of 5-methyltetrahydrofolate (5-MeTHF) by rat and human intestine is dose-dependently inhibited by the antidepressant drug fluoxetine (FLX). In rat jejunum rings, 0.2 mM FLX inhibited the uptake of 5-MeTHF (0.25 μM) by 32% (15 min) and 49% (45 min). In brush border membrane vesicles (BBMV) from rat jejunum, 0.2 mM FLX inhibited the folate uptake at the overshoot (90 s) by 40 %. Similar inhibition was observed with human Caco-2 cells and duodenal biopsies. FLX action is exerted on the active transport component of the folate uptake, since the drug has no effect when the passive diffusion component becomes prominent by high substrate concentration, or by 0-4 ºC incubation or by addition of the folate transport inhibitor DIDS (1mM). The kinetic analysis with rat BBMV suggests a non-competitive inhibition of the 5-MeTHF transport by FLX, with apparent values for K_M = 0.89 μM, Vmax = 1.89 pmol/mg prot./10 s, and K_I = 0.21 mM. After 21 days of treatment with FLX (10 mg/kg/day), the folate uptake by jejunum rings or by BBMV from the treated rats was diminished, and the folate levels in erythrocytes and serum were also decreased.     

Uranyl action on sugar transport across rat jejunum

The effect of uranyl on sugar transport across rat jejunum has been studied in vitro and in vivo. D-glucose and D-galactose accumulation in jejunum rings at pH 6.0 is inhibited about 40-65% by 1 mM uranyl nitrate. This inhibition is lower than that produced by 0.5 mM phlorizin. The effect was very similar when the incubation of the rings with the sugar was made in the absence of uranyl, after preincubation with the inhibitor. Washing with 10 mM EDTA reverted uranyl inhibition only slightly. D-fructose entry was weakly inhibited by uranyl. Glucose absorption in vivo along perfusion periods of 1 min was not affected by the presence of uranyl (0.001 to 1 mM) in the sugar solution, but the exposure of the mucosa to 0.1 mM uranyl at pH 6.5 for 10 min inhibited sugar absorption at the same pH in the subsequent periods of perfusion. Results suggest that uranyl impairs sugar transport by binding to protein chemical groups at the surface or in deeper sites of enterocyte membranes, a process that requires some minutes to be accomplished.     

Action of mercury on sugar transport across rat small intestine, in vivo

Absorption of galactose from in vivo perfused rat jejunum was inhibited by 0.1-0.5 mM Hg2+. A few minutes' delay was required for maximal inhibition values. The effects remained after saline solution washing but were in part reversed by EDTA and in higher proportion by dithioerythritol. Absorption inhibition could be ascribed to impairment of the sugar-Na phlorizin-sensitive cotransport component: The passive apparently diffusional component that remains under 0.5 mM phlorizin and absorption of L-sorbose were unaffected by the metal. Hg action is explained as due to its binding to thiol and perhaps other chemical groups of proteins, at different depths in the membrane, which are directly or indirectly related to the sugar transport system.     

Simultaneous uptake of galactose and glucose by Azotobacter vinelandii.

Azotobacter vinelandii growing on galactosides induced two distinct permeases for glucose and galactose. The apparent Vmax and Km of the galactose permease were 16 nmol galactose/min per 10(10) cells and 0.5 mM, respectively. The apparent Vmax and Km of the glucose permease were 7.8 nmol glucose/min per 10(10) cells and 0.04 mM, respectively. Excess glucose had no effect on the galactose uptake. However, excess galactose inhibited glucose transport. The galactosides-induced glucose permease also exhibited different uptake kinetics from that induced by glucose.     

Non-competitive inhibition of myo-inositol transport in cultured bovine retinal capillary pericytes by glucose and reversal by Sorbinil

myo-Inositol transport by retinal capillary pericytes in culture was characterized. The major myo-inositol transport process was sodium-dependent, ouabain-sensitive, and saturable at 40 mM, indicating a carrier-mediated process. The sodium ion concentration required to produce one-half the maximal rate of myo-inositol uptake ([Na+]0.5) did not show dependence on the external myo-inositol concentration (22.3 mM sodium for 0.005 mM myo-inositol; 18.2 mM sodium for 0.05 mM myo-inositol). myo-Inositol transport was an energy-dependent, active process functioning against a myo-inositol concentration gradient. The kinetics of the sodium-dependent system fitted a 'velocity type' co-transport model where binding of sodium ion to the carrier increased the velocity (Vmax 28 to 313 pmol myo-inositol/micrograms DNA per 20 min when [Na+] varied from 9 to 150 mM) but not the affinity for myo-inositol (Km 0.92 to 0.83 mM when [Na+] varied from 9 to 150 mM). Metabolizable hexoses (D-glucose or D-galactose; greater than 5 mM) inhibited myo-inositol uptake. Dixon-plot analysis indicated that the inhibition was non-competitive with a Ki of 22.7 mM for D-glucose and 72.6 mM for D-galactose. The inhibition was significantly reversed by Sorbinil (0.1 mM), an aldose reductase inhibitor. In contrast, high concentrations of non-metabolizable hexoses (L-glucose, 3-O-methyl-D-glucose), or partially metabolizable 2-deoxy-D-glucose, did not significantly inhibit myo-inositol uptake. The inhibitory effect of D-glucose or D-galactose on myo-inositol transport appeared to be related to glucose or galactose metabolism via the polyol pathway.     

Inhibition by sporidesmin of hepatocyte bile acid transport.

Exposure of isolated rat hepatocytes (approx. 2 x 10(7)--5 x 10(7) cells/10ml of incubation mixture) to 0.5 mg of the mycotoxin sporidesmin for 30--60 min at 37 degrees C produced loss of plasma-membrane microvilli with some disruption of organelle distribution in the sub-surface region. There was accompanying inhibition of [14C]cholate and [14C]taurocholate transport, but bile acid conjugation was not altered. Inhibition of cholate uptake was maximal after exposure of hepatocytes to sporidesmin for 1 min, and was not reversed by washing cells free of extracellular sporidesmin. N-Ethylmaleimide (0.1 mM) or dithiothreitol (1 mM) partially protected hepatocytes from sporidesmin inhibition of bile acid uptake. Significant protection was not given by other thiols or by zinc sulphate, cholesterol, ascorbate or alpha-tocopherol. The results are discussed in terms of sporidesmin action on cell membranes and the toxin's effect on bile secretion.     

Ileal resection and dietary content of sucrose influence the intestinal uptake of monosaccharides

The intestinal uptake of 0.5 and 40 mM glucose, galactose, and 3-O-methyl glucose (3-O-MG) was examined in vitro in rabbits fed a high (HS) or a low (LS) sucrose diet. In animals with an intact intestinal tract, the jejunal uptake of 0.5 mM 3-O-MG was unaffected by the dietary content of sucrose, whereas the uptake of 40 mM 3-O-MG was lower in LS than HS. The uptake of 40 mM galactose was higher in LS than HS and the uptake of 0.5 mM galactose was similar in HS and LS, whereas the uptake of 0.5 mM but not 40 mM glucose was lower in LS than HS. In animals subjected 6 weeks previously to an ileal resection, the adaptive changes in the jejunal uptake of the hexoses in response to alterations in the dietary content of sucrose differed from the changes observed in rabbits with an intact intestinal tract. For example, feeding HS to ileal resected animals was associated with increased jejunal uptake of 40 mM galactose, decreased uptake of 40 mM glucose, and unchanged uptake of 40 mM 3-O-MG; whereas in control animals with an intact intestinal tract, feeding HS resulted in increased uptake of 40 mM 3-O-MG, decreased uptake of 40 mM galactose, and no change in the uptake of 40 mM glucose. A similar adaptive pattern was noted in the jejunum and ileum for the effect of dietary sucrose on the uptake of 0.5 and 40 mM glucose.(ABSTRACT TRUNCATED AT 250 WORDS)     

Washing of Cadmium(II) from a Contaminated Soil Column

The washing of cadmium (from CdO_(s)) from a soil column employing either an acid solution or EDTA (a strong metal chelator) was examined. For Cd(II) levels of 50 to 1000 mg/kg, the fraction removed was essentially independent of the initial Cd(II) concentration. The most efficient washing of cadmium was achieved using an acid wash solution at pH 2.5. Lower Cd(II) removals were found at lower pH, apparently due to inhibition of CdO_(s) dissolution by constituents released from the soil under highly acidic conditions. EDTA wash solutions were employed at EDTA:cadmium molar ratios ranging from 1:1 to 10:1. Up to 90% removal of total Cd(II) was achieved at the 10:1 ratio after the passage of the first 50?PV of wash solution. Although higher chelate levels enhanced Cd(II) removal, the utilization efficiency of EDTA for cadmium decreased.     

Uptake of the components of phenylalanylphenylalanine and maltose by intestinal epithelium

The observed rate of phenylalanine absorption into rat intestinal rings with 0.5 or 5.0 mM phenylalanine is greater than that for absorption of phenylalanine from 0.25 or 2.5 mM Phe-Phe, respectively. With the amino acid phenylalanine, V for absorption is the same whether Na⁺ is present (149 mM) or absent, but the concentration at which the half-maximal transport rate occurred (K_t) is greater in the absence of Na⁺. For Phe-Phe, the V decreases in the absence of Na⁺ whilst K_t is not influenced by the Na⁺ concentration. The different effect of Na⁺ on Phe and Phe-Phe transport indicates that the absorptive mechanism for Phe-Phe is different from that for phenylalanine. Absorption of a mixture of [U-¹⁴C]Phe-Phe and Phe-[G-³H]Phe showed identical rates of uptake of the carboxyl and amino terminal amino acids.Studies of transport of radioactive maltose showed that the rates of uptake of the reducing and non-reducing glucosyl moieties are identical. Radioactive maltose absorption is not inhibited by glucose oxidase.These results provide evidence that in intestinal epithelium, hydrolysis of Phe-Phe and maltose does not occur on the cell surface with release of the hydrolyzed products to the medium. Rather, hydrolysis and release of the reaction products occur at a point on the cytosol side of a diffusion barrier located in the brush border membrane.     

Alternate models for shared carriers or a single maturing carrier in hexose uptake into rabbit jejunum in vitro

The uptake (tissue accumulation) of three hexoses into rabbit jejunum was measured in a flux chamber in conditions of effective stirring. Glucose uptake was inhibited by galactose or 3-O-methylglucose: 1-40 mM galactose caused a progressive decline in glucose uptake; 1-5 mM 3-O-methylglucose inhibited glucose uptake but higher concentrations of 3-O-methylglucose had no further effect. When 1-40 mM 3-O-methylglucose was added to glucose plus galactose there was a further decrease in the uptake of glucose; adding 1-40 mM galactose to glucose plus 3-O-methylglucose also produced a decrease in glucose uptake. Both glucose and 3-O-methylglucose inhibited uptake of galactose but the pattern of inhibition varied between the two sugars. The uptake of 3-O-methylglucose was also inhibited by glucose and by galactose, but the uptake of 3-O-methylglucose in the presence of either galactose or glucose was no further reduced by adding the third hexose. Graphical analysis and analysis by non-linear regression both showed that neither the single Michaelis-Menten function, nor the single Michaelis-Menten-plus-competitive-inhibition function was appropriate for any of these data. The results are consistent with the hypothesis that either there are multiple (at least three) intestinal carriers for hexoses; alternatively that there is a single carrier whose transport properties for the three hexoses change differentially during cell maturation and migration up the villus.     

Kinetic characteristics and regulation of hexose transport in a galactokinase-negative Chinese hamster fibroblast cell line: a good model for studies on sugar transport in cultured mammalian cells

We report the kinetic characteristics for D-galactose, 2-deoxy-D-glucose and 3-O-methyl-D-glucose transport in a galactokinase null-allele mutant of a Chinese hamster V79 cell line. GalKl cells exhibited a Km and Vmax for D-galactose, 2-deoxy-D-glucose, and 3-O-methyl-D-glucose transport of 8.6 +/- 2.6 mM and 26.1 +/- 7.2 nmol/mg p/min, 4.1 +/- 1.2 mM and 40.3 +/- 9.5 nmol/mg p/min, and 7.01 +/- .85 mM and 11.6 +/- 4.8 nmol/mg p/30 s, respectively. Nonsaturable hexose uptake was determined using cytochalasin B inhibition of galactose uptake (89.6 +/- 3.7% of galactose uptake was cytochalasin B inhibitable) and L-glucose uptake (7.5% of the galactose uptake). D-Galactose was not metabolized and effluxed rapidly from preloaded cells. The Kls for the inhibition of D-galactose transport were 4.5 +/- 2.5 mM for D-glucose, 7.0 +/- 2.0 mM for 2-deoxy-D-glucose, 6 mM for 2-deoxy-D-galactose and 6.0 +/- 0.6 mM for 3-O-methyl-D-glucose. This indicates the operation of a single common carrier. The hexose transport rate decreased 50-60% after 24 h serum deprivation. Addition of insulin was shown to increase hexose transport (more than twofold) in serum-deprived cells. Hexose transport rates increased substantially in glucose-deprived, D-fructose- or D-galactose-fed cells as compared to glucose-fed cells. Since GalKl does not metabolize galactose, the hexose transport increases induced by feeding cells galactose suggest that carrier interaction with ligand is not a significant factor in transport regulation in GalKl. The kinetic and regulatory characteristics of D-galactose transport in the GalKl cell line indicate that this system is a good model to study sugar transport from a mechanistic and regulatory point of view.     

Leptin effect on intestinal galactose absorption in ob/ob and db/db mice

Our previous works demonstrated that leptin inhibits galactose absorption in rat and mice intestinal rings. Here, we have studied the effect of exogenous leptin on intestinal galactose absorption in the genetically obese db/db (leptin-resistant) and ob/ob (leptin-deficient) mice. Assays were performed by incubating the intestinal rings in saline solution containing 5 mM galactose in the absence or presence of 0.2 or 0.4 nM leptin. Basal galactose uptake was similar in the wild-type and the two obese groups. Contrarily to what happens in wild-type mice, leptin increased galactose uptake in db/db animals; since these mice lack the functional long leptin receptor, the measured effect may be due to the short receptor signaling. In the ob/ob mice, 0.2 nM leptin also increased galactose absorption whereas 0.4 nM did not have any effect, suggesting that in the genetically obese animals the expression and regulation of leptin receptors may be altered.     

Divalent metals inhibit and lactose stimulates zinc transport across brush border membrane vesicles from piglets

Interactions between metals of similar coordination chemistry are of relevance to infant nutrition due to the highly variable metal:metal ratios found in formulas. Using ratios similar to those found in infant formulas, our objectives were to determine the effects of metals and of lactose and other saccharides on Zn(+2) transport across intestinal brush border membranes. Brush border membrane vesicles prepared from intestines of 5 preweaned piglets were used to determine whether Ca(+2), Mg(+2), Fe(+2), Cu(+2), Cd(+2), or Mn(+2) would antagonize Zn(+2) uptake. (65)Zn(+2) uptake by brush border membrane vesicles was measured over 20 min with metal concentrations constant, and at 1 min with increasing metal concentrations. Zn(+2) bound to the external surface of vesicles was removed with ethylenediamine-tetraacetic acid. Lactose induced Zn(+2) uptake to a greater extent than glucose polymer, whereas maltose, galactose, or galactose/glucose had no effect. Over 20 min, a 10:1 concentration of Fe(+2), Cd(+2), Cu(+2), and Mn(+2) lowered Zn(+2) uptake significantly (P < 0.05). Higher concentrations of divalent cation significantly lowered Zn(+2) (0.2 or 0.1 mM) uptake for all metals tested (P < 0.05), except for Mn(+2) (0.1 mM Zn(+2)). Inhibition constant determination quantified relative competitive potential with Mg(+2) < Ca(+2) < Mn(+2) < Fe(+2) < Zn(+2) < Cu(+2). Relative amounts of Ca(+2), Mg(+2), and Fe(+2) similar to those found in infant formulas reduced Zn(+2) uptake by at least 40%. Our data demonstrate that dietary minerals compete during brush border membrane transport, and may help explain antagonistic mineral interactions observed in vivo. Divalent metal concentrations and lactose content of milk affect zinc absorption in neonates and must be carefully considered in formula design.     

Characterization of cadmium uptake by the water lily Nymphaea aurora

This study characterizes cadmium (Cd) uptake by the waterlily Nymphaea aurora, (Nymphaeaceae) in two systems: a model hydroponic Cd solution and heavily polluted sludge from two sites in Israel. The uptake of Cd from hydroponic solution resulted in Cd storage in petioles and laminae of Nymphaea, as well as in the roots. The pH of the solution affected Cd solubility and availability, with pH 5.5 yielding maximum Cd content in the plant (140 mg Cd per g DW). Cd uptake was reduced by the addition of EDTA to the hydroponic growth medium, although EDTA enhanced heavy metal uptake by terrestrial plants. Nymphaea efficiently reduced the concentration of Cd in heavy metal polluted urban and industrial sludge and the amount of Cd uptake was enhanced by the addition of KCl to the sludge and by adjustment of the pH to 5.5. The inherent growth patterns of Nymphaea plants allowed Cd uptake by the shoot and root, and resulted in maximum contact between the various plant parts and the growth media. Thus, Nymphaea has potential as an optimal, highly effective phytoremediation tool for the removal of Cd from polluted waste sources.     

Influence of aerobic energy deficit, ouabain and harmaline on the phenylalanine and galactose active transport by snail intestine

The effect of anaerobiosis (N2 bubbling of the medium) or 10(-4) M dinitrophenol on the penetration of 0.5 mM Phe in snail and rat everted intestine, in 2 min and 30 min incubation periods, has been studied. The aerobic energy deficit inhibits the amino acid net entry in both species, but whereas the active transport is annulled in rat, snail intestine is capable of continuing to accumulate Phe against a gradient. The prolonged action (30 min of preincubation) of 1 mM ouabain inhibits 0.1 mM Phe and 0.1 mM galactose entry in snail intestine. Amino acid uptake is far higher than the one obtained in the absence of Na+, in which condition Phe keeps accumulating against a gradient in the tissue water. Galactose active transport, instead, becomes null in the presence of the glucoside or in the absence of Na+. One mM harmaline is able to inhibit the initial entry of galactose into the tissue, while higher than 5 mM concentrations are required to inhibit that of Phe. Results confirm that snail intestine is capable of easily carrying out active transport processes with energy from anaerobic origin. On the other hand Phe transport is less sensitive to the absence of Na+, presence of ouabain or harmaline than that of galactose, so that contrary to what has been observed for the sugar, the active transport of the amino acid is not annulled in any of the three conditions.