Effect of DTNB on rat intestinal galactose transport in vivo

The effect of the non-penetrating reagent of -SH groups: acid 5,5'-dithiobis (2-nitrobenzoic), (DTNB), on 1 mM galactose absorption in rat intestine in vivo has been studied. DTNB inhibits sugar absorption in about 35%, which is due to an action on the mediated transport component, but without affecting the diffusional passive one. Consequently it does not modify galactose absorption in the presence of 0.5 mM phlorizin or that of the non-transportable sugar 2-deoxy-glucose. Galactose transport inhibition appears after a not longer than 5 min preexposure period and it remains constant at least up to 30 min. The inhibitory effect does not vary between 0.1 and 1 mM DTNB and it reverses completely with 0.5 mM dithioerythritol. Protection by excess of substrate has not been observed. Results show that DTNB affects sulfhydryl groups very probably located at the luminal side and related to the proteins of the cotransport system.     

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.     

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.     

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.     

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.     

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.     

Effect of luminal Na+ on the kinetics of intestinal absorption of sugars in vivo

The effect of sodium concentration on the absorption kinetics of glucose, galactose and 3-o-methyl-glucose in rat and hamster jejunum in vivo has been studied. In consecutive 1 min periods the total absorption and absorption in presence of 0.5 mM phlorizin were measured. The difference between them was taken as the active transport rate. The perfusion rate value was 5.6 ml X min-1 and sugar concentrations in the perfusion solution ranged from 1 to 10 mM. The results for the different sodium concentrations show a nearly common Vmax for the same sugar and animal species, while the apparent KT values increase when the sodium concentration in the lumen decreases, mimicking a pure affinity-type activation system. The absorption of sugar when solutions without Na+ are perfused, is greater than that entering passively in the presence of phlorizin. An explanation may be that appreciable amounts of endogenous Na+ find their way to the intestinal lumen in favour of the gradient, making Na+-sugar cotransport possible.     

Transport and phosphorylation of D-galactose in renal cortical cells.

An improved analytical procedure for the extraction and determination of total, free and phosphorylated tissue sugar is described. This method, employing ZnSO4 plus Ba(OH)2 for the precipitation of sugar phosphates, yields values identical with those obtained by the more laborious separation of free and phosphorylated sugar by ion-exchange chromatography. Erroneous values for free sugar due to the action of a Zn2+ -activated phosphatase and/or the lability to acids of some sugar phosphates, are avoided. Using this technique for the sudy of transport and phosphorylation of D-galactose in rabbit renal cortical slices and tissue extracts, it was found: 1. The cellular uptake of D-galactose was associated with the appearance of both free and phosphorylated sugar whether or not external Na+ was present. At 1 mM sugar, galactose was accumulated in the cells against a modest concentration gradient of 1.445 +/- 0.097 (n = 17). Galactose phosphate appeared in the cells considerably faster than free sugar under conditions of net uptake as well as of steady-state exchange (pulse-labelling). 2. Increasing saline pH (6-8) increased the cellular levels of sugar phosphate without affecting the steady-state values of free sugar. With tissue extracts, increasing pH also stimulated the activity of galactokinase and the dephosphorylation of galactose 1-phosphate by a Zn2+ -activated phosphatase. 3. 0.5 mM phlorizin inhibited the tissue uptake of galactose and its subsequent oxidation to CO2 only to a minor degree (30 and 10%, respectively). The absence of external Na+ further depressed the phlorizin effect. Preincubation of the tissue with phlorizin and subsequent washing in part abolished the inhibitory effect. The data suggest that a major portion of the galactose uptake by the tissue proceeds by a mechanism with a low affinity for phlorizin. 4. Efflux studies showed that the wash-out of free galactose from slices was associated with a net decrease of both free and phosphorylated tissue sugar. 5. The above results suggest the possibility that phosphorylation may represent a step in the Na+ -independent, phloretin-sensitive transfer of D-galactose across the antiluminal cell membrane. The participation of intracellular galactokinase and a Zn2+ -activated alkaline phosphatase in the maintenance of the steady state of free and phosphorylated galactose in the cells has been demonstrated.     

Involvement of PKC and PKA in the inhibitory effect of leptin on intestinal galactose absorption

Studies from our laboratory have demonstrated that leptin inhibits galactose absorption in vitro by acting on the Na(+)/glucose cotransporter SGLT1. Since PKC and PKA are involved in the regulation of SGLT1 and leptin is able to activate these kinases, we have investigated the possible implication of PKC and PKA in the inhibition of sugar absorption by leptin in rat small intestinal rings. Inhibition of 1 mM galactose uptake by 0.2 nM leptin is blocked by 2 microM chelerythrine, a PKC inhibitor, which by itself does not affect galactose uptake. However, 1 microM H-89, a PKA inhibitor, inhibits galactose uptake and does not block leptin inhibition. Biochemical assays show that the inhibitory effect of leptin is accompanied by a approximately 2-fold increase in PKA and PKC activity. These findings indicate that the activation of PKC is more relevant than PKA activation in the inhibition of galactose absorption by leptin.     

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.     

Effect of thiol reagents and ionizing radiation on the permeability of erythrocyte membrane for spin-labeled non-electrolytes

Four different thiol reagents: p-chloromercuribenzoic acid (pCMB), mercuric chloride (HgCl2), N-ethylmaleimide (NEM), and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) were employed as agents modifying the transport of a hydrophilic and hydrophobic non-electrolyte spin labels: 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) and 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) into bovine erythrocytes. Gamma-irradiation of erythrocytes amplified the effects of pCMB, HgCl2 and NEM of inhibition of TEMPOL transport and attenuated them in the case of TEMPO transport. These results suggest that the transport of TEMPOL across the erythrocyte membrane is controlled by both superficially and more deeply located membrane -SH groups while only superficial -SH groups control the transport of TEMPO. The lower extent of inhibition of TEMPO transport indicates a higher contribution of diffusion through the lipid phase to the transport of TEMPO across the erythrocyte membrane as compared with TEMPOL.     

Inhibition by cadmium of D-galactose and L-phenylalanine transport by rat intestine in vitro

The effect of cadmium (CdCl2) on galactose and phenylalanine uptake by rat everted intestinal rings has been studied. The rings were preincubated (15 min) and incubated (5 min) in the presence of Cd. Galactose uptake (from 0.5 mM to 10 mM) was inhibited by 0.5 mM Cd about 25%. Only the phlorizin-dependent galactose transport was affected by cadmium, being a non-competitive type inhibition. A 15 min washing with saline solution significantly reduced the cadmium induced inhibition, which was practically reversed by washing with 5 mM EDTA. The uptake of 0.5 mM phenylalanine was not affected by 0.5 mM Cd but it was depressed by 1 mM Cd. Such inhibition was exerted on the sodium-dependent phenylaline transport. Washing with 5 mM EDTA diminished only slightly the inhibition of the transport by cadmium. It is suggested that the inhibition of intestinal transport of galactose and phenylalanine by cadmium may be due to its reversible interaction with metal-binding ligands, possibly sulfhydryl groups, related to the luminal transport systems.     

Transport of d-galactose by the gastrointestinal tract of the locust, Locusta migratoria

Due to exoskeleton, the absorption of nutrients in adult insects takes place across the gastrointestinal tract epithelium. In most physiological studies, sugar intestinal absorption has been described as a diffusional process and to date no sugar transporter has been cloned from the digestive tract of insects. In the present work, the existence of a saturable transport system for galactose in the gastric caeca of Locusta migratoria is clearly demonstrated. This transport shows a relatively high affinity for galactose (apparent K0.5=2-3 mM) and is inhibited by glucose, 2-deoxyglucose and with less potency by fructose and alpha-methyl-d-glucoside. The absence of sodium or the presence of phloridzin hardly affects galactose absorption, indicating that it is not mediated by a SGLT1-like transporter. The absence of K+, Cl-, Mg2+ and Ca2+ or changes in the pH do not modify galactose absorption either. Nevertheless, phloretin, cytochalasin B and theophylline (inhibitors of facilitative transporters) decrease sugar uptake around 50%. Xenopus laevis oocytes microinjected with poly A+ RNA isolated from gastric caeca show sodium-independent galactose uptake that is three times higher than in non-injected oocytes, further supporting the existence of a mRNA coding for at least one equilibrative sugar transporter in L. migratoria gastric caeca.     

Kinetics of intestinal sugar transport, in vivo

Sugar absorption by the small intestine has been studied in rat and hamster in vivo, with luminal perfusion, during 1 minute successive periods. Transport is calculated as the difference between absorption and diffusion. The diffusion component is evaluated in the presence of phlorizin or as absorption of sorbose. The resulting KT values for glucose and galactose (rat: 7.7 and 10 mM; hamster: 10 and 14 mM) and 3-0-methyl-glucose (hamster: 25-33 mM) are quite lower than those previously obtained in vivo, but still higher than those in vitro. The physiological levels of glucose in the intestine of normally fed animals imply that the diffusion component plays an important role in the proximal regions of the small intestine, especially in rat.     

Use of the paracellular way for the intestinal absorption of sugars

Passive absorption of D-Galactose (in the presence of 0.5 mM phlorizin), 2-deoxy-D-glucose and D-Mannitol by rat jejunum has been measured in vivo by perfusion of an intestinal segment with recirculation, along successive absorption periods of 5 or 10 min duration. In the range of 1 to 40 mM concentrations, the three solutes were absorbed at a very similar rate that varied as a lineal function of the concentrations in the perfusion solution. Absorption of 1 mM solute was not modified by the presence of 40 mM glucose or galactose. Passive absorption kinetics suggests processes of simple diffusion or solvent drag. The use of paracellular way for the passive absorption is supported by the fact that triaminopyrimidine (TAP) and protamine, which decrease the permeability through the tight junctions, also inhibit the absorption, with similar characteristics for both actions: TAP inhibition (53%) is very rapid and can be easily reversed, while that of protamine (30%) requires some time of previous exposure, lasts longer and can be reversed by heparin. The same analogy is shown by two actions that enhance the paracellular permeability: theophylline increases (30%) the passive absorption with lasting effect, while luminal hypertony enhances absorption transitorily. The passive absorption of the assayed solutes could be estimated to take place by the paracellular way in at least 50% and probably 70% or even more. The measure of net fluid fluxes reveals that solute fluxes must be prevailingly explained by simple diffusion, as the solvent drag can only play a very minor role.     

Effect of various coumarins on the intestinal absorption of galactose in vivo (author's transl)]

The effect of various coumarins on the active transport of galactose by small intestine in chick and rat was studied, using the in vivo technique of sucessive absorptions. A 10(-4) M concentration of the different coumarins inhibits the absorption of galactose in the chick. This effect persists in successive absorptions without coumarin. In rat, inhibition of galactose active transport by coumarins was observed at 10(-3) M concentration.     

Glucose Transport in Brucella abortus

Brucella abortus British strain 19 transported glucose with an apparent K(m) of 0.16 mM and an apparent V(max) of 250 nmol per min per mg of N. The only common glucose analogue transported was 2-deoxyglucose (2-DOG), with an apparent K(i) of 0.73 mM. Alpha- or beta-methyl glucosides and 3-O-methylglucose were not transported. Transport was linear for 70 to 90 s, depending on the concentration of substrate used. 2-Deoxyglucose was transported as the free sugar and was not further metabolized once inside the cell. There was no glucose phosphoenolpyruvate phosphotransferase system (PEP-PTS) present, and there were no inhibitors present in Brucella cell-free extract that inhibited the Escherichia coli glucose PEP-PTS. N-Ethylmaleimide (NEM) and p-chloromercuribenzoate (pCMB) completely inhibited transport of glucose and 2-DOG. Glutathione, dithiothreitol, and beta-mercaptoethanol reversed the effects of pCMB but not of NEM. A pH optimum of 7.2 and a temperature optimum of 37 to 45 C were observed for both K(m) and V(max). The glucose transport system appeared to be constitutive for glucose transport in cells grown on fructose, galactose, erythritol, or glucose. The electron transfer inhibitors carbonyl cyanide, m-chlorophenylhydrazone, NaN(3), 2,4-dinitrophenol, and KCN inhibited 2-DOG transport to a greater extent than did the metabolic energy inhibitors NaAsO(4), iodoacetate, KF, and 2-heptyl-4-hydroxyquinoline-N-oxide. Dicyclohexylcarbodiimide, an inhibitor of membrane-bound adenosine triphosphatases, inhibited transport by 100%.     

Effects of lanthanum on calcium-dependent phenomena in human red cells.

Lanthanum (0.25 mM) does not penetrate into fresh or Mg2+-depleted cells, whereas it does into ATP-depleted or ATP + 2,3-diphosphoglycerate-depleted cells, into cells containing more than 3 mM calcium, or cells stored for more than 4 weeks in acid/citrate/dextrose solution. In fresh cells loaded with calcium, extracellular lanthanum blocks the active Ca2+-efflux completely and inhibits (Ca2+ + Mg2+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity to about 50%. In Mg2+-depleted cells Ca2+-Ca2+ exchange is inhibited by lanthanum. Ca2+-leak is unaffected by lanthanum up to 0.25 mM concentration; higher lanthanum concentrations reduce leak rate. In NaCl medium Ca2+-leak +/ S.D. amounts to 0.28 +/ 0.08 mumol/1 of cells per min, whereas in KC1 medium to 0.15 +/ 0.04 mumol/1 of cells per min at 2.5 mM [Ca2+]e and 0.25 mM [La3+]e pH 7.1. Lanthanum inhibits Ca2+-dependent rapid K+ transport in ATP-depleted and propranolol-treated red cells, i.e. whenever intracellular calcium is below a critical level. The inhibition of the rapid K+ transport can be attributed to protein-lanthanum interactions on the cell surface, since lanthanum is effectively detached from the membrane lipids by propranolol. Lanthanum at 0.2--0.25 mM concentration has no direct effect on the morphology of red cells. The shape regeneration of Ca2+-loaded cells, however, is blocked by lanthanum owing to Ca2+-pump inhibition. Using lanthanum the transition in cell shape can be quantitatively correlated to intracellular Ca2+ concentrations.     

On the multiplicity of glucose analogues transport systems in rat intestine.

A study has been made to test if in intact epithelium of rat jejunum with in vivo and in vitro techniques, two transport systems for glucose and analogues, as those characterized in brush border membrane vesicles from guinea pig jejunum, are operative. The passive and mediated transport components of the D-galactose and methyl alpha-D-glucopyranoside intestinal absorption and the mutual inhibitions between both substrates at different relative concentrations have been measured. The effects of cytochalasin B and low temperature (20 degrees C) on the transport in vitro have also been observed. Cytochalasin B inhibits galactose and alpha-methylglucoside transport at 0.1 and 40 mM concentrations in similar percentage. Transport of 0.1 and 40 mM galactose is inhibited 61 and 77% respectively by low temperature (20 degrees C). The transport of galactose and alpha-methylglucoside could be explained by the assumption of just one transport system shared by both substrates, with a higher affinity for alpha-methylglucoside. Operation of two systems was not demanded by the results, due perhaps to species specificity or to the distorting action of the unstirred water layers.     

Conversion of 14C-galactose into amino acids in tissue culture cells and its inhibition by manganese

The incorporation of 14C-galactose into primary AGMK-cells was studied in the presence and absence of Mn2+. The transport of galactose into the cells is not influenced by Mn2+. 1 mM MnCl2 inhibits the incorporation of galactose into acid-precipitable material up to 50% after 6 hours incubation. In the absence of Mn2+ a substantial amount of galactose is converted to glucose, which is mainly metabolized into aspartic acid and serine. The conversion of galactose into glucose is inhibited by the addition of Mn2+. However, Mn2+ does not influence the activity of the UDP-galactose-4'-epimerase in vitro. Using the SDS-polyacrylamide electrophoresis the labelling of protein bands is similar with 14C-galactose or a 14C-amino acid mixture, respectively. In the presence of Mn2+ the incorporation of both galactose or amino acids is inhibited: With amino acids the inhibition is observed in all protein bands, whereas with galactose some bands remain unaffected. It is concluded that these are galactoproteins.