Active Transport Of D-glucose Research Articles

The use of a perfluorochemical emulsion as a vascular perfusate in drug absorption.

In-vitro simultaneous luminal and vascular perfusion using the perfluorochemical emulsion, FC-43 emulsion, as a vascular perfusate, was examined for drug absorption in rat jejunum. The intestinal membrane in this system was found to retain its normal barrier functions for drug transport, as evident from the following: (i) stable absorption clearance of tritiated water and salicylic acid at steady-state, (ii) agreement of this clearance with that by in-situ single-pass luminal perfusion, (iii) active transport of D-glucose and its inhibition by phloridzin and (iv) normal glutamate pyruvate transaminase activity in the intestinal mucosa. FC-43 emulsion gave a more normal absorption site blood flow than the usual vascular perfusate containing erythrocytes and albumin in Krebs-Henseleit bicarbonate buffer solution. Consequently, this emulsion was useful for examining the contribution of blood flow resistance toward drug absorption. The rate-limiting steps of absorption of tritiated water, antipyrine and salicylic acid were examined by perfusion using FC-43 emulsion. The absorption of tritiated water was almost completely blood flow-limited and its absorption clearance may possibly be an approximated absorption site blood flow. The contribution of blood flow resistance to total resistance for antipyrine absorption exceeded that for salicylic acid absorption.

Ketoprofen-induced intestinal permeability changes studied in side-by-side diffusion cells.

It is known that non-steroidal anti-inflammatory drugs (NSAIDs) increase intestinal permeability. Increased intestinal permeability is believed to result from the opening of tight junctions because of NSAID-induced reduction of prostaglandin synthesis and/or energy-depletion. In this study, ketoprofen-induced changes in intestinal permeability were evaluated by measuring tissue electrical parameters, namely tissue electrical resistance (TER), short circuit current (I(sc)) and transepithelial potential difference (PD), and the transport of a paracellular marker, fluorescein, across rat jejunum in-vitro. Ketoprofen, added to the mucosal side of the tissue, decreased TER and increased fluorescein transport in a concentration-dependent manner. I(sc) values and the active transport of D-glucose were not affected at ketoprofen concentrations of less than 5 mM. Higher ketoprofen concentrations decreased I(sc) values and diminished active transport of D-glucose, while transport of fluorescein increased markedly. Similar effects on intestinal properties were observed when the metabolic inhibitor sodium azide was added to the incubation medium. The results of this study suggest that the increased intestinal permeability observed at lower ketoprofen concentrations (< 5 mM) is most probably a consequence of reduced prostaglandin tight junction control, whereas at higher concentrations, ATP depletion caused by ketoprofen seems to be the major mechanism for increased intestinal permeability.

In-vitro and in-vivo studies of cefpirom using bile salts as absorption enhancers.

Cephalosporins have to be administered by injection because of the poor intestinal absorption of the orally delivered drugs. Because of the obvious drawbacks of drug delivery by injection, the development of alternatives with enhanced oral bioavailability is receiving much attention in pharmaceutical research. Cefpirom (Cp) is a new semi-synthetic amino-2-thiazolyl-methoxyimino cephalosporin that has been substituted in position 3 with a cyclopenteno-pyridinium group in order to create a zwitterionic compound. It exhibits highly hydrophilic properties, as shown from its extremely low partition coefficient, and therefore its lipophilicity was increased using bile salts. The effect of this on the partition coefficients determined in the n-octanol/buffer system was confirmed using an in-vitro transport model with artificial and biological membranes. The pharmacokinetic properties of Cp were investigated in rabbits after intraduodenal administration with and without bile salts. Furthermore, the physiological compatibility of the bile salts was investigated using active D-glucose transport.

Influence of enhancers on the absorption and on the pharmacokinetics of cefodizime using in-vitro and in-vivo models.

In the development of novel antibiotics, more and more compounds have been found that cannot be absorbed orally and, therefore, must be administered intravenously or intramuscularly. Because of the obvious drawbacks of drug delivery by injection, the development of alternatives with enhanced oral bioavailability has received much attention in pharmaceutical research. Cefodizime, a novel third-generation cephalosporin with significant advantages in the parenteral treatment of common infections, was used as a model drug. Cefodizime behaves as a highly hydrophilic compound, as shown from its extremely low partition coefficient. The effect of cationic absorption enhancers (hexadecyldimethylbenzylammonium chloride, N-hexadecylpyridinium bromide, dodecyltrimethylammonium bromide and hexadecyltrimethylammonium bromide) on the lipophilicity of cefodizime was investigated by means of the n-octanol/water system. Results showed that the counter-ions had a positive influence on the solubility of cefodizime. These results on partitioning coefficients in the n-octanol/buffer system were confirmed using an in-vitro transport model with artificial and biological membranes (Caco-2-cells). Furthermore, the physiological compatibility of the absorption enhancers was investigated using the active D-glucose transport. The pharmacokinetic profile of cefodizime was evaluated in rabbits after intraduodenal administration with and without an absorption enhancer.

Effect of exogenous ATP on Momordica charantia Linn. (Cucurbitaceae) induced inhibition of d-glucose, l-tyrosine and fluid transport across rat everted intestinal sacs in vitro

Momordica charantia (MC) is a common oriental vegetable with known antidiabetic, laxative and antimicrobial properties. This study investigates the effects of aqueous fruit extract of MC on the transport of d-glucose, l-tyrosine and fluid across rat-everted intestine in vitro. Everted intestinal sacs from rats were mounted in an organ bath containing Krebs–Henseleit bicarbonate buffer. Graded concentrations (1.5–12 mg/ml) of MC fruit extract were incubated in the mucosal solution with and without exogenous ATP in the mucosal bathing fluid. The serosal appearance and mucosal disappearance of d-glucose, l-tyrosine and the fluid absorptive capacity of the intestine were significantly inhibited ( p < 0.05) with increasing graded concentrations of MC. The concentration of d-glucose accumulated or metabolized by the enterocytes in the intestinal tissues were significantly higher ( p < 0.05) when incubated with MC. Increasing graded concentrations of exogenous ATP (25–200 μM) were incubated with 3.0 mg/ml MC to confirm inhibition of the ATP-dependent active transport of d-glucose, l-tyrosine and fluid across rat enterocytes. It was found that increasing concentrations of mucosal ATP from 25 to 100 μM significantly ( p < 0.05) reverses the MC-depression of the d-glucose, l-tyrosine and fluid uptake across rat everted intestinal sacs. It is hypothesized that bioactive phytochemicals such as saponins in MC fruit extract inhibits the active transport of d-glucose, l-tyrosine and fluid across rat intestine by inhibiting the production of ATP responsible for the active transport of these molecules. It is likely that MC can be a potential alternative drug therapy of postprandial hyperglycaemia via inhibition of glucose uptake across the small intestine.

Developmental and lesion-induced changes in the distribution of the glucose transporter Glut-1 in the central and peripheral nervous system.

The active transport of D-glucose from the vascular to the neural compartment requires the presence of a carrier molecule at the blood-brain and the blood-nerve barrier. The glucose transporter 1 (Glut-1) seems to be the main carrier in blood-tissue barriers of endothelial and perineurial type. The distribution of Glut-1 was assessed in the normal central and peripheral nervous system of young and adult animals and compared with changes after nerve injury. Immuno-histochemistry, in situ hybridization, and perfusions with Evans Blue were carried out. Glut- I was not expressed in the perineurium of peripheral nerves at birth, but appeared in the perineurium of peripheral nerves, spinal roots, in the capsule of dorsal-root ganglia, and in the pia mater of adult animals. The perineurium of peripheral nerves subjected to Wallerian degeneration presented a faint Glut-1 immunoreaction, which was restored after regeneration. Glut-1 was expressed in capillaries of the gray substance of the spinal cord. Perineurial-derived lamellar cells of Pacinian corpuscles exhibited a strong Glut-1-like immunoreactivity in response to denervation and during development. Merkel cells and Meissner corpuscles were found to be Glut-1 negative. Glut-1 seems to reflect the quality of an adult, mature perineurial and blood-nerve barrier.

Effect of sanguinarine on the transport of essential nutrients in an everted gut sac model: role of Na+,K(+)-ATPase.

The effect of the argemone alkaloid, sanguinarine, was studied on the active transport of D-glucose and some of the L-amino acids in everted sacs of the small intestine of rats. Sanguinarine (1.0 mumole) was found to inhibit (61%) the transport of D-glucose, while an alkaloid concentration of 0.1 mumole was ineffective. Both 0.1 and 1.0 mumole of sanguinarine had no effect on the transport of the L-amino acids including aspartic acid, lysine, and tyrosine. Sanguinarine showed a dose dependent inhibition of intestinal and hepatic Na+,K(+)-ATPase in a non-competitive manner. The inhibition of Na+,K(+)-ATPase by sanguinarine may in turn inhibit the active transport of D-glucose which requires a sodium pump.

Boosted mucosal immune responsiveness in the rat intestine by actively transported hexose

Anaphylaxis-mediated intestinal fluid secretion was measured in Trichinella spiralis- or ovalbumin-immunized rats challenged intraduodenally with T. spiralis somatic antigen (1 mg protein/0.5 mL saline) or ovalbumin (1 mg/0.5 mL saline), respectively. Intestinal fluid volume was measured 30 minutes after challenge as an index of net secretion. Challenge with the antigenic bolus containing 40 mmol/L d-glucose induced twice the fluid secretion as that induced by either antigen alone. l-Glucose was an ineffective substitute for d-glucose. The enhancement of secretion by d-glucose was dependent on prior immunologic sensitization, was diminished in the presence of phlorizin, and was mimicked by β-methyl glucoside. Results indicate that the active transport of d-glucose augments the antigen-mediated fluid secretion, possibly by enhancing permeation of the intestinal epithelium to antigen, thereby providing greater access to the mucosal immune system.

Effect of medium-chain glycerides on the membrane transport of D-glucose and sulfanilic acid in the intestinal brush-border membrane vesicles.

To clarify the influence of medium-chain glycerides (MCG) on a biological membrane, we investigated the membrane transport of D-glucose and sulfanilic acid in the brush-border membrane (BBM) vesicles pretreated with MCG. The size distribution of the BBM vesicles determined by electron microscopic observation was not significantly different between the vesicles incorporated with MCG and those of the control. However, the amount of D-glucose taken up by the vesicles at an equilibrated stage (30 min) was significantly decreased in the MCG-treated ones based on unit content of protein. Based on these results we estimated the membrane transport of D-glucose and sulfanilic acid in consideration of vesiculation or filter-capturing efficiency in MCG-treated vesicles. The rates of Na+ gradient-independent D-glucose transport and sulfanilic acid transport were significantly greater in MCG-treated vesicles than in the control. On the other hand, the magnitude of overshooting effect in Na+ gradient-dependent uptake of D-glucose in MCG-treated vesicles was maintained similar to the control. Comparison of kinetic parameters for active D-glucose transport at different concentrations indicated that Km and Vmax were not significantly different between MCG-treated and the control vesicles. These results indicated that passive diffusion of D-glucose and sulfanilic acid was significantly increased but Na(+)-glucose cotransporter was not significantly changed by the incorporation of MCG in the intestinal BBM vesicles.

A correlation of permeabilities for passively transported compounds in monkey and rabbit jejunum.

Permeability measurements were conducted for a series of compounds using in vitro tissue sections from monkey and rabbit jejunum. Jejunal segments were stripped of serosal musculature and mounted in a diffusion-cell system, using previously described methods and equipment. Permeability determinations of radiolabeled compounds ranging over two orders of magnitude in molecular weight were conducted. For the compounds examined, the permeability of the rabbit jejunum was approximately twice that of the monkey. This was in contrast to the relationship implied by the stripped tissue thickness measurements of 0.92 and 0.83 mm for rabbit and monkey, respectively. An investigation of the size of the paracellular space in the jejunum was undertaken to account for this apparent discrepancy in tissue permeability. Scanning electron micrographs of intestinal sections revealed a similar packing density of cells between species; however, a difference was noted in the shape and number of villi per unit area. Comparative measurements of the paracellular volume in both species using mannitol and methoxyinulin as extracellular space markers further suggests that the paracellular junctions are similar in size but more numerous per unit area of rabbit jejunum than that of the monkey. In contrast to passively transported compounds, the active transport of D-glucose was greater in monkey jejunum compared to rabbit tissue segments. When active transport was inhibited by blockade of the sodium pump with ouabain, the passive component of D-glucose transport for both rabbit and monkey tissue was in agreement with the relationship demonstrated above for compounds which are solely transported by passive processes.

Stimulation of intestinal Na+/d-glucose cotransport by monoclonal antibodies

The small intestinal brush border membrane is endowed with a number of transport systems. Monoclonal antibodies were produced against integral membrane proteins and tested for their ability to bind to such membranes. For this purpose papain-digested, deoxycholate-extracted BBMVs from rabbit small intestine were used to immunize mice. Of the 765 hybridoma supernatants tested, 119 gave a significantly higher extent of binding to the crude antigen preparation as compared with the background. The monoclonal antibodies were also tested for their ability to influence the sodium-dependent uptake of solutes into intact BBMVs. Two monoclonal antibodies clearly showed stimulation of secondary active D-glucose transport, whereas sodium-dependent uptake of L-alanine and L-proline was not affected. Hydrophobically labeled, i.e. intrinsic, membrane proteins of 175, 78 and 65 kilodaltons could be immunoprecipitated by both monoclonal antibodies, the 78 kDa band corresponding in all likelihood to the Na+/glucose cotransporter.

In vitro measurement of gastrointestinal tissue permeability using a new diffusion cell.

A new diffusion cell, derived from the Ussing chamber, was developed for the measurement of tissue permeability. This cell incorporates the attributes of using a single material and laminar flow across the tissue surface. In addition, the design allows the cell to be manufactured in a wide range of sizes to allow optimization of surface area to volume for a variety of tissues. The apparatus is applicable to the evaluation of transport of compounds through mucosal/epithelial barriers, i.e., gastrointestinal tissue. Active transport, permeability enhancers, enzymatic degradation, and absorption in various tissue sections can be explored. Preliminary data are consistent with the expected effects of molecular size and partition coefficient of a transported molecule on permeability in epithelial tissue. In addition, active transport of D-glucose and inhibition by phloridzin and ouabain can be demonstrated.

The Assessment of Recovery of the Intestine after Acute Radiation Injury

Several aspects of intestinal function and morphology are affected by acute radiation damage, including changes in the activity of proliferative cells in the crypts, immune cell populations, and the transport of various substrates. This study was designed to compare the time course of the recovery of intestinal proliferation, transport, and leukocyte population following radiation injury. Rats received a single dose of 6 Gy to the abdomen from a 137Cs source and were studied 3, 7, and 14 days later. No changes in the passive uptake of L-glucose or D-leucine were observed in the jejunum. Active transport of D-glucose and maximal water uptake were reduced at 3 days but had returned to normal by 7 days, whereas L-leucine uptake required more than 7 days to return to control levels. Mucosal permeability, assessed by an in vivo potential difference technique, remained increased 7 days after irradiation. Ornithine decarboxylase, an indicator of DNA synthetic activity, was elevated following radiation treatment and remained so even after 14 days. By comparison, myeloperoxidase activity, used as a quantitative monitor of granulocyte numbers, was still reduced after 7 days. These data indicate that while certain parameters of gut function may return to normal soon after radiation injury, the recovery of other factors is more prolonged. Thus the return of transport function to normal values post irradiation may be viewed as an adaptive change rather than simply the recovery of the tissue.

Heavy metal-induced inhibition of active transport in the rat small intestine in vitro. Interaction with other ions

1. 1. The effect of Hg 2+, Cd 2+ and Pb 2+ on the intestinal transport of d-glucose and l-tyrosine was studied in everted sacs of small intestine. 2. 2. Hg 2+, Cd 2+ and Pb 2+ (10 −5 and 10 −4) singly or in combination inhibit the active transport of d-glucose (10mM) and l-tyrosine (2mM) in rat and pigeon small intestine. 3. 3. The presence of other ions (Mg 2+, Zn 2+ and SeO 3 2) diminishes the toxic effect of heavy metal ions, rendering them less effective.

Forskolin-induced elevation of rat jejunal cyclic AMP levels and stimulation of active glucose transport in vitro.

The adenylate cyclase stimulator forskolin enhanced both mucosal cyclic AMP levels and D-aldohexose transport in rat jejunum in vitro in a concentration-dependent manner. With mucosal cyclic AMP elevated to 400% of basal values, active D-glucose transport was at 200% of control values. Transport parameters, as determined in a three compartment model using a dual label method, indicate a) increased "uphill" glucose transport at the brush border membrane with higher intracellular accumulation, b) unchanged serosal to mucosal glucose fluxes and passive permeabilities. Transport stimulating effects of forskolin were also present with D-galactose in the rat. In mouse jejunum active transport of D-glucose and 3-O-methylglucose was increased by forskolin 10 mumol/l. Phlorizin inhibited D-glucose transport as well as L-glucose transport in the rat were not changed by the persisting cyclic AMP elevation induced by forskolin. The results show a positive correlation of active intestinal hexose transport with a modest elevation of mucosal cyclic AMP. In accordance to current models of cyclic AMP-induced changes in intestinal fluid absorption, rat jejunal net fluid transport was reduced by 40% with 20 mumol/l forskolin.

Polyamines stimulate d-glucose transport in isolated renal brush-border membrane vesicles

Polyamines are natural constituents of most living organisms. However, their function in normal or pathological conditions is not fully understood. We have investigated in vitro effects of polyamines on characteristic properties of isolated renal brush-border membrane vesicles in order to determine whether polyamines have a regulatory role in membrane transport processes. The polyamines putrescine, spermidine and spermine were found to stimulate d-glucose uptake. Diffusional l-glucose uptake was not altered, indicating that the polyamines affected the active transport of d-glucose, rather than inducing nonspecific changes in membrane lipid properties. The amiloride-sensitive Na +/H + exchange was slightly inhibited by polyamines while Mg 2+-ATPase activity was stimulated. The polyamine effects could not be explained solely by the polycationic properties of these agents, since polycationic polypeptides had an opposite effect. For example, lysozyme was found to inhibit d-glucose transport. Spermine was incorporated into the trichloroacetic acid-insoluble fraction of brush-border membrane proteins. Results indicated that this incorporation process consisted of at least two components: a Ca 2+-independent component and a Ca 2+-dependent component, possibly as a result of transglutaminase activity which was present in the isolated renal brush-border membranes. By using SDS-polyacrylamide gel electrophoresis in conjunction with fluorography, | 3H|spermine was shown to be incorporated into several brush-border membrane proteins, mainly the 57 kDa, 74 kDa, 100kDa, a heavy molecular weight band (greater than 200 kDa) and a low molecular weight band (less than 10 kDa). Our results suggest that the polyamine effects on membrane function may be due to a covalent modification of membrane proteins, possibly via a transglutaminase-mediated incorporation of polyamines or to the crosslinking of membrane proteins.

Enzyme activities and sodium-dependent active d-glucose transport in apical membrane vesicles isolated from kidney epithelial cell line (LLC-PK 1)

Apical membrane vesicles were isolated from the confluent LLC-PK 1 cells by nitrogen cavitation and Mg/EGTA precipitation mfethods. The specific activities of marker enzymes for apical membranes were enriched 8- to 18-fold relative to those in the homogenate. d-[ 3H]Glucose uptake into the vesicles was stimulated in the presence of Na + gradient (overshoot phenomenon), and the values of apparent K m and V max for Na +-dependent component of d-glucose uptake were 0.3 mM and 5.8 nmol/mg protein per min, respectively.

Inhibition of the active transport of d-glucose and l-tyrosine by DDT and DDE in the rat small intestine

1. The effect of DDT and DDE on the active transport of d-glucose and l-tyrosine was studied in everted sacs of small intestine. 2. DDT and DDE (10 −5 and 10 −4 M) inhibit the active transport of d-glucose and l-tyrosine. 3. These organochlorine compounds also inhibit the (Na + + K +)-ATPase from the same tissue, to about the same extent. 4. These results suggest that the inhibition of the active transport is due to an action of DDT and DDE on the sodium pump.

Transport of d‐Glucose and 3‐O‐Methyl‐d‐Glucose in the Cyanobacteria Aphanocapsa 6714 and Nostoc strain Mac

1. The cyanobacterium Aphanocapsa 6714 which grow in the dark on D-glucose, will take up D-glucose and the analogue 3-O-methyl-D-glucose; uptake of each of these compounds was inhibited competitively by the other and by 6-deoxy-D-glucose. 2. This cyanobacterium accumulated 3-O-methyl-D-glucose up to 100-fold relative to the medium but did not modify or metabolize it to a significant degree. 3. Intracellular 3-O-methyl-D-glucose was rapidly displaced from Aphanocapsa 6714 by exogenous D-glucose and 3-O-methyl-D-glucose. 4. Although not characterized to the same extent, D-glucose and 3-O-methyl-D-glucose uptake by Nostoc strain Mac, another cyanobacterium capable of growth in the dark on D-glucose, was similar. 5. Other cyanobacteria that do not grow on D-glucose take up this compound at much lower rates which were unaffected by analogues of D-glucose that greatly reduced carbohydrate uptake by Aphanocapsa 6714 and Nostoc strain Mac. 6. It is therefore proposed that Aphanocapsa 6714 and Nostoc strain Mac possess a mechanism for the active transport of D-glucose. The absence of this transport mechanism is suggested as the reason why other strains fail to grow in the dark on this substrate. These latter organisms are therefore naturally cryptic with respect to D-glucose as a growth substrate.

Action of 5-thio- d-glucose and its 1-phosphate with hexokinase and phosphoglucomutase

Previous work from this laboratory has shown that 5-thio- d-glucose is a competitive inhibitor for active transport of d-glucose. The present work indicates that the thiosugar analog and its 1-phosphate can also interfere with d-glucose 6-P formation. 5-Thio- d-glucose serves as a substrate for yeast hexokinase with a K m of 4 m m, and V of 8.8 nmol/min/μg of protein. The analog competitively inhibits d-glucose phosphorylation with a K i of 20 m m. 5-Thio- d-glucose 1-P can act as a substrate for rabbit skeletal muscle phosphoglucomutase with a K m of 60 μ m and V of 0.17 μmol/min/μg of protein. Thus, 5-thio- d-glucose 1-P behaves as a near metabolic analog of d-glucose 1-P. 5-Thio- d-glucose 1-P is a competitive inhibitor of d-glucose 1-P conversion to the 6-P with a K i of 16.2 μ m. 5-Thio- d-glucose 6-P produced by phosphorylation of 5-thio- d-glucose and by conversion from 5-thio- d-glucose 1-P was identified by chromatographic mobility and by color reactions.