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Postnatal development of glucose transporter proteins in bovine skeletal muscle and adipose tissue.

Facilitated diffusion of glucose across the plasma membrane is mediated by a family of glucose transporter (GLUT). GLUT1 is ubiquitously present in all tissues and involved in cellular glucose uptake, while GLUT4 plays a key role in cellular glucose uptake stimulated by insulin in skeletal muscles and adipose tissue. To examine the postnatal change in the GLUTs of ruminants, the protein levels of GLUT1 and GLUT4 were measured by Western blot analysis of skeletal muscles, adipose tissue and brain of Holstein male calves aged from 0 to 12 months. Analysis of rumen short chain volatile fatty acids revealed that rumen fermentation increased around 2-3 months old. The GLUT1 level did not change in all tissues examined during the postnatal period, while the GLUT4 levels in skeletal muscle and subcutaneous adipose tissue decreased gradually, and at 12 month old, it was about 40% of those seen at 0 month old. These results are contrast to those in non-ruminant species, in which GLUT4 increases during postnatal development, and may be related to the insulin-resistance seen in adult ruminants.

Adipose Tissue↗

Nickel absorption and distribution from rat small intestine in situ.

The aim of this work was to study the absorption of nickel chloride in rats by means of the intestinal perfusion in situ technique at nickel concentrations of 1, 5, 10, 25, and 100 mg/L. Active transport and facilitated diffusion seem to play an important role in the intestinal absorption of nickel at concentrations < or = 10 mg/L. At higher concentrations, the absorption rate would be limited by saturation of the carriers. The distribution of the absorbed nickel was studied by intestinal perfusion of a 10-mg Ni/L solution for 30 or 60 min. Both in concentration and amount, the jejunum showed the higher values of absorbed nickel, followed by the kidneys and liver. When all of the collected organs (brain, heart, liver, lungs, spleen, kidneys, and testicles) and blood, but not the small intestine, are analyzed following a 60-min perfusion, it was found that 1% of the initial concentration had passed through the intestinal barrier.

Animals↗

Placental glucose transfer and fetal growth.

One of the primary regulators of maternofetal glucose transfer is the density of glucose transporter proteins in the placenta. These transporters, members of the GLUT gene family of facilitated-diffusion transporters, are embedded in the microvillous (maternal-facing) and basal (fetal-facing) membranes of the syncytiotrophoblast, the main placental barrier layer. Eight members of this family have been described in human placental tissue, but only GLUT1 protein has been identified in the syncytium, where its distribution is asymmetric. The microvillous membrane contains markedly more transporter than the basal, and, as a result, the basal membrane acts as the rate-limiting step in transplacental glucose transport; thus, changes in the density of basal membrane GLUT1 will have a significant impact on transplacental glucoseflux. What little is known about syncytial GLUT1 expression is restricted to factors associated with fetoplacental growth and metabolism; GLUT is inversely regulated by glucose concentration and basal membrane GLUT1 is positively regulated by insulin-like growth factor I, placental growth hormone, and hypoxia. In vivo, basal membrane GLUT1 is upregulated over gestation, increased in diabetic pregnancy, and decreased in chronic hypoxia, while microvillous membrane GLUT1 is unaffected. The contrast between in vitro and in vivo regulation and the specific changes in GLUT1 distribution suggest more complex regulatory interactions than those yet described.

Biological Transport↗

Regulation of the intracellular pH in the protozoan parasite Trypanosoma brucei brucei.

The mechanisms regulating the intracellular pH (pHi) in both forms of Trypanosoma brucei brucei (cultured cells) were investigated using the fluorescent probe 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). The pHi values measured were 7.22+/-0.03 in the procyclics and 7.40+/-0.05 in the bloodstream form. In the presence of 24mM HCO3-, pHi values were slightly higher in both forms of trypanosomes suggesting a bicarbonate-linked pH regulation. pHi was more stable in procyclics (between 7.15 and 7.30 in the external pH range 6.4-7.6) than in the bloodstream forms. The amiloride analogue tested decreased pHi, suggesting Na+-driven Na+/H+ antiporters. H+-ATPases also seem to be involved in pHi regulation since the inhibitors N-ethylmaleimide (1 mM) and N,N'-dicyclohexylcarbodiimide (80 microM) induced a rapid acidification in both forms of trypanosomes. Addition of pyruvate caused a cytosol acidification in the bloodstream form only confirming the existence of a diffusion-facilitated carrier for pyruvate, with the cotransport of H+. Our results show that, although similar pH regulation mechanisms seem to exist in both forms of trypanosomes, the procyclics can regulate efficiently their pHi and consequently their plasma membrane potential whereas the bloodstream forms cannot always maintain their pHi and are easily depolarized following a small acid load.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

Uptake of thymidine into isolated rat hepatocytes. Evidence for two transport systems.

Thymidine transport was studied in isolated rat hepatocytes. In these cells no phosphorylation of the substrate by thymidine kinase occurred subsequent to transport. Results from studies of the concentration-dependent uptake of thymidine indicated two transport systems with about 80-fold differences in their kinetic constants. These systems were denoted as high affinity [Km = 5.3 micron, V = 0.47 pmol/(10(6) cells X s)] and low affinity systems [Km = 480 micron, V = 37.6 pmol/(10(6) cells X s)]. From intracellular to extracellular distribution ratios of [3H]thymidine it could be concluded that the uptake by the high affinity system was a concentrative process while the transport by the low affinity system was non-concentrative. The uptake of [3H]-thymidine by the high affinity system could only be inhibited by unlabeled thymidine. In contrast, all other nucleosides tested (uridine, 2'-deoxycytidine, and 2'-deoxyguanosine) were equally effective in inhibiting the low affinity system competitively. The results would suggest that in hepatocytes lacking phosphorylation by thymidine kinase, thymidine is taken up by a high and a low affinity system working in tandem. The high affinity system seems to be an active transport process with narrow substrate specificity. Thymidine uptake by the low affinity system is a facilitated diffusion process. This system is considered to be a common transport route for nucleosides of different structures.

Animals↗

Pregabalin: a new anxiolytic.

Pregabalin (S-[+]-3-isobutylgaba) was designed as a lipophilic GABA (gamma-aminobutyric acid) analogue substituted at the 3'-position in order to facilitate diffusion across the blood-brain barrier. It was originally developed as an anticonvulsant agent, however it has been shown to be effective in the treatment of several disorders including hyperalgesia and behavioural disorders. Although its exact mode of action remains unclear, pregabalin interacts with the same binding site and has a similar pharmacological profile as its predecessor, gabapentin (1-[aminomethyl] cyclohexane acetic acid). Its main site of action appears to be on the alpha(2)delta subunit of voltage-dependent calcium channels, widely distributed throughout the peripheral and central nervous system. Pregabalin appears to produce an inhibitory modulation of neuronal excitability. In healthy volunteers, it is rapidly absorbed with peak blood concentrations within 1 h and it has a bioavailability of approximately 90%. In preclinical trials of anticonvulsant activity, pregabalin is three to ten times more potent than gabapentin. It is well-tolerated and associated with dose-dependent adverse effects (ataxia, dizziness, headache and somnolence) that are mild-to-moderate and usually transient. There are no known pharmacokinetic drug-drug interactions reported to date. Preliminary animal and human studies showed beneficial effects in both ethological and conflict models of anxiety, as well as having some sleep-modulating properties. In Phase II and III trials, pregabalin shows promising anxiolytic action when compared to placebo in generalised anxiety disorder, social phobia and panic disorder.

Animals↗

Glucose movement into rat interine fluid.

The uterine horns of anaesthetized rats were cannulated and 2.5 ml 0.9% NaCl at 37 degrees C were recirculated for up to 90 min. Glucose appeared in the medium, reaching a concentration of approximately 1 mM. Various aspects of the transport process were examined, and it is suggested that the glucose is mainly derived from the plasma, and probably enters the uterine lumen by a process involving facilitated diffusion.

Animals↗

Glucose transport and metabolism in rabbit oviduct epithelial cells.

The respiration of intact slices and of isolated epithelial cells from rabbit oviducts was compared. The oxygen consumption of the slices was approximately double that of the cells, but glucose was found to be a preferred substrate in each case. Isolated cells exhibited a Crabtree effect in that glucose concentrations greater than 5.55 mmol l-1 inhibited the respiratory rate. The oviduct epithelial cells grown in primary culture on collagen-impregnated filters formed a polarized monolayer, enabling the transepithelial movement of glucose to be studied under defined conditions. The cells, grown in this manner exhibited an asymmetry characteristic of an epithelium, transporting glucose preferentially in a basal to apical as opposed to apical to basal direction, and corresponding to net glucose transport into the oviduct lumen in vivo. Eighty-one per cent of glucose metabolized under these conditions could be accounted for by the formation of lactate, which appeared predominantly in the basal medium. Glucose was transported across the epithelial cells by facilitated diffusion in association with a smaller, passive component. The rate of glucose transport fell and the transepithelial resistance rose in cells removed and cultured 3 days after mating. The data indicate that rabbit oviduct epithelial cells may be grown as a polarized monolayer in primary culture and that endocrine changes induced in vivo persist under these conditions.

Animals↗

Glucose affects monocarboxylate cotransporter (MCT) 1 expression during mouse preimplantation development.

Cleavage-stage embryos have an absolute requirement for pyruvate and lactate, but as the morula compacts, it switches to glucose as the preferred energy source to fuel glycolysis. Substrates such as glucose, amino acids, and lactate are moved into and out of cells by facilitated diffusion. In the case of lactate and pyruvate, this occurs via H+-monocarboxylate cotransporter (MCT) proteins. To clarify the role of MCT in development, transport characteristics for DL-lactate were examined, as were mRNA expression and protein localisation for MCT1 and MCT3, using confocal laser scanning immunofluorescence in freshly collected and cultured embryos. Blastocysts demonstrated significantly higher affinity for DL-lactate than zygotes (Km 20 +/- 10 vs 87 +/- 35 mmol lactate/l; P = 0.03 by linear regression) but was similar for all stages. For embryos derived in vivo and those cultured with glucose, MCT1 mRNA was present throughout preimplantation development, protein immunoreactivity appearing diffuse throughout the cytoplasm with brightest intensity in the outer cortical region of blastomeres. In expanding blastocysts, MCT1 became more prominent in the cytoplasmic cortex of blastomeres, with brightest intensity in the polar trophectoderm. Without glucose, MCT1 mRNA was not expressed, and immunoreactivity dramatically reduced in intensity as morulae died. MCT3 mRNA and immunoreactivity were not detected in early embryos. The differential expression of MCT1 in the presence or absence of glucose demonstrates that it is important in the critical regulation of pH and monocarboxylate transport during preimplantation development, and implies a role for glucose in the control of MCT1, but not MCT3, expression.

Animals↗

Infected erythrocyte choline carrier inhibitors: exploring some potentialities inside Plasmodium phospholipid metabolism for eventual resistance acquisition.

We have developed a model for designing antimalarial drugs based on interference with an essential metabolism developed by Plasmodium during its intraerythrocytic cycle, phospholipid (PL) metabolism. The most promising drug interference is choline transporter blockage, which provides Plasmodium with a supply of precursor for synthesis of phosphatidylcholine (PC), the major PL of infected erythrocytes. Choline entry is a limiting step in this metabolic pathway and occurs by a facilitated-diffusion system involving an asymmetric carrier operating according to a cyclic model. Choline transport in the erythrocytes is not sodium dependent nor stereospecific as demonstrated using stereoisomers of alpha and beta methylcholine. These last two characteristics along with distinct effects of nitrogen substitution on transport rate demonstrate that choline transport in the infected erythrocyte possesses characteristics quite distinct from that of the nervous system. This indicates a possible discrimination between the antimalarial activity (inhibition of choline transport in the infected erythrocyte) and a possible toxic effect through inhibition of choline entry in synaptosomes. Apart from the de novo pathway of choline, PC can be synthesized by N-methylation from phosphatidylethanolamine (PE). There is a de novo pathway for PE biosynthesis from ethanolamine in infected cells but phosphatidylserine (PS) decarboxylation also occurs. In addition, PE can be directly and abundantly synthesized from serine decarboxylation into ethanolamine, a pathway which is absent from the host. The variety of the pathways that exist for the biosynthesis of one given PL led us to investigate whether an equilibrium can occur between all PL metabolic pathways.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Fluorine content of several brands of chocolate bars and chocolate cookies found in Brazil.

Chocolate bars and chocolate cookies are foodstuffs highly appreciated by children. The possibility of having fluorine (F) among their components, associated with an excessive consumption, may make them decisive contributors to the total daily F intake. Thus, they could participate in the establishment of dental fluorosis. The aim of this study was to analyze the fluorine concentration [F] of the chocolates bars (CB) Baton, Confeti, Garoto Ball, Kinder Ovo, M&M s, Milkybar, Nescau, Nescau Ball, Surpresa, Surpresa Bichos, Tortuguita; and of the chocolate cookies (CC) Danyt s, Hipop , Nescau, Passatempo, Pokémon, S tio do Pica-Pau Amarelo and Trakinas. Samples were purchased in Bauru, São Paulo, Brazil. Three grams of each product were previously ashed at 525 C (CB and cookies fillings) and at 550 C (cookies dough), during 4 hours. Fluorine was separated from the ash by hexamethyldisiloxane (HMDS)-facilitated diffusion. Fluorine analysis was carried out with the specific electrode. Mean [F]s SD and amplitude (unit mg/g) were: CB = 0.30 0.45 (0.07 - 1.60, n = 12) and CC = 1.08 2.64 (0.04 - 7.10, n = 7). It was concluded that some of the analyzed foods may be important contributors to the total daily F intake. As for the product that had the highest [F] (Danyt s), when only 3 units are consumed just once a day, they may supply up to 40% of the maximum recommended daily F intake (0.07 mg/kg body weight) for a 2-year-old child (12 kg). The [F] in these products should be informed on their labels.

Brazil↗

Analysis of fluoride concentration in mother's milk substitutes.

The aim of the present study was to determine the fluoride concentration in some brands of mother's milk substitutes and evaluate the possibility of developing dental fluorosis by consuming these products. The products, all powdered, were divided into 3 groups: infant formulae (group I, n = 7), milk-based (group M, n = 8) and soy-based (group S, n = 3). Samples from 3 cans of different batches of each brand were reconstituted in deionized water and analyzed using the specific electrode method, after hexamethyldisiloxane (HMDS) facilitated diffusion. The fluoride content (mg F/L) of the products ranged from 0.044 to 0.326 (I), 0.014 to 0.045 (M) and 0.253 to 0.702 (S). There was significant difference in the fluoride content of cans from distinct batches (p < 0.05) in most of the brands. The reconstitution of all products in water with optimal fluoride concentration for consumption during the mineralization phase of the primary teeth could result in daily fluoride intake above 0.07 mg F/kg body weight/day. Therefore, the consumption of these products, especially when reconstituted with optimally fluoridated water, could increase the risk of developing dental fluorosis.

Fluorides↗

Uptake of nicotinamide by rat pancreatic beta cells with regard to streptozotocin action.

Exposure of rat pancreatic beta cells in monolayer culture to 2 mmol streptozotocin (STZ)/l for 1 h followed by thorough washing inhibited their uptake of [14C]nicotinamide and [3H]2-deoxyglucose [( 3H]2-DG) to about 50% and also reduced the intracellular ATP concentration to 50% of that in control cells. These changes were not due to a lethal cytotoxic effect of STZ, because cell viability, as estimated by succinic dehydrogenase activity, was 90% of that of control cells. Oligomycin and carbonylcyanide-m-chlorophenylhydrazone (CCCP), an uncoupler of oxidative phosphorylation, caused a dose-dependent decrease in intracellular ATP concentration while maintaining high cell viability. These ATP-depleted cells showed a decrease in insulin release and an inhibition of the uptake of [14C]nicotinamide and [3H]2-DG in a dose-dependent manner. Therefore oligomycin and CCCP reproduced the same effects as those found in beta cells treated with STZ. These results suggest that the uptake of nicotinamide and 2-DG by beta cells might be regulated by their intracellular ATP concentration. The decreased uptake of nicotinamide in ATP-depleted beta cells caused by STZ might explain the lack of protective effect of nicotinamide against STZ cytotoxicity when administered after the latter. Furthermore, the radiotracer experiments demonstrated that the transport of nicotinamide by intact beta cells was inhibited in a dose-dependent manner by 2-DG and vice versa, i.e. the transport of 2-DG was inhibited by nicotinamide. These findings suggest the existence of a common transport mechanism in beta cells responsible for the uptake of nicotinamide and 2-DG, the transport of which is known to occur by facilitated diffusion.

Adenosine Triphosphate↗

1,25-dihydroxyvitamin D3 stimulates vesicular transport within 5 s in polarized intestinal epithelial cells.

Controversy remains regarding whether the seco-steroid hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) enhances calcium and phosphate movement across the intestinal epithelial cell by facilitated diffusion or a vesicular transport mechanism. In this study we investigated whether membrane trafficking, as judged by confocal microscopy, was sufficiently rapid in comparison to hormone-stimulated uptake of phosphate (32P). Primary cultures of chick intestinal cells were established overnight either in Petri dishes (uptake studies) or chambered coverslips (confocal microscopy). Addition of 130 pM 1,25(OH)2D3 resulted in an apparent increase in 32P uptake within 1 min, relative to controls, that was statistically significant from 3-10 min of incubation. Using the endocytic marker dye, FM1-43, confocal microscopy revealed a profound decrease in membrane-associated fluorescence (apical> basal) within 10 s of hormone treatment, a return of fluorescence at 15-65 s, followed by another round of decreasing and increasing fluorescence. Between 3-9 min of incubation, fluorescence intensity increased 50% (apical region) and 20% (basal region) over control conditions. An antibody (Ab 099) directed against a putative membrane receptor for 1,25(OH)2D3 (1,25D3-MARRS) inhibited both 32P uptake, and changes in fluorescence. In addition, the protein kinase C (PKC) inhibitor, calphostin C, inhibited both 32P uptake and the observed 1,25(OH)2D3-mediated changes in fluorescence. At the microscopic level, calphostin C pretreatment abolished the very rapid redistribution of the endocytic marker dye, although a slight increase in fluorescence was still observed. We conclude that 1,25(OH)2D3-stimulated vesicular trafficking is mediated by the 1,25D3-MARRS protein, implicates a PKC signaling mechanism, and occurs in a time frame that is commensurate with a role in ion transport.

Animals↗

Immunolocalization of tight junction proteins, occludin and ZO-1, and glucose transporter GLUT1 in the cells of the blood-nerve barrier.

Facilitated-diffusion glucose transporter GLUT1 is abundant in the blood-nerve barrier. To observe the relationship between glucose transfer across the barrier and the molecular architecture of the barrier, we examined the localization of GLUT1 and tight junction proteins, occludin and zonula occludens-1 (ZO-1), by immunofluorescence microscopy and immunogold electron microscopy in the rat sciatic nerve. GLUT1 was enriched at the whole aspects of the plasma membranes of the cells of the barrier: perineurial cells, and endothelial cells of the blood vessels in the endoneurium. These GLUT1-positive cells were also positive for occludin and ZO-1, both of which were localized at tight junctions. ZO-1 additionally was present in the GLUT1-negative cells not serving as the blood-nerve barrier. These observations suggest that occludin in the tight junctions and GLUT1 at the plasma membranes in the cells of the barrier may constitute a mechanism for the selective transfer of glucose across the barrier while preventing the non-specific flow of blood constituents.

Animals↗

Calbindin-D9k and parvalbumin are exclusively located along basolateral membranes in rat distal nephron.

There is strong evidence that vitamin D-dependent Ca(2+)-binding proteins, i.e., calbindin-D9k and calbindin-D28k, facilitate diffusion of Ca2+ through the cytosolic compartment of renal and intestinal cells, which transport Ca2+ transcellularly. In the study presented here, parvalbumin, calbindin-D9k, and calbindin-D28k were localized precisely by immunocytochemistry in rat kidney. Antisera recognizing specifically the thick ascending loop of Henle, the connecting tubules and collecting ducts, and the intercalated cells of the collecting ducts were used to identify different cell types. In rat kidney cortex, parvalbumin and calbindin-D9k colocalized in the thick ascending loop of Henle, the distal convoluted tubule, the connecting tubule, and the intercalated cells of the collecting duct. Strikingly, in all responsive cells, parvalbumin and calbindin-D9k were exclusively present in a thin layer along the basolateral membrane. In contrast, calbindin-D28k was only present in the distal convoluted and connecting tubule, where it was evenly distributed through the cytosol. In conclusion, the exclusive localization of parvalbumin and calbindin-D9k at the basolateral membrane of immunopositive renal cells implies their involvement in the regulation of transport processes located in these membranes rather than a role as intracellular Ca2+ buffer and Ca2+ shuttle between the two opposing membranes.

Animals↗

Uranium complexation and uptake by a green alga in relation to chemical speciation: the importance of the free uranyl ion.

The bioavailability and toxicity of dissolved metals are closely linked to the metals' chemical speciation in solution. Normally the complexation of a metal by a ligand would be expected to decrease its bioavailability. The aqueous speciation of uranium (U) undergoes tremendous changes in the presence of ligands commonly found in natural waters (carbonate, phosphate, hydroxide, and natural organic matter). In the present project, links between speciation, medium composition, and bioavailability of uranium toward Chlamydomonas reinhardtii, a unicellular green alga, were investigated. Short-term metal uptake rates were determined in simple inorganic media at constant low pH (5.0) and hardness with particular emphasis on the differentiation between adsorbed and intracellular metal. While intracellular uptake was fairly linear over 1 h, partly reversible adsorption reached steady-state within minutes. Both adsorption and absorption were saturable processes (with a half-saturation constant Km of 0.51 microM). Addition of phosphate, citrate, or ethylenediaminetetraacetic acid (EDTA) as ligands decreased uranium bioavailability. No evidence indicating the transport of intact uranyl complexes was found (i.e., facilitated diffusion of metal bound to an assimilable ligand such as uranium-phosphate complexes). Within these experimental conditions, uranium uptake was correlated with the free uranyl ion concentration as predicted by the free-ion activity model (FIAM) and biotic ligand model (BLM).

Absorption↗

Clinical pharmacokinetics of stavudine.

Stavudine (d4T) is a pyrimidine nucleoside analogue used in the treatment of human immunodeficiency virus (HIV) infection. It inhibits viral reverse transcriptase as do zidovudine (AZT), didanosine (ddI), zalcitabine (ddC) and lamivudine (3TC), which comprise the family of nucleoside HIV-reverse transcriptase inhibitors. Stavudine is currently approved by the US Food and Drug Administration for the treatment of patients who have become intolerant to or have failed to response to zidovudine, didanosine or zalcitabine therapy. Oral administration of stavudine results in maximal concentrations within 2 hours and increases linearly as doses increase. The absolute oral bioavailability is high, approaching 100%. There is evidence to suggest that stavudine does not accumulate in the plasma. It distributes into total body water and appears to enter cells by non-facilitated diffusion. Penetration into the cerebrospinal fluid occurs, as does the transfer of the drug across human placental tissue. Stavudine is cleared quickly by both renal and nonrenal processes. The pharmacokinetic properties of stavudine in children are similar to those of adults. The pharmacokinetic parameters of stavudine were not affected by simultaneous administration of didanosine. It appears that stavudine at doses < 2 mg/kg/day is most efficient at increasing CD4 + cell numbers. While stavudine is reported to be less cytotoxic than zidovudine, the principal toxicity in humans is peripheral neuropathy and appears to be related to daily, but not cumulative, doses.

Adult↗