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PubMed2000

D-glucose additive protects against osmotic-induced decrease in erythrocyte filterability.

Glucose has long-term effects on erythrocyte filterability owing to sorbitol accumulation and glycosylation of intracellular proteins, but glucose effects prior to these long-term metabolic consequences have not been studied to the same extent. D-glucose is osmotically inert in erythrocytes because of facilitated diffusion. Erythrocyte volume regulation, hemolysis and filterability were studied with reference to effects of glucose derivatives dissolved in water. Control situations were water alone or buffer. Salt-stock dilution by water, D-glucose, or 3-O-M-glucose in water (both 570 mmol/L additives) created volume increases of 4.0 +/- 0.2%, 3.4 +/- 0.5% and 3.3 +/- 0.2%, respectively, whereas L-glucose resulted in a 2.2 +/- 0.4% volume decrease (all p < 0.001 versus baseline, mean values +/- SEM). D-glucose at a final concentration 30 mmol/L did not display any osmotic properties. Despite erythrocyte swelling, electrolyte-free glucose did not induce any significant changes in filterability with either 3-microm or 5-microm filters. 3-O-M-glucose gave a 7.8 +/- 1.4% decrease in 3-microm filterability (mean value +/- SEM, p < 0.001), but not to the same extent as by water alone at the corresponding dilution rate (31.7 +/- 3.5%, mean value +/- SEM, p < 0.001). L-glucose caused a 2.0 +/- 0.8% decrease in filterability across the 5-microm (mean value +/- SEM, p < 0.05), but not with the 3-microm filters. Stressing the glucose transport system further, to simulate the water-like properties of electrolyte-free glucose during intravenous infusion, we found that glucose becomes osmotically active and prevents hemolysis at these extreme concentrations (> 114 mmol/L). We conclude that an in vivo concentration range of D-glucose protects the erythrocytes from a decreased filterability induced by osmotic swelling but without having any osmotic properties.

Cell Separation↗
PubMed1999

Disposition, toxicity, and intestinal absorption of cobaltous chloride in male Fischer 344 rats.

The absorption and disposition of inorganic cobalt salts after oral administration have not been well characterized. The objectives of this study were to compare in vivo results with cobalt transport through the in vitro everted small intestine and to relate the disposition results to a biochemical indicator of cobalt toxicity. Cobalt chloride was given to male Fischer 344 rats orally at 33.3 mg Co(II)/kg or intravenously at 4.16 mg Co(II)/kg. By 36 h, 74.5% of the oral dose was eliminated in the feces. The liver, kidney, and heart accumulated cobalt to the greatest extent. Following the single oral dose, the blood cobalt concentration-time curve was triphasic, peaked at 3.2 h, and had an absorptive half-life of 0.9 h, an elimination phase half-life of 3.9 h, and a terminal elimination half-life of 22.9 h. Following intravenous administration, 10.1% of the dose was excreted in the feces, indicating that cobalt can be secreted in the bile. Following a single intravenous injection, the concentration-time curve displayed three segments. The first segment, which occurred during the first 4 h, had a rapid half-life of 1.3 h. The second phase, from 4 to 12 h, demonstrated a slower clearance rate with a half-life of 4.3 h. The final and slowest phase, from 12 to 36 h, had a half-life of 19 h. Intestinal jejunal ring experiments indicated that cobalt transport has both active and passive components; however, cobalt transport through the in vitro rat everted duodenum indicated that cobalt transport had almost exclusively passive components with facilitated diffusion. The finding that uptake was saturable may explain the small extent of absorption following oral dosing. Heme oxygenase studies following subcutaneous and intravenous administration resulted in an increase in activity (twofold) over controls, while oral administration did not. We concluded that the extent of cobalt absorption across the gastrointestinal tract is incomplete, and that the concentration administered and the route of exposure may determine its systemic toxicity.

Animals↗
PubMed1977

The effects of X-radiation on thymidine-transport kinetics and DNA synthesis in Ehrlich ascites tumour cells in relation to extracellular thymidine concentration.

A TdR carrier-transport system, believed to be facilitated diffusion, has been shown to exist in Ehrlich ascites tumour cells. It is suggested that this system is the predominant transport mechanism at low extracellular concentrations (less than 1-5 micron). The transport system was damaged considerably by 5 krad X-radiation, resulting in a 30-35 per cent reduction in the initial total TdR uptake rat at low extracellular concentrations and 15-20 min after irradiation. The extent of the damage was dependent on the age of the cells as was reflected by relative decreases in V max and Km. It can be concluded that the enhanced depression in 14C-TdR incorporation into DNA of irradiated cells when low precursor concentrations were used for monitoring, is partly attributed to the radiation-induced damage to the carrier-transport system. The permeability constant for passive diffusion in asynchronous E.A.T. cells and the endogenous natural rate of dTTP synthesis in S-phase cells were estimated.

Animals↗
PubMed2000

Sources of dietary fluoride intake in 4-year-old children residing in low, medium and high fluoride areas in Iran.

Accurate estimation of fluoride dietary intake is desirable for optimising caries prevention. Little is known about the dietary fluoride intake of children aged 4 years, an age when many permanent teeth are forming. This study was undertaken in Fars Province, Iran, in 1995-1996, where there are big differences in temperature between winter and summer. The aims were to determine: (a) the relative contributions of different components of the diet to fluoride intake, (b) the effect of variation in fluoride concentration in drinking water, and (c) the effect of climate temperature. Temperature varied between +40 degrees C in summer to -5 degrees C in winter. The mean fluoride concentrations in drinking water in the three areas were 0.3, 0.6 and 4.0 mgF/L. Dietary information was obtained by two 3-day diet diaries with interview, validated with reference to international standards. The fluoride content of foods was measured using the silicon-facilitated diffusion method. One hundred and three 4-year-old children completed the study. The mean (and 95% confidence interval) dietary fluoride intakes in each of the three areas, respectively, were 413 (+/- 21), 698 (+/- 89) and 3472 (+/- 557) micrograms/day. Drinks provided 72 to 87% of dietary fluoride--this proportion increased with increasing water fluoride concentration and increasing climate temperature. Tea (infusion) was an important source of dietary fluoride, providing 31 to 38% of total dietary intake. Tap water was a more important source of fluoride than soft drinks. Cooked rice and bread were the most important food source of fluoride and the amount of fluoride they contributed increased as water fluoride concentration increased. The results of this first such survey in the Middle East showed (a) that water (as a drink) and tea were by far the most important contributors to dietary fluoride intake, (b) substantial increases in fluoride intake with increasing water fluoride concentrations, and (c) substantially higher fluoride intakes in summer than in the winter.

Beverages↗
PubMed1999

Fluoride concentration in foods from Iran.

Knowledge of fluoride intake is important in optimizing the caries-preventive role of fluoride, and the measurement of fluoride intake usually requires information on the fluoride concentration in foods and drinks. Most information comes from developed countries and there are no data on fluoride concentration in foods in Middle Eastern countries including Iran. The aim of the study was to: (a) describe a modification to the silicon-facilitated diffusion method for determining fluoride concentration, (b) provide information on fluoride content of foods in Iran to supplement food tables for the Middle East, and (c) determine the effect of variation in the fluoride concentration of drinking water on the fluoride concentration of prepared foods. Five hundred and ten samples of 84 popular foods and drinks were collected from three areas of Iran where water fluoride concentrations were 0.32, 0.58 and 4.05 mg/L. The mean recovery of fluoride added to food samples before diffusion was 98 +/- 5%. Values for duplicate analysis of 20 food samples were within 0.03 microgram F/g. Most of the samples of foods and drinks came from the area with 0.32 mg F/L in water supplies. For 30 of the 84 items, fluoride concentration was below 0.1 microgram/g. Fluoride concentrations in the cereals group (which constituted much of the diet) were mainly between 0.2 and 0.3 microgram/g, when prepared for consumption. It was concluded that: (a) modification of a published method for determining fluoride concentration of foods appeared to be an advance, (b) values for fluoride concentration of foods commonly consumed in Iran showed variation between groups but were in broad agreement with published data, and (c) concentration of fluoride in water influences positively fluoride concentration in foods cooked in water, but the increase in foods was less than the increase in fluoride concentration in water.

Fluoridation↗
PubMedDate not supplied

Vitamin C transport systems of mammalian cells.

Vitamin C is essential for many enzymatic reactions and also acts as a free radical scavenger. Specific non-overlapping transport proteins mediate the transport of the oxidized form of vitamin C, dehydroascorbic acid, and the reduced form, L-ascorbic acid, across biological membranes. Dehydroascorbic acid uptake is via the facilitated-diffusion glucose transporters, GLUT 1, 3 and 4, but under physiological conditions these transporters are unlikely to play a major role in the uptake of vitamin C due to the high concentrations of glucose that will effectively block influx. L-ascorbic acid enters cells via Na+-dependent systems, and two isoforms of these transporters (SVCT1 and SVCT2) have recently been cloned from humans and rats. Transport by both isoforms is stereospecific, with a pH optimum of approximately 7.5 and a Na+:ascorbic acid stoichiometry of 2:1. SVCT2 may exhibit a higher affinity for ascorbic acid than SVCT1 but with a lower maximum velocity. SVCT1 and SVCT2 are predicted to have 12 transmembrane domains, but they share no structural homology with other Na+ co-transporters. Potential sites for phosphorylation by protein kinase C exist on the cytoplasmic surface of both proteins, with an additional protein kinase A site in SVCT1. The two isoforms also differ in their tissue distribution: SVCT1 is present in epithelial tissues, whereas SVCT2 is present in most tissues with the exception of lung and skeletal muscle.

Amino Acid Sequence↗
PubMed1999

Endogenous nitric oxide synthesis: biological functions and pathophysiology.

Modern molecular biology has revealed vast numbers of large and complex proteins and genes that regulate body function. By contrast, discoveries over the past ten years indicate that crucial features of neuronal communication, blood vessel modulation and immune response are mediated by a remarkably simple chemical, nitric oxide (NO). Endogenous NO is generated from arginine by a family of three distinct calmodulin- dependent NO synthase (NOS) enzymes. NOS from endothelial cells (eNOS) and neurons (nNOS) are both constitutively expressed enzymes, whose activities are stimulated by increases in intracellular calcium. Immune functions for NO are mediated by a calcium-independent inducible NOS (iNOS). Expression of iNOS protein requires transcriptional activation, which is mediated by specific combinations of cytokines. All three NOS use NADPH as an electron donor and employ five enzyme cofactors to catalyze a five-electron oxidation of arginine to NO with stoichiometric formation of citrulline. The highest levels of NO throughout the body are found in neurons, where NO functions as a unique messenger molecule. In the autonomic nervous system NO functions NO functions as a major non-adrenergic non-cholinergic (NANC) neurotransmitter. This NANC pathway plays a particularly important role in producing relaxation of smooth muscle in the cerebral circulation and the gastrointestinal, urogenital and respiratory tracts. Dysregulation of NOS activity in autonomic nerves plays a major role in diverse pathophysiological conditions including migraine headache, hypertrophic pyloric stenosis and male impotence. In the brain, NO functions as a neuromodulator and appears to mediate aspects of learning and memory. Although endogenous NO was originally appreciated as a mediator of smooth muscle relaxation, NO also plays a major role in skeletal muscle. Physiologically muscle-derived NO regulates skeletal muscle contractility and exercise-induced glucose uptake. nNOS occurs at the plasma membrane of skeletal muscle which facilitates diffusion of NO to the vasculature to regulate muscle perfusion. nNOS protein occurs in the dystrophin complex in skeletal muscle and NO may therefore participate in the pathophysiology of muscular dystrophy. NO signalling in excitable tissues requires rapid and controlled delivery of NO to specific cellular targets. This tight control of NO signalling is largely regulated at the level of NO biosynthesis. Acute control of nNOS activity is mediated by allosteric enzyme regulation, by posttranslational modification and by subcellular targeting of the enzyme. nNOS protein levels are also dynamically regulated by changes in gene transcription, and this affords long-lasting changes in tissue NO levels. While NO normally functions as a physiological neuronal mediator, excess production of NO mediates brain injury. Overactivation of glutamate receptors associated with cerebral ischemia and other excitotoxic processes results in massive release of NO. As a free radical, NO is inherently reactive and mediates cellular toxicity by damaging critical metabolic enzymes and by reacting with superoxide to form an even more potent oxidant, peroxynitrite. Through these mechanisms, NO appears to play a major role in the pathophysiology of stroke, Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis.

Animals↗
PubMed1984

Vesicles with transport capability isolated from cultured fibroblasts.

Plasmalemmas from cultured human skin fibroblasts, isolated by a simple and reproducible method, can be converted to vesicles which are capable of active transport of aminoacid when glutathione is included within the vesicles. In the isolation, the plasmalemmas are stabilized with a Ricinus lectin, with preservation of the classic plasmalemma enzymes. The procedure has been applied successfully to a number of normal and abnormal human skin fibroblasts including those of myotonia dystrophica and progeria victims, and to the lung fibroblast WI-38. As part of a study of the characteristics of transport enzymes related to aging and to the muscular dystrophies in cultured fibroblasts, it was desirable to simplify the system by the use of vesicles prepared from the fibroblast plasmalemmas. The procedure described below is similar in some respects to that applied to another membranous system in the use of a lectin to stabilize the plasmalemma structure. The use of other stabilizing agents such as heavy metals and surfactant polymers which react with the membranes, but could compromise the reliability of the enzyme assays, was avoided. Since the focus of this study was on the enzymic systems of transport, the examination of facilitated diffusion or exchange was excluded. The well defined glutathione-dependent mechanism of aminoacid transport was examined to verify the competence of the vesicles for active transport and to confirm their sidedness. Other enzymes of transport, the ATPases, and membrane marker enzymes were also determined.

Adenosine Triphosphatases↗
PubMed1999

Synthesis, lipophilicity and anti-HIV activity of a new brominated analog of zidovudine.

A novel cyclic bromine zidovudine analog, (-)-trans-(5S,6S)-5-bromo-6,5'- epoxy-5,6-dihydro-3'-azido-3'-deoxythymidine (2), and its diastereoisomer (+)-trans-(5R,6R)-(3) were synthesized and characterized by spectroscopic methods, obtaining 3 in very low yields. The major product 2 presents a selectivity index (CCID50/IC50) similar to zidovudine but 55.5 times with higher lipophilicity, which should increase the ability of 2 to cross the blood-brain barrier by a non facilitated diffusion mechanism.

Anti-HIV Agents↗
PubMed1997

Functional expression cloning and characterization of SFT, a stimulator of Fe transport.

A stimulator of Fe transport (SFT) was identified by functional expression cloning in Xenopus oocytes. SFT-mediated transport has properties defined for transferrin-independent Fe uptake, but its cytolocalization in recycling endosomes and the observed stimulation of transferrin-bound Fe assimilation indicate a key role in intracellular Fe membrane transport as well. SFT has six predicted transmembranous domains and a functionally important RExxE motif that resembles domains involved in yeast Fe transport and Fe-binding by ferritin L-chains. The observation that SFT oligomerizes, along with other structural and mechanistic features, suggests it may be a member of either the ATP-binding cassette or cation diffusion facilitator families. The 3' untranslated region of SFT contains a translation inhibitory element and inhibition of SFT expression in Xenopus oocytes was found to be relieved by coinjection of transcripts from other defined cDNAs that are also described in this report. SFT is the first component of the mammalian Fe membrane transport machinery to be identified.

Amino Acid Sequence↗
PubMed2004

Zinc and the Msc2 zinc transporter protein are required for endoplasmic reticulum function.

In this report, we show that zinc is required for endoplasmic reticulum function in Saccharomyces cerevisiae. Zinc deficiency in this yeast induces the unfolded protein response (UPR), a system normally activated by unfolded ER proteins. Msc2, a member of the cation diffusion facilitator (CDF) family of metal ion transporters, was previously implicated in zinc homeostasis. Our results indicate that Msc2 is one route of zinc entry into the ER. Msc2 localizes to the ER when expressed at normal levels. UPR induction in low zinc is exacerbated in an msc2 mutant. Genetic and biochemical evidence indicates that this UPR induction is due to genuine ER dysfunction. Notably, we found that ER-associated protein degradation is defective in zinc-limited msc2 mutants. We also show that the vacuolar CDF proteins Zrc1 and Cot1 are other pathways of ER zinc acquisition. Finally, zinc deficiency up-regulates the mammalian ER stress response indicating a conserved requirement for zinc in ER function among eukaryotes.

Carrier Proteins↗
PubMedDate not supplied

Differential handling of urea and its analogues suggests carrier-mediated urea excretion in freshwater rainbow trout.

The possible presence of urea transport mechanisms in the gill and kidney of the freshwater rainbow trout (Oncorhynchus mykiss) was investigated in vivo by comparing the branchial and renal handling of analogues acetamide and thiourea with the handling of urea. Trout were fitted with indwelling dorsal aortic catheters and urinary catheters and injected with an isosmotic dose of [(14)C]-labeled urea analogue (acetamide or thiourea) calculated to bring plasma analogue concentrations close to plasma urea concentrations. Urea and analogue concentrations were significantly greater in the urine than in the plasma. Branchial clearance rate of acetamide was only 48% of urea clearance, whereas the clearance of thiourea was only 22%, a pattern that was also observed in branchial uptake of these substances and was similar to our previous observations in toadfish and midshipmen. The renal secretion clearance rates of urea and acetamide were similar, and on average, both substances were secreted on a net basis, although reabsorption did occur in some cases. In contrast, thiourea was neither reabsorbed nor secreted by the kidney tubule. The secretion clearance rates of both acetamide and urea were well correlated with the secretion clearance rates of Na(+), Cl(-), and water, whereas there was no relationship between thiourea and these substances. The pattern of acetamide, thiourea, and urea handling by the gill of the trout is similar to that found in the gills of the midshipman and the gulf toadfish and strongly suggests the presence of a UT-type facilitated diffusion urea transport mechanism. The pattern of differential handling in the kidney is unlike that in the gill and also unlike that in the kidney of the midshipman and the gulf toadfish, suggesting a different mechanism. In addition, renal urea secretion occurs against a concentration gradient, suggesting the involvement of an active transport mechanism.

Acetamides↗
PubMed2002

High-density lipoprotein-facilitated entry of thyroid hormones into cells: a mechanism different from the low-density lipoprotein-facilitated entry.

In human skin fibroblasts incubated with interstitial fluid concentrations of low-density lipoproteins (LDL) (22 microg/mL) that had been preincubated with [(125)I] thyroid hormone (TH), thyroxine (T(4)) whole-cell saturable up-take (CSU) is approximately 40% greater than in control fibroblasts. This effect of the LDL, which requires upregulation of low-density lipoprotein-receptors (LDL-R; K(d) approximately 20 microg/mL), is less for [(125)I]triiodothyronine (T(3)). We have evaluated high-density lipoproteins (HDL), and assessed whether lipoproteins target TH to the nucleus. In some experiments, fibroblasts were cholesterol-deprived (chol(-)) or cholesterol-enriched (chol(+)) to upregulate LDL-R or high-density lipoprotein-receptors (HDL-R), respectively. In chol(-) fibroblasts, 25 microg/mL of LDL increased both T(4) CSU and T(4) nuclear saturable uptake (NSU) by 48% or 30%, respectively; the latter becoming appreciable at approximately 180 minutes. Interstitial fluid concentrations of HDL (280 microg/mL or approximately 50-fold greater than K(d) of the HDL-R) inhibited both T(4) and T(3) CSU even in chol(+) fibroblasts. However, when chol(+) fibroblasts were incubated first with HDL, and after cell washings with [(125)I]T(4) or [(125)I]T(3), T(4) or T(3) CSU increased by 24% or 12%, respectively. The corresponding increase in chol(-) fibroblasts was 8% or 0%, and in nonmanipulated fibroblasts was 15% or 4%. Unlike LDL, the magnitude of the increase in T(4) or T(3) NSU caused by HDL matched the corresponding CSU, and was already evident at 30 minutes. In conclusion, cells have a LDL-facilitated, LDL-R-mediated mode of entry of TH (T(4) >> T(3)) that targets relatively late only part of TH to the nucleus, and an HDL-facilitated mode of entry (T(4) > T(3)) that targets immediately to the nucleus all of the TH, suggesting entry of TH in free form. This effect of HDL represents facilitated diffusion of TH through the cell membrane.

Cell Nucleus↗
PubMed1999

A local, high-density, single-nucleotide polymorphism map used to clone Caenorhabditis elegans cdf-1.

Ras-mediated signaling is required for induction of vulval cell fates during Caenorhabditis elegans development. By screening for suppressors of the multivulva phenotype caused by constitutively active let-60 ras, we identified the mutation n2527. To clone the gene affected by n2527, we developed a method for high-resolution mapping. We took advantage of the genomic DNA sequence of the N2 strain by using DNA sequencing to scan for single-nucleotide polymorphisms (SNPs) at defined genomic positions of the RC301 strain. An average of one polymorphism per 1.4 kb was detected in predicted intergenic regions. Because of this high frequency, DNA sequencing is an efficient method to scan for SNPs. By alternating between identifying SNPs and mapping n2527 using selected recombinants, we generated an SNP map of progressively higher density. An intensive search for SNPs resulted in a local map with an average marker spacing of approximately 4 kb. This was used to map n2527 to a 9.6-kb interval. The small size of this interval made it feasible to use DNA sequencing to identify the molecular lesion. In principle, this approach can be used for high-resolution mapping of any C. elegans mutation. Furthermore, this approach can be applied to other species as the genomic sequence becomes available. The n2527 mutation affects a previously uncharacterized gene that we named cdf-1, as it encodes a predicted protein with significant similarity to members of the cation diffusion facilitator family.

Animals↗
PubMedDate not supplied

Nail and bone surface as biomarkers for acute fluoride exposure in rats.

When acute exposure to fluoride is thought to be the cause of death, confirmation often depends on the analysis of some body fluid or tissue. The aim of this study was to evaluate the use of nails and the periosteal surface of bone as indicators of acute exposure to fluoride. Six groups of rats were given a single oral dose of fluoride (50 mg/kg body weight), while the control group was given deionized water. The rats were killed at 2, 4, 8, 16, 24, and 48 h after fluoride administration. Plasma and nails (the proximal halves) were collected and analyzed for fluoride with an ion-specific electrode after hexamethyldisiloxane-facilitated diffusion. A circular area of the femur (4.52 mm(2)) was etched with 0.5M HCl for 15 s, and, after the addition of a buffer, the solution was analyzed with an ion-specific electrode. Peak plasma concentration occurred at 2 h, followed by progressively declining concentrations. Peak nail fluoride concentrations occurred at 8 h. The mean nail concentrations at 8, 16, and 24 h were significantly higher than that of the control group. Bone surface concentrations were significantly higher than that of the control group at 4 h and thereafter. Thus, the proximal portion of nails and bone surface are suitable biomarkers for acute fluoride exposure in rats.

Animals↗
PubMedDate not supplied

Bone surface and whole bone as biomarkers for acute fluoride exposure.

This study compares fluoride concentrations ([F]) in surface and whole bone for up to 27 days following an acute oral dose of F. Four groups of rats received single oral F dose (50 mg/kg body weight), and the control group received deionized water (n = 10/group). Groups were euthanized at 1, 3, 9, or 27 days after F administration. Plasma and femurs were collected. F on the femur surface was removed from a circular area (4.52 mm(2)) by immersion in 0.5M HCl for 15 s. The solution was buffered with total ionic strength adjustment buffer and analyzed with an electrode. The subjacent bone was sectioned and ashed at 600 degrees C. Ash and plasma were analyzed for F with the electrode following hexamethyldisiloxane-facilitated diffusion. Data were analyzed by Kruskall-Wallis and Dunn's test and by linear regression (p < 0.05). Peak plasma and bone surface [F] occurred on day 1 (0.26 +/- 0.14 microg/mL and 1801 +/- 888 microg/g, respectively). Bone surface [F] at 3, 9, and 27 were not statistically different from control. A significant increase in whole bone [F] was observed 3 days after F administration and the [F] remained relatively constant thereafter. The mean (+/- SD) surface/whole bone [F] ratios for the control and F groups were 2.45 +/- 0.98, 3.92 +/- 1.32, 1.61 +/- 0.82, 1.73 +/- 0.39, and 1.09 +/- 0.28, respectively. Plasma and bone surface [F]s were positively correlated (r = 0.74). Thus, bone surface was found to be a suitable biomarker for acute, sublethal F exposure 1 day after F administration. Whole bone [F] were significantly increased at 3, 9, and 27 days after F administration.

Animals↗
PubMed1983

Uptake of riboflavin by isolated rat liver cells.

The effect of riboflavin status on uptake was investigated in hepatocytes isolated from control, riboflavin-sufficient and riboflavin-deficient rats. The uptake exhibited biphasic characteristics with an initial rapid phase [13.2 +/- 1.8 pmol/(10(6) cells X minute)] for the first couple of minutes followed by a second slower phase which continued for over an hour. The accumulation of riboflavin at near equilibrium conditions was 2.5- and 5.2-fold greater than external concentration in control and riboflavin-deficient cells, respectively. An apparent Km of 12 +/- 1.3 microM and Vmax of 82.3 +/- 9.1 pmol/(10(6) cells X minute) were obtained for control and riboflavin-sufficient rats while a similar Km but higher Vmax were obtained with deficient animals. Correspondence of the Km to that of flavokinase for riboflavin suggested the possibility that uptake of the vitamin may occur via metabolic trapping, i.e., phosphorylation. As substantiation of this, the rate of uptake was decreased by lumiflavin and 2'-hydroxyethylflavin, which are competitive inhibitors, and by 7,8-dichloroflavin, a substrate for flavokinase. Furthermore, the uptake was found to be temperature-dependent and studies with carbonylcyanide-p-trifluoro-methoxyphenylhydrazone (FCCP) and ethionine indicated a requirement for ATP. These results showed that overall, entry of riboflavin into hepatocytes occurs predominantly by a facilitated diffusion process followed by rapid trapping by flavokinase-catalyzed phosphorylation to FMN.

Adenosine Triphosphate↗
PubMed1987

Evidence for facilitated transport of biotin by hamster enterocytes.

The uptake of biotin by isolated hamster intestinal cells was investigated in the zone of physiological concentrations. Uptake of the vitamin was not a linear function of the external concentrations and kinetics could be saturated [Km = 1.12 microM, Vmax = 33.9 pmol/(mg protein X min)]. Metabolic inhibitors (antimycin A, 2,4-dinitrophenol) had no effect, so uptake is not energy dependent. Ouabain and N-ethylmaleimide, inhibitors of cation gradients, had no effect on biotin uptake, thus showing the absence of cotransport. The inhibition of uptake by analogs of biotin, that is, biocytin and DL-thioctic acid, indicates that the terminal carboxyl group and the thiophane ring play a role in the recognition of biotin. Counterflow experiments showed competitive inhibition of efflux when excess biotin was present in the cells. These findings are consistent with biotin uptake by isolated hamster enterocytes being a process of facilitated diffusion.

Animals↗