Non-mediated Uptake Research Articles

Mechanism and rate of glucose absorption differ between an Australian honeyeater (Meliphagidae) and a lorikeet (Loriidae)

Efficient mechanisms of glucose absorption are necessary for volant animals as a means of reducing mass during flight: they speed up gut transit time and require smaller volume and mass of gut tissue. One mechanism that may be important is absorption via paracellular (non-mediated) pathways. This may be particularly true for nectarivorous species which encounter large quantities of sugar in their natural diet. We investigated the extent of mediated and non-mediated glucose absorption in red wattlebirds Anthochaera carunculata (Meliphagidae) and rainbow lorikeets Trichoglossus haematodus (Loriidae) to test the hypothesis that paracellular uptake accounts for a significant proportion of total glucose uptake in these species. We found that routes of glucose absorption are highly dynamic in both species. In lorikeets, absorption of L-glucose (non-mediated uptake) is slower than that of D-glucose (mediated and non-mediated uptake), with as little as 10% of total glucose absorbed by the paracellular pathway initially (contrasting previous indirect estimates of approximately 80%). Over time, however, more glucose may be absorbed via the paracellular route. Glucose absorption by both mediated and non-mediated mechanisms in wattlebirds occurred at a faster rate than in lorikeets, and wattlebirds also rely substantially on paracellular uptake. In wattlebirds, we recorded higher bioavailability of L-glucose (96+/-3%) compared with D-glucose (57+/-2%), suggesting problems with the in vivo use of radiolabeled d-glucose. Further trials with 3-O-methyl-D-glucose revealed high bioavailability in wattlebirds (90+/-5%). This non-metabolisable glucose analogue remains the probe of choice for measuring uptake rates in vivo, especially in birds in which absorption and metabolism occur extremely rapidly.

Conserved Glutamate Residues Are Critically Involved in Na+/Nucleoside Cotransport by Human Concentrative Nucleoside Transporter 1 (hCNT1)

Human concentrative nucleoside transporter 1 (hCNT1), the first discovered of three human members of the SLC28 (CNT) protein family, is a Na+/nucleoside cotransporter with 650 amino acids. The potential functional roles of 10 conserved aspartate and glutamate residues in hCNT1 were investigated by site-directed mutagenesis and heterologous expression in Xenopus oocytes. Initially, each of the 10 residues was replaced by the corresponding neutral amino acid (asparagine or glutamine). Five of the resulting mutants showed unchanged Na+-dependent uridine transport activity (D172N, E338Q, E389Q, E413Q, and D565N) and were not investigated further. Three were retained in intracellular membranes (D482N, E498Q, and E532Q) and thus could not be assessed functionally. The remaining two (E308Q and E322Q) were present in normal quantities at cell surfaces but exhibited low intrinsic transport activities. Charge replacement with the alternate acidic amino acid enabled correct processing of D482E and E498D, but not of E532D, to cell surfaces and also yielded partially functional E308D and E322D. Relative to wild-type hCNT1, only D482E exhibited normal transport kinetics, whereas E308D, E308Q, E322D, E322Q, and E498D displayed increased K50(Na+) and/or Km(uridine) values and diminished Vmax(Na+) and Vmax(uridine) values. E322Q additionally exhibited uridine-gated uncoupled Na+ transport. Together, these findings demonstrate roles for Glu-308, Glu-322, and Glu-498 in Na+/nucleoside cotransport and suggest locations within a common cation/nucleoside translocation pore. Glu-322, the residue having the greatest influence on hCNT1 transport function, exhibited uridine-protected inhibition by p-chloromercuriphenyl sulfonate and 2-aminoethyl methanethiosulfonate when converted to cysteine.

Multiple effects of mercuric chloride on hexose transport in Xenopus oocytes

HgCl 2 had both stimulatory and inhibitory effects on [ 3H]2-deoxyglucose (DG) uptake in Xenopus laevis oocytes. The Hg dose response was complex, with 0.1–10 μM Hg increasing total DG uptake, 30–50 μM Hg inhibiting, and concentrations >100 μM increasing uptake. Analyses of the effects of Hg on DG transport kinetics and cell membrane permeability indicated that low concentrations of Hg stimulated mediated uptake, intermediate concentrations inhibited mediated uptake, but high Hg concentrations increased non-mediated uptake. 10 μM Hg increased the apparent V max for DG uptake, but caused little or no change in apparent K m. Phenylarsine oxide prevented the increase in DG uptake by 10 μM Hg, suggesting that the increase was due to transporter recruitment. Microinjecting low doses of HgCl 2 into the cell increased mediated DG uptake. Higher intracellular doses of Hg increased both mediated and non-mediated DG uptake. Both insulin and Hg cause cell swelling in isotonic media and, for insulin, this swelling has been linked to the mechanism of hormone action. Osmotically swelling Xenopus oocytes stimulated DG transport 2–5-fold and this increase was due to an increased apparent V max. Exposing cells to 10 μM Hg or 140 nM insulin both increased cellular water content by 18% and increased hexose transport 2–4-fold. These data indicate that low concentrations of Hg and insulin affect hexose transport in a similar manner and that for both an increase cellular water content could be an early event in signaling the increase in hexose transport.

Effect of Detergents on Ligand Binding and Translocation Activities of Solubilized/Reconstituted Nucleoside Transporters

Purification of equilibrative nucleoside transporters has been hampered by the functional instability of the detergent-solubilized proteins. A variety of nonionic detergents were compared with octylglucoside (the most commonly used detergent in this regard) for their abilities to solubilize functionally stable nucleoside transporter proteins from Ehrlich cell plasma membranes. Transporter stability was assessed through the binding of the specific probe [3H]nitrobenzylthi- oinosine to freshly solubilized and stored (48 h/6°C) preparations. The most promising detergents were decylmaltoside and cyclohexylbutylmaltoside, both of which, like octylglucoside, solubilized over 70% of the transporters from the membrane. Decylmaltoside- and cyclohexylbutylmaltoside-solubilized transport proteins retained 61 and 83%, respectively, of their [3H]nitrobenzylthioinosine binding activity upon storage, compared to about 30% using octylglucoside. Decylmaltoside was also superior to octylglucoside in its capacity to solubilize the transporter in a state that retained its high affinity for the transport inhibitors dilazep (Ki= 11 nM, vs 75 nMin octylglucoside) and dipyridamole (Ki= 260 nM, vs 12 μMin octylglucoside). Reconstitution studies indicated that both the de- cylmaltoside- and cyclohexylbutylmaltoside-solubilized transporters were capable of mediating the uptake of [3H]uridine. Decylmaltoside was superior to cyclohexylbutylmaltoside, however, in both the enhanced transport activity of the resulting proteoliposomes (Vi= 21 pmol/mg/s vs 13 pmol/mg/s, respectively) and the lower nonmediated uptake observed in the decylmaltoside-derived vesicles (27% of total uptake at 4 min incubation). Nevertheless, cyclohexylbutylmaltoside may be useful in initial solubilization procedures due to its ability to selectively solubilize the nucleoside transporter from the plasma membrane. The rational use of these detergents will enable a more extensive purification of functional nucleoside transporters.

Intestinal permeability and orocaecal transit time in elderly patients with Parkinson's disease.

The aetiology of weight loss in patients with Parkinson's disease is likely to be multifactorial. We studied 15 patients with Parkinson's disease and 15 age- and sex-matched controls and looked for evidence of malabsorption due to small bowel bacterial overgrowth or alteration of intestinal permeability. There was a marked increase in orocaecal transit time in the patients with Parkinson's disease, although lactulose hydrogen breath testing did not show evidence of small bowel bacterial contamination. Intestinal permeability measured by the differential sugar absorption test was also deranged. There was reduced absorption of mannitol in patients with Parkinson's disease while lactulose absorption was similar in both groups, suggesting decreased non-mediated uptake across the enterocyte brush border membrane in patients with Parkinson's disease.

Effect of starvation on neutral amino acid transport in isolated small-intestinal cells from guinea pigs

The effects of starvation on neutral amino acid transport were examined in isolated enterocytes. Starvation stimulated L-alanine transport by the Na(+)-dependent system A and the Na(+)-independent system L without producing any changes in either the Na(+)-dependent systems ASC or the passive non-mediated uptake. Starvation produces a twofold increase in Vmax of system A without any change in Kt. Starvation produces an increase in Vmax of system L of 1.7 times without any change in Kt. Activation of systems A and L by starvation was reversible with subsequent refeeding. The effects of a series of amino acids on systems A and L were evaluated. A different inhibition pattern was found in starved animals as compared to controls. Starvation increases Na(+)-dependent L-alanine uptake and Na(+)-independent cycloleucine uptake by small-intestinal brush-border membrane vesicles. These results suggest that starvation stimulates amino acid transport across the apical plasma membrane of the enterocytes by inducing specific carrier units.

Mechanisms of transport of L-histidine and beta-alanine in hamster small intestine.

Whole rings of hamster jejunum and ileum were used to study the uptake of L-histidine (L-His) and beta-alanine (beta-Ala), the constituents of the dipeptide carnosine. The rate of uptake of L-His and beta-Ala (1 mM) was not significantly different in the jejunum compared with the ileum. Results of total influx (2 min) of 0.5-100 mM L-His suggested that transport was by more than one pathway, and the contribution of nonmediated component was calculated to be 0.24 mumol X g-1 X 2 min-1 X mM-1 for both jejunum and ileum. The apparent affinity of L-His for a transporter was higher in the ileum (K iota, 8.0 mM) than the jejunum (Kt, 11.7 mM). Influx (2 min) of beta-Ala was found to be linearly related to substrate concentration over the range 0.5-100 mM. The Kd (rate constant for nonmediated uptake of beta-Ala) was 0.23 and 0.14 mumol X g-1 X 2 min-1 X mM-1 for jejunum and ileum, respectively. Steady-state (20-min) uptake of L-His was significantly higher in the ileum than jejunum at substrate concentrations of 75 mM. L-His accumulated in the tissue up to a medium concentration of 50 mM in the jejunum and 75 mM in the ileum. In contrast, no evidence of tissue accumulation of beta-Ala was found in 20-min incubations. beta-Ala steady-state uptake in the ileum was significantly higher than in the jejunum at substrate concentrations of 30 and 75 mM.

Kinetics of uptake of lysine and lysyl-lysine by hamster jejunum in vitro.

1. The kinetics of 2-min uptake of L-lysine and L-lysyl-L-lysine have been studied by using rings of everted hamster intestine in vitro, and values for Kt and Vmax. established. 2. On a molar basis, mediated uptake was more rapid for the amino acid than for the peptide. Non-mediated uptake was more rapid for the peptide than for the amino acid. 3. Comparison of relative rates of uptake of lysine from equivalent solutions of lysine and lysyl-lysine showed that at low concentrations, uptake of lysine was less rapid from the peptide than from the amino acid, whereas at high concentrations, uptake of lysine was more rapid from the peptide than from the amino acid. This type of effect of concentration on relative rates of uptake from equivalent solutions of amino acid and peptide has not previously been described.

Development of Peptide Transport Activity in Barley Scutellum during Germination

The scutella separated from nongerminated grains of barley (Hordeum vulgare L. cv. Pirkka) took up labeled glycylsarcosine (Gly-Sar), glycylglycine, and leucylleucine at a rate of about 2 nanomoles per scutellum per hour. About 55% of the uptake of Gly-Sar (2 millimolar) was due to nonmediated uptake, but the active component was similar to that operating in the scutella from germinating grains (pH optimum about 4.5, K(m) about 10 millimolar).When the grains were allowed to germinate aseptically on agar, the rate of uptake of Gly-Sar began to increase after a lag of about 5 hours. The increase continued until a maximum value of 70 nanomoles per scutellum per hour (35-fold increase) was attained after 3 days of germination. After this the activity began to decrease slowly. The uptake of glycylglycine and leucylleucine changed in a similar way. When embryo-containing halves of seeds or separated embryos were allowed to germinate for 12 hours on agar containing various inhibitors of RNA and protein synthesis, the increase in the peptide uptake activity was completely or partially inhibited. The increase was not affected by removal of the "embryo proper" or endosperm, or by additions of gibberellic or abscisic acid.Root tips and aleurone layers took up Gly-Sar very slowly; the rate of the mediated component of uptake was less than 1% of the uptake by scutella.The preferential localization of the peptide transport system in the scutellum and its rapid development at the beginning of germination strengthen the earlier conclusion that peptide transport plays an essential role in the mobilization of reserve proteins during germination.

Kinetics of uptake of an amino acid and a dipeptide into hamster jejunum and ileum: the effect of semistarvation and starvation.

1. This paper reports an investigation of the effects of semistarvation and starvation on the kinetics of uptake of an amino acid, l-leucine, and a hydrolysis-resistant dipeptide, Gly-Sar, by rings of everted hamster jejunum and ileum in vitro. The concentration range used was 0·1–100 mmol/l. Total uptake, non-mediated uptake and Kt and Vmax. for mediated influx were estimated. 2. At many concentrations, both semistarvation and starvation caused a decrease in uptake of the peptide and the amino acid. Uptake of the peptide was more severely depressed than that of the amino acid. In control animals, the jejunum was the site of maximal uptake of Gly-Sar, and the ileum the site of maximal uptake of leucine. In semistarved and starved animals, the ileum became the site of maximal uptake of Gly-Sar, as it was for leucine. The effects of semistarvation and starvation on uptake were similar whether this was expressed per unit wt. or per unit length, though they were accentuated when expressed per unit length, since the intestine lost weight per unit length. 3. The main effect of semistarvation and starvation on the kinetics of mediated influx of the amino acid and the peptide was to reduce Vmax. in both jejunum and ileum. This effect was compatible with a reduction in the number of mediated transport sites for both the amino acid and the peptide. The observation that mediated influx of the peptide was more severely affected than that of the amino acid supports the hypothesis of the independence of the mechanisms for intestinal uptake of peptides and amino acids.

Stimulation of active uptake of nucleosides and amino acids by cyclic adenosine 3' :5'-monophosphate in the yeast Schizosaccharomyces pombe.

In conditions of glucose starvation, the maximum velocity of the mediated transport of nonmetabolized and metabolized amino acids, uridine, adenosine, and sucrose across the plasma membrane is stimulated by a factor of two by the addition of 1 mM adenosine 3':5'-monophosphate to Schizosaccharomyces pombe 972h- wild strain, to the glucose-super-repressed and derepressed mutants COB5 and COB6, and to Saccharomyces cerevisiae strain IL 216-IA. The mediated uptake of 2-D-deoxyglucose and the apparently nonmediated uptake of guanosine are not stimulated by the cyclic nucleotide. N6,O2'-Dibutyryl adenosine 3':5'-monophosphate is also efficient, whereas theophylline, guanosine 3':5'-monophosphate, 5'-AMP, ATP, and adenosine are ineffective. The cellular ATP content of glycerol-grown S. pombe COB5 is about 10 nmol per mg of protein and is not decreased by further incubation in the starvation medium. The addition of 100 mM glucose markedly enhances transport without any increase of the cellular ATP content. The addition of antimycin A or Dio-9 decreases markedly both cellular ATP content and transport. The addition of 2.5 mM glucose to antimycin A-containing medium restores both transport is not necessarily of mitochondrial origin. The uptake of 2-D-deoxyglucose is unaffected by the respiratory inhibitors. Stimulation of uptake by cyclic adenosine 3':5'-monophosphate occurs only in glucose-deprived cells. The addition of 10 mM glucose elicits the disappearance of the stimulation and prevents the 30% decrease of the cellular adenosine 3':5'-monophosphate content produced by glucose starvation. Adenosine 3':5'-'monophosphate does not enhance the steady state ATP level but requires cellular ATP produced either by endogenous respiration or, in the absence of respiration blocked by antimycin A, by further addition of 2.5 mM glucose. Stimulation of active uptake by adenosine 3':5'-monophosphate does not require protein synthesis because the addition of cycloheximide or anisomycin does not prevent the stimulation of L-leucine uptake. In the absence of respiration, Dio-9, and ATPase inhibitor, suppresses instantaneously the cellular ejection of protons as well as the uptake of uridine and amino acids. It abolishes also the adenosine 3':5'-monophosphate-stimulated transport. In the presence of antimycin A, specific mitochondrial ATPase inhibitors such as venruricidin A do not inhibit metabolite uptakes and their stimulation by adenosine 3':5'-monophosphate. These results suggest that in these conditions, the target of Dio-9 is not the mitochondrial ATPase but a plasma membrane proton-translocating function generating an electrochemical gradient required for active transport. That adenosine 3':5'-monophosphate enhances the Dio-9-sensitive proton extrusion supports the view that the cyclic nucleotide might modulate the plasma membrane ATPase.