Studies on the permeability of rat liver lysosomes to carbohydrates.
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ABSTRACT:
SUBMITTER: Lloyd JB
PROVIDER: S-EPMC1185196 | biostudies-other | 1969 Dec
REPOSITORIES: biostudies-other
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Project description:1. By the osmotic-protection method, the penetration of sugars through the rat liver lysosomal membranes was studied with a view of determining whether sugar uptake was by facilitated diffusion. 2. The following criteria for this type of transport were established: sugar specificity, the order of uptake being 2-deoxy-D-glucose less than D-glucose less than D-mannose less than D-galactose less than D-ribose less than 2-deoxy-D-ribose; stereospecificity, the uptake of L-glucose and L-ribose being 50% slower than their D-stereoisomers; inhibition by 1 MM-phlorrhizin and 1 M-cytochalastin B; competition between sugars for uptake, and a Q10 (rate difference over a 10 degrees C temperature range) for uptake of approx. 2.8. 3. It is proposed that sugar uptake into lysosomes from rat liver is by facilitated diffusion.
| S-EPMC1186523 | biostudies-other
Project description:Amino acid efflux from highly purified rat liver lysosomes exposed to the methyl ester derivatives of leucine, methionine, tyrosine and cystine was examined. The lysosomal efflux of leucine, methionine and tyrosine was unaffected by the presence of MgATP, whereas cystine efflux was stimulated by MgATP. Exposure of lysosomes to 2 mM-MgATP resulted in lysosomal acidification and a 0.5 pH unit increase in the lysosomal pH gradient through the action of a proton-pumping ATPase. Cystine efflux was also stimulated when the lysosomal proton gradient was increased through changes in buffer pH. Decreasing the lysosomal proton gradient with ionophores resulted in diminished cystine efflux. Bivalent cations had no effect on the lysosomal efflux of leucine, methionine and tyrosine. However, cystine efflux was stimulated by the presence of bivalent cations even when the lysosomal proton gradient was minimized. Cation-stimulated cystine efflux was inhibited by the presence of the calcium ionophore A23187, which altered the lysosomal membrane potential. Cystine efflux from lysosomes appears to be uniquely dependent on pH gradients and cation concentrations.
| S-EPMC1146898 | biostudies-other
Project description:Transport of D-glucose was studied in Percoll-gradient-purified rat liver lysosomes. D-Glucose uptake had a Km of 22 mM and a t1/2 of approx. 30 s. D-Fucose, 2-deoxyglucose and methyl alpha-glucoside were the most effective competitors for uptake of D-glucose, although D-galactose, D-mannose, D-xylose and L-fucose also appeared to compete for uptake. L-Glucose was a poor competitor for uptake. No competition was observed with N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, D-glucuronic acid, N-acetylneuraminic acid, D-glucosamine or the amino acids L-glycine, L-lysine and L-proline. Uptake was unaffected by N-ethylmaleimide, dithiothreitol, KCl, NaCl, ATP/Mg or alteration of buffer pH. D-Glucose efflux from lysosomes was temperature-dependent, with a Q10 of 2.3, and was inhibited by cytochalasin B. Counter-transport could not be demonstrated. In contrast, L-fucose uptake had a Km of 65 mM and was largely unaffected by 5 M excess of neutral D-sugars. Both uptake and efflux of L-fucose were inhibited by cytochalasin B. It appears that lysosomes possess a facilitated transport system for D-glucose and perhaps other neutral D-sugars that is discrete from transport systems for acetylated and acidic sugars.
| S-EPMC1149702 | biostudies-other
Project description:The latency of the alpha-glucosidase activity of intact rat liver lysosomes was studied by using four substrates (glycogen, maltose, p-nitrophenyl, alpha-glucoside, alpha-fluoroglucoside) at a range of substrate concentrations. The results indicate that the entire lysosome population is impermeable to glycogen and maltose, but a proportion of lysosomes are permeable to alpha-fluoroglucoside and a still higher proportion permeable to p-nitrophenyl alpha-glucoside. Incubation at 37 degrees C in an osmotically protected buffer of of pH 5.0 caused lysosomes to become permeable to previously impermeant substrates and ultimately to release their alpha-glucosidase into the medium. The latencies of lysosomal beta-glucosidase and beta-galactosidase were examined by using p-nitrophenyl beta-glucoside and beta-galactoside as substrates. The results indicate permeability properties to these substrates similar to that to p-nitrophenyl alpha-glucoside. On incubation in an osmotically protected buffer of pH 5, lysosomes progressively released their beta-galactosidase in soluble form, but beta-glucosidase remained attached to sedimentable material. Lysosomal beta-glucosidase was inhibited by 0.1% Triton X-100; alpha-glucosidase and beta-galactosidase were not inhibited.
| S-EPMC1164280 | biostudies-other
Project description:(1) The hydrolysis of (32)P- or myo-[2-(3)H]inositol-labelled rat liver microsomal phospholipids by rat liver lysosomal enzymes has been studied. (2) The relative rates of hydrolysis of phospholipids at pH4.5 are: sphingomyelin>phosphatidylethanolamine>phosphatidylcholine> phosphatidylinositol. (3) The predominant products of phosphatidylcholine and phosphatidylethanolamine hydrolysis are their corresponding lyso-compounds, indicating a slow rate of total deacylation. (4) Ca(2+) inhibits the hydrolysis of all phospholipids, though only appreciably at high (>5mm) concentration. The hydrolysis of sphingomyelin is considerably less sensitive to Ca(2+) than that of glycerophospholipids. (5) Analysis of the water-soluble products of phosphatidylinositol hydrolysis (by using myo-[(3)H]inositol-labelled microsomal fraction as a substrate) produced evidence that more than 95% of the product is phosphoinositol, which was derived by direct cleavage from phosphatidylinositol, rather than by hydrolysis of glycerophosphoinositol. (6) This production of phosphoinositol, allied with negligible lysophosphatidylinositol formation and a detectable accumulation of diacylglycerol, indicates that lysosomes hydrolyse membrane phosphatidylinositol almost exclusively in a phospholipase C-like manner. (7) Comparisons are drawn between the hydrolysis by lysosomal enzymes of membrane substrates and that of pure phospholipid substrates, and also the possible role of phosphatidylinositol-specific lysosomal phospholipase C in cellular phosphatidylinositol catabolism is discussed.
| S-EPMC1161342 | biostudies-other
Project description:1. When rat liver slices were incubated aerobically with [U-(14)C]glutamate the concentration of (14)C within the slices remained lower (about 50%) than in the medium. The maximal concentration of (14)C in the liver was reached within minutes. In rat kidney-cortex slices by contrast, (14)C reached concentrations more than six times those of the medium. 2. In both liver and kidney (14)C appeared in the respiratory CO(2), indicating penetration of glutamate carbon into the mitochondria. In kidney slices the rate of glutamate oxidation per unit weight was about five times that in liver slices. 3. Taking into account the conversion of glutamate into glucose that occurs in the kidney but not in the liver, the flux rates of glutamate through the kidney were calculated to be about 15 times those through the liver when the external glutamate concentration was 5mm. 4. Anaerobically the glutamate concentrations in medium and tissue rapidly became equal in both liver and kidney. Thus the maintenance of concentration gradients depended on the expenditure of energy. 5. [U-(14)C]Succinate behaved similarly to glutamate. [U-(14)C]Serine was taken up more rapidly by the kidney than by the liver slices, but the concentrations reached in the liver did not remain below those of the medium. [(14)C]Urea was distributed evenly between medium and tissue water. 6. Incubation of liver slices with [(3)H]inulin indicated an extracellular space of liver slices of 26%. 7. When glutamate was generated within liver slices or the perfused liver on addition of oxaloacetate, pyruvate and a source of nitrogen, the concentration of glutamate in the tissue after 1hr. was 70-97 times that in the medium. Thus the exit of glutamate from the liver cell, like its entry, is restricted. This is borne out by measurements of the specific activity of extra- and intra-cellular glutamate on addition of [U-(14)C]glutamate medium. 8. Liver homogenates removed added glutamate and dicarboxylic acids 20-30 times as fast as did the perfused liver. 9. It is concluded that a major permeability barrier restricts the entry and exit through the outer liver cell membrane.
| S-EPMC1198752 | biostudies-other
Project description:NO and cGMP administered at reperfusion after ischaemia prevent injury to hepatocytes mediated by the MPT (mitochondrial permeability transition). To characterize further the mechanism of protection, the ability of hepatic cytosol in combination with cyclic nucleotides to delay onset of the calcium-induced MPT was evaluated in isolated rat liver mitochondria. Liver cytosol plus cGMP or cAMP dose-dependently inhibited the MPT, required ATP hydrolysis for inhibition and did not inhibit mitochondrial calcium uptake. Specific peptide inhibitors for PKA (protein kinase A), but not PKG (protein kinase G), abolished cytosol-induced inhibition of MPT onset. Activity assays showed a cGMP- and cAMP-stimulated protein kinase activity in liver cytosol that was completely inhibited by PKI, a PKA peptide inhibitor. Size-exclusion chromatography of liver cytosol produced a single peak of cGMP/cAMP-stimulated kinase activity with an estimated protein size of 180-220 kDa. This fraction was PKI-sensitive and delayed onset of the MPT. Incubation of active catalytic PKA subunit directly with mitochondria in the absence of cytosol and cyclic nucleotide also delayed MPT onset, and incubation with purified outer membranes led to phosphorylation of a major 31 kDa band. After ischaemia, administration at reperfusion of membrane-permeant cAMPs and cAMP-mobilizing glucagon prevented reperfusion injury to hepatocytes. In conclusion, PKA in liver cytosol activated by cGMP or cAMP acts directly on mitochondria to delay onset of the MPT and protect hepatocytes from cell death after ischaemia/reperfusion.
| S-EPMC3501339 | biostudies-literature
Project description:Increased intestinal or gastric permeability is one of the major hallmarks of liver cirrhosis. The current gold standard for diagnosis of aberrant gut permeability due to disease is the triple-sugar test, where carbohydrates are orally administered and urinary excretion is measured. Hereby, elevated lactulose levels indicate intestinal permeability, whereas increased sucrose levels reveal gastric permeability. However, reliable detection and quantification of these sugars in a complex biological fluid still remains challenging due to interfering substances. Here we used Nuclear Magnetic Resonance (NMR) spectroscopy with a simple and fast protocol, without any additional sample extraction steps, for straight-forward simultaneous quantification of sugars in urine in order to detect increased intestinal and gastric permeability. Collected urine samples were diluted in buffer and one- and two-dimensional proton spectra were recorded in order to reveal carbohydrate concentrations in individual urine samples containing mannitol, sucrose and/or lactulose. Overall, this article presents a fast and robust method for simultaneous quantification of different sugars down to low micro-molar concentrations for research studies and can be further extended for clinical studies with automation of the quantification process.
| S-EPMC6168465 | biostudies-literature
Project description:Glutathione (GSH) is transported into renal mitochondria by the dicarboxylate (DIC; Slc25a10) and 2-oxoglutarate carriers (OGC; Slc25a11). To determine whether these carriers function similarly in liver mitochondria, we assessed the effect of competition with specific substrates or inhibitors on GSH uptake in isolated rat liver mitochondria. GSH uptake was uniphasic, independent of ATP hydrolysis, and exhibited K(m) and V(max) values of 4.08 mM and 3.06 nmol/min per mg protein, respectively. Incubation with butylmalonate and phenylsuccinate inhibited GSH uptake by 45-50%, although the individual inhibitors had no effect, suggesting in rat liver mitochondria, the DIC and OGC are only partially responsible for GSH uptake. H4IIE cells, a rat hepatoma cell line, were stably transfected with the cDNA for the OGC, and exhibited increased uptake of GSH and 2-oxoglutarate and were protected from cytotoxicity induced by H(2)O(2), methyl vinyl ketone, or cisplatin, demonstrating the protective function of increased mitochondrial GSH transport in the liver.
| S-EPMC3840356 | biostudies-literature
Project description:Light-scattering measurements of osmotically induced changes in the size of rat liver microsomal vesicles pre-equilibrated in a low-osmolality buffer revealed the following. (1) The increase in extravesicular osmolality by addition of glucose 6-phosphate or mannose 6-phosphate (25 mM each) caused a rapid shrinking of microsomal vesicles. After shrinkage, a rapid swelling phase (t1/2 approx. 22 s) was present with glucose 6-phosphate but absent with mannose 6-phosphate, indicating that the former had entered microsomal vesicles, but the latter had not. (2) Almost identical results were obtained in the absence of any glucose 6-phosphate hydrolysis, i.e. with microsomes pre-treated with 100 microM-vanadate. (3) The anion-channel blocker 4,4'-di-isothiocyanostilbene-2,2'-disulphonic acid (DIDS) suppressed the glucose 6-phosphate-induced swelling phase. (4) The swelling phase was more prolonged as the glucose 6-phosphate concentration increased (t1/2 = 16 +/- 3, 22 +/- 3 and 35 +/- 4 s with 25 mM, 37.5 mM- and 50 mM-glucose 6-phosphate respectively). The behaviour of glucose-6-phosphatase activity of intact and disrupted microsomes measured in the presence of high concentrations (less than 30 mM) of substrate also indicated the saturation of the glucose 6-phosphate permeation system by extravesicular concentrations of glucose 6-phosphate higher than 20-30 mM. Additional experiments showed that vanadate-treated microsomes pre-equilibrated with 0.1 mM- and 1.0 mM-glucose 6-phosphate (and [1-14C]glucose 6-phosphate as a tracer) rapidly (t1/2 less than 20 s) released [1-14C]glucose 6-phosphate when diluted in a glucose 6-phosphate-free medium. The efflux of [1-14C]glucose 6-phosphate was largely prevented by DIDS, allowing an evaluation of the intravesicular space of glucose 6-phosphate of approx. 1.0 microliter/mg of microsomal protein.
| S-EPMC1132976 | biostudies-other