The effect of azaserine on glutamine uptake by rat renal brush-border membranes.
Ontology highlight
ABSTRACT:
SUBMITTER: Hsu BY
PROVIDER: S-EPMC1162314 | biostudies-other | 1980 Oct
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Uptake of L-cystine by brush-border membrane vesicles isolated from rat renal-cortical tissue was time-dependent and occurred in the absence of cystine reduction. A significant capacity for vesicular binding of cystine was observed. The amount bound increased with time of incubation and could be displaced by thiol reagents. At early time points, cystine uptake measured the transport of cystine into the intravesicular space. Total cystine uptake was mediated by multiple transport systems, including a low-Km high-affinity component which was shared by lysine, arginine, ornithine and glutamine and on which hetero-exchange diffusion of lysine and cystine was demonstrated.
| S-EPMC1162767 | biostudies-other
Project description:Glutamine uptake was examined in isolated renal brush-border and basolateral-membrane vesicles from control and acidotic rats. In brush-border vesicles from acidotic animals, there was a significant increase in the initial rate of glutamine uptake compared with that in controls. Lowering the pH of the medium increased the initial rate of glutamine uptake in brush-border vesicles from acidotic, but not from control, rats. In brush-border vesicles from both groups of animals, two saturable transport systems mediated glutamine uptake. There was a 2-fold increase in the Vmax. of the low-affinity high-capacity system in the brush-border vesicles from the acidotic animals compared with that from control animals, with no alteration in the other kinetic parameters. There was no difference in glutamine uptake by the two saturable transport systems in basolateral vesicles from control and acidotic animals. Lowering the incubation-medium pH increased the uptake of glutamine by basolateral vesicles from both control and acidotic rats to a similar extent. The data indicate that during acidosis there are alterations in glutamine transport by both the basolateral and brush-border membrane which could enhance its uptake by the renal-tubule cell for use in ammoniagenesis.
| S-EPMC1153148 | biostudies-other
Project description:Phosphate uptake by rat renal brush-border membrane vesicles was studied under experimental conditions where transmembrane electrical potential (delta psi) could be manipulated. Experiments were performed under initial rate conditions to avoid complications associated with the dissipation of ion gradients. First, phosphate uptake was shown to be strongly affected by the nature of Na+ co-anions, the highest rates of uptake being observed with 100 mM-NaSCN (1.010 +/- 0.086 pmol/5 s per micrograms of protein) and the lowest with 50 mM-Na2SO4 (0.331 +/- 0.046 pmol/5 s per micrograms of protein). Anion substitution studies showed that potency of the effect of the co-anions was in the order thiocyanate greater than nitrate greater than chloride greater than isethionate greater than gluconate greater than sulphate, which correlates with the known permeability of the membrane to these anions and thus to the generation of transmembrane electrical potentials of decreasing magnitude (inside negative). The stimulation by ion-diffusion-induced potential was observed from pH 6.5 to 8.5, indicating that the transport of both monovalent and divalent phosphate was affected. In addition, inside-negative membrane potentials were generated by valinomycin-induced diffusion of K+ from K+-loaded vesicles and showed a 57% stimulation of phosphate uptake, at pH 7.5. Similar experiments with H+-loaded vesicles, in the presence of carbonyl cyanide m-chlorophenylhydrazone gave a 50% stimulation compared with controls. Inside-positive membrane potentials were also induced by reversal of the K+ gradient (outside greater than inside) in the presence of valinomycin and gave 58% inhibition of phosphate uptake. The membrane-potential dependency of phosphate uptake was finally analysed under thermodynamic equilibrium, and a stimulation by inside-negative potential was observed. The transport of phosphate was thus driven against a concentration gradient by a membrane potential, implicating the net transfer of a positive charge during the translocation process. These results indicate a major contribution of electrical potential to phosphate uptake in renal brush-border membranes.
| S-EPMC1149218 | biostudies-other
Project description:Transport of glutamine by brush-border vesicles prepared from the renal cortex was studied. The transport system had both Na+-dependent and Na+-independent components. The presence of Na+ in the incubation resulted in an 'overshoot' at 30s at which time the rates of transport were approx. 8 times the values obtained in the absence of Na+. Variation of the glutamine concentration showed that the system obeyed Michaelis-Menten kinetics with Km and Vmax. values for the Na+-dependent system of 0.86 mM and 9.6 nmol/min per mg of protein respectively. Vesicles obtained from chronically acidotic rats showed similar kinetic characteristics. The Km and Vmax. values for the Na+-dependent system were 0.76 mM and 9.6 nmol/min per mg of protein respectively. There was increased uptake of glutamine by vesicles from acidotic rats and this increase was associated with increased activity of gamma-glutamyltransferase in these preparations. Vesicles from acidotic rats, however, showed no increase in glucose transport and no increase in the activity of maltase, another brush-border enzyme.
| S-EPMC1161307 | biostudies-other
Project description:The Ca2+ transport process by isolated renal brush-border membranes was characterized and the influence of the acidic phospholipid phosphatidic acid (PtdA) on this transport process was assessed. Ca2+ uptake by brush-border membranes exhibited saturation kinetics. It was inhibitable by a variety of multivalent cations, as well as by Ca2+-entry inhibitors, including verapamil, Ruthenium Red and gentamicin. It was selective for Ca2+ compared with Mg2+. This process was also electrophoretic since generation of K+ and anion-diffusion potentials, negative inside the vesicle, increased Ca2+ uptake. Elevations in PtdA content of brush-border membranes by either exogenous addition or endogenous generation of PtdA by incubating brush-border membranes with MgATP2- elevated the rate of Ca2+ uptake. This ATP effect could not be attributed to (Ca2+ + Mg2+)-dependent ATPase or contaminating membrane fragments. PtdA also increased the magnitude and rate of Ca2+ efflux from brush-border membranes preloaded with Ca2+. These modulations in uptake and efflux were not observed with phosphatidylcholine or phosphatidylinositol. In summary, these results are consistent with the presence of an electrophoretic uniport system for Ca2+ in renal brush-border membranes, and demonstrate that PtdA uniquely among phospholipids tested appears to facilitate transmembrane flux of Ca2+ across this membrane preparation.
| S-EPMC1152207 | biostudies-other
Project description:INTRODUCTION:Radiolabeled, low-molecular-weight prostate-specific membrane antigen (PSMA) inhibitors based on the Glu-ureido pharmacophore show promise for the detection and treatment of castration-resistant prostate cancer; however, high renal retention of activity, related in part to overexpression of PSMA in kidneys can be problematic. The goal of the current study was to investigate the use of brush border enzyme-cleavable linkers as a strategy for reducing kidney activity levels from radiolabeled PSMA inhibitors. METHODS:PSMA-769 (6), a derivative of the prototypical PSMA inhibitor (((S)?1?carboxy?5?(4?iodobenzamido)pentyl)carbamoyl)glutamate (12) modified to contain a Gly-Tyr linker, and its protected tin precursor (11) were synthesized starting from the basic pharmacophore molecule Lys-urea-Glu. An analogue of 6 containing d?tyrosine in lieu of l?tyrosine (PSMA-769-d-tyrosine) and the corresponding tin precursor (d-11) also were synthesized. Both radioiodinated and 211At-labeled 6 were synthesized by radiohalogenation of 11 and deprotection in situ. Similarly, radioiodinated d-6 was synthesized from d-11. Paired label biodistribution of [125I]12 and [131I]6 was performed in normal mice and in SCID mice bearing both PC3 PIP (PSMA+) and PC3 flu (PSMA-) subcutaneous prostate carcinoma xenografts. The biodistribution of [131I]6 and [211At]6 was also evaluated in this tumor model. Biodistribution of the two radioiodinated diastereomers of 6 was evaluated in normal mice and urine samples were analyzed for the presence of 4?iodohippuric acid. RESULTS:Compounds [131I]6 and [211At]6 were synthesized from 11 in overall radiochemical yields of 32.5?±?0.1% (n?=?4) and 22% (n?=?1), respectively; radiochemical purity was >95%. In normal mice, renal uptake of [131I]6 was 1.4-, 2.8- and 161-fold lower than that seen for co-injected [125I]12 at 1?h, 4?h and 21?h, respectively. In tumor-bearing mice, kidney uptake of [131I]6 was similar to that for [125I]12 (P?>?0.05) at 1?h and 4?h but was 6- to 7-fold lower at 21?h; however, [131I]6 uptake in PC3 PIP tumors was also lower than that seen for [125I]12 at 21?h (12.6?±?3.4%ID/g vs. 36.8?±?12.4%ID/g). Uptake of [211At]PSMA-769 in PC3 PIP tumors was slightly higher than that seen for [131I]PSMA-769 at 4?h (9.6?±?1.6%ID/g versus 7.8?±?1.6%ID/g; P?=?0.002); its uptake in a number of normal tissues also was higher. In normal mice, kidney uptake of [125I]PSMA-769 at 4?h was about 73% of that seen for [131I]PSMA-769-d-tyrosine. Activity in the urine of mice receiving [125I]PSMA-769 contained mainly 4?[125I]iodohippuric acid while unmetabolized intact molecule was present in the case of [125I]PSMA-769-d-tyrosine. CONCLUSION:Use of this brush border enzyme-cleavable linker reduced kidney uptake and resulted in improved tumor:kidney uptake ratios. Although further structural improvements are needed, this linker approach might be useful as a component in strategies for reducing renal uptake of radiolabeled PSMA inhibitors.
| S-EPMC6054815 | biostudies-literature
Project description:Using highly specific mass assays, concentrations of inositol lipids and 1,2-diacylglycerol (DAG) were determined in plasma membranes isolated from rat kidney cortex. Significantly higher concentrations of inositol lipids were determined in brush-border (BBM) than in basal-lateral (BLM) plasma membranes, although DAG concentrations were similar in both. After unilateral nephrectomy, a decrease in PtdIns(4,5)P2 and PtdIns4P, with a concomitant increase in DAG and translocation of protein kinase C (PKC), were observed in BBM but not in BLM isolated from the remaining kidney. On the other hand, stimulation of renal cortical slices with insulin-like growth factor II (IGF-II) or phenylephrine caused similar effects in BLM but not in BBM. Stimulation of phospholipase C activity with translocation of PKC only to BBM in one kidney was also induced by occlusion of blood flow through the contralateral kidney for 15 min. At 30 min after the occlusion was removed and reflow established, DAG concentration and the amount of PKC in BBM returned to control values. These results suggest that an early signal after unilateral nephrectomy is transmitted to cells through BBM and can be switched on and off by blood occlusion and reflow through the contralateral kidney, while hormonal signals caused by IGF-II and phenylephrine are transmitted to cells through BLM.
| S-EPMC1134923 | biostudies-other
Project description:Renal brush-border membrane vesicles isolated from rats kept for 6-8 weeks on a low-phosphate diet (0.15% of dry matter) showed a markedly faster Na(+)-dependent phosphate uptake than did membrane vesicles isolated from animals kept on a high-phosphate diet (2% of dry matter). Phosphate-uptake rate by brush-border membrane vesicles isolated from animals on a low-phosphate diet remained significantly increased after acute parathyroidectomy. Dietary adaptation was also observed in animals that had been parathyroidectomized before exposure to the different diets. In animals on the low-phosphate diet parathyrin administration inhibited phosphate uptake by brush-border vesicles only if the animals were repleted with P(i) (5ml of 20mm-NaH(2)PO(4)) 1h before being killed. After acute phosphate loading and parathyrin administration the difference in the transport rate between the two dietary groups remained statistically significant. The results suggest that the adaptation of proximal-tubule phosphate transport to dietary intake of phosphate is reflected in the Na(+)/phosphate co-transport system located in the luminal membrane of the proximal-tubule cell. Since the dietary effects on phosphate transport by brush-border membranes are only partially reversed by acute changes in parathyrin concentration and are also observed in chronically parathyroidectomized animals, the adaptation of the Na(+)/phosphate co-transport system to dietary phosphate intake seems to involve an additional mechanism independent of parathyrin.
| S-EPMC1161083 | biostudies-other
Project description:Purified rat renal brush-border membrane vesicles possess a heat-labile enzyme activity which hydrolyses NAD+. A reciprocal relationship exists between the disappearance of NAD+ and the appearance of adenosine; 2 mol of Pi are liberated from each mol of NAD+ incubated with brush-border membrane vesicles. Freezing and thawing brush-border membrane vesicles does not enhance the initial rate of NAD+ hydrolysis. Preincubation of brush-border membrane vesicles with NAD+ results in inhibition of Na+-dependent Pi-transport activity, whereas Na+-dependent glucose transport is not affected. EDTA, which prevents the release of Pi from NAD+ and which itself has no direct effect on brush-border membrane Pi transport, reverses the NAD+ inhibition of Na+-dependent Pi transport. These results suggest that it is the Pi liberated from NAD+ and not NAD+ itself that inhibits Na+-dependent Pi transport.
| S-EPMC1158401 | biostudies-other
Project description:An extract of soluble proteins was prepared from a rat kidney brush-border membranes by Triton X-100 solubilization followed by centrifugation for 1 h at 100000g. Its protein composition was markedly different from that of the brush-border membranes. Proteoliposomes were formed by co-sonication of the Triton X-100-free extract with a naturally occurring mixture of phospholipids extracted from rat kidney. These proteoliposomes were shown to contain Na+-stimulated D-glucose-, L-alanine- and phosphate-transport systems.
| S-EPMC1183766 | biostudies-other