Pamela D. McNamara

Sodium gradient dependence of proline and glycine uptake in rat renal brush-border membrane vesicles

The sodium-dependent entry of proline and glycine into rat renal brush-border membrane vesicles was examined. The high Km system for proline shows no sodium dependence. The low Km system for glycine entry is strictly dependent on a Na+ gradient but shows no evidence of the carrier system having any affinity for Na+. The low Km system for proline and high Km system for glycine transport appear to be shared. Both systems are stimulated by a Na+ gradient and appear to have an affinity for the Na+. The effect of decreasing the Na+ concentration in the ionic gradient is to alter the Km for amino acid entry and, at low Na+ concentrations, to inhibit the V for glycine entry.

On the maleic acid induced Fanconi syndrome: Effects on transport by isolated rat kidney brushborder membrane vesicles

Abstract The association of maleic acid with isolated rat kidney brushborder membranes and the effect of maleic acid on uptake of amino acids and α-methyl-D-glucoside by brushborder membrane vesicles has been studied. Although maleic acid associates with the membrane, no alteration of sodium dependent transport of these substrates into the vesicles was observed. These findings indicate that the maleic acid induced Fanconi syndrome in the rat with aminoaciduria and glycosuria is not due to a direct, adverse effect on the renal tubule brushborder membrane per se .

Ion dependence of cystine and lysine uptake by rat renal brush-border membrane vesicles

The shared transport system for uptake of L-cystine and L-lysine was examined in isolated rat renal brush-border membrane vesicles for the ionic requirements for activation of the system. No requirement for sodium was seen for either cystine or lysine influx. However, the efflux of lysine from the vesicle was stimulated by Na+. Therefore, the transport system appears to be asymmetric in its requirement for sodium. Two different divalent cations were used in the membrane isolations which resulted in different responses of cystine uptake to the electrogenic movement of K+ out of the vesicle. Membranes prepared by Mg-aggregation showed no stimulation of cystine influx by the imposition of a transient interior negative potential while vesicles prepared by Ca-aggregation did respond to electrogenic stimulation by an outwardly directed K-diffusion potential in the presence of valinomycin. Lysine influx was stimulated by electrogenic potassium efflux in both Mg-prepared and Ca-prepared membranes. No difference in sodium requirement for cystine influx was seen between the vesicles isolated by different cation-aggregation methods.

Cystine uptake by rat jejunal brushborder membrane vesicles

The presence of a sodium-stimulated, saturable uptake process for L-cystine is described in brushborder membrane vesicles isolated from rat jejunal mucosa. Concentration-dependence studies indicate the presence of a single transport system for cystine withKm=0.053 mM andVmax=0.633 nmol/mg/15 s. Lysine completely inhibits the uptake of cystine.

An improved method for isolation of basolateral membranes from rat kidney

Abstract We have described a method using differential centrifugation and free-flow electrophoresis for the isolation of basolateral membranes from rat renal cortical tissue. Purity of the membrane preparation was assessed by determination of enzymic constituents and substantiated by observation of transport characteristics for d - and l -glucose, l -proline and l -glutamine. Specific activity of ( Na + + K + )- ATPase in the pooled electrophoretic fractions was 15.4 times that in the cortical homogenate, and the relative purification of ( Na + + K + )- ATPase compared to alkaline phosphatase was 25-fold. l -Glucose showed no Na+-gradient dependence and was used as a measure of diffusion. Uptake of d -glucose as well as l -proline followed the same time course in the presence or absence of an Na+ gradient. Only with l -glutamine was uptake stimulated by the presence of an Na+ gradient. Our data indicate we have sucessfully isolated basolateral membranes which are free of brush border contamination in superior yield and with greater facility than previously reported.

Cystinuria in dogs: Comparison of the cystinuric component of the Fanconi syndrome in Basenji dogs to isolated cystinuria☆

Two animal models for cystinuria have been examined: the Basenji dog with Fanconi syndrome and cystine stone-forming dogs of various breeds. Brush-border membranes were isolated from these animals and uptake of D-glucose and L-cystine was characterized. Experiments with isolated brush-border vesicles from Basenji dogs with cystinuria as a component of the Fanconi syndrome showed diminished sodium-dependent D-glucose uptake but no decrease in L-cystine uptake even though the cystine defect in vivo was as high as 94% (ie, 6% reabsorption). In contrast, brush-border vesicles isolated from the kidney of a cystine stone-forming dog (Welsh Corgi) with a cystine defect of only 16% (ie, 84% reabsorption) had decreased uptake of cystine compared to values found for Beagle and Basenji vesicles. Thus, cystinuria found in Basenji dogs with the Fanconi syndrome differs from that in classic stone-forming cystinuric dogs. The alteration responsible for the cystinuria of Basenji dogs with Fanconi syndrome does not appear to have a membrane locus and may reflect altered energetics for transport, which are not detected in isolated vesicles. The cystine defect in cystinuric stone-forming dogs does appear to be reflected in the isolated membrane.

Uptake of proline by renal brushborder vesicles: A mathematical analysis

Abstract A mathematical model for amino acid uptake by membrane vesicles is described which includes two components, a Na + dependent and a Na + independent system. Uptake in the model is a function of both initial external Na + and amino acid concentrations. Sodium dependence of amino acid transport in the model is manifested by changing affinity constants for amino acid uptake under different Na + concentrations. The differing affinities for influx and efflux caused by increasing internal Na + concentrations with time during transport incubations result in an “overshoot” for amino acid accumulation. For inwardly directed Na + gradients, the model predicts the dependence of the occurrence of the overshoot on initial external substrate concentration and the dependence of the height of the overshoot on initial external Na + concentration. This model has been used to describe experimental data on proline uptake by rat renal brushborder membrane vesicles.

Assessment of binding of L-cystine and L-lysine by rat renal brush-border membranes.

Cystine and lysine bind to isolated rat renal brush-border vesicles. Three methods to determine the extent of amino acid binding to the membranes have been compared, one relying on the osmotic reactivity of the vesicle, a second by trichloroacetic acid precipitation of membrane-bound material and a third by initial rate analysis. For cystine, all methods yield comparable results at early time points, indicating the trichloroacetic acid method is a simple and valuable tool for binding estimation under initial-rate or near initial-rate conditions. For lysine, initial rate analysis and osmotic perturbation are the methods of choice since lysine co-precipitates with trichloroacetic acid.

Uptake of proline by brushborder vesicles isolated from human kidney cortex

Proline transport into renal brushborder membrane vesicles isolated from human kidney is mediated by two uptake systems. The high-affinity system is stimulated by a Na gradient and appears to be shared with glycine while the low-affinity system is not. Uptake curves of low concentrations of proline exhibit a Na-gradient-dependent overshoot indicative of electrogenic transport. The proline transport systems observed in isolated human renal brushborder membrane vesicles appear to have characteristics similar to those in rat kidney membranes.

The effect of diamide on amino acid transport by rat renal cortex slices.

Diamide directly added to renal cortical slices inhibits the uptake of amino acids. Steady-state kinetic analysis indicates an inhibition of alpha-amino acid influx without effect on efflux. The effect could be reversed by addition of pyruvate to the incubation medium. Although there was a good correlation of the transport effect of diamide with its ability to decrease cellular reduced glutathione concentration, there did not appear to be a necessary connection between them. This was shown by the fact that renal cortical slices stored at 4 degrees C have no alteration in amino acid uptake despite the fact that GSH concentration is as low as that seen with diamide. Diamide was shown to have a direct effect on the uptake of glycine by isolated renal brush border membrane vesicles.