Stanton Segal

Transport of amino acids by slices of rat-kidney cortex

Abstract This report presents data showing that α-aminoisobutyric acid, glycine, L -phenylalanine, and L -histidine are accumulated against a concentration gradient by slices of rat-kidney cortex in vitro . The process(es) involved depend on oxidative metabolism as shown by inhibition when anaerobic conditions or 2,4-dinitrophenol are substituted for the basal system described. The rate and extent of transport have been shown to depend on the initial concentration of the amino acids studied. The potential value of and hazards inherent in the technique are described and relationship to renal tubular transport of amino acids in the intact animal discussed.

Ionic requirements for amino acid transport in the rat kidney cortex slice: I. Influence of extracellular ions

Abstract 1. 1. Active transport of amino acids in rat kidney cortex slices diminished as the Na+ concentration of the medium was decreased below physiologic levels. In Na+-free media, active transport of glycine and α-amino [ i -14C]isobutyric acid was abolished, but active transport of lysine persisted. 2. 2. Lysine transport was found to be mediated by two mechanisms—one Na+ dependent and ouabain sensitive, and the other independent of Na+ and insensitive to ouabain. 3. 3. Maximal transport of amino acids occurred over a narrow range of medium K+ concentrations, falling off at higher and lower K+ levels. 4. 4. Substitution of other ions for Na+ in the medium caused significant alterations of the intracellular and extracellular fluid spaces of the tissues. 5. 5. Replacement of medium Na+ by K+ resulted in tissue swelling and suppression of amino acid transport beyond that caused by the absence of Na+. 6. 6. The common ionic requirements of kidney ATPase systems and of the mechanisms for active transport of certain amino acids suggest that these processes may be intimately related.

Cystinuria: Defective Intestinal Transport of Dibasic Amino Acids and Cystine*

The clinical manifestations of cystinuria are localized to the urinary tract and result from the formation of cystine calculi. The findings of increased excretion of cystine, lysine, arginine, and ornithine in the urine at a time when the plasma levels of these amino acids were normal or low suggested to Dent and Rose (2) that a renal tu-1)ular reabsorptive site, shared by the involved amino acids, was defective. Investigations from our laboratory (3), using slices of human kidney, failed to confirm the hypothesis of Dent and Rose. Cystine did not compete with the dibasic amino acids in vitro, and although the transport of ly-sine and arginine was defective in cystinuria, cys-tine transport was unimpaired. Although evidence had been accumulating for over 60 years (4-6), it was not until 1960 that the concept of an intestinal transport defect in cystinuria emerged. Milne, Asatoor, and co-workers (7, 8) noted that urinary and fecal ex-cretion of the diamines, cadaverine and putrescine, was elevated in cystinuric subjects fed lysine and ornithine, respectively. These diamines are formed by bacterial decarboxylation of the dibasic amino acids. Hence, it was postulated that lysine and ornithine were poorly absorbed from the small intestine in cystinuria and were presented to colonic bacteria in increased amounts, resulting in excessive diamine formation and excretion. These authors provided further evidence for de-A portion of this work was reported in a preliminary communication (1). fective gut absorption by showing that oral in-gestion of arginine in cystinurics was followed by minimal elevations of plasma arginine compared to results obtained with control subjects. We have previously reported a defect in the uptake of lysine and cystine by jejunal mucosa from cys-tinuric subjects (1), an observation made simnul-taneously by McCarthy and his associates (9). The present studies extend our previous observations and show that lysine and cystine are accumulated by saturable, energy-dependent processes in the human jejunum and that these two amino acids are mutually inhibitory in the gut. Furthermore, it is shown that some patients with cystinuria do not share the intestinal transport defect for cystine and lysine. Methods After an overnight fast, peroral biopsy of the intestinal mucosa was performed with a Rubin tube placed at the ligament of Treitz under fluoroscopic control. A total of 75 biopsies was performed on 18 normal volunteers and 12 patients with cystinuria. The 14 male and four female volunteers were between the ages of 18 and 36 …

Studies of the kinetics of amino acid transport, incorporation into protein and oxidation in kidney-cortex slices

Abstract The present report describes an approach to the study of the steady-state kinetics of amino acid transport and protein synthesis in rat-kidney-cortex slices, using multi-compartment models. The following conclusions seem justified from the data presented: 1. 1. Using inulin and the non-metabolizable amino acid, α-aminoisobutyric acid, amino acid uptake could be characterized by a three-compartment “parallel” model representing the medium, extracellular space and intracellular space. Appropriate influx and efflux rate constants were calculated. 2. 2. 2,4-Dinitrophenol and incubation at 27° significantly effected the rate of efflux of α-aminoisobutyric acid from the intracellular space, as well as the rate of influx, suggesting that efflux phenomena are metabolically linked. 3. 3. Kinetic studies of combined transport and protein synthesis with glycine and l -lysine indicated that equilibration of exogenous amino acid with the intracellular pool need not occur before incorporation into protein takes place. 4. 4. The kinetics of 14 CO 2 evolution from labeled l -lysine indicated that the rate of amino acid oxidation reflected the buildup of the intracellular lysine pool. 5. 5. The results suggest that the mathematical approach, used in the analysis of the experimental data, provides means of quantitating and understanding membrane phenomena and the relationship between rates of membrane transfer and subsequent intracellular utilization.

The quantitative determination of galactose—An enzymic method using galactose oxidase, with applications to blood and other biological fluids

Abstract Techniques are described for the quantitative analysis of biological solutions for galactose content, involving incubation of unknown samples with the enzyme galactose oxidase in the presence of peroxidase and benzidine. Some characteristics of the reaction are discussed and conditions are defined for two procedural variations, depending upon the sensitivity and range desired. Directions are stated for the application of the method to blood, urine, and other biological fluids, and illustrations of a number of these applications are presented.

Dibasic aminod acid transport in rat-kidney cortex slices

Abstract The effect of anaerobiosis, Na+ deprivation and pH alteration on the accumulation of l -lysine, l -arginine, l -diaminobutyric acid and l -cystine was studied at physiological levels of substrate in rat-kidney cortex slices. Lysine, arginine and diaminobutyric acid were capable of being actively transported despite oxygen and Na+ lack in the incubation medium. Initial rates of accumulation of lysine and diaminobutyric acid appeared to be normal under the latter conditions although the steady-state concentration gradients were reduced. Cystine and arginine transport was inhibited initially and there was impairment of the ability to form a concentration gradient, most marked for cystine in Na+-free medium. The variation of accumulation with pH differed for each of the amino acids, the curve of cystine being more like that of glycine, than the dibasic amino acids. The results lend further support to the concept that cystine transport in kidney slices occurs by a system separate from that of dibasic amino acids and indicate that for the other dibasic amino acids, variation of intra-group transport characteristics is present.

Exchange diffusion of dibasic amino acids in rat-kidney cortex slices

Abstract 1. 1.|Lysine, arginine, ornithine, and diaminobutyric acid were found to participate in autoexchange and heteroexchange diffusion in rat-kidney cortex slices. 2. 2.|Neither cystine nor cysteine exchanged with the dibasic amino acids. However, at late incubation time points, tissues which had been preloaded with ornithine or lysine accumulated higher intracellular concentrations of cysteine than unloaded controls. This effect appeared to be due to a specific inhibition of cysteine efflux by ornithine and lysine.

Effect of Alterations of Coronary Blood Flow on the Oxygen Consumption of the Nonworking Heart

The effect of varying CBF and myocardial O2 delivery on MVO2 was studied in 23 experiments. In 14 of the experiments an isolated dog heart was perfused, while in the others the heart of a dog, whose systemic circulation was maintained on cardiopulmonary bypass, was studied. The ventricles were kept empty, developed no pressure and performed no external work, while their temperature was held constant. CBF and myocardial O2 delivery were controlled by pumping blood into the coronary arteries, total coronary venous return was collected from the right side of the heart and MVO2, was calculated during a steady state by the Fick principle. Myocardial anoxia was avoided by maintaining the coronary venous O2 content above 4 vol % and myocardial O2 extraction below 78%. A comparison of MVO2 at two levels of CBF (and O2 delivery) was made in 42 instances, and in 32 of them 11 MV increased substantially as CBF was elevated, or vice versa. The ten exceptions all occurred when O2 delivery greatly exceeded MVO2 with O2 extraction ratios below 35%. The fundamental mechanisms responsible for these findings are not clear, but a number of possible explanations are discussed.

Observations on cataract formation in the newborn offspring of rats fed a high-galactose diet

A high incidence of cataracts has been demonstrated in the newborn of Sprague-Dawley rats fed a 40 per cent galactose diet. Galactose present in maternal blood has rapid and extensive access to the fetal circulation. These findings if extrapolated to man offer strong evidence for in utero damage of a galactosemic fetus.

Hexose inhibition of amino acid uptake in the rat-kidney-cortex slice

Abstract Glucose, galactose and fructose have been shown to inhibit the intracellular accumulation of amino acids by the rat-kidney-cortex slice. The uptake of glycine, α-amino- [1−14C]isobutyric acid, valine and cycloleucine was inhibited by these hexoses but the accumulation of lysine, histidine and phenylalanine was unaffected. The inhibitory effect has been shown to be dependent upon the length of incubation and upon the sugar concentration. Evidence has been presented to indicate that sugars reduce the rate of influx of amino acid into cells but have no effect upon the apparent affinity of transport sites for amino acids.