Properties of carnitine transport in rat kidney cortex slices

The properties of carnitine transport were studied in rat kidney cortex slices. Tissue: medium concentration gradients of 7.9 for L-[methyl-¹⁴C]carnitine were attained after 60-min incubation at 37°C in 40 μM substrate. L- and D-carnitine uptake showed saturability. The concentration curves appeared to consist of (1) a high-affinity component, and (2) a lower affinity site. When corrected for the latter components, the estimated K_m for L-carnitine was 90 μM and $\text{V = 22}\text{nmol/min}$ per ml intracellular fluid; for D-carnitine, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 166}\text{μM}$ and $\text{V = 15}\text{nmol/min}$ per ml intracellular fluid. The system was stereospecific for L-carnitine. The uptake of L-carnitine was inhibited by (1) D-carnitine, γ-butyrobetaine, and (2) acetyl-L-carnitine. γ-Butyrobetaine and acetyl-L-carnitine were competitive inhibitors of L-carnitine uptake. Carnitine transport was not significantly reduced by choline, betaine, lysine or γ-aminobutyric acid. Carnitine uptake was inhibited by 2,4-dinitrophenol, carbonyl cyanide $\text{m-}\text{chlorophenylhydrazone}$, N₂ atmosphere, KCN, $\text{N-}\text{ethylmaleimide}$, low temperature (4°C) and ouabain. Complete replacement of Na⁺ in the medium by Li⁺ reduced L- and D-carnitine uptake by 75 and 60%, respectively. Complete replacement of K⁺ or Ca²⁺ in the medium also significantly reduces carnitine uptake. Two roles for the carnitine transport system in kidney are proposed: (1) a renal tubule reabsorption system for the steady-state maintenance of plasma carnitine; and (2) maintenance of normal carnitine levels in kidney cells, which is required for fatty acid oxidation.     

The uptake of methonium compounds by isolated slices of rat cerebral cortex

Abstract— The uptakes of a series of methonium compounds, C2, C4, C6, C7, C8, C9 and C10, by isolated slices of rat cerebral cortex were measured. Analyses of the kinetics of uptake of C7, C8, C9 and C10 indicate that their initial influxes consist of two components. A Michaelis-Menten type uptake by carrier can account for the saturable fraction. C7 had the lowest affinity for the carrier, with values increasing for C8, C9 and C10, while C10 had the smallest maximum influx with values increasing for C9, C8 and C7. The unsaturable fraction corresponded to the slow continuous swelling of the tissue. The uptake of C8 was inhibited by other methonium compounds, choline, ACh, physostigmine, ouabain and morphine. C8 could be counter-transported by C7. There is evidence that the main driving force for uptake is the transmembrane potential, but the steric factor may be a restricting influence.     

Tryptophan uptake by glia and synaptosomes of rabbit cerebral cortex

The authors studied the engulfment of L-tryptophane-14C by gliacytes and synaptosomes of the rabbit cerebral cortex. The system of engulfment of the gliacytes was characterized by a high affinity to tryptophane (Km = 0.8 micrometer). Engulfment of tryptophane by synaptosomes had a lower affinity (Km = 50 micrometer). Psychotropic substances--chlorpromazine and imipramine produced an inhibitory influence on glial engulfment. The leading role of gliacytes in the trophic provision of the neurons and the normal course of neurodynamic processes is confirmed.     

Roles of sodium and potassium ions on p-aminohippurate transport in rabbit kidney slices.

This investigation was principally undertaken to test the ionic gradient hypothesis as applied to active p-aminohippurate uptake in the rabbit kidney cortical slice preparation. Efflux of p-aminohippurate from the slice was shown to be independent of external Na+ concentration. Transferring slices from a low sodium preincubation to a high sodium incubation medium containing p-aminohippurate increased intracellular concentrations of both Na+ and K+, and p-aminohippurate accumulation occurred. Transferring slices from a low sodium preincubation to a high sodium incubation medium containing ouabain and p-aminohippurate resulted in a net increase in intracellular Na+ concentration but no p-aminohippurate accumulation occurred. Different combinations of preincubation and incubation media gave a high to low array of intracellular Na+ concentrations and these directly reflected their respective p-aminohippurate uptake. These results suggest that the Na+ gradient hypothesis does not adequately explain the transport of organic acids in rabbit kidney. These results also suggest that Na+ possibly has an intracellular role through its stimulation of (Na+ + K+)-ATPase channeled to energizing the p-aminohippurate accumulative mechanism.     

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 α-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°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.     

Carnitine transport by rat kidney cortex slices: stimulation by dibutyryl cyclic AMP+.

The transport of carnitine by rat kidney cortex slices against a concentration gradient has been demonstrated. Similarities to other transport systems included a linear period of uptake, as well as indications of saturability of the system with increasing concentrations of substrate. The transport of carnitine was inhibited by anoxia, and carbonyl cyanide-m-chloro-phenylhydroxazone (CCC1P), an uncoupler of oxidative phosphorylation. Carnitine uptake was stimulated approximately 50% when kidney slices were treated with dibutyryl cAMP.     

Characterization of transport systems for the transfer of 3,4-L-dihydroxyphenylalanine into slices of rat cerebral cortex.

1. Slices of rat cerebral cortex incubated aerobically at 37 degrees C in Krebs-Ringer-bicarbonate solution accumulated 3,4-L-dihydroxyphenylalanine (L-DOPA) against its concentration gradient. With 1 mM L-DOPA in the medium, tissue-water/medium concentration ratios of about 6 : 1 are reached, which are modified by the presence of other amino acids in the medium. 2. Kinetic analysis suggested that L-DOPA influx into brain cells occurred by at least two saturable processes, which show apparent Km values in the range of 10(-3) M and 10(-5) M, respectively. 3. Prior incubation of the slices in Na+-free (choline-containing) medium at 37 degrees C depressed their subsequent uptake of L-DOPA in normal Na+-containing medium; this inhibition did not appear when the preincubation was carried out at 0-4 degrees C. Besides this effect of preincubation, most of L-DOPA influx into brain slices was independent of the actual concentration of Na+ in the medium; the two saturable processes described in this article behaved similarly in this respect. 4. Most of L-DOPA uptake by the high-Km process is mediated by an agency that resembles the Na+-independent L system described in Ehrlich cells (Oxender, D. L. and Christensen, H. N. (1963) J. Biol. Chem. 238, 2686-2699), both in its specificity and in its participation in exchange phenomena. A lesser component of uptake by a type A mediation is also suggested as contributing to the high-Km process . 5. The kinetic and specificity properties of the low-Km process of L-DOPA uptake suggest a similarity between its mediation and that of the high-affinity systems for L-tyrosine and L-tryptophan found in brain tissue preparations (Belin, M. F. and Pujol, J. F. (1973) Experientia 29, 411-413; Bauman, A., Bourgoin, S., Benda, P., Glowinski, J. and Hamon, M. (1971 Brain Res. 66, 253-263).     

Thiosulfate accumulation by rat renal cortex slices

S₂O₃²⁻ excretion in the dog has been well documented, and its sensitivity to certain hormones has been reported. For example, pretreatment of the female dog with cortisone promotes renal tubular secretion of S₂O₃²⁻. This study was designed to establish the parameters for S₂O₃²⁻ transport by isolated rat renal tissue prior to investigating the hormone effects. Standard renal slice procedures were employed with incubations performed in modified Krebs-Ringer phosphate solutions. Rat cortex slices accumulate ³⁵SSO₃²⁻ to steady-state distribution ratios (concentration in cell water/concentration in bathing solution) of about 3.0 in 90–120 min. Steady-state distribution ratios were depressed as the concentration of S₂O₃²⁻ in the bathing solution was increased. No metabolic substrates were found to increase accumulation. Specifically, glucose had no effect, while a large number of organic acid intermediates (e.g. acetate, succinate) reduced accumulation. The uptake was energy dependent, i.e. it was reduced by metabolic inhibitors. S₂O₃²⁻ accumulation was influenced by the K⁺ concentration of the bathing solution, and perhaps to a slight extent by the N⁺ concentration. Specific activity measurements and high-voltage electrophoresis analyses indicated that ³⁵SSO₃²⁻ was neither metabolized by this slice system nor bound significantly to macromolecules. However, a small amount of trichloroacetic acid-insoluble label was detected.     

Effect of hydrocortisone on phosphoinositide metabolism in rat cerebral cortex slices]

Hydrocortisone administered in a dose of 1 and 5 mg per 100 g of mass has increased the rate of 32P turnover in di- and triphosphatidyl inositides up to 170-230% and has no influence on the content of these phospholipids.     

Mechanism of l-histidine-induced stimulation of amino acid transport in slices of rat cerebral cortex

Effects of l-histidine on the transport of other amino acids were studied with slices of rat cerebral cortex. Histidine (0.5 mM) significantly increased the 60-min accumulation of large neutral and basic amino acids (0.5 mM). The effect was dependent on sodium ions and could be demonstrated in slices from both adult and newborn rats. Other amino acids tested were either ineffective or inhibitory; in particular, l-phenylalanine was strongly inhibitory. The 5-min influx of amino acids into slices was also enhanced by preincubation with histidine. This effect was stereospecific for l-histidine, sodium-independent and not produced by histidine metabolites or activation of histamine H₁ and H₂ receptors. Kinetic analysis of leucine influx showed that the maximal velocity of transport (V) increased relatively more than the other transport parameters. The results could be explained by stimulation of amino acid exchange by intracellular l-histidine. The opposite effects of histidine and phenylalanine on the accumulation of other amino acids are in keeping with the generally less severe impairment of cerebral functions in clinical histidinemia as compared to that in phenylketonuria.     

Metal ions affect neuronal membrane fluidity of rat cerebral cortex

The effect of various metal ions on neuronal membrane fluidity was examined using 2-(14-carboxypropyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxy, which has been used for the examination of membrane fluidity in hydrophobic areas by electron spin resonance spectrometry. Potassium, cobalt, calcium, magnesium, nickel, copper, ferric, and aluminium ions decreased the membrane fluidity while ferrous ions increased it at each high concentration. Sodium and zinc ions had no effect. Ethylenediaminetetraacetic acid decreased membrane fluidity at high concentrations. Nicardipine lowered membrane fluidity and flunarizine elevated it at each high concentration. There was no change in membrane fluidity by other calcium antagonists, nimodipine and nifedipine.