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.     

alpha-Methyl-D-glucoside uptake in renal cortical slices of normal and alloxan diabetic rabbits.

The uptake of alpha-methyl-D-glucoside by slices of renal cortex was compared in normal and alloxan diabetic rabbits. Alloxanized rabbit tissue showed significantly higher levels of sugar accumulation than normal tissue. Diamide, which is known to oxidize intracellular glutathione (GSH), inhibited the uptake of alpha-methyl-D-glucoside by renal cortical slices. GSH stimulated sugar uptake and was also capable of reversing the inhibition of sugar accumulation caused by diamide. Mercaptoethanol and dithiothreitol, which are commonly used thiol compounds, were not as effective as GSH in stimulating sugar uptake. The level of GSH found in normal and alloxan diabetics rabbit kidneys shows a slightly decreased cortical GSH content in alloxanized animals. One can conclude that GSH participates in sugar uptake in kidney slices from both diabetic and normal rabbits.     

The influence of glutathione oxidation on renal cortex taurine transport

The interaction of diamide, a rapidly reversible thioloxidizing re reagent, with the taurine accumulation system was examined in rat kidney cortex slices from animals of different ages. Diamide at 10 mM lowered renal cortex glutathione content by 80% at a time that taurine accumulation was inhibited by 65%. Although the addition of equimolar GSH overcame diamide inhibition of taurine uptake, GSH per se inhibited taurine accumulation at 0.01 mM, but not at 0.2 or 1.0 mM. Dithiothreitol (DTT) also overcame diamide inhibition of uptake. As previously shown by Pillion et al (Eur. J. Biochem. $\text{79}$, 73, 1977) diamide inhibited gluconeogenesis by cortex slices.Diamide inhibited taurine accumulation by 85% by the low Km taurine transport site in cortex from newborn, 2 week, 4 week and adult animals, but only 50% at the high Km site. In contrast to the situation in adult tissue, efflux of taurine from preloaded slices of immature animals was not increased by diamide. Accordingly, one maturational event identified by these studies is that diamide-enhanced efflux was found only in mature cortex.     

Inhibition of protein kinase activity and amino acid and α-methyl-d-glucoside transport by diamide

Dizene dicarboxylic acid $\text{bis}\text{-(N,N-}\text{dimethylamide}\text{)}$, commonly called diamide, is known to oxidize stoichiometrically intracellular pools of reduced glutathione and inhibit the accumulation of sugars and amino acids by rat kidney slices. Incubation of rat renal cortical slices in diamide also leads to a significant decrease in the level of endogenous protein kinase activity. The inhibition of sugar and amino acid transport and protein kinase activity by diamide is partially reversible by the addition of exogenous glutathione or other thiols. A comparison of protein kinase activity with amino acid and sugar transport at various concentrations of diamide indicates that there is a high degree of correlation between these two processes.     

Inhibition of protein kinase activity and amino acid and alpha-methyl-D-glucoside transport by diamide.

Dizene dicarboxylic acid bis-(N,N-dimethylamide), commonly called diamide, is known to oxidize stoichiometrically intracellular pools of reduced glutathione and inhibit the accumulation of sugars and amino acids by rat kidney slices. Incubation of rat cortical slices in diamide also leads to a significant decrease in the level of endogenous protein kinase activity. The inhibition of sugar and amino acid transport and protein kinase activity by diamide is partially reversible by the addition of exogenous glutathione or other thiols. A comparison of protein kinase activity with amino acid and sugar transport at various concentrations of diamide indicates that there is a high degree of correlation between these two processes.     

The role of glutathione in renal cortical tissue. effects of diamide on Na+ and GSSG levels,amino acid transport and Na+-K+-ATPase activity

Summary The effects of diamide were studied in rat kidney cortical tissue. It was found that diamide increased oxidized glutathione levels and inhibited Na⁺-K⁺-ATPase activity. Consistent with this finding was the observation that diamide compromised the sodium gradients maintained in renal cortical slices. Amino acid transport studies with ouabain or a sodium-free buffer indicated that diamide interferes with both Na⁺-dependent and Na⁺-independent transport systems. These results indicate that diamide has a number of different effects on renal cortical tissue and emphasize the important role of glutathione in maintaining control of a number of key metabolic pathways.     

The effect of diamide and glutathione on the uptake of α-methyl-d-glucoside by slices of rat kidney cortex

The uptake of α-methyl-d-glucoside was stimulated in slices of rat kidney cortex by pretreatment with reduced glutathione. Diamide, an oxidizing agent with high specificity for GSH, caused an inhibition of α-methyl-d-glucoside uptake. These effects appeared to be related specifically to GSH, since dithiothreitol and mercaptoethanol did not increase α-methyl-d-glucoside uptake, and were not as effective as GSH in reversing the effects of diamide. GSH and diamide had no effect on the uptake of another sugar analog, $\text{3-O-}\text{methylglucose}$, which is not actively transported. Kinetic studies indicated that GSH increased the apparent $\text{V}$ without affecting $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$. The results are discussed in terms of the possible role of GSH in the process of sugar transport.     

Inhibition of protein secretion and protein kinase activity in the locust fat body by diamide (Azodicarboxylic acid-bis-dimethylamide)

Protein secretion from locust fat body incubated in vitro is instantaneously halted in the presence of diamide (azodicarboxylic acid-bis-dimethylamide). Independent inhibition of amino acid uptake, protein synthesis (verified in cell-free systems), and protein secretion is demonstrated. Diamide inhibits cyclic AMP- and cyclic GMP-dependent protein kinase activity in locust fat body.     

Effect of piperonyl butoxide on organic anion and cation transport in rabbit kidneys

Piperonyl butoxide has been shown to reduce accumulation of cephaloridine in rabbit renal cortex; however, the mechanism responsible for this effect remains unclear. Cephaloridine is a zwitterion and its accumulation in renal cortex has been suggested to be regulated by both organic anion and cation transport systems. Thus, it was of interest to determine the effect of piperonyl butoxide on renal transport of p-aminohippurate (PAH, an organic anion) and tetraethylammonium (TEA, an organic cation). Although pretreatment with piperonyl butoxide markedly inhibited renal cortical uptake of cephaloridine, the same treatment had less inhibitory effect on either PAH or TEA uptake. Efflux of PAH from preloaded renal cortical slices was enhanced by pretreatment with piperonyl butoxide; however, TEA efflux was unaffected. Thus, piperonyl butoxide appears to have effects on renal membrane functions which result in differential effects on PAH, TEA, and cephaloridine transport.     

Prostaglandins and renin release: I. Stimulation of renin release from rabbit renal cortical slices by PGI2.

Prostaglandins have been shown to be involved in the mechanism of renin secretion in a variety of situations. Both arachidonic acid and prostaglandin endoperoxide have been shown to release renin from cortical slices and to be converted to PGI2 by cortical microsomes. In the present studies PGI2 was found to cause a time dependent increase in renin release from rabbit renal cortical slices, a system isolated from any indirect effects that result from the administration of prostaglandins in vivo. The stimulation was linear up to 30 minutes and effective over a range of concentrations from 10(7 M to 10(-5) M. At similar concentrations 6-keto-prostaglandin F1alpha was not active on these slices. Thus, it is proposed that PGI2 exerts a direct effect on the release of renin from cortical cells and may be the mediator of arachidonate or prostaglandin endoperoxide stimulated renin secretion.     

HYPEROSMOLALITY-INDUCED GABA RELEASE FROM RAT BRAIN SLICES: STUDIES OF CALCIUM DEPENDENCY AND SOURCES OF RELEASE

Abstract— The effects of hyperosmolal superfusion upon the release of preloaded, radio-labeled GABA has been studied, using both first cortical and first pontine brain slices. GABA release was stimulated with either hyperosmolal Na⁺ or sucrose superfusion in cortical slices. This stimulated release of radio-labeled GABA was partially Ca²⁺-dependent in cortical slices. When barium ions replaced Ca²⁺ in hyperosmolal medium, a similar effect was seen. High concentration of magnesium in Ca²⁺ -free hyperosmolal medium did not induce stimulation. The increased release of α-aminoisobutyric acid (AIBA), a non-metabolized amino acid induced by hyperosmolality, was not Ca²⁺-dependent.
GABA release was also stimulated with hyperosmolal sucrose superfusion in pontine slices. The effect of pre-treatment of cortical and pontine slices with β-alanine or L-2,4-diaminobutyric acid (DABA) was used to study the source of exogenous GABA release induced by hyperosmolality. In cortical slices, β-alanine blocked the hyperosmolal release of GABA and also slightly inhibited GABA uptake. DABA did not change hyperosmolal GABA release, although it inhibited GABA uptake. In pontine slices, both DABA and β-alanine inhibited GABA uptake, but were unable to inhibit the hyperosmolal release of GABA.
The data suggest that hyperosmolality causes increased release of GABA from neurons, analogous to that seen with K⁺-depolarization. AIBA, unlike GABA, is released from brain cells as a non-Ca²⁺ -dependent response to osmotic equilibration. The observation that pre-treatment with β-alanine inhibits the hyperosmolal release of GABA suggests that hyperosmolality alters glial cell function.     

Effect of L-leucine on p-aminohippurate accumulation by mammalian renal cortical slices

The effect of several amino acids (L-glutamate, L-phenylalanine, L-leucine, glycine, L-tryptophan, L-histidine, L-valine) on p-aminohippurate accumulation was evaluated in rat, mouse and rabbit kidneys. Only leucine was found to enhance p-aminohippurate accumulation in rat and mouse renal cortical slices but not in rabbit slices. Leucine had no effect on tetraethylammonium accumulation. In rat renal cortical slices, leucine increased the uptake and decreased the runout of p-aminohippurate, each effect contributing to the increase in p-aminohippurate accumulation. The apparent K_m of p-aminohippurate uptake was decreased by leucine with no change in the apparent V. Aminooxyacetate (an inhibitor of transamination of leucine) partially depressed the stimulating effect of leucine on p-aminohippurate accumulation, whereas α-ketoisocaproic acid (a metabolite of leucine formed by transamination) enhanced p-aminohippurate accumulation, suggesting that the metabolism of leucine in kidney slices may be necessary for the stimulating effect on p-aminohippurate transport.     

Thiol-dependent K:Cl transport in sheep red cells: VIII. Activation through metabolically and chemically reversible oxidation by diamide

The sulfhydryl (SH) oxidant diamide activated in a concentration-dependent manner ouabain-resistant (OR), Cl-dependent K flux in both low potassium (LK) and high potassium (HK) sheep red cells as determined from the rate of zero-trans K efflux into media with Cl or Cl replaced by NO3 or methane sulfonate (CH3SO3). Diamide did not alter the OR Na efflux into choline Cl. The diamide effect on K efflux appeared after 80% of cellular glutathione (GSH) was oxidized to GSSG, its disulfide. The stimulation of K efflux was completely reversed during metabolic restitution of GSH, a process that depended on the length of exposure to and the concentration of diamide. The action of diamide on both the K:Cl transporter and GSH was also fully reversed by the reducing agent dithiothreitol (DTT). Diamide apparently oxidized the same SH groups alkylated by N-ethylmaleimide (NEM) (Lauf, P.K. 1983. J. Membrane Biol. 73:237-246). Like NEM, diamide activated K:Cl transport several-fold more in LK cells than in HK cells, and the effect on LK cells was partially inhibited by anti-L1, the allo-antibody known to inhibit OR K fluxes.     

Renal tubular transport of delta-aminolevulinic acid in rat

delta-Aminolevulinic acid (ALA) interferes with cell membrane and metabolic functions in a variety of tissues. To determine if ALA interacts with renal tubular transport functions, we examined concentrative transport of this heme precursor in rat kidneys. ALA was accumulated against a concentration gradient in rat renal cortical slices. Section freeze-dry autoradiography demonstrated selective accumulation in cells of proximal tubules. Concentrative uptake of ALA was inhibited by KCN, probenecid and p-aminohippurate (PAH). ALA inhibited slice uptake of PAH but failed to block slice accumulation of galactose, cycloleucine, lysine, glycine, proline, or alpha-aminoisobutyric acid and did not alter O2 utilization. Massive intraperitoneal injection of ALA did not increase 24 hr fractional excretion of amino acids in vivo. Concentrative transport of ALA in proximal tubules does not lead to generalized renal tubular transport defects but ALA appears to share the organic acid secretory system in rat kidney.     

Stimulation of septation in mitochondria by diamide,a thiol oxidising agent

Summary Diamide (10^–4M), a thiol oxidizing agent, rapidly promotes septation in isolated frog liver mitochondria and also in situ in liver slices. The effect is partially inhibited by dithioerythritol. DNP does not have this effect, and it is concluded that diamide does not promote septation via an uncoupling action. The septate mitochondria have a different appearance from typical dividing mitochondria previously described; it is suggested that (1) diamide may act by favouring the fusion of the internal membranes, and (2) -SH oxidation is of importance in mitochondria in ageing and in various pathological conditions. The ways in which septa may develop in mitochondria in the orthodox and condensed configuration are discussed.     

Differential in vitro and in vivo behavior of mouse ascorbate/Fe3+ and diamide oxidized erythrocytes

Chemical oxidation of mouse erythrocytes has been carried out using two different oxidizing systems namely: Diamide and Ascorbate/Fe³⁺ together with different concentrations of the oxidant. These oxidation treatments produced different extents of modification in membrane proteins as was observed by electrophoretic analyses that showed a possible formation of high molecular weight aggregates. Lipid peroxidation was also observed as the result of these chemical treatments. The action of these two oxidation treatments produced different extents of lipid peroxidation in which the effect Ascorbate/Fe³⁺ reached higher values than that shown by diamide treatments. To study the resulting in vitro behavior of such oxidized erythrocytes, we have evaluated the recognition of oxidized erythrocytes by peritoneal macrophages. In the conditions used, diamide oxidized erythrocytes were more highly recognized by macrophages than Ascorbate/Fe³⁺ treated erythrocytes. However, in both cases an influence of serum factors in the recognition process can be inferred. Additionally, we have correlated on one side the action of different oxidation systems on mouse erythrocytes with different in vivo behavior and organ uptake of the oxidized erythrocytes. On the other side, differential targeting of oxidized erythrocytes to a liver or spleen was observed on dependence of the oxidant used.     

THE DISTRIBUTION OF AMINO ACIDS, Na+ AND K+ FROM SURFACE TO CENTRE IN INCUBATED SLICES OF MOUSE BRAIN

—The effect of tissue damage on the uptake of amino acids by brain slices was investigated by measuring uptake in slices of different thickness and measuring the distribution of [¹⁴C]-labelled amino acid on the surface and in the centre of incubated slices. The uptake of glutamate, aspartate, and GABA was greater in 0.1 mm-thick slices than in 0.42 mm-thick slices in short and in long (up to 120 min) incubations; the uptake of other amino acids was equal or greater in the 0.42 mm-thick slices. The water content of incubated slices did not change greatly from surface to centre; inulin space was greater at the surface, and in slices from cortex, especially higher at the cut surface. Na⁺ and K⁺ concentrations were also higher at the surface. In the rest of the slice space, inulin, Na⁺ and K⁺ distribution was quite uniform. The distribution of ATP was inhomogeneous: in thinner slices the centre concentration was higher; in thicker slices the centre concentration was lower. Amino acid uptake initially (at 5 min) was higher at the surface, especially in the thicker slices; after longer time (30 min) incubation, the distribution of lysine and leucine was uniform, and glutamate uptake was greater at the surface. The inhomogeneity of distribution increased with increasing thickness of the slices. We concluded that the uptake of some amino acids (perhaps those for which, beside a low affinity transport, also a higher affinity transport system exists) is greater in thinner slices and greater on the surface of slices, and there is an initially inhomogeneous distribution during amino acid uptake. The uptake on the surface constitutes only a small portion of the total uptake, and tissue damage does not explain the greater uptake of amino acids by slices in comparison to the brain in vivo. This shows the higher transport capacity of cells in the brain and emphasizes the importance of mechanisms controlling the metabolite composition of the extracellular fluid in finally influencing the metabolite composition of the brain itself.     

Uptake of mercury and mercury-amino acid complexes by rat renal cortex slices.

This study was undertaken in order to assess the effects of metabolism and complexations with amino acids on the renal uptake of mercury using rat renal cortex slices as the experimental system. Mercury levels attained in the slices after 60 min of incubation were 50% higher with mercuric cysteine than with mercuric chloride. This enhancement of uptake with mercuric cysteine was reduced in the presence of a tenfold molar excess of histidine or lysine, but not by serine. Excess cysteine markedly increased mercury uptake. Incubation at 25 degrees significantly reduced uptake of mercuric cysteine, but not mercuric chloride. Anaerobic conditions and incubation in the presence of DNP each reduced mercuric cysteine uptake to the control level of mercuric chloride without affecting uptake of mercuric chloride. The differential aspects of metabolism on the uptake of mercuric cysteine and mercuric chloride and the competitive effects obtained with amino acids known to compete with cysteine in renal reabsorption support the hypothesis that a portion of the renal uptake of mercury operates through amino acid transport mechanisms acting on mercury-amino acid complexes.     

Thiol modification in H2O2- and thromboxane-induced vaso- and bronchoconstriction in rat perfused lung.

Hydrogen peroxide (H2O2), arachidonic acid (AA), and U-44069, a thromboxane analogue, all induced vaso- and bronchoconstriction in the isolated perfused rat lung. The role of protein sulfhydryl modifications in these processes was investigated. The thiol oxidizing agent diamide inhibited both vaso- and bronchoconstriction induced by H2O2, AA, or U-44069. Diamide had only a marginal effect on glutathione and protein thiol levels and no effect on lung mechanics. The diamide inhibition was reversible, and H2O2-induced vaso- and bronchoconstriction was almost maximal after 10 min of perfusion with buffer. The recovery was more rapid if dithiothreitol, a thiol reducing agent, was used in the buffer. H2O2- and AA-induced vaso- and bronchoconstriction is caused by thromboxane release. Diamide did not influence H2O2- or AA-dependent thromboxane formation, indicating that neither AA release nor AA metabolism to thromboxane is sensitive to thiol oxidation. Thus our results indicate that the site of diamide-induced thiol oxidation is the thromboxane receptor or its signal transduction.