L-arginine uptake and L-phosphoarginine synthesis in Trypanosoma cruzi

A very specific L-arginine transporter showing high affinity has been characterized in Trypanosoma cruzi epimastigotes. Uptake was found to be dependent on L-arginine concentration and it was saturable. Values for maximum velocity and Km ranged between 48.1-57.5 pmol.min-1 per 3 x 10(7) cells and between 4.2-5.5 microM, respectively. The calculated activation energy and Q10 were 31.1 KJ.mol-1, and 1.7, respectively. Uptake velocity significantly increased when cells were preincubated in the absence of L-arginine. Cells retained the labeled amino acid independently of the presence or absence of exogenous L-arginine. The specificity of L-arginine uptake was demonstrated by competition assays in the presence of 80-fold molar excess of natural amino acids and several L-arginine derivatives. The highest levels of inhibition were caused by L-homoarginine, D-arginine, L-canavanine, L-ornithine, and L-citrulline. L-arginine uptake by T. cruzi epimastigotes was not affected by the presence of potassium or sodium ions in the incubation mixture or by pH changes in the range between 5.5-8.5. The major product of L-arginine uptake was characterized as phosphoarginine. Moreover, arginine kinase activity was detected in soluble extracts from T. cruzi epimastigotes.     

Amino acid repletion does not decrease muscle protein catabolism during hemodialysis

Intradialytic protein catabolism is attributed to loss of amino acids in the dialysate. We investigated the effect of amino acid infusion during hemodialysis (HD) on muscle protein turnover and amino acid transport kinetics by using stable isotopes of phenylalanine, leucine, and lysine in eight patients with end-stage renal disease (ESRD). Subjects were studied at baseline (pre-HD), 2 h of HD without amino acid infusion (HD-O), and 2 h of HD with amino acid infusion (HD+AA). Amino acid depletion during HD-O augmented the outward transport of amino acids from muscle into the vein. Increased delivery of amino acids to the leg during HD+AA facilitated the transport of amino acids from the artery into the intracellular compartment. Increase in muscle protein breakdown was more than the increase in synthesis during HD-O (46.7 vs. 22.3%, P < 0.001). Net balance (nmol.min(-1).100 ml (-1)) was more negative during HD-O compared with pre-HD (-33.7 +/- 1.5 vs. -6.0 +/- 2.3, P < 0.001). Despite an abundant supply of amino acids, the net balance (-16.9 +/- 1.8) did not switch from net release to net uptake. HD+AA induced a proportional increase in muscle protein synthesis and catabolism. Branched chain amino acid catabolism increased significantly from baseline during HD-O and did not decrease during HD+AA. Protein synthesis efficiency, the fraction of amino acid in the intracellular pool that is utilized for muscle protein synthesis decreased from 42.1% pre-HD to 33.7 and 32.6% during HD-O and HD+AA, respectively (P < 0.01). Thus amino acid repletion during HD increased muscle protein synthesis but did not decrease muscle protein breakdown.     

Modulation of the L-arginine/nitric oxide signalling pathway in vascular endothelial cells

Nitric oxide (NO) is synthesized from L-arginine, and in endothelial cells influx of L-arginine is mediated predominantly via Na+-independent cationic amino acid transporters. Constitutive, Ca2+-calmodulin-sensitive eNOS (endothelial nitric oxide synthase) metabolizes L-arginine to NO and L-citrulline. eNOS is present in membrane caveolae and the cytosol and requires tetrahydrobiopterin, NADPH, FAD and FMN as additional cofactors for its activity. Supply of L-arginine for NO synthesis appears to be derived from a membrane-associated compartment distinct from the bulk intracellular amino acid pool, e.g. near invaginations of the plasma membrane referred to as 'lipid rafts' or caveolae. Co-localization of eNOS and the cationic amino acid transport system y+ in caveolae in part explains the 'arginine paradox', related to the phenomenon that in certain disease states eNOS requires an extracellular supply of L-arginine despite having sufficient intracellular L-arginine concentrations. Vasoactive agonists normally elevate [Ca2+]i (intracellular calcium concentration) in endothelial cells, thus stimulating NO production, whereas fluid shear stress, 17beta-oestradiol and insulin cause phosphorylation of the serine/threonine protein kinase Akt/protein kinase B in a phosphoinositide 3-kinase-dependent manner and activation of eNOS at basal [Ca2+]i levels. Adenosine causes an acute activation of p42/p44 mitogen-activated protein kinase and NO release, with membrane hyperpolarization leading to increased system y+ activity in fetal endothelial cells. In addition to acute stimulatory actions of D-glucose and insulin on L-arginine transport and NO synthesis, gestational diabetes, intrauterine growth retardation and pre-eclampsia induce phenotypic changes in the fetal vasculature, resulting in alterations in the L-arginine/NO signalling pathway and regulation of [Ca2+]i. These alterations may have significant implications for long-term programming of the fetal cardiovascular system.     

Taste responses to amino acids in the southern leopard frog, Rana sphenocephala

Integrated taste recordings of the glossopharyngeal (IX) nerve innervating the tongue of the southern leopard frog were studied in response to various amino acids and quinine hydrochloride. Amino acids and quinine hydrochloride elicited primarily phasic taste responses. Acidic (L-aspartic and L-glutamic) and basic (L-lysine and L-arginine) amino acids, adjusted to pH8, were effective taste stimuli. All glossopharyngeal nerve twigs that responded to amino acid stimuli also responded to quinine; however, not all quinine-sensitive IX nerve bundles were responsive to amino acids. Electrophysiological thresholds for amino acids were estimated to be 2.5-10 mM, whereas threshold for quinine hydrochloride averaged approximately 10 microM.     

Deprivation of L-Arginine Induces Oxidative Stress Mediated Apoptosis in Leishmania donovani Promastigotes: Contribution of the Polyamine Pathway

The growth and survival of intracellular parasites depends on the availability of extracellular nutrients. Deprivation of nutrients viz glucose or amino acid alters redox balance in mammalian cells as well as some lower organisms. To further understand the relationship, the mechanistic role of L-arginine in regulation of redox mediated survival of Leishmania donovani promastigotes was investigated. L-arginine deprivation from the culture medium was found to inhibit cell growth, reduce proliferation and increase L-arginine uptake. Relative expression of enzymes, involved in L-arginine metabolism, which leads to polyamine and trypanothione biosynthesis, were downregulated causing decreased production of polyamines in L-arginine deprived parasites and cell death. The resultant increase in reactive oxygen species (ROS), due to L-arginine deprivation, correlated with increased NADP+/NADPH ratio, decreased superoxide dismutase (SOD) level, increased lipid peroxidation and reduced thiol content. A deficiency of L-arginine triggered phosphatidyl serine externalization, a change in mitochondrial membrane potential, release of intracellular calcium and cytochrome-c. This finally led to DNA damage in Leishmania promastigotes. In summary, the growth and survival of Leishmania depends on the availability of extracellular L-arginine. In its absence the parasite undergoes ROS mediated, caspase-independent apoptosis-like cell death. Therefore, L-arginine metabolism pathway could be a probable target for controlling the growth of Leishmania parasites and disease pathogenesis.     

Regulation of amino acid transport in L6 muscle cells: I. Stimulation of transport system a by amino acid deprivation

The regulation of amino acid transport in L6 muscle cells by amino acid deprivation was investigated. Proline uptake was Na⁺-dependent, saturable and concentrative, and was predominantly through system A. Proline uptake was inhibited by alanine, α-amino isobutyric acid (AIB), and by α-methylamino isobutyric acid, but not by lysine or valine. At 25°C, Km of proline uptake was 0.5 mM. Amino acid-deprivation resulted in a progressive increase in the rate of proline uptake, reaching up to 6-fold stimulation after 6 hours. The basal and stimulated transport were equally Na⁺-dependent, and both were inhibited by competition with the same amino acids. Kinetic analysis showed that Km decreased by a factor of 2.4 and Vmax increased 1.9-fold in deprived cells. Amino acid-deprivation did not stimulate amino acid uptake through systems other than system A. This suggests that the higher Km in proline-supplemented cells is not due to release of intracellular amino acids into unstirred layers surrounding the cells. The presence of amino acids which are substrates of system A (including AIB) during proline-deprivation, prevented stimulation of proline uptake, whereas those transported by systems Ly⁺ or L exclusively were ineffective. The stimulation of the transport-rate in deprived cells could be reversed by subsequent exposure to proline or other substrates of system A. L6 cells, deprived of proline for 6 hours, retained the stimulation of transport after detachment from the monolayers with trypsin. Uptake rates were comparable in suspended and attached cells in monolayer culture. Thus, amino acid-depreivation of L6 cells results in an adaptive increase in proline uptake, which is not due to unstirred layers but appears to be mediated by other mechanisms of selective transport regulation.     

Tuning adenosine A1 and A2A receptors activation mediates L-citrulline-induced inhibition of [3H]-acetylcholine release depending on nerve stimulation pattern

The influence of nerve stimulation pattern on transmitter release inhibition by L-citrulline, the co-product of NO biosynthesis by nitric oxide synthase (NOS), was studied in the rat phrenic nerve-hemidiaphragm. We also investigated the putative interactions between NOS pathway and the adenosine system. L-citrulline (10-470 microM), the NOS substrate L-arginine (10-470 microM) and the NO donor 3-morpholinylsydnoneimine (SIN-1, 1-10 microM), concentration-dependently inhibited [(3)H]-acetylcholine ([(3)H]-ACh) release from rat motor nerve endings. Increasing stimulus frequency from 5 Hz-trains to 50 Hz-bursts enhanced [(3)H]-ACh release inhibition by l-arginine (47 microM) and L-citrulline (470 microM), whereas the effect of SIN-1 (10 microM) remained unchanged. NOS inhibition with N(omega)-nitro-L-arginine (100 microM) prevented the effect of L-arginine, but not that of L-citrulline. Adenosine deaminase (2.5 U/ml) and the adenosine transport inhibitor, S-(p-nitrobenzyl)-6-thioinosine (10 microM), attenuated release inhibition by L-arginine and L-citrulline. With 5 Hz-trains, blockade of A(1) receptors with 1,3-dipropyl-8-cyclopentyl xanthine (2.5 nM), but not of A(2A) receptors with ZM241385 (10nM), reduced the inhibitory action of l-arginine and L-citrulline; the opposite was verified with 50 Hz-bursts. Blockade of muscarinic M(2) autoreceptors with AF-DX116 (10 nM) also attenuated the effects of L-arginine and L-citrulline with 50 Hz-bursts. L-citrulline (470 microM) increased basal adenosine outflow via the equilibrative nucleoside transport system sensitive to NBTI (10 microM), without significantly (P>0.05) changing the nucleoside release subsequent to nerve stimulation. Data indicate that NOS-derived L-citrulline negatively modulates [(3)H]-ACh release by increasing adenosine outflow channelling to A(1) and A(2A) receptors activation depending on the stimulus paradigm. While adenosine acts predominantly at inhibitory A(1) receptors during 5 Hz-trains, inhibition of ACh release by L-citrulline at 50 Hz-bursts depends on the interplay between adenosine A(2A) and muscarinic M(2) receptors.     

THE EFFECT OF SLEEP DEPRIVATION UPON THE IN VIVO AND IN VITRO INCORPORATION OF TRITIATED AMINO ACIDS INTO BRAIN PROTEINS IN THE RAT AT THREE DIFFERENT AGE LEVELS

Abstract— The effect of sleep deprivation on the in vivo and in vitro tritiated amino acid incorporation into brain proteins was studied in the rat at three age levels. Sleep deprivation was induced either by water tank or handling methods. Three experimental groups of animals were used: control, sleep deprived and post deprivation sleeping rats.
A significant decrease of protein synthesis was found in the cerebellum, telencephalon and in crude subcellular fractions of brainstem of adult rats selectively deprived of paradoxical sleep. However, no alteration of protein synthesis was observed either in vivo or in vitro , in the same brain regions or in the liver after the rebound of paradoxical sleep following deprivation.
In four crude subcellular protein fractions a specific increase of the in vitro labelled amino acid incorporation was observed in the brain stem of 24-day-old rats allowed to recuperate after sleep deprivation as compared with the deprived rats. No significant changes were seen in the telencephalon.
No alteration of incorporation was found in 7-day-old rats deprived of sleep.
The possible functional significance of these results is discussed in relation to stress and to variations in the size of the precursor pool for protein synthesis.     

Free amino acids in claw muscle and haemolymph from Australian freshwater crayfish at different stages of the moult cycle

Amino acids were measured in claw muscle and haemolymph in the freshwater decapod crustacean, Cherax destructor, at different stages of the moult cycle. The total pool of amino acids in muscles from animals in intermoult (97+/-13 mmol kg(-1) muscle), premoult (80+/-20 mmol kg(-1)) and postmoult (97+/-19 mmol kg(-1)) were not significantly different. Despite the relatively stable total pool of amino acids, there were changes in the concentrations of alanine, glutamine and proline over the moult cycle. Compared to intermoult, claw muscles from animals in premoult had a lower concentration of proline, and animals in postmoult had higher concentrations of alanine and glutamine, but lower concentrations of proline. Concentrations of alanine and glutamine in claw muscle of animals in postmoult were higher and proline concentrations lower than in the same animals during the premoult stage. The concentration of proline in haemolymph was lower in animals in premoult and postmoult compared to intermoult. The total amino acid pool in the claw muscle of Cherax destructor did not change significantly over the moult which is distinctly different to the changes in amino acids reported in the claw muscles of marine decapod crustaceans.     

Protein synthesis in central nervous tissue: studies on retina in vitro

Rabbit retinas were maintained in vitro in medium that resembled CSF but with leucine varied from 2 to 1000 microM. Both leucine and threonine were isotopically labelled. When leucine in the medium was 100-1000 microM, leucine was incorporated into protein at 2.03 +/- 0.04 (S.E.M.) mumol/g dry wt./h, a turnover per h of 0.55% of the leucine in retinal protein. Incorporation was constant for at least 7 h. It was reduced 34% when the other amino acids were omitted from the medium and 24% when they were increased 15 fold above physiological levels. When medium leucine was reduced to 2 microM with other amino acids constant, 14C-leucine incorporation fell 70% without significant change in 3H-threonine incorporation, indicating a fall in intracellular specific activity of leucine. The intracellular/extracellular concentration ratio of labelled leucine was 4:1 with medium leucine 23 microM. It fell markedly when medium leucine was reduced to 2 microM or increased to 1000 microM. The concentration ratio of labelled threonine was 15:1 with medium leucine at physiological levels but fell to 6:1 when medium leucine was increased to 1000 microM. Decarboxylation removed 1.5% of free intracellular leucine per min and, at physiological concentrations, was 7.7% the rate of protein incorporation. The ratio of protein synthesis/breakdown, estimated from changes in leucine and 7 other essential amino acids in the medium, was nearly unity. The potential of this preparation for study of CNS protein metabolism is discussed.     

Functional characterization of Glu298Asp mutant human endothelial nitric oxide synthase purified from a yeast expression system.

The Glu298Asp polymorphism of human endothelial nitric oxide synthase (eNOS) has been reported to be associated with several cardiovascular diseases, including hypertension and myocardial infarction. Therefore, we investigated the effect of the Glu298Asp (E298D) mutation on the function of purified recombinant eNOS expressed in the yeast Pichia pastoris. Wild type (WT) and mutant exhibited comparable affinities for L-arginine (K(m) values 4.4+/-0.6 and 5.2+/-0.8 microM, respectively) and V(max) values (142+/-36 and 159+/-29 nmol of L-citrulline/mg min, respectively). The E298D mutation affected neither electron transfer through the reductase domain (measured as cytochrome c reduction) nor reductive O(2) activation (measured either as NADPH oxidation or as H(2)O(2) formation in the absence of L-arginine and tetrahydrobiopterin (BH4)). The mutant was activated by BH4 with an EC(50) of 0.24+/-0.04 microM, a value comparable to that obtained with WT eNOS (0.22+/-0.02 microM). Activation of the enzyme by Ca(2+) was not affected (EC(50)=0.50+/-0.04 and 0.49+/-0.02 microM for WT and E298D eNOS, respectively). Calmodulin (CaM) affinity, studied by radioligand binding using 125I-labeled CaM, revealed virtually identical K(D) (3.2+/-0.5 and 4.0+/-0.3nM) and B(max) (1.4+/-0.2 and 1.2+/-0.3 pmol/pmol subunit) values for WT and E298D eNOS, respectively. Furthermore, E298D eNOS did not differ from the WT enzyme with respect to heme and flavin content or the ability to form SDS-resistant dimers. To summarize, we obtained no evidence for altered enzyme function of the eNOS mutant that could explain endothelial dysfunction associated with the E298D polymorphism.     

N omega-hydroxy-L-arginine is an intermediate in the biosynthesis of nitric oxide from L-arginine.

Authentic N omega-hydroxy-L-arginine was synthesized and used to determine whether it is an intermediate in nitric oxide (.NO) synthesis from L-arginine by macrophage .NO synthase. The apparent Km (6.6 microM) and Vmax (99 nmol x min-1 x mg-1) observed with N omega-hydroxy-L-arginine were similar to those observed with L-arginine (Km = 2.3 microM; Vmax = 54 mumol x min-1 x mg-1). N omega-Hydroxy-D-arginine was not a substrate. Stable isotope studies showed that .NO synthase exclusively oxidized the hydroxylated nitrogen of N omega-hydroxy-L-arginine, forming .NO and L-citrulline. As with L-arginine, O2 was the source of the ureido oxygen in L-citrulline from N omega-hydroxy-L-arginine. In the presence of excess N omega-hydroxy-L-arginine, .NO synthase generated a metabolite of L-[14C]arginine that cochromatographed with authentic N omega-hydroxy-L-arginine. The labeled metabolite exhibited identical chromatographic behavior in three solvent systems and generated the same product (L-citrulline) upon alkaline hydrolysis as authentic N omega-hydroxy-L-arginine. Experiments were then run to identify which redox cofactor (NADPH or tetrahydrobiopterin) participated in the enzymatic synthesis of N omega-hydroxy-L-arginine. Both cofactors were required for synthesis of .NO from either N omega-hydroxy-L-arginine or L-arginine. However, with L-arginine, the synthesis of 1 mol of .NO was coupled to the oxidation of 1.52 +/- 0.02 mol of NADPH; whereas with N omega-hydroxy-L-arginine, only 0.53 +/- 0.04 mol of NADPH was oxidized per mol of .NO formed. These results support a mechanism in which N omega-hydroxy-L-arginine is generated as an intermediate in .NO synthesis through an NADPH-dependent hydroxylation of L-arginine.     

Regulation of amino acid transport in L6 myoblasts. II. Different chemical properties of transport after amino acid deprivation

The mechanism of stimulation of amino acid transport system A caused by amino acid deprivation in L6 cells was investigated. In cells loaded with alpha-aminoisobutyric acid (AIB), amino acid deprivation increased the rate of proline uptake only after the intracellular [AIB] dropped below 7 mM. Efflux of proline was not sensitive to the presence of proline in the outer medium (with or without external Na+), suggesting that efflux through system A (and possibly uptake) is not susceptible to transinhibition. Transport (stimulated uptake) into amino acid-deprived cells and that into amino acid-supplemented cells differed in several chemical properties: 1) In the former group, transport was higher at lower pH values than in the latter, and the optimum pH values were 7.5 and 7.8, respectively. 2) Unlike proline uptake in supplemented cells, uptake in deprived cells was inhibited by 50% with N-ethylmaleimide (1 mM) or by 50 microM p-chloromercuribenzoate (PCMBS). Inhibition by PCMBS was not due to collapse of the Na+ gradient. The mercurial inhibited only the deprivation-induced stimulation of transport, bringing the rate of proline uptake to the "basal" uptake level observed in amino acid-supplemented cells. Proline uptake was not stimulated by a second deprivation following treatment with PCMBS and a supplementation-deprivation cycle. However, in untreated cells, or by reversing mercaptide formation with dithiotreitol, the second deprivation stimulated transport. Deprivation at 4 degrees C did not elicit stimulation of proline uptake. Cycloheximide prevented the stimulation and decreased the rate of proline uptake in deprived cells more efficiently than in supplemented cells. Actinomycin D prevented stimulation when added at the onset of deprivation. The above data indicate that stimulation of transport by deprivation is protein synthesis-dependent and that the stimulated transport had chemical properties distinct from the "basal" transport in supplemented cells. The evidence presented is consistent with a model of activation of a finite pool of transporters upon deprivation, the chemical characteristics of which differ from those of the "basal" transport system.     

Amino acid deprivation induces AKT activation by inducing GCN2/ATF4/REDD1 axis

Amino acid availability is sensed by various signaling molecules, including general control nonderepressible 2 (GCN2) and mechanistic target of rapamycin complex 1 (mTORC1). However, it is unclear how these sensors are associated with cancer cell survival under low amino acid availability. In the present study, we investigated AKT activation in non-small cell lung cancer (NSCLC) cells deprived of each one of 20 amino acids. Among the 20 amino acids, deprivation of glutamine, arginine, methionine, and lysine induced AKT activation. AKT activation was induced by GCN2/ATF4/REDD1 axis-mediated mTORC2 activation under amino acid deprivation. In CRISPR-Cas9-mediated REDD1-knockout cells, AKT activation was not induced by amino acid deprivation, indicating that REDD1 plays a major role in AKT activation under amino acid deprivation. Knockout of REDD1 sensitized cells cultured under glutamine deprivation conditions to radiotherapy. Taken together, GCN2/ATF4/REDD1 axis induced by amino acid deprivation promotes cell survival signal, which might be a potential target for cancer therapy.Subject terms: Cancer metabolism, Cell death     

Methods using stable isotopes to measure nitric oxide (NO) synthesis in the L-arginine/NO pathway in health and disease

Nitric oxide (NO) is an important gaseous radical involved in many physiological processes. It is produced from the amino acid L-arginine by the action of nitric oxide synthases (NOS) in what is called the L-arginine/NO pathway. Tracking its metabolic fate in biological fluids is of particular interest as it may indicate how the human body responds in health and disease. However, due to its short life span (a few seconds) it is very difficult to accurately monitor any up- or down-regulation in body fluids in vivo. As a consequence, methods have been developed based on the measurement of the NO-derived products nitrite and nitrate or on the substrate of NO, L-arginine and its simultaneously generated product, L-citrulline. Considering only a fraction of the endogenous L-arginine pool is used for the synthesis of NO, NO-production cannot be estimated by measuring changes in the concentrations of L-arginine and/or L-citrulline alone. Instead, to estimate NO-related changes in the L-arginine and/or L-citrulline pools a form of tagging these metabolites for the NOS-mediated reaction is required. The application of stable isotopes is an elegant way to track NOS-mediated changes. The present paper is focussed on the application of various combinations of chromatography and mass spectrometry to measure isotopic enrichments resulting from the conversion of L-arginine to NO and L-citrulline in a one-to-one stoichiometry. In addition, the various aspects and principles involved in the application of stable isotopes in metabolic studies in general and the study of the activity of NOS in particular are discussed.     

L-arginine uptake and release by cultured avian retinal cells: differential cellular localization in relation to nitric oxide synthase

The availability of L-arginine is of pivotal importance for the synthesis of nitric oxide, a signaling molecule in the CNS. Here we show the presence of a high-affinity L-arginine uptake system (Km of 4.4 +/- 0.5 microM and a Vmax of 26.0 +/- 0.9 fmol/well/min) in cultured chick retinal cells. Different compounds, such as N(G)-mono-methyl-L-arginine and L-lysine, were able to inhibit the uptake that was also inhibited 60-70% in the absence of sodium and/or calcium ions. No trans stimulation was observed when cells were preloaded with L-lysine. The data indicate that the L-arginine uptake in cultured retinal cells is partially mediated by the y+ system, but has a great contribution of the B(0,+) system. Autoradiographic studies revealed that the uptake is predominant in glial cells and can also be detected in neurons, whereas immunocytochemistry of nitric oxide synthase and L-citrulline showed that the enzyme is present in neurons and photoreceptors, but not in glial cells. L-[3H]Arginine is released from purified glial cultures incubated with high concentrations of potassium in the extracellular medium. Moreover, the amino acid released from preloaded glial cells was taken up by purified neuronal cultures. These results indicate that L-arginine released from glial cells is taken up by neurons and used as substrate for the synthesis of nitric oxide.     

The effects of hypoosmotic infusion on the composition of renal tissue of the Australian brush-tailed possum Trichosurus vulpecula

Although the occurrence of organic osmolytes in the inner medulla of the marsupial kidney has been recently reported [Comp. Biochem. Physiol. (2002) 132B 635-644], changes in these substances, in response to water loading in vivo, has not been studied. Adult Trichosurus vulpecula, the Australian brush-tailed possum, were subjected to water deprivation for 48 h. Following anaesthesia and unilateral nephrectomy, the animals were perfused with hypo-osmotic saline (80 mmol l(-1); 1.5 ml min(-1)) for 60 min. This resulted in a rapid increase in urine volume and a corresponding fall in urine osmolality. At the end of the infusion the animals were killed and the second kidney removed. Analysis of the renal tissue revealed that water content of cortical, outer and inner medullary regions of the kidney increased slightly following infusion, while sodium, and chloride contents of all three regions fell. Potassium contents, on the other hand, were barely changed. Of the organic osmolytes determined, very significant decreases in the inner medulla, following infusion, were found for sorbitol (from 397+/-79 to 266+/-49 mmol kg(-1) protein), inositol (247+/-23 to 190+/-25 mmol kg(-1) protein), and betaine (464+/-70 to 356+/-21 mmol kg(-1) protein), while only inositol was significantly decreased in the outer medulla (197+/-22 to 150+/-16 mmol kg(-1) protein). Glycerophosphorylcholine levels were low throughout the kidney and were not significantly affected by the infusion. It was concluded that inositol and sorbitol play a significant role as compatible organic osmolytes in the possum kidney, while betaine functions as the principal counteracting osmolyte. Amino acid levels in the cortex and outer medulla showed no overall change in amount following infusion, although there were highly significant changes in individual amino acids. In the inner medulla there was a highly significant reduction in total amino acids with infusion, largely due to a fall in amounts of taurine (104+/-4 to 75+/-17 mmol kg(-1) protein), and glycine (97+/-15 to 71+/-18 mmol kg(-1) protein). A fall in free amino acid levels in the inner medulla appears to significantly contribute to the process of intracellular osmotic adjustment during an induced diuresis.     

Release of γ-Amino[3H]butyric Acid from Cultured Amacrine-Like Neurons Mediated by Different Excitatory Amino Acid Receptors

The release of preaccumulated gamma-amino[3H]butyric acid ([3H]GABA) from putative GABAergic amacrine cells was studied in neuronal monolayer cultures made from embryonic chick retina. Release was specifically stimulated by excitatory amino acid agonists. N-Methyl-D-aspartate (NMDA; EC50, 19.1 +/- 5.0 microM), kainic acid (EC50, 15.6 +/- 2.3 microM), and the presumptive endogenous ligand glutamate (EC50, 3.6 +/- 0.5 microM) showed the same efficacy. Quisqualic acid, although the most potent agonist (EC50, 0.56 +/- 0.12 microM), was only half as efficacious. The time course of [3H]GABA release and autoradiographic visualization of responsive GABA-accumulating cells suggest that approximately 50% of the [3H]GABA-accumulating cells possess no or very low responsiveness to quisqualic acid. Depolarization (56 mM KCl)-induced release was fivefold lower than the maximal effect elicited by excitatory amino acids. Release of [3H]GABA and of endogenous GABA was entirely independent of extracellular Ca2+ but was completely abolished after replacement of Na+ by choline or Li+. The effects of NMDA and low concentrations of glutamate (up to 10 microM) were blocked by 2-amino-5-phosphonovaleric acid, by MK 801, and (in a voltage-dependent manner) by Mg2+. The reduction of NMDA responses by kynurenic acid was reversed by D-serine, and quisqualic acid competitively inhibited kainic acid-evoked release. Our results show that the cultured [3H]GABA-accumulating neurons, which probably represent the in vitro counterparts of GABAergic amacrine cells, express at least two types of excitatory amino acid receptors (of the NMDA and non-NMDA type), both of which can mediate a Ca2(+)-independent but Na2(+)-dependent release of GABA.