Neuronal glutamine utilization: pathways of nitrogen transfer studied with [15N]glutamine.

Gas chromatography-mass spectrometry was used to evaluate the metabolism of [15N]glutamine in isolated rat brain synaptosomes. In the presence of 0.5 mM glutamine, synaptosomes accumulated this amino acid to a level of 25-35 nmol/mg protein at an initial rate greater than 9 nmol/min/mg of protein. The metabolism of [15N]glutamine generated 15N-labelled glutamate, aspartate, and gamma-aminobutyric acid (GABA). An efflux of both [15N]glutamate and [15N]aspartate from synaptosomes to the medium was observed. Enrichment of 15N in alanine could not be detected because of a limited pool size. Elimination of glucose from the incubation medium substantially increased the rate and amount of [15N]aspartate formed. It is concluded that: (1) With 0.5 mM external glutamine, the glutaminase reaction, and not glutamine transport, determines the rate of metabolism of this amino acid. (2) The primary route of glutamine catabolism involves aspartate aminotransferase which generates 2-oxoglutarate, a substrate for the tricarboxylic acid cycle. This reaction is greatly accelerated by the omission of glucose. (3) Glutamine has preferred access to a population of synaptosomes or to a synaptosomal compartment that generates GABA. (4) Synaptosomes maintain a constant internal level of glutamate plus aspartate of about 70-80 nmol/mg protein. As these amino acids are produced from glutamine in excess of this value, they are released into the medium. Hence synaptosomal glutamine and glutamate metabolism are tightly regulated in an interrelated manner.

Role of alpha-adrenergic stimuli in the control of rat renal ammoniagenesis.

The effects of phenylephrine on renal ammoniagenesis and the involvement of Ca2+ in phenylephrine action were investigated in isolated proximal fragments of rat-kidney tubules. Phenylephrine stimulated renal ammoniagenesis from 1 and 2 mM glutamine whereas no significant changes took place at a higher concentration of glutamine (20 mM). Stimulation of ammonia synthesis by phenylephrine was found to be linear with time and dose-dependent between 10(-9) and 10(-4) M. Phenylephrine-stimulated ammoniagenesis was blocked by phentolamine (10 microM) but not by propranolol (10 microM) confirming that the effect is mediated by alpha-adrenergic stimuli. No stimulatory effect of phenylephrine was observed in Ca2+ depleted proximal tubule fragments, suggesting that Ca2+ is required in this adrenergic response.

N2O reduction and HD formation by nitrogenase from a nifV mutant of Klebsiella pneumoniae.

Dinitrogenase from a nifV mutant of Klebsiella pneumoniae contains an altered form of iron-molybdenum cofactor (FeMoco) that lacks a biologically active homocitric acid molecule. Change in the composition of FeMoco led to substantial variation in the kinetics of nitrogenase action. The KmS of the mutant enzyme for N2 and N2O were 0.244 and 0.175 atm (24,714 and 17,726 kPa), respectively. The km for N2 was higher and the Km for N2O was lower than that for the wild-type enzyme. The mutant enzyme was ineffective in N2 fixation, in N2O reduction, and in HD formation, as indicated by the low Vmax of these reactions with saturating levels of substrate and under conditions of saturating electron flux. These observations provide further support for the concept that N2, N2O, and D2 interact with the same form of dinitrogenase. H2 evolution by the mutant enzyme is only partially inhibited by CO. Observation that different numbers of electrons are stored in CO-inhibited than in noninhibited dinitrogenase before H2 is released suggests that the mutant enzyme has more sites responsible for H2 evolution than the wild-type enzyme, whose H2 evolution is not inhibited by CO.

Ammoniagenesis by cultured human renal cortical epithelial cells: study with 15N.

The metabolic fate of 15N-labeled glutamine and glutamate in cultured human renal cortical epithelial cells was investigated. The main goal was to elucidate the major pathways of ammoniagenesis depending on varying H+ concentration. Incubations at pH 7.4 or 6.8 were conducted with either 1 mM [5-15N]glutamine, [2-15N]glutamine, [15N]glutamate, or L-[2-15N]-gamma-glutamylmethylamide. The results demonstrate that acute acidosis had little effect on total ammonia generation from glutamine. However, 15NH3 formation from [5-15N]glutamine was significantly higher at pH 7.4 compared with pH 6.8. Conversely, at pH 6.8, 15NH3 production from either [2-15N]-glutamine or [15N]glutamate was twofold higher than at pH 7.4. Thus the observations indicate that acute acidosis had little effect on net ammonia production from glutamine due to decreased flux through glutaminase and concomitant increased flux through glutamate dehydrogenase. When L-[2-15N]-gamma-glutamylmethylamide was utilized as the sole substrate, significantly higher amounts of 15NH3 and 15N-labeled amino acids were formed at pH 6.8 compared with pH 7.4. Addition of either 1 mM pyruvate or alpha-ketoglutarate significantly decreased 15NH3 and increased 15N-amino acid formation from either [2-15N]glutamine or [2-15N]-gamma-glutamylmethylamide. The metabolism of either substrate via transamination reaction was significantly stimulated at acidic pH, presumably due to a depleted pool of alpha-ketoglutarate during the course of the incubations. The data indicate that in addition to glutaminase I and glutamate dehydrogenase, the glutamine aminotransferase (glutaminase II) pathway exists in cultured human renal cells. The data suggest that glutamate dehydrogenase flux and/or the alpha-ketoglutarate dehydrogenase reaction may have an important regulatory role in ammoniagenesis from glutamine and/or glutamate in human kidney during acute acidosis.

Glycolysis and glutaminolysis in perifused Ehrlich ascites tumour cells.

A perifusion system was designed in order to study glucose and glutamine metabolism by freshly harvested Ehrlich ascites tumour cells in steady state conditions. Cells were perifused in the presence of 5 mM glucose, 0.5 mM glutamine or 5 mM glucose and 0.5 mM glutamine. The results in steady state reveal that both substrates glucose and glutamine are continuously wasted by tumour cells, excreting two moles of lactate per mol of glucose and one mol of glutamate and ammonia per mol of glutamine consumed into the medium. Glutamine consumption in the presence of glucose was higher than with glutamine alone.

Glucose and synaptosomal glutamate metabolism: studies with [15N]glutamate.

The metabolism of [15N]glutamate was studied with gas chromatography-mass spectrometry in rat brain synaptosomes incubated with and without glucose. [15N]Glutamate was taken up rapidly by the preparation, reaching a steady-state level in less than 5 min. 15N was incorporated predominantly into aspartate and, to a much lesser extent, into gamma-aminobutyrate. The amount of [15N]ammonia formed was very small, and the enrichment of 15N in alanine and glutamine was below the level of detection. Omission of glucose substantially increased the rate and amount of [15N]aspartate generated. It is proposed that in synaptosomes (a) the predominant route of glutamate nitrogen disposal is through the aspartate aminotransferase reaction; (b) the aspartate aminotransferase pathway generates 2-oxoglutarate, which then serves as the metabolic fuel needed to produce ATP; (c) utilization of glutamate via transamination to aspartate is greatly accelerated when flux through the tricarboxylic acid cycle is diminished by the omission of glucose; (d) the metabolism of glutamate via glutamate dehydrogenase in intact synaptosomes is slow, most likely reflecting restriction of enzyme activity by some unknown factor(s), which suggests that the glutamate dehydrogenase reaction may not be near equilibrium in neurons; and (e) the activities of alanine aminotransferase and glutamine synthetase in synaptosomes are very low.

Real importance of alanine in renal metabolism: in vitro studies in rat and dog.

In vitro studies were performed on cortical renal tubules to clarify possible differences between dog and rat with regard to alanine production and to define more precisely the role of alanine on ammonia and glucose production by the kidney. It was established that glutamate-pyruvate transaminase has an activity that is seven times lower in the rat than in the dog kidney. At the same time, alanine production from lactate, pyruvate, and glutamate is three times lower in the rat than in the dog kidney. The enzymatic reaction could be completely inhibited in a competitive fashion with aminooxyacetate. O2 consumption and CO2 production by the renal tubules were lower than that observed with glutamine. CO2 production in the rat was lowest. Production of ammonia and glucose by the kidney from alanine during acidosis averaged less than 20% of that produced with L-glutamine. Furthermore, during metabolic acidosis the production of ammonia and glucose from alanine was not augmented and failed to be influenced by increasing the concentration of alanine in the incubation medium.

Gamma glutamyltransferase contribution to renal ammoniagenesis in vivo.

The role of gamma-glutamyltransferase (gamma-GT) in renal ammoniagenesis and glutamine utilization was evaluated in the intact functioning rat kidney. Total NH4+ released, as the sum of renal venous and urinary NH4+, was measured under conditions of chronic metabolic acidosis and paraminohippurate infusion. Ammonia derived from extracellular gamma-GT hydrolysis of glutamine was differentiated from that produced by intracellular phosphate dependent glutaminase (PDG) by employing acivicin, a gamma-GT inhibitor. In non-acidotic animals acivicin administration inhibited gamma-GT 95% and renal venous NH4+ release 48%; NH4+ release into the urine was not inhibited. Chronic metabolic acidosis elevated total NH4+ release 2.5fold, associated with adaptive increase in both gamma-GT and PDG; acivicin reduced total NH4+ released 36% with both renal venous and urinary release effected. The contribution of gamma-GT to total NH4+ production doubles in metabolic acidosis in agreement with the adaptive rise in the in vitro assayed gamma-GT activity. Luminal ammoniagenesis increases in chronic acidosis associated with a fall in urinary glutamine concentration and a rise in the blood to urine glutamine concentration gradient; gamma-GT inhibition eliminates this gradient suggesting luminal ammoniagenesis is largely dependent upon the paracellular glutamine flux. In support of this, paraminohippurate (PAH) infusion increased total renal NH4+ release due entirely to enhanced NH4+ excretion. PAH stimulated luminal ammoniagenesis was associated with an acceleration of renal glutamine extraction and a steeper blood to urine glutamine diffusion gradient; acivicin blocked this response consistent with PAH secretion coupled to activation of intraluminal gamma-GT and glutamine hydrolysis.

Control by pH of urea synthesis in isolated rat hepatocytes.

Control by pH of urea synthesis has been studied in isolated rat hepatocytes incubated with a physiological mixture of amino acids. Inhibition of urea synthesis by decreasing the pH of the medium was caused by diminished production of ammonia and not, as suggested in the literature, by inhibition of entry of ammonia into the ornithine cycle. The decrease by low pH of the rate of degradation of the added amino acids, that of alanine being quantitatively the most important, was accompanied by a decrease in their intracellular concentration. It is concluded that inhibited transport of amino acids across the plasma membrane of the hepatocyte is responsible, at least in part, for the fall in urea synthesis with decreasing pH. It is proposed that inhibition by low pH of other steps in the ureogenic pathway, distal to the production of ammonia, does not affect flux through the ornithine cycle per se, but rather contributes to the buffering of the intrahepatic concentration of ammonia.

Valproate-induced stimulation of renal ammonia production and excretion in the rat.

Administration of valproate, a widely used antiepileptic drug, markedly stimulated the production of ammonia by the rat kidney, resulting in an increase in both the renal venous release and the urinary excretion of ammonia. These effects were associated with a diminution of the plasma concentration of urea which was not accompanied by a stimulation of the urinary excretion of urea. These results indicate that the kidneys, together with the liver, might contribute to the hyperammonaemia caused by valproate.

Ammonia production from intraluminal amino acids in canine jejunum.

Dietary protein increases the blood ammonia concentration when hepatic metabolic function is impaired, but the site of ammonia production and its specific precursors have not been clearly defined. The purpose of this study is to determine if individual luminal amino acids are metabolized to ammonia by the jejunum during the process of absorption. In anesthetized, fasted dogs, a cannula was inserted into the mesenteric vein draining a segment of the jejunum weighing approximately 18 g, and the ends of the segment were ligated to isolate its blood flow. Ammonia and amino acids were determined in luminal fluid as well as arterial and mesenteric venous blood. One of six amino acids (10 mM) was luminally perfused for a 15-min equilibration period and two 15-min collection periods, and the results were compared with control periods that preceded and followed the amino acid perfusion. Alanine, leucine, and glutamine significantly (P less than 0.01) increased ammonia release into mesenteric venous blood by 37, 42, and 106%, respectively, whereas threonine, serine, and glycine had no effect. Net jejunal uptake of glutamine from arterial blood, which accounts for ammonia release by the jejunum in the basal state, was not altered by perfusions other than with glutamine. Luminal glycine perfusion also caused an increased release of serine into mesenteric venous blood and alanine perfusion increased the release of glutamate. Glutamine perfusion caused increased release of glutamate, alanine, proline, and citrulline. These results indicate that some, but not all, luminal amino acids are partially metabolized to ammonia during the process of absorption in the small intestine.

Renal ammoniagenic response to chronic acid loading: role of glucocorticoids.

Adrenalectomized (ADX) animals exhibit a blunted renal response to chronic acid loading. To determine whether this response truly reflects impaired renal ammoniagenesis from glutamine, urinary ammonium excretion was compared with acid intake in ADX, intact, and ADX rats supplemented with either a low dose (4 micrograms.100 g-1.day-1) or a high dose (40 micrograms.100 g-1.day-1) of triamcinolone. ADX rats consumed similar amounts of acid as did intact controls yet excreted only 37% of the load as ammonium; in contrast intact controls returned 86% and triamcinolone-supplemented animals returned 98 and 88% for low and high doses, respectively. Nor could the reduced ammonium excretion be attributed to increased renal venous release, since total ammonia production, the sum of renal venous and urine ammonium, was reduced to 49% of the intact controls; low- and high-dose triamcinolone restored and markedly increased the production rate. Underlying the impaired ammonia production rate in ADX rats was a reduced rate of glutamine extraction, 350 +/- 49 vs. 896 +/- 102 and 1,260 +/- 247 and 1,448 +/- 112 nmol.min-1.100 g-1 for intact and low and high doses, respectively. Unlike intact acidotic and glucocorticoid-supplemented ADX acidotic rats, glutamine extraction was disassociated from the delivered glutamine load consonant with the role of glucocorticoid in coupling cellular glutamine transport to its metabolic utilization.

Luminal secretion of ammonia in the mouse proximal tubule perfused in vitro.

A major portion of the total ammonia (tNH3 = NH3 + NH+4) produced by the isolated perfused mouse proximal tubule is secreted into the luminal fluid. To assess the role of Na+-H+ exchange in net tNH3 secretion, rates of net tNH3 secretion and tNH3 production were measured in proximal tubule segments perfused with control pH 7.4 Krebs-Ringer bicarbonate (KRB) buffer or with modified KRB buffers containing 10 mM sodium and 0.1 mM amiloride. Net tNH3 secretion was inhibited by 90% in proximal tubule segments perfused with the pH 7.4 modified KRB buffer while tNH3 production remained unaffected. The inhibition of net tNH3 secretion by perfusion with the modified KRB buffer was only partially reversed by acidifying the modified KRB luminal perfusate from 7.4 to as low as 6.2. These data indicate that the Na+-H+ exchanger facilitates a major portion of net tNH3 secretion by the proximal tubule and that luminal acidification may play only a partial role in the mechanism by which the Na+-H+ exchanger mediates net tNH3 secretion.

Intestinal ammonium production in the rat: the role of the colon, small intestine, and circulating glutamine.

The intestinal ammonium production and the intestinal uptake of circulating glutamine were investigated in anesthetized intact rats and rats with resected small intestine or colon by simultaneous measurements performed on portal and arterial blood. It was shown that ammonium release into the portal blood by the small intestine is of equal magnitude to that released by the colon, and that circulating glutamine participates in ammonium production by the small intestine. Increased levels of circulating glutamine induced by its i.v. infusion to intact rats were not accompanied by an increase in intestinal ammonium production.

Insulin stimulates ammoniagenesis in canine renal proximal tubular segments.

To characterize the effect of insulin on ammoniagenesis in renal proximal tubule, ammonia productions were measured in suspensions of canine renal proximal tubular segments incubated with 10 mM L-glutamine and varying concentrations of insulin. Productions of ammonia were linear functions of time for 120 min. Insulin (10(-6) M) increased ammonia production at 2 h by 34 +/- 5%. At the same time, gluconeogenesis, as measured by glucose production, was decreased by 16 +/- 2%. Significant enhancement of ammoniagenesis occurred in suspensions of segments incubated with as little as 10(-9) M insulin. Half-maximal stimulation occurred at between 10(-9) and 10(-8) M insulin. Oxidation of L-glutamine in cells within segments was also increased by insulin in a concentration-dependent manner. Insulin increased ammonia productions in segments incubated at pH 7.5 but not at 7.0. Under the former conditions, insulin enhanced ammoniagenesis in proximal tubular segments under conditions such that extracellular [Na+] was greater than intracellular [Na+], but not when extracellular [Na+] equaled intracellular [Na+]. We conclude that insulin stimulates ammonia production in suspensions of canine renal proximal tubular segments. Stimulation of ammonia production in vitro could reflect an action of insulin to enhance proximal tubular ammoniagenesis in vivo.

Effect of xylitol-containing carbohydrate mixtures on acid and ammonia production in suspensions of salivary sediment.

pH changes and the production of lactic acid, acetic acid and ammonia were studied in suspensions of salivary sediment supplemented with mixtures of xylitol and other carbohydrate sweeteners. The only mixtures which increased the pH values of the suspensions were those containing xylitol alone or mixtures of xylitol and sorbitol. Mixtures of xylitol and Lycasin 80/55 caused a relatively small pH reduction. Xylitol was not able to inhibit the acid production from the easily fermented glucose, fructose and Lycasin 05/60. The levels of lactic acid, determined in the incubation mixtures, directly reflected these pH changes. The levels of acetic acid and ammonia were, however, relatively similar in all incubation mixtures. The results suggest that the inhibitory effects of xylitol on acid production of oral flora should be retained, provided that xylitol is used either alone or in mixtures with slowly fermentable carbohydrates, such as sorbitol and Lycasin 80/55.

Bicarbonate is a recycling substrate for cyanase.

Cyanase is an inducible enzyme in Escherichia coli that catalyzes bicarbonate-dependent decomposition of cyanate to ammonia and bicarbonate. Previous studies provided evidence that carbamate is an initial product and that the kinetic mechanism is rapid equilibrium random (bicarbonate serving as substrate as opposed to activator); the following mechanism was proposed (Anderson, P. M. (1980) Biochemistry 19, 2282-2888; Anderson, P. M., and Little, R. M. (1986) Biochemistry 25, 1621-1626). (formula; see text) Direct evidence for this mechanism was obtained in this study by 1) determining whether CO2 or HCO3- serves as substrate and is formed as product, 2) identifying the products formed from [14C]HCO3- and [14C] OCN-, 3) identifying the products formed from [13C] HCO3- and [12C]OCN- in the presence of [18O]H2O, and 4) determining whether 18O from [18O]HCO3- is incorporated into CO2 derived from OCN-. Bicarbonate (not CO2) is the substrate. Carbon dioxide (not HCO3-) is produced in stoichiometric amounts from both HCO3- and OCN-. 18O from [18O]H2O is not incorporated into CO2 formed from either HCO3- or OCN-. Oxygen-18 from [18O]HCO3- is incorporated into CO2 derived from OCN-. These results support the above mechanism, indicating that decomposition of cyanate catalyzed by cyanase is not a hydrolysis reaction and that bicarbonate functions as a recycling substrate.