Hepatocyte heterogeneity in glutamate metabolism and bidirectional transport in perfused rat liver

1. The metabolic fate of infused [1-14C]glutamate was studied in perfused rat liver. The 14C label taken up by the liver was recovered to 85 +/- 2% as 14CO2 and [14C]glutamine. Whereas 14CO2 production accounted for about 70% of the [1-14C]glutamate taken up under conditions of low endogenous rates of glutamine synthesis, stepwise stimulation of glutamine synthesis by NH4Cl increased 14C incorporation into glutamine at the expense of 14CO2 production. Extrapolation to maximal rates of hepatic glutamine synthesis yielded an about 100% utilization of vascular glutamate taken up by the liver for glutamine synthesis. This was observed in both, antegrade and retrograde perfusions and suggests an almost exclusive uptake of glutamate into perivenous glutamine-synthetase-containing hepatocytes. 2. Glutamate was simultaneously taken up and released from perfused rat liver. At a near-physiological influent glutamate concentration (0.1 mM), the rates of unidirectional glutamate influx and efflux were similar (about 100 and 120 nmol g-1 min-1, respectively). 3. During infusion of [1-14C]oxoglutarate (50 microM), addition of glutamate (2 mM) did not affect hepatic uptake of [1-14C]oxoglutarate. However, it increased labeled glutamate release from the liver about 10-fold (from 9 +/- 2 to 86 +/- 20 nmol g-1 min-1; n = 4), whereas 14CO2 production from labeled oxoglutarate decreased by about 40%. This suggests not only different mechanisms of oxoglutarate and glutamate transport across the plasma membrane, but also points to a glutamate/glutamate exchange. 4. Oxoglutarate was recently shown to be taken up almost exclusively by perivenous glutamine-synthetase-containing hepatocytes [Stoll, B & H?ussinger, D. (1989) Eur. J. Biochem. 181, 709-716] and [1-14C]oxoglutarate (9 microM) was used to label selectively the intracellular glutamate pool in this perivenous cell population. The specific radioactivity of this intracellular (perivenous) glutamate pool was assessed by measuring the specific radioactivity of newly synthesized glutamine which is continuously released from these cells into the perfusate. Comparison of the specific radioactivities of glutamine and glutamate released from perivenous cells indicates that about 60% of total glutamate release from the liver is derived from the perivenous glutamine-synthetase-containing cell population. Following addition of unlabeled glutamate (0.1 mM), unidirectional glutamate efflux from perivenous cells increased from about 30 to 80 nmol g-1 min-1, whereas glutamate efflux from non-perivenous (presumably periportal) hepatocytes remained largely unaltered (i.e. 20-30 nmol g-1 min-1). 5. It is concluded that, in the intact liver, vascular glutamate is almost exclusively taken up by the small perivenous hepatocyte population containing glutamine synthetase.     

Characteristics of glutamine metabolism by rat kidney tubules: a carbon and nitrogen balance.

The metabolism of glutamine by a suspension of rat kidney tubules was studied in vitro. The influence of duration of incubation, glutamine concentration, and metabolic state of the donor animals was investigated. The relative importance of glucose synthesis, amino acid production, and oxidation to CO2 was estimated by drawing a complete balance of the nitrogens and the carbon chains of the extracted glutamine. It was found that the initial (first 15 min) rate of glutamine utilization was significantly greater than the subsequent rate due to an initial, but transient, extracellular accumulation of glutamate. This phenomenon was suppressed when a small amount of glutamate was added to the incubation medium. Glucose production constitutes the major fate for glutamine metabolism. No net oxidation of glutamine could be detected with 1 mM glutamine during the first 30 min. However, glutamine oxidation becomes significant after prolonged incubation (16% at 120 min). The metabolic fate of glutamine differs when 5 or 10 mM are presented to the tubules, glutamate production and oxidation to CO2 becoming more important. Metabolic acidosis or a 48-h fast increases glutamine extraction and enhances its utilization glucose synthesis while they depress glutamate accumulation and oxidation to CO2. Metabolic alkalosis has the opposite effect. It is concluded that the metabolism of glutamine in vitro is dependent on the conditions of the study. Furthermore, total oxidation to CO2 is not a major fate for glutamine metabolism at physiological concentration and is not enhanced by acidosis in the rat kidney in vitro.     

Calcium-ion transport by intact synaptosomes. Intrasynaptosomal compartmentation and the role of the mitochondrial membrane potential.

1. The pathways and the fate of glutamate carbon and nitrogen were investigated in isolated guinea-pig kidney-cortex tubules. 2. At low glutamate concentration (1 mM), the glutamate carbon skeleton was either completely oxidized or converted into glutamine. At high glutamate concentration (5 mM), glucose, lactate and alanine were additional products of glutamate metabolism. 3. At neither concentration of glutamate was there accumulation of ammonia. 4. Nitrogen-balance calculations and the release of 14CO2 from L-[1-14C]glutamate (which gives an estimation of the flux of glutamate carbon skeleton through alpha-oxoglutarate dehydrogenase) clearly indicated that, despite the absence of ammonia accumulation, glutamate metabolism was initiated by the action of glutamate dehydrogenase and not by transamination reactions as suggested by Klahr, Schoolwerth & Bourgoignie [(1972) Am. J. Physiol. 222, 813-820] and Preuss [(1972) Am. J. Physiol. 222, 1395-1397]. Additional evidence for this was obtained by the use of (i) amino-oxyacetate, an inhibitor of transaminases, which did not decrease glutamate removal, or (ii) L-methionine DL-sulphoximine, an inhibitor of glutamine synthetase, which caused an accumulation of ammonia from glutamate. 5. Addition of NH4Cl plus glutamate caused an increase in both glutamate removal and glutamine synthesis, demonstrating that the supply of ammonia via glutamate dehydrogenase is the rate-limiting step in glutamine formation from glutamate. NH4Cl also inhibited the flux of glutamate through glutamate dehydrogenase and the formation of glucose, alanine and lactate. 6. The activities of enzymes possibly involved in the glutamate conversion into pyruvate were measured in guinea-pig renal cortex. 7. Renal arteriovenous-difference measurements revealed that in vivo the guinea-pig kidney adds glutamine and alanine to the circulating blood.     

Intrabiliary glutathione hydrolysis. A source of glutamate in bile

High concentrations of glutathione (GSH) and two of its constituent amino acids, glutamate and glycine, are normally found in rat bile. To examine the role of intrabiliary GSH hydrolysis as a source of these amino acids, as well as of cystine in bile, the biliary excretion of GSH and free amino acids was measured in normal male Sprague-Dawley rats; in animals given either phenol 3,6-dibromphthalein disulfonate or diethyl maleate, inhibitors of GSH secretion into bile; and after a retrograde intrabiliary infusion of (alpha S, 5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125), an irreversible inhibitor of gamma-glutamyl transferase activity. Total concentration of amino acids in normal rat bile ranged from 4 to 7 mM and was more than double the concentration in plasma (2-3 mM). Although most amino acids were detected in bile, glutamate and glycine were the most prevalent (1.2 and 1.0 mM, respectively), followed by the branched chain amino acids valine and leucine. The administration of phenol 3,6-dibromphthalein disulfonate (180 mumol/kg, intravenous), or of diethyl maleate (1 mmol/kg, intraperitoneal), resulted in a marked decrease in the biliary excretion of GSH, as well as a decrease in the excretion of glutamate, cystine, and glycine; however, the effects of these agents were not specific for the amino acid constituents of GSH. Following retrograde intrabiliary infusion of AT-125 (10 mumol/kg), there was an immediate and sustained doubling in the rate of biliary excretion of both GSH and glutathione disulfide and a marked decrease in the rate of excretion of glutamate. Varying the dose of AT-125 (0-20 mumol/kg) resulted in an inverse linear relation between hepatic gamma-glutamyl transferase activity and the biliary excretion of intact GSH. These findings suggest that most, if not all, of the free glutamate in excreted bile is formed from the intrabiliary hydrolysis of GSH. Prior to hydrolysis within the biliary tree, substantial concentrations of GSH must be transported from liver cells into bile; minimal canalicular concentrations of this tripeptide are estimated at 5 mM.     

Benzoate stimulates glutamate release from perfused rat liver.

In isolated perfused rat liver, benzoate addition to the influent perfusate led to a dose-dependent, rapid and reversible stimulation of glutamate output from the liver. This was accompanied by a decrease in glutamate and 2-oxoglutarate tissue levels and a net K+ release from the liver; withdrawal of benzoate was followed by re-uptake of K+. Benzoate-induced glutamate efflux from the liver was not dependent on the concentration (0-1 mM) of ammonia (NH3 + NH4+) in the influent perfusate, but was significantly increased after inhibition of glutamine synthetase by methionine sulphoximine or during the metabolism of added glutamine (5 mM). Maximal rates of benzoate-stimulated glutamate efflux were 0.8-0.9 mumol/min per g, and the effect of benzoate was half-maximal (K0.5) at 0.8 mM. Similar Vmax. values of glutamate efflux were obtained with 4-methyl-2-oxopentanoate, ketomethionine (4-methylthio-2-oxobutyrate) and phenylpyruvate; their respective K0.5 values were 1.2 mM, 3.0 mM and 3.8 mM. Benzoate decreased hepatic net ammonia uptake and synthesis of both urea and glutamine from added NH4Cl. Accordingly, the benzoate-induced shift of detoxication from urea and glutamine synthesis to glutamate formation and release was accompanied by a decreased hepatic ammonia uptake. The data show that benzoate exerts profound effects on hepatic glutamate and ammonia metabolism, providing a new insight into benzoate action in the treatment of hyperammonaemic syndromes.     

Carbon-13 and phosphorus-31 NMR study of hepatic metabolism in the perfused rat liver

Phosphorus-31 nuclear magnetic resonance (NMR) has been used to determine non-invasively absolute concentrations of phosphorylated metabolites in the perfused rat liver. It has been shown that the NMR method does detect cytoplasmic ATP and ADP (ATP:ADP ratio of 15 +/- 3) with no contribution from mitochondrial adenine nucleotides. The concentration of ATP was 7.2 +/- 0.3 mM in the cytosol of well-oxygenated liver, after two hours of perfusion with a Krebs-Ringer buffer. Other phosphorylated metabolites were detected, mainly inorganic phosphate (1.1 mumol/g liver wet weight), phosphorylcholine (1.0 mumol/g wet weight), glycerophosphorylethanolamine (0.34 mumol/g wet weight) and glycerophosphorylcholine (0.30 mumol/g wet weight). The intracellular pH measured from the position of the Pi resonance has a value of 7.2 +/- 0.1. It is likely that the detectable Pi originates from the cytosolic compartment since a pH value of 7.4-7.6 would be expected for the mitochondrial matrix. Natural abundance carbon-13 NMR has also been used to follow the glycogen breakdown in situ by measuring the intensity of the glycogen C-1 resonance in the perfused liver spectrum as a function of the perfusion time. The glycogenolytic process has been studied as a function of the glucose content of the perfusate. Rate of glycogenolysis from 2.7 to 0.16 muEq glycosyl units g wet weight-1 min-1 were found when glucose concentration in the perfusate was varied from 0 to 50 mM. The fate of 90% enriched [2-13C] acetate has been studied in the perfused rat liver by 13C-NMR in order to investigate the mitochondrial metabolism and the interrelations between cytosolic and mitochondrial pools of metabolites. Some compounds of the intermediary metabolism where found to be extensively labelled, e.g. glutamate, glutamine, acetoacetate and beta-hydroxybutyrate. Under our experimental conditions, labelling of glutamate reached a steady-state within 30 min after the onset of perfusion of 20 mM acetate. In addition, the observed incorporation of carbon-13 isotope into glutamine can be linked to the operation of the glutamate-glutamine antiporter and to the high activity of the cytosolic glutamate synthetase. The finding of both active glutaminase and glutamine synthetase activity in the same liver cells is an evidence of the existence of an active glutamine-glutamate futile cycle.     

Signal transduction in the ductuli efferentes testis of the rat: inhibition of fluid reabsorption by cyclic adenosine 3', 5'-monophosphate

It is important to identify the signal transduction pathway involved in the regulation of fluid reabsorption by the ductuli efferentes of the testis because they reabsorb most of the fluid leaving the testis and are essential for male fertility. Microperfusion studies of the ducts in vivo showed that 0.1 or 1.0 mM dibutyryl (db)-cGMP in the perfusate had no effect on fluid reabsorption, but 0.1 mM db-cAMP significantly reduced fluid reabsorption, 0.25 mM abolished reabsorption, and 0.5-1.0 mM caused secretion. The inhibitory effect of db-cAMP was reversible. Although the presence of db-cAMP in the perfusate did not affect the concentration of Na+ in the collectate, the concentrations of K(+) and Cl(-) increased, indicating that their transport is at least partly regulated by cAMP. Including the phosphodiesterase inhibitor pentoxifylline in the perfusate decreased fluid reabsorption by the ducts in a dose-dependent manner, and it also increased the concentration of cAMP (5.5-fold) in collectate. Pentoxifylline also increased the production of cAMP (4-fold) by ducts incubated in vitro. It is concluded that cAMP, but probably not cGMP, is an intracellular messenger regulating fluid reabsorption in the efferent ducts.     

Effect of valproate on lactate and glutamine metabolism by rat renal cortical tubules

The metabolic effects of sodium valproate (VPA) on rat renal cortical tubules have been examined. When 1 or 5 mM lactate was used as substrate in the incubation medium, VPA decreased markedly the lactate uptake by the tubules. When 1 or 5 mM glutamine was used, the addition of VPA accelerated glutamine uptake, ammoniagenesis, but also stimulated markedly the accumulation of lactate and pyruvate produced from glutamine. VPA had a dose-dependent inhibitory effect on gluconeogenesis from both glutamine and lactate. With 5 mM glutamine, VPA also induced a significant accumulation of glutamate in the medium. The oxygen consumption by the tubules was diminished by 40% following VPA addition. It is concluded that VPA modifies the metabolism of rat cortical tubules by interfering with the oxidation of natural substrates and stimulates in this fashion the production of ammonia by kidney tubules.     

Gamma glutamyltransferase ammonia production from glutamine: effect of physiological glutathione concentration

The formation of ammonia from physiological glutamine concentration catalyzed by gamma glutamyltransferase was studied in the presence of physiological glutathione concentration. The apparent Km for ammonia formation from glutamine was 1.6 mM some 2 fold greater than the actual plasma concentration. In the presence of 30 microM glutathione neither the apparent Km or Vmax were changed. At supraphysiological glutathione concentration, 1mM, the apparent Km was increased while the Vmax decreased to one third. Hippurate the physiological modulator of the enzyme's glutaminase activity reduced the Km to 0.9 mM, the physiological range, and elevated the Vmax 2.7 fold.     

Evidence indicating that pig renal phosphate-activated glutaminase has a functionally predominant external localization in the inner mitochondrial membrane

Phosphate-activated glutaminase in intact pig renal mitochondria was inhibited 50-70% by the sulfhydryl reagents mersalyl and N-ethylmaleimide (0.3-1.0 mM), when assayed at pH 7.4 in the presence of no or low phosphate (10 mM) and glutamine (2 mM). However, sulfhydryl reagents added to intact mitochondria did not inhibit the SH-enzyme beta-hydroxybutyrate dehydrogenase (a marker of the inner face of the inner mitochondrial membrane), but did so upon addition to sonicated mitochondria. This indicates that the sulfhydryl reagents are impermeable to the inner membrane and that regulatory sulfhydryl groups for glutaminase have an external localization here. The inhibition observed when sulfhydryl reagents were added to intact mitochondria could not be attributed to an effect on a phosphate carrier, but evidence was obtained that pig renal mitochondria have also a glutamine transporter, which is inhibited only by mersalyl and not by N-ethylmaleimide. Mersalyl and N-ethylmaleimide showed nondistinguishable effects on the kinetics of glutamine hydrolysis, affecting only the apparent Vmax for glutamine and not the apparent Km calculated from linear Hanes-Woolf plots. Furthermore, both calcium (which activates glutamine hydrolysis), as well as alanine (which has no effect on the hydrolytic rate), inhibited glutamine transport into the mitochondria, indicating that transport of glutamine is not rate-limiting for the glutaminase reaction. Desenzitation to inhibition by mersalyl and N-ethylmaleimide occurred when the assay was performed under optimal conditions for phosphate activated glutaminase (i.e. in the presence of 150 mM phosphate, 20 mM glutamine and at pH 8.6). Desenzitation also occurred when the enzyme was incubated with low concentrations of Triton X-100 which did not affect the rate of glutamine hydrolysis. Following incubation with [14C]glutamine and correction for glutamate in contaminating subcellular particles, the specific activity of [14C]glutamate in the mitochondria was much lower than that of the surrounding incubation medium. This indicates that glutamine-derived glutamate is released from the mitochondria without being mixed with the endogenous pool of glutamate. The results suggest that phosphate-activated glutaminase has a functionally predominant external localization in the inner mitochondrial membrane.     

Glutamine cycling in isolated working rat heart

To what extent does glutamine turnover keep pace with oxidative metabolism in the rat heart? To address this question, the following groups of substrates were presented to the isolated, working rat heart: 1) glucose (5 mM), insulin (40 microU/ml), and [2-13C]acetate (5 mM; high workload, n = 5); 2) pyruvate (2.5 mM) and [2-13C]acetate (5 mM; normal workload, n = 5); or 3) propionate (1 mM) and [2-13C]acetate (2.5 mM; normal workload, n = 3). In a subset of these experiments, the exchange of glutamate and glutamine was quantified by separation with ion exchange chromatography and analysis by GC-MS. There was an apparent equilibration of mass isotopomers of glutamate and glutamine after 50 min of perfusion, although the extent of equilibration was not determined. The fractional enrichment in glutamine was 31% of the enrichment of glutamate with the three different perfusates. From high-resolution nuclear magnetic resonance spectra, we found a ratio of glutamine to glutamate content of 94.1, 53.4, and 96.9%, respectively, for each experimental group. In experiments for which l-[1-13C]glutamine (5 mM) was included in the perfusate of group 2, [1-13C]glutamine was detected in the heart, but transfer of 13C from glutamine to glutamate was not detected (n = 4). We conclude that, in the perfused working heart, production of glutamine by amidation of glutamate takes place and can be detected, whereas the reverse process, generation of glutamate from glutamine, remains undetected.     

Metabolism of glutamine and glutamate by rat renal tubules. Study with 15N and gas chromatography-mass spectrometry

Gas chromatography-mass spectrometry was utilized to study the metabolism of [15N]glutamate, [2-15N]glutamine, and [5-15N]glutamine in isolated renal tubules prepared from control and chronically acidotic rats. The main purpose was to determine the nitrogen sources utilized by the kidney in various acid-base states for ammoniagenesis. Incubations were performed in the presence of 2.5 mM 15N-labeled glutamine or glutamate. Experiments with [5-15N]glutamine showed that in control animals approximately 90% of ammonia nitrogen was derived from 5-N of glutamine versus 60% in renal tubules from acidotic rats. Experiments with [2-15N]glutamine or [15N]glutamate indicated that in chronic acidosis approximately 30% of ammonia nitrogen was derived either from 2-N of glutamine or glutamate-N by the activity of glutamate dehydrogenase. Flux through glutamate dehydrogenase was 6-fold higher in chronic acidosis versus control. No 15NH3 could be detected in renal tubules from control rats when [2-15N]glutamine was the substrate. The rates of 15N transfer to other amino acids and to the 6-amino groups of the adenine nucleotides were significantly higher in normal renal tubules versus those from chronically acidotic rats. In tubules from chronically acidotic rats, 15N abundance in 15NH3 and the rate of 15NH3 appearance were significantly higher than that of either the 6-amino group of adenine nucleotides or the 15N-amino acids studied. The data indicate that glutamate dehydrogenase activity rather than glutamate transamination is primarily responsible for augmented ammoniagenesis in chronic acidosis. The contribution of the purine nucleotide cycle to ammonia formation appears to be unimportant in renal tubules from chronically acidotic rats.     

Metabolic effects of valproate on dog renal cortical tubules

The effect of valproate (0.01-10 mM), an antiepileptic drug inducing hyperammonemia in humans, was studied in vitro on a suspension of renal cortical tubules (greater than 85% proximal tubules) obtained from six normal dogs. When these tubules were incubated with 1 mM glutamine, the addition of valproate accelerated glutamine uptake, ammoniagenesis, and the production of alanine, lactate, and pyruvate. With 5 mM glutamine, a rise in glutamate accumulation, a much greater synthesis of alanine, an important aspartate production, and a striking accumulation of lactate and pyruvate were observed. With 1 or 5 mM lactate, lactate utilization and gluconeogenesis were markedly reduced with increasing concentrations of valproate. Oxygen consumption was reduced by only 15-20% by 10 mM valproate. The accelerated glutamine utilization resulting from valproate could not be prevented by aminooxyacetate, an inhibitor of transamination. Valproate also reduced various enzymatic activities, a finding that could not explain its metabolic effects. Four sites of action may explain these various metabolic changes: (i) a stimulation of mitochondrial glutamine transport, (ii) an increase in the flux of glutamate to malate, and (iii) a reduction in the net oxidation of pyruvate and (iv) in the flux through pyruvate carboxylase.     

Glutamine metabolism in endothelial cells: ornithine synthesis from glutamine via pyrroline-5-carboxylate synthase

L-Glutamine (the most abundant free amino acid in plasma and the body) is a potent inhibitor of endothelial NO synthesis. However, little is known about glutamine metabolism in endothelial cells (EC). As an initial step toward understanding the role of glutamine in endothelial physiology, the present study was conducted to quantify glutamine catabolism in microvascular, aortic and venous EC. For metabolic studies, EC were incubated for 1 h in Krebs bicarbonate buffer containing 5 mM glucose and 0.5-4 mM L-[U-(14)C]-glutamine. For enzymological studies, cell extracts and mitochondrial fractions were prepared to determine the activities of glutamine-degrading enzymes. Our results reveal extensive hydrolysis of glutamine to glutamate and ammonia in a concentration-dependent manner via phosphate-dependent glutaminase in all EC studied. In addition, both metabolic and enzymological evidence indicate a novel pathway for endothelial synthesis of ornithine from glutamine via pyrroline-5-carboxylate synthase. This new knowledge of glutamine metabolism may pave a new path for understanding the physiological role of glutamine in vascular function.     

Effect of the antiepileptic drug sodium valproate on glutamine and glutamate metabolism in isolated human kidney tubules

We studied the effects of sodium valproate, a widely used antiepileptic drug and a hyperammonemic agent, on L-[1-14C]glutamine and L-[1-14C]glutamate metabolism in isolated human kidney-cortex tubules. Valproate markedly stimulated glutamine removal as well as the formation of ammonia, 14CO2, pyruvate, lactate and alanine, but it inhibited glucose synthesis; the increase in ammonia formation was explained by a stimulation by valproate mainly of flux through glutaminase (EC 3.5.1.2) and to a much lesser extent of flux through glutamate dehydrogenase (EC 1.4.1.3). By contrast, valproate did not stimulate glutamate removal or ammonia formation, suggesting that the increase in flux through glutamate dehydrogenase observed with glutamine as substrate was secondary to the increase in flux through glutaminase. Accumulation of pyruvate, alanine and lactate in the presence of valproate was less from glutamate than from glutamine. Inhibition by aminooxyacetate of accumulation of alanine from glutamine caused by valproate did not prevent the acceleration of glutamine utilization and the subsequent stimulation of ammonia formation. It is concluded from these data, which are the first concerning the in vitro metabolism of glutamine and glutamate in human kidney-cortex tubules, that the stimulatory effect of valproate is primarily exerted at the level of glutaminase in human renal cortex.     

Effect of glutamine on water and sodium absorption in human jejunum at baseline and during PGE1-induced secretion.

Glutamine, a major fuel for enterocytes, stimulates water and sodium absorption in animal models of secretory diarrhea, but data in humans are still limited. The aim of this study was to investigate the effect of glutamine on jejunal absorption during hypersecretion in humans. In six healthy adults, the effects of glutamine on jejunal absorption were assessed with a triple-lumen tube on two occasions, at baseline and during PGE(1)-induced hypersecretion (0.1 microg.kg(-1).min(-1)) in a random order. Isoosmolar solutions containing polyethylene glycol 4000 as nonabsorbable marker were infused in the jejunum at 10 ml/min over 1-h periods: saline (sodium chloride 308 mmol/l), glucose-mannitol 45:45 mM, glucose 90 mM, alanine-glucose 45:45 mM, glutamine-glucose 45:45 mM, and glutamine 90 mM. Net absorptive and secretory fluxes were measured at steady state. At baseline, glutamine- and alanine-containing solutions induced a threefold increase of water and sodium absorption (P < 0.05); 90 mM glutamine stimulated water absorption more than 90 mM glucose (3.6 +/- 0.6 vs. 1.9 +/- 0.3 ml.min(-1).30 cm(-1), P < 0.05). PGE(1)-induced hypersecretion was reduced (P < 0.05) by solutions of alanine-glucose, glutamine-glucose, and glutamine 90 mM (P < 0.05) and reversed to absorption by alanine-glucose and glutamine-glucose. Glutamine and alanine absorption was nearly complete and was not influenced by PGE(1). In conclusion, glutamine stimulates water and electrolyte absorption in human jejunum, even during experimental hypersecretion. In addition to the metabolic effects of glutamine, these results support the evaluation of glutamine-containing solutions for the rehydration and the nutritional support of patients with secretory diarrhea.     

Glutamine metabolism in AS-30D hepatoma cells. Evidence for its conversion into lipids via reductive carboxylation

A study was undertaken to assess the role of a physiological concentration of glutamine in AS-30D cell metabolism. Flux of¹⁴C-glutamine to¹⁴CO₂ and of¹⁴C-acetate to glutamate was detected indicating reversible flux between glutamate and TCA cycle -ketoglutarate. These fluxes were transaminase dependent. A flux analysis was compared using data from three tracers that label -ketoglutarate carbon 5, [2-¹⁴C]glucose, [1-¹⁴C]acetate and [5-¹⁴C]glutamine. The analysis indicated that the probability of flux of TCA cycle -ketoglutarate to glutamate was, at minimum, only slightly less than the probability of flux of -ketoglutarate through -ketoglutarate dehydrogenase. The apparent Km for oxidative flux of [¹⁴C]glutamine to¹⁴CO₂, 0.07 mM, indicated that this flux was at a maximal rate at physiological, 0.75 mM, glutamine. Although oxidative flux through -ketoglutarate dehydrogenase was the major fate of glutamine, flux of glutamine to lipid via reductive carboxylation of -ketoglutarate was demonstrated by measuring incorporation of [5-¹⁴C]glutamine into¹⁴C-lipid. In media containing glucose (6 mM), and glutamine (0.75 mM) 47 per cent of the lipid synthesized from substrates in the media was derived from glutamine via reductive carboxylation and 49 per cent from glucose. These findings of nearly equal fluxes suggest that lipogenesis via reductive carboxylation may be an important role of glutamine in hepatoma cells.     

Regulation of mitochondrial glutamine/glutamate metabolism by glutamate transport: studies with (15)N

We focused on the role of plasma membrane glutamate uptake in modulating the intracellular glutaminase (GA) and glutamate dehydrogenase (GDH) flux and in determining the fate of the intracellular glutamate in the proximal tubule-like LLC-PK(1)-F(+) cell line. We used high-affinity glutamate transport inhibitors D-aspartate (D-Asp) and DL-threo-beta-hydroxyaspartate (THA) to block extracellular uptake and then used [(15)N]glutamate or [2-(15)N]glutamine to follow the metabolic fate and distribution of glutamine and glutamate. In monolayers incubated with [2-(15)N]glutamine (99 atom %excess), glutamine and glutamate equilibrated throughout the intra- and extracellular compartments. In the presence of 5 mM D-Asp and 0.5 mM THA, glutamine distribution remained unchanged, but the intracellular glutamate enrichment decreased by 33% (P < 0.05) as the extracellular enrichment increased by 39% (P < 0.005). With glutamate uptake blocked, intracellular glutamate concentration decreased by 37% (P < 0.0001), in contrast to intracellular glutamine concentration, which remained unchanged. Both glutamine disappearance from the media and the estimated intracellular GA flux increased with the fall in the intracellular glutamate concentration. The labeled glutamate and NH formed from [2-(15)N]glutamine and recovered in the media increased 12- and 3-fold, respectively, consistent with accelerated GA and GDH flux. However, labeled alanine formation was reduced by 37%, indicating inhibition of transamination. Although both D-Asp and THA alone accelerated the GA and GDH flux, only THA inhibited transamination. These results are consistent with glutamate transport both regulating and being regulated by glutamine and glutamate metabolism in epithelial cells.     

In vivo alteration in hypothalamic amino acid synthesis during perfusion of ethanol and morphine in unrestrained rat

In the freely moving rat [U-¹⁴C]glucose was microinjected through a guide tube to label a discrete site in the hypothalamus. After 10 min, a push-pull cannula was used to perfuse an artificial CSF within the site at a rate of 25 l/min. During the fourth 5 min perfusion of each series, one of three concentrations of either ethanol (94–471 mM) or morphine SO₄ (0.13–1.3 mM) was added to the perfusate. Each sample of perfusate was assayed for its content of GABA, glutamate, alanine, aspartate, glycine and glutamine by two-dimensional, thin-layer chromatography. The results show that within a circumscribed region of the dorsal hypothalamus, the synthesis of [¹⁴C]glycine and [¹⁴C]glutamine was enhanced by ethanol and morphine, respectively. Ethanol generally augmented also the synthesis of GABA, glutamate, and glutamine at sites reactive to the compound. Within the same sites, morphine increased the synthesis of glycine. Other amino acids were not significantly different from the control. Thus, anatomically specific and selective changes in amino acid activity are produced within the rat's hypothalamus in response to the localized presence of ethanol or morphine suggesting the involvement of certain amino acids in the action of these addictive compounds within the hypothalamus.     

The distinction between γ-glutamylhydroxamate synthetase and l-glutamine–hydroxylamine glutamyltransferase activities in rat tissues. Studies in vitro

Two common ways of measuring the potential for glutamine synthesis in a tissue are the rates of formation of gamma-glutamylhydroxamate either by synthesis from glutamate (the glutamylhydroxamate synthetase reaction) or by transfer from glutamine (the glutamyltransferase reaction); it has not been established, however, that either reaction is a specific measure of glutamine synthetase. By differential extraction of glutamylhydroxamate synthetase and glutamyltransferase activities from water homogenates of several rat tissues I obtained an extract, rich in glutamylhydroxamate synthetase activity but nearly devoid of glutamyltransferase activity, and a fraction, solubilized by deoxycholate from the pellet, which contained virtually no glutamylhydroxamate synthetase activity but most of the original glutamyltransferase activity. Synthesis of glutamine, quantitatively similar to the gamma-glutamylhydroxamate formed by glutamylhydroxamate synthetase, is catalysed in the water extract but not in the particulate fraction. gamma-Glutamylhydroxamate formation by glutamylhydroxamate synthetase and glutamyltransferase shows discrepant substrate and metal specificities and can be differentially inhibited by l-methionine sulphoximine, phosphate and adenine nucleotides. The concordance between the formation of glutamine and gamma-glutamylhydroxamate by glutamylhydroxamate synthetase but not by glutamyltransferase and the different solubilities of the glutamylhydroxamate synthetase and glutamyltransferase enzyme activities demonstrate that these two activities are not inextricably associated; they therefore cannot be catalysed exclusively by the same protein.