glutaminase and Alkalosis

Although the liver was long known to play a major role in the uptake, synthesis, and disposition of glutamine, metabolite balance studies across the whole liver yielded apparently contradictory findings suggesting that little or no net turnover of glutamine occurred in this organ. Efforts to understand the unique regulatory properties of hepatic glutaminase culminated in the conceptual reformulation of the pathway for glutamine synthesis and turnover, especially as regards the role of sub-acinar distribution of glutamine synthetase and glutaminase. This chapter describes these processes as well as the role of glutamine in hepatocellular hydration, a process that is the consequence of cumulative, osmotically active uptake of glutamine into cells. This topic is also examined in terms of the effects of cell swelling on the selective stimulation or inhibition of other far-ranging cellular processes. The pathophysiology of the intercellular glutamine cycle in cirrhosis is also considered.

Topics: Alkalosis; Ammonia; Biological Transport; Cell Membrane; Glutamate-Ammonia Ligase; Glutaminase; Glutamine; Hormones; Humans; Hydrogen-Ion Concentration; Liver; Mitochondria; Models, Biological; Transaminases

Despite a marked reduction of the urea cycle capacity, patients with well-compensated chronic liver disease excrete near-normal amounts of urea. Compensation of the urea cycle defect apparently occurs through the activation of liver glutaminase, as suggested by an inverse relationship between the in vitro ureagenic capacity and the flux through glutaminase in liver tissue from patients with a normal, fatty, or cirrhotic liver. In these patients, the flux through glutaminase, as determined in vitro, increases in parallel with the plasma bicarbonate level and plasma pH determined in vivo. In view of this and results from previous studies, the following hypothesis is suggested: The decrease of urea cycle enzyme activities in liver cirrhosis produces metabolic alkalosis due to an impaired bicarbonate elimination. Alkalosis in turn activates and stabilizes hepatic glutaminase and accordingly mitochondrial ammonia provision for carbamoylphosphate synthetase. This results in a compensatory stimulation of the urea cycle flux in the cirrhotic patient to near-normal rates, despite the marked reduction of urea cycle enzyme activity. Accordingly, alkalosis is an important driving force for urea synthesis in the cirrhotic patient. With respect to clinical medicine, attention must be paid to acid-base disturbances in the hyperammonemic patient.

Topics: Alkalosis; Bicarbonates; Enzyme Activation; Fatty Liver; Glutaminase; Humans; Liver Cirrhosis; Urea

We studied mechanism(s) by which adaptations of renal TCA cycle metabolism abet ammoniagenesis from glutamine in altered acid-base states. Renal tubules from control, acidotic, or alkalotic rats were incubated at pH 7.4 with 1 mM [3-13C,5-15N]glutamine or 2 mM [3-13C]pyruvate. In acidosis there was a significantly higher flux through glutaminase and through glutamate, 2-oxoglutarate, succinate and malate dehydrogenases as well as markedly enhanced 13C-glucose formation. Alkalosis was associated with little change in 13C flux from glutamine to TCA cycle intermediates compared with control but production of 15NH3 and 13C glucose was significantly diminished. The current studies indicate that renal ammoniagenesis might be regulated at the sites of citrate synthetase (CS) and/or alpha-ketoglutarate dehydrogenase (KGDH). Thus, in chronic metabolic acidosis decreased flux through CS and increased flux through KGDH resulted in enhanced flux through glutamate dehydrogenase and glutaminase pathway. The opposite occurred in alkalosis. The data suggest that in various acid-base states the rate of renal gluconeogenesis is linearly correlated with malate efflux from the mitochondria. In renal tissue, inhibition occurs at one site of the TCA cycle there is an augmentation of fluxes through pathways beyond that site in order to maintain the respiratory process and the redox state in the mitochondria.

Topics: Acid-Base Imbalance; Acidosis; Adaptation, Physiological; Alkalosis; Ammonia; Animals; Aspartic Acid; Citrate (si)-Synthase; Citric Acid Cycle; Glucose; Glutamate Dehydrogenase; Glutaminase; Glutamine; Ketoglutarate Dehydrogenase Complex; Kidney; Kinetics; Male; Rats; Rats, Inbred Strains

A gluconeogenic strain of renal epithelial cells (LLC-PK1-F+) was used to characterize the effect of pH and bicarbonate concentration on the levels of phosphoenolpyruvate carboxykinase (PCK) and glutaminase (GA) mRNAs. The levels of both mRNAs are markedly dependent upon medium glucose concentration. The level of PCK mRNA is increased with increasing glucose concentration from 0 to 40 mM, whereas the level of GA mRNA is maximal between 3 and 5 mM glucose. When LLC-PK1-F+ cells are grown with 5 mM glucose and then subjected to an acute decrease in pH (from 7.4 to 6.9) and bicarbonate concentration (from 25 to 10 mM), the level of PCK mRNA exhibits a biphasic response. The PCK mRNA is initially increased 4-fold within 3 h, then decreases slightly and subsequently increases between 10 and 20 h to a level that is 17-fold greater than normal. Only the initial increase parallels the changes observed in vivo. In contrast, after onset of acidosis, the level of GA mRNA initially remains unchanged, is then increased 8-fold between 10 and 16 h, and then decreases slightly. This response closely mimics the results obtained in vivo. A decrease in media pH at constant bicarbonate causes a marked increase in both mRNAs. However, the levels of the two mRNAs are also elevated by decreasing bicarbonate at a constant pH. Thus, both parameters independently affect the level of the two mRNAs. The use of actinomycin D to measure the half-lives of PCK and GA mRNAs at pH 7.4 and 6.9 indicates that stabilization may fully account for the induction of GA mRNA and contributes to the inductive effects of decreased pH and/or bicarbonate on PCK mRNA. Following recovery from acidic conditions, the two mRNAs exhibit a rapid and coordinate decrease (t1/2 approximately 20 min). Dexamethasone had no effect on the level of either mRNA, whereas cAMP increased only PCK mRNA. The latter effect was additive with the increase caused by decreased pH and/or bicarbonate and was reversed by incubating in alkalotic media. Thus, the induction of PCK and GA mRNAs during acidosis is initiated in direct response to a decrease in extracellular pH and/or bicarbonate.

Topics: Acidosis; Alkalosis; Bicarbonates; Blotting, Northern; Cell Line; Cyclic AMP; Dexamethasone; Epithelium; Gene Expression Regulation, Enzymologic; Glutaminase; Kidney; Phosphoenolpyruvate Carboxykinase (GTP); RNA, Messenger

The effects of short- and long-term diabetes on the maximal activities of phosphate-dependent glutaminase and glutamine metabolism were studied in the colon and the small intestine of streptozotocin-diabetic rats. The maximal activity of colonic phosphate-dependent glutaminase was decreased [44% in mucosal scrapings (p less than 0.01); 29% in whole colon (p less than 0.001)] or unchanged in short- or long-term diabetes respectively. That of the small intestine was increased in both short- (110%) and long-term (200%-500%) diabetes; insulin treatment corrected this increase. Acute insulin-deficiency (using anti-insulin serum) resulted in the increase (18%, p less than 0.05) of the activity of only intestinal glutaminase. Chemically-induced acidosis and alkalosis decreased (46%, p less than 0.001) and increased (24%, p less than 0.001), respectively, the activity of intestinal glutaminase, but had no effect on the colonic enzyme. Changes in glutaminase of the enlarged colon and small intestine were only detectable when activities were measured in whole organ. Arteriovenous-difference measurements showed diminished metabolism of plasma glutamine by the gut which correlated with the duration of the state of diabetes, and was accompanied by enhanced release by skeletal muscle and increased uptake by both kidney and liver. It is concluded that insulin is directly or indirectly involved in the regulation of glutamine metabolism of the gut.

Topics: Acidosis; Alkalosis; Animals; Colon; Diabetes Mellitus, Experimental; Glutaminase; Glutamine; Intestinal Mucosa; Intestine, Small; Male; Phosphates; Rats; Rats, Inbred Strains

Topics: Acidosis; Alkalosis; Ammonia; Ammonium Chloride; Animals; Biological Transport, Active; Glutamate Dehydrogenase; Glutaminase; Glutamine; Ketoglutaric Acids; Kidney; Phosphoenolpyruvate Carboxykinase (GTP); Rats; Rats, Inbred Strains

Topics: Acidosis; Alkalosis; Animals; Cecum; Colon; Gastric Mucosa; Glutaminase; Glutamine; Ileum; Intestinal Mucosa; Intestine, Small; Jejunum; Ketones; Kidney; Male; Mitochondria; Organ Specificity; Phosphates; Rats; Species Specificity

Experiments performed with isolated mitochondria from dog renal cortex provide evidence for a carrier for glutamine located in the inner mitochondrial membrane. This carrier transfers glutamine to glutaminase located in the inner membrane or matrix space and provides a site for regulation of glutamine metabolism and ammoniagenesis. Examination of glutamate formation by the carrier-glutaminase system in mitochondria and in submitochondrial preparations from acidotic and alkalotic dogs shows enhanced glutamate formation without accompanying alteration in glutaminase levels in preparations form acidotic animals. These findings suggest that the increased renal ammonia formation from glutamine during metabolic acidosis results from an adaptive increase in transport of glutamine by the inner membrane carrier.

Topics: Acidosis; Alkalosis; Ammonia; Animals; Biological Transport; Cell Membrane; Deamination; Dogs; Glutamates; Glutaminase; Glutamine; Kidney; Kidney Cortex; Mitochondria; Phosphates

Topics: Acid-Base Equilibrium; Acidosis; Alkalosis; Ammonia; Animals; Bicarbonates; Dogs; Glutamates; Glutaminase; Glutamine; Kidney; Liver; Mannitol; Mitochondria; Quaternary Ammonium Compounds

Topics: Acidosis; Alkalosis; Animals; Drug Stability; Enzyme Activation; Glutamate Dehydrogenase; Glutaminase; Hot Temperature; Hydrogen-Ion Concentration; Isoenzymes; Kidney; Kidney Cortex; Kidney Medulla; Kidney Tubules, Distal; Kidney Tubules, Proximal; Male; Maleates; Nephrons; Phosphates; Rats; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet

Topics: Acidosis; Alkalosis; Ammonia; Animals; Fluorometry; Glutamates; Glutaminase; Glutamine; In Vitro Techniques; Kidney Glomerulus; Kidney Medulla; Kidney Tubules; Kidney Tubules, Distal; Kidney Tubules, Proximal; Male; Rats; Time Factors

Topics: Acid-Base Equilibrium; Acidosis; Alkalosis; Ammonia; Ammonium Chloride; Animals; Carbon Dioxide; Cell Membrane Permeability; Depression, Chemical; Gluconeogenesis; Glutamate Dehydrogenase; Glutamates; Glutaminase; Glutamine; Hydrogen-Ion Concentration; Ketoglutaric Acids; Kidney; Kidney Tubules; Urine

Topics: Acid-Base Equilibrium; Acidosis; Alkalosis; Ammonia; Ammonium Chloride; Carbon Dioxide; Cell Membrane Permeability; Depression, Chemical; Gluconeogenesis; Glutamate Dehydrogenase; Glutamates; Glutaminase; Glutamine; Humans; Hydrogen-Ion Concentration; Ketoglutaric Acids; Kidney; Kidney Tubules; Male; Urine

Studies of the metabolism of glutamine and glutamate by renal cortex slices from acidotic, alkalotic, and control rats were performed. 88-95% of the glutamine and 104-115% of the glutamate taken up from the medium could be accounted for by the products found. Acidosis increased glutamine uptake and conversion to ammonia, CO(2), glucose, lactate, pyruvate, lipid, and protein. The increase in glutamine conversion to ammonia after acidosis could be completely accounted for by the associated increase in its conversion to glucose, glutamate, lactate, and pyruvate. When glutamate metabolism was examined, acidosis did not affect substrate uptake but did increase its conversion to ammonia, glucose, lactate, CO(2), and lipid. The increase in (14)CO(2) from U-(14)C-glutamine and U-(14)C-glutamate found with cortex slices from acidotic animals could be explained by the CO(2) production calculated to be associated with the enhanced conversion of these substrates to other products during acidosis. (14)CO(2) production from 1.2-(14)C-acetate was found to be significantly increased in alkalosis rather than acidosis. These studies suggest that in the rat, the rate at which glutamine is completely oxidized in the Krebs cycle is not a factor regulating renal ammonia production. A comparison of the effects of acidbase status on glutamine and glutamate metabolism suggests that either glutamine transport or glutamine transaminase activity are significantly increased by acidosis.

Topics: Acidosis; Alkalosis; Amino Acids; Ammonia; Animals; Carbon Dioxide; Carbon Isotopes; Glucose; Glutamates; Glutaminase; Glutamine; Kidney; Lactates; Lipid Metabolism; Male; Proteins; Pyruvates; Rats

Topics: Acidosis; Alkalosis; Ammonia; Animals; Glutaminase; Humans; Hydronephrosis; Kidney; Kidney Failure, Chronic; Kidney Neoplasms; Phosphates; Pyelonephritis; Rabbits; Rats; Tuberculosis, Renal; Ureteral Obstruction

Topics: Acid-Base Equilibrium; Acidosis; Alkalosis; Amides; Ammonia; Animals; Carbon Isotopes; Dogs; Female; Glutamates; Glutaminase; Glutamine; Kidney

Glutamate is an inhibitor of phosphate dependent glutaminase (PDG), and renal cortical glutamate is decreased in metabolic acidosis. It has been postulated previously that the rise in renal production of ammonia from glutamine in metabolic acidosis is due primarily to activation of cortical PDG as a consequence of the fall in glutamate. The decrease in cortical glutamate has been attributed to the increase in the capacity of cortex to convert glutamate to glucose in acidosis. In the present study, administration of ammonium chloride to rats in an amount inadequate to decrease cortical glutamate increased the capacity of cortex to produce ammonia from glutamine in vitro and increased cortical PDG. Similarly, cortex from potassium-depleted rats had an increased capacity to produce ammonia and an increase in PDG, but glutamate content was normal. The glutamate content of cortical slices incubated at pH 7.1 was decreased, and that at 7.7 was increased, compared to slices incubated at 7.4, yet ammonia production was the same at all three pH levels. These observations suggest that cortical glutamate concentration is not the major determinant of ammonia production. In potassium-depleted rats there was a 90% increase in the capacity of cortex to convert glutamate to glucose, yet cortical glutamate was not decreased. In vitro, calcium more than doubled conversion of glutamate to glucose by cortical slices without affecting the glutamate content of the slices, and theophylline suppressed conversion of glutamate to glucose yet decreased glutamate content. These observations indicate that the rate of cortical gluconeogenesis is not the sole determinant of cortical glutamate concentration. The increase in cortical gluconeogenesis in acidosis and potassium depletion probably is not the primary cause of the increase in ammonia production in these states, but the rise in gluconeogenesis may contribute importantly to the maintenance of increased ammoniagenesis by accelerating removal of the products of glutamine degradation.

Topics: Acid-Base Equilibrium; Acidosis; Adenine Nucleotides; Alkalosis; Ammonia; Animals; Calcium; Culture Media; Cyclic AMP; Food Deprivation; Gluconeogenesis; Glutamates; Glutaminase; Glutamine; Kidney; Male; Methods; Nucleosides; Potassium Deficiency; Rats; Theophylline

Topics: Acidosis; Adrenalectomy; Alkalosis; Ammonia; Animals; Gluconeogenesis; Glutamate Dehydrogenase; Glutamates; Glutaminase; Glutamine; In Vitro Techniques; Kidney; Male; Rats

Topics: Acidosis; Alkalosis; Ammonia; Animals; Carbon Isotopes; Glomerular Filtration Rate; Glutamates; Glutaminase; Glutamine; Ketoglutaric Acids; Kidney; Kinetics; Ligases; Male; Rats

Experiments were done on rats to investigate the nature of the renal response to metabolic acidosis and the changes in enzyme activity associated with increased ammoniagenesis. When metabolic acidosis was induced with oral feeding of ammonium chloride for 48 hr, there was an increase of activity of the enzyme phosphoenolpyruvate carboxykinase (PEPCK) in whole kidneys as well as in the kidney cortex. There was no change in PEPCK in liver, and glucose-6-phosphatase showed no change in kidney or liver in response to metabolic acidosis. The increase in PEPCK activity in kidney cortex varied with the degree of acidosis and there was a close correlation between cortical PEPCK activity and urinary ammonia. Kidney cortex mitochondrial PEPCK did not change in response to metabolic acidosis. An increase in PEPCK occurred as early as 6 hr after NH(4)Cl feeding, before there was any increase in kidney glutaminase I activity. Rats fed sodium phosphate, or given triamcinolone intramuscularly, developed a metabolic alkalosis, but there was increased urinary ammonia and an increase in activity of renal cortical PEPCK. Triamcinolone plus ammonium chloride induced a greater increase of PEPCK activity than triamcinolone by itself; on the contrary, the rise of glucose-6-phosphatase induced by triamcinolone was not enhanced by acidosis. Glucose-6-phosphatase from control and acidotic rats had identical kinetic characteristics. The results indicate that increased PEPCK activity is constantly related to increases of urinary ammonia. It is proposed that the increase of PEPCK activity is the key event in the ammoniagenesis and gluconeogenesis which follow on metabolic acidosis.

Topics: Acid-Base Equilibrium; Acidosis; Alkalosis; Ammonia; Ammonium Chloride; Animals; Gluconeogenesis; Glucose-6-Phosphatase; Glutaminase; Kidney; Kinetics; Liver; Mitochondria; Phosphates; Phosphotransferases; Rats; Triamcinolone Acetonide

Topics: Acidosis; Alkalosis; Ammonia; Animals; Dactinomycin; Glutamates; Glutaminase; Hydrogen-Ion Concentration; Kidney; Kinetics; Phosphates; Rats

Topics: Acidosis; Alkalosis; Ammonia; Animals; Enzymes; Glutamates; Glutaminase; In Vitro Techniques; Kidney; Mitochondria; Rats

Topics: Acidosis; Alkalies; Alkalosis; Amidohydrolases; Glutaminase; Guinea Pigs; Kidney; Psychomotor Agitation