glutaminase and Acidosis

The renal response to metabolic acidosis is mediated, in part, by increased expression of the genes encoding key enzymes of glutamine catabolism and various ion transporters that contribute to the increased synthesis and excretion of ammonium ions and the net production and release of bicarbonate ions. The resulting adaptations facilitate the excretion of acid and partially restore systemic acid-base balance. Much of this response may be mediated by selective stabilization of the mRNAs that encode the responsive proteins. For example, the glutaminase mRNA contains a direct repeat of 8-nt AU sequences that function as a pH-response element (pHRE). This element is both necessary and sufficient to impart a pH-responsive stabilization to chimeric mRNAs. The pHRE also binds multiple RNA-binding proteins, including zeta-crystallin (zeta-cryst), AU-factor 1 (AUF1), and HuR. The onset of acidosis initiates an endoplasmic reticulum (ER)-stress response that leads to the formation of cytoplasmic stress granules. zeta-cryst is transiently recruited to the stress granules, and concurrently, HuR is translocated from the nucleus to the cytoplasm. On the basis of the cumulative data, a mechanism for the stabilization of selective mRNAs is proposed. This hypothesis suggests multiple experiments that should define better how cells in the kidney sense very slight changes in intracellular pH and mediate this essential adaptive response.

Topics: Acidosis; Gene Expression Regulation, Enzymologic; Glutaminase; Glutamine; Humans; Hydrogen-Ion Concentration; Kidney; Response Elements; RNA Stability; RNA-Binding Proteins; RNA, Messenger

Increased renal catabolism of plasma glutamine during metabolic acidosis generates two ammonium ions that are predominantly excreted in the urine. They function as expendable cations that facilitate the excretion of acids. Further catabolism of alpha-ketoglutarate yields two bicarbonate ions that are transported into the venous blood to partially compensate for the acidosis. In rat kidney, this adaptation is sustained, in part, by the induction of multiple enzymes and various transport systems. The pH-responsive increases in glutaminase (GA) and phosphoenolpyruvate carboxykinase (PEPCK) mRNAs are reproduced in LLC-PK(1)-fructose 1,6-bisphosphatase (FBPase) cells. The increase in GA activity results from stabilization of the GA mRNA. The 3'-untranslated region of the GA mRNA contains a direct repeat of an eight-base AU sequence that functions as a pH-response element. This sequence binds zeta-crystallin/NADPH:quinone reductase with high affinity and specificity. Increased binding of this protein during acidosis may initiate the pH-responsive stabilization of the GA mRNA. In contrast, induction of PEPCK occurs at the transcriptional level. In LLC-PK(1)-FBPase(+) kidney cells, a decrease in intracellular pH leads to activation of the p38 stress-activated protein kinase and subsequent phosphorylation of transcription factor ATF-2. This transcription factor binds to cAMP-response element 1 within the PEPCK promoter and may enhance its transcription during metabolic acidosis.

Topics: Acidosis; Animals; Fructose-Bisphosphatase; Gene Expression Regulation, Enzymologic; Glutaminase; Humans; Kidney; Phosphoenolpyruvate Carboxykinase (GTP); Transcription, Genetic

During normal acid-base balance, the kidney extracts very little of the plasma glutamine. However, during metabolic acidosis, as much as one third of the plasma glutamine is extracted and metabolized in a single pass through this organ. The substantial increase in renal utilization occurs solely within the proximal convoluted tubule and is sustained by compensating adaptations in the intraorgan metabolism of glutamine. The primary pathway for renal glutamine metabolism involves its transport into mitochondria and its deamidation and deamination by glutaminase (GA) and glutamate dehydrogenase (GDH), respectively. The resulting ammonium ions are excreted predominantly in the urine where they function as expendable cations to facilitate the excretion of acids. The resulting alpha-ketoglutarate is further metabolized to phosphoenolpyruvate and subsequently to glucose or CO2. The intermediate steps yield two bicarbonate ions that are selectively transported into the venous blood to partially compensate the metabolic acidosis. In rat kidney, this adaptation is sustained in part by the cell-specific induction of the glutaminase that results primarily from stabilization of the GA mRNA. The 3'-nontranslated region of the GA mRNA contains a direct repeat of an 8-base AU-sequence that functions as a pH-response element. This sequence exhibits a high affinity and specificity for zeta (z)-crystallin. The same protein binds to two separate, but homologous, 8-base AU-sequences within the 3'-nontranslated region of the GDH mRNA. The apparent binding activity of z-crystallin is increased significantly during onset of metabolic acidosis. Thus, increased binding of z-crystallin may initiate the pH-responsive stabilization of the two mRNAs.

Topics: Acid-Base Equilibrium; Acidosis; Animals; Cells, Cultured; Glutamate Dehydrogenase; Glutaminase; Glutamine; Ketoglutaric Acids; Kidney Tubules, Proximal; Mitochondria; Models, Chemical; Promoter Regions, Genetic; Rats; RNA, Messenger

Topics: Acidosis; Animals; Aspartate Aminotransferases; Aspartic Acid; Brain; Cells, Cultured; Energy Metabolism; Glutamate Dehydrogenase; Glutamate-Ammonia Ligase; Glutamates; Glutaminase; Glutamine; Hepatic Encephalopathy; Humans; Intestine, Small; Intracellular Membranes; Kidney; Liver; Mitochondria; Mitochondria, Liver; Muscles; Neoplasms; Organ Specificity; Oxidation-Reduction; Rats

In chronic metabolic acidosis in the rat, there is increased ammoniagenesis, gluconeogenesis and renal extraction of glutamine with induction of renal phosphate-dependent glutaminase (PDG). Because the stimulus for these changes is not yet clear and also because acute acidosis is the more common clinical problem, the present study deals mainly with the metabolism of glutamine in acute metabolic acidosis. When acute metabolic acidosis is produced in rats by administration of mineral acid or by causing them to swim, thus inducing a severe lactic acidosis, a factor is found in the plasma which stimulates renal glutamine uptake and ammoniagenesis in vivo as well as in vitro. Acute acidosis does not induce synthesis of PDG in the kidney but causes a change in enzyme kinetics. The plasma factor not only enhances glutamine entry into cells, but apparently causes a conformational change in PDG, as shown by an increase in V1.0mM/Vmax. Intestinal metabolism of glutamine is also stimulated in vivo and in vitro by the plasma factor of acute acidosis.

Topics: Acidosis; Acute Disease; Ammonia; Animals; Chronic Disease; gamma-Glutamyltransferase; Glutamate Dehydrogenase; Glutaminase; Glutamine; Intestinal Mucosa; Kidney; Kidney Tubules; Mitochondria; Physical Exertion

Topics: Acidosis; Animals; Cells, Cultured; Chemical Phenomena; Chemistry; Energy Metabolism; Glutaminase; Glutamine; Intestinal Absorption; Intestine, Small; Kidney; Liver; Phosphates

Topics: Acidosis; Adenosine Triphosphatases; Adenosine Triphosphate; Adenylyl Cyclases; Ammonia; Animals; Biological Transport; Cell Membrane; Citric Acid Cycle; Fatty Acids; Gluconeogenesis; Glucose; Glutamates; Glutaminase; Glutamine; Glycolysis; In Vitro Techniques; Kidney Cortex; Kidney Medulla; Kidney Tubules; Kidney Tubules, Proximal; Lactates; Natriuresis; Oxygen Consumption; Sodium

Topics: Acidosis; Ammonia; Animals; Biological Transport; Chronic Disease; Dactinomycin; Deamination; Diazooxonorleucine; Enzyme Inhibitors; Glutamate Synthase; Glutaminase; Glutamine; Humans; Kidney; Mitochondria; Phosphates

Topics: Acidosis; Ammonia; Animals; Biological Transport, Active; Blood Pressure; Blood Volume; Chlorides; Cyclic AMP; Diuresis; Extracellular Space; Glomerular Filtration Rate; Gluconeogenesis; Glutaminase; Glutamine; Hormones; Hydrostatic Pressure; Kidney; Kidney Glomerulus; Kidney Tubules, Proximal; Models, Biological; Osmotic Pressure; Sodium; Sympathetic Nervous System; Water-Electrolyte Balance

Topics: Acid-Base Equilibrium; Acidosis; Acids; Amino Acids; Ammonia; Ammonium Chloride; Animals; Bicarbonates; Chlorides; Dogs; Glutamate Dehydrogenase; Glutaminase; Humans; Hydrogen-Ion Concentration; Kidney; Rats

A glutaminase-asparaginase enzyme from Achromobacter sp has antitumour activity in vitro and in animals. Glutaminase was administered in doses of 3500-20 000 IU/m2 body surface area/day to six patients with acute lymphoblastic leukaemia (ALL) and three patients with acute myeloid leukaemia (AML). The enzyme had a blood half life of 80 minutes but depletion of blood glutamine persisted for 12 hours after single doses. Seven patients, including four (two with AML and two with ALL) resistant to asparaginase, received repeated doses of glutaminase. Antileukaemic effects were observed in all seven; one elderly patient developed metabolic acidosis. Study of this new antileukaemic agent in patients with acute leukaemia at an earlier stage of their disease is now justified.

Topics: Acidosis; Adolescent; Adult; Aged; Alcaligenes; Asparagine; Child; Clinical Trials as Topic; Female; Glutaminase; Glutamine; Half-Life; Humans; Leukemia, Lymphoid; Leukemia, Myeloid, Acute; Male

Advanced chronic kidney disease (CKD) is associated with the development of renal metabolic acidosis. Metabolic acidosis per se may represent a trigger for progression of CKD. Renal acidosis of CKD is characterized by low urinary ammonium excretion with preserved urinary acidification indicating a defect in renal ammoniagenesis, ammonia excretion or both. The underlying molecular mechanisms, however, have not been addressed to date.. We examined the Han:SPRD rat model and used a combination of metabolic studies, mRNA and protein analysis of renal molecules involved in acid-base handling.. We demonstrate that rats with reduced kidney function as evident from lower creatinine clearance, lower haematocrit, higher plasma blood urea nitrogen, creatinine, phosphate and potassium had metabolic acidosis that could be aggravated by HCl acid loading. Urinary ammonium excretion was highly reduced whereas urinary pH was more acidic in CKD compared with control animals. The abundance of key enzymes and transporters of proximal tubular ammoniagenesis (phosphate-dependent glutaminase, PEPCK and SNAT3) and bicarbonate transport (NBCe1) was reduced in CKD compared with control animals. In the collecting duct, normal expression of the B1 H(+)-ATPase subunit is in agreement with low urinary pH. In contrast, the RhCG ammonia transporter, critical for the final secretion of ammonia into urine was strongly down-regulated in CKD animals.. In the Han:SPRD rat model for CKD, key molecules required for renal ammoniagenesis and ammonia excretion are highly down-regulated providing a possible molecular explanation for the development and maintenance of renal acidosis in CKD patients.

Topics: Acidosis; Amino Acid Transport Systems, Neutral; Ammonia; Animals; Bicarbonates; Creatinine; Disease Models, Animal; Gene Expression Regulation; Glutaminase; Heterozygote; Hydrogen-Ion Concentration; Intracellular Signaling Peptides and Proteins; Kidney; Phosphates; Phosphoenolpyruvate Carboxykinase (GTP); Rats; Renal Insufficiency, Chronic; RNA, Messenger; Sodium-Hydrogen Exchanger 3; Sodium-Hydrogen Exchangers

Vacuolar-Type H(+)-ATPase (V-ATPase) takes the central role in pumping H(+) through cell membranes of diverse organisms, which is essential for surviving acid-base fluctuating lifestyles or environments. In mammals, although glucose is believed to be an important energy source to drive V-ATPase, and phosphoenolpyruvate carboxykinase (PEPCK), a key enzyme for gluconeogenesis, is known to be activated in response to acidosis, the link between acid secretion and PEPCK activation remains unclear. In the present study, we used zebrafish larva as an in vivo model to show the role of acid-inducible PEPCK activity in glucose production to support higher rate of H(+) secretion via V-ATPase, by utilizing gene knockdown, glucose supplementation, and non-invasive scanning ion-selective electrode technique (SIET). Zebrafish larvae increased V-ATPase-mediated acid secretion and transiently expression of Pck1, a zebrafish homolog of PEPCK, in response to acid stress. When pck1 gene was knocked down by specific morpholino, the H(+) secretion via V-ATPase decreased, but this effect was rescued by supplementation of glucose into the yolk. By assessing changes in amino acid content and gene expression of respective enzymes, glutamine and glutamate appeared to be the major source for replenishment of Krebs cycle intermediates, which are subtracted by Pck1 activity. Unexpectedly, pck1 knockdown did not affect glutamine/glutamate catalysis, which implies that Pck1 does not necessarily drive this process. The present study provides the first in vivo evidence that acid-induced PEPCK provides glucose for acid-base homeostasis at an individual level, which is supported by rapid pumping of H(+) via V-ATPase at the cellular level.

Topics: Acidosis; Amino Acids; Ammonium Compounds; Animals; Citric Acid Cycle; Gene Knockdown Techniques; Glucose; Glutamate Dehydrogenase; Glutaminase; Malates; Phosphoenolpyruvate Carboxykinase (ATP); Protons; RNA, Messenger; Stress, Physiological; Vacuolar Proton-Translocating ATPases; Zebrafish; Zebrafish Proteins

Label-free quantitative strategies are commonly used in shotgun proteomics to detect differences in protein abundance between biological sample groups. Here, we have employed a combination of two such approaches, spectral counting (SpC) and average MS/MS total ion current (MS(2) TIC), for the analysis of rat kidney mitochondria in response to metabolic acidosis. In total, 49 proteins were observed to be significantly altered in response to metabolic acidosis (p-value < 0.05). Of these, 32 proteins were uniquely observed as significantly different by SpC, 14 by MS(2) TIC, and only 3 by both approaches. Western blot analysis was performed on a subset of these proteins to validate the observed abundance differences. This study illustrates the utility and ease of combining these two label-free quantitative approaches to increase the number of detected protein abundance differences in the shotgun analysis of complex biological samples.

Topics: Acidosis; Animals; Blotting, Western; Glutamate Dehydrogenase; Glutaminase; Kidney; Mitochondria; Mitochondrial Proteins; Rats; Tandem Mass Spectrometry

The Rhesus factor protein, Rh C glycoprotein (Rhcg), is an ammonia transporter whose expression in the collecting duct is necessary for normal ammonia excretion both in basal conditions and in response to metabolic acidosis. Hypokalemia is a common clinical condition associated with increased renal ammonia excretion. In contrast to basal conditions and metabolic acidosis, increased ammonia excretion during hypokalemia can lead to an acid-base disorder, metabolic alkalosis, rather than maintenance of acid-base homeostasis. The purpose of the current studies was to determine Rhcg's role in hypokalemia-stimulated renal ammonia excretion through the use of mice with collecting duct-specific Rhcg deletion (CD-Rhcg-KO). In mice with intact Rhcg expression, a K(+)-free diet increased urinary ammonia excretion and urine alkalinization and concurrently increased Rhcg expression in the collecting duct in the outer medulla. Immunohistochemistry and immunogold electron microscopy showed hypokalemia increased both apical and basolateral Rhcg expression. In CD-Rhcg-KO, a K(+)-free diet increased urinary ammonia excretion and caused urine alkalinization, and the magnitude of these changes did not differ from mice with intact Rhcg expression. In mice on a K(+)-free diet, CD-Rhcg-KO increased phosphate-dependent glutaminase (PDG) expression in the outer medulla. We conclude that hypokalemia increases collecting duct Rhcg expression, that this likely contributes to the hypokalemia-stimulated increase in urinary ammonia excretion, and that adaptive increases in PDG expression can compensate for the absence of collecting duct Rhcg.

Topics: Acidosis; Ammonia; Animals; Cation Transport Proteins; Female; Gene Deletion; Glutaminase; Hydrogen-Ion Concentration; Hypokalemia; Kidney Medulla; Kidney Tubules, Collecting; Male; Membrane Glycoproteins; Mice; Mice, Knockout; Phosphates; Potassium, Dietary; Urine

Since the pioneering work of Claude Bernard, the scientific community has considered the liver to be the major source of endogenous glucose production in all postabsorptive situations. Nevertheless, the kidneys and intestine can also produce glucose in blood, particularly during fasting and under protein feeding. The aim of this study was to better define the importance of the three gluconeogenic organs in glucose homeostasis.. We investigated blood glucose regulation during fasting in a mouse model of inducible liver-specific deletion of the glucose-6-phosphatase gene (L-G6pc(-/-) mice), encoding a mandatory enzyme for glucose production. Furthermore, we characterized molecular mechanisms underlying expression changes of gluconeogenic genes (G6pc, Pck1, and glutaminase) in both the kidneys and intestine.. We show that the absence of hepatic glucose release had no major effect on the control of fasting plasma glucose concentration. Instead, compensatory induction of gluconeogenesis occurred in the kidneys and intestine, driven by glucagon, glucocorticoids, and acidosis. Moreover, the extrahepatic action of glucagon took place in wild-type mice.. Our study provides a definitive quantitative estimate of the capacity of extrahepatic gluconeogenesis to sustain fasting endogenous glucose production under the control of glucagon, regardless of the contribution of the liver. Thus, the current dogma relating to the respective role of the liver and of extrahepatic gluconeogenic organs in glucose homeostasis requires re-examination.

Topics: Acidosis; Animals; Blood Glucose; Blotting, Western; Chromatin Immunoprecipitation; Fasting; Glucagon; Gluconeogenesis; Glucose; Glucose-6-Phosphatase; Glutaminase; Insulin; Intestinal Mucosa; Intestines; Kidney; Liver; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Phosphoenolpyruvate Carboxykinase (GTP)

Kidneys produce ammonium to buffer and excrete acids through metabolism of glutamine. Expression of the glutamine transporter Slc38a3 (SNAT3) increases in kidney during metabolic acidosis (MA), suggesting a role during ammoniagenesis. Potassium depletion and high dietary protein intake are known to elevate renal ammonium excretion. In this study, we examined SNAT3, phosphate-dependent glutaminase (PDG), and phosphoenolpyruvate carboxykinase (PEPCK) regulation during a control (0.36%) or low-K(+) (0.02%) diet for 7 or 14 days or a control (20%) or high-protein (50%) diet for 7 days. MA was induced in control and low-K(+) groups by addition of NH(4)Cl. Urinary ammonium excretion increased during MA, after 14-day K(+) restriction alone, and during high protein intake. SNAT3, PDG, and PEPCK mRNA abundance were elevated during MA and after 14-day K(+) restriction but not during high protein intake. SNAT3 protein abundance was enhanced during MA (both control and low K(+)), after 14-day low-K(+) treatment alone, and during high protein intake. Seven-day dietary K(+) depletion alone had no effect. Immunohistochemistry showed SNAT3 staining in earlier parts of the proximal tubule during 14-day K(+) restriction with and without NH(4)Cl treatment and during high protein intake. In summary, SNAT3, PDG, and PEPCK mRNA expression were congruent with urinary ammonium excretion during MA. Chronic dietary K(+) restriction, high protein intake, and MA enhance ammoniagenesis, an effect that may involve enhanced SNAT3 mRNA and protein expression. Our data suggest that SNAT3 plays an important role as the glutamine uptake mechanism in ammoniagenesis under these conditions.

Topics: Acidosis; Amino Acid Transport Systems, Neutral; Ammonium Chloride; Animals; Caseins; Disease Models, Animal; Glutaminase; Kidney; Kidney Tubules, Proximal; Male; Mice; Phosphoenolpyruvate Carboxykinase (GTP); Potassium Deficiency; Potassium, Dietary; Quaternary Ammonium Compounds; RNA, Messenger; Time Factors; Up-Regulation

Proximal tubules were isolated from control and acidotic rats by collagenase digestion and Percoll density gradient centrifugation. Western blot analysis indicated that the tubules were approximately 95% pure. The samples were analyzed by two-dimensional difference gel electrophoresis (DIGE) and DeCyder software was used to quantify the temporal changes in proteins that exhibit enhanced or reduced expression. The mass-to-charge ratios and the amino acid sequences of the recovered tryptic peptides were determined by MALDI-TOF/TOF mass spectrometry and the proteins were identified using Mascot software. This analysis confirmed the well-characterized adaptive responses in glutaminase (GA), glutamate dehydrogenase (GDH), and phosphoenolpyruvate carboxykinase (PEPCK). This approach also identified 17 previously unrecognized proteins that are increased with ratios of 1.5 to 5.6 and 16 proteins that are decreased with ratios of 0.67 to 0.03 when tubules from 7-day acidotic vs. control rats were compared. Some of these changes were confirmed by Western blot analysis. Temporal studies identified proteins that were induced either with rapid kinetics similar to PEPCK or with more gradual profiles similar to GA and GDH. All of the mRNAs that encode the latter proteins contain an AU sequence that is homologous to the pH response element found in GA mRNA. Thus selective mRNA stabilization may be a predominant mechanism by which protein expression is increased in response to acidosis.

Topics: Acidosis; Adaptation, Physiological; Animals; Blotting, Western; Chronic Disease; Electrophoresis, Gel, Two-Dimensional; Glutamate Dehydrogenase; Glutaminase; Hydrogen-Ion Concentration; In Vitro Techniques; Kidney Tubules, Proximal; Kinetics; Male; Phosphoenolpyruvate Carboxykinase (GTP); Proteins; Proteomics; Rats; Rats, Sprague-Dawley; RNA, Messenger; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

During chronic metabolic acidosis, the adaptive increase in rat renal ammoniagenesis is sustained, in part, by increased expression of mitochondrial glutaminase (GA) and glutamate dehydrogenase (GDH) enzymes. The increase in GA activity results from the pH-responsive stabilization of GA mRNA. The 3'-untranslated region (3'-UTR) of GA mRNA contains a direct repeat of an eight-base AU-rich element (ARE) that binds zeta-crystallin/NADPH:quinone reductase (zeta-crystallin) with high affinity and functions as a pH-response element. RNA EMSAs established that zeta-crystallin also binds to the full-length 3'-UTR of GDH mRNA. This region contains four eight-base sequences that are 88% identical to one of the two GA AREs. Direct binding assays and competition studies indicate that the two individual eight-base AREs from GA mRNA and the four individual GDH sequences bind zeta-crystallin with different affinities. Insertion of the 3'-UTR of GDH cDNA into a beta-globin expression vector (pbetaG) produced a chimeric mRNA that was stabilized when LLC-PK1-F+ cells were transferred to acidic medium. A pH-responsive stabilization was also observed using a betaG construct that contained only the single GDH4 ARE and a destabilizing element from phosphoenolpyruvate carboxykinase mRNA. Therefore, during acidosis, the pH-responsive stabilization of GDH mRNA may be accomplished by the same mechanism that affects an increase in GA mRNA.

Topics: 3' Untranslated Regions; Acidosis; Ammonia; Animals; Gene Expression Regulation, Enzymologic; Glutamate Dehydrogenase; Glutaminase; Hydrogen-Ion Concentration; Kidney; LLC-PK1 Cells; Male; Rats; Rats, Sprague-Dawley; Recombinant Fusion Proteins; RNA Processing, Post-Transcriptional; RNA, Messenger; Swine; zeta-Crystallins

Hippurate (Hip), an endogenous conjugate, belongs to the group of uremic toxins. Hip stimulates P-independent glutaminase (PIG) localized at the proximal luminal membrane, desamidating glutamine with the formation of ammonia, a dominant and adaptive elimination product of H+. This appears to be important because metabolic acidosis (MAC) does not stimulate PIG. Moreover, Hip inhibits ammonia production by P-dependent mitochondrial glutaminase (PDG) that is primarily stimulated by MAC. By this mechanism, it shifts the ammonia production from mitochondria to proximal tubular lumen. MAC stimulates Hip synthesis in the liver and kidney and increases Hip plasma concentration and even fractional excretion by the kidney, which creates an effective regulatory loop of ammoniagenesis. Thus, it appears that Hip by its participation in the correction of MAC possesses the modulatory function.

Topics: Acid-Base Equilibrium; Acidosis; Ammonia; Animals; Glutaminase; Hippurates; Humans; Kidney; Kidney Failure, Chronic; Liver; Rats; Toxins, Biological; Uremia

LLC-PK(1)-FBPase(+) cells, which are a gluconeogenic substrain of porcine renal LLC-PK(1) cells, exhibit enhanced oxidative metabolism and increased levels of phosphate-dependent glutaminase (PDG) activity. On adaptation to acidic medium (pH 6.9, 9 mM HCO(-)(3)), LLC-PK(1)-FBPase(+) cells also exhibit a greater increase in ammonia production and respond with an increase in assayable PDG activity. The changes in PDG mRNA levels were examined by using confluent cells grown on plastic dishes or on permeable membrane inserts. The latter condition increased the state of differentiation of the LLC-PK(1)-FBPase(+) cells. The levels of the primary porcine PDG mRNAs were analyzed by using probes that are specific for the 5.0-kb PDG mRNA (p2400) or that react equally with both the 4.5- and 5.0-kb PDG mRNAs (p930 and r1500). In confluent dish- and filter-grown LLC-PK(1)-FBPase(+) cells, the predominant 4.5-kb PDG mRNA is increased threefold after 18 h in acidic media. However, in filter-grown epithelia, which sustain an imposed pH and HCO(-)(3) gradient, this adaptive increase is observed only when acidic medium is applied to both the apical and the basolateral sides of the epithelia. Half-life experiments established that induction of the 4. 5-kb PDG mRNA was due to its stabilization. An identical pattern of adaptive increases was observed for the cytosolic PEPCK mRNA. In contrast, no adaptive changes were observed in the levels of the 5. 0-kb PDG mRNA in either cell culture system. Furthermore, cultures were incubated in low-potassium (0.7 mM) media for 24-72 h to decrease intracellular pH while maintaining normal extracellular pH. LLC-PK(1)-FBPase(+) cells again responded with increased rates of ammonia production and increased levels of the 4.5-kb PDG and PEPCK mRNAs, suggesting that an intracellular acidosis is the initiator of this adaptive response. Because all of the observed responses closely mimic those characterized in vivo, the LLC-PK(1)-FBPase(+) cells represent a valuable tissue culture model to study the molecular mechanisms that regulate renal gene expression in response to changes in acid-base balance.

Topics: Acid-Base Equilibrium; Acidosis; Animals; Gluconeogenesis; Glutaminase; LLC-PK1 Cells; Phosphoenolpyruvate Carboxykinase (ATP); Potassium; Rats; RNA, Messenger; Swine

The specificity and the functional significance of the binding of a specific cytosolic protein to a direct repeat of an eight-base AU sequence within the 3'-nontranslated region of the glutaminase (GA) mRNA were characterized. Competition experiments established that the protein that binds to this sequence is not an AUUUA binding protein. When expressed in LLC-PK(1)-F(+) cells, the half-life of a beta-globin reporter construct, betaG-phosphoenolpyruvate carboxykinase, was only slightly affected (1.3-fold) by growth in acidic (pH 6.9, 10 mM HCO(-)(3)) vs. normal (pH 7.4, 25 mM HCO(-)(3)) medium. However, insertion of short segments of GA mRNA containing the direct repeat or a single eight-base AU sequence was sufficient to impart a fivefold pH-responsive stabilization to the chimeric mRNA. Furthermore, site-directed mutation of the direct repeat of the 8-base AU sequence in a betaG-GA mRNA, which contains 956 bases of the 3'-nontranslated region of the GA mRNA, completely abolished the pH-responsive stabilization of the wild-type betaG-GA mRNA. Thus either the direct repeat or a single eight-base AU sequence is both sufficient and necessary to create a functional pH-response element.

Topics: Acidosis; Animals; Base Sequence; Binding Sites; DNA; DNA Primers; Genes, Reporter; Globins; Glutaminase; Kidney; LLC-PK1 Cells; RNA Stability; RNA, Messenger; Swine

The pH-responsive stabilization of the rat renal glutaminase (GA) mRNA during metabolic acidosis is mediated by a pH-response element (pH-RE). The primary pH-RE within the GA mRNA consists of a direct repeat of an 8-base adenosine and uridine-rich sequence, which binds a specific cytosolic protein, the pH-response element binding protein (REBP). The functional analysis of this system was performed in LLC-PK(1)-F(+) cells, a pH-responsive line of porcine proximal tubule-like cells. Cytosolic extracts of LLC-PK(1)-F(+) cells also contain a protein that binds with high affinity to the rat GA mRNA pH-RE. The apparent binding of this protein is increased threefold in cytosolic extracts prepared from LLC-PK(1)-F(+) cells that were grown in acidic medium (pH = 6.9, HCO(3)- = 10 mM). Extracts prepared from the renal cortex of rats that were made acutely acidotic also exhibit a similar increase in binding to the RNA probe that contains the direct repeat of the pH-RE. The temporal increase in binding correlates with the temporal increase in GA mRNA. Scatchard analysis indicates that the increased binding is due to an increase in both the affinity and the maximal binding of the pH-REBP. Thus, increased binding of the pH-REBP to the GA mRNA may initiate its stabilization and increased expression during acidosis.

Topics: Acidosis; Acids; Amino Acid Sequence; Animals; Cell Extracts; Culture Media; Cytosol; Glutaminase; Hydrogen-Ion Concentration; Kidney; Kidney Cortex; LLC-PK1 Cells; Rats; Response Elements; RNA-Binding Proteins; RNA, Messenger; Swine; Time Factors; Tissue Extracts

Three groups of rats were given either a standard protein diet, a protein-free diet, or a protein-free diet with the inclusion of 0.28 M NH4Cl in their drinking water, for 10 days. Body, liver and kidney masses were decreased similarly in the protein-free and protein-free NH4Cl groups. Ingestion of protein-free diet resulted in profound systemic acidosis in both groups, the simultaneous consumption of NH4Cl having no further effect. The activities of the urea-cycle enzymes carbamoyl-phosphate synthease, ornithine transcarbamoylase, arginosuccinate lyase and arginase were significantly reduced in the protein-free group, and the simultaneous ingestion of NH4Cl had no further effect. These results indicate that ammonium ingestion does not prevent the decrease in urea cycle enzyme activities during a period of dietary-protein deprivation. Renal phosphate-dependent glutaminase activity was unchanged in the protein-free group, but was significantly higher with simultaneous NH4Cl consumption, suggesting that the renal adaptation to acid ingestion is not compromised by a lack of dietary protein. Urinary ammonia excretion also increased in rats consuming protein-free diet and NH4Cl. Urinary urea excretion was greater in rats receiving protein-free diet and NH4Cl than in rats receiving protein-free diet only, at all time-points examined. These data demonstrate that urea synthesis is driven primarily by the need to dispose of protein-derived ammonia rather than bicarbonate.

Topics: Acidosis; Ammonium Chloride; Animals; Bicarbonates; Blood; Dietary Proteins; Female; Glutaminase; Hydrogen-Ion Concentration; Kidney; Liver; Nitrogen; Rats; Rats, Sprague-Dawley; Urea

The key regulatory enzymes of kidney ammoniagenesis appear to be P-dependent (PDG) and P-independent (PIG) glutaminases. While the participation of PDG has been satisfactorily elucidated, the significance of PIG remains doubtful. Rat kidney cortex slices synthesized ammonia even under basal conditions. Metabolic acidosis, hippurate and insulin stimulated ammonia production. Under basal conditions, PDG activity in kidney homogenate, was twice as high as PIG activity. Metabolic acidosis stimulated ammonia production by the stimulation of both PDG (100%) and PIG (57%) activities. Hippurate stimulated only PIG activity both under basal conditions (90%) and in metabolic acidosis (52%), while it inhibited PDG activity only insignificantly under basal conditions and markedly (53%) in metabolic acidosis. Insulin stimulated both PIG and PDG activities under basal conditions as well as in metabolic acidosis and potentiated the PIG stimulation by hippurate while it potentiated the hippurate inhibition of PDG both under basal conditions and in acidotic rats. In conclusion, both PDG and PIG participate in ammoniagenesis and are stimulated by metabolic acidosis and insulin. Hippurate stimulates PIG, while it inhibits PDG in metabolic acidosis and even after insulin administration. The effect of hippurate appears to be of physiological interest.

Topics: Acidosis; Ammonia; Animals; Glutaminase; Hippurates; Insulin; Kidney; Kidney Cortex; Male; Rats; Rats, Wistar

The metabolic effects of an ammonium salt on the liver and kidney were investigated. Rats were allowed free access to a 0.28 M ammonium chloride (NH4Cl) solution for 7- and 8-day periods. Serum urea concentration was significantly increased after 8 days of NH4Cl ingestion. However the following hepatic urea cycle enzymes remained unchanged: CPS, OTC, ASS and ASL. The pattern of urinary urea excretion was variable. When the data for the 7-day period were pooled, there was no significant difference between the control and acidotic groups. However, when they were examined on a daily basis, acidosis significantly decreased urea excretion on day 2. Urea excretion then began to increase, reached the control value on day 4 and was significantly greater than the control value on day 7. Urinary ammonium excretion of the acidotic group was significantly increased on day 2 and continued to rise throughout the 7-day period. Renal phosphate-dependent glutaminase of the acidotic group was significantly increased on the eighth day. These data indicate that NH4Cl ingestion alters the pattern of urea excretion in a manner not previously demonstrated.

Topics: Acidosis; Ammonium Chloride; Animals; Drug Evaluation, Preclinical; Female; Glutaminase; Kidney; Liver; Rats; Rats, Sprague-Dawley; Urea

Changes in protein and mRNAs for enzymes of glutamine metabolism were determined in rat kidney cortex at different times after induction of NH4Cl acidosis. After NH4Cl, phosphoenolpyruvate carboxykinase (PEPCK) mRNA increased 16-fold by 10 h (P < 0.05) and then returned to control levels by 30 h. In situ hybridization (ISH) showed that PEPCK mRNA was confined to medullary rays; after NH4Cl, expression of PEPCK expanded throughout the cortex, reaching a maximal intensity at 10 h. Phosphate-dependent glutaminase (PDG) and glutamate dehydrogenase (GDH) mRNAs increased 8- and 2.6-fold, respectively (both P < 0.05), by 10 h before decreasing; the increased expression was confirmed by ISH. Immunohistochemistry showed that increased PEPCK, PDG, and GDH protein occurred at variable times after the rise in mRNAs. The increase was confined to proximal tubules and was sustained, a finding noted also by Western blot analysis. In contrast, glutamine synthase protein and mRNA, confined to deep cortex and outer medullar, did not change after NH4Cl. These studies reveal striking changes in PEPCK and PDG mRNAs in rat renal cortex during acidosis. The ISH pattern suggested that increased amounts of PEPCK were synthesized in recruited cells which contained little enzyme under physiological conditions. mRNA levels for PEPCK, PDG, and GDH peaked at 10 h before returning to control levels. Despite the decrease in mRNAs, a sustained increase in proteins was noted.

Topics: Acidosis; Ammonium Chloride; Animals; Enzymes; Glutamate Dehydrogenase; Glutamate-Ammonia Ligase; Glutaminase; Glutamine; Kidney; Liver; Male; Phosphates; Phosphoenolpyruvate Carboxykinase (GTP); Rats; Rats, Wistar; RNA, Messenger

Topics: Acidosis; Animals; Glutamate Dehydrogenase; Glutamate-Ammonia Ligase; Glutaminase; Glutamine; Kidney; Male; Phosphoenolpyruvate Carboxykinase (GTP); Rats; Rats, Wistar; RNA, Messenger

Topics: Acidosis; Animals; Female; Glutaminase; Hydrochloric Acid; Kidney; Liver; Rats; Rats, Sprague-Dawley; Urea

Topics: Acidosis; Animals; Gene Expression Regulation, Enzymologic; Glutamate-Ammonia Ligase; Glutaminase; Glutamine; Male; Rats; Rats, Inbred Strains

During chronic acidosis, the levels of the rat renal mRNAs that encode the mitochondrial glutaminase (GA) and cytosolic phosphoenolpyruvate carboxykinase (PCK) are increased 6-fold. Following acute recovery of chronic acidosis, the levels of the two mRNAs are rapidly and coordinately decreased, returning to normal within 13-17 h. In contrast, the increases in GA and PCK mRNAs during acute onset of acidosis occur with very different kinetics. The increase in PCK mRNA occurs rapidly and reaches a maximum within 7 h, whereas the GA mRNA is increased after a 4-7-h lag and then plateaus at 14-17 h. Treatment with dexamethasone or with cAMP analogs significantly increases the level of renal PCK mRNA but has no effect on the level of GA mRNA. Nuclear run-on experiments indicate that the acute induction of PCK mRNA is primarily due to an increased rate of transcription. However, transcription of GA mRNA is unaffected by acute acidosis. Therefore, the changes in the two mRNAs are temporally coordinated but occur through different mechanisms. Furthermore, the inductive effects of acidosis are not mediated solely through glucocorticoid or cAMP regulatory elements.

Topics: Acidosis; Animals; Blotting, Northern; Gene Expression Regulation, Enzymologic; Glucocorticoids; Glutaminase; Kidney; Male; Mitochondria; Phosphoenolpyruvate Carboxylase; Rats; Rats, Inbred Strains; RNA; Transcription, Genetic

Topics: Acidosis; Animals; Bicarbonates; Cells, Cultured; Glutaminase; Hydrogen-Ion Concentration; Kidney; Phosphoenolpyruvate Carboxykinase (GTP); Rats; RNA, Messenger

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

We describe the kinetic modifications to mitochondrial-membrane-bound phosphate-dependent glutaminase in various types of rat tissue brought about by acute metabolic acidosis. The activity response of phosphate-dependent glutaminase to glutamine was sigmoidal, showing positive co-operativity, the Hill coefficients always being higher than 2. The enzyme from acidotic rats showed increased activity at subsaturating concentrations of glutamine in kidney tubules, as might be expected, but not in brain, intestine or liver tissues. Nevertheless, when brain and intestine from control rats were incubated in plasma from acutely acidotic rats enzyme activity increased at 1 mM glutamine in the same way as in kidney cortex. The enzyme from liver tissue remained unaltered. S0.5 and nH values decreased significantly in kidney tubules, enterocytes and brain slices preincubated in plasma from acidotic rats. The sigmoidal curves of phosphate-dependent glutaminase shifted to the left without any significant changes in Vmax. The similar response of phosphate-dependent glutaminase to acute acidosis in the kidney, brain and intestine confirms the fact that enzymes from these tissues are kinetically identical and reaffirms the presence of an ammoniagenic factor in plasma, either produced or concentrated in the kidneys of rats with acute acidosis.

Topics: Acidosis; Animals; Brain; Glutaminase; Isoenzymes; Kidney Cortex; Kinetics; Liver; Male; Phosphates; Rats; Rats, Inbred Strains; Stomach

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

Increased rat renal ammoniagenesis is sustained during chronic metabolic acidosis by the cell-specific induction of the regulatory enzymes of glutamine catabolism and of gluconeogenesis. A glutaminase-specific cDNA hybridizes to 6.0- and 3.4-kb mRNAs that are contained in total or poly(A)+ RNA isolated from rat kidney. When translated in a rabbit reticulocyte lysate, each of the fractionated mRNAs produces the 72-kDa precursor of the mitochondrial glutaminase. The levels of both mRNAs are increased 5-fold within 1 day following onset of chronic acidosis and reach a maximum (8-fold) after 5 days. During recovery from chronic acidosis, the levels of the glutaminase mRNAs are returned to normal within 1 day. The observed changes in mRNA levels correlate with equivalent changes in the relative levels of translatable glutaminase mRNA. Nuclear run-on assays indicate that the rate of transcription of the renal phosphoenolpyruvate carboxykinase gene is increased and decreased in response to onset and recovery from chronic acidosis, respectively. In contrast, the rates of transcription of the glutaminase and beta-actin genes are unaffected by alterations in acid-base balance. Thus, the increase in renal glutaminase activity during chronic acidosis results from an equivalent increase in the levels of total and translatable glutaminase mRNAs which apparently results from an increased stability of the glutaminase mRNA.

Topics: Acidosis; Actins; Animals; Blotting, Northern; Chronic Disease; Enzyme Induction; Glutaminase; Kidney; Kinetics; Male; Phosphoenolpyruvate Carboxykinase (GTP); Rats; Rats, Inbred Strains; RNA, Messenger; Transcription, Genetic

Phosphate-dependent glutaminase (PDG) was measured in kidney cortex homogenates and mitochondria from control and acutely acidotic rats. The effect of plasma from acutely acidotic rats on PDG activity in homogenates from normal rats was also studied. Acidosis or incubation in acidotic plasma enhanced enzyme activity when measured at 1.0 mM but not at 20.0 mM glutamine. This effect was not due to increased mitochondrial permeability since similar results were obtained after solubilization of the enzyme with Triton X-100. Increased enzyme activity was observed with either the Tris (monomer) form or the borate (polymer) form of the enzyme, indicating that enhanced activity is not due to polymerization but probably to a conformational change in the enzyme such that the Km for glutamine is lowered.

Topics: Acidosis; Acute Disease; Animals; Enzyme Activation; Glutaminase; Glutamine; Kidney Cortex; Kinetics; Male; Mitochondria; Polymers; Rats; Rats, Inbred Strains

Glutamine transport was studied in submitochondrial particles (SMP) to avoid interference from glutamine metabolism. Phosphate-dependent glutaminase activity in SMP was only 0.04% of that in intact mitochondria. The uptake of glutamine in SMP represented both the transport into vesicles and membrane binding (about one-third of total uptake). Sulfhydryl reagents inhibited glutamine uptake in SMP. The uptake of L-[3H]glutamine increased more than twofold in SMP preloaded with 1 mM L-glutamine, an effect that was not seen with 1 mM D-glutamine. The uptake of L-[3H]glutamine was inhibited in the presence of either L-glutamine or L-alanine in the incubation medium. Other amino acids did not inhibit glutamine uptake. Alanine was also shown to trans-stimulate glutamine transport in SMP and cis-inhibit glutamine transport in both SMP and intact mitochondria. Glutamine transport showed a positive cooperativity effect with a Hill coefficient of 1.45. Metabolic acidosis increased the affinity of the transporter for glutamine without any change in other kinetic parameters. These data indicated that mitochondrial glutamine transport occurs via a specific carrier with multiple binding sites and that the transport of glutamine into mitochondria has an important role in increased ammoniagenesis during metabolic acidosis.

Topics: Acidosis; Amino Acids; Animals; Biological Transport; Glutaminase; Glutamine; Kidney; Kidney Cortex; Kinetics; Male; Mitochondria; Radioisotope Dilution Technique; Rats; Rats, Inbred Strains; Rotenone; Submitochondrial Particles; Tritium

Topics: Acidosis; Animals; Gene Expression Regulation; Glutaminase; Kidney; Rats; 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

The amount of the mitochondrial glutaminase present within rat kidney is increased 5-fold during chronic metabolic acidosis. This adaptive response is due to a corresponding increase in the relative rate of glutaminase synthesis. Poly(A+) RNA was purified from the kidneys of control, 7-day acidotic, and 2-day recovered rats and then fractionated by electrophoresis on a low melting temperature agarose gel. Translation of the fractionated RNA in a rabbit reticulocyte lysate yields a 72,000-dalton protein that is specifically precipitated by anti-glutaminase IgG. The level of this protein is at least 3-fold greater in the translation products of the fractionated poly(A+) RNA derived from the acidotic vs. control or recovered rats. Therefore, the 72,000-dalton product of translation is the apparent precursor to the 68,000- and 65,000-dalton proteins that are contained in the mitochondrial glutaminase. From its relative electrophoretic mobility, the size of the glutaminase mRNA was estimated to be approximately 6.5 kilobases. The relative levels of translatable glutaminase mRNA were determined by using unfractionated poly(A+) RNA prepared from rats at various times following onset and recovery from acidosis. The observed increase occurred gradually, requiring 7 days to reach a maximal induction of 4.2-fold. The increase could be due to the increased transcription of a stable mRNA (t1/2 approximately 3 days). However, 2 days of recovery was sufficient to return the level of translatable glutaminase mRNA to normal. Thus, the selective inactivation or the altered stability of the glutaminase mRNA must also contribute to the regulation of the glutaminase gene expression.

Topics: Acidosis; Animals; Glutaminase; Half-Life; Kidney; Kinetics; Male; Mitochondria; Protein Biosynthesis; Rats; Rats, Inbred Strains; RNA, Messenger

The effects of chronic uraemia on glucose production and nitrogen release (urea plus ammonia formation) from alanine, glutamine or serine in isolated rat hepatocytes were studied. Uraemia increased the rate of formation of urea plus ammonia from all three amino acids by 38-93% when they were present at a final concentration of 10 mmol/l. At lower concentrations (2 mmol/l) the rate of nitrogen release was not significantly increased. Hepatocytes from normal rats whose food intake had been restricted to the level of that of uraemic rats did not show the increased rates of nitrogen release. The increased rates of nitrogen release with hepatocytes from uraemic rats were not accompanied by increased rates of glucose synthesis. Instead, accumulation of metabolic intermediates occurred: lactate and pyruvate (alanine or serine as substrates) and glutamate (glutamine as substrate). Livers of uraemic rats had increased activities of glutaminase (30%) and serine dehydratase (100%). Hepatocytes from normal rats treated with phlorhizin to increase the plasma glucagon/insulin ratio behaved in a similar manner to hepatocytes from uraemic rats. They had increased serine dehydratase activity, and increased rates of utilization of serine or glutamine. The possible implications of these findings for human uraemia are discussed.

Topics: Acidosis; Alanine; Amino Acids; Ammonia; Animals; Gluconeogenesis; Glutaminase; Glutamine; In Vitro Techniques; L-Serine Dehydratase; Liver; Male; Phlorhizin; Rats; Rats, Inbred Strains; Serine; Urea; Uremia

Regulation of the mitochondrial phosphate-dependent glutaminase activity is an essential component in the control of renal ammoniagenesis. Alterations in acid-base balance significantly affect the amount of the glutaminase that is present in rat kidney, but not in brain or small intestine. The relative rates of glutaminase synthesis were determined by comparing the amount of [35S]methionine incorporated into specific immunoprecipitates with that incorporated into total protein. In a normal animal, the rate of glutaminase synthesis constitutes 0.04% of the total protein synthesis. After 7 days of metabolic acidosis, the renal glutaminase activity is increased to a value that is 5-fold greater than normal. During onset of acidosis, the relative rate of synthesis increases more rapidly than the appearance of increased glutaminase activity. The increased rate of synthesis reaches a plateau within 5 days at a value that is 5.3-fold greater than normal. Recovery from chronic acidosis causes a rapid decrease in the relative rate of glutaminase synthesis, but a gradual decrease in glutaminase activity. The former returns to normal within 2 days, whereas the latter requires 11 days. The apparent half-time for glutaminase degradation was found to be 5.1 days and 4.7 days for normal and acidotic rats respectively. These results indicate that the increase in renal glutaminase activity associated with metabolic acidosis is due primarily to an increase in its rate of synthesis. From the decrease in activity that occurs upon recovery from acidosis, the true half-life for the glutaminase was estimated to be 3 days.

Topics: Acidosis; Animals; Brain; Chemical Precipitation; Glutaminase; Half-Life; Intestine, Small; Kidney; Male; Methionine; Phosphates; Rats; Rats, Inbred Strains

Ammonia production and excretion are elevated in potassium depletion alkalosis, although normally they are reduced in alkalosis and elevated in acidosis. Studies were conducted with or without acute acid loading in normokalemic rats or rats made chronically hypokalemic with deoxycorticosterone acetate and a potassium-deficient diet to examine the role of phosphate-dependent glutaminase (PDG) in regulating ammonia excretion. Renal cortical PDG rose fourfold, and urinary ammonia excretion (UAE) doubled in potassium depletion compared to potassium-repleted controls. Following acid challenge PDG and urinary ammonia increased four- to sevenfold in both normokalemic and hypokalemic animals, but the rise in UAE did not correspond to the increase in PDG. Thus, PDG levels in acidotic normokalemic rats were one half those seen in potassium-depleted rats, but UAE in the acidotic rats was six times greater. These results could not be explained solely by changes in blood pH. The poor correlation between PDG and UAE also could not be explained by limited substrate availability, since blood glutamine levels were unaffected by potassium depletion. The disparity between UAE and PDG in potassium depletion was studied further during 9 days of potassium repletion of depleted rats. UAE was again increased by depletion but, after only 3 days of potassium repletion, UAE fell to levels found in normokalemic rats. The renal PDG activity, however, remained three times normal. Indeed, PDG remained significantly elevated even after 9 days of potassium replacement. Other enzymes involved in renal ammoniagenesis, including delta glutamyl transferase, glutamine transferase and omega deamidase, were assayed, and alterations in their activities could not account for the changes in UAE.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acidosis; Ammonia; Animals; Citrates; Glutaminase; Kidney; Male; Phosphates; Potassium Chloride; Potassium Deficiency; Rats; Rats, Inbred Strains

Topics: Acidosis; Ammonia; Animals; Gluconeogenesis; Glutaminase; Glutamine; Male; Nephrons; Rats; Rats, Inbred Strains

Rats develop metabolic acidosis acutely after exercise by swimming. Renal cortical slices from exercised rats show an increase in both ammoniagenesis and gluconeogenesis from glutamine. In addition, plasma from the exercised rats also stimulates ammoniagenesis in renal cortical slices from normal rats. In exercised rats renal phosphate dependent glutaminase shows a 200% activation when the enzyme activity is measured at subsaturating concentration of glutamine (1 mM) while only an increase of 12% in Vmax is observed. When kidney slices from normal rats are incubated in plasma from exercised rats an activation of phosphate dependent glutaminase is obtained with a 1.0 mM (100%) but not with 20 mM glutamine as substrate. This activation of phosphate dependent glutaminase at subsaturating levels of substrate may indicate a conformational change in PDG effected by a factor present in the plasma of exercised acidotic rats.

Topics: Acidosis; Ammonia; Animals; Enzyme Activation; Gluconeogenesis; Glutaminase; Kidney; Kidney Cortex; Phosphates; Physical Exertion; Rats; Rats, Inbred Strains; Swimming

Phosphate-dependent glutaminase was present at approximately similar activities in lymph nodes from mammals other than rat, and in thymus, spleen, Peyer's patches and bone marrow of the rat. This suggests that glutamine is important in all lymphoid tissues. Phosphate-dependent glutaminase activity was shown to be present primarily in the mitochondria of rat mesenteric lymph nodes, and most of the activity could be released by detergents. The properties of the enzyme in mitochondrial extracts were investigated. The pH optimum was 8.6 and the Km for glutamine was 2.0 mM. The enzyme was activated by phosphate, other phosphorylated compounds including phosphoenolpyruvate, and also leucine: 50% activation occurred at 5, 0.2 and 0.6 mM for phosphate, phosphoenolpyruvate and leucine respectively. The enzyme was inhibited by glutamate, 2-oxoglutarate, citrate and ammonia, and by N-ethylmaleimide and diazo-5-oxo-L-norleucine; 50% inhibition was observed at 0.7 and 0.1 mM for glutamate and 2-oxoglutarate respectively. Some of these properties may be important in the control of the enzyme activity in vivo.

Topics: Acidosis; Animals; Centrifugation, Density Gradient; Cricetinae; Enzyme Activation; Gerbillinae; Glutaminase; Guinea Pigs; Lymph Nodes; Lymphocytes; Male; Mesentery; Mice; Mitochondria; Phosphates; Rabbits; Rats; Rats, Inbred Strains; Species Specificity; Tissue Distribution

Topics: Acidosis; Ammonia; Animals; Bicarbonates; Glutamate Dehydrogenase; Glutaminase; Glutamine; Kidney; Male; Phosphoenolpyruvate Carboxykinase (GTP); Rats; Rats, Inbred Strains; Sodium Bicarbonate

Topics: Acidosis; Animals; Glutaminase; Glutamine; Kidney Cortex; Mitochondria; Phosphates; Rats

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

Phosphate- independent glutaminase (PIG) and gamma-glutamyl transpeptidase (gamma GT) activities were evaluated in renal cortex homogenates of rats under normal acid-base balance or chronic metabolic acidosis (CMA). An incubation medium containing glutamine 2 mM, no phosphate, pH 7,40, was used. PIG activity was measured as glutamate production and total gamma GT activity as ammonia production. In normal rats gamma GT activity was significantly higher (0,84 +/- 0,05 mumol/min/g wet wt.) than PIG activity (0,48 +/- 0,06 mumol/min/g wet wt.) (p less than 0,01). In CMA there was a significant increase in PIG activity and an even higher increase in gamma GT activity (p less than 0,01 compared to controls in both cases). The glutamate production/ammonia production ratio was 0,57 +/- 0,05 in normal rats, and 0,37 +/- 0,03 in CMA ( p less than 0,025). These data suggest that the increase in PIG activity and, to a further extent, the increase in gamma GT activity may play an important role in augmenting renal ammonia production in CMA.

Topics: Acidosis; Ammonia; Animals; Chronic Disease; gamma-Glutamyltransferase; Glutaminase; Glutamine; Kidney Cortex; Male; Phosphates; Rats; Rats, Inbred Strains

Ammonia is quantitatively the major buffer for hydrogen ion in the urine. Further, the excretion of ammonia can be varied by acid base status and is therefore of homeostatic importance. Acid base status exerts its effect on ammonia excretion both directly and also via an effect on renal ammonia production from glutamine. The mechanism of the effect of acid base status on glutamine deamidation and deamination is uncertain. Apart from its homeostatic role in health and disease alterations in renal ammonia production may assume pathological importance in potassium depletion and uric acid urolithiasis.

Topics: Acid-Base Equilibrium; Acidosis; Ammonia; Animals; Dogs; Glutaminase; Glutamine; Humans; Hydrogen-Ion Concentration; Kidney; Kidney Calculi; Potassium Deficiency; Rats; Uric Acid

Topics: Acidosis; Ammonia; Animals; Glutaminase; Kidney; Male; Phosphates; Rats

Topics: Acidosis; Animals; Carbon Tetrachloride; Carbon Tetrachloride Poisoning; Glutamate-Ammonia Ligase; Glutaminase; Glutamine; Kinetics; Liver; Male; Rats

Topics: Acidosis; Adaptation, Physiological; Animals; gamma-Glutamyltransferase; Glutaminase; Glutamine; In Vitro Techniques; Kidney; Male; Molecular Weight; Rats; Subcellular Fractions

Topics: Acidosis; Ammonia; Animals; Gluconeogenesis; Glutamate Dehydrogenase; Glutaminase; Hypoxia; Kidney; Male; Phosphoenolpyruvate Carboxylase; Phosphoric Monoester Hydrolases; Rats

Topics: Acidosis; Animals; Glutaminase; Ketone Oxidoreductases; Kidney; Male; Rats; Xanthine Dehydrogenase

Topics: Acidosis; Ammonia; Animals; Glutamate Dehydrogenase; Glutaminase; Glutamine; Humans; In Vitro Techniques; Kidney; Kidney Tubules; Mitochondria; Rats; Regeneration

Topics: Acidosis; Acids; Adrenal Glands; Adrenalectomy; Animals; Carbonic Anhydrases; Glutaminase; Hydrocortisone; Kidney; Rats

Topics: Acidosis; Animals; Female; Glutamates; Glutaminase; Kidney; Kinetics; Male; Mitochondria; Phosphates; Protein Conformation; Rats; Sodium Chloride; Thermodynamics

Topics: Acidosis; Acute Disease; Adaptation, Physiological; Ammonia; Animals; Bicarbonates; Chronic Disease; Female; Glutaminase; Kidney; Phosphates; Rats

Topics: Acidosis; Ammonia; Animals; Cytosol; gamma-Glutamyltransferase; Glutaminase; Glutamine; Kidney; Male; Methionine Sulfoximine; Mitochondria; Perfusion; Rats; Solubility; Subcellular Fractions

We found no overall correlation between mitochondrial swelling and PDG activity under many different conditions. We conclude that augmented PDG activity in acidosis is not related, at least to any great extent, to increased anion permeability produced by mitochondrial swelling.

Topics: Acidosis; Animals; Enzyme Activation; Glutaminase; Kidney; Mitochondrial Swelling; Phosphates; Rats

Topics: Acidosis; Animals; Biological Transport; Cell-Free System; Glutaminase; Glutamine; Kidney; Kinetics; Male; Mitochondria; Rats; Temperature

The response of renal ammonia excretion to acidosis was examined in adult rats with reduced renal mass (SNX). Three days after surgical ablation of 70% of renal mass, the activity of renal phosphate-dependent glutaminase (PDG) in SNX rats was 7.7 +/- 1.5 mu moles of ammonia/100 mg of protein min or approximately 50% of the activity in normal rats (14.5 +/- 2.6 mu moles of ammonia/100 mg of protein min), but enhanced ammonia excretion per unit weight was observed in SNX rats (7.2 +/- 0.7 in control vs. 14.6 +/- 3.2 mumoles/g of kidney.hr in SNX rats). The cause (s) of the reduction in the specific activity of PDG (as well as the increase in ammonia excretion) is unknown. The PDG decrease was not due to apparent tissue damage and appeared to be a specific change as the activity of renal succinate dehydrogenase, another mitochondrial inner-membrane enzyme, did not decrease (from the control level) in SNX rats. Ammonia excretion showed no significant response to an acute acid load (ammonium chloride, 5 mmoles/kg of body wt) in SNX rats. Ammonia excretion, however, did adapt to repeated acid-loading (10 mmoles of ammonium chloride per kg of body wt per day for 3 days); ammonia excretion increased more than two-fold by third day of treatment. This adaptive response was associated with a two-fold rise in renal PDG. Administration of actinomycin D, at a dose which produced no gross toxic signs (100 microgram/kg/day i.p.) inhibited virtually all the increase in both ammonia excretion and PDG activity. The correlation of ammonia excretion and PDG adaptations in acidotic SNX rats was similar to that previously observed in infant rats.

Topics: Acidosis; Adaptation, Physiological; Animals; Dactinomycin; DNA; Glutaminase; Kidney; Male; Nephrectomy; Phosphates; Quaternary Ammonium Compounds; Rats; RNA; Urine

Experiments were carried out on rats to evaluate the possible regulatory roles of renal glutaminase activity, mitochondrial permeability to glutamine, phosphoenolpyruvate carboxykinase activity and systemic acid-base changes in the control of renal ammonia (NH(3) plus NH(4) (+)) production. Acidosis was induced by drinking NH(4)Cl solution ad libitum. A pronounced metabolic acidosis without respiratory compensation [pH=7.25; HCO(3) (-)=16.9mequiv./litre; pCO(2)=40.7mmHg (5.41kPa)] was evident for the first 2 days, but thereafter acid-base status returned towards normal. This improvement in acid-base status was accompanied by the attainment of maximal rates of ammonia excretion (onset phase) after about 2 days. A steady rate of ammonia excretion was then maintained (plateau phase) until the rats were supplied with tap water in place of the NH(4)Cl solution, whereupon pCO(2) and HCO(3) (-) became elevated [55.4mmHg (7.37kPa) and 35.5mequiv./litre] and renal ammonia excretion returned to control values within 1 day (recovery phase). Renal arteriovenous differences for glutamine always paralleled rates of ammonia excretion. Phosphate-dependent glutaminase and phosphoenolpyruvate carboxykinase activities and the rate of glutamine metabolism (NH(3) production and O(2) consumption) by isolated kidney mitochondria all increased during the onset phase. The increases in glutaminase and in mitochondrial metabolism continued into the plateau phase, whereas the increase in the carboxykinase reached a plateau at the same time as did ammonia excretion. During the recovery phase a rapid decrease in carboxykinase activity accompanied the decrease in ammonia excretion, whereas glutaminase and mitochondrial glutamine metabolism in vitro remained elevated. The metabolism of glutamine by kidney-cortex slices (ammonia, glutamate and glucose production) paralleled the metabolism of glutamine in vivo during recovery, i.e. it returned to control values. The results indicate that the adaptations in mitochondrial glutamine metabolism must be regulated by extra-mitochondrial factors, since glutamine metabolism in vivo and in slices returns to control values during recovery, whereas the mitochondrial metabolism of glutamine remains elevated.

Topics: Acid-Base Equilibrium; Acidosis; Acids; Ammonia; Animals; Glutaminase; Glutamine; In Vitro Techniques; Kidney; Male; Mitochondria; Oxygen Consumption; Rats

NH4Cl-induced acidosis in rats resulted in renal enlargement and increase in activities of phosphate-dependent glutaminase and glutamic dehydrogenase. The renal enlargement was associated with protein synthesis but not deoxyribonucleic acid synthesis. In control rats histochemical activity of glutamic dehydrogenase was seen dominantly in the proximal straight tubule. In acidotic rats high activity was noted in the proximal convoluted tubule as well as in the proximal straight tubule. By electron microscopy reaction product was in mitochondria. The results suggest that urine ammonia is produced in mitochondria of epithelial cells in the proximal straight tubule in both normal and acidotic rats. Increased enzyme activity in acidotic rats is largely associated with epithelial cells of the proximal convoluted tubule.

Topics: Acidosis; Ammonia; Ammonium Chloride; Animals; Glutamate Dehydrogenase; Glutamate Synthase; Glutaminase; Histocytochemistry; Hydrogen-Ion Concentration; Kidney; Male; Rats

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

1. Slices of duodenum and jejunum produce ammonia from glutamine in vitro. 2. Ammoniagenesis does not increase in response to acidosis or potassium deficiency, two conditions known to cause enhanced ammoniagenesis in the kidney. 3. Gut contains glutaminase 1 as well as gamma-glutamyl transpeptidase. 4. These enzymes do not show any increase during starvation.

Topics: Acidosis; Acute Disease; Ammonia; Animals; Chronic Disease; Duodenum; gamma-Glutamyltransferase; Glutaminase; Glutamine; In Vitro Techniques; Intestine, Small; Jejunum; Organ Specificity; Rats; Starvation

NH3 production by renal cortical mitochondria was studied under conditions of metabolic acidosis induced in vivo and with pH manipulations of the media bathing mitochondria from normal rats. A HCO3- medium equilibrated with O2 and CO2 was utilized with glutamine concentrations of either 10 or 0.5 mM. With chronic acidosis NH3 production increased significantly at either substrate concentration. Similar results were obtained with rotenone in the media, both with chronic acidosis and with acidosis of 3 h duration, indicating that increased glutamine entry and/or phosphate-dependent glutaminase (PDG) activity accounts for the increased ammoniagenesis. In contrast to acidosis induced in vivo, mitochondria from normal rats subjected to a diminution in medium pH, either by manipulation of HCO3 concentration or PCO2, significantly decrease NH3 production. Mitochondrial studies with rotenone, as well as studies of solubilized PDG, suggest that a low pH diminishes NH3 production by directly altering PDG activity. Furthermore, regardless of the specifics of the mechanism, these studies indicate that adaptation to metabolic acidosis is not the immediate, direct result of a change in pH.

Topics: Acidosis; Ammonia; Animals; Glutaminase; Glutamine; Hydrogen-Ion Concentration; In Vitro Techniques; Kidney Cortex; Male; Mitochondria; Rats; Rotenone

Topics: Acidosis; Ammonia; Animals; Enzyme Activation; Glutaminase; Glutamine; Hydrogen-Ion Concentration; Kidney; Male; Mitochondria; Potassium; Rats; Triamcinolone

Previous studies have demonstrated that the adrenal glands were necessary for acidosis activation of the mitochondrial glutaminase I pathway. The present studies were undertaken to determine if corticosterone levels are elevated in acidotic rats and if so, whether acidosis stimulates the adrenal glands directly or via the pituitary-adrenal axis. Metabolic acidosis induced by NH4Cl, either acute or chronic, increased corticosterone levels 100 to 130% in intact rats. Acute metabolic acidosis did not activate the mitochondrial pathway in adrenalectomized rats; corticosterone levels were not elevated in hypophysectomized rats nor did activation of the mitochondrial pathway occur in response to acidosis. Therefore, acidosis does not stimulate the adrenal gland directly; rather, it requires the intact pituitary. Administering exogenous corticotropin to hypophysectomized rats resulted in elevation of plasma corticosterone levels and activation of the mitochondrial pathway. The pituitary-adrenal cortex-renal glutaminase I axis apparently operates as a functional unit in the homeostatic response to metabolic acidosis.

Topics: Acidosis; Adrenal Glands; Adrenocorticotropic Hormone; Ammonia; Animals; Corticosterone; Glutaminase; Kidney; Male; Mitochondria; Pituitary Gland; Rats

Glutaminase activity was assayed in homogenates and mitochondria of kidneys from normal and acidotic rats. These preparations were also subjected to ultrasonic disintegration and the enzyme was assayed in the pellets and supernatants resulting from ultracentrifugation. Glutaminase activity was recovered mainly in the mitochondrial fraction of unsonicated tissue. Sonication released some of the glutaminase from the mitochondria. The increase in glutaminase activity due to the addition of phosphate was greater for the enzyme released from the mitochondria by sonication than for the enzyme recovered in the mitochondrial pellet after centrifugation. Acidosis did not significantly alter glutaminase activity when assayed in Tris buffer. However, when phosphate was present in the incubation medium acidosis increased glutaminase activity whether or not it remained attached to the mitochondrial membrane during sonication. The data indicates that there is more than one isoenzyme of glutaminase in kidney mitochondria and that the sensitivities of these isoenzymes to phosphate are not identical.

Topics: Acidosis; Ammonia; Animals; Enzyme Activation; Glutaminase; Kidney; Mitochondria; Phosphates; Rats; Sonication; Subcellular Fractions

Topics: Acidosis; Animals; Glutaminase; Kidney; Male; Mitochondria; Phosphates; Polyethylene Glycols; Precipitin Tests; Rats; Solubility

The activities of renal lactate and malate dehydrogenases, glutaminase, and Na-K-ATPase were determined in aging male C57BL/6 mice. Urine concentrating ability in these mice and renal response to metabolic acidosis were also studied. Total enzyme activities were measured in vitro in tissue homogenates from mice that were 120, 400, 500, 600, 700, and 800 days old. Urine concentrating ability was determined in these mice prior to sacrifice. Lactate and malate dehydrogenase activities decreased between 120 and 700 days with only male dehydrogenase activity increasing between 700 and 800 days. Age did not affect glutaminase or Na-K-ATPase activities and urine concentrating ability was decreased only at 700 days. Both urine ammonia excretion and renal glutaminase activity increased at 120 and 600 days in response to metabolic acidosis. However, only 5 of 12 animals tested at 600 days survived the acid stress for a full 7 days.

Topics: Acidosis; Adenosine Triphosphatases; Aging; Ammonia; Animals; Body Weight; Glutaminase; Kidney; Kidney Concentrating Ability; L-Lactate Dehydrogenase; Liver; Magnesium; Malate Dehydrogenase; Male; Mice; Mice, Inbred C57BL; Organ Size; Potassium; Sodium; Testis

The purpose of this investigation was to determine the role of enzyme adaptation in the response of ammonia excretion to acidosis in developing rats. The response of renal ammonia excretion was low in infant rats (7-12 days old) following administration of a single dose of acidifying salt (5 mmol NH4CL/kg). However, repeated administration (2 times daily) of the salt increased ammonia excretion two- to threefold within 2 days. This adaptive response was associated with a concomitant rise in renal phosphate-dependent glutaminase (PDG) activity; PDG activity increased from approximately 36% adult level in untreated infants to 79% adult level in infants given NH4Cl for 2 days. Ammonia excretion and PDG activity decreased in parallel following cessation of NH4Cl treatment. Administration of the antibiotic, actinomycin D (100 mug/kg, ip, 2 times daily for 2 days) completely inhibited the response of PDG to repeated NH4Cl administration. In contrast to the situation previously observed in adult rats, actinomycin D treatment prevented the acid-induced rise in renal ammonia excretion. These results suggest that the level of renal PDG plays a more direct role in the adaptation of ammonia excretion to acidosis in infant rats than in adults.

Topics: Acid-Base Equilibrium; Acidosis; Adaptation, Physiological; Age Factors; Ammonia; Ammonium Chloride; Analysis of Variance; Animals; Animals, Newborn; Dactinomycin; Glutaminase; Kidney; Phosphates; Rats; Time Factors

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: Acidosis; Adenosine Triphosphate; Ammonia; Animals; Energy Metabolism; Glutamate Dehydrogenase; Glutaminase; Humans; Hydrogen; Kidney; Kidney Cortex; Kidney Tubules; Phosphoenolpyruvate Carboxykinase (GTP); Postural Balance

Topics: Acidosis; Ammonia; Animals; Cell Fractionation; Centrifugation, Zonal; Ficoll; Glutaminase; Glutamine; Kidney; Mitochondria; Rats; Succinates; Sucrose

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

Topics: Acidosis; Ammonia; Animals; Chronic Disease; Glutamates; Glutaminase; Glutamine; Kidney; Rats

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

Glutamate is known to inhibit the activity of isolated glutaminase I; however, its actual physiologic importance in regulating renal ammoniagenesis has not been established. To determine the regulatory role of glutamate on the metabolism of glutamine by rat kidney slices, we followed the effects on glutamine (2 mM) deamidation of increased removal of glutamate via augmented deamination. Three agents (malonate, 2,4-dinitrophenol, and methylene blue) were known to and shown here to hasten exogenous glutamate deamination. In slices from 10 control rats, 21.5+/-1.7 (SEM) mumol/g of ammonia were formed from amide nitrogen and 9.3+/-0.5 (SEM) mumol/g from the amino nitrogen of glutamine in vitro. Over 90% of the glutamine deamidated formed glutamate at one point in its catabolism. After addition of malonate (10 mM), 2,4-dinitrophenol (0.1 mM), or methylene blue (0.5 mM), the production of ammonia from the amino group rose to 29.3+/-6.0 (SEM) mumol/g, 20.0+/-1.8 (SEM) mumol/g, and 15.5+/-4.2 (SEM) mumol/g, respectively; ammonia production from the amide nitrogen rose also, 45.1+/-7.3 (SEM) mumol/g, 39.7+/-2.6 (SEM) mumol/g, and 41.9+/-3.7 (SEM) mumol/g. In the case of the former two, a minimum of 99% and 75% of the glutamine catabolized formed glutamate. Despite increased glutamine catabolism, there was no build up of glutamate in the media. A correlation between the formation of ammonia from the amino and amide nitrogen was apparent. Since none of the three agents selected affected phosphate activated glutaminase I activity directly or appeared to affect glutamine transport, we interpret the increase in deamidation as an expression of deinhibition of glutaminase I activity secondary to lowered glutamate concentrations at the deamidating sites through more rapid removal of glutamate via hastened deamination. Interestingly, slices removed from acidotic rats produced more ammonia from both the amino 29.1+/-3.8 (SEM) and amide nitrogens 45.9+/-4.3 (SEM) of glutamine, without a buildup of glutamate in the medium. At least 90% of the glutamine deamidated formed glutamate. A common mechanism is proposed to explain these results and the previous ones.

Topics: Acidosis; Ammonia; Animals; Dinitrophenols; Glutamates; Glutaminase; Glutamine; In Vitro Techniques; Kidney; Malonates; Methylene Blue; Mitochondrial Swelling; Nitrogen; Rats

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; Ammonia; Animals; Dehydration; Dogs; Extracellular Space; Glutamates; Glutaminase; Glutamine; Hydrogen-Ion Concentration; Kidney; Kidney Cortex; Kidney Medulla; Male; Phosphates; Rats; Vasopressins; Water

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

Topics: Acidosis; Ammonia; Ammonium Chloride; Animals; Cell Membrane Permeability; Disease Models, Animal; Dogs; Female; Glutaminase; Glutamine; Ketoglutaric Acids; Kidney; Male; Membranes; Mitochondria; Permeability

Topics: Acidosis; Ammonia; Ammonium Chloride; Animals; Blood; Deamination; Gluconeogenesis; Glutamates; Glutaminase; Glutamine; Guinea Pigs; Hydrogen-Ion Concentration; In Vitro Techniques; Kidney; Ligases; Male; Rats; 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: Acetone; Acidosis; Ammonia; Animals; Animals, Newborn; Female; Gluconeogenesis; Glucose; Glutamates; Glutaminase; Glutamine; Glyceraldehyde; Glycerol; Hydrogen-Ion Concentration; In Vitro Techniques; Ketoglutaric Acids; Kidney; Malates; Male; Oxaloacetates; Pyruvates; Rats; Trioses

Topics: Acidosis; Acids; Ammonia; Animals; Escherichia coli; Glutaminase; Glutamine; Guinea Pigs; Humans; Hydroxybutyrates; In Vitro Techniques; Kidney; Methods; Rabbits; Rats; Sodium; Swine

Topics: Acidosis; Adenosine Triphosphate; Ammonia; Animals; Biochemical Phenomena; Biochemistry; Cell-Free System; Edetic Acid; Enzyme Activation; Female; Glutaminase; Glutamine; Kidney; Microscopy, Electron; Mitochondria; Mitochondrial Swelling; Phlorhizin; Phosphates; Proteins; Rats; Spectrum Analysis

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

Topics: Acidosis; Alanine; Amino Acids; Animals; Aspartic Acid; Fasting; Gluconeogenesis; Glutamates; Glutaminase; Glutamine; Glycine; Kidney; Liver; Lysine; Male; Muscles; Physical Exertion; Rats; Serine; Swimming

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; Alkaline Phosphatase; Ammonia; Ammonium Chloride; Animals; Enzymes; Glutaminase; Hydrogen-Ion Concentration; Ischemia; Kidney Tubules; Rats

Topics: Acidosis; Ammonia; Animals; Dogs; Female; Glutamates; Glutaminase; Ketoglutaric Acids; Kidney; Male; Regional Blood Flow

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; Adenosine Triphosphate; Animals; Edetic Acid; Glutaminase; Kidney; Microscopy, Electron; Mitochondria; Phlorhizin; Rats; Stimulation, Chemical

Topics: Acidosis; Animals; Cycloheximide; Dietary Proteins; DNA; Female; Glutaminase; Kidney; Mice; Nephrectomy; Organ Size; Phosphates; Proteins

Topics: Acidosis; Ammonia; Ammonium Chloride; Animals; Carbon Isotopes; Dactinomycin; Female; Glutaminase; Glutamine; Hyperplasia; Kidney; Leucine; Methods; Nephrectomy; Protein Biosynthesis; Proteins; Rats; Regional Blood Flow; RNA

Topics: Acetates; Acidosis; Acids; Ammonia; Animals; Dicarboxylic Acids; Ethionine; Female; Glutaminase; Glutamine; Hydrogen-Ion Concentration; Kidney; Mitochondria; Oxaloacetates; Phosphates; Pyruvates; Rats

Topics: Acidosis; Alanine Transaminase; Ammonia; Animals; Antimetabolites; Glutaminase; Glutamine; In Vitro Techniques; Kidney; Male; Phosphates; Rats

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; Alanine Transaminase; Amino Acid Oxidoreductases; Amino Acids; Ammonia; Ammonium Chloride; Aspartate Aminotransferases; Body Fluids; Carbonic Anhydrases; Citrates; Dogs; Fluids and Secretions; Glutamate Dehydrogenase; Glutaminase; Hydrogen-Ion Concentration; Kidney; L-Lactate Dehydrogenase; Liver; Lyases; Malate Dehydrogenase; Research; Urine

Topics: Acidosis; Alanine; Amino Acids; Ammonia; Ammonium Chloride; Animals; Aspartic Acid; Body Fluids; Dogs; Fluids and Secretions; Glomerular Filtration Rate; Glutamates; Glutaminase; Glutamine; Glycine; Kidney; Kidney Tubules; Lactates; p-Aminohippuric Acid; Physiology; Research; Serine; Urine

Topics: Acidosis; Ammonia; Ammonium Chloride; Biochemical Phenomena; Biochemistry; DNA; Fluids and Secretions; Glutaminase; Glutamine; Hypertrophy; Ketoglutaric Acids; Kidney; Metabolism; Nephrectomy; Physiology; Rats; Research; RNA; Urine

Topics: Acidosis; Amidohydrolases; Ammonia; Ammonium Chloride; Body Fluids; Glutaminase; Kidney; Pharmacology; Potassium Deficiency; Rats; Research; Urine

Topics: Acidosis; Adaptation, Physiological; Ammonia; Animals; Anura; Carbonic Anhydrases; Glutaminase; Kidney; Metabolism; Research; Urine

Topics: Acid-Base Equilibrium; Acidosis; Adaptation, Physiological; Ammonia; Blood Chemical Analysis; Body Fluids; Carbonic Anhydrases; Dogs; Glutaminase; Histocytochemistry; Kidney; Metabolism; Research; Urine

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