scholarly journals Release and fixation of CO2 by guinea-pig kidney tubules metabolizing aspartate

1992 ◽  
Vol 284 (3) ◽  
pp. 697-703
Author(s):  
G Martin ◽  
C Michoudet ◽  
N Vincent ◽  
G Baverel
Keyword(s):  
Guinea Pig ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Co2 Fixation ◽  
Carbon Oxidation ◽  
Kidney Tubules ◽  
Pig Kidney ◽  
Glutamine Synthesis ◽  
Oxoglutarate Dehydrogenase

1. The metabolism of L-[U-14C]aspartate, L-[1-14C]aspartate and L-[4-14C]aspartate was studied in isolated guinea-pig kidney tubules. 2. Oxidation of C-1 plus that of C-4 of aspartate accounted for 90-92% of the CO2 released from aspartate, whereas oxidation of the inner carbon atoms of aspartate (which occurs beyond the 2-oxoglutarate dehydrogenase step) represented only 8-10% of aspartate carbon oxidation. 3. The formation of [1-14C]glutamine and [1-14C]glutamate from [1-14C]aspartate and [4-14C]aspartate indicated that about one-third of the oxaloacetate synthesized from aspartate underwent randomization at the level of fumarate. 4. With [U-14C]aspartate as substrate, the percentage of the C-1 of glutamate and glutamine found radiolabelled after 60 min of incubation was 92.7% and 47.5% in the absence and the presence of bicarbonate respectively. 5. That CO2 fixation occurred at high rates in the presence of bicarbonate was demonstrated by incubating tubules with aspartate plus [14C]bicarbonate; under this condition, the label fixed was found in C-1 of glutamate, glutamine and aspartate, as well as in C-4 of aspartate, demonstrating not only randomization of aspartate carbon but also aspartate resynthesis secondary to oxaloacetate cycling via phosphoenolpyruvate carboxykinase, pyruvate kinase and pyruvate carboxylase. 6. The importance of CO2 fixation in glutamine synthesis from aspartate is discussed in relation to the possible role of the guinea-pig kidney in systemic acid-base regulation in vivo.

scholarly journals Glutamine synthesis from glucose and ammonium chloride by guinea-pig kidney tubules

1994 ◽  
Vol 297 (1) ◽  
pp. 69-74 ◽  
Author(s):  
C Michoudet ◽  
M F Chauvin ◽  
G Baverel
Keyword(s):  
Glucose Metabolism ◽  
Guinea Pig ◽  
Carbon Skeleton ◽  
Kidney Cortex ◽  
Physiological Concentration ◽  
Pig Kidney ◽  
Glucose Carbon ◽  
Glutamine Synthesis ◽  
Stimulation Of

1. At a physiological concentration (5 mM), glucose was found to be metabolized by isolated kidney cortex tubules prepared from fed guinea pigs. 2. The release of 14CO2 from [U-14C]glucose indicated that oxidation of the glucose carbon skeleton represented about 50% of the glucose removed; significant amounts of lactate and glutamine also accumulated. 3. Addition of 0.1-10 mM NH4Cl led to a dose-dependent stimulation of glucose metabolism which was accompanied by a large increase in lactate and glutamine accumulation and, to a lesser extent, in glucose oxidation. 4. Comparison of the release of 14CO2 from [1-14C]- and [6-14C]glucose indicates that, in both the absence and the presence of NH4Cl, the pentose phosphate shunt was only a minor pathway of glucose metabolism. 5. The central role of pyruvate carboxylase in the conversion of glucose carbon into glutamine carbon was demonstrated by using a bicarbonate-free medium and measuring the fixation of 14CO2 from [14C]bicarbonate, which was recovered mostly at C-1 of glutamine plus glutamate. 6. The NH4Cl-induced stimulation of glucose removal was secondary not only to increased glutamine synthesis, as shown by the effect of methionine sulphoximine, an inhibitor of glutamine synthetase, but also to the stimulation of phosphofructokinase activity by NH4Cl. 7. Renal arterio-venous difference measurements revealed that, in vivo, the guinea-pig kidney removed glucose from the circulating blood, which suggests that glucose carbon may contribute to the carbon skeleton of the glutamine released by this organ.

scholarly journals Fate of glutamate carbon and nitrogen in isolated guinea-pig kidney-cortex tubules. Evidence for involvement of glutamate dehydrogenase in glutamine sythesis from glutamate

1980 ◽  
Vol 188 (3) ◽  
pp. 873-880 ◽  
Author(s):  
G Baverel ◽  
C Genoux ◽  
M Forissier ◽  
M Pellet
Keyword(s):  
Guinea Pig ◽  
Glutamate Dehydrogenase ◽  
Carbon Skeleton ◽  
Kidney Cortex ◽  
Glutamate Metabolism ◽  
Carbon And Nitrogen ◽  
Pig Kidney ◽  
Glutamate Concentration ◽  
Rate Limiting Step

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

scholarly journals The subcellular localization of di- and tri-peptide hydrolase activity in guinea-pig small intestine

1970 ◽  
Vol 120 (1) ◽  
pp. 195-203 ◽  
Author(s):  
T. J. Peters
Keyword(s):  
Guinea Pig ◽  
Brush Border ◽  
Soluble Fraction ◽  
Subcellular Fractionation ◽  
Border Region ◽  
Protein Digestion ◽  
Kinetic Assay ◽  
Brush Borders

1. Two different subcellular fractionation techniques were applied to guinea-pig intestinal mucosa and the composition of the brush borders prepared by the two methods were compared. 2. By using a kinetic assay system the subcellular distribution of activity against ten dipeptides and five tripeptides was studied. 3. Only small amounts (5–10%) of activity against dipeptides were found in the brush-border region, the enzymes being concentrated in the cytosol. 4. Significant amounts (10–60%) of activity against tripeptides were found in the brush border with the remainder largely present in the soluble fraction. 5. The relevance of these studies to the localization in vivo and the possible role of peptidases in protein digestion is discussed.

scholarly journals Glutamine synthesis from aspartate in guinea-pig renal cortex

1990 ◽  
Vol 268 (2) ◽  
pp. 437-442 ◽  
Author(s):  
G Baverel ◽  
G Martin ◽  
C Michoudet
Keyword(s):  
Glutamine Synthetase ◽  
Guinea Pig ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Tricarboxylic Acid Cycle ◽  
Kidney Cortex ◽  
Carbon And Nitrogen ◽  
Ammonia Release ◽  
Glutamine Synthesis ◽  
Methionine Sulphoximine ◽  
Main Carbon

1. Glutamine was found to be the main carbon and nitrogen product of the metabolism of aspartate in isolated guinea-pig kidney-cortex tubules. Glutamate, ammonia and alanine were only minor products. 2. Carbon-balance calculations and the release of 14CO2 from [U-14C]aspartate indicate that oxidation of the aspartate carbon skeleton occurred. 3. A pathway involving aspartate aminotransferase, glutamate dehydrogenase, glutamine synthetase, phosphoenolpyruvate carboxykinase, pyruvate kinase, pyruvate dehydrogenase and enzymes of the tricarboxylic acid cycle is proposed for the conversion of aspartate into glutamine. 4. Evidence for this pathway was obtained by: (i) inhibiting aspartate removal by amino-oxyacetate, an inhibitor of transaminases, (ii) the use of methionine sulphoximine, an inhibitor of glutamine synthetase, which induced a large increase in ammonia release from aspartate, (iii) the use of quinolinate, an inhibitor of phosphoenolpyruvate carboxykinase, which inhibited glutamine synthesis from aspartate, (iv) the use of alpha-cyano-4-hydroxycinnamate, an inhibitor of the mitochondrial transport of pyruvate, which caused an accumulation of pyruvate from aspartate, and (v) the use of fluoroacetate, an inhibitor of aconitase, which inhibited glutamine synthesis with concomitant accumulation of citrate from aspartate.

scholarly journals Molecular, Biological, and In Vivo Characterization of the Guinea Pig Cytomegalovirus (CMV) Homologs of the Human CMV Matrix Proteins pp71 (UL82) and pp65 (UL83)

2004 ◽  
Vol 78 (18) ◽  
pp. 9872-9889 ◽  
Author(s):  
Alistair McGregor ◽  
Fenyong Liu ◽  
Mark R. Schleiss
Keyword(s):  
Guinea Pig ◽  
Guinea Pigs ◽  
Matrix Proteins ◽  
Wild Type ◽  
Viral Dna ◽  
Guinea Pig Cytomegalovirus ◽  
Human Cmv ◽  
Hsv 1

ABSTRACT We recently identified the genes encoding the guinea pig cytomegalovirus (GPCMV) homologs of the upper and lower matrix proteins of human CMV, pp71 (UL82) and pp65 (UL83), which we designated GP82 and GP83, respectively. Transient-expression studies with a GP82 plasmid demonstrated that the encoded protein targets the nucleus and that the infectivity and plaquing efficiency of cotransfected GPCMV viral DNA was enhanced by GP82. The transactivation function of GP82 was not limited to GPCMV, but was also observed for a heterologous virus, herpes simplex virus type 1 (HSV-1). This was confirmed by its ability to complement the growth of an HSV-1 VP16 transactivation-defective mutant virus in an HSV viral DNA cotransfection assay. Study of a GP82 “knockout” virus (and its attendant rescuant), generated on a GPCMV bacterial artificial chromosome construct, confirmed the essential nature of the gene. Conventional homologous recombination was used to generate a GP83 mutant to examine the role of GP83 in the viral life cycle. Comparison of the one-step growth kinetics of the GP83 mutant (vAM409) and wild-type GPCMV indicated that GP83 protein is not required for viral replication in tissue culture. The role of GP83 in vivo was examined by comparing the pathogenesis of wild-type GPCMV, vAM409, and a control virus, vAM403, in guinea pigs. The vAM409 mutant was significantly attenuated for dissemination in immunocompromised strain 2 guinea pigs, suggesting that the GP83 protein is essential for full pathogenicity in vivo.

scholarly journals Studies on the activation of rat liver pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase by adrenaline and glucagon. Role of increases in intramitochondrial Ca2+ concentration

1985 ◽  
Vol 231 (3) ◽  
pp. 597-608 ◽  
Author(s):  
J G McCormack
Keyword(s):  
Rat Liver ◽  
Pyruvate Dehydrogenase ◽  
Liver Mitochondria ◽  
Active Enzyme ◽  
Female Rats ◽  
Rat Liver Mitochondria ◽  
Na Ions ◽  
Oxoglutarate Dehydrogenase

The administration in vivo of either adrenaline or glucagon alone resulted in increases of about 2-fold in the amounts of active, non-phosphorylated, pyruvate dehydrogenase in the livers of fed male or female rats, whereas when administered together increases of about 4-fold were obtained. Ca2+-dependent increases in the amount of active enzyme of up to about 5-fold could be achieved in isolated rat liver mitochondria by incubating them with increasing extramitochondrial [Ca2+]; from this, two conditions of Ca loading were chosen which caused increases in active enzyme similar to those with the hormone treatments given above. The increases in enzyme activity owing to these Ca loads persisted through the ‘re-isolation’ of mitochondria and their incubation in Na+-free KCl-based media containing EGTA. Differences from values obtained with unloaded controls could be diminished by adding Na+ ions to cause the egress of Ca2+ from the mitochondria, or enough extramitochondrial Ca2+ to saturate the enzyme in its Ca2+-dependent activation; the effects of Na+ could be blocked by diltiazem, an inhibitor of mitochondrial Na+/Ca2+ exchange. The re-isolated, Ca-preloaded, mitochondria also exhibited enhanced activities of 2-oxoglutarate dehydrogenase when assayed at non-saturating [2-oxoglutarate] by two different methods; effects of Na+, Ca2+ or diltiazem on the persistent activations of this enzyme were similar to those for pyruvate dehydrogenase. Na+ caused a marked depletion, which could be blocked by diltiazem, of the 45Ca content of re-isolated mitochondria which had pre-loaded with Ca, containing 45Ca, to the same degrees as above. The activities of pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase in incubated liver mitochondria prepared from rats subjected to the hormone treatments given above were found to behave in a very similar manner to those exhibited in the re-isolated, Ca-preloaded, mitochondria. It is concluded that these hormones each bring about the activations of these rat liver enzymes by causing increases in intramitochondrial [Ca2+], and that their effects, as such, are additive.

L-649,923, Sodium (βS*, γR*)-4-(3-(4-acetyl-3-hydroxy-2-propylphenoxy)-propylthio)-γ-hydroxy-β-methylbenzenebutanoate, a selective, orally active leukotriene receptor antagonist

1986 ◽  
Vol 64 (8) ◽  
pp. 1068-1075 ◽  
Author(s):  
T. R. Jones ◽  
R. Young ◽  
E. Champion ◽  
L. Charette ◽  
D. Denis ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Competitive Inhibition ◽  
Competitive Inhibitor ◽  
Plot Analysis ◽  
Prostaglandin F ◽  
Leukotriene Receptor ◽  
Orally Active ◽  
Intraduodenal Administration

L-649,923, Sodium (βs*, γR*)-4-(3-(4-acetyi-3-hydroxy-2-propylphenoxy)propylthio)-γ-hydroxy-β-methylbenzenebutanoate is a selective and competitive inhibitor of [3H]leukotriene D4 (Ki value of 400 nM) and to a lesser extent [3H]leukotriene C4 (Ki value of 8.6 μM) binding in guinea-pig lung homogenates. Functionally, it selectively antagonized contractions of guinea pig trachea induced by leukotriene C4, D4, E4, and F4 but not those induced by acetylcholine, histamine, serotonin, prostaglandin F2α, or U-44069 (stable endoperoxide analogue). Schild plot analysis indicated a competitive inhibition of contractions of guinea-pig ileum induced by leukotriene D4 (pA2 8.1) and contractions of guinea-pig trachea induced by leukotrienes E4 and F4 (pA2 7.1 and 6.9, respectively). In contrast, contractions of guinea-pig trachea induced by leukotrienes C4 (pA2 7.2; slope 0.6) and D4 (pA2 7.2; slope 0.7) were inhibited in a noncompetitive fashion. In vivo, intravenously administered L-649,923 selectively blocked bronchoconstriction induced in anesthetized guinea pigs by leukotriene C4 and D4 (ED50 values i.v. 0.38 and 0.26 mg/kg, respectively) but not that induced by histamine, arachidonic acid, serotonin, U-44069, or acetylcholine. Following intraduodenal administration, L-649,923, blocked leukotriene D4 induced bronchoconstriction (5 and 10 mg/kg). The present findings indicate that selective antagonists, such as L-649,923, may be useful for defining the role of leukotrienes in diseases such as bronchial asthma.

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