Ammonia formation from glutamine isomers by nonacidotic and acidotic perfused rat kidneys.

The following points summarize these findings: (i) there are 2 glutamine utilizing enzyme systems in the rat kidney; (ii) the cytoplasmic glutamyltransferase system hydrolyzes either glutamine isomer while the mitochondrial localized glutaminase 1 is specific for the L-isomer; (iii) the cytoplasmic pathway contributes 70% of the total renal ammonia production in the normal kidney; (iv) chronic metabolic acidosis results in a 20-fold activation of the mitochondrial glutaminase 1 pathway.     

Ammonia production and pathways of glutamine utilization in rat kidney slices.

Ammonia production from glutamine was studied in slices from non-acidotic and acidotic rat kidneys. Slices from non-acidotic kidneys made 53% as much ammonia from D-glutamine as from L-glutamine during the initial 15 min of incubation. Thereafter the production rate from the L-isomer accelerated while that from the D-isomer remained constant. The accelerated rate of ammonia production from L-glutamine was dependent upon tissue swelling since prevention of swelling reduced the production rate. Swelling activates the mitochondrial glutaminase I pathway as evidenced by the rise in ammonia produced per glutamine utilized ratio as well as by the accelerated rate of CO2 production derived from the oxidative disposal of glutamin's carbon skeleton. Cortical slice swelling activates the mitochondrial pathway in a manner not unlike that seen in vivo during chronic acidosis and may reflect increased permeability to glutamine. Acidotic rat kidneys are not swollen in vivo while cortical slices initially produce 4-fold more ammonia than do non-acidotic slices. After 15 min, this 4-fold difference in total ammonia production drops to only a 2-fold difference due to the swelling-induced activation of the mitochondrial pathway. Consequently, slice swelling obliterates the important fact that ammonia production by the mitochondrial pathway is 15-fold greater in acidotic than in non-acidotic kidneys.     

Ammonia production and pathways of glutamine utilization in rat kidney slices

Ammonia production from glutamine was studied in slices from non-acidotic and acidotic rat kidneys. Slices from non-acidotic kidneys made 53% as much ammonia from d-glutamine as from l-glutamine during the initial 15 min of incubation. Thereafter the production rate from the l-isomers accelerated while that from the d-isomers remained constant. The accelerated rate of ammonia production from l-glutamine was dependent upon tissue swelling since prevention of swelling reduced the production rate. Swelling activates the mitochondrial glutaminase I pathway as evidenced by the rise in ammonia produced per glutamine utilized ratio as well as by the accelerated rate of CO₂ production derived from the oxidative disposal of glutamine's carbon skeleton. Cortical slice swelling activates the mitochondrial pathway in a manner not unlike that seen in vivo during chronic acidosis and may reflect increased permeability to glutamine.Acidotic rat kidneys are not swollen in vivo while cortical slices initially produce 4-fold more ammonia than do non-acidotic slices. After 15 min, this 4-fold difference in total ammonia production drops to only a 2-fold difference due to the swelling-induced activation of the mitochondrial pathway. Consequently, slice swelling obliterates the important fact that ammonia production by the mitochondrial pathway is 15-fold greater in acidotic than in non-acidotic kidneys.     

The transport and metabolism of glutamine by kidney-cortex mitochondria from normal and acidotic rats.

1. The oxidation of glutamine by kidney-cortex mitochondria from normal and acidotic rats was not inhibited by avenaciolide, which did inhibit glutamate uptake and oxidation. The oxidation of glutamine by these mitochondria was always greater than that of glutamate. Direct measurements of the metabolism of [1-14C]glutamine in the presence of glutamate, and of [1-14C]glutamate in the presence of glutamine, demonstrated that the uptake and metabolism of external glutamate is insufficient to account for the observed rate of glutamine uptake and metabolism. Thus the postulated glutamine/glutamate antiport does not play a quantitatively important role in the metabolism of glutamine by renal mitochondria. 2. Rapid swelling of these mitochondria was observed in iso-osmotic solutions of L-glutamine and L-glutamyl-gamma-monohydroxamate but not in D-glutamine or L-isoglutamine (1-amido-2-aminoglutaric acid). Thus a relatively specific glutamine uniport exists in these mitochondria. 3. The utilization of glutamine was increased about 3-fold in mitochondria from chronically acidotic rats. Thus mitochondrial adaptations play an important part in the renal response to metabolic acidosis.     

Glutamine release from hindlimb and uptake by kidney in the acutely acidotic rat.

The arteriovenous difference (release) for glutamine across the hindlimb increases significantly during acute HCl-induced acidosis. This additional amount release by muscle tissue can account for the extra glutamine taken up by the kidney.     

Renal glutamine utilization: glycylglycine stimulation of gamma-glutamyltransferase

Glycylglycine stimulation of renal glutamine utilization was studied on the homogenate, subcellular and purified enzyme level. The results clearly establish the existence of two glutamine utilizing pathways, the mitochondrial dependent L-glutamine amidohydrolase (PDG) and a second, extramitochondrial pathway. In contrast to the mitochondrial pathway which produces stoichiometric amounts of ammonia and glutamate, this second pathway hydrolyzes glutamine to produce ammonia and transfers the gamma-glutamyl moiety, producing gamma-glutamyl peptides. In the crude systems, containing cyclotransferase, the gamma-glutamyl moiety appears mainly as 5-oxoproline; however, in the enzyme preparation, purified 112-fold, gamma-glutamyl peptides (transpeptidation) and a small amount of glutamate (hydrolysis) appear. D-Glutamine was also hydrolyzed, in contrast to the stereospecific PDG, but at less than one-half the rate of the L-isomer. The molecular weight of this extramitochondrial D- and L-glutamine utilizing enzyme was estimated by gel filtration on a Sephadex G-200 column and found to be approximately 70 000. Based on product formation, molecular weight estimation and copurification with the activity responsible for p-nitroanilide release from gamma-glutamyl-p-nitroanilide, we conclude that this reaction is catalyzed by gamma-glutamyltranspeptidase. Glycylglycine stimulated this enzyme to produce more ammonia while decreasing the appearance of glutamate; in contrast, the mitochondrial glutaminase was unaffected by glycylglycine. This extramitochondrial glutamine utilizing pathway can make a significant contribution to in vivo renal ammoniagenesis.     

Redox control of glutamine utilization in cancer

Glutamine utilization promotes enhanced growth of cancer cells. We propose a new concept map of cancer metabolism in which mitochondrial NADH and NADPH, in the presence of a dysfunctional electron transfer chain, promote reductive carboxylation from glutamine. We also discuss why nicotinamide nucleotide transhydrogenase (NNT) is required in vivo for glutamine utilization by reductive carboxylation. Moreover, NADPH, generated by both the pentose phosphate pathway and the cancer-specific serine glycolytic diversion, appears to sustain glutamine utilization for amino-acid synthesis, lipid synthesis, and for ROS quenching. The fact that the supply of NAD⁺ precursors reduces tumor aggressiveness suggests experimental approaches to clarify the role of the NADH-driven redox network in cancer.     

Adenylate cyclase from rat-liver plasma membrane: inhibition by mersalyl and other mercurial derivatives.

The adenylate cyclase activity from a rat liver plasma membrane preparation was inhibited by low concentrations (1-10 muM) of the mercurial diuretic mersalyl. Complete inhibition was obtained with 0.1 mM mersalyl. Similar effects were observed whether the adenylate cyclase preparation was assayed in the presence of 10 muM GTP, 0.1 muM glucagon, 10 mM NaF or without any addition. The effect of mersalyl was not due to inhibition of the regenerating system present in the incubation medium, since the effect of mersalyl was preserved and even enhanced in its absence. The inhibition brought about by mersalyl was due to both a decrease of the maximal velocity of the reaction and of the affinity of the enzyme for the substrate. It was immediate, and irreversible spontaneously, but it was reversed by the simultaneous additions of 2-mercaptoethanol, in a dose-dependent fashion. Other -SH reagents were found to have an effect equal to, or lower than, that of mersalyl. Mersalyl had no effect upon Mg2+-ATPase, although it inhibited the (Na+-K+) activated ATPase. Since mersalyl is known to be a 'non-penetrant' reagent, it is postulated that a catalytically important, mercurial-sensitive, part of adenylate cyclase is at the surface of the plasma membrane. This view is supported by the following facts: (a) mersalyl acted with a similar dose-response curve upon an intact as well as a detergent-dispersed cyclase preparation while no effect was observed upon a solubilized Mg2+-ATPase preparation; (b) a covalent p-chloromercuribenzoate-Sephadex preparation (but not its supernatant) inhibited the cyclase from intact membranes. It is proposed that mercurial derivatives, by their relative specificity of action (no effect on Mg2+-ATPase), can serve as useful probes in the elucidation of the multicomponent structure of the cyclase system.     

Role of ADH in ethylketocyclazocine-induced diuresis: studies in the Brattleboro rat

Kappa opioids produce diuresis presumably through ADH. We investigated further the role of ADH in kappa-induced diuresis by utilizing the Brattleboro rat, a strain lacking endogenous ADH. Ethylketocyclazocine (EKC), a kappa opioid prototype, increased urine formation in Sprague-Dawley, but not in Brattleboro rats. Furthermore, EKC pretreatment abolished the antidiuretic response to ADH administered exogenously to Brattleboro rats. Our study suggests that, in addition to a fall in plasma ADH reported previously, kappa opioids have direct effects on the renal response to ADH.     

Glucose and glutamine utilization by rat lymphocytes, monocytes and neutrophils in culture: a comparative study

Glucose and glutamine utilization and production of glutamate and lactate were determined for up to 48 h in lymphocytes, monocytes and neutrophils cultured in medium rich in metabolites and vitamins. Glucose was utilized by the three cell types in culture in the following order: neutrophils > monocytes > lymphocytes, whereas lactate was produced in the order: monocytes > neutrophils > lymphocytes. The consumption of glucose followed the activity of glucose-6-phosphate dehydrogenase but it was not related to hexokinase activity. Glutamine was consumed by the three leukocyte types in culture as follows: neutrophils > lymphocytes > or = monocytes. The consumption of glutamine was not fully related to the activity of phosphate-dependent glutaminase. The production of glutamate was not remarkably different among the three cell types. For comparison, glutamine and glucose utilization and glutamate and lactate production were also evaluated using 1-h incubated leukocytes. Under this condition, only glucose or glutamine was added to the medium. Glucose was utilized as follows: neutrophils > monocytes > lymphocytes, whereas lactate was produced in the following order: monocytes > or = neutrophils > lymphocytes. Glutamine was consumed as follows: neutrophils > lymphocytes > monocytes, whereas glutamate was produced as follows: neutrophils > or = monocytes = lymphocytes. The ratio of the amount of glucose/glutamine consumed by 1-h incubated cells was 0.5 for neutrophils, 1.5 for monocytes, and 0.3 for lymphocytes. However, the three cell types cultured for 48 h utilized glucose to a much higher degree than glutamine. The ratio of the amount of glucose/glutamine utilized by the cultured cells was 8.9 for neutrophils, 16.4 for monocytes, and 6.7 for lymphocytes. These observations support the proposition that glutamine is required in much higher amounts than glucose to accomplish the total metabolic requirement of leukocytes. Under conditions closer to physiological when the availability of a variety of metabolites and vitamins is not restricted, glucose is the preferred substrate for lymphocytes, monocytes and neutrophils.