Role of alpha-adrenergic stimuli in the control of rat renal ammoniagenesis

The effects of phenylephrine on renal ammoniagenesis and the involvement of Ca2+ in phenylephrine action were investigated in isolated proximal fragments of rat-kidney tubules. Phenylephrine stimulated renal ammoniagenesis from 1 and 2 mM glutamine whereas no significant changes took place at a higher concentration of glutamine (20 mM). Stimulation of ammonia synthesis by phenylephrine was found to be linear with time and dose-dependent between 10(-9) and 10(-4) M. Phenylephrine-stimulated ammoniagenesis was blocked by phentolamine (10 microM) but not by propranolol (10 microM) confirming that the effect is mediated by alpha-adrenergic stimuli. No stimulatory effect of phenylephrine was observed in Ca2+ depleted proximal tubule fragments, suggesting that Ca2+ is required in this adrenergic response.     

The relationship of 4-aminobutyric acid metabolism to ammoniagenesis in renal cortex

Mitochondrial 4-aminobutyrate aminotransferase in rat kidney can utilize pyruvate as the acceptor for the amino group of 4-aminobutyrate. Renal 4-aminobutyrate aminotransferase activity at saturating equimolar concentration of 4-aminobutyrate and 5 mM pyruvate is 42.8 ± 2.5 μmol/g protein per h (mean ± S.E.M.) or 70% of 4-aminobutyrate aminotransferase activity with equimolar α-ketoglutarate. 4-Aminobutyrate aminotransferase in brain does not transaminate with pyruvate. Since pyruvate is an important mitochondrial metabolite in kidney, net disposal of glutamate via the 4-aminobutyrate pathway is possible. The renal 4-aminobutyrate pathway in the rat has other distinctive features when compared with the pathway in rat brain. Most inhibitors of rat neuronal glutamate decarboxylase were ineffective against the renal form of the enzyme, but 20 mM semicarbazide inhibited the latter form by 80% (P < 0.001) in vitro and reduced renal 4-aminobutyrate content by 75% (P < 0.001) in vivo. In the presence of 20 mM semicarbazide, ammoniagenesis by rat renal cortex slices incubated in 1 mM glutamine was inhibited 26% (P < 0.01). Semicarbazide was proportionately less effective (15% inhibition) when ammoniagenesis was stimulated (+243%) in slices prepared from chronically acidotic animals, and was no deterrant to ammoniagenesis when non-acidotic slices were incubated in supraphysiologic concentrations of 10 mM glutamine. We conclude that whereas integrity of the renal 4-aminobutyrate pathway may contribute to glutamate disposal and thus ammoniagenesis under physiologic conditions, the pathway is a passive participant in the overall process of ammoniagenesis.     

Regulation of renal ammoniagenesis in the rabbit

• 1.1. In order to identify sites of regulation of ammoniagenesis in rabbit renal cortex, the activities of the key steps in this pathway were measured.
• 2.2. Results indicate that the flux through the mitochondrial dicarboxylate transporter could limit ammoniagenesis in this species.

     

Effects of acid challenge on in vivo and in vitro rat renal ammoniagenesis.

This study compares the ability of different strengths of NH₄Cl, CaCl₂, and HCl to affect the termporal excretion of ammonium in rats. Oral NH₄Cl given in a single dose of 0.5 mmole, 1.0 mmole and 1.5 mmole/100 g BW steadily increases ammonium excretion in rats. The majority of the augmented ammonium excretion is secondary to increased renal production — not to changes in urine pH or urine volume. Acute challenges greater than 1.5 mmole/100 g BW do not increase ammonium excretion further. Results were similar when chronic acid challenge was investigated — greater NH₄Cl challenges cause greater ammonium excretion. Challenges beyond 1.5 mmole/100 g BW bid frequently cause death unless the rats are preconditioned (made mildly acidotic) prior to initiation of this dose. At the 1.5 mmole/100 g BW dose, maximal ammonium excretion is reached by day 2 or 3. Thus, maximal renal ammoniagenesis during acid stress occurs rapidly, and at different times depending on the strength of the acid challenge. CaCl₂ or HCl offer no advantages over NH₄Cl as acidifying agents. In addition to the above, there is a significant correlation between ammonium excretion $\text{in vivo}$ and the ability of rat renal slices to produce ammonia from glutamine or glutamate $\text{in vitro}$.     

Adaptive ammoniagenesis in chronic renal failure.

The role of gamma-glutamyltransferase (gamma-GT) in renal ammoniagenesis, glutamine (Gln), and glutathione (GSH) utilization was evaluated in the intact functioning rat kidney of subtotal nephrectomy (SNX) model of chronic renal failure (CRF). NH4+ derived from extracellular gamma-GT hydrolysis of Gln and GSH was differentiated from the intramitochondrial phosphate-dependent glutaminase by using acivicin, a gamma-GT-specific inhibitor. In the control (C) group Gln extraction accounted for 61% of total NH4+ production (sum of renal venous and urinary NH4+), but only 41% in SNX group. In the SNX group GSH extraction accounted for 10% of total NH4+ production, but only 1% in the C group. Acivicin inhibited 44% and 33% of total NH4+ production in SNX and C group respectively, as compared to baseline before acivicin. In CRF, gamma-GT a key enzyme of the gamma-glutamyl cycle plays a significant role in adaptive ammoniagenesis.     

Altered oxidative metabolism in selenium-deficient rat granulocytes

Rats fed a selenium-deficient diet for 12 to 15 wk became selenium-depleted, measured by the selenium content of liver and granulocytes. The activity of granulocyte glutathione peroxidase, a selenoenzyme, in deficient rats was 11% of the activity in replete rat granulocytes. When stimulated with an H2O2 generating system, the HMPS activity of the deficient granulocytes was 50% of replete; however, when stimulated with methylene blue, the HMPS activity in deficient and replete granulocytes was the same. When granulocytes were incubated with PMA or OPZ, deficient granulocytes initially had the same O-2-generating activity as replete granulocytes; however, with increasing duration of stimulation, granulocytes from deficient rats generated less O-2 than replete rats. After 20 min in an H2O2-generating system, deficient granulocytes stimulated with PMA or OPZ generated less O-2 than replete granulocytes. These results indicate that deficient granulocytes did not metabolize H2O2 as well as replete granulocytes and that H2O2 caused damage to the O2-generating system. Measurement of O-2 generation in membrane-enriched particles showed the above effects were due to inactivation of the NADPH-dependent O2-generating system. Deficient granulocytes stimulated with OPZ for 20 min had 70% less membrane O-2-generating activity than controls. In addition, when membrane-enriched particles were made from cells that had been stressed with an H2O2-generating system, NADPH-dependent O-2-generating activity in deficient granulocytes was 50% of replete. In selenium-deficient granulocytes with low GSH-Px activity, prolonged incubation with stimulants and prior incubations with an H2O2-generating system caused loss of activity of the membrane-bound, NADPH-dependent, O-2-generating system.     

Lipid peroxidation and alterations to oxidative metabolism in mitochondria isolated from rat heart subjected to ischemia and reperfusion.

Ischemia-reperfusion injury to cardiac myocytes involves membrane damage mediated by oxygen free radicals. Lipid peroxidation is considered a major mechanism of oxygen free radical toxicity in reperfused heart. Mitochondrial respiration is an important source of these reactive oxygen species and hence a potential contributor to reperfusion injury. We have examined the effects of ischemia (30 min) and ischemia followed by reperfusion (15 min) of rat hearts, on the kinetic parameters of cytochrome c oxidase, on the respiratory activities and on the phospholipid composition in isolated mitochondria. Mitochondrial content of malonyldialdheyde (MDA), an index of lipid peroxidation, was also measured. Reperfusion was accompanied by a significant increase in MDA production. Mitochondrial preparations from control, ischemic and reperfused rat heart had equivalent Km values for cytochrome c, although the maximal activity of the oxidase was 25 and 51% less in ischemic and reperfused mitochondria than that of controls. These changes in the cytochrome c oxidase activity were associated to parallel changes in state 3 mitochondrial respiration. The cytochrome aa3 content was practically the same in these three types of mitochondria. Alterations were found in the mitochondrial content of the major phospholipid classes, the most pronounced change occurring in the cardiolipin, the level that decreased by 28 and by 50% as function of ischemia and reperfusion, respectively. The lower cytochrome c oxidase activity in mitochondria from reperfused rat hearts could be almost completely restored to the level of control hearts by exogenously added cardiolipin, but not by other phospholipids nor by peroxidized cardiolipin. It is proposed that the reperfusion-induced decline in the mitochondrial cytochrome c oxidase activity can be ascribed, at least in part, to a loss of cardiolipin content, due to peroxidative attack of its unsaturated fatty acids by oxygen free radicals. These findings may provide an explanation for some of the factors that lead to myocardial reperfusion injury.     

Adenosine metabolism in a rat hippocampal slice preparation: incorporation into S-adenosylhomocysteine

Abstract: The incorporation of [¹⁴C]adenosine into various metabolites was studied in a hippocampal slice preparation in order to assess the extent of adenosine metabolism via synthesis of S -adenosylhomocysteine, a potent inhibitor of transmethylation reactions. Highest incorporation of ¹⁴C occurred into nucleotides, with only a few percent being recovered in inosine + hypoxanthine, S -adenosylhomocysteine, and the free adenosine pool. Labeling of S -adenosylhomocysteine did not significantly increase with higher concentrations of added adenosine despite greater accumulation of free [¹⁴C]adenosine in the tissue. Addition of l -homocysteine significantly increased the labelling of S -adenosylhomocysteine. The results indicate that S -adenosylhomocysteine synthesis is a minor pathway of adenosine metabolism in brain tissue under steady-state conditions. Further, changes in adenosine concentration, without a concomitant change in l -homocysteine availability, are unlikely to lead to a significant accumulation of S -adenosylhomocysteine. S -Adenosylhomocysteine is therefore not likely to play a significant role in mediating the biological effects of adenosine in the CNS via inhibition of transmethylations.     

Effects of malonate administration on renal ammoniagenesis in intact dogs

In the dog kidney in vivo, malonate augmented ammoniagenesis from both amide and nonamide nitrogen sources, similar to previous in vitro investigations using incubating canine renal tubules. This was highly significant in alkalotic dogs, where it was accompanied by decreased renal tissue concentrations of glutamate. Changes in renal ammonia metabolism were less evident in acidotic dogs where a markedly decreased glomerular filtration rate was noted following malonate administration. Under conditions of complete ureteric obstruction which effectively abolished glomerular filtration, malonate significantly augmented ammoniagenesis above baseline in acidotic dogs. These in vivo results with malonate have similarities to those seen in dogs subjected to an acid challenge alone and suggest that the adaptation in renal ammoniagenesis under both circumstances occurs via enhanced deamination of glutamate pools.     

5-aminolevulinic acid-induced alterations of oxidative metabolism in sedentary and exercise-trained rats.

5-Aminolevulinic acid (ALA), a heme precursor that accumulates in acute intermittent porphyria patients and lead-exposed individuals, has previously been shown to autoxidize with generation of reactive oxygen species and to cause in vitro oxidative damage to rat liver mitochondria. We now demonstrate that chronically ALA-treated rats (40 mg/kg body wt every 2 days for 15 days) exhibit decreased mitochondrial enzymatic activities (superoxide dismutase, citrate synthase) in liver and soleus (type I, red) and gastrocnemius (type IIb, white) muscle fibers. Previous adaptation of rats to endurance exercise, indicated by augmented (cytosolic) CuZn-superoxide dismutase (SOD) and (mitochondrial) Mn-SOD activities in several organs, does not protect the animals against liver and soleus mitochondrial damage promoted by intraperitoneal injections of ALA. This is suggested by loss of citrate synthase and Mn-SOD activities and elevation of serum lactate levels, concomitant to decreased glycogen content in soleus and the red portion of gastrocnemius (type IIa) fibers of both sedentary and swimming-trained ALA-treated rats. In parallel, the type IIb gastrocnemius fibers, which are known to obtain energy mainly by glycolysis, do not undergo these biochemical changes. Consistently, ALA-treated rats under swimming training reach fatigue significantly earlier than the control group. These results indicate that ALA may be an important prooxidant in vivo.     

Renal metabolism and ammoniagenesis during acute respiratory alkalosis in the dog

Acute respiratory alkalosis (blood pH, 7.60; arterial PCO2, 15 mmHg (1 mmHg = 133.322 Pa); plasma bicarbonate, 14 mM) was induced in nine anesthetized dogs by increasing their respiratory rate and depth. Renal glutamine extraction and ammonia production expressed per 100 mL of glomerular filtration rate did not change during acute hypocapnia, whereas arterial glutamine concentration decreased significantly from 0.47 to 0.36 mM. Hypocapnia did not change plasma potassium concentration and its urinary excretion. Acute hypocapnia increased lactate extraction and pyruvate production, whereas citrate extraction and glutamate and alanine production did not change. Citraturia remained minimal. Renal cortical glutamine concentration fell from 0.64 to 0.38 mM during hypocapnia while alpha-ketoglutarate, glutamate, malate, oxaloacetate, and citrate did not change. Lactate concentration rose from 1.1 to 2.0 mM. Glutamine concentration in the liver and muscle decreased following acute hypocapnia. Our data are compatible with the hypothesis that an acute respiratory alkalosis might not result in any change in the hydrogen ion concentration and (or) gradient between the mitochondrial matrix and the cytosol. Consequently, renal glutamine extraction and ammonia production are not reduced, renal cortical concentrations of relevant metabolites in the ammoniagenic pathway are not changed, and renal handling of citrate remains unaffected.     

The purine nucleotide cycle and ammoniagenesis in rat kidney tubules

The contribution of the purine nucleotide cycle to renal ammoniagenesis was examined in cortical tubule suspensions prepared from acidotic rats and incubated with [alpha-15N]glutamine, [15N]glutamate, or [15N]aspartate. Labeling of ammonia and adenine nucleotides was determined after enzymatic transformations designed to circumvent the technical problem that 15NH3 and H2O have the same nominal mass. Labeling of the adenine nucleotide was undetectable (less than 10%) even after 1 h of incubation. From the measured concentrations of adenine nucleotides and ammonia and the labeling of the ammonia, the flux through the purine nucleotide cycle was calculated to account for less than 1% of the deamination of alpha-amino groups from all three substrates. The glutamate dehydrogenase reaction is therefore the likely pathway for deamination. The rate of 15NH3 production from [alpha-15N]glutamine was two or three times greater than from added [15N]glutamate, indicating a preference for intracellularly generated glutamate. 15NH3 production from added [15N]aspartate was similar to and perhaps slightly greater than that from added [15N]glutamate.     

6-Mercaptopurine-induced alterations in mineral metabolism and teratogenesis in the rat

The relationship between 6-mercaptopurine-induced alterations in mineral metabolism and the teratogenic effects of the drug were investigated. Pregnant Sprague-Dawley rats were fed diets containing 4.5, 100, or 1,000 micrograms Zn per 1 g diet. On day 11 of gestation, dams were given intraperitoneal injections of 6-mercaptopurine (27.5 mg/kg). At term, dams fed the 1,000-micrograms Zn per 1 g diet showed fewer drug-induced deleterious effects on reproduction and embryogenesis than did those fed lower levels of zinc. Mineral analysis of maternal and fetal tissues revealed pronounced effects of 6-mercaptopurine on metabolism of zinc, copper, iron, calcium, and magnesium. The results of this study indicate that 6-mercaptopurine teratogenesis may be due in part to drug-induced changes in mineral metabolism.     

Phospholipid metabolism in the rat renal inner medulla

In view of the importance of phospholipids as a source of precursor fatty acids for the high prostaglandin synthesis in the renal inner medulla, we studied pathways of phospholipid esterification and degradation in the rat inner medulla. De novo acylation of [14C]arachidonate occurred predominantly in position 2 of phosphatidylcholine in the microsomal fraction. This newly esterified [14C]arachidonate was accessible to deacylation by a microsomal phospholipase A2 (EC 3.1.1.4) with alkaline optimum which was Ca2+-dependent and resistant to 0.1% deoxycholate. No phospholipase A1 (EC 3.1.1.32) activity against endogenous labeled phosphatidylcholine could be demonstrated in the microsomal fraction. When exogenous phosphatidylcholine labeled at position 2 was deacylated by renomedullary homogenates, labeled free fatty acid but no labeled lysophosphatidylcholine was recovered in the reaction products. This could be attributed to further degradation of generated lysophosphatidylcholine by a cytosolic lysophospholipase (EC 3.1.1.5). Sodium deoxycholate at a concentration of 0.1% or higher inhibited the lysophospholipase and allowed the demonstration of both A2 and A1 alkaline phospholipase activities in the homogenate. The major in vitro pathway of lysophosphatidylcholine disposition is further degradation by a cytosolic lysophospholipase, while reutilization for phosphatidylcholine synthesis through the action of a predominantly microsomal acyltransferase appears to be a minor pathway. In the presence of several acyl-CoAs, reutilization of lysophosphatidylcholine is significantly increased by an acyl-CoA:lysophosphatidylcholine acyltransferase (EC 2.3.1.23) but there is no preferential transfer of arachidonyl-CoA compared to other acyl-CoAs.     

Carnosine treatment largely prevents alterations of renal carnosine metabolism in diabetic mice

Recently, we identified an allelic variant of human carnosinase 1 (CN1) that results in increased enzyme activity and is associated with susceptibility for diabetic nephropathy in humans. Investigations in diabetic (db/db) mice showed that carnosine ameliorates glucose metabolism effectively. We now investigated the renal carnosinase metabolism in db/db mice. Kidney CN1 activity increased with age and was significantly higher in diabetic mice compared to controls. Increased CN1 activity did not affect renal carnosine levels, but anserine concentrations were tenfold lower in db/db mice compared to controls (0.24±0.2 vs. 2.28±0.3 nmol/mg protein in controls; p<0.001). Homocarnosine concentrations in kidney tissue were low in both control and db/db mice (below 0.1 nmol/mg protein, p=n.s.). Carnosine treatment for 4 weeks substantially decreased renal CN1 activity in diabetic mice (0.32±0.3 in non-treated db/db vs. 0.05±0.05 μmol/mg/h in treated db/db mice; p<0.01) close to normal activities. Renal anserine concentrations increased significantly (0.24±0.2 in non-treated db/db vs. 5.7±1.2 μmol/mg/h in treated db/db mice; p<0.01), while carnosine concentrations remained unaltered (53±6.4 in non-treated vs. 61±15 nmol/mg protein in treated db/db mice; p=n.s.). Further, carnosine treatment halved proteinuria and reduced vascular permeability to one-fifth in db/db mice. In renal tissue of diabetic mice carnosinase activity is significantly increased and anserine concentrations are significantly reduced compared to controls. Carnosine treatment largely prevents the alterations of renal carnosine metabolism.