Inhibition of urea synthesis by pent-4-enoate associated with decrease in N-acetyl-L-glutamate concentration in isolated rat hepatocytes

Pent-4-enoate at 0.1 to 1.0 mM strongly inhibited urea synthesis in isolated rat hepatocytes. Pent-4-enoate at the same concentrations markedly decreased concentrations of $\text{N-}\text{acetyl-}\text{L}\text{-glutamate}$, an essential activator of carbamoyl-phosphate synthase-I (EC 2.7.2.5), and the decrease was well parallel with the inhibition of urea synthesis by pent-4-enoate. This compound also lowered cellular concentrations of acetyl-CoA, a substrate of acetylglutamate synthase (EC 2.3.1.1). Pent-4-enoate in a dose of 1 mM did not significantly affect cellular concentrations of ATP, and had no direct effect on acetylglutamate synthase activity. These results suggest that the inhibition of urea synthesis by pent-4-enoate is due to decrease in $\text{N-}\text{acetyl-}\text{L}\text{-glutamate}$ concentration and that the decrease is probably brought about by decreased rate of its synthesis due to the lowered concentration of cellular acetyl-CoA.     

Sources of ammonia for urea synthesis in isolated rat liver cells

l-Leucine inhibits urea synthesis in rat hepatocytes from a number of nitrogen sources, including ammonia. The inhibition by l-leucine is largely overcome by addition of 1 mM l-ornithine, suggesting that the main site of l-leucine action is at ornithine transcarbamylase, rather than at glutamate dyhydrogenase. l-Norvaline is a more potent inhibitor of urea synthesiss than is l-leucine, but again the inhibition is largely counteracted by l-ornithine. Addition of aminooxyacetate and l-norvaline strongly suppresses the formation of glucose and lactate from l-asparagine, suggesting that an alternate pathway of aspartate metabolism, the purine nucleotide cycle, in not a major pathway. Hadacidin, an inhibitor of adenylosuccinate synthetase, an enzyme of the purine nucleotide cycle, has no effect on urea synthesis in rat liver cells.     

土壤盐渍化对尿素与磷酸脲氨挥发的影响

氨挥发是肥料氮素损失的重要途径之一,肥料类型、土壤类型、肥料用量以及土壤全盐量均影响氨挥发损失率及挥发特征。本文采用通气法测定了磷酸脲和尿素两种肥料六个施肥量处理分别施入六个不同盐渍化程度（1.7、9.9、16.4、23.2、29.1、37.9 g/kg）的土壤后氨挥发累积状况和动力学特性,以及土壤氨挥发累积量与土壤电导值之间的相关性。结果表明：（1）在土壤总盐介于1.66 -37.9 g/kg的范围内,随着土壤含盐量增加,尿素与磷酸脲处理的氨挥发累积量显著增加;土壤含盐量对氨挥发速率有显著的促进作用。（2）各处理二次线性函数拟合的二项式系数a均为负值,表明：在不同盐渍化条件下肥料的挥发速率是随着时间增长而降低的;一次线性函数和Elovich 方程的斜率a随土壤含盐量增加而增大,表明：土壤盐渍化将加剧土壤的氨挥发速率。（3）土壤氨挥发累积量与电导值拟合结果符合logistic方程（︱R︱分别为0.9732,0.9815,0.965,0.9182,0.9817,0.9971︱R︱>r0.01=0.9172, n=6）,氨挥发累积量随土壤电导值呈“S”型增长。     

Effects of a slow-release urea source on absorption of ammonia and endogenous production of urea by cattle

Three experiments were conducted with Angus or Holstein steers to evaluate effects of dietary urea–calcium (a slow rumen-release urea source) on absorption of ammonia N from the gut and urea N production in the liver. Steers were fed a high-grain diet (Experiment 1) or an all-forage diet (Experiments 2 and 3). Urea or urea–calcium (0.25 g/kg body weight) was dosed into the esophagus (Experiments 1 and 2) or rumen (Experiment 3), and blood samples were serially collected for 180 min. Blood concentrations of ammonia N and urea N were measured in all experiments, and net flux of metabolites across splanchnic tissues was measured in Experiment 3. Compared to urea, urea–calcium reduced (P<0.05) plasma concentrations of ammonia N in steers fed all-forage diets, and tended (P<0.06) to reduce arterial glucose concentrations in Experiment 3. Plasma concentrations of urea N were not affected by treatment in any experiment. Treatment and time post-dosing interactions (P<0.05) in Experiment 3 were due to increased ruminal fluid concentrations of ammonia N, net release of ammonia N by portal-drained viscera and total splanchnic tissues with urea versus urea–calcium treatment shortly after dosing. Similar interactions (P<0.05) indicated that urea caused higher hepatic glucose release and increased l-lactate release by total splanchnic tissues after dosing than urea–calcium. Urea–calcium was effective in mitigating rapid ammonia release in the rumen and subsequent effects on glucose and lactate metabolism.     

Mitochondrial compartmentation of metabolic CO2 resulting from its site of origin in relation to urea synthesis

In isolated hepatocytes the entry into urea of metabolic ¹⁴CO₂; derived from [¹⁴C] formate is modified by the addition of dichloroacetate and hydroxypyruvate. An explanation is that this results from changes in the cytoplasmic/mitochondrial pH gradient. ¹⁴CO₂, derived from [1-¹⁴C]alanine enters into urea more readily than ¹⁴CO₂ arising from [1-¹⁴C]glutamate. It is proposed that the difference, which is more than 4-fold, is indicative of a preferred pathway for metabolic CO₂ in liver mitochondria from pyruvate dehydrogenase to carbamoylphosphate synthetase than form oxoglutarate dehydrogenase. Acetazolamide inhibition of carbonic anhydrase is without effect on this observed incorporation into urea.     

Urea synthesis in the liver and kidney of developing sheep

In order to establish if the urea found in foetal fluids in sheep could be of foetal origin and whether there are changes in the ability of ovine liver to synthesise urea during foetal and postnatal development, the rates of urea production from ammonium and bicarbonate ions have been measured in liver and kidney slices from animals aged from 50 days conceptual age to 16 weeks after birth, and in pregnant and non-pregnant ewes. The activities of five enzymes directly involved in the biosynthesis of urea have also been determined.Urea was found to be synthesised by foetal liver from at least 50 days conceptual age at rates similar to those observed in adult ewes. Highest rates of urea synthesis per unit weight of liver were found immediately after birth. In the liver there were significant positive correlations between the rates of urea synthesis by slices and the activities of carbomoyl phosphate synthase (ammonia) (EC 2.7.2.5), argininosuccinate synthetase (EC 6.3.4.5) and argininosuccinate lyase EC 4.3.2.1). Ornithine carbomoyl transferase (EC 2.1.3.3) activity was highest in the livers of ruminating animals. Hepatic arginase activity (EC 3.5.3.1) was highest during the late foetal life and in the mature foetuses the activity was ten-fold greated than that in maternal liver.Urea was not synthesised from ammonia and bicarbonate in kidney slices and neither ornithine carbomoyl transferase activity nor argininosuccinate synthetase activity could be detected. The activity of renal arginase was at least 70 times less than that found in the liver and the highest activity was found in ruminating lambs.The changes observed in the activities of the urea cycle enzymes during development have been contrasted with those reported to occur in other species. It is concluded that there is no single factor regulating the activities of the five enzymes directly concerned with urea synthesis during development. The results support the hypothesis that in mammals the ability of the liver to synthesise urea in foetal life is related to renal development.     

Arginine uptake by isolated rat liver mitochondria

The question of arginine uptake by mitochondria is important in that arginine is an allosteric effector of N-acetylglutamate synthetase. Thus, changes in mitochondrial arginine concentration have the potential for acutely modifying levels of N-acetylglutamate, a compound necessary for maximal activity of carbamyl phosphate synthesis. Mitochondria were isolated from chow-fed rats, incubated with [guanido-¹⁴C]arginine and were centrifuged through silicon oil into perchloric acid for determination of intramitochondrial metabolites. Arginine was separated from urea by cation-exchange resin. Mitochondrial water space was determined by [¹⁴C]urea arising from arginase activity associated with the mitochondrial preparations. Extramatrix space was determined by parallel incubations with [inulin-¹⁴C]carboxylic acid or [¹⁴C]sucrose There was considerable degradation of arginine by arginase associated with the mitochondrial preparation. This was inhibited by 7 mM ornithine and 7 mM lysine. Arginine was concentrated intramitochondrially to 4-times the extramitochondrial levels. The concentration ratio was decreased in the presence of ornithine and lysine but not with citrulline, NH₄Cl, glutamate, glutamate or leucine. No uptake was observed when mitochondria were incubated at 0°C. Mitochondria did not concentrate citrulline.     

The effect of dichloroacetate and hydroxypyruvate on the entry of C from [1-C]alanine into urea in rat hepatocytes

Entry of metabolic ¹⁴CO₂ into urea is shown to be decreased by dichloroacetate although the production of ¹⁴CO₂ is stimulated 2-fold. Hydroxypyruvate, a product of dichloroacetate metabolism, increases the incorporation of metabolic ¹⁴CO₂ into urea. It is proposed that these effects result from changes in the cytoplasmic-mitochondrial pH gradient.     

Preincubation accelerates taurocholate uptake into isolated liver cells

Initial rates of taurocholate uptake into isolated hepatocytes stored at 0°C increased 3-fold during a 25 min preincubation. Concomitantly, $\text{V}$ increased while $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ remained unaffected. There are several possible explanations for the preincubation effects, such as new synthesis of carrier protein, altered fluidity of the membrane or stimulation of the sodium-dependent taurocholate uptake via a change in the cation distribution. The experiments presented strongly favor the latter explanation as the sodium gradient as well as the uptake of the bile acid reach their steady state within 20–30 min and replacement of sodium by potassium in the medium abolished the effect.     

The effect of urea synthesis on extracellular pH in isolated perfused rat liver.

In a non-recirculating system of isolated liver perfusion, stimulation of urea synthesis by NH4Cl is followed by a decrease of effluent pH by up to 0.2 pH unit. This effect is not observed when urea synthesis is inhibited by amino-oxyacetate or norvaline. When the urea formed by the liver is immediately hydrolysed with urease before the effluent perfusate reaches the pH electrode, the urea-synthesis-induced acidification is no longer observed. This indicates that accompanying alterations in hepatic metabolism after stimulation of urea synthesis, such as increased energy provision and consumption, are not responsible for the extracellular acidification, but that the effect is due to the formation of urea itself. The acidification of the extracellular space after stimulation of urea synthesis by NH4Cl is quantitatively explained by the consumption of 2 mol of HCO3-/mol of urea formed: 1 mol being incorporated into urea, the other being protonated to yield CO2 and H2O. The data match the theoretically predicted HCO3- consumption during ureogenesis and underline the role of hepatic urea synthesis for disposal of HCO3- by converting it into the excretable products CO2 and urea.     

The binding of cytochrome b5 to plasma membranes of rat liver. Its implication for membrane specificity and biogenesis

The in vitro incorporation of cytochrome $\text{b}{}_5$ into purified plasma membranes was investigated by biochemical and immunological methods. Plasma membrane preparations incorporated three times less cytochrome $\text{b}{}_5$ than did microsomal preparations; 60% of this cytochrome $\text{b}{}_5$ could not be reduced by the NADH-cytochrome $\text{b}{}_5$ reductase and was considered as being bound to the plasma membrane. The morphological observations made after the immunochemical labeling of cytochrome $\text{b}{}_5$ clearly showed a good but asymmetrical distribution of the ferritin labeling: only the inner face of the plasma membrane incorporated cytochrome $\text{b}{}_5$. These results are discussed with respect to theories which concern the subcellular membrane relationships in the cell.     

The time of urea treatment and its effects on the succession of ammonia fungi in two warm temperate forests in Japan

We studied the effects of the timing of urea treatment on the succession of ammonia fungi. In two evergreen Castanopsis cuspidata forests and in one deciduous Quercus serrata forest, we applied 343g urea to 25 and 15 plots of 0.5m², respectively, at three different times of the year. Ten of the early-phase (EP) species, considered to be saprotrophic, and 6 of the late-phase (LP) ones, considered ectomycorrhizal, fruited. In both phases, the commencement, peak, and cessation of fruiting took place simultaneously among all the plots treated at the same time. The fruiting occurred in summer and autumn. Quantity and size of the fruit bodies was larger in the LP than in the EP species. Fruiting of EP species was affected by the treatment time and that of LP species by interaction of the treatment time and vegetation type. EP was short and occurred as one period, whereas LP was long and occurred as two or more fruiting seasons. We found that species composition, dominant species, and degree of its dominance in fruiting of the ammonia fungi are predictable for different treatment times of the year and different vegetation types.     

The effects L-leucine on the synthesis of urea, glutamate and glutamine by isolated rat liver cells.

With either alanine or a mixture of 15 different amino acids as nitrogen source, the addition of L-leucine inhibited the synthesis of urea by isolated rat liver cells. With alanine present leucine promoted the production of glutamate and glutamine. Comparison of effects of leucine on soluble glutamate dehydrogenase, mitochondria and isolated cells supports the postulate that leucine exerts its effect through activation of glutamate dehydrogenase. It is suggested that this latter enzyme may not be as important for the production of NH3 for carbamoyl phosphate synthesis as has been considered hitherto.     

Long-term effectiveness of high-dosed ornithine-aspartate on urea synthesis rate and portal hypertension in human liver cirrhosis

Summary The effectiveness of ammonia reducing amino acids on hyperammonemia and hepatic encephalopathy is well known in patients suffering from liver cirrhosis. Data concerning long-term therapy on hepatic function and urea synthesis rate (UNSR) are still lacking. According to Vilstrup/Poulsen it is a good standard for functioning liver mass. Therefore, 25 patients with histologically proven liver cirrhosis and distinct portal hypertension were treated daily with 9 gr. ornithinasparte over 13 years (8–20 years). Shunt operations, esophageal varicosis sclerosis, or portal pressure reducing medication were not applied. Rigorous alcohol abstinence and 60 gr protein/day were prescribed. During the investigation, 3 laparoscopies and 4 liver biopsies were performed, on the average, on each individual. Significant improvements of clinical and biochemical results (Child-Pugh-Index; Composite Clinical and Laboratory Index) were obtained during the long-term therapy with ornithine-aspartate. Esophageal varicosis II–III was either reduced to 0-I or totally eliminated. Also significant was an increased urea synthesis rate and a decreased hyperammonemia.A plausible explanation for the long-term therapy effectiveness with ornithine-aspartate is the possible recovery of the functioning mass without hepatic size increase. Also important is the rigorous alcohol abstinence. It leads to a significant reduction of portal hypertension in patients suffering from alcohol induced liver cirrhosis (Reynolds, own observations).Additional favorable factors are intensive muscle training and absence of gastrointestinal bleeds.     

Effect of phenylphosphorodiamidate on urea hydrolysis,ammonia volatilization and rice growth in an alkali soil

Summary In order to improve nitrogen recovery by rice, the effect of a urease inhibitor phenylphosphorodiamidate (PPD) on the efficiency of fertilizer urea was studied in laboratory and greenhouse. Addition of PPD to urea (5% w/w) delayed urea hydrolysis by 3 to 4 days and reduced ammonia volatilization from 45% (without PPD) to 8.5% (with PPD). Ammonia volatilization obeyed first order kinetics. Urea hydrolysis was sufficiently strongly inhibited to match the nitrification potential of the soil. N application to rice by three different modes showed that a delayed mode (4 splits) was superior to two conventional modes (3 splits) in nitrogen recovery and fertilizer efficiency since it met nitrogen requirement of plants at reproductive stage. In 2 out of 3 modes of application, there was a 14% increase (relative) in grain yields and dry matter, and 6.8% increase in N uptake efficiency on application of PPD along with urea. The results indicate that urease inhibitors like PPD can be effectively used to block urea hydrolysis, reduce ammonia volatilization losses and improve N use efficiency by rice.     

Hepatocyte heterogeneity in ammonia metabolism: impairment of glutamine synthesis in CCl4 induced liver cell necrosis with no effect on urea synthesis

After induction of a perivenous liver cell necrosis by CCl4 pretreatment of the rat, ammonia uptake by perfused liver is decreased. This was due to an inhibition of glutamine synthesis from added ammonia, whereas urea synthesis was not affected by CCl4 pretreatment. The data confirm recent findings on hepatocyte heterogeneity in ammonia metabolism and are explained by an impairment of perivenous glutamine synthetase, but not of periportal urea synthesis, by the perivenous liver cell necrosis induced by CCl4. Regarding the pathogenesis of hyperammonemia in acute severe liver disease like CCl4 poisoning, the data point to a role of an impaired glutamine synthesis, but not to an impairment of urea synthesis.     

The effect of nisin on murein synthesis

Nisin inhibits murein synthesis with concomitant accumulation of undecaprenyl-pyrophospho-MurNAc(pentapeptide) (lipid intermediate I). This inhibition is caused by the formation of a complex between the antibiotic and lipid intermediate I. Undecaprenyl-pyrophospho-MurNAc(pentapeptide)-GlcNAc (lipid intermediate II) also forms a complex with nisin. However, when murein synthesis is inhibited by nisin, this latter complex is not formed since lipid intermediate II is no longer synthesized.Abbreviations GlcNAc N-acetylglucosamine - MurNAc N-acetylmuramyl - Pentapeptide Ala--DGlu-Lys-DAla-DAla - C₅₅ undecaprenol Dedicated to Professor Otto Kandler on occasion of his 60th birthday     

Body fluid osmolytes and urea and ammonia flux in the colon of two chondrichthyan fishes, the ratfish, Hydrolagus colliei, and spiny dogfish, Squalus acanthias

The present study has examined the role of the colon in regulating ammonia and urea nitrogen balance in two species of chondrichthyans, the ratfish, Hydrolagus colliei (a holocephalan) and the spiny dogfish, Squalus acanthias (an elasmobranch). Stripped colonic tissue from both the dogfish and ratfish was mounted in an Ussing chamber and in both species bi-directional urea flux was found to be negligible. Urea uptake by the mucosa and serosa of the isolated colonic epithelium through accumulation of ¹⁴C-urea was determined to be 2.8 and 6.2 fold greater in the mucosa of the dogfish compared to the serosa of the dogfish and the mucosa of the ratfish respectively. Furthermore, there was no difference between serosal and mucosal accumulation of ¹⁴C-urea in the ratfish. Through the addition of 2 mM NH₄Cl to the mucosal side of each preparation the potential for ammonia flux was also examined. This was again found to be negligible in both species suggesting that the colon is an extremely tight epithelium to the movement of both urea and ammonia. Plasma, chyme and bile fluid samples were also taken from the agastric ratfish and were compared with solute concentrations of equivalent body fluids in the dogfish. Finally molecular analysis revealed expression of 3 isoforms of the urea transport protein (UT) and an ammonia transport protein (Rhbg) in the gill, intestine, kidney and colon of the ratfish. Partial nucleotide sequences of the UT-1, 2 and 3 isoforms in the ratfish had 95, 95 and 92% identity to the equivalent UT isoforms recently identified in another holocephalan, the elephantfish, Callorhinchus milii. Finally, the nucleotide sequence of the Rhbg identified in the ratfish had 73% identity to the Rhbg protein recently identified in the little skate, Leucoraja erinacea.     

Amino acid inhibition of the autophagic/lysosomal pathway of protein degradation in isolated rat hepatocytes

Protein degradation in isolated rat hepatocytes, as measured by the release of [¹⁴C]valine from pre-labelled protein, is partly inhibited by a physiologically balanced mixture of amino acids. The inhibition is largely due to the seven amino acids leucine, phenylalanine, tyrosine, tryptophan, histidine, asparagine and glutamine.When the amino acids are tested individually at different concentrations, asparagine and glutamine are the strongest inhibitors. However, when various combinations are tested, a mixture of the first five amino acids as well as a combination of leucine and asparagine inhibit protein degradation particularly strongly.The inhibition brought about by asparagine plus leucine is not additive to the inhibition by propylamine, a lysosomotropic inhibitor; thus indicating that the amino acids act exclusively upon the lysosomal pathway of protein degradation.Following a lag of about 15 min the effect of asparagine plus leucine is maximal and equal to the effect of propylamine, suggesting that their inhibition of the lysosomal pathway is complete as well as specific.Degradation of endocytosed ¹²⁵I-labelled asialofetuin is not affected by asparagine plus leucine, indicating that the amino acids do not affect lysosomes directly, but rather inhibit autophagy at a step prior to the fusion of autophagic vacuoles with lysosomes.The aminotransferase inhibitor, aminooxyacetate, does not prevent the inhibitory effect of any of the amino acids, i.e. amino acid metabolites are apparently not involved.