Regulation of messenger ribonucleic acid levels for five urea cycle enzymes in cultured rat hepatocytes. Requirements for cyclic adenosine monophosphate, glucocorticoids, and ongoing protein synthesis

In adult rat liver, amounts of the urea cycle enzymes are regulated by diet, glucocorticoids, and cAMP. Rat hepatocytes cultured in chemically defined medium were used to precisely define the roles of glucocorticoids and cAMP in regulation of these enzymes at the pretranslational level. With the exception of ornithine transcarbamylase mRNA, cultured rat hepatocytes retain the capacity to express mRNAs for the urea cycle enzymes at the same level observed for liver of intact rats. In the absence of added hormones, mRNAs for argininosuccinate synthetase and argininosuccinate lyase remained at or above normal in vivo levels, while mRNAs for the other three enzymes declined to very low levels. Messenger RNAs for carbamyl phosphate synthetase I, argininosuccinate synthetase, argininosuccinate lyase, and arginase increased in response to either dexamethasone or 8-(4-chlorophenylthio) cAMP (CPT-cAMP). Half-maximal responses occurred at 2-3 nM dexamethasone and at 2-7 microM CPT-cAMP. Cycloheximide abolished the response to dexamethasone but not to CPT-cAMP, suggesting that dexamethasone induced expression of an intermediate gene product required for induction of these mRNAs. The effects of a combination of both hormones were additive for argininosuccinate lyase mRNA and synergistic for carbamyl phosphate synthetase I, argininosuccinate synthetase, and arginase mRNAs. Messenger RNA for ornithine transcarbamylase showed little or no response to any condition tested. Depending on the particular mRNA and hormonal condition tested, increases in mRNA levels ranged from 1.4- to 70-fold above control values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of inducible nitric oxide synthase and enzymes of arginine metabolism in Fusarium kyushuense-exposed mouse lung.

Expression of inducible nitric oxide (NO) synthase (iNOS) and related enzymes of arginine metabolism in the mouse lung exposed to filamentous fungus Fusarium kyushuense was studied by RNA blot, immunoblot, and histological analyses. When mice were exposed intranasally to the fungi only once, no induction of iNOS mRNA was observed. However, when the animals were infected again 6 days after the first exposure, iNOS mRNA was induced, reached a maximum 12-24 h after the exposure, and decreased to an undetectable level at 48 h. mRNAs for cationic amino acid transporter-2 (CAT2) and argininosuccinate synthetase were induced gradually, reached a maximum at 24 h, and decreased at 48 h. Arginase II mRNA increased at 24 h and decreased markedly at 48 h. On the other hand, arginase I mRNA started to increase at 24 h and reached to a much higher level at 48 h. Ornithine decarboxylase and ornithine aminotransferase mRNAs were also induced. Immunoblot analysis showed that iNOS, argininosuccinate synthetase, and arginase I and II proteins were induced with similar kinetics as those of their respective mRNAs. In histological examination, fungal elements were observed in the bronchoalveolar lumen at 3-6 h, decreased at 12 h, and almost disappeared at 48 h. Small granuloma appeared 3 h after the infection and their size increased with time. These results suggest that NO is produced in the mouse lung in response to F. kyushuense exposure and that the NO production is regulated by CAT2, the citrulline-NO cycle, and arginase isoforms. Enhanced synthesis of polyamines and proline (and thus collagen) is also suggested.     

Immunohistochemical Localization of Arginase II and Other Enzymes of Arginine Metabolism in Rat Kidney and Liver

Arginine is a precursor for the synthesis of urea, polyamines, creatine phosphate, nitric oxide and proteins. It is synthesized from ornithine by argininosuccinate synthetase and argininosuccinate lyase and is degraded by arginase, which consists of a liver-type (arginase I) and a non-hepatic type (arginase II). Recently, cDNAs for human and rat arginase II have been isolated. In this study, immunocytochemical analysis showed that human arginase II expressed in COS-7 cells was localized in the mitochondria. Arginase II mRNA was abundant in the rat small intestine and kidney. In the kidney, argininosuccinate synthetase and lyase were immunostained in the cortex, intensely in proximal tubules and much less intensely in distal tubules. In contrast, arginase II was stained intensely in the outer stripes of the outer medulla, presumably in the proximal straight tubules, and in a subpopulation of the proximal tubules in the cortex. Immunostaining of serial sections of the kidney showed that argininosuccinate synthetase and arginase II were collocalized in a subpopulation of proximal tubules in the cortex, whereas only the synthetase, but not arginase II, was present in another subpopulation of proximal tubules. In the liver, all the enzymes of the urea cycle, i.e. carbamylphosphate synthetase I, ornithine transcarbamylase, argininosuccinate synthetase and lyase and arginase I, showed similar zonation patterns with staining more intense in periportal hepatocytes than in pericentral hepatocytes, although zonation of ornithine transcarbamylase was much less prominent. The implications of these results are discussed.     

Growth hormone and rat liver mitochondria effects on urea cycle enzymes

Effects of hypophysectomy and subsequent growth hormone administration on mitochondrial enzymes of the urea cycle were investigated in rat liver. Hypophysectomy increased the activities of the two mitochondrial enzymes, carbamyl phosphate synthetase and ornithine transcarbamylase but not of the cytosolic enzyme, argininosuccinate synthetase. The activity of mitochondrial phosphate dependent glutaminase was not affected. Administration of bovine growth hormone (100 μg/100 g body weight) for two weeks decreased the activities of carbamyl phosphate synthetase and ornithine transcarbamylase almost to the normal level. These results suggest a specific effect of growth hormone on mitochondrial enzymes of the urea cycle and serve to explain the increased urea formation in hypopituitarism.     

Channeling of urea cycle intermediates in situ in permeabilized hepatocytes

Preferential use of endogenously generated intermediates by the enzymes of the urea cycle was observed using isolated rat hepatocytes made permeable to low molecular weight compounds with alpha-toxin. The permeabilized cells synthesized [14C]urea from added NH4Cl, [14C]HCO3-, ornithine, and aspartate, using succinate as a respiratory substrate; with all substrates saturating, about 4 nmol of urea were formed per min/mg dry weight of cells. Urea usually accounted for about 40-50% of the total (NH3 + ornithine)-dependent counts, arginine for less than 10%, and citrulline for about 30%. Very tight channeling of arginine between argininosuccinate lyase and arginase was shown by the fact that the addition of a 200-fold excess of unlabeled arginine to the incubations did not decrease the percentage of counts found in urea or increase that found in arginine, even though a substantial amount of the added arginine was hydrolyzed inside the cells. The channeling of argininosuccinate between its synthetase and lyase was demonstrated by similar observations; unlabeled argininosuccinate added in 200-fold excess decreased the percentage of counts in urea by only 25%. Channeling of citrulline from its site of synthesis by ornithine transcarbamylase in the mitochondrial matrix to argininosuccinate synthetase in the cytoplasmic space was also shown. These results strongly suggest that the three "soluble" cytoplasmic enzymes of the urea cycle are grouped around the mitochondria and are spatially organized within the cell in such a way that intermediates can be efficiently transferred between them.     

Influence of Forced-Feeding of Individual Essential Amino Acids on the Activities of All Urea Cycle Enzymes in Rat Liver

The response of all urea cycle enzymes, i.e. carbamyl phosphate synthetase, ornithine transcarbamylase, argininosuccinate synthetase, argininosuccinase and arginase, has been determined in the liver of protein-depleted young rats which were forcibly fed individual essential l-amino acids along with or without caloric sources. The feeding of individual amino acids produced different effects on the level of each of the enzymes, and generally the response of carbamyl phosphate synthetase, argininosuccinate synthetase, argininosuccinase and arginase was greater than that of ornithine transcarbamylase. Of all the essential amino acids tested tryptophan was most effective on the elevation of these enzymes. Several amino acids, phenylalanine, leucine, threonine and methionine had also somewhat effect on the increase of some enzyme activities, but other amino acids had little or no effect on the response of these enzymes. On the contrary, histidine and lysine caused appreciable decrease of arginase activity. These enzyme activities in rats fed tryptophan alone were extremely higher than those of animals fed it along with caloric sources. The response level of the enzymes was essentially dependent on the tryptophan content in diets under the proper conditions. Tryptophan feeding did not produce any increase in both levels of urine and plasma urea despite the elevation of all urea cycle enzyme activities occured.     

Nutritional and hormonal regulation of mRNA abundance for arginine biosynthetic enzymes in kidney

Argininosuccinate synthetase and argininosuccinate lyase catalyze the synthesis of arginine from citrulline in kidney and also serve as components of the urea cycle in liver of ureotelic animals. Dietary and hormonal regulation of mRNAs encoding these enzymes have been well studied in liver but not in kidney. Messenger RNAs for these enzymes are localized within the renal cortex. Starvation and extreme variations in dietary protein content (0% vs 60% casein) produced 2.6- to 3.5-fold increases in mRNA abundance for these two enzymes in rat kidney. Argininosuccinate lyase mRNA was not induced by dibutyryl cAMP, dexamethasone, or a combination of the two agents. In contrast, argininosuccinate synthetase mRNA was induced 2-fold by dibutyryl cAMP but was unresponsive to dexamethasone. Thus, diet and hormones regulate levels of these mRNAs in rat kidney, but the responses are both qualitatively and quantitatively distinct from the responses previously reported for rat liver.     

Evidence for urea cycle activity in Sporosarcina ureae

Sporosarcina ureae BS 860, a motile, sporeforming coccus, possesses the enzymes required for a functioning urea (ornithine) cycle. This is only the second known example of urea cycle activity in a prokaryote. Specific activities are reported for ornithine carbamoyltransferase, argininosuccinase, arginase, and urease. Although argininosuccinate synthetase activity could not be detected directly in crude cell extracts, indirect evidence from radiocarbon tracing data for arginine synthesis from the substrate, l-[1-¹⁴C]-ornithine, strongly suggest the presence of this or other similar enzyme activity. Furthermore, good growth in defined media containing either 1.0% glutamine, ornithine, or citrulline as sole carbon sources suggests argininosuccinate synthetase activity is necessary for arginine synthesis. The effect of varying pH on arginase and urease activities indicate that these two enzymes may function within the context of the urea cycle to generate ammonia for amino acid synthesis, as well as for raising the pH of the growth micro-environment.     

Abundance of mRNAs encoding urea cycle enzymes in fetal and neonatal mouse liver

The relative abundances of mRNAs encoding the five urea cycle enzymes during development of mouse liver have been determined and compared with those of mRNAs encoding four other liver-specific proteins (phosphoenolpyruvate carboxykinase, tyrosine aminotransferase, alpha-fetoprotein, and albumin). Urea cycle enzyme mRNAs in fetal liver are expressed at 2-14% of the abundance in adult liver as early as 6 days before birth. Expression of the urea cycle enzyme mRNAs is not coordinate during the fetal and neonatal period. However, profiles of three urea cycle enzyme mRNAs are quite similar to that of alpha-fetoprotein mRNA, suggesting the possibility of a common response to regulatory signals during fetal development. With the exception of ornithine transcarbamylase mRNA, the urea cycle enzyme mRNAs have been shown previously to be inducible by cAMP and glucocorticoids. However, only argininosuccinate lyase mRNA exhibits any significant change in abundance at birth, resembling postnatal expression of tyrosine aminotransferase mRNA. The results indicate that the urea cycle enzyme mRNAs are potentially useful markers for elucidating various features of hepatocyte differentiation in mammals.     

Subcellular location of glutamine synthetase and urea cycle enzymes in liver of spiny dogfish (Squalus acanthias)

Glutamine synthetase and glutamine- and acetylglutamate-dependent carbamoyl-phosphate synthetase, both of which are present in high concentrations in liver of urea-retaining elasmobranchs, have been found to be located exclusively in the mitochondria in liver from the representative elasmobranch Squalus acanthias. This observation is consistent with the view that the function of this unique carbamoyl-phosphate synthetase is related to urea synthesis, and that the initial nitrogen-donating substrate for urea synthesis in these species is glutamine rather than ammonia. The urea cycle enzymes, ornithine carbamoyltransferase and arginase, are also located in the mitochondria, whereas argininosuccinate synthetase and argininosuccinate lyase are located in the cytosol. Glutamine synthetase and arginase are mitochondrial enzymes in uricotelic species, but are normally found in the cytoplasm in ureotelic species. the properties of the elasmobranch arginase, however, are characteristic of arginases from ureotelic species (e.g. the Km for arginine is 1.2 mM, and the enzyme has an Mr congruent to 100,000).     

Ureogenesis in a freshwater teleost: an unusual sub-cellular localization of ornithine-urea cycle enzymes in the freshwater air-breathing teleost Heteropneustes fossilis.

Sub-cellular localization of different ornithine-urea cycle enzymes was studied in the liver and kidney of a freshwater air-breathing teleost. Carbamyl phosphate synthetase, ornithine transcarbamylase, and arginase were found to be localized inside the mitochondria, and argininosuccinate synthetase and argininosuccinate lyase were found in the soluble fraction. Mitochondrial localization of arginase, a feature known in marine elasmobranchs and toadfishes, indicates the evolutionary position of H. fossilis to be different from that of present day freshwater teleosts.     

Modulation of intestinal urea cycle by dietary spermine in suckling rat

Argininosuccinate synthetase, an ubiquitous enzyme in mammals, catalyses the formation of argininosuccinate, the precursor of arginine. Arginine is recognised as an essential amino acid in foetuses and neonates, but also as a conditionally essential amino acid in adults. Argininosuccinate synthetase is initially expressed in enterocytes during the developmental period, it disappeared from this organ then appeared in the kidneys. Although the importance of both intestinal and renal argininosuccinate synthetases has been recognised for a long time, nutrients have not yet been identified as inducers of the gene expression. In the context of a proteomic screening of intestinal modifications induced by dietary spermine in suckling rats, we showed that argininosuccinate synthetase and carbamoyl phosphate synthase disappeared from enterocytes after this treatment. The disappearance of argininosuccinate synthetase in small intestine was confirmed by immunodetection. Expression of carbamoyl phosphate synthase and argininosuccinate synthetase coding genes decreased also after spermine administration. Expression of other urea cycle enzyme coding genes was modulated by spermine administration: argininosuccinate lyase decreased and arginase increased. Our results fit with the developmental variation of argininosuccinate synthetase and carbamoyl phosphate synthase. Modulation of the gene expression for several urea cycle enzymes suggests a coordination between all the pathway steps and switch toward polyamine (or proline and glutamate) biosynthesis from ornithine.     

STUDIES ON FUNCTIONAL AND METABOLIC ROLE OF UREA CYCLE INTERMEDIATES IN BRAIN

Abstract— The distribution of argininosuccinate synthetase, argininosuccinase and arginase, and the synthesis of urea in cerebullum. cerebral cortex and brain stem have been studied. Cerebral cortex had high levels of argininosuccinate synthetase and argininosuccinase. and a high ability to synthesize urea from aspartic acid and citrulline. Of the three regions, cerebullum had the highest arginase activity. The activities of the enzymes transamidinase and ornithine aminotransferase in the metabolism of arginine and ornithine in pathways other than urea formation have been studied in the three regions of the rat brain. The activity of creatine phosphokinase in all regions was the same: carbamylphosphatase activity was highest in cerebullum. Cerebral cortex had a high activity of aspartic acid transcarba-mylase. The brain stem, among the three regions, had the lowest activities of glutamine synthetase and glutaminase. The activities of these enzymes in the different regions are discussed in relation to urea production and the utilization of the urea cycle intermediates.
Intraperitoneal injection of high amounts of citrulline brought about a rise in the glutamine synthetase activity of cerebellum and brain stem and a rise in ornithine aminotransferase in cerebral cortex and liver. These results are discussed in relation to the mechanism of action of citrulline in alleviating the toxicity in hyperammonaemic states.     

Urea biosynthesis in normal human fetuses

Normal human fetuses at different gestation periods were collected on ice after hysterotomy and the enzymes of the urea cycle were measured in the liver. The activity of all enzymes increased with increasing gestational age towards the adult value, however, in no case did the values reach the normal adult level. The bladder fluid of these fetuses contained urea and ammonia nitrogen at concentrations which were akin to the concentrations found in fetal blood. The ornithine transcarbamylase activity was the lowest when compared to the adult values and appeared to be the rate-limiting enzyme in the cycle, along with argininosuccinic acid synthetase activity, which was also very low. The activity of arginase was found to be the highest in the cycle. The very low ornithine transcarbamylase and argininosuccinic acid synthetase activities and the comparatively higher arginase activity migh lead to the channeling of ornithine into alternate metabolic pathways.     

Regulation of nitric oxide production by arginine metabolic enzymes

Nitric oxide (NO) is synthesized from arginine by NO synthase (NOS), and the availability of arginine is one of the rate-limiting factors in cellular NO production. Citrulline, which is formed as a by-product of the NOS reaction, can be recycled to arginine by successive actions of argininosuccinate synthetase (AS) and argininosuccinate lyase (AL), forming the citrulline-NO cycle. AS and sometimes AL have been shown to be coinduced with inducible NOS (iNOS) in various cell types including activated macrophages, vascular smooth muscle cells, glial cells, neuronal PC12 cells, and pancreatic beta-cells. Cationic amino acid transporter (CAT)-2 is induced in activated macrophages but not in PC12 cells. On the other hand, arginase can downregulate NO production by decreasing intracellular arginine concentrations. iNOS and arginase activities are regulated reciprocally in macrophages by cytokines, and this may guarantee the efficient production of NO. In contrast, iNOS and arginase isoforms (type I and II) are coinduced in lipopolysaccharide (LPS)-activated macrophages. These results indicate that NO production is modulated by the uptake, recycling, and degradation of arginine.     

Urea cycle disorder in C3H-H-2 degree mice with juvenile steatosis of viscera

We determined the activities of urea cycle enzymes in the liver of C3H-H-2 degree-jsv mice. The activities of all urea cycle enzymes decreased in the latter period of lactation. The activities of carbamylphosphate synthetase and ornithine transcarbamylase in some affected mice were undetectable. On the other hand, the activities of enzymes other than urea cycle enzymes were normal. We consider that the decrease in the urea cycle enzymes is caused by an abnormality in the mechanism of gene expression.     

Changes in the activities of the enzymes of urea synthesis caused by dexamethasone and dibutyryladenosine 3'':5''-cyclic monophosphate in foetal rat liver maintained in organ culture.

Liver explants from 19-day foetal rats were maintained in organ culture, in a defined medium, for up to 48h. Both 6-N,2'-O-dibutyryl cyclic AMP, in the presence of theophylline, and dexamethasone caused an increase in the activities of carbamoyl phosphate synthase, argininosuccinate synthetase, argininosuccinate lyase and arginase. These increases could be abolished by simultaneously incubating the explants with cycloheximide. No change in the activity of ornithine transcarbamoylase was found with either hormone. Previous work has shown that injection of corticosteroids into 19.5-day foetal rats in utero did not cause an increase in the arginine synthetase system. Present results suggest that this lack of effect is not due to any incompetence of the foetal rat liver at this stage to respond to this agent. The observations on ornithine transcarbamoylase activity suggest that this enzyme is induced in the liver of the perinatal rat by neither corticosteroids nor hormones acting via cyclic AMP, and it may be that all the enzymes of the urea cycle are induced physiologically by an agent or agents as yet unidentified.     

Thyroxine elicits divergent changes in mRNA levels for two urea cycle enzymes and one gluconeogenic enzyme in tadpole liver

Thyroxine-induced metamorphosis of the tadpole to the frog (Rana catesbeiana) is marked by increased activities of the urea cycle enzymes in liver. Cloned cDNAs for two mammalian urea cycle enzymes--carbamyl-phosphate synthetase I and argininosuccinate synthetase--were shown to cross-hybridize with the corresponding mRNAs in tadpole liver. Thyroxine treatment produced nearly 10-fold, coordinate increases in hybridizable mRNA levels for these two enzymes in tadpole liver. This increase is sufficient to account for reported increases in enzyme levels and synthesis rates, demonstrating that thyroxine largely regulates concentrations of these enzymes at a pretranslational step(s). In contrast, levels of phosphoenolpyruvate carboxykinase mRNA in tadpole liver decreased by more than 90% following thyroxine treatment. This differs from the thyroxine-induced increases in synthesis rates of enzyme and mRNA reported for phosphoenolpyruvate carboxykinase in rat liver. However, the decreased levels of this mRNA in tadpole liver may represent a secondary response due to thyroxine-stimulated release of insulin.