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.     

Hormonal regulation of four urea cycle enzymes in postnatal rat liver in organ culture

The administration of hydrocortisone to 3- to 15-day-old rats increased the levels of hepatic argininosuccinate synthetase (ASS) and arginase. In 13-day-old rat liver explants maintained in organ culture, ornithine carbamoyltransferase (OTC), carbamoylphosphate synthetase (CPS) and arginase were stimulated by betamethasone. Actinomycin D prevented the responses of the latter two enzymes. Dibutyryl cyclic AMP raised OTC, CPS, ASS and arginase in vitro. The responses of the latter three enzymes were blocked by cycloheximide and puromycin and partially inhibited by actinomycin D. The simultaneous presence of betamethasone and dibutyryl cyclic AMP in the culture medium raised CPS and OTC in an additive manner. The sequential treatment of the cultures with betamethasone followed by dibutyryl cyclic AMP increased CPS and arginase synergistically and amplified the response of ASS to dibutyryl cyclic AMP.     

Expression of nuclear genes encoding the urea cycle enzymes, carbamoyl-phosphate synthetase I and ornithine carbamoyl transferase, in rat liver and intestinal mucosa

RNA dot-blot, quantitative electron microscope immunocytochemistry, and electrophoretic immunoblotting techniques were employed to investigate the expression of carbamoyl-phosphate synthetase I (CPS) and ornithine carbamoyl transferase (OCT) genes in rat liver and intestinal mucosa. Comparing only those cell types in the two tissues which express these enzymes, we show that the concentration of CPS and OCT in hepatocyte mitochondria is 2.3-times and 1.2-times greater, respectively, than in intestinal epithelial cell mitochondria. As a percentage of total tissue protein, however, liver homogenates contain 10-20 times more CPS and 5-10 times more OCT than is found in intestinal mucosa. These relatively large differences in enzyme protein levels between the two tissues are not reflected by differences in their mRNA levels. As a percentage of total translational activity in vitro (based on incorporation of [35S]methionine), total liver mRNA directed synthesis of about twice as much precursor CPS (pCPS) and precursor OCT (pOCT) than did equivalent amounts of mRNA from intestinal mucosa. The ratio of pCPS and pOCT mRNA levels between the two tissues (2:1, liver:intestinal mucosa) was confirmed by dot-blot and Northern hybridizations employing specific cDNA probes. The sizes of the respective mRNAs were the same for the two tissues: about 6000 residues for pCPS mRNA and about 1700 residues for pOCT mRNA.     

Characterization of novel mRNAs encoding enzymes involved in peptide alpha-amidation

The COOH-terminal alpha-amidation of bioactive peptides is a 2-step process catalyzed by two separable enzymatic activities both derived from the peptidylglycine alpha-amidating monooxygenase (PAM) precursor. Two forms of PAM mRNA (rPAM-1 and -2), differing by the presence or absence of optional Exon A, were previously characterized; both encode precursors predicted to have an NH2-terminal signal sequence, an intragranular domain containing both enzymatic activities, and a single transmembrane domain followed by a short, cytoplasmic COOH-terminal domain. In this report, two novel types of PAM mRNA were identified in adult rat atrium. A cDNA of each type was sequenced, and the results indicate that rPAM-3 and -4 could be related to each other and to the previously characterized rat PAM cDNAs by alternative mRNA splicing. Deletion of a 258-nucleotide segment (optional Exon B) encoding the transmembrane domain from rPAM-3 and the presence of a novel 3'-exon in rPAM-4 mean that both rPAM-3 and -4 mRNAs encode precursor proteins that have an NH2-terminal signal peptide but lack a transmembrane domain. The rPAM-4 precursor protein lacks the region of the PAM precursor catalyzing the second step in the alpha-amidation reaction. Low levels of rPAM-3 and -4 type mRNA were detected in atrium. Utilizing the polymerase chain reaction, two major patterns of distribution of forms of PAM mRNA were found. In the heart and central nervous system, PAM mRNAs both containing and lacking optional Exon A were prevalent and almost all of the PAM mRNAs detected contained optional Exon B. In the pituitary and submaxillary glands, PAM mRNAs lacking optimal Exon A were prevalent, as were PAM mRNAs lacking all or part of optional Exon B. Since the distribution of PAM activity between soluble and membrane fractions is tissue-specific and developmentally regulated and since rPAM-4 lacks an enzymatic portion of the PAM precursor, the tissue-specific expression of these forms of rat PAM mRNA is expected to be of functional significance.     

Simultaneous activation of genes encoding urea cycle enzymes and gluconeogenetic enzymes coincides with a corticosterone surge period before metamorphosis in Xenopus laevis

Amphibian tadpoles are postulated to excrete ammonia as nitrogen metabolites but to shift from ammonotelism to ureotelism during metamorphosis. However, it is unknown whether ureagenesis occurs or plays a functional role before metamorphosis. Here, the mRNA-expression levels of two urea cycle enzymes (carbamoyl phosphate synthetase I [CPSI] and ornithine transcarbamylase [OTC]) were measured beginning with stage-47 Xenopus tadpoles at 5 days post-fertilization (dpf), between the onset of feeding (stage 45, 4 dpf) and metamorphosis (stage 55, 32 dpf). CPSI and OTC expression levels increased significantly from stage 49 (12 dpf). Urea excretion was also detected at stage 47. A transient corticosterone surge peaking at stage 48 was previously reported, supporting the hypothesis that corticosterone can induce CPSI expression in tadpoles, as found in adult frogs and mammals. Stage-46 tadpoles were exposed to a synthetic glucocorticoid, dexamethasone (Dex, 10–500 nM) for 3 days. CPSI mRNA expression was significantly higher in tadpoles exposed to Dex than in tadpoles exposed to the vehicle control. Furthermore, glucocorticoid receptor mRNA expression increased during the pre-metamorphic period. In addition to CPSI and OTC mRNA upregulation, the expression levels of three gluconeogenic enzyme genes (glucose 6-phosphatase, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase 1) increased with the onset of urea synthesis and excretion. These results suggest that simultaneous induction of the urea cycle and gluconeogenic enzymes coincided with a corticosterone surge occurring prior to metamorphosis. These metabolic changes preceding metamorphosis may be closely related to the onset of feeding and nutrient accumulation required for metamorphosis.     

Inhibition of urea cycle enzymes by lysine and saccharopine

Crude and purified preparations of argininosuccinate synthetase, argininosuccinate lyase and arginase were subjected to inhibition studies with L-lysine and saccharopine. Saccharopine proved to be the more potent inhibitor of argininosuccinate synthetase and lyase, whereas lysine had more effect on arginase. Similar results were found with pure enzyme and crude preparations. Computer analysis of the results suggested that inhibition of urea cycle enzymes by saccharopine and lysine might have contributed to the high levels of citrulline found in a human patient with saccharopinuria, a defect of saccharopine metabolism, but that this was unlikely to be the sole explanation.     

Expression of aldolase isozyme mRNAs in fetal rat liver

The regulation of aldolase isozyme expression during development was studied by measuring the concentrations of mRNAs coding for aldolase A and B subunits in fetal and adult rat liver. Poly(A)-containing RNAs were extracted from livers at various stages of development of fetal rats, and the aldolase A and B subunits in the in vitro translation products of these RNAs were analyzed immunologically. The content of aldolase B mRNA in 14-day fetal liver, measured quantitatively as translational activity, was somewhat smaller than that of aldolase A mRNA; immunologically precipitable aldolase B and A amounted to 0.06% and 0.25% respectively, of the total products. Similar experiments using RNAs from fetuses at later stages, however, showed that aldolase B mRNA increased during development, whereas aldolase A mRNA decreased. In newborn rat liver, aldolase B constituted 0.56% of the total translation products of mRNA, but there was little detectable aldolase A (0.03%). The changes of aldolase mRNA levels were analyzed further by northern blot and dot-blot hybridization experiments using cloned aldolase A and B cDNAs. The content of aldolase B mRNA increased in the fetal stage, and that in newborn rat liver was about 12 times that in 14-day fetal liver. In contrast, the aldolase A mRNA content decreased during gestation and that in newborn rat liver was about one-eighth of that in 14-day fetal liver. These observations suggest that the switch of aldolase isozyme expression in fetal liver is controlled by the levels of the respective mRNAs.     

Primary culture of normal adult rat liver cells which maintain stable urea cycle enzymes.

Normal adult rat liver cells have been successfully cultured as monolayers without subjecting donor animals to a partial hepatectomy before cell isolation. Coating plastic tissue culture dishes with acid soluble calf skin collagen increases the efficiency of cell attachment. Hepatocytes form a monolayer in 24 hr in serum-free L-15 medium although 10% fetal calf serum for 24 hr increases efficiency of attachment. In serum-free medium the mono-layer remains viable for at least one week without added insulin. Cultured cells maintain tyrosine transaminase and four of the five urea cycle enzymes at levels above or equal to freshly isolated cells for 72 hr. Adenylyl cyclase activities are maintained for at least 72 hr, and are stimulated by epinephrine, glucagon and fluoride. Tyrosine transaminase activity is increased in cultured cells by glucagon and dexamethasone, but urea cycle enzymes are not.     

Regulation of genes for inducible nitric oxide synthase and urea cycle enzymes in rat liver in endotoxin shock

Arginine is an intermediate of the urea cycle in the liver. It is synthesized by the first four enzymes of the cycle, carbamylphosphate synthetase I, ornithine transcarbamylase, argininosuccinate synthetase, and argininosuccinate lyase, and is hydrolyzed to urea and ornithine by arginase I, forming the cycle. In endotoxemia shock, inducible nitric oxide (NO) synthase (iNOS) is induced in hepatocytes and arginine is utilized for NO production. Regulation of the genes for iNOS and the urea cycle enzymes was studied using lipopolysaccharide (LPS)-treated rat livers. When rats were injected intraperitoneally with LPS, iNOS mRNA was markedly induced. Cationic amino acid transporter-2 and C/EBPbeta mRNAs were also highly increased. In contrast, mRNAs for all the urea cycle enzymes except ornithine transcarbamylase were gradually decreased and reached 16-28% of controls at 12 h. However, all these enzymes remained unchanged at protein level up to 24 h. In light of these results, we suggest that synthesis of urea cycle enzymes is downregulated and that the protein synthetic capacity is directed to synthesis of proteins required for defense against endotoxemia.     

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).     

Effect of streptozotocin diabetes on some urea cycle enzymes

Streptozotocin induced diabetes in rats increased the activities of the three mitochondrial enzymes, carbamylphosphate synthetase, ornithine transcarbamylase and N-acetylglutamate synthetase, but not of the cytosolic N-acetylglutamate deacylase. Levels of both N-acetylglutamate and arginine, which are activators of carbamylphosphate synthetase and N-acetylglutamate synthetase respectively, increased in diabetes. These results serve to explain the increase both of mitochondrial citrulline and urea formation in hepatocytes and the increased urea excretion in diabetes.