Modulation of L-Arginine-Arginase Metabolic Pathway Enzymes: Immunocytochemistry and mRNA Expression in Peripheral Blood and Tissue Levels in Head and Neck Squamous Cell Carcinomas in North East India.

BACKGROUND: Arginine may play important roles in tumor progression by providing ornithine for polyamine biosynthesis, required for cell growth. The aim of this work was to determine the expression of arginine metabolic pathway enzymes in head and neck squamous cell carcinoma (HNSCC) in northeast India. MATERIALS AND METHODS: The expressions of arginase isoforms (ARG1 and ARG2), ornithine aminotransferase (OAT) and ornithine decarboxylase (ODC) were examined in fifty paired HNSCC and adjacent non-tumor tissues by immunohistochemistry. Immunocytochemistry, semiquantitative reverse transcription sq-PCR and quantitative real-time qPCR were used to assess protein and mRNA expressions in peripheral blood of fifty HNSCC patients and hundred controls. RESULTS: ARG1 and ODC protein and mRNA were strongly expressed in peripheral blood from HNSCC patients. No ARG2 expression was observed. In vivo, expression of ARG1, ARG2 and ODC was significantly higher in tumor than in non-tumor tissues. Most tumors expressed low levels of OAT, with no difference in tissues or blood, compared to controls. The absolute extent of maximal ARG1 upregulation with qPCR showed 6.23 fold increase in HNSCC. CONCLUSIONS: These findings strongly suggest that in HNSCCs, the ARG1 pathway is stimulated leading to the formation of polyamines as indicated by higher ODC expression, which promote tumor growth.

Overexpression of ornithine aminotransferase: consequences on amino acid homeostasis.

Ornithine aminotransferase (OAT) is a reversible enzyme expressed mainly in the liver, kidney and intestine. OAT controls the interconversion of ornithine into glutamate semi-aldehyde, and is therefore involved in the metabolism of arginine and glutamine which play a major role in N homeostasis. We hypothesised that OAT could be a limiting step in glutamine-arginine interconversion. To study the contribution of the OAT enzyme in amino acid metabolism, transgenic mice that specifically overexpress human OAT in the liver, kidneys and intestine were generated. The transgene expression was analysed by in situ hybridisation and real-time PCR. Tissue (liver, jejunum and kidney) OAT activity, and plasma and tissue (liver and jejunum) amino acid concentrations were measured. Transgenic male mice exhibited higher OAT activity in the liver (25 (sem 4) v. 11 (sem 1) nmol/min per microg protein for wild-type (WT) mice; P < 0.05) but there were no differences in kinetic parameters (i.e. Km and maximum rate of reaction (Vmax)) between WT and transgenic animals. OAT overexpression decreased plasma and liver ornithine concentrations but did not affect glutamine or arginine homeostasis. There was an inverse relationship between ornithine levels and OAT activity. We conclude that OAT overexpression has only limited metabolic effects, probably due to the reversible nature of the enzyme. Moreover, these metabolic modifications had no effect on phenotype.

Ornithine deficiency in the arginase double knockout mouse.

Knockout mouse models have been created to study the consequences of deficiencies in arginase AI and AII, both individually and combined. The AI knockout animals die by 14 days of age from hyperammonemia, while the AII knockout has no obvious phenotype. The double knockout (AI(-/-)/AII(-/-)) exhibits the phenotype of the AI-deficient mice, with the additional absence of AII not exacerbating the observed phenotype of the AI knockout animals. Plasma amino acid measurements in the double knockout have shown arginine levels increased roughly 100-fold and ornithine decreased roughly 10-fold as compared to wildtype. Liver ornithine levels were reduced to 2% of normal in the double knockout with arginine very highly elevated. Arginine and ornithine were also altered in other tissues in the double knockout mice, such as kidney, brain, and small intestine. This is the first demonstration that the fatal hyperammonemia in the AI knockout mouse is almost certainly due to ornithine deficiency, the amino acid needed to drive the urea cycle. Others have shown that the expression of ornithine aminotransferase (OAT) rapidly decreases in the intestine at the same age when the AI-deficient animals die, indicating that this enzyme is critical to the maintenance of ornithine homeostasis, at least at this early stage of mouse development. Although most human AI-deficient patients have no symptomatic hyperammonemia at birth, it is possible that clinically significant ornithine deficiency is already present.

Hepatic response to right ventricular pressure overload.

BACKGROUND & AIMS: Modifying the afferent blood supply to the liver does not change the zonal expression pattern of hepatic enzymes in the rat. METHODS: We used pulmonary trunk banding (PTB) to study the effect of an efferent hindrance of blood flow on hepatic architecture and zonation of gene expression. RESULTS: Most PTB rats developed right ventricular hypertrophy and congestive heart failure. The hepatic response to PTB developed concomitantly with the decline in heart function. Enzyme expression in the periportal region was not affected, but the pericentral rim of hepatocytes expressing glutamine synthetase, ornithine aminotransferase, and NADPH cytochrome P-450 reductase (CYPred) first declined in diameter, then became discontinuous, and finally disappeared. Meanwhile, ornithine aminotransferase and especially CYPred, became re-expressed in the periportal zone. These changes occurred without appreciable cell death or fibrotic changes; the expression of fibronectin and alpha-smooth muscle actin increased perisinusoidally, but that of collagen did not. Electron microscopic analysis revealed normal fenestration of the sinusoidal endothelial cells without detectable deposition of basement membrane material, but both the width of the space of Disse and the length and number of hepatic microvilli were significantly reduced, implying a decreased flow of fluid in the space of Disse. CONCLUSIONS: The reprogramming of gene expression in livers with a postsinusoidal hindrance of blood flow results from declining access of the hepatocytes to intrasinusoidal signal-transduction molecules and suggest that the impaired biotransformation that accompanies right ventricular failure is caused by a central-to-portal shift in expression of the corresponding enzymes.

Assay of ornithine aminotransferase with ninhydrin.

We developed an assay system for ornithine aminotransferase (EC 2.6.1.13) using ninhydrin. Pyrroline 5-carboxylate, a product of enzymatic transamination, reacts with ninhydrin under hot acidic conditions to form a reddish pigment soluble in ethanol. The millimolar extinction coefficient of reaction product dissolved in ethanol was 16.5 at 510 nm. Acidification with perchloric acid effectively abolished the interfering color development by L-ornithine and L-glutamate. The paired activity measurement in mouse tissues by ninhydrin and o-aminobenzaldehyde methods showed a good correlation (gamma = 0.985). In our ninhydrin method, stable ninhydrin replaced unstable o-aminobenzaldehyde, and sensitivity was much higher than that with the conventional o-aminobenzaldehyde method.

Changes in ornithine metabolic enzymes induced by dietary protein in small intestine and liver: intestine-liver relationship in ornithine supply to liver.

Compared with the activity obtained with a high-protein diet in rats, a low-protein diet doubled the activity of ornithine aminotransferase [EC 2.6.1.13] (OAT), a key enzyme for citrulline synthesis, in the small intestine. The induction of ornithine aminotransferase in the small intestine by the low-protein diet and its suppression by the high-protein diet, and the converse in the liver, were immunohistochemically verified with anti-OAT antiserum. The immunohistochemical studies revealed that ornithine aminotransferase molecules localized in the villous surface epithelia, but not in the cryptic epithelia, were most responsive to the changes in dietary conditions, these results indicating that intestinal ornithine aminotransferase may be involved in the ornithine supply to the liver, with the reversal of the enzyme reaction occurring with a low-protein diet. Reconstituted model experiments on citrulline synthesis revealed that the addition of ornithine carbamoyl-transferase and carbamoyl phosphate was essential to overcome the unfavorable equilibrium of the reverse reaction, and the further addition of glutamate dehydrogenase and ammonia resulted in a stimulating effect.

Schistosoma mansoni: higher free proline levels in the livers of infected mice.

The concentration of L-hydroxyproline in the liver of ICR female mice increased rapidly during the 8th to 11th weeks of Schistosoma mansoni infection. Free L-proline concentration began to increase about the 7th week and reached its maximum at the 8th to 9th weeks of the infection, when the granulomatous response to the schistosome eggs in the liver was most prominent, as indicated by the increase in liver wet weight and its deoxyribonucleic acid concentration. A significant increment in the total activity of ornithine-delta-transaminase (EC 2.6.1.13) and the decrease in the specific activity of proline oxidase (EC 1.4.3.2) became detectable in the liver homogenate of infected mice on the 8th week. However, changes in these enzymatic activities were not parallel to that of the hepatic free L-proline content. Intraperitoneal administration of S. mansoni egg granulomas or 15,000g x 30 min supernatant fluid of their extracts into uninfected, normal mice significantly increased the hepatic free L-proline content without any appreciable effect on the enzymatic activities of proline oxidase and ornithine-delta-transaminase. These findings suggest that S. mansoni egg granulomas contain a factor(s) which may be responsible for the elevation of free L-proline content in the fibrotic liver caused by experimental schistosomiasis mansoni.

Complete amino acid sequence of rat kidney ornithine aminotransferase: identity with liver ornithine aminotransferase.

The complete amino acid sequence of rat kidney ornithine aminotransferase [EC 2.6.1.13] is presented. The 404-residue sequence was determined by analysis of peptides generated by digestion of the S-carboxyamidomethylated protein with CNBr, Achromobacter protease I, arginylendopeptidase, or Staphylococcus aureus V8 protease. Mueckler and Pitot have reported the amino acid sequence of the rat liver enzyme (440 residues) as predicted from the nucleotide sequence of the cDNA [Mueckler, M.M. & Pitot, H.C. (1985) J. Biol. Chem. 260, 12993-12997]. The amino acid sequence of the rat kidney enzyme presented herein coincides with residue 36 (Gly) through 440 (Phe) of the predicted precursor protein, indicating that the liver and kidney enzymes are identical, and that the enzyme is processed at the amino-terminal region after translation.

A glutamate dehydrogenase-based method for the assay of L-glutamic acid: formation of pyridine nucleotide fluorescent derivatives.

A method for the quantitation of L-glutamic acid in the picomole range was developed by finding conditions which allowed the production of NADH by the action of the L-glutamate dehydrogenase (EC 1.4.1.3) and its subsequent transformation to a highly fluorescent derivative. The method measures linearly glutamate from 250 pmol to 5 nmol. For its simplicity and low cost it is ideally suited to the assay of a large number of samples within a single working day. Its application to the determination of regional glutamate levels in the rat brain, as well as to the measurement of ornithine aminotransferase (EC 2.6.1.13) activity from several tissues is described. The results are similar to those obtained by different methodologies in several laboratories, but the present method offers additional advantages.

[Immunohistochemical localization and significance of ornithine aminotransferase in gut and liver in rats].

This study was performed to elucidate immunohistochemical localization of OAT in the gut mucosa and liver of rats using the ABC method. In the normal rat gut mucosa, strong immunoreactivity of OAT as fine granules was present in the surface epithelial cells and apical portion of intestinal glands. In the liver, this reaction was observed in the range of zone 2 to 3 of the pericentral vein and periportal vein. The changes in the intestinal glands of rats with a hyperprotein intake were observed to peak at 2 weeks, but in the liver a strongly positive diffuse reaction throughout whole lobules was detected at 4 weeks. This immunoreactive morphological localization of OAT suggests the location of absorption of amino acids in the small intestine and the distribution of the urea cycle enzyme OAT in the liver. The relationship between absorption of amino acids in the small intestine and the distribution of OAT in the liver was assumed to facilitate feedback repression. The changes in OAT in the mitochondria showed many diverse patterns by immunochemical electron microscopy, and these reactions are presumed to facilitate various types of metabolism in a single cells.

Immunohistochemical localization of ornithine aminotransferase in normal rat tissues by Fab'-horseradish peroxidase conjugates.

Immunohistochemical localization of ornithine aminotransferase (L-ornithine: 2-oxo-acid aminotransferase, EC 2.6.1.13), a mitochondrial enzyme whose hereditary absence induces gyrate atrophy of the choroid and retina, was elucidated by a direct immunoperoxidase method using Fab'-horseradish peroxidase conjugates. In immunodiffusion studies, the antibodies raised with the re-crystallized enzyme were highly specific to ornithine aminotransferase. To show localization of ornithine aminotransferase in normal rat tissues, clear immunohistochemical staining of this enzyme through the inner mitochondrial membrane in paraffin sections was achieved with Fab'-horseradish peroxidase conjugates. Strong immunoreactivity was present in cerebral neurons, hepatocytes, and epithelial cells of renal tubuli, gut mucous membranes, and ocular tissues. Specific distribution of ornithine aminotransferase was found in ependymal cell groups: namely, epithelial cells of the choroid plexus, pigmented and nonpigmented epithelial cells of the ciliary body. and Müller cells and pigment epithelium of the retina.

The primary structure of ornithine aminotransferase. Identification of active-site sequence and site of post-translational proteolysis.

Tentative assignments of functional residues in rat liver mitochondrial ornithine aminotransferase have recently been made using the amino acid sequence deduced from a cDNA clone [(1985) J.Biol.Chem. 260, 12993-12997]. Partial sequences obtained using the pure mature protein demonstrate that one of these assignments, that of Lys 292 as the residue that binds the coenzyme pyridoxal phosphate, is correct. However, the identification of the Glu 34-Gln 35 bond as the site of post-translational proteolysis is in error. This cleavage occurs instead at Ala 25-Thr 26.

Arginine catabolism in Agrobacterium strains: role of the Ti plasmid.

We present a study of the enzymatic activities involved in the pathway for arginine catabolism by Agrobacterium tumefaciens. Nitrogen from arginine is recovered through the arginase-urease pathway; the genes for these two activities are probably chromosomally born. Arginase was found to be inducible during growth in the presence of arginine or ornithine. Urease was constitutively expressed. Ornithine, resulting from the action of arginase on arginine, could be used as a nitrogen source via transamination to delta 1-pyrroline-5-carboxylate and reduction of the latter compound to proline by a reductase (both enzymatic activities are probably chromosomally encoded). Ornithine could also be used as a carbon source. Thus, we identified an ornithine cyclase activity that was responsible for direct conversion of ornithine to proline. This activity was found to be Ti plasmid encoded and inducible by growth in medium containing octopine or nopaline. The same activity was also chromosomally encoded in some Agrobacterium strains. In such strains, this activity was inducible during growth in arginine-containing medium.

Intracellular localization of Aspergillus nidulans ornithine carbamoyltransferase in native host cells and in Saccharomyces cerevisiae cells harbouring its cloned structural gene.

Differential centrifugation of the Aspergillus nidulans cell lysate shows that ornithine carbamoyltransferase (EC 2.1.3.3) appears mainly in the particulate (organellar) fraction. The enzyme was located to the mitochondria by co-sedimentation with cytochrome oxidase in isopycnic density gradient and by cytochemical-electron microscopic means. Arginase (EC 3.5.3.1) and ornithine delta-aminotransferase (E.C. 2.6.1.13) were found to reside in cytosol. The release of ornithine carbamoyltransferase from the organellar fraction by various agents indicates that the enzyme resides in the mitochondrial matrix. In Saccharomyces cerevisiae the plasmid pSAL43, carrying cloned Aspergillus nidulans ornithine carbamoyltransferase gene, directs the synthesis of the enzyme partially associated with yeast mitochondria even though the homologous yeast enzyme is exclusively cytosolic. The implications of these findings are discussed.

A one-step assay for pyrroline-5-carboxylate synthase using a short AG1-X8 column.

A rapid and simple method for assay of pyrroline-5-carboxylate synthase is presented. In this method, the incubation is terminated by raising the pH of incubation mixture to 10, and [14C]pyrroline 5-carboxylate produced from the substrate, [14C]glutamate, is first converted quantitatively to [14C]proline by reduction with NaBH4 at pH 10 and then the proline is allowed to pass through column of AG1-X8 anion exchanger under the conditions where the glutamate is completely retained by the column. Radioactive counting of the eluate gives the synthase activity. The entire procedure takes only one hour.

A radioisotopic assay for delta 1-pyrroline-5-carboxylate synthase activity.

A radioisotopic assay is described for measuring the activity of delta 1-pyrroline-5-carboxylate synthase, the enzyme that catalyzes the formation of delta 1-pyrroline-5-carboxylic acid from glutamic acid. Pyrroline-5-carboxylic acid is a common intermediate in the pathways through which glutamic acid, proline, and ornithine are interconverted. To determine pyrroline-5-carboxylate synthase activity, cell homogenates are incubated with [14C]-glutamic acid, the products of the reaction are converted quantitatively to proline by sodium borohydride, and proline is isolated by cation-exchange column chromatography. Cofactor requirements have been defined, and the activity of pyrroline-5-carboxylate synthase in several different cultured fibroblast lines is reported.

A new sensitive and convenient assay of ornithine aminotransferase.

A new radioisotopic assay for ornithine aminotransferase with [U-14C]ornithine as a substrate was developed. The dihydroquinazolinium compound formed was extracted from the reaction mixture with n-butanol and an addition of high concentration of sodium sulfate. This assay is sensitive and convenient and more than 50 assays can be done in one day without any special apparatus. The activities of ornithine aminotransferase in rat liver and human lymphoblastoid cells could be measured at nanomole levels by this method. Thus, the assay should be useful for measurement of OAT activity in cultured cells and for studies on the metabolic bases of the different types of ornithine aminotransferase deficiency: vitamin B6-responsive and -nonresponsive types.

Effect of tumors with different growth rates on enzymes in host liver.

In response to extrahepatic neoplasms, ornithine aminotransferase, malic enzyme, alanine aminotransferase and glucokinase activity of the 'uninvolved' liver is diminished and that of hexokinase is increased. Comparison of rats at various times after the implantation of ascites tumor, mammary carcinoma, fibrosarcoma and Morris hepatomas indicate that the faster the growth rate of tumors, the earlier the onset of these hepatic changes. The results also show that, when the different tumors are the same size, the magnitude of the enzymic deviations in the liver is directly related to characteristic growth rate of the tumor lines. These and previous observations on other host tissues suggest that tumor-doubling time, which is a known factor in metastatic spread and survival, may also be a variable in the production of systemic agents through which neoplasms affect the metabolic state of the cancer host.