Masataka Mori

Distinct arginase isoforms expressed in primary and transformed macrophages : regulation by oxygen tension

Experiments were performed to identify arginase isoforms expressed in primary and transformed rodent macrophages and to determine the molecular mechanisms for the previously observed increase in arginase activity in macrophages cultured in hypoxia or anoxia. Results demonstrate the following: 1) mRNA and protein for hepatic-type AI arginase are expressed in primary cultures of rat and mouse peritoneal macrophages and are enhanced seven- and ninefold, respectively, by lipopolysaccharide (LPS). 2) mRNA for extrahepatic-type AII arginase is constitutively expressed in mouse, but not rat, peritoneal macrophages and is detected in RAW264.7 cells after LPS treatment; neither J774A.1 nor P388D1 cells contain arginase mRNA. 3) AI arginase mRNA, arginase activity in cell lysates, andl-arginine flux through arginase in intact cells are all increased in rat wound-derived and mouse peritoneal macrophages by hypoxic or anoxic culture; AII arginase mRNA is, in contrast, suppressed >50% by O2deprivation. 4) Expression of thel-arginine transporter mCAT-2 is increased greater than twofold by reduced O2 culture. These results demonstrate substantial variability in arginase isoform expression among primary and transformed rodent macrophages. They also identify AI and AII arginase and the mCAT-2 l-arginine transporter as O2-regulated genes.

L-arginine uptake, the citrulline-NO cycle and arginase II in the rat brain : an in situ hybridization study

Nitric oxide (NO) is synthesized from a unique precursor, arginine, by nitric oxide synthase (NOS). In brain cells, arginine is supplied by protein breakdown or extracted from the blood through cationic amino acid transporters (CATs). Arginine can also be recycled from the citrulline produced by NOS activity, through argininosuccinate synthetase (AS) and argininosuccinate lyase (AL) activities, and metabolized by arginase. NOS, AS and AL constitute the so-called citrulline-NO cycle. In order to better understand arginine transport, recycling and degradation, we studied the regional distribution of cells expressing CAT1, CAT3, AS, AL, neuronal NOS (nNOS) and arginase II (AII) in the adult rat brain by non-radioisotopic in situ hybridization (ISH). CAT1, AL and AII presented an ubiquitous neuronal and glial expression, whereas CAT3 and AS were confined to neurons. nNOS was restricted to scattered neurons and a few brain nuclei and layers. We demonstrate by this study that cells expressing nNOS all appear to express the entire citrulline-NO cycle, whereas numerous cells expressing AL do not express AS. The differential expression of these genes within the same anatomical structure could indicate that intercellular exchanges of citrulline-NO cycle metabolites are relevant. Thus vicinal interactions should be taken into account to study their regulatory mechanisms.

Regulation of the genes for arginase isoforms and related enzymes in mouse macrophages by lipopolysaccharide

Arginase exists in two isoforms, the hepatic (arginase I) and extrahepatic types (arginase II). Arginase I is markedly induced in rat peritoneal macrophages and rat tissues in vivo by bacterial lipopolysaccharide (LPS). In contrast, both arginase I and arginase II are induced in LPS-activated mouse peritoneal macrophages. In the present study, expression of arginase isoforms and related enzymes was studied in mouse tissues in vivo and in peritoneal macrophages with RNA blot and immunoblot analyses and enzyme assay. When mice were injected intraperitoneally with LPS, inducible nitric oxide synthase (iNOS) and arginase II were induced early in the lung and spleen. mRNAs for argininosuccinate synthase (AS) and ornithine decarboxylase (ODC) were also induced early. In comparison, arginase I was induced later in the lung. Early induction of iNOS, arginase II, AS, ODC, and cationic amino acid transporter 2 and late induction of arginase I were observed in LPS-activated peritoneal macrophages. These results indicate that the genes for the two arginase isoforms are regulated differentially. Possible roles of the arginase isoforms in the regulation of nitric oxide production and in polyamine synthesis are discussed.Arginase exists in two isoforms, the hepatic (arginase I) and extrahepatic types (arginase II). Arginase I is markedly induced in rat peritoneal macrophages and rat tissues in vivo by bacterial lipopolysaccharide (LPS). In contrast, both arginase I and arginase II are induced in LPS-activated mouse peritoneal macrophages. In the present study, expression of arginase isoforms and related enzymes was studied in mouse tissues in vivo and in peritoneal macrophages with RNA blot and immunoblot analyses and enzyme assay. When mice were injected intraperitoneally with LPS, inducible nitric oxide synthase (iNOS) and arginase II were induced early in the lung and spleen. mRNAs for argininosuccinate synthase (AS) and ornithine decarboxylase (ODC) were also induced early. In comparison, arginase I was induced later in the lung. Early induction of iNOS, arginase II, AS, ODC, and cationic amino acid transporter 2 and late induction of arginase I were observed in LPS-activated peritoneal macrophages. These results indicate that the genes for the two arginase isoforms are regulated differentially. Possible roles of the arginase isoforms in the regulation of nitric oxide production and in polyamine synthesis are discussed.

Precise distribution of neuronal nitric oxide synthase mRNA in the rat brain revealed by non-radioisotopic in situ hybridization.

Regional distribution of neurons expressing neuronal nitric oxide synthase mRNA in the rat brain was examined by non-radioisotopic in situ hybridization, using digoxigenin-labeled complementary RNA probes. Clustering of intensely positive neurons was observed in discrete areas including the main and accessory olfactory bulbs, the islands of Calleja, the amygdala, the paraventricular nucleus of the thalamus, several hypothalamic nuclei, the lateral geniculate nucleus, the magnocellular nucleus of the posterior commissure, the superior and inferior colliculi, the laterodorsal and pedunculopontine tegmental nuclei, the nucleus of the trapezoid body, the nucleus of the solitary tract and the cerebellum. Strongly-stained isolated neurons were scattered mainly in the cerebral cortex, the basal ganglia and the brain stem, especially the medulla reticular formation. In the hippocampus, an almost uniform distribution of moderately stained neurons was observed in the granular cell layer of the dentate gyrus and in the pyramidal cell layer of the Ammons horn, while more intensely stained isolated neurons were scattered over the entire hippocampal region. These observations can serve as a good basis for studies on function and gene regulation of neuronal nitric oxide synthase.

Hypoglycemia-associated hyperammonemia caused by impaired expression of ornithine cycle enzyme genes in C/EBPalpha knockout mice

Ammonia produced by amino acid metabolism is detoxified through conversion into urea by the ornithine cycle in the liver, whereas carbon skeletons of amino acids are converted to glucose by gluconeogenic enzymes. Promoter and enhancer sequences of several genes for ornithine cycle enzymes interact with members of the CCAAT/enhancer-binding protein (C/EBP) transcription factor family. Disruption of the C/EBPα gene in mice causes hypoglycemia associated with the impaired expression of gluconeogenic enzymes. Here we examined the expression of ornithine cycle enzyme genes in the livers of C/EBPα-deficient mice. mRNA levels for the first, third, fourth, and fifth enzymes of five enzymes in the cycle were decreased in C/EBPα-deficient mice. Protein levels for the first, second, fourth, and fifth enzymes were also decreased. In situhybridization analysis revealed that the enzyme mRNAs were distributed normally in the periportal region but were disordered in C/EBPα-deficient mice with relatively higher mRNA levels in the midlobular region. Blood ammonia concentrations in the mutant mice were severalfold higher than in wild-type mice. Thus, C/EBPα is crucial for ammonia detoxification by ornithine cycle enzymes and for coordination of gluconeogenesis and urea synthesis.

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.

Regulation of the urea cycle enzyme genes in nitric oxide synthesis

Nitric oxide (NO) is synthesized from arginine by nitric-oxide synthase (NOS), and citrulline that is generated can be recycled to arginine by argininosuccinate synthase (AS) and argininosuccinate lyase (AL). Rats were injected with bacterial lipopolysaccharide (LPS) and expression of the inducible isoform of NOS (iNOS), AS and AL was analysed. In RNA blot analysis, iNOS mRNA was induced by LPS in the lung, heart, liver and spleen, and less strongly in the skeletal muscle and testis. AS and AL mRNAs were induced in the lung and spleen. Kinetic studies showed that iNOS mRNA increased rapidly in both spleen and lung, reached a maximum 2–5 h after the treatment, and decreased thereafter. On the other hand, AS mRNA increased more slowly and reached a maximum in 6–12 h (by about 10-fold in the spleen and 2-fold in the lung). AL mRNA in the spleen and lung increased slowly and remained high upto 24 h. In immunohistochemical analysis, macrophages in the spleen that were negative for iNOS and AS before LPS treatment were strongly positive for both iNOS and AS after this treatment. As iNOS, AS and AL were co-induced in rat tissues and cells, citrulline–arginine recycling seems to be important in NO synthesis under the conditions of stimulation.Arginine is a common substrate of NOS and arginase. Rat peritoneal macrophages were cultured in the presence of LPS and expression of iNOS and liver-type arginase (arginase I) was analysed. mRNAs for iNOS and arginase I were induced by LPS in a dose-dependent manner. iNOS mRNA appeared 2 h after LPS treatment and increased up to a near-maximum at 8–12 h. On the other hand, arginase I mRNA began to increase after 4 h with a lag time and reached a maximum at 12 h. Immunoblot analysis showed that iNOS and arginase I proteins were also induced. Induction of iNOS and arginase I mRNAs were also observed in LPS-injected rats in vivo. Thus, arginase I appears to have an important role in downregulating NO synthesis in murine macrophages by decreasing the availability of arginine.A cDNA for human arginase II, an arginase isozyme, was isolated. A polypeptide of 354 amino acid residues including the putative NH2-terminal presequence for mitochondrial import was predicted. It was 59% identical with arginase I. mRNA for human arginase II was present in the kidney and other tissues but was not detected in the liver. Arginase II mRNA was co-induced with iNOS mRNA in murine macrophage-like RAW 264.7 cells by LPS. This induction was enhanced by dexamethasone and dibutyrul cAMP, and was prevented by interferon-γ.These results indicate that NO synthesis is regulated by arginine-synthesizing and -degrading enzymes in a complicated manner.

Clinical evaluation of serum ornithine carbamoyltransferase by enzyme-linked immunosorbent assay in patients with liver diseases.

We developed a sensitive enzyme-linked immunosorbent assay (ELISA) for serum ornithine carbamoyltransferase (OCT) protein, and examined serum OCT concentrations in patients with various liver diseases. OCT concentrations were markedly elevated in cases of hepatic encephalopathy, acute on chronic, and those with the acute phase of acute hepatitis, moderately in chronic hepatitis, liver cirrhosis, hepatocellular carcinoma, primary biliary cirrhosis, and slightly in those with a fatty liver. High percentages (92-98%) of patients with chronic hepatitis, liver cirrhosis and hepatocellular carcinoma had higher than normal concentrations of serum OCT protein. There was a close correlation with aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities and moderate correlations with those of mitochondrial AST, glutamate dehydrogenase and gamma-glutamyltranspeptidase. The OCT/ALT ratio was higher in patients with liver cirrhosis than in those with chronic hepatitis (p < 0.001), and was still higher in cases of hepatocellular carcinoma (p < 0.05). In 2 patients with acute on chronic disease, OCT concentrations decreased similarly with or more rapidly than AST or ALT activities after admission. In 2 patients with hepatic encephalopathy, the OCT concentrations changed similarly with AST and ALT activities. This OCT ELISA system will aid in diagnosing various liver diseases and in the follow-up of the patients, and the OCT/ALT ratio may serve for a differential diagnosis of liver diseases.

Immunoelectron microscopical localization of ornithine transcarbamylase in hepatic parenchymal cells of the rat

SummaryThe electron microscopical localization of ornithine transcarbamylase in rat liver was investigated by a protein A—gold technique applied to thin sections of Lowicryl K4M- or LR gold-embedded materials and to ultracryosections. Gold particles were exclusively confined to mitochondria of the parenchymal cells but not of sinus-lining cells. In mitochondria, gold particles were present in the matrix and closely associated with the inner membrane. The most intensive labelling was obtained from ultracryosections, while weaker labelling was noted in sections of materials embedded in both Lowicryl K4M and LR gold. The association of the enzyme with the inner membrane was confirmed by quantitative analysis of distribution pattern.

A SENSITIVE ENZYME-LINKED IMMUNOSORBENT ASSAY OF SERUM ORNITHINE CARBAMOYLTRANSFERASE

Ornithine carbamoyltransferase (OCT), a urea cycle enzyme, is located almost exclusively in liver mitochondria. We designed a sensitive enzyme-linked immunosorbent assay (ELISA) of serum OCT protein using an antibody against purified bovine enzyme. OCT protein measured using this method showed a good correlation with OCT activity (r = 0.961), and was much higher in patients with liver diseases than in the controls. Measurements of serum OCT protein in 442 healthy blood donors gave the upper limit of normal range of 23 ng Eq/ml (equivalent to the bovine enzyme) for males, 8 ng Eq/ml for females, and 16 ng Eq/ml for males plus females. The values differ significantly between the sexes and depending on ages. This ELISA system is expected to be used as a pertinent liver function test.