Arginase exists in two isoforms. Liver-type arginase ciency [24]. Recently, arginase activity as well as nitric oxide (arginase I) is expressed almost exclusively in the liver and synthase (NOS) activity was found to be induced in murine catalyzes the last step of urea synthesis, whereas the nonhepatic precursor synthesized in vitro was imported into isolatedWe report here the cloning of a full-length cDNA for humitochondria and proteolytically processed, mRNA for human arginase II was present in the kidney and other tissues, but was man arginase II and a partial cDNA for the rat enzyme. The not detected in the liver. Arginas¢ II mRNA was coinduced with human enzyme contains 354 amino acid residues including the nitric oxide synthase mRNA in routine macrophage-like RAW putative NH2-terminal mitochondria-targeting presequence. cells by lipopolysaccharide. This induction was enhancedMitochondrial import of the arginase II precursor synthesized by dexamethasone and dibutyryl cAMP, and was prevented by in vitro with concomitant proteolytic processing was shown. interferon-y. Possible roles of arginase II in NO synthesis areInduction of mRNAs for arginase II and inducible form of discussed.NOS (iNOS or NOS2) by LPS and other compounds in RAW 264.7 cells was also reported.
Nitric oxide is synthesized by nitric-oxide synthase from arginine, a common substrate of arginase. Rat peritoneal macrophages were cultured in the presence of bacterial lipopolysaccharide (LPS), and expression of the inducible isoform of nitric-oxide synthase (iNOS) and liver-type arginase (arginase I) was analyzed. 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 to a near maximum at 8 -12 h. On the other hand, arginase I mRNA that was undetectable prior to the treatment 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. mRNA for arginase II, an arginase isozyme, was not detected in the LPS-activated peritoneal cells. mRNA for CCAAT/enhancer-binding protein  (C/EBP), a transactivator of the arginase I gene, was also induced, and the induction was more rapid than that of arginase I mRNA. Changes in iNOS and arginase I mRNAs were also examined in LPS-injected rats in vivo. iNOS mRNA increased rapidly in the lung and spleen, reached a maximum 2-6 h after the LPS treatment, and decreased thereafter. Arginase I mRNA was induced markedly and more slowly in both tissues, reaching a maximum in 12 h. Thus, arginase I appears to have an important role in down-regulating nitric oxide synthesis in murine macrophages by decreasing the availability of arginine, and the induction of arginase I is mediated by C/EBP. Nitric oxide (NO)1 is a major molecule regulating blood vessel dilatation and immune response and functions as a neurotransmitter in the brain and peripheral nervous system (see Refs. 1-3 for reviews). NO is synthesized from arginine by nitric-oxide synthase (NOS), generating citrulline. Cellular NO production is absolutely dependent on the availability of arginine. This amino acid can be obtained from exogenous sources via the blood circulation, from intracellular protein degradation, or by the endogenous synthesis of arginine. Major sites of arginine synthesis in ureotelic animals are the liver, where arginine generated in the urea cycle (ornithine cycle) is rapidly converted to urea and ornithine by arginase, and the kidney, where arginine is synthesized from citrulline and released into the blood circulation (see Ref. 4 for a review). In other tissues and cell types, arginine can be generated from citrulline, which is produced as a coproduct of the NOS reaction, forming a cycle that is composed of NOS, argininosuccinate synthetase, and argininosuccinate lyase and that is termed the "citrulline-NO cycle" (5-10). The inducible isoform of NOS (iNOS) and argininosuccinate synthetase are coinduced in activated murine macrophage-like RAW 264.7 cells (8), in cultured vascular smooth muscle cells (9), and in vivo (10, 11). Argininosuccinate lyase is also induced in vivo (10, 11).On the other hand, arginine is utilized for both the arginase and NOS reactions. Thus, these two enzymes compete for arginine. At least two isoforms of ...
Background — Excessive production of nitric oxide (NO) by the inducible isoform of NO synthase (iNOS) is critically involved in endotoxin (ET)-induced hypotension. Tumor necrosis factor-α (TNF-α) plays an important role in induction of iNOS. Because activated protein C (APC), a physiological anticoagulant, inhibits TNF-α production, it might prevent hypotension by inhibiting excessive production of NO. In this study, we examined this possibility using a rat model of septic shock. Methods and Results — Intravenous administration of APC prevented both ET-induced hypotension and the increases in plasma levels of NO 2 − /NO 3 − . The hypotension was also inhibited when APC was administered 30 minutes after ET administration. APC inhibited the increases in lung levels of iNOS activity by inhibiting expression of iNOS mRNA in animals given ET. APC significantly inhibited the increases in lung tissue levels of TNF-α and expression of TNF-α mRNA in animals given ET. Neither DEGR-F.Xa, a selective inhibitor of thrombin generation, nor DIP-APC, an active site-blocked APC, showed any effect on these ET-induced changes. Both inhibition of TNF-α production by leukocytopenia and treatment with anti-rat TNF-α antibody produced effects similar to those induced by APC. Aminoguanidine, a selective inhibitor of iNOS, inhibited both the hypotension and the increases in plasma levels of NO 2 − /NO 3 − in this animal model. Conclusions — These observations strongly suggest that APC inhibits iNOS induction by decreasing TNF-α production, leading to the prevention of ET-induced hypotension. Furthermore, such effects of APC were not dependent on its anticoagulant effects but rather on its serine protease activity.
Nitric oxide (NO) is synthesized from arginine by nitric oxide synthase (NOS), and citrulline which is generated can be recycled to arginine by argininosuccinate synthetase (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 analyzed. In RNA blot analysis, iNOS mRNA was undetectable before the LPS treatment but was induced by LPS in the lung, heart, liver, and spleen, and less strongly in the skeletal muscle and testis. AS mRNA was induced in the lung and spleen, and AL mRNA was weakly induced in these tissues. AS and AL mRNAs were abundant in the control liver and remained unchanged after the treatment. 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 up to 24 h. In immunoblot analysis, increase of iNOS protein was evident in the lung, liver, and spleen, and there was an increase of AS protein in the lung and spleen. 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 coinduced in rat tissues and cells, citrulline-arginine recycling seems to be important in NO synthesis under the conditions of stimulation.Nitric oxide (NO) is a major messenger molecule regulating blood vessel dilatation and immune function and functions as a neurotransmitter in the brain and peripheral nervous system (see Refs. 1-3 for reviews). NO is synthesized from arginine by nitric oxide synthase (NOS), 1 generating citrulline as another product. Cellular NO production is absolutely dependent on availability of arginine. This amino acid can be obtained from exogenous sources via the blood circulation, from intracellular protein degradation, or by endogenous synthesis of arginine.Major sites of arginine synthesis in ureotelic animals are the liver, where arginine generated in the urea cycle (ornithine cycle) is rapidly converted to urea and ornithine by arginase, and the kidney, where arginine is synthesized from citrulline and released into the blood circulation (see Ref. 4 for a review). However, other tissues and cell types also contain low levels of argininosuccinate synthetase (AS) and argininosuccinate lyase (AL), which together synthesize arginine from citrulline (5-8). Therefore, arginine can be generated from citrulline which is produced as a co-product of the NOS reaction, forming a cycle which could be termed the "citrulline-NO cycle" (9) or "arginine-citrulline cycle" (10). Vascular endothelial cells can convert citrulline to arginine (11), and this conversion is increased when cells are stimulated to produce NO (12). Cytokine-activated macrophages, which produ...
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.
In the brain, three isoforms of nitric oxide (NO) synthase (NOS), namely neuronal NOS (nNOS, NOS1), inducible NOS (iNOS, NOS2), and endothelial NOS (eNOS, NOS3), have been implicated in biological roles such as neurotransmission, neurotoxicity, immune function, and blood vessel regulation, each isoform exhibiting in part overlapping roles. Previous studies showed that iNOS is induced in the brain by systemic treatment with lipopolysaccharide (LPS), a Gram-negative bacteria-derived stimulant of the innate immune system. Here we found that eNOS mRNA is induced in the rat brain by intraperitoneal injection of LPS of a smaller amount than that required for induction of iNOS mRNA. The induction of eNOS mRNA was followed by an increase in eNOS protein. Immunohistochemical analysis revealed that eNOS is located in astrocytes of both gray and white matters as well as in blood vessels. Induction of eNOS in response to a low dose of LPS, together with its localization in major components of the blood-brain barrier, suggests that brain eNOS is involved in early pathophysiologic response against systemic infection before iNOS is induced with progression of the infection.
Nitric oxide (NO) is synthesized from arginine by nitric oxide synthase, generating citrulline as another product, which can be recycled to arginine by argininosuccinate synthetase and argininosuccinate lyase. Rat argininosuccinate synthetase was expressed in Escherichia coli as a fusion protein with maltose binding protein, cleaved from binding protein, and purified. The purified synthetase had no enzyme activity. Rat argininosuccinate lyase was expressed in E. coli using pET-3a as a vector, and purified. The purified enzyme had a specific enzyme activity of arginine formation of 2.6 mumol/min/mg protein at 37 degrees C, the value being somewhat lower than those of the enzyme purified from various tissues. Antibodies against these enzymes were produced in rabbits. Immunoblot analyses showed that the two enzymes are most abundant in the liver, followed by kidney and testis. Smaller amounts of the enzyme proteins were present in other tissues. RNA blot analysis showed that the argininosuccinate synthetase mRNA was most abundant in the liver and kidney, followed by testis and other tissues. On the other hand, argininosuccinate lyase mRNA was most abundant in the testis, followed by kidney and liver, and by other tissues. These results show that argininosuccinate synthetase and argininosuccinate lyase are expressed both tissue-specifically and ubiquitously, and that practically all tissues have activities to convert citrulline to arginine.
We examined interspecies reactivities of eight anti-human monocgte/maaophage monoclonal antibodies (MAbs), AM-3K, PM-2K, X4, X14, Bet-MAC3, GHIl61, EBMl11, and KP1, with various animal h e s including rats, guinea pigs, rabbits, cats, dogs, goats, pigs, M e s , horses, andmonkeys. All MAbs recognized monkey macrophages. Pig macrophages were detected by most MAbs except for EBMlll and KP1. Ofthe eight antibodies, AM3K showed the widest intespecies reactivity. It reacted with macrophages of all animal species examined, except for rats. Westem blot analysis revealed a similarity in the antigens recognized by AM-3K among guinea pigs, rabbits, and humans. Other anti-human MAbs demonstrated distinct reactive patterns against mac-
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