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Arginase II downregulates nitric oxide (NO) production and prevents NO-mediated apoptosis in murine macrophage-derived RAW 264.7 cells.

Gotoh T, Mori M - J. Cell Biol. (1999)

Bottom Line: An arginase I expression plasmid was also effective.On the other hand, transfection with the arginase II plasmid did not prevent apoptosis when a NO donor SNAP or a high concentration (12 mM) of arginine was added.These results indicate that arginase II prevents NO-dependent apoptosis of RAW 264.7 cells by depleting intracellular arginine and by decreasing NO production.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Kumamoto University School of Medicine, Kumamoto 862-0976, Japan.

ABSTRACT
Excess nitric oxide (NO) induces apoptosis of some cell types, including macrophages. As NO is synthesized by NO synthase (NOS) from arginine, a common substrate of arginase, these two enzymes compete for arginine. There are two known isoforms of arginase, types I and II. Using murine macrophage-like RAW 264.7 cells, we asked if the induction of arginase II would downregulate NO production and hence prevent apoptosis. When cells were exposed to lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma), the inducible form of NOS (iNOS) was induced, production of NO was elevated, and apoptosis followed. When dexamethasone and cAMP were further added, both iNOS and arginase II were induced, NO production was much decreased, and apoptosis was prevented. When the cells were transfected with an arginase II expression plasmid and treated with LPS/IFN-gamma, some cells were rescued from apoptosis. An arginase I expression plasmid was also effective. On the other hand, transfection with the arginase II plasmid did not prevent apoptosis when a NO donor SNAP or a high concentration (12 mM) of arginine was added. These results indicate that arginase II prevents NO-dependent apoptosis of RAW 264.7 cells by depleting intracellular arginine and by decreasing NO production.

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Effect of various reagents  on iNOS protein (A), arginase activity (B), and NO2/NO3 production  (C) in RAW cells. The cells were  treated with or without 1 μM Dex  and 1 mM dibutyryl cAMP for 24 h  and then with LPS (150 μg/ml) and  IFN-γ (100 U/ml) as indicated at  the bottom for 12 h (A and B) or 18 h  (C). (A) Cell extracts (30 μg protein) were subjected to immunoblot  analysis for iNOS protein and the  immunoblots were quantitated and  are shown by means ± SE (n = 3).  Maximal value is set at 100%. (B)  Arginase activity of the cell extracts  was measured and the results are  shown by means ± SE (n = 3). (C)  NO2 plus NO3 in the medium was  measured and the results are shown  by means ± SE (n = 3).
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Figure 5: Effect of various reagents on iNOS protein (A), arginase activity (B), and NO2/NO3 production (C) in RAW cells. The cells were treated with or without 1 μM Dex and 1 mM dibutyryl cAMP for 24 h and then with LPS (150 μg/ml) and IFN-γ (100 U/ml) as indicated at the bottom for 12 h (A and B) or 18 h (C). (A) Cell extracts (30 μg protein) were subjected to immunoblot analysis for iNOS protein and the immunoblots were quantitated and are shown by means ± SE (n = 3). Maximal value is set at 100%. (B) Arginase activity of the cell extracts was measured and the results are shown by means ± SE (n = 3). (C) NO2 plus NO3 in the medium was measured and the results are shown by means ± SE (n = 3).

Mentions: We next examined the effect of arginase II induction on NO production in RAW cells (Fig. 5). NO production measured by NO2 plus NO3 in the culture medium was not detected in untreated cells. When the cells were treated with LPS/IFN-γ, iNOS protein was induced and NO production was markedly increased. On the other hand, when Dex plus cAMP were included with LPS/IFN-γ, iNOS protein was further induced, whereas arginase activity was highly induced, and NO production was markedly decreased. These results suggest strongly that the induced arginase II competes with iNOS for arginine and suppresses NO production.


Arginase II downregulates nitric oxide (NO) production and prevents NO-mediated apoptosis in murine macrophage-derived RAW 264.7 cells.

Gotoh T, Mori M - J. Cell Biol. (1999)

Effect of various reagents  on iNOS protein (A), arginase activity (B), and NO2/NO3 production  (C) in RAW cells. The cells were  treated with or without 1 μM Dex  and 1 mM dibutyryl cAMP for 24 h  and then with LPS (150 μg/ml) and  IFN-γ (100 U/ml) as indicated at  the bottom for 12 h (A and B) or 18 h  (C). (A) Cell extracts (30 μg protein) were subjected to immunoblot  analysis for iNOS protein and the  immunoblots were quantitated and  are shown by means ± SE (n = 3).  Maximal value is set at 100%. (B)  Arginase activity of the cell extracts  was measured and the results are  shown by means ± SE (n = 3). (C)  NO2 plus NO3 in the medium was  measured and the results are shown  by means ± SE (n = 3).
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Related In: Results  -  Collection

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Figure 5: Effect of various reagents on iNOS protein (A), arginase activity (B), and NO2/NO3 production (C) in RAW cells. The cells were treated with or without 1 μM Dex and 1 mM dibutyryl cAMP for 24 h and then with LPS (150 μg/ml) and IFN-γ (100 U/ml) as indicated at the bottom for 12 h (A and B) or 18 h (C). (A) Cell extracts (30 μg protein) were subjected to immunoblot analysis for iNOS protein and the immunoblots were quantitated and are shown by means ± SE (n = 3). Maximal value is set at 100%. (B) Arginase activity of the cell extracts was measured and the results are shown by means ± SE (n = 3). (C) NO2 plus NO3 in the medium was measured and the results are shown by means ± SE (n = 3).
Mentions: We next examined the effect of arginase II induction on NO production in RAW cells (Fig. 5). NO production measured by NO2 plus NO3 in the culture medium was not detected in untreated cells. When the cells were treated with LPS/IFN-γ, iNOS protein was induced and NO production was markedly increased. On the other hand, when Dex plus cAMP were included with LPS/IFN-γ, iNOS protein was further induced, whereas arginase activity was highly induced, and NO production was markedly decreased. These results suggest strongly that the induced arginase II competes with iNOS for arginine and suppresses NO production.

Bottom Line: An arginase I expression plasmid was also effective.On the other hand, transfection with the arginase II plasmid did not prevent apoptosis when a NO donor SNAP or a high concentration (12 mM) of arginine was added.These results indicate that arginase II prevents NO-dependent apoptosis of RAW 264.7 cells by depleting intracellular arginine and by decreasing NO production.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Kumamoto University School of Medicine, Kumamoto 862-0976, Japan.

ABSTRACT
Excess nitric oxide (NO) induces apoptosis of some cell types, including macrophages. As NO is synthesized by NO synthase (NOS) from arginine, a common substrate of arginase, these two enzymes compete for arginine. There are two known isoforms of arginase, types I and II. Using murine macrophage-like RAW 264.7 cells, we asked if the induction of arginase II would downregulate NO production and hence prevent apoptosis. When cells were exposed to lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma), the inducible form of NOS (iNOS) was induced, production of NO was elevated, and apoptosis followed. When dexamethasone and cAMP were further added, both iNOS and arginase II were induced, NO production was much decreased, and apoptosis was prevented. When the cells were transfected with an arginase II expression plasmid and treated with LPS/IFN-gamma, some cells were rescued from apoptosis. An arginase I expression plasmid was also effective. On the other hand, transfection with the arginase II plasmid did not prevent apoptosis when a NO donor SNAP or a high concentration (12 mM) of arginine was added. These results indicate that arginase II prevents NO-dependent apoptosis of RAW 264.7 cells by depleting intracellular arginine and by decreasing NO production.

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