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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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Induction of NO-mediated apoptosis of RAW cells by  treatment with LPS and IFN-γ. (A) RAW cells were cultured for  24 h with (d–f) or without (a–c) 150 μg/ml LPS and 100 U/ml  IFN-γ. After fixation, the cells were analyzed for apoptosis by  the TUNEL method (b and e) or stained with a DNA-specific  fluorochrome Hoechst dye 33258 (c and f). Phase-contrast images (a and d) and fluorescence images (b and e) of the same  fields are shown. Original magnifications: a, b, d, and e, ×400; c  and f, ×1,000. Bars, 10 μm. When the cells were treated with  LPS/IFN-γ, a portion of the cells was apparently dead and detached from coverslips and the number of attached cells decreased. (B) RAW cells were cultured for 24 h with 150 μg/ml  LPS and 100 U/ml IFN-γ. At the same time of the addition of  LPS/IFN-γ, a NO scavenger carboxy-PTIO (300 μM) was added  to the medium where indicated. After fixation, the cells were analyzed for apoptosis by the TUNEL method. Phase-contrast images (a and c) and fluorescence images (b and d) of the same  fields are shown. Original magnifications: ×400. Bars, 10 μm.
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Figure 1: Induction of NO-mediated apoptosis of RAW cells by treatment with LPS and IFN-γ. (A) RAW cells were cultured for 24 h with (d–f) or without (a–c) 150 μg/ml LPS and 100 U/ml IFN-γ. After fixation, the cells were analyzed for apoptosis by the TUNEL method (b and e) or stained with a DNA-specific fluorochrome Hoechst dye 33258 (c and f). Phase-contrast images (a and d) and fluorescence images (b and e) of the same fields are shown. Original magnifications: a, b, d, and e, ×400; c and f, ×1,000. Bars, 10 μm. When the cells were treated with LPS/IFN-γ, a portion of the cells was apparently dead and detached from coverslips and the number of attached cells decreased. (B) RAW cells were cultured for 24 h with 150 μg/ml LPS and 100 U/ml IFN-γ. At the same time of the addition of LPS/IFN-γ, a NO scavenger carboxy-PTIO (300 μM) was added to the medium where indicated. After fixation, the cells were analyzed for apoptosis by the TUNEL method. Phase-contrast images (a and c) and fluorescence images (b and d) of the same fields are shown. Original magnifications: ×400. Bars, 10 μm.

Mentions: When mouse macrophage-like RAW 264.7 cells were treated with LPS/IFN-γ for 24 h, morphological changes characteristic of apoptosis occurred (Fig. 1 A). Round-shaped cells and apoptotic bodies were observed in phase-contrast images, and chromatin condensation and nucleus fragmentation were seen in the case of Hoechst dye 33258 staining. Most cells were positive for TUNEL staining. These apoptotic changes were strongly prevented by adding a NO scavenger carboxy-PTIO (Akaike et al., 1993; Fig. 1 B). These results show that LPS/IFN-γ induces apoptosis of RAW cells, and this apoptosis is mediated by NO. LPS alone was much less effective in inducing apoptosis (data not shown).


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)

Induction of NO-mediated apoptosis of RAW cells by  treatment with LPS and IFN-γ. (A) RAW cells were cultured for  24 h with (d–f) or without (a–c) 150 μg/ml LPS and 100 U/ml  IFN-γ. After fixation, the cells were analyzed for apoptosis by  the TUNEL method (b and e) or stained with a DNA-specific  fluorochrome Hoechst dye 33258 (c and f). Phase-contrast images (a and d) and fluorescence images (b and e) of the same  fields are shown. Original magnifications: a, b, d, and e, ×400; c  and f, ×1,000. Bars, 10 μm. When the cells were treated with  LPS/IFN-γ, a portion of the cells was apparently dead and detached from coverslips and the number of attached cells decreased. (B) RAW cells were cultured for 24 h with 150 μg/ml  LPS and 100 U/ml IFN-γ. At the same time of the addition of  LPS/IFN-γ, a NO scavenger carboxy-PTIO (300 μM) was added  to the medium where indicated. After fixation, the cells were analyzed for apoptosis by the TUNEL method. Phase-contrast images (a and c) and fluorescence images (b and d) of the same  fields are shown. Original magnifications: ×400. Bars, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132906&req=5

Figure 1: Induction of NO-mediated apoptosis of RAW cells by treatment with LPS and IFN-γ. (A) RAW cells were cultured for 24 h with (d–f) or without (a–c) 150 μg/ml LPS and 100 U/ml IFN-γ. After fixation, the cells were analyzed for apoptosis by the TUNEL method (b and e) or stained with a DNA-specific fluorochrome Hoechst dye 33258 (c and f). Phase-contrast images (a and d) and fluorescence images (b and e) of the same fields are shown. Original magnifications: a, b, d, and e, ×400; c and f, ×1,000. Bars, 10 μm. When the cells were treated with LPS/IFN-γ, a portion of the cells was apparently dead and detached from coverslips and the number of attached cells decreased. (B) RAW cells were cultured for 24 h with 150 μg/ml LPS and 100 U/ml IFN-γ. At the same time of the addition of LPS/IFN-γ, a NO scavenger carboxy-PTIO (300 μM) was added to the medium where indicated. After fixation, the cells were analyzed for apoptosis by the TUNEL method. Phase-contrast images (a and c) and fluorescence images (b and d) of the same fields are shown. Original magnifications: ×400. Bars, 10 μm.
Mentions: When mouse macrophage-like RAW 264.7 cells were treated with LPS/IFN-γ for 24 h, morphological changes characteristic of apoptosis occurred (Fig. 1 A). Round-shaped cells and apoptotic bodies were observed in phase-contrast images, and chromatin condensation and nucleus fragmentation were seen in the case of Hoechst dye 33258 staining. Most cells were positive for TUNEL staining. These apoptotic changes were strongly prevented by adding a NO scavenger carboxy-PTIO (Akaike et al., 1993; Fig. 1 B). These results show that LPS/IFN-γ induces apoptosis of RAW cells, and this apoptosis is mediated by NO. LPS alone was much less effective in inducing apoptosis (data not shown).

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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