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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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Immunocytostaining of arginase II in RAW cells.  RAW cells were treated with LPS (150 μg/ml), 1 μM Dex, and  1 mM dibutyryl cAMP for 24 h to induce arginase II (B and C),  or transfected with human arginase II expression plasmid  pCAGGS-hAII and cultured for 36 h (D and E). The cells were  then fixed and immunostained with rabbit antiserum against arginase II, as described in Materials and Methods. A is an untreated  control. Original magnifications: A, B, and D, ×400; C and E,  ×1000. Bars, 10 μm.
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Figure 3: Immunocytostaining of arginase II in RAW cells. RAW cells were treated with LPS (150 μg/ml), 1 μM Dex, and 1 mM dibutyryl cAMP for 24 h to induce arginase II (B and C), or transfected with human arginase II expression plasmid pCAGGS-hAII and cultured for 36 h (D and E). The cells were then fixed and immunostained with rabbit antiserum against arginase II, as described in Materials and Methods. A is an untreated control. Original magnifications: A, B, and D, ×400; C and E, ×1000. Bars, 10 μm.

Mentions: To examine the effect of arginase on apoptosis even more directly, transfection experiments were done. In a control experiment, RAW cells were treated with LPS plus Dex/ cAMP to induce arginase II. After fixation, the cells were immunostained with an antibody against arginase II (Fig. 3). Little immunoreactivity was observed in untreated cells. On the other hand, when the cells were treated with LPS plus Dex/cAMP, the cells became positive for arginase II immunoreactivity. Thread-like structures were stained, results reflecting localization of arginase II in the mitochondria (Gotoh et al., 1996). When the cells were transiently transfected with the arginase II plasmid, some cells that were apparently transfected with the plasmid were strongly immunostained. Thread-like mitochondrial structures were visible in most of the positive cells, whereas whole cells were stained in some positive cells, probably due to a high overexpression of the enzyme.


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)

Immunocytostaining of arginase II in RAW cells.  RAW cells were treated with LPS (150 μg/ml), 1 μM Dex, and  1 mM dibutyryl cAMP for 24 h to induce arginase II (B and C),  or transfected with human arginase II expression plasmid  pCAGGS-hAII and cultured for 36 h (D and E). The cells were  then fixed and immunostained with rabbit antiserum against arginase II, as described in Materials and Methods. A is an untreated  control. Original magnifications: A, B, and D, ×400; C and E,  ×1000. Bars, 10 μm.
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Related In: Results  -  Collection

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Figure 3: Immunocytostaining of arginase II in RAW cells. RAW cells were treated with LPS (150 μg/ml), 1 μM Dex, and 1 mM dibutyryl cAMP for 24 h to induce arginase II (B and C), or transfected with human arginase II expression plasmid pCAGGS-hAII and cultured for 36 h (D and E). The cells were then fixed and immunostained with rabbit antiserum against arginase II, as described in Materials and Methods. A is an untreated control. Original magnifications: A, B, and D, ×400; C and E, ×1000. Bars, 10 μm.
Mentions: To examine the effect of arginase on apoptosis even more directly, transfection experiments were done. In a control experiment, RAW cells were treated with LPS plus Dex/ cAMP to induce arginase II. After fixation, the cells were immunostained with an antibody against arginase II (Fig. 3). Little immunoreactivity was observed in untreated cells. On the other hand, when the cells were treated with LPS plus Dex/cAMP, the cells became positive for arginase II immunoreactivity. Thread-like structures were stained, results reflecting localization of arginase II in the mitochondria (Gotoh et al., 1996). When the cells were transiently transfected with the arginase II plasmid, some cells that were apparently transfected with the plasmid were strongly immunostained. Thread-like mitochondrial structures were visible in most of the positive cells, whereas whole cells were stained in some positive cells, probably due to a high overexpression of the enzyme.

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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