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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 transfection  of arginase expression plasmids.  RAW cells were transfected  with insertless pCAGGS (e and  f), human arginase II expression  plasmid pCAGGS-hAII (g–j), or  rat arginase I expression plasmid  pCAGGS-rAI (k and l). 24 h after transfection, LPS (150 μg/ml)  and IFN-γ (100 U/ml) were  added to the medium and cultured for 12 h. The cells were  then fixed and stained with  Hoechst dye 33258 except i and j  that were analyzed for apoptosis  by the TUNEL method after fixation. Phase-contrast images (a,  c, e, g, i, and k) and fluorescence  images (b, d, f, h, j, and l) of the  same fields are shown. Original  magnifications: ×400. Bars, 10  μm. A portion of the cells was  detached from coverslips by  treatment with LPS/IFN-γ (c–l).  Arrows indicate cells rescued  from apoptosis. The percentage  of total cells which were determined to be apoptotic is shown  on the bottom of each panel.
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Figure 4: Effect of transfection of arginase expression plasmids. RAW cells were transfected with insertless pCAGGS (e and f), human arginase II expression plasmid pCAGGS-hAII (g–j), or rat arginase I expression plasmid pCAGGS-rAI (k and l). 24 h after transfection, LPS (150 μg/ml) and IFN-γ (100 U/ml) were added to the medium and cultured for 12 h. The cells were then fixed and stained with Hoechst dye 33258 except i and j that were analyzed for apoptosis by the TUNEL method after fixation. Phase-contrast images (a, c, e, g, i, and k) and fluorescence images (b, d, f, h, j, and l) of the same fields are shown. Original magnifications: ×400. Bars, 10 μm. A portion of the cells was detached from coverslips by treatment with LPS/IFN-γ (c–l). Arrows indicate cells rescued from apoptosis. The percentage of total cells which were determined to be apoptotic is shown on the bottom of each panel.

Mentions: Fig. 4 shows the effect of arginase II expression on apoptosis of RAW cells. Apoptotic changes were observed by the addition of LPS/IFN-γ. When the arginase II plasmid was transfected, some cells were protected from apoptotic change. Transfection of an expression plasmid for arginase I, a cytosolic enzyme, was also effective in protecting cells from LPS/IFN-γ–dependent apoptosis. Transfection of the insertless plasmid was without effect. These results support our hypothesis that arginase prevents apoptosis by depleting intracellular arginine and thus decreasing production of NO. Both mitochondrial arginase II and cytosolic arginase I were effective.


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 transfection  of arginase expression plasmids.  RAW cells were transfected  with insertless pCAGGS (e and  f), human arginase II expression  plasmid pCAGGS-hAII (g–j), or  rat arginase I expression plasmid  pCAGGS-rAI (k and l). 24 h after transfection, LPS (150 μg/ml)  and IFN-γ (100 U/ml) were  added to the medium and cultured for 12 h. The cells were  then fixed and stained with  Hoechst dye 33258 except i and j  that were analyzed for apoptosis  by the TUNEL method after fixation. Phase-contrast images (a,  c, e, g, i, and k) and fluorescence  images (b, d, f, h, j, and l) of the  same fields are shown. Original  magnifications: ×400. Bars, 10  μm. A portion of the cells was  detached from coverslips by  treatment with LPS/IFN-γ (c–l).  Arrows indicate cells rescued  from apoptosis. The percentage  of total cells which were determined to be apoptotic is shown  on the bottom of each panel.
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Related In: Results  -  Collection

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Figure 4: Effect of transfection of arginase expression plasmids. RAW cells were transfected with insertless pCAGGS (e and f), human arginase II expression plasmid pCAGGS-hAII (g–j), or rat arginase I expression plasmid pCAGGS-rAI (k and l). 24 h after transfection, LPS (150 μg/ml) and IFN-γ (100 U/ml) were added to the medium and cultured for 12 h. The cells were then fixed and stained with Hoechst dye 33258 except i and j that were analyzed for apoptosis by the TUNEL method after fixation. Phase-contrast images (a, c, e, g, i, and k) and fluorescence images (b, d, f, h, j, and l) of the same fields are shown. Original magnifications: ×400. Bars, 10 μm. A portion of the cells was detached from coverslips by treatment with LPS/IFN-γ (c–l). Arrows indicate cells rescued from apoptosis. The percentage of total cells which were determined to be apoptotic is shown on the bottom of each panel.
Mentions: Fig. 4 shows the effect of arginase II expression on apoptosis of RAW cells. Apoptotic changes were observed by the addition of LPS/IFN-γ. When the arginase II plasmid was transfected, some cells were protected from apoptotic change. Transfection of an expression plasmid for arginase I, a cytosolic enzyme, was also effective in protecting cells from LPS/IFN-γ–dependent apoptosis. Transfection of the insertless plasmid was without effect. These results support our hypothesis that arginase prevents apoptosis by depleting intracellular arginine and thus decreasing production of NO. Both mitochondrial arginase II and cytosolic arginase I were effective.

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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