Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of induction of  arginase II on apoptosis of  RAW cells. (A) Time course  of treatment of RAW cells is  shown. RAW cells were cultured in the medium with or  without 1 μM Dex and/or 1 mM  dibutyryl cAMP (cAMP) for  24 h, and then treated with various combinations of LPS (150  μg/ml) and IFN-γ (100 U/ml)  for 6 h for RNA blot analysis  (B–D) or 12 h for immunoblot  analysis and apoptosis assays  (E–H). (B) RAW cells were  treated with reagents as indicated and total RNAs (2 μg)  were subjected to blot analysis  for iNOS and arginase II (AII)  mRNAs. The positions of 18S  and 28S rRNAs are shown on  the right. The bottom panels in  B show ethidium bromide staining of 18S and 28S rRNAs. (C)  The results in B were quantitated and are shown by means ±  ranges (n = 2). Maximal values  are set at 100%. (D) Experiments were performed as in B  and the results for arginase II  (AII) mRNA were quantitated  and are shown by means ± SE  (n = 3). Maximal value is set at  100%. (E) RAW cells were  treated with reagents as indicated on the top and the cell extracts (30 μg) were subjected to  immunoblot analysis for the arginase II (AII) protein. The molecular mass marker on the right  was ovalbumin (46 kD). (F) The results in E (n = 2) and a parallel experiment (n = 2) were quantitated and are shown by means ± SE  (n = 4). Maximal value is set at 100%. (G) RAW cells were cultured in the medium with or without 1 μM Dex and/or 1 mM dibutyryl  cAMP for 24 h, and then treated with LPS (150 μg/ml) and IFN-γ (100 U/ml) for 12 h. After fixation, the cells were stained with Hoechst  dye 33258. Phase-contrast images (a, c, e, and g) and fluorescence images (b, d, f, and h) of the same fields are shown. Original magnifications: ×400. Bars, 10 μm. The percentage of total cells which were determined to be apoptotic is shown on the bottom of each panel.  (H) RAW cells were treated with reagents as indicated on the top. DNAs were isolated, resolved on an agarose gel, stained with SYBR  Green I, and visualized for DNA fragmentation by UV transillumination.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132906&req=5

Figure 2: Effect of induction of arginase II on apoptosis of RAW cells. (A) Time course of treatment of RAW cells is shown. RAW cells were cultured in the medium with or without 1 μM Dex and/or 1 mM dibutyryl cAMP (cAMP) for 24 h, and then treated with various combinations of LPS (150 μg/ml) and IFN-γ (100 U/ml) for 6 h for RNA blot analysis (B–D) or 12 h for immunoblot analysis and apoptosis assays (E–H). (B) RAW cells were treated with reagents as indicated and total RNAs (2 μg) were subjected to blot analysis for iNOS and arginase II (AII) mRNAs. The positions of 18S and 28S rRNAs are shown on the right. The bottom panels in B show ethidium bromide staining of 18S and 28S rRNAs. (C) The results in B were quantitated and are shown by means ± ranges (n = 2). Maximal values are set at 100%. (D) Experiments were performed as in B and the results for arginase II (AII) mRNA were quantitated and are shown by means ± SE (n = 3). Maximal value is set at 100%. (E) RAW cells were treated with reagents as indicated on the top and the cell extracts (30 μg) were subjected to immunoblot analysis for the arginase II (AII) protein. The molecular mass marker on the right was ovalbumin (46 kD). (F) The results in E (n = 2) and a parallel experiment (n = 2) were quantitated and are shown by means ± SE (n = 4). Maximal value is set at 100%. (G) RAW cells were cultured in the medium with or without 1 μM Dex and/or 1 mM dibutyryl cAMP for 24 h, and then treated with LPS (150 μg/ml) and IFN-γ (100 U/ml) for 12 h. After fixation, the cells were stained with Hoechst dye 33258. Phase-contrast images (a, c, e, and g) and fluorescence images (b, d, f, and h) of the same fields are shown. Original magnifications: ×400. Bars, 10 μm. The percentage of total cells which were determined to be apoptotic is shown on the bottom of each panel. (H) RAW cells were treated with reagents as indicated on the top. DNAs were isolated, resolved on an agarose gel, stained with SYBR Green I, and visualized for DNA fragmentation by UV transillumination.

Mentions: We then asked whether LPS/IFN-γ–induced apoptosis would be prevented by arginase II induction. RAW cells were cultured in the presence or absence of Dex/cAMP for 24 h, and then treated with LPS or IFN-γ, or their combination for 6 or 12 h (Fig. 2 A). Fig. 2, B and C, shows effects of various combinations of reagents on iNOS and arginase II mRNAs. iNOS mRNA of ∼4.5 kb was not evident in the untreated cells but was induced by LPS, and this induction was enhanced by IFN-γ or Dex/cAMP, and even more strongly by their combination. Dex/cAMP was without effect. Arginase II mRNA of ∼1.8 kb that was low in untreated cells was induced by LPS or Dex/cAMP and even more so by their combination. IFN-γ strongly prevented the induction of arginase II by LPS, whereas it had little effect on the induction by LPS plus Dex/cAMP. In other words, inhibition of LPS induction of arginase II mRNA by IFN-γ was alleviated by Dex/cAMP. cAMP alone had some effect in such cases, whereas Dex alone was ineffective (Fig. 2 D).


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 induction of  arginase II on apoptosis of  RAW cells. (A) Time course  of treatment of RAW cells is  shown. RAW cells were cultured in the medium with or  without 1 μM Dex and/or 1 mM  dibutyryl cAMP (cAMP) for  24 h, and then treated with various combinations of LPS (150  μg/ml) and IFN-γ (100 U/ml)  for 6 h for RNA blot analysis  (B–D) or 12 h for immunoblot  analysis and apoptosis assays  (E–H). (B) RAW cells were  treated with reagents as indicated and total RNAs (2 μg)  were subjected to blot analysis  for iNOS and arginase II (AII)  mRNAs. The positions of 18S  and 28S rRNAs are shown on  the right. The bottom panels in  B show ethidium bromide staining of 18S and 28S rRNAs. (C)  The results in B were quantitated and are shown by means ±  ranges (n = 2). Maximal values  are set at 100%. (D) Experiments were performed as in B  and the results for arginase II  (AII) mRNA were quantitated  and are shown by means ± SE  (n = 3). Maximal value is set at  100%. (E) RAW cells were  treated with reagents as indicated on the top and the cell extracts (30 μg) were subjected to  immunoblot analysis for the arginase II (AII) protein. The molecular mass marker on the right  was ovalbumin (46 kD). (F) The results in E (n = 2) and a parallel experiment (n = 2) were quantitated and are shown by means ± SE  (n = 4). Maximal value is set at 100%. (G) RAW cells were cultured in the medium with or without 1 μM Dex and/or 1 mM dibutyryl  cAMP for 24 h, and then treated with LPS (150 μg/ml) and IFN-γ (100 U/ml) for 12 h. After fixation, the cells were stained with Hoechst  dye 33258. Phase-contrast images (a, c, e, and g) and fluorescence images (b, d, f, and h) of the same fields are shown. Original magnifications: ×400. Bars, 10 μm. The percentage of total cells which were determined to be apoptotic is shown on the bottom of each panel.  (H) RAW cells were treated with reagents as indicated on the top. DNAs were isolated, resolved on an agarose gel, stained with SYBR  Green I, and visualized for DNA fragmentation by UV transillumination.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132906&req=5

Figure 2: Effect of induction of arginase II on apoptosis of RAW cells. (A) Time course of treatment of RAW cells is shown. RAW cells were cultured in the medium with or without 1 μM Dex and/or 1 mM dibutyryl cAMP (cAMP) for 24 h, and then treated with various combinations of LPS (150 μg/ml) and IFN-γ (100 U/ml) for 6 h for RNA blot analysis (B–D) or 12 h for immunoblot analysis and apoptosis assays (E–H). (B) RAW cells were treated with reagents as indicated and total RNAs (2 μg) were subjected to blot analysis for iNOS and arginase II (AII) mRNAs. The positions of 18S and 28S rRNAs are shown on the right. The bottom panels in B show ethidium bromide staining of 18S and 28S rRNAs. (C) The results in B were quantitated and are shown by means ± ranges (n = 2). Maximal values are set at 100%. (D) Experiments were performed as in B and the results for arginase II (AII) mRNA were quantitated and are shown by means ± SE (n = 3). Maximal value is set at 100%. (E) RAW cells were treated with reagents as indicated on the top and the cell extracts (30 μg) were subjected to immunoblot analysis for the arginase II (AII) protein. The molecular mass marker on the right was ovalbumin (46 kD). (F) The results in E (n = 2) and a parallel experiment (n = 2) were quantitated and are shown by means ± SE (n = 4). Maximal value is set at 100%. (G) RAW cells were cultured in the medium with or without 1 μM Dex and/or 1 mM dibutyryl cAMP for 24 h, and then treated with LPS (150 μg/ml) and IFN-γ (100 U/ml) for 12 h. After fixation, the cells were stained with Hoechst dye 33258. Phase-contrast images (a, c, e, and g) and fluorescence images (b, d, f, and h) of the same fields are shown. Original magnifications: ×400. Bars, 10 μm. The percentage of total cells which were determined to be apoptotic is shown on the bottom of each panel. (H) RAW cells were treated with reagents as indicated on the top. DNAs were isolated, resolved on an agarose gel, stained with SYBR Green I, and visualized for DNA fragmentation by UV transillumination.
Mentions: We then asked whether LPS/IFN-γ–induced apoptosis would be prevented by arginase II induction. RAW cells were cultured in the presence or absence of Dex/cAMP for 24 h, and then treated with LPS or IFN-γ, or their combination for 6 or 12 h (Fig. 2 A). Fig. 2, B and C, shows effects of various combinations of reagents on iNOS and arginase II mRNAs. iNOS mRNA of ∼4.5 kb was not evident in the untreated cells but was induced by LPS, and this induction was enhanced by IFN-γ or Dex/cAMP, and even more strongly by their combination. Dex/cAMP was without effect. Arginase II mRNA of ∼1.8 kb that was low in untreated cells was induced by LPS or Dex/cAMP and even more so by their combination. IFN-γ strongly prevented the induction of arginase II by LPS, whereas it had little effect on the induction by LPS plus Dex/cAMP. In other words, inhibition of LPS induction of arginase II mRNA by IFN-γ was alleviated by Dex/cAMP. cAMP alone had some effect in such cases, whereas Dex alone was ineffective (Fig. 2 D).

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.

Show MeSH
Related in: MedlinePlus