Article Figures & Data
Figures
- Figure 1.
Motor neurons were cultured for 3 d with (A, C) and without (B, D) trophic factors and stained for arginase 1 using mouse monoclonal (A, B) and rabbit polyclonal (C, D) antibodies as described in Materials and Methods. The images were captured after 3 d in culture. Scale bars: (in B) A, B, 100 μm; (in D) C, D, 50 μm.
- Figure 2.
Production of nitric oxide by motor neurons in culture. After 3 d in culture, the culture medium was replaced, and the cells were incubated in L15 without phenol red supplemented with DAF-FM (Control) or treated with 100 μm nitro-l-arginine (LNAME), 1 μm NOHA, or 1 μm NOHA plus 100 μm LNAME for 2 h before the fluorescence was measured as described in Materials and Methods. The 100% is the fluorescence intensity in the cultures without treatment. The values are the mean ± SD of three experiments performed in quintuplicate. *p < 0.001 versus Control; **p < 0.001 versus NOHA (one-way ANOVA followed by Bonferroni’s post test).
- Figure 3.
A, NOHA-induced death of trophic factor-deprived motor neurons. Motor neurons were cultured for 3 d with BDNF (filled bar) and without trophic support (open bar). NOHA was added during plating alone at the indicated concentrations (▪) or in combination with 1 ng/ml BDNF (·). The relation between NOHA concentration and motor neuron survival was fitted to a line with slope of 0 for BDNF and to a sigmoid curve using nonlinear regression (goodness of fit R2 = 0.91 with 57 degrees of freedom). NOHA reduced motor neuron survival in a dose-dependent manner with an EC50 of 0.7 ± 0.7 μm (mean ± SD), the top best-fit value equal to 48 ± 10% (mean ± SD), and the bottom best-fit value equal to 6 ± 10%. Values are the mean ± SD of three independent experiments performed in quadruplicate. *p < 0.001 versus BDNF analyzed first by ANOVA and then by the Newman–Keuls multiple-comparison test. B, Inhibition of NOS and scavenging of superoxide and peroxynitrite reversed the toxic effects of NOHA. Motor neurons were cultured for 3 d with (open bars) or without (filled bar) NOHA (1 μm). Trophic factor-deprived motor neurons were incubated with NOHA (1 μm) or with NOHA and either nitro-l-arginine methyl ester (LNAME) (100 μm), iron porphyrin (FeTCPP) (10 μm), or Mn(III) tetra(4-benzoic acid) porphyrin chloride (MnTBAP) (100 μm). Motor neuron survival was determined after 3 d in culture. Values are the mean ± SD of three independent experiments performed in quadruplicate. *p < 0.001 versus NOHA; **p < 0.01 versus NOHA; ***p < 0.05 versus NOHA.
- Figure 4.
Overexpression of arginase prevented motor neuron death induced by trophic factor deprivation. Motor neurons were electroporated with the pEGFP.C1 vector alone (filled bars) or in combination with the pEIF/Myc-His C vector containing the gene for the arginase 1 (open bars). Motor neurons were incubated in the presence of trophic factors overnight. Motor neuron survival was determined 3 d after trophic factor deprivation by counting all fluorescent cells. The protection provided by overexpression of arginase 1 was reversed by incubation with 1 μm NOHA or 20 μm DETANONOate [(Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl) amino]diazen-1-ium-1,2-diolate]. Overexpression of arginase 1 did not affect the survival of motor neurons incubated with 1 ng/ml BDNF at the moment of changing the medium. Values are the mean ± SD from at least three independent experiments performed in quadruplicate. *p < 0.01 versus trophic factor deprivation (TFD); **p < 0.01 versus arginase 1.
