J Clin Neurol.  2005 Apr;1(1):81-91. 10.3988/jcn.2005.1.1.81.

Comparison of the Protective Effect of Indole beta-carbolines and R-(-)-deprenyl Against Nitrogen Species-Induced Cell Death in Experimental Culture Model of Parkinson's Disease

• Han YS
• Kim JM
• Cho JS
• Lee CS
• Kim DE

• KMID: 2287717
• DOI: http://doi.org/10.3988/jcn.2005.1.1.81

Abstract

BACKGROUND
The membrane permeability transition of mitochondria has been suggested to be involved in toxic and oxidative forms of cell injury. Mitochondrial dysfunction is considered to play a critical role in neurodegeneration in Parkinson's disease. Despite the suggestion that indole beta-carbolines may be neurotoxic, these compounds provide a protective effect against cytotoxicity of other neurotoxins. In addition, the effect of indole beta-carbolines on change in the mitochondrial membrane permeability due to reactive nitrogen species (RNS), which may lead to cell death, has not been clarified.
METHODS
Differentiated PC12 cells were used as the experimental culture model for the investigation of neuronal cell injury, which occurs in Parkinson's disease. The effect of indole beta-carbolines (harmalol and harmine) on differentiated PC12 cells against toxicity of S-nitroso-N-acetyl-DL-penicillamine (SNAP) was determined by measuring the effect on the change in transmembrane potential, cytochrome c release, formation of ROS, GSH contents, caspase-3 activity and cell viability, and was compared to that of R-(-)-deprenyl.
RESULTS
Specific inhibitors of caspases (z-LEHD.fmk, z-DQMD.fmk) and antioxidants (N-acetylcysteine, dithiothreitol, melatonin, carboxy-PTIO and uric acid) depressed cell death in PC12 cells due to SNAP. beta-Carbolines and R-(-)-deprenyl attenuated the SNAP-induced cell death and GSH depletion concentration dependently with a maximal inhibitory effect at 25-50 microM. The compounds inhibited the nuclear damage, decrease in mitochondrial transmembrane potential, cytochrome c release and formation of reactive oxygen species caused by SNAP in PC12 cells. beta-Carbolines and R-(-)-deprenyl attenuated the H2O2-induced cell death and depletion of GSH.
CONCLUSIONS
The results suggest that indole beta-carbolines attenuate the SNAP-induced viability loss in PC12 cells by inhibition of change in the mitochondrial membrane permeability, which may be caused by free radicals. Indole beta-carbolines appear to exert a protective effect against the nitrogen species-mediated neuronal cell injury in Parkinson's disease comparable to R-(-)-deprenyl.

Keyword

Indole beta-carbolines; Nitrogen species; Mitochondrial membrane permeability; Cell death; Differentiated PC12 cells

MeSH Terms

Animals
Antioxidants
Carbolines*
Caspase 3
Caspases
Cell Death*
Cell Survival
Cytochromes c
Dithiothreitol
Free Radicals
Melatonin
Membrane Potentials
Membranes
Mitochondria
Mitochondrial Membranes
Neurons
Neurotoxins
Nitrogen*
Parkinson Disease*
PC12 Cells
Permeability
Reactive Nitrogen Species
Reactive Oxygen Species
Antioxidants
Carbolines
Caspase 3
Caspases
Cytochromes c
Dithiothreitol
Free Radicals
Melatonin
Neurotoxins
Nitrogen
Reactive Nitrogen Species
Reactive Oxygen Species

Figure

• 

  Figure 1 Effect of NO scavengers on cell death due to SNAP. PC12 cells were treated with 500 µM SNAP in the presence of caspase inhibitors (40 µM z-LEHD.fmk [LEHD], 40 µM z-DQMD.fmk [DQMD]) or antioxidants (1 mM N-acetylcysteine [NAC], 1 mM dithiothreitol [DTT], 100 µM melatonin, 25 µM carboxy-PTIO [PTIO] and 250 µM uric acid) for 24 hours at 37℃ and mixtures were treated with 0.5 mg/ml MTT for 2 hours. Data are expressed as the percentage of control and represent means±SEM of 6 replicate values in two separate experiments. †P<0.05, compared to control. *P<0.05, compared to SNAP alone.

• 

  Figure 2 Effect of β-carbolines and R-(-)-deprenyl on SNAP-induced loss of cell viability. PC12 cells were treated with 500 µM of SNAP in the presence of 5-100 µM β-carbolines (harmalol and harmine) or R-(-)-deprenyl for 24 hours. Data are expressed as the percentage of control and represent means±SEM of 6 replicate values in two separate experiments. †P<0.05, compared to control. *P<0.05, compared to SNAP alone.

• 

  Figure 3 Effect of β-carbolines and R-(-)-deprenyl on SNAP-induced nuclear damage. PC12 cells were treated with 500 µM SNAP in the presence of 25 µM β-carbolines or R-(-)-deprenyl for 24 hours. In the experiment (A) cells were observed by fluorescence microscopy after nuclei staining with Hoechst 33258. a: control cells, b: cells treated with SNAP alone, c: cells treated with SNAP and harmine, (d) cells treated with harmine alone. All the subparts are representative of four different experiments. In the experiment (B) the 3'-ends of DNA fragments were detected as described in Materials and Methods. Data are expressed as absorbance and represent the means±SEM of 6 replicate values in two separate experiments. †P<0.05, compared to control. *P<0.05, compared to SNAP alone.

• 

  Figure 4 Effect of harmine and R-(-)-deprenyl on loss of the mitochondrial transmembrane potential due to SNAP. PC12 cells were treated with 500 µM SNAP in the presence of 25 µM harmine for 24 hours and mixtures were treated with 40 nM DiOC6(3). Data represent means±SEM of the percentage-depolarized cells in three independent experiments. †P<0.05, compared to control. *P<0.05, compared to SNAP alone.

• 

  Figure 5 Effect of β-carbolines on cytochrome c release and caspase-3 activation. PC12 cells were treated with 500 µM SNAP in the presence of β-carbolines or R-(-)-deprenyl (each 25 µM) for 24 hours. Data are expressed as nanograms/ml for cytochrome c release (A) and units for caspase-3 activity (B). The values represent means±SEM of 4-6 replicate values in two separated experiments. †P<0.05, compared to control. *P<0.05, compared to SNAP alone.

• 

  Figure 6 Effect of β-carbolines and R-(-)-deprenyl on ROS formation and GSH depletion. PC12 cells were treated with 500 µM of SNAP in the presence of 5-100 µM β-carbolines (harmalol and harmine) or R-(-)-deprenyl for 24 hours. The values are expressed as arbitrary units of fluorescence for ROS formation (A) and nmol for GSH contents (B). Data represent means±SEM of 5-6 replicate values in two separated experiments. †P<0.05, compared to control. *P<0.05, compared to SNAP alone.

• 

  Figure 7 Effect of β-carbolines and R-(-)-deprenyl on the H2O2-induced cell death and GSH depletion. PC12 cells were treated with 100 µM of H2O2 in the presence of compounds (25 µM β-carbolines, 25 µM R-(-)-deprenyl and 10 µg/ml catalase) for 24 hours. Data are expressed as the percentage of control for cell viability and nmol/µg protein for the GSH contents. The values represent means±SEM of 6 replicate values in two separate experiments. †P<0.05, compared to control. *P<0.05, compared to H2O2 alone.

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