Nitric oxide-induced apoptosis is mediated by Bax/Bcl-2 gene expression, transition of cytochrome c, and activation of caspase-3 in rat vascular smooth muscle cells

Clin Chim Acta. 2004 Mar;341(1-2):83-91. doi: 10.1016/j.cccn.2003.11.009.

Abstract

Background: In contrast to the anti-apoptotic action of nitric oxide (NO) on endothelial cells, NO exerts a pro-apoptotic effect on vascular smooth muscle cells (VSMCs). This study was designed to elucidate the mechanism underlying NO-induced apoptosis in rat VSMCs.

Methods and results: (1) Using the terminal deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick end labeling (TUNEL) assay and fluorescence activated cell sorter (FACS) analyses, apoptosis of rat VSMCs were confirmed after exposure to sodium nitroprusside (SNP) (0.5 to 4 mmol/l), an exogenous NO donor. The effects of SNP were blocked by hemoglobin. (2) A universal caspase inhibitor, z-VAD-fmk, dose-dependently inhibited NO-induced apoptosis. VSMCs degraded Ac-DEVD-pNA rather than Ac-WHED-pNA after exposure to SNP, which suggested that the activation of caspase 3 rather than caspase 1 was involved in the process. Immunoblot analysis confirmed the activation of caspase-3. (3) Exposure to SNP induced the release of cytochrome c from the mitochondria to the cytosol, which was detected by immunoblot analysis of mitochondrial and cytosol fractions. (4) SNP exposure increased the ratio of Bax/Bcl-2 protein expression twofold by immunoblot analysis.

Conclusions: The mechanism of NO-induced apoptosis in rat VSMCs involves an increase in the ratio of Bax/Bcl-2 gene expression, which leads to the release of cytochrome c from the mitochondria to the cytosol, finally activating caspase-3 and resultant apoptosis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Aorta, Thoracic / cytology
  • Aorta, Thoracic / enzymology
  • Aorta, Thoracic / metabolism
  • Apoptosis / physiology*
  • Blotting, Western
  • Caspase 3
  • Caspases / metabolism*
  • Cell Count
  • Cell Survival / physiology
  • Cells, Cultured
  • Cytochromes c / genetics*
  • Cytoplasm / enzymology
  • Enzyme Activation / physiology
  • Flow Cytometry
  • Gene Expression / genetics
  • Gene Expression / physiology*
  • Genes, bcl-2 / genetics*
  • In Situ Nick-End Labeling
  • Male
  • Mitochondria, Muscle / enzymology
  • Muscle, Smooth, Vascular / enzymology*
  • Nitric Oxide / physiology*
  • Proto-Oncogene Proteins / genetics*
  • Proto-Oncogene Proteins c-bcl-2*
  • Rats
  • Rats, Sprague-Dawley
  • Tetrazolium Salts
  • Thiazoles
  • bcl-2-Associated X Protein

Substances

  • Bax protein, rat
  • Proto-Oncogene Proteins
  • Proto-Oncogene Proteins c-bcl-2
  • Tetrazolium Salts
  • Thiazoles
  • bcl-2-Associated X Protein
  • Nitric Oxide
  • Cytochromes c
  • Casp3 protein, rat
  • Caspase 3
  • Caspases
  • thiazolyl blue