Studies on the molecular mechanisms of carbon monoxide on the induction or protection of cell death.

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Carbon monoxide (CO), either exogenous or endogenous, has been shown to protect the myocardial and vascular cells against injuries due to ischemia or lipopolysaccharide, through NF-kB or mitochondrial ATP-dependent K^+-channel. Heme-oxygenase (HO)-1, generates CO, thereby protecting the cells. We have shown that a Ca^<2+>-dependent protease calpain promotes necrotic death in the cardiogenic H9c2 cells under hypoxia through α-fodrin proteolysis (Aki, T., Yoshida, K., and Fujimiya, T. (2002) J.Biochem. 132, 921-926). Here, we show the first line of evidence that CO inhibits Ca^<2+>-influx, as detected by fluo-3 fluorescenece, which was enhanced by a L-type Ca^<2+>-channel agonist BAYK8644. The ischemic death was characterized as necrotic either by dye exclusion, LDH release, or propidium iodide permeabilization. The Ca^<2+>-influx, α-fodrin proteolysis, as detected by western blotting, and the ischemic death, were inhibited by CO ora L-type Ca^<2+.-channel inhibitor verapamil. Ischemia also induced mitochondrial depolarization, as detected by JC-1, which was inhibited by CO or verapamil. Additionally, reactive oxygen species (ROS) generation, as detected by DCF, hydroethidine, or Amplex Red Hydrogen Peroxide, was enhanced by ischemia, but unaffected by Co. Hemin treatment increased the HO-1 expression in the hypoxic cells, as detected by western blotting. The HO-1 induction reduced the Ca^<2+>-influx and cell death after ischemia. Thus, exogenous and endogenous CO protect the cardiomyogenic cells against ischemia by inhibiting Ca^<2+>-influx through L-type Ca^<2+> channel and calpain activation.