We previously demonstrated that both mitogenicity and anabolism in rat L6 myoblasts even in the presence of insulin could be markedly reduced by milimolar concentrations of H202, SIN-1 (3-mor- pholinosydnonimine) and SNP (sodium nitroprusside) - donors of peroxynitrite (ONOO ) and nitric oxide (NO), respectively (Orzechowski and Grzelkowska 2000). In this paper we described the results of the experiment, which was carried out in order to confirm the assumption that muscle cell death occurred after treatment with high (milimolar) doses of ROS/RNS. Hydrogen peroxide (H202) or SIN-1, or SNP, all induced cell death in rat L6 myoblasts when given at 1 mM. Symptoms of cell shrinking with nuclear chromatin condensation collapse of chromatin into patches along nuclear membrane and formation of apoptotic bodies occurred within 24 hours of the study. Apoptosis was evaluated in situ on the basis of apoptotic index. The process of oligonucleosomal fragmentation of nuclear DNA was demonstrated whenever apoptotic cell death was observed. The verification of cell viability was monitored and the above-mentioned index confirmed the lack of cell respiration in dead myoblasts, especially those treated with SNP. On average 92%, 80%, 77% and 65% of cells were found apoptotic after 1 mM H202, 1 mM SIN-1 or 0.5 mM SNP and 1 mM SNP, respectively. However, necrotic cell death significantly contributed to overall cell death in cultures treated with NO donor and amounted to 6% and 21% for 0.5 mM SNP and 1 mM SNP, respectively. Taken together, these results indicate massive apoptosis induced by H202 and SIN-1, but, with regard to apoptotic action of NO, cell respiration was additionally attenuated and associated with necrotic cell death.
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