Preliminary Study On Apoptosis Induced By Endothelin-1 In Cultured Rat Cortical Neurons

Objective: To investigate whether endothelin (ET) -1 can directly induce apoptosis in primary cultured rat brain neurons, and the ET receptor subtype(s) involved in this action.Method: Primary neuron cultures were obtained from neonatal rats cerebral cortex and incubated for 5 days After the treatment of ET-1 (0.2 nM or 20 nM) for 24 h, apoptosis was semi-quantitatively evaluated with Annexin V staining, Hoechst33258 staining. Thereafter, ET-1 (20 nM), with BQ123 or BQ788, respectively, was added into the culture medium simultaneously for 24 h, and the neuron apoptosis was quantitatively measured with flow cytometer.Result: After the treatment with lower dose of ET-1 (0.2 nM), no obvious change was found in the rate of positive stained cell (Annexin V or Hoechest 33258). By contrast, after incubation with the higher dose of ET-1 (20 nM), much higher rates of apoptosis were evident when measured with Annexin V, Hoechest33258 and flow cytometer (P<0.05~ 0.001). BQ123 (selective antagonist for ET receptor A, 1 mM) and BQ788 (selective antagonist for ET receptor B, ImM) partially blocked the effect of ET-1 inducing neuron apoptosis (P<0.01), respectively. However, the blocking effect of BQ123 or BQ788 was not thoroughly, while the rates of apoptosis after the treatment were still higher than the blank control (p<0.01).Conclusion: The high dose of ET-1 (20 nM) can directly induce apoptosis in primary cultured rat brain neurons, but the low dose of ET-1. The effect of ET-1 inducing neuronal apoptosis was mediated via both ET receptor A and B, smimutanously. Our results indicate that the super-elevated ET-1 level in the central nervous system after different pathaphysiological conditions may directly harm neurons via ET receptors, while the ET antangonist may protect neurons.