| Objectives: To confirm endothelin (ET)-1's effect of inducing apoptosis in primary cultured rat brain neurons; to investigate whether nitric oxide (NO) and glutamate are involved in this action.Methods: Primary neuron cultures were obtained from rat cerebral cortex and incubated for 5 days. The neuron cultures were treated with ET-1 (20nM), ET-1+L-NAME (a NO synthase inhibitor, 100mM), ET-1+APV (an antagonist for glutamate receptors, 100μM), or PBS (control) for 24 hours, respectively. The neuron apoptosis rates were quantitatively measured with flow cytometer. The cultured medium was thereafter collected to measure the concentrations of NO (enzymatic conversion of nitrate to nitrite) and glutamate (high performance liquid chromatography).Results: After the treatment with ET-1(20nM) for 24h, significant higher apoptosis rates were evident when compared with control (p<0.001), together with significantly elevated levels of NO and glutamate in the culture medium (p<0.001, respectively). Coincubation with L-NAME completely inhibited the elevation of NO level in the medium, however, only partially inhibited the apoptotic effect of ET-1 to cultured neurons (apoptosis rate from 31.02 ±9.77% to 18.2 ±2.62%, p<0.05, n=4). APV significantly blocked the apoptotic effect of ET-1, making the apoptosis rate to 6.23 ±1.54% (VS ET-1 group, p<0.001, n=4)., which, however was still higher than the control (0.10±0.07%). Meanwhile, the inhibitory action of APV on apoptotic effect of ET-1 was much stronger than L-NAME.Conclusion: ET-1 (20nM) can induce apoptosis in primary cultured rat brain neurons, and increase the release of endogenous NO and glutamate from neurons. The apoptotic effect of ET-1 to cultured neurons may be partially inhibited by NO synthase inhibitor and significantly blocked by glutamate NMDA receptor antagonist. It is concluded that ET-1 induces apoptosis of cultured brain neurons via NO and predominantly glutamate pathway. |
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