Roles Of Chloride Channels In Neuroprotection Induced By H₂O₂ Preconditioning In PC12 Cells

Objective:1) To assess the roles of chloride channels in apoptosis induced by hydrogen peroxide in pheochromocytoma cells (PC 12).2) To investigate the roles and mechanism of volume-activated chloride channels in neuroprotection induced by H2O2 preconditioning.Methods:1) Apoptosis was monitored by flow cytometery.2) Nuclear staining was evaluated by Hoechst 33258.3) The whole-cell patch clamp technique was used to record volume-activated chloride currents.4) The Scion Image processor and analysis software were used to detect and calculate the volume (μm3) of PC 12 cells.5) The expression ClC-3 protein was tested by Westernblot and Immunofluorescence techniques.Results:1. In the control, apoptotic cells were only (3.0±0.2)%in the population. However, exposure to 300μmol/L H2O2 for 24h increased apoptotic rate greatly to (70.2±3.2)%(n=4, P <0.01). With the treatment of chloride channel blocker NPPB (5-nitro-2-(3-phenylpropylamino, 100μmol/L) or H2O2 preconditioning, the apoptosis were greatly inhibited.2. When challenged with H2O2, cell volume decreased gradually and was worsened with the increase of H2O2 concentration. NPPB suppressed H2O2-induced apoptotic cell volume decrease (AVD).3. Hoechst33258 showed that most of cells emitted strong fluorescence 1h after exposed to 300μmol/L H2O2 while in those treated with the mixture of 100μmol/L NPPB and 300μmol/L H2O2, only a small portion of cells emitted bright fluorescence.4. The current was significantly enhanced upon the application of H2O2 (300μmol/L) for 3-10min and inhibited by NPPB.5. Hypotonic challenges swelled H2O2 preconditioning PC 12 cells and induced a RVD, which was stronger than that in non-preconditioning cells, while the degree of cell swelling was reduced. NPPB almost completely blocked the RVD.6.47% hypotonic solution activated a volume-activated chloride current in the H2O2 preconditioning PC 12 cells. Compared with the non-preconditioning cells, the property of this current and the sensitivity to Cl" channel blockers NPPB were changed. 7. With the application of H2O2 (100μmol/L) at preconditionging concentration for 90 min, only 55% cells can activate chloride currents. Compare with H2O2 (300μmol/L), there were some similarities and differences of current characteristics.8. The expression of ClC-3 protein was increased in H2O2 preconditioning PC12 cells and dispersed in both kinds of cells.Conclusion:1. Chloride channels may contribute to H2O2-induced apoptosis by ways of elevation of membrane permeability and AVD in PC 12 cells.2. Volume-activated chloride channels may play an important physiological role in the H2O2 preconditioning through enhances cell volume regulation which substantially reduces cells swelling during subsequent long oxidative period, which suggests that the neuroprotective effect may be through improved cell volume regulation. The properties of the volume-activated chloride channels were changed.and capacity of RVD was increased.3. The expression of chloride protein (ClC-3) in H2O2 preconditioning PC12 cells was upregulated.4. Cl" channel activation exerts dual but reciprocal roles in neuroprotection, serving as:(1) An AVD-inducing anion exit pathway under oxidative stress, leading to the cells dead.(2) A RVD anion efflux pathway after preconditioning under hypotonic insults, rescuing the cells from being injured.