Inactivation Of Endothelial Nitric Oxide Synthase By Sodium Chloride And The Mechanism

ObjectiveIn this experiment, we tested whether changes in sodium concentration, within the physiological range, can alter eNOS activity. Using eNOS activity assay, we demonstrate that a small increase in sodium chloride concentration would decrease the activity of eNOS significantly. Our findings indicate that eNOS is highly sensitive to changes in sodium chloride concentration. A small increase in sodium chloride levels has a large impact on eNOS activity. By using Ach and A23187, further investigate the molecule mechanism of how sodium chloride inactive eNOS.Methods1. Cell treating experiment: Using CHO-vector cells, primary bovine endothelial cells (BAEC) and CHO-eNOS cells, we did Western Blot and eNOS activity assay.2. Exclude the negative interferences: We used BEAC to determine whether the eNOS activity was unaffected in the presence of equal milliosmol of mannitol. Using cell viability assay (WST staining), we next determined whether the increase in sodium chloride levels affects the viability of cells. In addition, we did 3H-L-arginine uptake assay to exclude the System y+-mediated arginine uptake was unchanged with the increase in sodium chloride concentration.3. The aortic angiogenesis assay: Using eNOS inhibitor L-NAME or sodium chloride incubates mice aorta endothelial cells. Both groups of cells grow very slowly. This experiment indicates that the proliferation of endothelial cells is nitric oxide-dependent.4. Further experiment: Using eNOS activity material Ach, Ach plus sodium chloride, A23187 and A234187 plus sodium chloride we did eNOS activity assay. Ach and A23187 could active eNOS but after plused sodium chloride, eNOS activity was decreased significantly and in a does-dependent manner. Results1. Western Blot of BAEC, CHO-eNOS and CHO-vector demonstrated that CHO-vector could not express eNOS, while BAEC and CHO-eNOS expressed eNOS, so we could use BAEC and CHO-eNOS did eNOS activity assay.2. Using BAEC and CHO-eNOS cells, we demonstrated that a small increase in sodium concentration (137～142 mmol/L), which was still considered to be within the physiological range, caused a decrease in eNOS activity in 15minutes. Importantly, the decrease in eNOS activity was sodium concentration-dependent.3. The eNOS activity was unaffected in the presence of equal milliosmol of mannitol, which excluded osmotic effect. WST assay of BAEC which was incubated by sodium chloride demonstrated that the cell vibality was unaffected by sodium chloride. The System y+-mediated arginine uptake was unchanged with the increase in sodium chloride concentration, indicating that sodium chloride specifically suppressed eNOS but did not universally impair other functions of the cells.4. The aortic angiogenesis assay indicated that the proliferation of endothelial cells was nitric oxide-dependent.5. The molecule mechanism of how sodium chloride inactive eNOS: After incubated cells with Ach and A23187, eNOS activity was increased. But after plusing sodium chloride incubated cells together, we found that eNOS was inactived by sodium chloride. This finding demonstrated that sodium chloride could affect the transport of Calcium (Ca2+ ).Conclusion1. A small increase in sodium chloride levels has a large impact on eNOS activity. This kind of inactivity is acutely (in 15minutes) and in a dose dependent manner.2. The hyphothesis of the molecule mechanism of how sodium chloride inactive eNOS is sodium chloride affects the transport of Ca2+ into cells.