The Role And Mechanism Of Extracellular Calcium Sensing Receptor In Hypoxic Pulmonary Vasoconstriction

Part1The role and mechanism of the extracellular calcium-sensing receptor in hypoxia-induced cytoplasmic calcium concentration increase in pulmonary arterial smooth muscle cellsObjective:To study the role and mechanism of the extracellular calcium-sensing receptor (CaSR) in hypoxia induced [Ca2+]i increase in pulmonary artery smooth muscle cells (PASMCs).Methods:Primary culture of SD rat PASMCs were used in this study. The expression of CaSR in PASMCs was detected with immunohistochemistry and western blot. The function of CaSR was detected by using different concentrations of extracellular Ca2+to handle the PASMCs. The calcium fluorescence probe Fura-2/AM and fluorescence imaging system were used to detect [Ca2+]i change. H2-DCFDA and mito-RoGFP were used to detect the generation of intracellular reactive oxygen species (ROS). ShRNA gene technology was used to knock down the CaSR, STIM1and RyR3expression, and the interference efficiency was detected with western blot at the same time.Results:(1) Both immunohistochemistry and western blot have detected CaSR expression in PASMCs, and its distribution is reflected in within the cell membrane and the cytoplasm with molecular weights of55-72kD and170kD.(2) By using4,6and7.5mM extracellular Ca2+to handle PASMCs, the phosphorylation level of ERK1/2is enhanced significantly and gradually, then6mM and7.5mM extracellular Ca2+can induce [Ca2+]i significant increase, while this effect is inhibited by Calhex231(CaSR inhibitor).(3) With different concentrations of exogenous H2O2and hypoxia to stimulate PASMCs, and the varying levels of ROS were detected, it is found that15.6μM H2O2and hypoxia-induced ROS change has the same effect.(4) Both hypoxia and15.6μM H2O2can cause PASMCs [Ca2+]i rise, and Calhex231and CaSR gene knockdown can inhibit hypoxia and15.6μM H2O2-induced [Ca2+]i rise.(5)Mitochondrial DNA depletion can not only inhibit hypoxia-induced ROS generation, but also eliminate the hypoxia-induced [Ca2+]i increase, this effect can be reversed by exogenous15.6μM H2O2, and Calhex231and CaSR gene knockout are able to stop this reversal effect. PEG-Catalase pretreatment the PASMCs is able to inhibit hypoxia-induced [Ca2+]i increase, but PEG-SOD pretreatment does not have this effect.(5)100μM ryanodine inhibiting RyR function can reduce hypoxia and15.6μM H2O2caused [Ca2+]; rise, and IP3R inhibitor20μM XeC can be failed to reduce hypoxia and15.6μM H2O2caused [Ca2+]i rise, but if the application of PKA inhibitor H-89is able to make100μM ryanodine and20μM XeC produce opposite effect at the same time. SOCE inhibitor50μM SKF96365can inhibit hypoxia and15.6μM H2O2induced [Ca2+]i rise significant. STIM1and RyR3knockout can also reduce hypoxia and15.6μM H2O2induced [Ca2+]i rise.Conclusion:Hypoxia induced the production of mitochondria-derived H2O2increase in the PASMCs, the increased H2O2can sensitize CaSR, and CaSR activated by extracellular Ca2+, which caused the cytoplasm of Ca2+from intracellular calcium stores release by RyR, inducing SOCE opening and extracellular Ca2+inflow through STIM1, the combined action has induced [Ca2+]i rise in the PASMC. Part2The role of the extracellular calcium-sensing receptor in hypoxic pulmonary vasoconstriction in vitro and vivoObjective:To explore the key role of CaSR in hypoxic pulmonary vasoconstriction (HPV) in vitro and vivo.Methods:The lentiviral was used to infect SD rats, aiming to knock down the expression of CaSR protein. The Power lab four conductivity meters was used to monitor the tension of pulmonary artery and rat pulmonary artery pressure and cardiac output under different conditions. Results:(1) The lentiviral packaging of CaSR specific interference plasmid was injected into SD rat from tail vein,72hours later, western blot was used to detect the expression level CaSR protein, the expression level was significantly decreased.(2) In vitro experiments, both hypoxia and15.6μM H2O2can cause increased pulmonary vascular tone, the same time Calhex231can inhibit hypoxia and15.6μM H2O2cause increase in pulmonary vascular tone. In addition, the CaSR gene knockdown also can eliminate the increase in hypoxia-induced pulmonary vascular tone.(3) In vivo experiments, hypoxia can cause SD rat pulmonary artery pressure increased significantly, while Calhex231and CaSR knockout can inhibit hypoxia-induced increase in pulmonary artery pressure.Conclusion:The CaSR has involved in hypoxia-induced [Ca2+]i rise in pulmonary arterial smooth muscle cells, which resulted in hypoxic pulmonary vasoconstriction and pulmonary hypertension.