Role Of Novel Mechanism Of Extracellular Calcium Sensing In The Change Of Cytoplasm Calcium Concentration Induced By Hypoxia In Pulmonary Artery Smooth Muscle Cells

Partâ… The Expression of Calcium Sensing Receptor in RatPulmonary Artery Smooth Muscle and Its FunctionObjectives: (1) To explore the expression and distribution of calcium sensing receptorin rat pulmonary artery smooth muscle; (2) to explore the role of calcium sensing receptorin the changes of calcium concentration induced by extracellular high calcium inpulmonary artery smooth muscle cells.Methods: Primary culture of rat pulmonary artery smooth muscle cells was used inthis study. Western blot analysis and immunofluorescence were used to detect theexpression and distribution of calcium sensing receptor in pulmonary artery smooth musclecells. Calcium fluorescence probe (Fura-2/AM) combined with microscopic imagingsystem to detect the concentration of calcium.Results: (1) The morphology of the cells and the specific expression of smooth muscleα-actin confirmed that the cultures cells were rat pulmonary artery smooth muscle cells. (2)The immunostaining revealed that calcium sensing receptor was expressed in pulmonaryartery Smooth muscle cells, it mainly distributed in membrane and cytoplasm of cells. Themolecular weight of calcium is around 55-72kD and170 kD. (3) 6 mM and 7.5 mM [Ca²⁺]₀increased the concentration of calcium in cytoplasm of pulmonary artery smooth musclecells from 76.53±2.23 nM to 238.78±7.26 and 82.74±9.87 nM (p＜0.01, N=23), respectively. The concentration was kept at a high level until the concentration ofextracellular calcium restored to normal level; However, 4 mM [Ca²⁺]0 could not induce asignificant change of cytoplam calcium concentration (p＞0.05, N=23). (4) The inhibitor ofcalcium sensing receptor Calhex 231(1μM + 6 mM [Ca²⁺]₀ or Calhex 231 1μM+ 7.5 mM[Ca²⁺]₀ increased cytoplasm calcium concentration from 75.54±6.48 nM to 139.26±11.25nM and 198.89±18.39 nM (p＜0.01, N=21), respectively; 3μM Calhex 231 + 6 mM or 7.5mM Ca²⁺ increased cytoplasm calcium concentration from 77.91±3.65 nM to 99.15±6.18nM and 119.07±10.63 nM (p＜0.01, N=20), respectively; However, 0.1μM Calhex 231had no significant effect on increased cytoplasm calcium concentration induced by 6 mMand 7.5 mM [Ca²⁺]₀ (p＞0.05, N=23).Conclusions: (1) pnlmonary artery smooth muscle cells expressed calcium sensingreceptor, which was mainly distributed in membrane and cytoplasm in the cells; (2) Theinhibitor of calcium sensing receptor Calhex 231 exerted inhibitory effects on increasedcytoplasm calcium concentration induced by extracellular high concentration calcium. It isindicated that calcium sensing receptor might be involved in extracellular highcalcium-induced increase in cytoplasm calcium concentrationPartâ…¡Role of Novel Mechanism of Extracellular Calcium Sensingin the Change of Cytoplasm Calcium Concentration Induced byHypoxia in Pulmonary Artery Smooth Muscle CellsObjectives: (1) To confirm the change of cytoplasm calcium concentration andreactive oxygen species induced by hypoxia; (2) to explore the role. of calcium sensingreceptor in hypoxia-triggered calcium signaling change; (3) to determine the relationshipsbetween the changes of reactive oxygen species and cytoplasm calcium concentrationduring hypoxia in pulmonary artery smooth muscle cells Methods: Nitrogen gas was filled in the perfusion liquid to reproduce the cell hypoxiamodel. Fluorescence probe Fura-2/AM and ROS sensitive probe H2-DCFDA were used todetect the changes of cytoplasm calcium concentration and reactive oxygen species; RNAiwas used to inhibit expression of calcium sensing receptor and Western blot analysis wasused to determine the interference efficiency.Results: (1) The treatment of hypoxia of pulmonary artery smooth muscle cells for 3discontinuous times induced significant increase in cytoplasm calcium concentration from77.66±1.74 nM to 161.13±11.25 nM,133.03±8.44 nM,109.94±4.64 nM, respectively(p＜0.01, N=51). The peak value of cytoplasm calcium concentration for the last timehypoxia treatment was significantly lower the one for the previous hypoxia treatment(p＜0.01). The perfusion liquid with low oxygen and no calcium decreased the cytoplasmcalcium concentration to 69.65±3.59 nM, which is slightly lower the baseline level (78.09±2 nM, N=43), but no statistically significantly difference was observed (p＞0.05).However, when it was compared with the treatment of low oxygen and extracellularcalcium, the cytoplasm calcium concentration is significantly lower than the later one(133.03±8.44 nM, N=51) (p＜0.01). (3) and (6) The experiment includes three groups:blank control, ShRNA controlå’ŒShRNA targeted. The base levels of [Ca²⁺]_I were 80.67±3.49 nM(N=14),79.3±4.17 nM (N=11) and 80.53±4.3 nM (N=9), respectively. Therewere no significant differences for the levels among all groups (p＞0.05). Hypoxia increased[Ca²⁺]_I to 109.16±8.41 nM in ShRNA targeted group. The concentration was significantlylowed than the ones in blank control (148.22±8.89 nM) and ShRNA control (150.68±10.48 nM) (p＜0.01). 100μM histamine treatment increased [Ca²⁺]_I in pulmonary arterysmooth muscle cells to 222.21±15.33 nM,223.12±16.85 nM,224.48±18.76 nM inblank control,ShRNA control and ShRNA targeted group, respectively. There were nosignificant differences for the levels among all groups (p＞0.05). (4) Hypoxia (PO₂:14.3±2.5 mmHg) increased reactive oxygen species from 824.44±26.43 to 2590.66±97.37 inpulmonary artery smooth muscle cells (around 3 fold increase, 3.14±0.13 (p＜0.01, N=22).(5) The treatment of 0.1,1,10,20,30,50 and 100μM H₂O₂ to mimic hypoxia treatment induced the changes of reactive oxygen species and a regression curve was achieved:y=0.064x+1.9023, R²=0.9897 [x represents fluorescence variation rate (%) and y representsthe concentration of H₂O₂ (μM)]. The concentration of reactive oxygen species in the modelwas figured out to be 15.61±1.24μM (N=22) based on this formula. (6) Treatment of15.61μM H₂O₂ in pulmonary artery smooth muscle cells fro 3 minutes in blank control,ShRNA control and ShRNA targeted increased [Ca²⁺]_I to 151.27±9.7 nM (N=12),149.83±10.05 nM (N=8),105.7±7.83 nM (N=7). The concentration in ShRNA targeted group wassignificantly lower than the ones in blank control and ShRNA control (p＜0.01).Conclusions: Hypoxia activates calcium sensing receptor and increases its sensitivityvia increasing reactive oxygen species. Under normal extracellular calcium, the receptor isactivated to trigger the increase in cytoplasm calcium concentration in pulmonary arterysmooth muscle cells.