The Study Of The Mechanism Of TRPV4-mediated Mouse Airway Smooth Muscle Relaxation

Objective The aim of the present study was to identify the mechanism how14,15-epoxyeicosatrienoic acid(14, 15-EET), which is a product of cytochrome P450 epoxygenase regulates mouse airway smooth muscle(ASM) contraction. We investigated the effect of 14, 15-EET on mice ASM contraction induced by carbachol and the interaction of TRPV4 and SKCa in ASM via isolated tracheal ring tension measurement and co-immunoprecipitation experiments.Methods1. Isolation of Mouse Bronchi Kunming mice(SPF grade), 6 weeks old, weighing 20~25g were used. After sacrificed with overdose of carbon dioxide, the main bronchial were quickly cut out and placed in an oxygenated Krebs-Henseleit solution bubbled with a mixed gas(95%CO2 and 5% O2) to maintain a p H at 7.4±0.05. The connective tissues and adhering parenchyma were dissected quickly. The bronchial was cut into about 2 mm rings. The epithelial layer was removed with a metal wire carefully.2. Isometric Tension Measurements The tissues were allowed to treated with different inhibitors and 300 nmol/L 14,15-EET together. After then, we used carbachol(CCh) to induce reproducible concentration-dependent tension response curves. The response was recorded with a acquisition and analysis system(BL-420S).3. Co- immunoprecipitation Co-immunoprcipitation was used to detect the interaction of TRPV4 and SKCa in ASM.4. Statistical analyses The experimental data were presented as mean ± S.E.M. Multi factor analysis of variance were performed using Sigma Plot 12.5 software and P < 0.05 was considered as the level of significance.Results1.CCh(10-10~10-3 mol/L) induced a concentration-dependent contraction in mouse ASM. Compared to the control groups, CCh-induced contraction was significantly reduced in the pretreatment of 14, 15-EET(300 nmol/L) groups(*P < 0.05).2. The inhibitor of large conductance Ca2+-activated K+ channels: 50 μmol/L Ib TX and the inhibitor of intermediate conductance Ca2+-activate K+ channels: 10 μmol/L TRAM-34 did not significantly affect the effect of 300 nmol/L 14, 15-EET on CCh-induced mouse ASM contraction; While the inhibitor of small conductance Ca2+-activated K+ channels: 1 μmol/L Apamin blocked the effect of 14,15-EET on ASM contraction remarkably(*P < 0.05).3. Compared with the control groups, TRPV4 channel inhibitor : 20 μmol/L RN-1734 significantly decreased the inhibitive effect of 300 nmol/L 14, 15-EET on CCh-induced mouse ASM contraction(*P < 0.05).4. TRPV4 and SK3 :Co-immunoprecipitation result indicated that TRPV4 pulled down SK3 each other.Conclusion14, 15-EET regulated CCh-induced tracheal contraction through TRPV4-SKCa signal complex in mouse ASM.Objective Transient receptor potential vanilloid 4(TRPV4) channel is one of the nonselective cation channels which preferably permeate Ca2+.TRPV4 channels are widely expressed in various tissues. When TRPV4 is activated by related stimulators, TRPV4 will mediate Ca2+ influx and increase intracellular Ca2+ concentration, which then directly regulates the contraction and relaxation of airway smooth muscle(ASM). IP3 can activate IP3 R to medicate Ca2+ release from endoplasmic reticulum. At the same time,TRPV4-mediated Ca2+ influx will induce localized Ca2+ rise and enhance sodium/calcium enchanger. Therefore, in this study we used isometric tension measurements, co-immunopercipitation et al. To observe the interaction of TRPV4-IP3R1-NCX2 and the possible function of it in ASM contraction.Methods1. Isolation of Mouse Bronchi Same as part one.2. Isometric Tension Measurements Using carbachol(CCh) to induce reproducible concentration-dependent tension response curves and record the data with a data record system(BL-420S).GSK1016790A-induced bronchi contraction with concentration-dependent manner was recorded.3. Co- immunoprecipitation Co-immunoprcipitation was used to detect the interaction of TRPV4, Na+/Ca2+exchanger and IP3R1 in mouse ASM.4. Statistical analyses The experimental data were presented as mean ± S.E.M. Multiple comparisons were analyzed using Sigma plot 12.5 and those at P < 0.05 were considered as the level of significance.Results1.Diastolic response was induced by GSK106790 A in CCh-induced ASM contraction.Compaired to the control group, the relaxation response of ASM was significantly reduced in the pretreatment of NCX inhibitor 5 μmol/L KB-R7943 and Li Cl(*P < 0.05).2.Compared with the control groups, the pretreatment of 10 μmol/L HC067047 reduced the relaxation of ASM notably(*P < 0.05).3.Compared with the control group, the pretreatment of 4 μmol/L TG reduced the relaxation of ASM notably(*P < 0.05).4.Compared with the control group, the pretreatment of 10 μmol/L Ryanodine didn’t have obvious effect on relaxation of ASM very much.5.Co-immunoprecipitation result indicated that TRPV4, IP3R1 and NCX2 pulled down each other in ASM..Conclusion We conclude that TRPV4,NCX2 and IP3R1 form a signal complex in tracheal smooth muscle cells to regulate tracheal tension and respiration.