Cytochrome P450 Epoxygenase Dependent Activation Of TRPV4 Channel Participates In Enhanced Serotonin-induced Pulmonary Vasoconstriction In Chronic Hypoxia

Introduction:Transient receptor potential(TRP)channels are a group of non-selective cation channels expressed widely in the respiratory system.Transient receptor potential vanilloid 4(TRPV4)channel is a mechanosensitive ion channel expressed in pulmonary blood vessels.TRPV4 contributes to serotonin(5-HT)-induced pulmonary vascular contraction,and it is partly responsible for the enhanced 5-HT response in pulmonary arteries of chronic hypoxia mice.Epoxyeicosatrienoic acid(EET)is an endogenous agonist of TRPV4 and is known to regulate vasoreactivity.The levels of EETs and the expression of cytochrome P450(CYP)epoxygenase,an enzyme for EET production,are significantly increased under chronic hypoxia.We hypothesize that the EET-dependent TRPV4 activation contributes to the 5-HT mediated pulmonary vasoconstriction.Methods:Male wild type(WT)and trpv4-/-C57BL/6J mice were age-matched.Mice were placed in a hypoxic chamber and exposed to hypoxia(10%O2)for 3-4 weeks to induce chronic hypoxic pulmonary hypertension.Control group were housed in the same condition but exposed to room air.The mice pulmonary arteries(PAs)were isolated,cut into segments,de-endothelialized and placed in Krebs solution.PA rings were fixed on a wire-myograph chamber and their isometric tension was recorded.After a 60-minute equilibration,PA rings were exposed to 60 mM KCl to establish maximum contraction.5-HT,HC-067047,a specific TRPV4 inhibitor,and MS-PPOH were applied to PA rings and isometric tension was recorded.The active tension induced was normalized to maximum contraction generated by 60 mM KCl.Results:Application of MS-PPOH,the CYP epoxygenase inhibitor,caused no significant change of Emax or EC50 in PAs of normoxic WT mice.Inhibition of TRPV4 with HC-067047 had no effect on Emax,but it caused a right shift of the concentration-response curve(-logEC50 7.425±0.0884 vs.7.780±0.1134,P<0.05)5-HT-induced PA contraction was significantly potentiated in endothelium-denuded CH PAs compared to normoxic PAs(Emax 178.1 ± 9.767%vs.114.7 ± 2.266%,P<0.05)Significant suppression of the enhanced 5-HT response was observed after HC-067047 treatment(P<0.05),and the inhibitory effect was similar to PAs treated with MS-PPOH(P>0.05).Moreover,5-HT-activated maximum contractile response in trpv4-/-mice was identical to those in WT PAs after TRPV4 inhibition(153.4±6.207%vs.142.5 ±4.603%).Blockage of CYP epoxy genase showed no effect on neither EmaX nor EC50 in trpv4 null mice.We further evaluated the percentage increase in maximal response to 5-HT.Basically,CH caused 55%increase in maximum response to serotonin.The percent enhancement was dramatically attenuated in HC-067047 and MS-PPOH treated PAs of CH WT mice.Moreover,the Emax(HC-067047:24.24 ±4.013%vs.MS-PPOH:35.16±3.336%,P>0.05)and-Log EC50(HC-067047:7.436±0.1673 vs.MS-PPOH:7.311 ± 0.07788,P>0.05)of 5-HT-induced contractions were comparable in PAs of CH mice treated with HC-067047 and MS-PPOH.Conclusions:These results suggest that TRPV4 contributes to the enhanced 5-HT-induced vasoconstriction in chronic hypoxic PAs,partly via the CYP-EET-TRPV4 pathway.Our results further support the notion that regulation of TRPV4 function may offer therapeutic strategies for the treatment of chronic hypoxic pulmonary hypertension.