Alteration Of Ca²⁺ Signaling Mediated By TRPM8Channels In Pulmonary Arterial Smooth Muscle Cells Of Pulmonary Hypertension Rats

Objective:We applied pulmonary hypertension（PH） rat models by means of chronichypoxia （CH） and monocrotaline（MCT）-injection to discuss the alteration of Ca²⁺signaling mediated by transient receptor potential melastatin8（TRPM8） channel in thedevelopment of PH.Methods: We employed2procedures to establish the PH rat models, i.e. exposure toa long period of hypoxia and administration of monocrotaline hydrochloride（50mg/kg） in rats. We observe:â‘ mean right ventricular pressure （mRVP）,rightventricular systolic pressure（RVSP） and right ventricular mass index （RVMI）②pulmonary artery smooth muscle cells （PASMCs） cultured and TRPM8confirmedexisting in PASMCs by immunohistochemistryâ‘¢the rate and magnitude ofMn²⁺quenching in different pre-treatment PASMCs before and after the impact ofmentholâ‘£the variation of [Ca²⁺]iinduced by menthol and the effect of BCTC on it indisparate pre-disposed PASMCs superfused with Ca²⁺-free or Ca²⁺-containing （2mM）Tyrode solutions.Results:â‘ RVSP,mRVP and RVMI in CH-and MCT-rat model were much higher（P<0.01）than CON,suggesting that CH-and MCT-treated could induce PH.â‘¡Pulmonary artery smooth muscle cells （PASMCs） were smoothly cultivatedand we had prove that TRPM8exists in PASMCs through immunohistochemistry.â‘¢In control group，the character of Ca²⁺response induced by menthol wasdifferent （P<0.01） when PASMCs were superfused with unequal(Ca²⁺-free or Ca²⁺-normal) Tyrode solutions. BCTC could notably block （P<0.01） the rise of [Ca²⁺]i caused by menthol in the condition of both Ca²⁺-free and2Ca²⁺-Tyrode solutions.Thepresent data indicated that in/extracellular Ca²⁺ were jointly involved in the Ca²⁺ transients mediated by menthol in control PASMCs.â‘£The increment of [Ca²⁺]ielicited through menthol in two model PASMCssuperfused both with2Ca²⁺-and0Ca²⁺-Tyrode solutions were respectively decreased（P<0.01） compared to the CON.In addition, the elevation of [Ca²⁺]iinduced bymenthol was obviously inhibited（P<0.01） in normal Ca²⁺-Tyrode solutions or0Ca²⁺-Tyrode solutions,implying that CH-and MCT-preprocessed could respectivelyreduce the rise of [Ca²⁺]imediated by TRPM8channel in PASMCs.⑤Compared with CON, the rest rate and magnitude of Mn²⁺ quenchingin pre-treatment（CH and MCT） PASMCs superfused with0Ca²⁺-Tyrode solutions were slightly increased（P<0.05）,hinting that CH-and MCT-pretreated wasable to strengthen the Ca²⁺ infiux aroused by nonslective cation channels. Onthe contrary,the rate and magnitude of Mn²⁺ quenching caused by menthol intwo model PASMCs superfused with0Ca²⁺-Tyrode solutions were remarkablydeclined（P<0.01）, furthermore, in normal Ca²⁺-Tyrode solutions BCTC couldstrikingly suppress the Ca²⁺ response（P<0.01）generated by menthol,pointing outthat CH-and MCT-pretreated could respectively down-regulate the Ca²⁺ entrymediated by TRPM8channel.Conclusion:â‘ CH-and MCT-treated could respectively induce PH and lead to theupregulation of nonselective cation channels function in rest state.â‘¡TRPM8was also able to mediate Ca²⁺ release apart from Ca²⁺ influx incontrol PASMCs.â‘¢CH-and MCT-preprocessed could respectively down-regulate the Ca²⁺ entry and Ca²⁺ release mediated by TRPM8channel in PASMCs.