Roles Of NADPH Oxidase On Calcium Response Of Pulmonary Arterial Smooth Muscle Cells Induced By 5-hydroxytryptamine And The Change In The Process Of Pulmonary Hypertension

Pulmonary hypertension?PH?is a pulmonary circulatory disease,a clinical and pathophysiological syndrome caused by a variety of etiologies and pathogenesis,and its main feature is the progressive increase of pulmonary vascular resistance.In the chronic hypoxia-induced pulmonary hypertension?CHPH?model,the oxidation and reduction systems in the body are in an unbalanced state,leading to the accumulation of reactive oxygen species?ROS?in the body or cells,which promotes the occurrence of oxidative stress and then causes oxidative damage.The increase of intracellular ROS can cause imbalance of calcium homeostasis in vascular smooth muscle,causing vasoconstriction and proliferation of smooth muscle cells.The main source of ROS is NADPH oxidase?NOX?,and TRPM2 in the transient receptor potential?TRP?superfamily acts as an oxidative stress sensor which involves the increases of intracellular Ca²⁺concentration induced by oxidative stress and participates in the physiological/pathological processes of many cells.Studies have shown that 5-HT can activate NOX to produce ROS and promote vasoconstriction,cell proliferation,and cardiac hypertrophy on arteries of systemic circulation and vascular smooth muscle cells.In this study,we established CHPH model to compare the 5-HT-induced calcium responses in pulmonary artery smooth muscle cells?PASMCs?of rat with control?CON?group,and used nonspecific and specific NOX inhibitors,and TRPM2inhibitor to observe their inhibitory effects to determine which NOX subtype plays a major role in the reaction,and verify whether it is mediated by TRPM2,and then provide a new theoretical basis for the study of the pathogenesis of PH.OBJECTIVE:To observe the 5-HT-induced calcium response in PASMCs from CON and CH rat and the effect of different NOX inhibitors on calcium responses,and to identify the main NOX subtypes in the pathogenesis of PH and whether it is mediated by TRPM2.METHODS:Male SD rats were exposed in broiler box with the oxygen pressure?9.5%¹0.5%?within 21days to establish the rat model of CH-induced chronic pulmonary hypertension.Measure the following indicators:1.Right ventricular systolic pressure?RVSP?;2.Right ventricular mass index?RVMI?;3.Incubate primary PASMCs with enzymatic hydrolysis;4.Detect intracellular calcium concentration with fluorescence in CON and CH rats:Use different NOX inhibitors and TRPM2 inhibitor to observe the 5-HT-induced calcium response and then determine if the response is mediated by NOXs-TRPM2.Results:1.mRVSP and RVMI were significantly increased in CHPH model rats compared with CON group,suggesting that the model was successfully prepared;2.Effects of NOX inhibitors on calcium responses in PASMCs induced by 5-HT in CON rats.NOX non-specific inhibitor?APO,30?M?can reduce the calcium release and calcium entry of PASMCs induced by 5-HT in CON rats,suggesting that NOX participates in this reaction process;After applying three specific NOX inhibitory,5-HT-induced PASMCs calcium entry was decreased,and its rate was reduced,among which the inhibitory effects of NOX 1/4 inhibitor?GKT137831,10?M?and NOX1inhibitor?ML171,10?M?were obvious;as for 5-HT-induced PASMCs calcium release,the inhibitory effect of gp91ds-tat?10?M?was not obvious,while GKT137831 and ML171 can significantly inhibit the release.It is suggested that NOX1,NOX2,and NOX4 subtypes all participate in the 5-HT-induced calcium response.Among them,NOX1 and 4 subtypes play an important role in this reaction.3.The effect of NOX inhibitor on 5-HT-induced calcium responses in PASMCs of CH rats.Compared with CON group,5-HT-induced calcium release and calcium entry in PASMCs were increased in CHPH model rats,and the rate of calcium entry was also increased,suggesting that the PASMCs of CHPH was highly reactive to 5-HT;in CH group,both GKT137831 and ML171 can inhibit the 5-HT-induced calcium responses in PASMCs,which was similar to that in the CON group,suggesting that the Nox1,4isoforms play a major role in the calcium higher response of PASMCs in CH rats.4.The effect of TRPM2 inhibitor on the calcium response of PASMCs induced by5-HT in CON rats.TRPM2 inhibitor?ACA?reduced 5-HT-induced calcium release and calcium entry in PASMCs of CON rats,suggesting that TRPM2 participates in the process;5-HT-induced calcium responses in PASMCs of CON rats may be mediated by TRPM2 channels.Conclusions:1.In CON rats,NOX is involved in 5-HT-induced calcium responses in PASMCs;and NOX1 and NOX4 isoforms play an important role in this reaction.2.In CHPH model rats,NOX1 and NOX4 may participate in the 5-HT-induced calcium higher response of PASMCs.3.5-HT-induced calcium response may be mediated by TRPM2 channels.4.The results of this study can provide new ideas for the elucidation of PH pathogenesis and the prevention and therapy of PH.