The Effect Of Two Pore Domain Potassium Channels TASK-1on Acute And Chronic Hypoxic Human Pulmonary Vasoconstriction And Related Mechanism Research

Objective: To observe the changes of two pore domain potassium channels TASK-1expression in human pulmonary artery smooth muscle cells during acute and chronichypoxia,as well as the impacts of tyrosine kinase c-Src inhibitor and other drugs.Then to explore the role of two pore domain potassium channels TASK-1in acute andchronic hypoxic human pulmonary vasoconstriction，and the molecular mechanism ofthe regulation of c-Src on two pore domain potassium channels TASK-1mediatedhypoxic human pulmonary vasoconstriction.Methods: The primary human pulmonary artery smooth muscle cells (hPASMCs) forthis study were isolated from human pulmonary arteries of lung cancer patientsundergoing lung surgery without a history of pulmonary vascular disease or arterialhypoxemia, and then were cultured. SMC identity was verified by characteristicappearance in phase–contrast microscopy. The purity of hPASMCs was confirmed byimmunohistochemical antibody staining for smooth muscle–specific isoforms of a-actin(at least95%of cells stained positive). Cells were sub-cultured in Petri dishes to70–90%confluent at passages3–6for molecular biology studies. The classical hypoxiamodel was used to treat the cultured hPASMCs,and then divided into:Normal group(Con), hypoxia30minutes group (Hypoxia30’), hypoxia6hours group (Hypoxia6h)and hypoxia48hours group (Hypoxia48h). Furthermore, hypoxia48hours grouprespectively was treated with tyrosine kinase c-Src inhibitors PP2, PP2analogue PP3and tyrosine phosphate inhibitor bpV pre-incubation, and then divided into: hypoxia48hours+PP2group (Hypoxia48h+PP2), hypoxia48hours+PP3group (Hypoxia48h+PP3), hypoxia48hours+bpV group (Hypoxia48h+bpV). Flow cytometry was used to analyzethe cell cycle, RT-PCR technique was carried out to detect the expression of TASK-1mRNA, and Western Blot method was utilized to examine the expression of TASK-1protein in different groups.Results:(1) Changes in the cell cycle: Compared with normal control group, withprolonged hypoxia, the percentages of hypoxia30minutes group, hypoxia6hoursgroup and hypoxia48hours group human pulmonary artery smooth muscle cells in Sphases were increased（P <0.001）. While compared with hypoxia48hours group, thepercentages of hypoxia48hours+PP2group human pulmonary artery smooth musclecells in S phases were decreased（P <0.001）.(2) The changes of two pore domain potassium channels TASK-1mRNA andprotein expression in human pulmonary artery smooth muscle cells during acuteand chronic hypoxia: On the mRNA level, compared with the normal control group,the expression of TASK-1mRNA in hypoxia30min group had no significant difference(P>0.05), the expression of TASK-1mRNA in hypoxia6h group was increased (P<0.05), the expression of TASK-1mRNA in hypoxia48h group was significantlydecreased (P <0.001). On the protein level, compared with the normal control group,with prolonged hypoxia, the expression of TASK-1protein in hypoxia30minutes group,hypoxia6hours group and hypoxia48hours group was decreased (P <0.001).(3) The changes of two pore domain potassium channels TASK-1mRNA andprotein expression in human pulmonary artery smooth muscle cells during chronichypoxia and the drug intervention was given: Compared with the hypoxia48hoursgroup, the expression of TASK-1mRNA and protein in hypoxia48hours+PP2groupwas increased(P <0.01), the expression of TASK-1protein in hypoxia48hours+bpVgroup was decreased(P <0.01),while the expression of TASK-1protein in hypoxia48hours+PP3group had no significant difference（P>0.05）.Conclusions:(1) Hypoxia promotes human pulmonary artery smooth muscle cellproliferation,and tyrosine kinase c-Src regulates the proliferation of hypoxic pulmonarysmooth muscle cells.(2) Two pore domain potassium channels TASK-1are expressed in human pulmonary vascular smooth muscle cell membrane, participated in acute and chronic hypoxicpulmonary vasoconstriction,and chronic hypoxia inhibits the expression of two poredomain potassium channels TASK-1.(3) The tyrosine kinase c-Src regulates the expression of two pore domain potassiumchannels TASK-1mediated hypoxic pulmonary vasoconstriction.(4) Two pore domain potassium channels TASK-1may be a potential target for chronichypoxic pulmonary vascular disease treatment, and the tyrosine kinase c-Src may beacted as the related regulatory factors for guiding us to further study the mechanism ofhypoxic pulmonary hypertension.