西地那非通过上调电压依赖性钾通道抗低氧刺激的人肺动脉平滑肌细胞增殖

目的探讨西地那非抗低氧刺激的人肺动脉平滑肌细胞(pulmonary artery smooth muscle cells,PASMCs)增殖的机制与电压依赖性钾通道(voltage-dependent potassium channels,Kv)及环鸟甘酸依赖性蛋白激酶(cGMP-dependent protein kinase,PKG)的关系。方法采用MTT法及细胞免疫荧光法等检测细胞增殖情况;应用膜片钳技术记录低氧刺激PASMCs前后及西地那非或KT-5823干预前后的Kv通道电流,运用Kv1.5抗体透析细胞后的Kv通道电流;应用siRNA干扰技术沉默Kv1.5基因。结果低氧下西地那非组的细胞增殖水平较低氧对照组明显降低;西地那非反转了低氧对细胞K +通道电流,尤其Kv1.5通道电流的降低作用;siRNA组的Kv1.5蛋白表达明显减低,沉默Kv1.5通道基因逆转了西地那非抗低氧刺激的细胞增殖作用;PKG抑制剂KT-5823阻断了西地那非抗低氧刺激的细胞增殖作用,并且反转了西地那非对低氧下的Kv通道电流的上调作用。 结论 西地那非可通过上调Kv通道亚型主要是Kv1.5通道,抑制低氧刺激的人PASMCs增殖,且可能与PKG有关。

Sildenafil inhibits hypoxia-induced proliferation of human pulmonary artery smooth muscle cells by up-regulation of voltage-dependent potassium channels

Objective To investigate the inhibitory effect of sildenafil on hypoxia-induced proliferation of human pulmonary artery smooth muscle cells(PASMCs)and the mechanism of Kv1.5 channel and PKG.Methods The cell proliferation was detected by cell counting,DAPI staining,MTT and cyto-immunofluorescence.Patch clamp technique was used to detect Kv channel currents of PASMCs before and after hypoxia stimulation and sildenafil or KT-5823 intervention.The Kv1.5 antibody was used for dialysis and Kv channel current was recorded.The siRNA interference technique was used to silence the Kv1.5 channel gene.Results Sildenafil inhibited hypoxia-induced proliferation of human PASMCs compared with hypoxic control.Sildenafil reversed the decrease of K+channel currents,especially Kv1.5 channel currents of hypoxic PASMCs compared with hypoxic control.siRNA-Kv1.5 reversed the inhibition of sildenafil on hypoxia-induced cell proliferation.The PKG inhibitor KT-5823 blocked the inhibition of sildenafil on hypoxia-induced cell proliferation and reversed the up-regulation of sildenafil on Kv channel currents under hypoxia.Conclusions Sildenafil could inhibit hypoxia-induced proliferation of human PASMCs by up-regulating Kv channel subtypes,primarily Kv1.5 channel,and might be associated with PKG.