摘要
目的以人体肺动脉平滑肌细胞(HPASMCs)为研究对象,研究线粒体膜上ATP敏感的钾通道(MitoKATP)对HPASMCs膜电位及电压门控钾通道(KV1.5)表达的影响,以及蛋白激酶C(PKC)在其信号转导中的可能作用。方法从人正常肺组织分离出HPASMCs进行培养。利用激光共聚焦显微镜技术检测细胞线粒体膜电位(ΔΨm),全细胞膜片钳技术记录细胞膜电流变化,Western blot检测KV1.5蛋白和PKC-α的表达情况。结果①Diazoxide组肺动脉平滑肌细胞PKC-α的表达比正常对照组明显增强(P<0.05);这种作用可被5-羟基癸酸盐(5-HD)的作用所逆转;单独应用5-HD,平滑肌细胞PKC-α的表达与对照组比较差异无显著性意义。②Diazoxide作用24h可明显抑制Kv电流以及下调Kv1.5蛋白的表达(P<0.05);佛波酯(PMA)作用24h也可明显抑制Kv电流以及下调Kv1.5蛋白的表达(P<0.05),而加入R0-31-8220可以逆转PMA的这一作用;Diazoxide和PMA同时应用较其中之一单独应用对人肺动脉平滑肌细胞Kv电流和Kv1.5表达有更加明显的抑制作用(均P<0.05);Diazoxide与R0-31-8220同时应用则部分逆转了Diazoxide对肺动脉平滑肌细胞Kv电流和Kv1.5表达的抑制(均P<0.05)。结论MitoKATP的开放和ΔΨm的增加可抑制Kv通道的活性和表达,可能参与并影响了肺动脉高压的发生发展,PKC在其中可能起介导作用。
Objective To investigate the effect of MitOKATP-PKC on voltage-gated potassium （Kv） channels in human pulmonary artery smooth muscle cells （HPASMCs）. Methods Fresh human lung tissues were obtained from the Division of Chest Surgery. HPASMCs were isolated and cultured. Whole cell patch-clamp technique was used to trace the cell membrane K^＋ currents. The expression of cell membrane Kvl. 5 protein was detected by Western blot. Results （1）After exposed to diazoxide for 24 h, the expression of PKC-α protein in normoxic HPASMCs was significantly increased as compared with control group （P〈 0.05）, but there were no significant changes in these tests after the HPASMCs were exposed to 5-HD for 24 h. 5-HD could offset the inhibitory effect of diazoxide on the expression of PKC-α protein （P〈0.05） ; （2）After exposed to diazoxide or PMA for 24 h, the cell membrane K^＋ currents and the expression of cell membrane Kvl. 5 protein in normoxic HPASMCs were significantly decreased as compared with control group （P〈0.05）. Diazoxide＋PMA could decrease the cell membrane K^＋ currents and the expression of Kvl. 5 protein （P〈0.05）, and the changes were more significant in diazoxide＋PMA group than in diazoxide or PMA group （P〈0.05）. R0-31-8220 could partly weaken the inhibitory effect of diazoxide and PMA on the cell membrane K^＋ currents and the expression of Kv1. 5 protein （P〈0.05）. Conclusion The opening of MitoKArP followed by an increase of ΔΨm might contribute to the inhibition of cell membrane Kv1. 5 channel, leading to the change in the cell membrane potential of HPASMCs. The signal transduction pathway of PKC might play an important role in the relationship between Kv channel and ΔΨm.
出处
《华中科技大学学报（医学版）》
CAS
CSCD
北大核心
2008年第5期565-570,F0002,共7页
Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
基金
国家自然科学基金资助项目(No.30700340
No.30370623)
