HCN1通道基因敲除下调小鼠膀胱Cajal间质细胞中BK通道的表达及功能

Knockout of HCN1 channels down-regulates expression and function of BK channels in interstitial cells of Cajal in mouse urinary bladder

目的观察小鼠HCN1通道基因敲除对其膀胱Cajal间质细胞(interstitial cells of Cajal,ICCs)中BK通道的表达和功能的影响,探讨这种影响对膀胱兴奋性调控的意义。方法健康清洁成年的野生型C57BL/6J小鼠和HCN1通道基因敲除的C57BL/6J小鼠各48只(雌雄各半)分别记为正常组和敲基因组,反转录PCR(RT-PCR)、荧光定量PCR(Q-PCR)、Western blot和免疫荧光双标检测其膀胱ICCs中BK通道各亚基的表达变化,离体逼尿肌肌条实验检测加入BK通道阻滞剂IBTX前后肌条收缩的变化,激光共聚焦显微镜下检测分别加入BK通道激动剂NS1619、阻滞剂IBTX前后小鼠膀胱ICCs内钙荧光的变化。结果 Q-PCR显示敲基因组小鼠膀胱中BK通道α、β1、β2、β3、β4各亚基表达均降低(P<0.01);Western blot显示敲基因组小鼠膀胱中BK通道α、β1、β2、β3、β4亚基表达均降低(其中α、β3、β4:P<0.01;β1、β2:P<0.05);免疫荧光双标显示敲基因组小鼠膀胱ICCs中BK通道α亚基表达降低(P<0.01);离体逼尿肌肌条实验显示加入IBTX后敲基因组和正常组肌条收缩幅度均变大(P<0.01,P<0.05),且敲基因组肌条收缩幅度的变化值小于正常组(P<0.05);激光共聚焦显微镜下可见加入NS1619后两组ICCs内钙荧光均降低(P<0.05)、加入IBTX后两组ICCs内钙荧光均增强(P<0.01),且不论加入激动剂或阻滞剂,敲基因组加药前后ICCs内钙荧光的变化值均小于正常组(P<0.01)。结论小鼠HCN1通道基因敲除下调了其膀胱ICCs中BK通道的表达及功能,这种下调可能是对HCN1通道基因敲除后膀胱收缩减弱的一种代偿。

Objective To determine the effects of the knockout of hyper-polarization-activated,cyclic nucleotide-gated cation channels（ HCN1 channels） on the expression and function of large-conductance calcium-activated potassium channels（ BK channels） in interstitial cells of Cajal（ ICCs） in the mouse bladder,and investigate the significance of these effects to the excitatory regulation of bladder. Methods Forty-eight healthy clean adult wild-type C57 BL /6J mice（ HCN1 WT） and 48 HCN1 channels knockout C57 BL /6J mice（ HCN1 KO） were employed in this study. The expression of BK channels and subunits in ICCs at mRNA and protein levels was detected by RT-PCR, Q-PCR, Western blotting and immunofluorescence staining. The inhibitor of BK channel,IBTX,was added in the isolated detrusor muscle strips to observe the effect on muscle contraction. While,intracellular calcium concentration in ICCs were measured with laser confocal microscopy after the treatment of IBTX and the agonist of BK channels,NS1619,respectively. Results Q-PCR and Western blot results indicated that the expression of 5 BK channels ’subunits,α,β1,β2,β3 and β4 were significantly lower at mRNA level（ P 〈 0. 01） and at protein level（ α,β3,β4： P 〈 0. 01; β1,β2： P 〈 0. 05） in the bladder of HCN1 KO mice than HCN1 WT mice. Doublelabeling immunofluorescence staining revealed that the expression of α subunit was also lower in ICCs of HCN1 KO mice than HCN1 WT mice（ P 〈 0. 05）. IBTX increased the amplitudes of detrusor contraction in the detrusor strip of HCN1 KO and WT mice（ P 〈 0. 01,P 〈 0. 05）,but the KO mice detrusor had a lower sensitivity to IBTX（ P 〈 0. 05）. NS1619 decreased while IBTX increased the intracellular calcium concentration of ICCs in both WT and KO mice（ P 〈 0. 05,P 〈 0. 01）,but the actions of NS1619 and IBTX on KO mice were lower than WT mice（ P 〈 0. 01）. Conclusion Knockout of HCN1 channels decreases the expression and function of BK channels in ICCs,and it might be a compensational mechanism of the decreased contractility induced by the knockout of HCN1 channels.