大鼠血管平滑肌细胞IκB激酶2对血管舒缩调控的作用及机制
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摘要
目的探讨血管平滑肌细胞IκB激酶2(IKK2)对血管平滑肌肌球蛋白轻链(MLC)激酶(MLCK)的调控作用。方法去除血管内皮的大鼠主动脉环由血管收缩剂去氧肾上腺素、KCl、U-46619、离子霉素、花萼海绵体诱癌素A预处理后,分别加入选择性IKK2抑制剂IMD0354、竞争性IKK2抑制剂SC-514,并设置IKK2抑制多肽对照组,采用Multi Myograph System计算机生物信号采集分析系统记录血管张力变化。将MCL与EGTA(为MLCK抑制剂)或对照组中分别加入IKK2、MLCK共孵育1 h,通过Western blotting检测MLC第19号丝氨酸的磷酸化水平。用SC-514舒张血管,观察SC-514舒血管效应的可逆性。结果 IKK2抑制剂IMD-0354和SC-514均能对常见缩血管因素引起的主动脉环的收缩产生舒张效应(P均<0.05)。MLCK抑制剂EGTA可显著抑制MLCK对MLC磷酸化作用(P<0.05)。IKK2亦可使MLC磷酸化,而加入EGTA对IKK2的磷酸化无抑制作用(P>0.05)。经SC-514处理产生血管舒张效应主动脉环,再被去氧肾上腺素处理后舒血管效应可逆转。结论 IKK2对MLC的收缩调控作用不依赖MLCK,且其抑制剂对血管平滑肌的舒张功能存在可逆性。
Objective To investigate the regulation effect of vascular smooth muscle cells IκB kinase 2( IKK2) on vascular smooth muscle myosin light chain kinase( MLCK). Methods After preconditioning with vasoconstrictor deoxyadrenaline,KCl,U-46619,iycomycin and calyx cavernous lure A,the selective IKK2 inhibitor IMD0354 and the competitive IKK2 inhibitor SC-514 were added to the aorta of the vascular endothelium,and the IKK2 inhibition polypeptide was set up as the control group. and Multi Myograph System was used to record the changes of blood vessel tension. MCL and EGTA( MLCK inhibitor) or control group were added with IKK2 and MLCK for incubation of 1 h,and the phosphorylation level of the nineteenth serine of MLC was detected by Western blotting. SC-514 vasodilator was used to observe the reversibility of vasodilation effect of IKK2 inhibitor SC-514. Results IKK2 inhibitors IMD-0354 and SC-514 had vasodilator effects on the contraction of aortic rings caused by common vasoconstrictor factors( both P < 0. 05). MLCK antagonist EGTA significantly inhibited the phosphorylation of MLCK on MLC( P < 0. 05). IKK2 also made MLC phosphorylate,while the addition of EGTA had no inhibitory effect on IKK2 phosphorylation( P > 0. 05). The vasodilatory effect of aortic rings after SC-514 treatment was reversed by the treatment of phenylephrine. Conclusion IKK2 is a phosphorylated kinase that is contractile to MLC without MLCK and has a reversible effect on the relaxation function of vascular smooth muscle cells.
Objective To investigate the regulation effect of vascular smooth muscle cells IκB kinase 2( IKK2) on vascular smooth muscle myosin light chain kinase( MLCK). Methods After preconditioning with vasoconstrictor deoxyadrenaline,KCl,U-46619,iycomycin and calyx cavernous lure A,the selective IKK2 inhibitor IMD0354 and the competitive IKK2 inhibitor SC-514 were added to the aorta of the vascular endothelium,and the IKK2 inhibition polypeptide was set up as the control group. and Multi Myograph System was used to record the changes of blood vessel tension. MCL and EGTA( MLCK inhibitor) or control group were added with IKK2 and MLCK for incubation of 1 h,and the phosphorylation level of the nineteenth serine of MLC was detected by Western blotting. SC-514 vasodilator was used to observe the reversibility of vasodilation effect of IKK2 inhibitor SC-514. Results IKK2 inhibitors IMD-0354 and SC-514 had vasodilator effects on the contraction of aortic rings caused by common vasoconstrictor factors( both P < 0. 05). MLCK antagonist EGTA significantly inhibited the phosphorylation of MLCK on MLC( P < 0. 05). IKK2 also made MLC phosphorylate,while the addition of EGTA had no inhibitory effect on IKK2 phosphorylation( P > 0. 05). The vasodilatory effect of aortic rings after SC-514 treatment was reversed by the treatment of phenylephrine. Conclusion IKK2 is a phosphorylated kinase that is contractile to MLC without MLCK and has a reversible effect on the relaxation function of vascular smooth muscle cells.
引文
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[3]Zandi E,Rothwarf DM,Delhase M,et al.The Ikappa B kinase complex(IKK)contains two kinase subunits,IKKalpha and IKK-beta,necessary for Ikappa B phosphorylation and NF-kappa B activation[J].Cell,1997,91(91):243-252.
[4]Tran LT,Macleod KM,Mcneill JH.Chronic etanercept treatment prevents the development of hypertension in fructose-fed rats[J].Mol Cell Biochem,2009,330(1):219-228.
[5]Guzik TJ,Hoch NE,Brown KA,et al.Role of the T cell in the genesis of angiotensin II-induced hypertension and vascular dysfunction[J].J Exp Med,2007,204(10):2449-2460.
[6]Hai CM,Murphy RA.Ca2+,crossbridge phosphorylation,and contraction[J].Annu Rev Physiol,1989,51(51):285-298.
[7]PadróT,Pe a E,García-Arguinzonis M,et al.Low-density lipoproteins impair migration of human coronary vascular smooth muscle cells and induce changes in the proteomic profile of myosin light chain[J].Cardiovasc Res,2008,77(1):211-220.
[8]Somlyo AV,Wang H,Choudhury N,et al.Myosin light chain kinase knockout[J].J Muscle Res Cell M,2004,25(3):241-242.
[9]Uehata M,Ishizaki T,Satoh H,et al.Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension[J].Nature,1997,389(6654):990-994.
[10]Wilson DP,Sutherland C,Borman MA,et al.Integrin-linked kinase is responsible for Ca2+-independent myosin diphosphorylation and contraction of vascular smooth muscle[J].Biochem J,2005,392(3):641-648.
[11]Raymond K,Cagnet S,Kreft M,et al.Control of mammary myoepithelial cell contractile function by alpha3beta1 integrin signaling[J].Embo J,2011,30(10):1896-1906.
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[13]Ying Z,do Carmo JM,Xiang L,et al.InhibitorκB kinase 2 is a myosin light chain kinase in vascular smooth muscle[J].Circ Res,2013,113(5):562.
[14]Liu Q,Wu H,Chim SM,et al.SC-514,a selective inhibitor of IKK beta attenuates RANKL-induced;osteoclastogenesis and NF-kappa B activation[J].Biochem Pharmacol,2013,86(12):1775-1783.