肌球蛋白轻链激酶和Rho激酶在兔脑血管平滑肌细胞迁移中的作用

Roles of Myosin Light Chain Kinase and Rho Kinase in Migration of Rabbit Intracranial Vascular Smooth Muscle Cells

目的探讨肌球蛋白轻链激酶(MLCK)和Rho激酶在颅内血管平滑肌细胞(VSMC)迁移中的作用。方法体外培养兔大脑中动脉VSMC。构建携带反义MLCK cDNA的腺病毒载体并转染VSMC以降低MLCK表达。应用Rho激酶特异性抑制剂Y-27632来抑制Rho激酶的活性。免疫印迹法测定细胞内MLCK和Rho激酶的表达水平;改良Boyden小室法检测血管紧张素Ⅱ(ATⅡ)诱导的VSMC迁移;甘油凝胶电泳和免疫印迹法检测肌球蛋白轻链(MLC)的磷酸化水平。结果反义MLCK cDNA转染后,MLCK表达量较LacZ基因转染组下降(84.6±5.8)%(P<0.01),但ATⅡ刺激引起的MLC磷酸化和VSMC迁移未受明显影响(P均>0.05)。Y-27632预处理反义MLCK cDNA转染后的VSMC可明显降低基础水平以及ATⅡ刺激引起的MLC磷酸化,并部分阻断VSMC迁移(P均<0.05)。结论颅内、外动脉VSMC迁移的分子机制不同,在ATⅡ诱导的颅内VSMC迁移中MLCK作用有限,而Rho激酶可能通过非钙依赖型MLC磷酸化途径发挥重要作用。

Objective To explore the roles of myosin light chain kinase(MLCK) and Rho kinase in the migration of intracranial vascular smooth muscle cells(VSMCs).Methods VSMCs derived from the middle cerebral artery of rabbit were cultured in vitro.To examine the involvement of MLCK and Rho kinase in VSMC migration,an adenovirus vector carrying antisense MLCK cDNA was transfected into VSMCs to down-regulate MLCK expression,and Y-27632,a selective inhibitor of Rho kinase,was used to inhibit Rho kinase activity.The expression of MLCK and Rho kinase was detected by Western blot.VSMC migration in response to angiotensin Ⅱ(AT Ⅱ) was determined by using a modified Boyden chamber method.The myosin light chain(MLC) phosphorylation assay was performed using glycerol-PAGE coupled with Western blot.Results Transfection of the antisense MLCK cDNA reduced the expression of two MLCK isoforms by(84.6±5.8)%(P<0.01 versus LacZ gene-transfection VSMCs).However,the downregulation of MLCK by antisense cDNA hardly affected AT Ⅱ-induced MLC phosphorylation and VSMC migration(P>0.05,respectively).In contrast,pretreatment of the MLCK-downregulated cells with Y-27632,markedly suppressed AT Ⅱ-induced(as well as basal) MLC phosphorylation,and partially but significantly blocked AT Ⅱ-directed VSMC migration(P<0.05 versus AT Ⅱalone,respectively).Conclusion The molecular mechanism of VSMC migration is different between the intra-and extracranial arteries.MLCK appears to have little contribution in ATⅡ-induced migration of intracranial VSMC,while Rho kinase might play a crucial role via mechanism of Ca2+-independent MLC phosphorylation.