Park Sun Young, Song Hyun Ju, Sohn Uy Dong
Department of Pharmacology, College of Pharmacy, Chung-Ang University, Seoul 156-756, Republic of Korea.
Eur J Pharmacol. 2009 Apr 1;607(1-3):220-5. doi: 10.1016/j.ejphar.2009.02.027. Epub 2009 Feb 23.
The RhoA/Rho-associated kinase (ROCK) signaling pathway has been known to play a critical role in Ca(2+)-sensitization of smooth muscle contraction. In this study, we investigated the role of ROCK in feline esophageal body smooth muscle contraction induced by electrical field stimulation and exogenous acetylcholine in vitro. Y-27632 [(+)-(R)-trans-4-(1-aminoethyl)-(4-pyridyl) cyclohexanecarboxamide dihydrochloride], ROCK inhibitor, and specific antibodies to ROCK1 and ROCK2 proteins, which are two isoforms of ROCK, were used. Electrical field stimulation induced off-contraction and on-contraction in the presence of N(G)-nitro-L-arginine methylester, originating from the cholinergic nerve. Y-27632 inhibited both excitatory contractions in a concentration-dependent manner. Exogenous acetylcholine concentration-dependently induced two types of contractions: an initial contraction which occurred immediately after the addition of acetylcholine during short periods, and a sustained contraction which sluggishly continued after the initial contraction. Maximal initial and sustained contractions were reached at 10(-5) M acetylcholine. Y-27632 significantly inhibited both acetylcholine-induced contractions in a concentration-dependent manner. Western blot analysis revealed that acetylcholine maximally increased the level of phosphorylation in the 20 kDa regulatory light chain of myosin II (MLC(20)) at Ser(19) from 0.25 min to 1 min, and then declined after 2 min. The level changes of MLC(20) phosphorylation during the 5 min paralleled with those of acetylcholine-induced contractions. The expression of ROCK1 and ROCK2 in membrane fractions of muscle was increased by acetylcholine; more specifically, ROCK2 continually expressed up to 5 min. Taken together, ROCK may be involved in neural-evoked and acetylcholine-induced contraction via translocation to the membrane in feline esophageal smooth muscle.
已知RhoA/ Rho相关激酶（ROCK）信号通路在平滑肌收缩的钙敏化过程中起关键作用。在本研究中，我们调查了ROCK在体外电场刺激和外源性乙酰胆碱诱导的猫食管体平滑肌收缩中的作用。使用了ROCK抑制剂Y-27632 [（+）-（R）-反式-4-（1-氨基乙基）-（4-吡啶基）环己烷甲酰胺二盐酸盐]以及针对ROCK的两种亚型ROCK1和ROCK2蛋白的特异性抗体。电场刺激在存在N（G）-硝基-L-精氨酸甲酯的情况下诱导出源于胆碱能神经的舒张期收缩和收缩期收缩。Y-27632以浓度依赖性方式抑制这两种兴奋性收缩。外源性乙酰胆碱浓度依赖性地诱导两种类型的收缩：一种是在短时间内添加乙酰胆碱后立即发生的初始收缩，另一种是在初始收缩后缓慢持续的持续性收缩。在10（-5）M乙酰胆碱时达到最大初始收缩和持续性收缩。Y-27632以浓度依赖性方式显著抑制两种乙酰胆碱诱导的收缩。蛋白质印迹分析显示，乙酰胆碱在0.25分钟至1分钟内最大程度地增加了肌球蛋白II（MLC（20））20 kDa调节轻链在Ser（19）处的磷酸化水平，然后在2分钟后下降。5分钟内MLC（20）磷酸化水平的变化与乙酰胆碱诱导的收缩变化平行。乙酰胆碱增加了肌肉膜部分中ROCK1和ROCK2的表达；更具体地说，ROCK2持续表达长达5分钟。综上所述，在猫食管平滑肌中，ROCK可能通过转位到膜上参与神经诱发和乙酰胆碱诱导的收缩。
