在肠系膜小动脉内毒素血症期间，Rho-A/ Rho激酶信号通路被上调，但仍受环磷酸鸟苷依赖性机制的抑制。
The Rho-A/Rho-kinase pathway is up-regulated but remains inhibited by cyclic guanosine monophosphate-dependent mechanisms during endotoxemia in small mesenteric arteries.
作者信息
da Silva-Santos J Eduardo, Chiao Chin-Wei, Leite Romulo, Webb R Clinton
机构信息
Department of Physiology, Medical College of Georgia, Augusta, GA, USA.
出版信息
Crit Care Med. 2009 May;37(5):1716-23. doi: 10.1097/CCM.0b013e31819efb43.
OBJECTIVE
We investigated whether a reduced activity in the Rho-A/Rho-kinase pathway could be involved in the impaired vascular reactivity observed in septic shock.
DESIGN
Ex vivo animal study.
SETTING
University research laboratory.
SUBJECTS
Male Wistar rats.
INTERVENTIONS
Rats received an intraperitoneal injection of lipopolysaccharide (LPS, 10 mg/kg) either 6 or 24 hours before the onset of our experiments. The effects of Y-27632 (a Rho-kinase inhibitor) were assessed in first-order mesenteric rings taken from these animals using wire myograph. The expression of Rho-A, Rho-kinases I and II, and the total and phosphorylated myosin phosphatase targeting subunit 1 (MYPT1) were assessed by Western blotting.
MEASUREMENTS AND MAIN RESULTS
The EC50 to Y-27632 was reduced from 2.10 microM (1.22-3.66 microM) (control) to 0.21 microM (0.09-0.44 microM), and 9.54 (0.82-110.30) nM in LPS-treated groups 6 and 24 hours, respectively. The increased potency of Y-27632 was partially reversed by endothelium removal at both 6 and 24 hours. Incubation of Nomega-nitro-l-arginine methyl ester hydrochloride or 1400W (a nonselective and an inducible nitric oxide synthase inhibitor, respectively) normalized the responses to Y-27632 seen 6 hours after LPS. However, 1400W had no effect, whereas Nomega-nitro-l-arginine methyl ester hydrochloride caused a partial reduction in the enhanced potency of Y-27632 found 24 hours after LPS. The soluble guanylate cyclase inhibitor oxadiazolo[4,3-alpha]quinoxalin-1-one was able to bring the Y-27632 response back to normal both 6 and 24 hours after LPS. Rho-A, Rho-kinase I, Rho-kinase II, and MYPT1 were increased in mesenteric arteries from endotoxemic rats, but the phosphorylated MYPT1 was significantly reduced. However, incubation with oxadiazolo[4,3-alpha]quinoxalin-1-one circumvented the inhibition of MYPT1 phosphorylation found in preparations from LPS-treated animals.
CONCLUSIONS
Our findings revealed an impaired Rho-A/Rho-kinase-mediated phosphorylation of MYPT1 in vessels from endotoxemic animals in a cyclic guanosine monophosphate-dependent manner, suggesting that changes in mechanisms involved in calcium sensitization play a pivotal role in cardiovascular changes observed in septic shock.
目的
我们研究了Rho-A/ Rho激酶途径活性降低是否与脓毒性休克中观察到的血管反应性受损有关。
设计
离体动物研究。
单位
大学研究实验室。
对象
雄性Wistar大鼠。
干预措施
在我们的实验开始前6小时或24小时，给大鼠腹腔注射脂多糖（LPS，10 mg/kg）。使用线肌张力测定仪评估Y-27632（一种Rho激酶抑制剂）对取自这些动物的一级肠系膜环的作用。通过蛋白质印迹法评估Rho-A、Rho激酶I和II以及肌球蛋白磷酸酶靶向亚基1（MYPT1）的总表达和磷酸化表达。
测量指标和主要结果
Y-27632的半数有效浓度（EC50）在LPS处理6小时和24小时的组中分别从2.10 microM（1.22 - 3.66 microM）（对照组）降至0.21 microM（0.09 - 0.44 microM）和9.54（0.82 - 110.30）nM。在6小时和24小时时，去除内皮部分逆转了Y-27632增强的效力。孵育盐酸N-硝基-L-精氨酸甲酯或1400W（分别为非选择性和诱导型一氧化氮合酶抑制剂）可使LPS处理6小时后对Y-27632的反应恢复正常。然而，1400W没有效果，而盐酸N-硝基-L-精氨酸甲酯使LPS处理24小时后Y-27632增强的效力部分降低。可溶性鸟苷酸环化酶抑制剂恶二唑并[4,3-α]喹喔啉-1-酮能够使LPS处理6小时和24小时后Y-27632的反应恢复正常。在内毒素血症大鼠的肠系膜动脉中，Rho-A、Rho激酶I、Rho激酶II和MYPT1增加，但磷酸化的MYPT1显著降低。然而，用恶二唑并[4,3-α]喹喔啉-1-酮孵育可避免在LPS处理动物的制剂中发现的MYPT1磷酸化抑制。
结论
我们的研究结果揭示了内毒素血症动物血管中Rho-A/ Rho激酶介导的MYPT1磷酸化受损，且呈环磷酸鸟苷依赖性，这表明钙敏化相关机制的变化在脓毒性休克中观察到的心血管变化中起关键作用。
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