Impaired agonist-dependent myosin phosphorylation and decreased RhoA in rat portal hypertensive mesenteric vasculature.
Impaired agonist-dependent myosin phosphorylation and decreased RhoA in rat portal hypertensive mesenteric vasculature.
复制标题
大鼠门脉高压肠系膜血管系统中激动剂依赖性肌球蛋白磷酸化受损并降低 RhoA。
DOI：
10.1152/ajpgi.00116.2004
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Benoit,JosephN
中科院分区：
文献类型：
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作者：
Zhang,Hai-Ying;Shirasawa,Yuichi;Chen,Xuesong;Yu,Hong;Benoit,JosephN
本研究的目的是研究门脉高压对血管平滑肌中激动剂诱导的肌球蛋白磷酸化和RhoA表达的影响。还研究了与camp依赖性事件的可能联系。门静脉狭窄引起门静脉高压症。用或不加50 μM的PKA抑制剂Rp-cAMPS处理血管段30分钟，随后用10−4M的苯肾上腺素刺激。免疫印迹法检测肌球蛋白调节轻链磷酸化。利用RhoA和GAPDH引物，从肠系膜一级动脉和门静脉中分离总RNA并进行RT-PCR扩增。采用Western blot方法检测一级肠系膜动脉中RhoA蛋白的表达。门脉高压动物最大刺激一级肠系膜动脉肌球蛋白磷酸化水平（19.9±2.86%）明显低于假手术对照组（43.8±3.53%）。抑制PKA选择性地使肌球蛋白磷酸化增加34.7±4.18%。rp - camp不影响门静脉或肠系膜上动脉的磷酸化。与对照组相比，门脉高压一级肠系膜动脉中RhoA mRNA和膜相关RhoA蛋白的表达显著降低。急性抑制PKA对RhoA mRNA表达无影响。然而，它使门脉高压血管中膜相关RhoA蛋白的表达恢复到控制水平。结果表明，膜相关RhoA表达的减少，似乎是由camp依赖事件调节的，导致肌球蛋白磷酸化减少，可能是门静脉高压肠抵抗血管收缩剂有效性降低的基础。
The purpose of the present study was to examine the effects of portal hypertension on agonist-induced myosin phosphorylation and RhoA expression in vascular smooth muscle. A possible link to cAMP-dependent events was also examined. Portal hypertension was produced by stenosis of the portal vein. Vessel segments were treated with or without 50 μM of the PKA inhibitor Rp-cAMPS for 30 min and subsequently stimulated with 10−4M phenylephrine. Myosin regulatory light-chain phosphorylation was detected by immunoblotting. Total RNA from first-order mesenteric arteries and portal veins was isolated and amplified by RT-PCR using RhoA and GAPDH primers. RhoA protein expression was also measured in first-order mesenteric arteries using Western blot analysis. Myosin phosphorylation in maximally stimulated first-order mesenteric arteries was significantly lower in portal hypertensive animals (19.9 ± 2.86%) when compared with sham-operated control (43.8 ± 3.53%). Inhibition of PKA selectively increased myosin phosphorylation to 34.7 ± 4.18%. Rp-cAMPS did not affect the phosphorylation of the portal veins or superior mesenteric arteries. RhoA mRNA and membrane-associated RhoA protein expression in portal hypertensive first-order mesenteric arteries were significantly lower when compared with controls. Acute inhibition of PKA had no effect on RhoA mRNA expression. However, it restored membrane-associated RhoA protein expression in portal hypertensive vessels to control levels. The results suggest that reductions in membrane-associated RhoA expression, which appear to be regulated by cAMP-dependent events, lead to reduced myosin phosphorylation and may underlie the reduced vasoconstrictor effectiveness in the resistance vasculature of portal hypertensive intestine.