Roberts Richard E
Institute of Cell Signalling and Department of Obstetrics and Gynaecology, University of Nottingham, Medical School, Queen's Medical Centre, Nottingham NG7 2UH, UK.
J Pharmacol Exp Ther. 2004 Nov;311(2):742-7. doi: 10.1124/jpet.104.071100. Epub 2004 Jul 1.
Alpha2-adrenoceptor-mediated vasoconstriction in the porcine palmar lateral vein is dependent upon activation of the extracellular signal-regulated kinase-mitogen-activated protein (ERK-MAP) kinase signal transduction pathway. Recent studies have shown that alpha2-adrenoceptor-mediated vasoconstriction in the rat aorta is also dependent upon activation of Rho kinase. The aim of this study was to determine whether Rho kinase and ERK-MAP kinase are part of the same signaling pathway. The Rho kinase inhibitor Y27632 (trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride) (10 microM) almost completely inhibited the contractile response to the alpha2-adrenoceptor agonist UK14304 (5-bromo-6-[2-imidazolin-2-ylamine]-quinoxaline bitartrate) in segments of porcine palmar lateral vein [maximum response 2.9 +/- 2.3% of 60 mM KCl response (mean +/- S.E.M.) in the presence of Y27632, compared with 64.9 +/- 7.1% in control tissues, n = 4]. However, Y27632 had no effect on alpha2-adrenoceptor-mediated ERK activation, as measured by Western blotting. Alpha2-adrenoceptor-mediated vasoconstriction was associated with an increase in phosphorylation of the myosin phosphatase-targeting subunit (MYPT) at Thr696 (the Rho kinase phosphorylation site). This phosphorylation was inhibited by 10 microM Y27632. In contrast, inhibition of ERK activation with the MAP kinase kinase inhibitor PD98059 (2-amino-3-methoxyflavone) (50 microM) had no effect on MYPT phosphorylation. Both Y27632 and PD98059 inhibited myosin light chain phosphorylation. These data indicate that alpha2-adrenoceptor-mediated vasoconstriction in the porcine palmar lateral vein is dependent upon both Rho kinase and ERK activation, although these are separate pathways. Rho kinase causes vasoconstriction through inhibition of myosin phosphatase and an increase in myosin light chain phosphorylation, whereas ERK causes vasoconstriction through a myosin phosphatase-independent pathway.
α2-肾上腺素能受体介导的猪掌外侧静脉血管收缩依赖于细胞外信号调节激酶-丝裂原活化蛋白（ERK-MAP）激酶信号转导途径的激活。最近的研究表明，α2-肾上腺素能受体介导的大鼠主动脉血管收缩也依赖于Rho激酶的激活。本研究的目的是确定Rho激酶和ERK-MAP激酶是否属于同一信号通路。Rho激酶抑制剂Y27632（反式-4-[(1R)-1-氨基乙基]-N-4-吡啶基环己烷甲酰胺二盐酸盐）（10微摩尔）几乎完全抑制了猪掌外侧静脉段对α2-肾上腺素能受体激动剂UK14304（5-溴-6-[2-咪唑啉-2-基胺]-喹喔啉酒石酸盐）的收缩反应[在Y27632存在下，最大反应为60毫摩尔氯化钾反应的2.9±2.3%（平均值±标准误），而对照组织中为64.9±7.1%，n = 4]。然而，通过蛋白质印迹法检测，Y27632对α2-肾上腺素能受体介导的ERK激活没有影响。α2-肾上腺素能受体介导的血管收缩与肌球蛋白磷酸酶靶向亚基（MYPT）在苏氨酸696（Rho激酶磷酸化位点）的磷酸化增加有关。这种磷酸化被10微摩尔的Y27632抑制。相反，用MAP激酶激酶抑制剂PD98059（2-氨基-3-甲氧基黄酮）（50微摩尔）抑制ERK激活对MYPT磷酸化没有影响。Y27632和PD98059均抑制肌球蛋白轻链磷酸化。这些数据表明，α2-肾上腺素能受体介导的猪掌外侧静脉血管收缩依赖于Rho激酶和ERK的激活，尽管它们是独立的途径。Rho激酶通过抑制肌球蛋白磷酸酶和增加肌球蛋白轻链磷酸化引起血管收缩，而ERK通过不依赖肌球蛋白磷酸酶的途径引起血管收缩。
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