Failure of Bay K 8644 to induce RhoA kinase-dependent calcium sensitization in rabbit blood vessels
Failure of Bay K 8644 to induce RhoA kinase-dependent calcium sensitization in rabbit blood vessels
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DOI：
10.1111/j.1476-5381.2010.00751.x
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发表时间：
2010-07-01
影响因子：
7.3
通讯作者：
Ratz, P. H.
中科院分区：
文献类型：
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作者：
Alvarez, S. M.;Miner, A. S.;Ratz, P. H.
背景与目的：RhoA激酶（ROCK）参与K+去极化（KCl）引起的Ca ~（2+）敏感性收缩。无论是组成性的，去极化或Ca 2+激活的ROCK在这个信号系统中发挥主要作用仍有待确定。在这里，我们确定是否湾K 8644，二氢吡啶，促进Ca 2+通道集群在一个持久的Ca 2+内流模式，可能会导致ROCK依赖的Ca 2 + sensitivity.Experimental方法：肾和股动脉环从新西兰白色兔收缩湾K 8644。冷冻并处理组织以测量活性RhoA和ROCK底物（肌球蛋白磷酸酶靶向亚基，MYPT 1）和肌球蛋白轻链（MLC）磷酸化，或加载fura-2以测量细胞内游离Ca 2+（[Ca 2 +]（i））。在静息（基底）组织和Bay K 8644收缩的组织中评估选择性收缩抑制剂的作用。关键结果：Bay K 8644强烈增加[Ca 2 +]（i）、MLC磷酸化和张力，但不增加MYPT 1磷酸化。ROCK抑制H-1152取消基础MYPT 1-pT 853，减少基础MLC磷酸化和抑制海湾K 8644诱导的MLC磷酸化和张力的增加。MLC激酶抑制渥曼青霉素废除湾K 8644诱导的收缩和MLC磷酸化的增加，但不抑制基础MYPT 1-pT 853。H-1152和渥曼青霉素没有影响MYPT 1-pT 696，但1 μ M staurosporine抑制基础MYPT 1-pT 853，MYPT 1-pT 696和MLC的磷酸化。结论和影响：这些数据表明，组成活动的ROCK和staurosporine敏感的激酶调节MYPT 1的基础磷酸化，参与沿着与MLC激酶的激活，在确定强度的收缩诱导的Ca 2+激动剂，湾K 8644。
Background and purpose:RhoA kinase (ROCK) participates in K+ depolarization (KCl)-induced Ca2+ sensitization of contraction. Whether constitutive, depolarization- or Ca2+-activated ROCK plays the major role in this signalling system remains to be determined. Here, we determined whether Bay K 8644, a dihydropyridine that promotes Ca2+ channel clusters to operate in a persistent Ca2+ influx mode, could cause ROCK-dependent Ca2+ sensitization.Experimental approach:Renal and femoral artery rings from New Zealand white rabbits were contracted with Bay K 8644. Tissues were frozen and processed to measure active RhoA and ROCK substrate (myosin phosphatase targeting subunit, MYPT1) and myosin light chain (MLC) phosphorylation, or loaded with fura-2 to measure intracellular free Ca2+ ([Ca2+](i)). Effects of selective inhibitors of contraction were assessed in resting (basal) tissues and those contracted with Bay K 8644.Key results:Bay K 8644 produced strong increases in [Ca2+](i), MLC phosphorylation and tension, but not in MYPT1 phosphorylation. ROCK inhibition by H-1152 abolished basal MYPT1-pT853, diminished basal MLC phosphorylation and inhibited Bay K 8644-induced increases in MLC phosphorylation and tension. MLC kinase inhibition by wortmannin abolished Bay K 8644-induced contraction and increase in MLC phosphorylation but did not inhibit basal MYPT1-pT853. H-1152 and wortmannin had no effect on MYPT1-pT696, but 1 mu M staurosporine inhibited basal MYPT1-pT853, MYPT1-pT696 and MLC phosphorylation.Conclusions and implications:These data suggest that the constitutive activities of ROCK and a staurosporine-sensitive kinase regulate basal phosphorylation of MYPT1, which participates along with activation of MLC kinase in determining the strength of contraction induced by the Ca2+ agonist, Bay K 8644.