Eaton P, Fuller W, Bell J R, Shattock M J
The Centre for Cardiovascular Biology and Medicine, King's College London, The Rayne Institute, St Thomas' Hospital, London, SE1 7EH, UK.
J Mol Cell Cardiol. 2001 Sep;33(9):1659-71. doi: 10.1006/jmcc.2001.1418.
In this program of studies we have characterized in detail the translocation (assessed by Triton-insolubility) and phosphorylation (using serine-45 or -59 phosphospecific antibodies) of alphaB crystallin during myocardial ischemia [both with or without ischemic preconditioning (IPC)]. Pharmacological activators and inhibitors allowed us to characterize the signaling pathways involved in alphaB crystallin phosphorylation during ischemia. Ischemic preconditioning alone caused 30% of the heart's alphaB crystallin pool to translocate, providing a significant translocation 'head-start' in protected tissue. This enhanced translocation is coupled with increased (3-fold) alphaB crystallin phosphorylation at both serine residues. The possible role of alphaB crystallin in the protection afforded by ischemic preconditioning is supported by the signal transduction data; which showed preconditioning-induced alphaB crystallin phosphorylation can be blocked by tyrosine kinase inhibition (using genistein) and by p38 MAP kinase or PKC inhibition (using SB203580 or bisindolylmaleimide, respectively). The activation of both p38 MAP kinase and PKC are recognized requirements for the induction of preconditioning and their inhibition is known to block protection. Western immunoblotting analysis after isoelectric focusing electrophoresis, confirmed the observations made with the phosphospecific antibodies; but also showed that 27+/-4% of total cardiac crystallin was phosphorylated after 30 min of ischemia. AlphaB crystallin exists as large polymeric aggregates in cardiac tissue under basal conditions (approximately 1 MDa as determined by gel filtration chromatography). We induced phosphorylation of alphaB crystallin during aerobic perfusion by the administration of phenylephrine. However this treatment did not alter the molecular aggregate size of alphaB crystallin. It appears that alphaB crystallin molecular aggregate size is not simply regulated by phosphorylation. AlphaB crystallin may have a role to play in the myocardial protection induced by ischemic preconditioning, as both translocation and phosphorylation are both accelerated and enhanced by ischemic preconditioning.
在本研究项目中，我们详细表征了心肌缺血期间（无论有无缺血预处理[IPC]）αB晶状体蛋白的易位（通过Triton不溶性评估）和磷酸化（使用丝氨酸-45或-59磷酸特异性抗体）。药理学激活剂和抑制剂使我们能够表征缺血期间参与αB晶状体蛋白磷酸化的信号通路。单独的缺血预处理导致心脏中30%的αB晶状体蛋白池发生易位，在受保护的组织中提供了显著的易位“领先优势”。这种增强的易位与两个丝氨酸残基处αB晶状体蛋白磷酸化增加（3倍）相关。信号转导数据支持了αB晶状体蛋白在缺血预处理提供的保护中的可能作用；数据显示，预处理诱导的αB晶状体蛋白磷酸化可被酪氨酸激酶抑制（使用染料木黄酮）以及p38丝裂原活化蛋白激酶或蛋白激酶C抑制（分别使用SB203580或双吲哚马来酰胺）阻断。p38丝裂原活化蛋白激酶和蛋白激酶C的激活是诱导预处理的公认条件，已知对它们的抑制会阻断保护作用。等电聚焦电泳后的蛋白质免疫印迹分析证实了磷酸特异性抗体的观察结果；但也表明，缺血30分钟后，心脏总晶状体蛋白中有27±4%发生了磷酸化。在基础条件下，αB晶状体蛋白在心脏组织中以大的聚合物聚集体形式存在（通过凝胶过滤色谱法测定约为1 MDa）。我们通过给予去氧肾上腺素在有氧灌注期间诱导αB晶状体蛋白磷酸化。然而，这种处理并未改变αB晶状体蛋白的分子聚集体大小。看来αB晶状体蛋白分子聚集体大小并非简单地由磷酸化调节。αB晶状体蛋白可能在缺血预处理诱导的心肌保护中发挥作用，因为缺血预处理会加速并增强易位和磷酸化。
