Naitoh Kazuyuki, Yano Toshiyuki, Miura Tetsuji, Itoh Takahito, Miki Takayuki, Tanno Masaya, Sato Takahiro, Hotta Hiroyuki, Terashima Yoshiaki, Shimamoto Kazuaki
Second Department of Internal Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan.
Am J Physiol Heart Circ Physiol. 2009 Feb;296(2):H396-403. doi: 10.1152/ajpheart.00448.2008. Epub 2008 Dec 19.
Ischemic preconditioning (PC) suppresses chemical coupling of cardiomyocytes via gap junctions (GJs) during ischemia, which is an adjunct mechanism of protection. The aim of this study was to characterize roles of protein kinases in PC-induced GJ modulation. In isolated rat hearts, ventricular tissues were sampled before and after ischemia with or without PC, and intercalated disc-rich fractions were separated for immunoprecipitation and immunoblotting. Levels of protein kinase C (PKC)-epsilon, p38mitogen-activated protein kinase (MAPK)-alpha, and Src coimmunoprecipitated with connexin-43 (Cx43) were increased after ischemia, whereas p38MAPKbeta was not detected in the Cx43 immunoprecipitates. PC did not modify the level of Cx43-Src complex after ischemia. However, PC enhanced Cx43-PKCepsilon complex formation, which was abolished by PKCepsilon translocation inhibitory peptide (TIP). In contrast, PC reduced Cx43-p38MAPKalpha complex level and p38MAPK activity in the Cx43 immunoprecipitates after ischemia. The effect of PC on Cx43-p38MAPKalpha interaction was mimicked by SB-203580, a p38MAPK inhibitor. PC reduced permeability of GJs to Lucifer yellow in the myocardium at 25 min after ischemia, and this effect was abolished by PKCepsilon-TIP. SB-203580 increased the GJ permeability at 15 min after ischemia compared with that in untreated controls, but the difference became insignificant 25 min after ischemia. In conclusion, PC has distinct effects on interaction of GJ Cx43 with PKCepsilon, p38MAPKalpha, and Src during ischemia. Suppression of GJ permeability during ischemia by PC is primarily achieved by enhanced interaction of Cx43 with PKCepsilon, which overwhelms the counterbalancing effect of reduced Cx43-p38MAPKalpha interaction.
缺血预处理（PC）在缺血期间通过缝隙连接（GJ）抑制心肌细胞的化学偶联，这是一种辅助保护机制。本研究的目的是阐明蛋白激酶在PC诱导的GJ调节中的作用。在离体大鼠心脏中，在缺血前后对心室组织进行取样，有无PC处理，分离富含闰盘的组分进行免疫沉淀和免疫印迹。缺血后，蛋白激酶C（PKC）-ε、p38丝裂原活化蛋白激酶（MAPK）-α和与连接蛋白-43（Cx43）共免疫沉淀的Src水平升高，而在Cx43免疫沉淀物中未检测到p38MAPKβ。缺血后PC未改变Cx43-Src复合物的水平。然而，PC增强了Cx43-PKCε复合物的形成，这被PKCε转位抑制肽（TIP）所消除。相反，缺血后PC降低了Cx43免疫沉淀物中Cx43-p38MAPKα复合物水平和p38MAPK活性。PC对Cx43-p38MAPKα相互作用的影响被p38MAPK抑制剂SB-203580模拟。缺血25分钟后，PC降低了心肌中GJ对荧光黄的通透性，PKCε-TIP消除了这种作用。与未处理的对照组相比，SB-203580在缺血15分钟时增加了GJ通透性，但在缺血25分钟后差异变得不显著。总之，PC在缺血期间对GJ Cx43与PKCε、p38MAPKα和Src的相互作用有不同影响。缺血期间PC对GJ通透性的抑制主要通过增强Cx43与PKCε的相互作用来实现，这超过了Cx43-p38MAPKα相互作用减弱的平衡作用。
