Okubo Shinji, Tanabe Yujirou, Fujioka Nakaba, Takeda Kenji, Takekoshi Noboru
Department of Cardiology, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Kahoku, Ishikawa 920-0293, Japan.
Jpn J Physiol. 2003 Jun;53(3):173-80. doi: 10.2170/jjphysiol.53.173.
The objective of the present study was to investigate the differential activation of protein kinase C between ischemic (IPC) and pharmacological preconditioning (PPC) in the rabbit heart. Control, IPC, diazoxide (Diaz), and chelerythrine (Chel)+IPC groups underwent prolonged coronary artery occlusion (CAO) for 30 minutes followed by 180 minutes' reperfusion (protocol I). In protocol II, sham, IPC-only, Diaz-only, and Chel+IPC-only groups did not undergo prolonged CAO. IPC was induced with 4 cycles of 5-min regional ischemia and 10-min reperfusion before prolonged CAO. Diaz (5 mg/kg) was administered 30 min before prolonged CAO. Chel (5 mg/kg) was administered 5 min before the IPC procedure. Infarct size was determined by tetrazolium staining. Assessment of protein kinase C (PKC) isoforms from a left ventricular (LV) sample was conducted by western blotting. Apoptosis in situ was determined by TUNEL assay. The infarction area in the IPC (11.6 +/- 1.0%) and Diaz (19.5 +/- 3.8%) groups was reduced significantly (p< 0.01, p< 0.05) relative to the control group (40.0 +/- 3.8%). The reduction by IPC was abolished by pretreatment with Chel. Apoptosis was significantly decreased (p< 0.01) in the IPC and diazoxide groups compared with the control and Chel+IPC groups (control: 4.78 +/- 0.56% vs. IPC: 2.00 +/- 0.38% vs. Diaz: 2.20 +/- 0.32% vs. Chel+IPC: 4.32 +/- 0.41%) and DNA laddering was attenuated in the IPC and Diaz groups. Membrane PKC-epsilon levels in the IPC and Diaz groups increased significantly relative to the control and Chel+IPC groups. Membrane PKC-epsilon levels in the IPC-only group showed greater increases than the Diaz-only and Chel+IPC-only groups. These findings suggest that whereas PPC suppresses apoptosis when diazoxide opens mitochondrial K(ATP) channels and then activates PKC-epsilon through ischemia-reperfusion, IPC activates PKC-epsilon in the particulate fraction prior to continuous ischemia-reperfusion. We concluded that the difference between IPC and PPC appears to consist in the difference in the timing of PKC-epsilon activation, though both IPC and PPC provide the cardioprotection in ischemia-reperfusion injury.
本研究的目的是探讨兔心脏中缺血预处理（IPC）和药物预处理（PPC）之间蛋白激酶C的差异激活情况。对照组、IPC组、二氮嗪（Diaz）组和白屈菜红碱（Chel）+IPC组经历30分钟的冠状动脉长时间闭塞（CAO），随后进行180分钟的再灌注（方案I）。在方案II中，假手术组、单纯IPC组、单纯Diaz组和单纯Chel+IPC组未进行长时间CAO。在长时间CAO之前，通过4个循环的5分钟局部缺血和10分钟再灌注诱导IPC。在长时间CAO前30分钟给予Diaz（5mg/kg）。在IPC操作前5分钟给予Chel（5mg/kg）。通过四氮唑染色确定梗死面积。通过蛋白质印迹法对左心室（LV）样本中的蛋白激酶C（PKC）亚型进行评估。通过TUNEL法测定原位凋亡。与对照组（40.0±3.8%）相比，IPC组（11.6±1.0%）和Diaz组（19.5±3.8%）的梗死面积显著减小（p<0.01，p<0.05）。Chel预处理消除了IPC的梗死面积减小作用。与对照组和Chel+IPC组相比，IPC组和二氮嗪组的凋亡显著减少（p<0.01）（对照组：4.78±0.56% vs. IPC组：2.00±0.38% vs. Diaz组：2.20±0.32% vs. Chel+IPC组：4.32±0.41%），并且IPC组和Diaz组的DNA梯状条带减弱。与对照组和Chel+IPC组相比，IPC组和Diaz组的膜PKC-ε水平显著升高。单纯IPC组的膜PKC-ε水平升高幅度大于单纯Diaz组和单纯Chel+IPC组。这些发现表明，当二氮嗪打开线粒体K（ATP）通道并通过缺血再灌注激活PKC-ε时，PPC可抑制凋亡，而IPC在持续缺血再灌注之前激活微粒体部分的PKC-ε。我们得出结论，IPC和PPC之间的差异似乎在于PKC-ε激活时间的不同，尽管IPC和PPC均可对缺血再灌注损伤提供心脏保护作用。
