Department of Physiology and Morphology, Institute of Medicinal Chemistry, Hoshi University, Shinagawa-ku, Tokyo 142-8501, Japan.
Clin Sci (Lond). 2012 Sep;123(6):375-86. doi: 10.1042/CS20110621.
In the present sutdy, we have examined the relationship between the CaMKII (Ca(2+)/calmodulin-dependent protein kinase II) pathway and endothelial dysfunction in aortas from GK (Goto-Kakizaki) Type 2 diabetic rats. The ACh (acetylcholine)-induced relaxation and NO production were each attenuated in diabetic aortas (compared with those from age-matched control rats). ACh-stimulated Ser(1177)-eNOS (endothelial NO synthase) phosphorylation was significantly decreased in diabetic aortas (compared with their controls). ACh markedly increased the CaMKII phosphorylation level within endothelial cells only in control aortas (as assessed by immunohistochemistry and Western blotting). ACh-stimulated Thr(286)-CaMKII phosphorylation within endothelial cells was significantly decreased in diabetic aortas (compared with their controls). The ACh-induced relaxations, NO production, eNOS phosphorylation, and CaMKII phosphorylation were inhibited by KN93 and/or by lavendustin C (inhibitors of CaMKII) in control aortas, but not in diabetic ones. Pre-incubation of aortic strips with a PP (protein phosphatase)-1 inhibitor, PPI2 (protein phosphatase inhibitor 2), or with a PP2A inhibitor, CA (cantharidic acid), corrected the above abnormalities in diabetic aortas. The expression of PP2A type A subunit was increased in diabetic aortas. The ACh-stimulated Thr(320)-phosphorylation level of PP1α was lower in diabetic aortas than in their controls, but the total PP1α protein level was not different. These results suggest that the aortic relaxation responses, NO production, and eNOS activity mediated by CaMKII phosphorylation are decreased in this Type 2 diabetic model, and that these impairments of CaMKII signalling may be, at least in part, due to enhancements of PP1α activity and PP2A expression.
在本研究中，我们检查了 CaMKII（Ca(2+)/钙调蛋白依赖性蛋白激酶 II）通路与 GK（Goto-Kakizaki）2 型糖尿病大鼠主动脉内皮功能障碍之间的关系。与年龄匹配的对照组大鼠相比，糖尿病大鼠主动脉对 ACh（乙酰胆碱）诱导的舒张和 NO 产生均减弱。与对照组相比，糖尿病大鼠主动脉中 ACh 刺激的 Ser(1177)-eNOS（内皮型一氧化氮合酶）磷酸化显著降低。免疫组织化学和 Western blot 分析显示，ACh 仅在对照组主动脉内皮细胞中明显增加 CaMKII 磷酸化水平。与对照组相比，糖尿病大鼠主动脉内皮细胞中 ACh 刺激的 Thr(286)-CaMKII 磷酸化显著减少。在对照组主动脉中，KN93 和/或 lavendustin C（CaMKII 抑制剂）抑制 ACh 诱导的舒张、NO 产生、eNOS 磷酸化和 CaMKII 磷酸化，但在糖尿病大鼠中则不然。用蛋白磷酸酶-1 抑制剂 PPI2 或蛋白磷酸酶 2A 抑制剂 CA（斑蝥素）预先孵育主动脉条，可纠正糖尿病大鼠上述异常。与对照组相比，糖尿病大鼠主动脉中 PP2A 型 A 亚基表达增加。与对照组相比，糖尿病大鼠主动脉中 ACh 刺激的 Thr(320)-PP1α 磷酸化水平较低，但总 PP1α 蛋白水平无差异。这些结果表明，在该 2 型糖尿病模型中，CaMKII 磷酸化介导的主动脉舒张反应、NO 产生和 eNOS 活性降低，而 CaMKII 信号转导的这些损伤至少部分归因于 PP1α 活性和 PP2A 表达的增强。
