Department of Chemistry , University of Pittsburgh , Pittsburgh , Pennsylvania 15260 , United States.
ACS Chem Neurosci. 2018 Mar 21;9(3):535-544. doi: 10.1021/acschemneuro.7b00326. Epub 2017 Nov 16.
It has been known for over a century that the hippocampus, the center for learning and memory in the brain, is selectively vulnerable to ischemic damage, with the CA1 being more vulnerable than the CA3. It is also known that leucine enkephalin, or YGGFL, is neuroprotective. We hypothesized that the extracellular hydrolysis of YGGFL may be greater in the CA1 than the CA3, which would lead to the observed difference in susceptibility to ischemia. In rat organotypic hippocampal slice cultures, we estimated the Michaelis constant and the maximum velocity for membrane-bound aminopeptidase activity in the CA1 and CA3 regions. Using electroosmotic push-pull perfusion and offline capillary liquid chromatography, we inferred enzyme activity based on the production rate of GGFL, a natural and inactive product of the enzymatic hydrolysis of YGGFL. We found nearly 3-fold higher aminopeptidase activity in the CA1 than the CA3. The aminopeptidase inhibitor bestatin significantly reduced hydrolysis of YGGFL in both regions by increasing apparent K. Based on propidium iodide cell death measurements 24 h after oxygen-glucose deprivation, we demonstrate that inhibition of aminopeptidase activity using bestatin selectively protected CA1 against delayed cell death due to oxygen-glucose deprivation and that this neuroprotection occurs through enkephalin-dependent pathways.
一个多世纪以来，人们已经知道大脑中的海马体（学习和记忆的中心）易受缺血性损伤的影响，其中 CA1 比 CA3 更易受影响。此外，亮氨酸脑啡肽（或 YGGFL）具有神经保护作用。我们假设 YGGFL 的细胞外水解在 CA1 比 CA3 中可能更大，这将导致对缺血易感性的观察到的差异。在大鼠器官型海马切片培养物中，我们估计了 CA1 和 CA3 区细胞膜结合氨肽酶活性的米氏常数和最大速度。使用电渗透推挽灌流和离线毛细管液相色谱，我们根据 GGFL 的产生速率推断出酶活性，GGFL 是 YGGFL 酶水解的天然和非活性产物。我们发现 CA1 中的氨肽酶活性比 CA3 高近 3 倍。氨肽酶抑制剂 bestatin 通过增加表观 K，显著减少了两种区域中 YGGFL 的水解。基于氧葡萄糖剥夺 24 小时后的碘化丙啶细胞死亡测量，我们证明使用 bestatin 抑制氨肽酶活性可以选择性地保护 CA1 免受氧葡萄糖剥夺引起的迟发性细胞死亡，并且这种神经保护作用是通过脑啡肽依赖途径发生的。
