Fernando Ruani N, Luff Susan E, Albiston Anthony L, Chai Siew Yeen
Howard Florey Institute, The University of Melbourne, Parkville, Victoria, Australia.
J Neurochem. 2007 Aug;102(3):967-76. doi: 10.1111/j.1471-4159.2007.04659.x. Epub 2007 May 14.
Angiotensin IV and LVV-hemorphin 7 promote robust enhancing effects on learning and memory. These peptides are also competitive inhibitors of the insulin-regulated membrane aminopeptidase, suggesting that the biological actions of these peptides may result from inhibition of IRAP activity. However, the normal function of IRAP in the brain is yet to be determined. The present study investigated the sub-cellular distribution of IRAP in four neuronal cell lines and in the mouse brain. Using sub-cellular fractionation, IRAP was found to be enriched in low density microsomes, while lower levels of IRAP were also present in high density microsomes, plasma membrane and mitochondrial fractions. Dual-label immunohistochemistry confirmed the presence of IRAP in vesicles co-localized with the vesicular maker VAMP2, in the trans Golgi network co-localized with TGN 38 and in endosomes co-localized with EEA1. Finally using electron microscopy, IRAP specific immunoreactivity was predominantly associated with large 100-200 nm vesicles in hippocampal neurons. The location, appearance and size of these vesicles are consistent with neurosecretory vesicles. IRAP precipitate was also detected in intracellular structures including the rough endoplasmic reticulum, Golgi stack and mitochondrial membranes. The sub-cellular localization of IRAP in neurons demonstrated in the present study bears striking parallels with distribution of IRAP in insulin responsive cells, where the enzyme plays a role in insulin-regulated glucose uptake. Therefore, we propose that the function of IRAP in neurons may be similar to that in insulin responsive cells.
血管紧张素IV和LVV-血啡肽7对学习和记忆具有显著的增强作用。这些肽也是胰岛素调节膜氨肽酶的竞争性抑制剂，这表明这些肽的生物学作用可能源于对胰岛素调节膜氨肽酶（IRAP）活性的抑制。然而，IRAP在大脑中的正常功能尚未确定。本研究调查了IRAP在四种神经元细胞系和小鼠大脑中的亚细胞分布。通过亚细胞分级分离，发现IRAP在低密度微粒体中富集，而在高密度微粒体、质膜和线粒体组分中也存在较低水平的IRAP。双标记免疫组织化学证实，IRAP存在于与囊泡标记物VAMP2共定位的囊泡中、与TGN 38共定位的反式高尔基体网络中以及与EEA1共定位的内体中。最后，使用电子显微镜观察，IRAP特异性免疫反应主要与海马神经元中100 - 200 nm的大囊泡相关。这些囊泡的位置、外观和大小与神经分泌囊泡一致。在包括粗面内质网、高尔基体堆栈和线粒体膜在内的细胞内结构中也检测到了IRAP沉淀。本研究中所展示的IRAP在神经元中的亚细胞定位与IRAP在胰岛素反应性细胞中的分布具有显著的相似性，在胰岛素反应性细胞中，该酶在胰岛素调节的葡萄糖摄取中发挥作用。因此，我们推测IRAP在神经元中的功能可能与在胰岛素反应性细胞中的功能相似。
