Haug L S, Walaas S I, Ostvold A C
Neurochemical Laboratory, University of Oslo, Oslo, Norway.
J Neurochem. 2000 Nov;75(5):1852-61. doi: 10.1046/j.1471-4159.2000.0751852.x.
The type I inositol 1,4,5-trisphosphate (IP(3)) receptor is selectively down-regulated in several neurodegenerative diseases, including Alzheimer's disease, Huntington's chorea, and ischemia, all conditions in which apoptotic neuronal loss occurs. In the present study, we used a neuronal cell line, human neuroblastoma SH-SY5Y cells, to investigate whether the levels of IP(3) receptor are changed during apoptosis in these cells. Following induction of apoptosis by staurosporine, the immunoreactivity of the type I IP(3) receptor in microsome preparations from SH-SY5Y cells was reduced within 2 h, with a further reduction during subsequent hours. Immunoblot analyses, using antibodies to poly(ADP-ribose) polymerase and spectrin breakdown products, revealed proteolysis of these caspase-3 substrates within 3 h, confirming that IP(3) receptor cleavage is an early consequence of apoptosis. In vitro incubation of SH-SY5Y microsomes or immunopurified IP(3) receptor from rat cerebellum with recombinant caspase-3 led to generation of immunoreactive breakdown products similar to those observed in intact cells, suggesting that the type I IP(3) receptor is a potential substrate for caspase-3. Preincubation of the neuroblastoma cells with the caspase-3 inhibitor Z-Asp-Glu-Val-Asp-fluoromethyl ketone prevented IP(3) receptor degradation. These results show that the type I IP(3) receptor is a substrate for caspase-3 in neuronal cells and indicate that apoptotic down-regulation of IP(3) receptor levels may contribute to the pathology of neurodegenerative conditions.
I型肌醇1,4,5 -三磷酸（IP(3)）受体在包括阿尔茨海默病、亨廷顿舞蹈病和局部缺血在内的几种神经退行性疾病中选择性下调，所有这些病症都会发生凋亡性神经元丢失。在本研究中，我们使用一种神经细胞系，即人神经母细胞瘤SH-SY5Y细胞，来研究在这些细胞凋亡过程中IP(3)受体水平是否发生变化。在用星形孢菌素诱导凋亡后，SH-SY5Y细胞微粒体制剂中I型IP(3)受体的免疫反应性在2小时内降低，随后数小时内进一步降低。使用针对聚（ADP-核糖）聚合酶和血影蛋白降解产物的抗体进行免疫印迹分析，发现在3小时内这些caspase-3底物发生了蛋白水解，证实IP(3)受体的裂解是凋亡的早期结果。用重组caspase-3对SH-SY5Y微粒体或从大鼠小脑中免疫纯化的IP(3)受体进行体外孵育，导致产生与完整细胞中观察到的类似的免疫反应性降解产物，表明I型IP(3)受体是caspase-3的潜在底物。用caspase-3抑制剂Z-Asp-Glu-Val-Asp-氟甲基酮对神经母细胞瘤细胞进行预孵育可防止IP(3)受体降解。这些结果表明I型IP(3)受体是神经细胞中caspase-3的底物，并表明IP(3)受体水平的凋亡性下调可能导致神经退行性疾病的病理变化。
