Nicotera Thomas M, Hu Bo Hua, Henderson Donald
Molecular and Cellular Biophysics, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.
J Assoc Res Otolaryngol. 2003 Dec;4(4):466-77. doi: 10.1007/s10162-002-3038-2. Epub 2003 Oct 13.
We previously reported that intense noise exposure causes outer hair cell (OHC) death primarily through apoptosis. Here we investigated the intracellular signal pathways associated with apoptotic OHC death. Chinchillas were exposed to a 4 kHz narrowband noise at 110 dB SPL for 1 h. After the noise exposure, the cochleas were examined for the activity of each of three caspases, including caspase-3, -8, or -9 with carboxyfluorescein-labeled fluoromethyl ketone (FMK)-peptide inhibitors. The cochleas were further examined for cytochrome c release from mitochondria by immunohistology and for DNA degradation by the TUNEL method. The results showed that the noise exposure triggered activation of caspase-3, an important mediator of apoptosis. The noise exposure also caused the activation of caspase-8 and caspase-9, each of which is associated with a distinct signaling pathway that leads to activation of caspase-3. Caspase activation occurred only in the apoptotic OHCs and not in the necrotic OHCs. These results indicate that multiple signaling pathways leading to caspase-3 activation take place simultaneously in the apoptotic OHCs. In addition to caspase activation, noise exposure caused the release of cytochrome c from mitochondria, resulting in a punctate fluorescence in the cytosol. In contrast to activation of caspases, the release of cytochrome c took place in both apoptotic and necrotic OHCs. Moreover, the release of cytochrome c in a subpopulation of OHCs took place early in the cell death process, prior to any outward signs of necrosis or apoptosis. These data suggest that in this subpopulation there exists a common step that is shared by cell death pathways before entering either necrosis or apoptosis. Lastly, use of the TUNEL assay in combination with PI labeling provides a more accurate discrimination between apoptosis and necrosis.
我们之前报道过，强烈噪声暴露主要通过凋亡导致外毛细胞（OHC）死亡。在此，我们研究了与凋亡性OHC死亡相关的细胞内信号通路。将龙猫暴露于110 dB SPL的4 kHz窄带噪声中1小时。噪声暴露后，使用羧基荧光素标记的氟甲基酮（FMK）肽抑制剂检测耳蜗中三种半胱天冬酶（包括半胱天冬酶-3、-8或-9）各自的活性。通过免疫组织学进一步检测耳蜗中线粒体细胞色素c的释放情况，并通过TUNEL法检测DNA降解情况。结果显示，噪声暴露引发了半胱天冬酶-3的激活，半胱天冬酶-3是凋亡的重要介质。噪声暴露还导致了半胱天冬酶-8和半胱天冬酶-9的激活，它们各自与导致半胱天冬酶-3激活的不同信号通路相关。半胱天冬酶激活仅发生在凋亡的OHC中，而未发生在坏死的OHC中。这些结果表明，在凋亡的OHC中，导致半胱天冬酶-3激活的多条信号通路同时发生。除了半胱天冬酶激活外，噪声暴露还导致线粒体细胞色素c的释放，在细胞质中产生点状荧光。与半胱天冬酶激活不同，细胞色素c的释放在凋亡和坏死的OHC中均有发生。此外，在OHC的一个亚群中，细胞色素c的释放在细胞死亡过程的早期就已发生，早于任何坏死或凋亡的外在迹象。这些数据表明，在这个亚群中，在进入坏死或凋亡之前，细胞死亡途径存在一个共同步骤。最后，将TUNEL检测与PI标记结合使用，能更准确地区分凋亡和坏死。
