D'Mello S R, Aglieco F, Roberts M R, Borodezt K, Haycock J W
Department of Physiology and Neurobiology, University of Connecticut, Storrs 06269, USA.
J Neurochem. 1998 May;70(5):1809-18. doi: 10.1046/j.1471-4159.1998.70051809.x.
Cultured cerebellar granule neurons undergo apoptosis when switched from a medium containing depolarizing levels of K+ (25 mM KCl) to medium containing lower levels of K+ (5 mM KCl). We used this paradigm to investigate the role of caspases in the death process. Two broad-spectrum caspase inhibitors, tert-butoxycarbonyl-Asp x (O-methyl) x fluoromethyl ketone and benzyloxycarbonyl-Val-Ala-Asp x fluoromethyl ketone, significantly reduced cell death (90 and 60%, respectively) at relatively low concentrations (10-25 microM), suggesting that caspase activation is involved in the apoptotic process. DNA fragmentation, a hallmark of apoptosis, was also reduced by these caspase inhibitors, suggesting that caspase activation occurred upstream of DNA cleavage in the sequence of events leading to cell death. As a step toward identifying the caspase(s) involved, the effects of N-acetyl Tyr-Val-Ala-Asp x chloromethyl ketone (YVAD x cmk), an interleukin-1beta converting enzyme-preferring inhibitor, and N-acetyl Asp-Glu-Val-Asp x fluoromethyl ketone (DEVD x fmk), a CPP32-preferring inhibitor, were also evaluated. YVAD x cmk provided only modest (<20%) protection and only at the highest concentration (100 microM) tested, suggesting that interleukin-1beta converting enzyme and/or closely related caspases were not involved. In comparison, DEVD x fmk inhibited cell death by up to 50%. Western blot analyses, however, failed to detect an increase in processing/activation of CPP32 or in the proteolysis of a CPP32 substrate, poly(ADP-ribose) polymerase, during the induction of apoptosis in granule neurons. Similarly, the levels of Nedd2, a caspase that is highly expressed in the brain and that is partially inhibited by DEVD x fmk, also remained unaffected in apoptotic neurons undergoing apoptosis. These results suggest that a DEVD-sensitive caspase other than CPP32 or Nedd2 mediates the induction of apoptosis in K+-deprived granule neurons.
培养的小脑颗粒神经元从含有去极化水平钾离子（25 mM KCl）的培养基转换到含有较低水平钾离子（5 mM KCl）的培养基时会发生凋亡。我们利用这一模式来研究半胱天冬酶在死亡过程中的作用。两种广谱半胱天冬酶抑制剂，叔丁氧羰基 - 天冬氨酸 - （O - 甲基） - 氟甲基酮和苄氧羰基 - 缬氨酸 - 丙氨酸 - 天冬氨酸 - 氟甲基酮，在相对低的浓度（10 - 25 microM）下显著减少了细胞死亡（分别为90%和60%），这表明半胱天冬酶的激活参与了凋亡过程。DNA片段化是凋亡的一个标志，这些半胱天冬酶抑制剂也减少了DNA片段化，这表明在导致细胞死亡的事件序列中，半胱天冬酶的激活发生在DNA切割的上游。作为确定所涉及的半胱天冬酶的一个步骤，还评估了白细胞介素 - 1β转化酶偏好抑制剂N - 乙酰 - 酪氨酰 - 缬氨酰 - 丙氨酰 - 天冬氨酸 - 氯甲基酮（YVAD - cmk）和CPP32偏好抑制剂N - 乙酰 - 天冬氨酰 - 谷氨酰 - 缬氨酰 - 天冬氨酸 - 氟甲基酮（DEVD - fmk）的作用。YVAD - cmk仅提供了适度的（<20%）保护，并且仅在测试的最高浓度（100 microM）下，这表明白细胞介素 - 1β转化酶和/或密切相关的半胱天冬酶未参与其中。相比之下，DEVD - fmk抑制细胞死亡高达50%。然而，蛋白质印迹分析未能检测到在颗粒神经元凋亡诱导过程中CPP32的加工/激活增加或CPP32底物聚（ADP - 核糖）聚合酶的蛋白水解增加。同样，在经历凋亡的凋亡神经元中，在大脑中高度表达且部分被DEVD - fmk抑制的半胱天冬酶Nedd2的水平也未受影响。这些结果表明，除了CPP32或Nedd2之外，一种对DEVD敏感的半胱天冬酶介导了钾离子剥夺的颗粒神经元中的凋亡诱导。
