Real-time visualization of cytoplasmic calpain activation and calcium deregulation in acute glutamate excitotoxicity.
Real-time visualization of cytoplasmic calpain activation and calcium deregulation in acute glutamate excitotoxicity.
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急性谷氨酸兴奋性毒性中细胞质钙蛋白酶激活和钙失调的实时可视化。
DOI：
10.1111/j.1471-4159.2009.06194.x
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发表时间：
2009-08
影响因子：
4.7
通讯作者：
Polster BM
中科院分区：
文献类型：
--
作者：
Gerencser AA;Mark KA;Hubbard AE;Divakaruni AS;Mehrabian Z;Nicholls DG;Polster BM
虽然钙蛋白酶（EC 3.4.22）蛋白酶激活被认为有助于兴奋毒性迟发性钙失调（DCD）通过蛋白水解的Na+/Ca 2+交换器3（NCX 3），细胞质钙蛋白酶激活与DCD从来没有被可视化在真实的时间。我们采用钙蛋白酶荧光共振能量转移（FRET）基板，同时图像钙蛋白酶激活和钙失调活皮质神经元。在持续暴露于谷氨酸（100 μM）的神经元中，在DCD发生后39 ± 5 min观察到FRET的钙蛋白酶底物敏感性下降。钙蛋白酶抑制剂抑制钙蛋白酶没有延迟DCD的发作、从DCD样可逆性钙升高（RCE）的恢复或细胞死亡，尽管抑制α-血影蛋白加工> 90%。Na+/Ca 2+交换器在谷氨酸暴露期间逆转，NCX拮抗剂KB-R7943延长了DCD的时间，并且在谷氨酸暴露90 min后未观察到显著的NCX 3裂解。我们的研究结果表明，与急性谷氨酸中毒相关的钙蛋白酶激活只有在钙稳态持续丧失后才发生。NCX 3或其他钙蛋白酶底物的加工不太可能是皮质神经元急性兴奋性毒性的主要原因。然而，不排除钙蛋白酶作为一个促成因素或响应于轻度谷氨酸损伤的作用。
Although calpain (EC 3.4.22) protease activation was suggested to contribute to excitotoxic delayed calcium deregulation (DCD) via proteolysis of Na+/Ca2+ exchanger 3 (NCX3), cytoplasmic calpain activation in relation to DCD has never been visualized in real time. We employed a calpain fluorescence resonance energy transfer (FRET) substrate to simultaneously image calpain activation and calcium deregulation in live cortical neurons. A calpain inhibitor-sensitive decline in FRET was observed at 39 ± 5 min after DCD occurred in neurons exposed to continuous glutamate (100 μM). Inhibition of calpain by calpeptin did not delay the onset of DCD, recovery from DCD-like reversible calcium elevations (RCE), or cell death despite inhibiting α-spectrin processing by >90%. Na+/Ca2+ exchangers reversed during glutamate exposure, the NCX antagonist KB-R7943 prolonged the time to DCD, and significant NCX3 cleavage following 90 min of glutamate exposure was not observed. Our findings suggest that robust calpain activation associated with acute glutamate toxicity occurs only after a sustained loss in calcium homeostasis. Processing of NCX3 or other calpain substrates is unlikely to be the primary cause of acute excitotoxicity in cortical neurons. However, a role for calpain as a contributing factor or in response to milder glutamate insults is not excluded.
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影响因子：
4.7
作者：
Bevers, Matthew B.;Lawrence, Eric;Neumar, Robert W.
通讯作者：
Neumar, Robert W.
影响因子：
4
作者：
Bolshakov, Alexey P.;Mikhailova, Maria M.;Khodorov, Boris I.
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Khodorov, Boris I.
影响因子：
12.4
作者：
Araujo, I. M.;Carreira, B. P.;Carvalho, C. M.
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Carvalho, C. M.
影响因子：
8.3
作者：
BARTUS, RT;HAYWARD, NJ;POWERS, J
通讯作者：
POWERS, J
影响因子：
4.2
作者：
Buki, A;Farkas, O;Povlishock, JT
通讯作者：
Povlishock, JT