Haskew Renée E, Mongin Alexander A, Kimelberg Harold K
Center for Neuropharmacology and Neuroscience, Albany Medical College, Albany, New York 12208, USA.
J Neurochem. 2002 Aug;82(4):903-12. doi: 10.1046/j.1471-4159.2002.01037.x.
Volume-regulated anion channels (VRACs) are critically important for cell volume homeostasis, and under pathological conditions contribute to neuronal damage via excitatory amino (EAA) release. The precise mechanisms by which brain VRACs are activated and/or modulated remain elusive. In the present work we explored the possible involvement of nitric oxide (NO) and NO-related reactive species in the regulation of VRAC activity and EAA release, using primary astrocyte cultures. The NO donors sodium nitroprusside and spermine NONOate did not affect volume-activated d-[3H]aspartate release. In contrast, the peroxynitrite (ONOO-) donor 3-morpholinosydnomine hydrochloride (SIN-1) increased volume-dependent EAA release by approx. 80-110% under identical conditions. Inhibition of ONOO- formation with superoxide dismutase completely abolished the effects of SIN-1. Both the volume- and SIN-1-induced EAA release were sensitive to the VRAC blockers NPPB and ATP. Further pharmacological analysis ruled out the involvement of cGMP-dependent reactions and modification of sulfhydryl groups in the SIN-1-inducedmodulation of EAA release. The src family tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo [3,4-d]pyrimidine (PP2), but not its inactive analog PP3, abolished the effects of SIN-1. A broader spectrum tyrosine kinase inhibitor tyrphostin A51, also completely eliminated the SIN-1-induced EAA release. Our data suggest that ONOO- up-regulates VRAC activity via a src tyrosine kinase-dependent mechanism. This modulation may contribute to EAA-mediated neuronal damage in ischemia and other pathological conditions favoring cell swelling and ONOO- production.
容积调节性阴离子通道（VRACs）对于细胞容积稳态至关重要，并且在病理条件下通过兴奋性氨基酸（EAA）释放导致神经元损伤。脑VRACs被激活和/或调节的精确机制仍然不清楚。在本研究中，我们使用原代星形胶质细胞培养物探讨了一氧化氮（NO）和NO相关活性物质在VRAC活性调节和EAA释放中的可能作用。NO供体硝普钠和精胺NONOate不影响容积激活的d-[3H]天冬氨酸释放。相反，过氧亚硝酸盐（ONOO-）供体盐酸3-吗啉代 sydnonimine（SIN-1）在相同条件下使容积依赖性EAA释放增加约80-110%。用超氧化物歧化酶抑制ONOO-形成完全消除了SIN-1的作用。容积诱导和SIN-1诱导的EAA释放对VRAC阻滞剂NPPB和ATP均敏感。进一步的药理学分析排除了cGMP依赖性反应和巯基修饰参与SIN-1诱导的EAA释放调节。src家族酪氨酸激酶抑制剂4-氨基-5-（4-氯苯基）-7-（叔丁基）吡唑并[3,4-d]嘧啶（PP2）而非其无活性类似物PP3消除了SIN-1的作用。一种更广泛谱的酪氨酸激酶抑制剂 tyrphostin A51也完全消除了SIN-1诱导的EAA释放。我们的数据表明，ONOO-通过src酪氨酸激酶依赖性机制上调VRAC活性。这种调节可能在缺血和其他有利于细胞肿胀和ONOO-产生的病理条件下导致EAA介导的神经元损伤。
