KB-R7943, an inhibitor of the reverse Na+/Ca2+exchanger, blocks N-methyl-D-aspartate receptor and inhibits mitochondrial complex I
KB-R7943, an inhibitor of the reverse Na+/Ca2+exchanger, blocks N-methyl-D-aspartate receptor and inhibits mitochondrial complex I
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DOI：
10.1111/j.1476-5381.2010.01054.x
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发表时间：
2011-01-01
影响因子：
7.3
通讯作者：
Brustovetsky, Nickolay
中科院分区：
文献类型：
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作者：
Brustovetsky, Tatiana;Brittain, Matthew K.;Brustovetsky, Nickolay
背景和结论异噻唑烷衍生物（2-[2-[4-（4-硝基苄氧基）苯基]乙基]异噻唑烷甲烷磺酸盐（KB-R7943），一种广泛使用的反向Na+/Ca 2+交换器（NCXrev）抑制剂，有助于确定NCXrev在神经元中谷氨酸诱导的Ca 2+失调中的作用。实验方法采用荧光显微镜、电生理膜片钳技术和Seahorse XF 24细胞呼吸仪测定海马神经元的N-甲基-D-天冬氨酸（N-methyl-D-aspartate，NMDA）受体和线粒体复合物I的活性;膜电位成像，KEYSTSKB-R7943抑制NCXrev，IC 50 = 5.7 +/- 2.1 μ M，阻断NMDAR介导的离子电流，抑制NMDA诱导的胞浆Ca 2+增加，IC 50 = 13.4 +/- 3.6 μ M，但加速谷氨酸处理的神经元中的钙失调和线粒体去极化。KB-R7943以Ca 2+非依赖性方式使线粒体去极化。NMDA受体的刺激引起NAD（P）H氧化，其与ATP合成偶联或解偶联取决于浴溶液中Ca 2+的存在。KB-R7943或鱼藤酮在静息条件下增加NAD（P）H自发荧光，并在谷氨酸盐应用后抑制NAD（P）H氧化。KB-R7943抑制2，4-二硝基苯酚刺激的培养神经元呼吸，IC 50 = 11.4 +/- 2.4 μ M。与分离的脑线粒体，KB-R7943抑制呼吸，去极化细胞器和抑制Ca 2+摄取线粒体氧化复合物I基板时，但线粒体与琥珀酸，一个复杂的II substrate.CONCLUSIONS和IMPLICATIONSKB-R7943，除了NCXrev，阻断NMDA受体在培养的海马神经元和抑制复合物I在线粒体呼吸链时是无效的。这些发现对于正确解释KB-R7943获得的实验结果以及更好地理解其神经保护作用至关重要。
BACKGROUND AND PURPOSEAn isothiourea derivative (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methane sulfonate (KB-R7943), a widely used inhibitor of the reverse Na+/Ca2+ exchanger (NCXrev), was instrumental in establishing the role of NCXrev in glutamate-induced Ca2+ deregulation in neurons. Here, the effects of KB-R7943 on N-methyl-D-aspartate (NMDA) receptors and mitochondrial complex I were tested.EXPERIMENTAL APPROACHFluorescence microscopy, electrophysiological patch-clamp techniques and cellular respirometry with Seahorse XF24 analyzer were used with cultured hippocampal neurons; membrane potential imaging, respirometry and Ca2+ flux measurements were made in isolated rat brain mitochondria.KEY RESULTSKB-R7943 inhibited NCXrev with IC50 = 5.7 +/- 2.1 mu M, blocked NMDAR-mediated ion currents, and inhibited NMDA-induced increase in cytosolic Ca2+ with IC50 = 13.4 +/- 3.6 mu M but accelerated calcium deregulation and mitochondrial depolarization in glutamate-treated neurons. KB-R7943 depolarized mitochondria in a Ca2+-independent manner. Stimulation of NMDA receptors caused NAD(P)H oxidation that was coupled or uncoupled from ATP synthesis depending on the presence of Ca2+ in the bath solution. KB-R7943, or rotenone, increased NAD(P)H autofluorescence under resting conditions and suppressed NAD(P)H oxidation following glutamate application. KB-R7943 inhibited 2,4-dinitrophenol-stimulated respiration of cultured neurons with IC50 = 11.4 +/- 2.4 mu M. With isolated brain mitochondria, KB-R7943 inhibited respiration, depolarized organelles and suppressed Ca2+ uptake when mitochondria oxidized complex I substrates but was ineffective when mitochondria were supplied with succinate, a complex II substrate.CONCLUSIONS AND IMPLICATIONSKB-R7943, in addition to NCXrev, blocked NMDA receptors in cultured hippocampal neurons and inhibited complex I in the mitochondrial respiratory chain. These findings are critical for the correct interpretation of experimental results obtained with KB-R7943 and a better understanding of its neuroprotective action.