KB-R7943, an inhibitor of the reverse Na+/Ca2+exchanger, blocks N-methyl-D-aspartate receptor and inhibits mitochondrial complex I

被引:50
作者
Brustovetsky, Tatiana [1 ]
Brittain, Matthew K. [1 ]
Sheets, Patrick L. [1 ,2 ]
Cummins, Theodore R. [1 ,2 ]
Pinelis, Vsevolod [3 ]
Brustovetsky, Nickolay [1 ,2 ]
机构
[1] Indiana Univ, Sch Med, Dept Pharmacol & Toxicol, Indianapolis, IN 46202 USA
[2] Indiana Univ, Sch Med, Stark Neurosci Res Inst, Indianapolis, IN USA
[3] Russian Acad Med Sci, Res Ctr Childrens Hlth, Moscow, Russia
关键词
glutamate; excitotoxicity; calcium deregulation; cultured hippocampal neurons; Na; Ca2+exchanger; NMDA receptor; mitochondria; mitochondrial complex I; CYTOCHROME-C RELEASE; CORD WHITE-MATTER; NA+/CA2+ EXCHANGE; PERMEABILITY TRANSITION; HIPPOCAMPAL-NEURONS; NA+-CA2+ EXCHANGE; HYPOXIC/HYPOGLYCEMIC INJURY; CALCIUM DEREGULATION; NMDA EXCITOTOXICITY; CALPAIN ACTIVATION;
D O I
10.1111/j.1476-5381.2010.01054.x
中图分类号
R9 [药学];
学科分类号
1007 ;
摘要
BACKGROUND AND PURPOSE An 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 APPROACH Fluorescence 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 RESULTS KB-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 IMPLICATIONS KB-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.
引用
收藏
页码:255 / 270
页数:16
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