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

Abstract

An isothiourea derivative (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methane sulfonate (KB-R7943), a widely used inhibitor of the reverse Na + /Ca 2+ exchanger (NCXrev), was instrumental in establishing the role of NCXrev in glutamate-induced Ca 2+ 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 Ca 2+ flux measurements were made in isolated rat brain mitochondria. KEY RESULTSKB-R7943 inhibited NCXrev with IC50 = 5.7 Ϯ 2.1 mM, blocked NMDAR-mediated ion currents, and inhibited NMDA-induced increase in cytosolic Ca 2+ with IC50 = 13.4 Ϯ 3.6 mM but accelerated calcium deregulation and mitochondrial depolarization in glutamate-treated neurons. KB-R7943 depolarized mitochondria in a Ca 2+ -independent manner. Stimulation of NMDA receptors caused NAD(P)H oxidation that was coupled or uncoupled from ATP synthesis depending on the presence of Ca 2+ 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 Ϯ 2.4 mM. With isolated brain mitochondria, KB-R7943 inhibited respiration, depolarized organelles and suppressed Ca 2+ 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. AbbreviationsDy, mitochondrial membrane potential; [Ca 2+ ]c, cytosolic Ca 2+ concentration; CNQX, 6-cyano-7-nitroquinoxaline-2, 3-dione; DMSO, dimethyl sulfoxide; FCCP, carbonylcyanide-p-trifluoromethoxyphenylhydrazone; phenyl]ethyl]isothiourea methane sulfonate; MK801 (5R,10S)-(+)-5-methyl-10,11-dihydro-BJP British Journal of Pharmacology DOI:10.1111DOI:10. /j.1476DOI:10. -5381.2010 British Journal of Pharmacology (2011) IntroductionIn neurons exposed to glutamate, prolonged stimulation of glutamate receptors, particularly the N-methyl-D-aspartate (NMDA) subtype, results in massive Ca 2+ influx into the cell, disruption of calcium homeostasis and eventual cell death (Choi, 1988;Manev et al. 1989 (Blaustein and Lederer, 1999). Predictably, inhibition of NCX forward mode by removing external Na + leads to increased [Ca 2+ ]c (Mattson et al., 1989). However, under conditions of prolonged exposure to glutamate or NMDA, when cytosolic Na + is elevated and the plasma membrane is depol...

Involvement of the sodium–calcium exchanger 3 (NCX3) in ziram-induced calcium dysregulation and toxicity

“…4A). This effect may be caused by the ability of KB-R7943 to inhibit complex I in the mitochondrial respiratory chain (Brustovetsky et al, 2011). …”

“…4A). We can speculate that the observed potentiation with KBBR-7943 was caused by its fair specificity for the NCX3 reverse mode of action (Ca 2+ entry mode of exchange) and its ability to inhibit the complex I in the mitochondrial respiratory chain (Brustovetsky et al, 2011). This speculation is supported by our observation in Na + -free buffer, where NCX3 activity in its reverse mode of action is totally abolished by the addition of ziram and/or the inhibitor KB-7943.…”

Calcium/Calmodulin-dependent Protein Kinase II (CaMKII) Inhibition Induces Neurotoxicity via Dysregulation of Glutamate/Calcium Signaling and Hyperexcitability

“…Calcium Imaging-Hippocampal neurons 10 -12 DIV were loaded with 2.6 M Fura-2FF-AM (Invitrogen) and 1.7 M Rhodamine 123 and subsequently imaged as described previously (20,22). During imaging, the neurons were maintained in a bath solution containing 10 mM HEPES, pH 7.4, 139 mM NaCl, 3 mM KCl, 0.8 mM MgCl 2 , 1.8 mM CaCl 2 , 5 mM glucose, and 65 mM sucrose.…”

Ca2+ toxicity due to reverse Na+/Ca2+ exchange contributes to degeneration of neurites of DRG neurons induced by a neuropathy-associated Nav1.7 mutation

“…Off-target effects of ] fluxes and 2,4-dinitrophenol-stimulated respiration; however, these occur with higher concentrations (IC 50 ϭ 13.4 M and 11.4 M, respectively; Brustovetsky et al 2011) than the 0.5 M concentration used in our experiments, a level that has been shown to inhibit reverse operation of NCX (Watanabe et al 2006). A role of NCX in mitochondria cannot be ruled out, since pharmacological modulation could cause changes in calcium flux from this pool of calcium (Svichar et al 1999).…”