KB-R7943, an inhibitor of the reverse Na +/Ca 2+ exchanger, blocks N-methyl-D-aspartate receptor and inhibits mitochondrial complex i

Tatiana Brustovetsky, Matthew K. Brittain, Patrick Sheets, Theodore Cummins, Vsevolod Pinelis, Nikolai Broustovetski

Research output: Contribution to journalArticle

35 Citations (Scopus)

Abstract

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 +/Ca 2+ exchanger (NCX rev), was instrumental in establishing the role of NCX rev 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 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 Ca 2+ flux measurements were made in isolated rat brain mitochondria. Key Results KB-R7943 inhibited NCX rev with IC 50= 5.7 ± 2.1 μM, blocked NMDAR-mediated ion currents, and inhibited NMDA-induced increase in cytosolic Ca 2+ with IC 50= 13.4 ± 3.6 μM 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 IC 50= 11.4 ± 2.4 μM. 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 Implications KB-R7943, in addition to NCX rev, 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.

Original languageEnglish
Pages (from-to)255-270
Number of pages16
JournalBritish Journal of Pharmacology
Volume162
Issue number1
DOIs
StatePublished - Jan 2011

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Sodium-Calcium Exchanger
N-Methyl-D-Aspartate Receptors
Mitochondria
Neurons
NAD
Glutamic Acid
Respiration
Smegmamorpha
2,4-Dinitrophenol
2-(2-(4-(4-nitrobenzyloxy)phenyl)ethyl)isothiourea methanesulfonate
Rotenone
Methane
Brain
Succinic Acid
Patch-Clamp Techniques
N-Methylaspartate
Electron Transport
Baths
Fluorescence Microscopy
Organelles

Keywords

  • calcium deregulation
  • cultured hippocampal neurons
  • excitotoxicity
  • glutamate
  • mitochondria
  • mitochondrial complex I
  • Na /Ca exchanger
  • NMDA receptor

ASJC Scopus subject areas

  • Pharmacology

Cite this

KB-R7943, an inhibitor of the reverse Na +/Ca 2+ exchanger, blocks N-methyl-D-aspartate receptor and inhibits mitochondrial complex i. / Brustovetsky, Tatiana; Brittain, Matthew K.; Sheets, Patrick; Cummins, Theodore; Pinelis, Vsevolod; Broustovetski, Nikolai.

In: British Journal of Pharmacology, Vol. 162, No. 1, 01.2011, p. 255-270.

Research output: Contribution to journalArticle

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abstract = "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 +/Ca 2+ exchanger (NCX rev), was instrumental in establishing the role of NCX rev 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 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 Ca 2+ flux measurements were made in isolated rat brain mitochondria. Key Results KB-R7943 inhibited NCX rev with IC 50= 5.7 ± 2.1 μM, blocked NMDAR-mediated ion currents, and inhibited NMDA-induced increase in cytosolic Ca 2+ with IC 50= 13.4 ± 3.6 μM 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 IC 50= 11.4 ± 2.4 μM. 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 Implications KB-R7943, in addition to NCX rev, 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.",
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T1 - KB-R7943, an inhibitor of the reverse Na +/Ca 2+ exchanger, blocks N-methyl-D-aspartate receptor and inhibits mitochondrial complex i

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AU - Brittain, Matthew K.

AU - Sheets, Patrick

AU - Cummins, Theodore

AU - Pinelis, Vsevolod

AU - Broustovetski, Nikolai

PY - 2011/1

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N2 - 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 +/Ca 2+ exchanger (NCX rev), was instrumental in establishing the role of NCX rev 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 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 Ca 2+ flux measurements were made in isolated rat brain mitochondria. Key Results KB-R7943 inhibited NCX rev with IC 50= 5.7 ± 2.1 μM, blocked NMDAR-mediated ion currents, and inhibited NMDA-induced increase in cytosolic Ca 2+ with IC 50= 13.4 ± 3.6 μM 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 IC 50= 11.4 ± 2.4 μM. 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 Implications KB-R7943, in addition to NCX rev, 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.

AB - 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 +/Ca 2+ exchanger (NCX rev), was instrumental in establishing the role of NCX rev 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 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 Ca 2+ flux measurements were made in isolated rat brain mitochondria. Key Results KB-R7943 inhibited NCX rev with IC 50= 5.7 ± 2.1 μM, blocked NMDAR-mediated ion currents, and inhibited NMDA-induced increase in cytosolic Ca 2+ with IC 50= 13.4 ± 3.6 μM 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 IC 50= 11.4 ± 2.4 μM. 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 Implications KB-R7943, in addition to NCX rev, 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.

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KW - mitochondria

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KW - Na /Ca exchanger

KW - NMDA receptor

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