Background: Genetically encoded sensors developed on the basis of green fluorescent protein (GFP)-like proteins are becoming more and more popular instruments for monitoring cellular analytes and enzyme activities in living cells and transgenic organisms. In particular, a number of Ca 2+ sensors have been developed, either based on FRET (Fluorescence Resonance Energy Transfer) changes between two GFP-mutants or on the change in fluorescence intensity of a single circularly permuted fluorescent protein (cpFP).

Background: Accumulated evidence suggests that hydrogen peroxide (H 2 O 2 ) generated in cells during insulin stimulation plays an integral role in insulin receptor signal transduction. The role of insulin-induced H 2 O 2 in neuronal insulin receptor activation and the origin of insulin-induced H 2 O 2 in neurons remain unclear. The aim of the present study is to test the following hypotheses (1) whether insulin-induced H 2 O 2 is required for insulin receptor autophosphorylation in neurons, and (2) whether mitochondrial respiratory chain is involved in insulin-stimulated H 2 O 2 production, thus playing an integral role in insulin receptor autophosphorylation in neurons.

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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...

Glutamate excitotoxicity is implicated in the pathogenesis of numerous diseases, such as stroke, traumatic brain injury, and Alzheimer’s disease, for which insulin resistance is a concomitant condition, and intranasal insulin treatment is believed to be a promising therapy. Excitotoxicity is initiated primarily by the sustained stimulation of ionotropic glutamate receptors and leads to a rise in intracellular Ca2+ ([Ca2+]i), followed by a cascade of intracellular events, such as delayed calcium deregulation (DCD), mitochondrial depolarization, adenosine triphosphate (ATP) depletion that collectively end in cell death. Therefore, cross-talk between insulin and glutamate signaling in excitotoxicity is of particular interest for research. In the present study, we investigated the effects of short-term insulin exposure on the dynamics of [Ca2+]i and mitochondrial potential in cultured rat cortical neurons during glutamate excitotoxicity. We found that insulin ameliorated the glutamate-evoked rise of [Ca2+]i and prevented the onset of DCD, the postulated point-of-no-return in excitotoxicity. Additionally, insulin significantly improved the glutamate-induced drop in mitochondrial potential, ATP depletion, and depletion of brain-derived neurotrophic factor (BDNF), which is a critical neuroprotector in excitotoxicity. Also, insulin improved oxygen consumption rates, maximal respiration, and spare respiratory capacity in neurons exposed to glutamate, as well as the viability of cells in the MTT assay. In conclusion, the short-term insulin exposure in our experiments was evidently a protective treatment against excitotoxicity, in a sharp contrast to chronic insulin exposure causal to neuronal insulin resistance, the adverse factor in excitotoxicity.