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 Ca2+ 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 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 (NCX_rev), was instrumental in establishing the role of NCX_revin 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 NCX_revwith IC₅₀= 5.7 ± 2.1 µM, blocked NMDAR‐mediated ion currents, and inhibited NMDA‐induced increase in cytosolic Ca²⁺with IC₅₀= 13.4 ± 3.6 µM 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 µM. 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 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.

Background: Accumulated evidence suggests that hydrogen peroxide (H₂O₂) generated in cells during insulin stimulation plays an integral role in insulin receptor signal transduction. The role of insulin-induced H₂O₂in neuronal insulin receptor activation and the origin of insulin-induced H₂O₂in neurons remain unclear. The aim of the present study is to test the following hypotheses (1) whether insulin-induced H₂O₂is required for insulin receptor autophosphorylation in neurons, and (2) whether mitochondrial respiratory chain is involved in insulin-stimulated H₂O₂production, thus playing an integral role in insulin receptor autophosphorylation in neurons.Results: Insulin stimulation elicited rapid insulin receptor autophosphorylation accompanied by an increase in H₂O₂release from cultured cerebellar granule neurons (CGN). N-acetylcysteine (NAC), a H₂O₂scavenger, inhibited both insulin-stimulated H₂O₂release and insulin-stimulated autophosphorylation of insulin receptor. Inhibitors of respiratory chain-mediated H₂O₂production, malonate and carbonyl cyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP), inhibited both insulin-stimulated H₂O₂release from neurons and insulin-stimulated autophosphorylation of insulin receptor. Dicholine salt of succinic acid, a respiratory substrate, significantly enhanced the effect of suboptimal insulin concentration on the insulin receptor autophosphorylation in CGN.Conclusion: Results of the present study suggest that insulin-induced H₂O₂is required for the enhancement of insulin receptor autophosphorylation in neurons. The mitochondrial respiratory chain is involved in insulin-stimulated H₂O₂production, thus playing an integral role in the insulin receptor autophosphorylation in neurons.