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Role of membrane cholesterol in spontaneous exocytosis at frog neuromuscular synapses: reactive oxygen species–calcium interplay
Author(s) -
Petrov Alexey M.,
Yakovleva Anastasiya A.,
Zefirov Andrey L.
Publication year - 2014
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.2014.279695
Subject(s) - nadph oxidase , chemistry , exocytosis , capsazepine , apocynin , ruthenium red , reactive oxygen species , trpv , okadaic acid , biophysics , biochemistry , endocrinology , calcium , transient receptor potential channel , medicine , trpv1 , biology , receptor , phosphatase , phosphorylation , organic chemistry , secretion
Key pointsCholesterol depletion increases reactive oxygen species (ROS) levels in extra‐ and intracellular space through NAPDH oxidase activation, which is accompanied by synaptic lipid oxidation. ROS production due to extraction of cholesterol involves both an enhancement of synaptic vesicle exocytosis and an increase in cytosolic [Ca 2+ ] i . An ROS‐dependent rise in [Ca 2+ ] i is suppressed by inhibitors of the transient receptor potential vanilloid channel and leads to an increase in synaptic vesicle exocytosis. The ROS–calcium pathway might influence synaptic vesicle exocytosis via activation of phosphatase protein 2B (calcineurin). The results help us better understand why a decrease of membrane cholesterol increases spontaneous synaptic vesicle exocytosis.Abstract Using electrophysiological and optical techniques, we studied the mechanisms by which cholesterol depletion stimulates spontaneous transmitter release by exocytosis at the frog neuromuscular junction. We found that methyl‐β‐cyclodextrin (MCD, 10 m m )‐mediated exhaustion of cholesterol resulted in the enhancement of reactive oxygen species (ROS) production, which was prevented by the antioxidant N ‐acetyl cysteine (NAC) and the NADPH oxidase inhibitor apocynin. An increase in ROS levels occurred both extra‐ and intracellularly, and it was associated with lipid peroxidation in synaptic regions. Cholesterol depletion provoked a rise in the intracellular Ca 2+ concentration, which was diminished by NAC and transient receptor potential vanilloid (TRPV) channel blockers (ruthenium red and capsazepine). By contrast, the MCD‐induced rise in [Ca 2+ ] i remained unaffected if Ca 2+ release from endoplasmic stores was blocked by TMB8 (8‐(diethylamino)octyl‐3,4,5‐trimethoxybenzoate hydrochloride). The effects of cholesterol depletion on spontaneous release and exocytosis were significantly reduced by the antioxidant, intracellular Ca 2+ chelation with BAPTA‐AM and blockers of TRPV channels. Bath application of the calcineurin antagonist cyclosporine A blocked MCD‐induced enhancement of spontaneous release/exocytosis, whereas okadaic acid, an inhibitor of phosphatases PP1 and PP2A, had no effect. Thus, our findings indicate that enhancement of spontaneous exocytosis induced by cholesterol depletion may depend on ROS generation, leading to an influx of Ca 2+ via TRPV channels and, subsequently, activation of calcineurin.