Hydrogen peroxide regulates a cation channel to control neuroendocrine cell excitability
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Authors
Chauhan-Puri, Alamjeet
Date
Type
thesis
Language
eng
Keyword
cation channel , bursting , peptidergic neuron , H2O2 , mollusk , reproduction , diacylglycerol , inositol trisphosphate , oxidation
Alternative Title
Abstract
Non-selective cation channels promote neuronal spiking in many animals. For the hermaphroditic marine snail, Aplysia californica, synaptic input to neuroendocrine bag cell neurons triggers various cation channels, causing an ~30-min afterdischarge of action potentials and egg-laying hormone secretion. During the afterdischarge, protein kinase C (PKC) elevates hydrogen peroxide (H2O2), likely by stimulating nicotinamide adenine dinucleotide phosphate oxidase (NOX). Here, I show that in whole-cell voltage-clamped single, cultured bag cell neurons, H2O2 elicited a prolonged, concentration- and voltage-dependent inward current, associated with an increase in membrane conductance, an ~+30 mV reversal potential, and a requirement for extracellular cations. This current was inhibited by cation channel blockers, and boosted or lessened by preventing or enhancing cellular reduction, respectively. Under current-clamp, H2O2 stimulated bursting in single neurons and evoked afterdischarges from bag cell neurons in intact clusters.
During the afterdischarge, phospholipase C (PLC) hydrolyzes phosphatidylinositol-4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol trisphosphate (IP3). Pharmacologically activating PLC, or adding a DAG analogue plus IP3, enhanced both the current as well as the frequency of firing brought about by H2O2. Stimulating PKC with phorbol ester caused both a voltage-dependent current, that reversed at ~+30 mV, and augmented H2O2-induced spiking.
Because H2O2 is produced intracellularly, it may gate the cation channel at a cytoplasmic site. Consistent with this, the H2O2-induced current had a relatively slow activation latency and was sensitive to redox modifiers delivered intracellularly but not extracellularly. In addition, intracellular activation of NOX evoked a current that occluded the H2O2 response. Moreover, H2O2 enhanced cation channel activity in excised, inside-out patches, where the intracellular face is exposed.
The mitochondria are a potential source of H2O2 as well as being a Ca2+ reservoir. Releasing mitochondrial Ca2+ with a protonophore augmented the H2O2-induced current, and this was prevented by buffering Ca2+ with high intracellular EGTA. Futhermore, the H2O2-induced current was lessened by a mitochondrial-targeted antioxidant.
These results suggest that oxidation by H2O2 activates a voltage-dependent cation channel in bag cell neurons. The synergy of H2O2, phosphoinostide metabolites, and mitochondrial Ca2+ in promoting bursting has implications for neuroendocrine function in Aplysia and the wider animal kingdom.
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This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner.
Attribution-NonCommercial-NoDerivs 3.0 United States
ProQuest PhD and Master's Theses International Dissemination Agreement
Intellectual Property Guidelines at Queen's University
Copying and Preserving Your Thesis
This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner.
Attribution-NonCommercial-NoDerivs 3.0 United States