Dextromethorphan inhibition of voltage-gated proton currents in BV2 microglial cells
Highlights
► Dextromethorphan inhibits voltage-gated proton currents in BV2 microglial cells. ► Dextromethorphan does not change the reversal potential. ► Dextromethorphan does not change the voltage dependence of the gating. ► Dextrorphan and 3-hydroxymorphinan are ineffective to inhibit proton currents. ► Dextromethorphan methiodide is ineffective to inhibit proton currents.
Introduction
Activated microglia produce pro-inflammatory cytokines, reactive oxygen species (ROS) and nitric oxide (NO), which damage neurons, leading to neurodegeneration. Dextromethorphan is an antitussive drug showing a neuroprotective property in various models of central nervous system injury [19], [20]. The neuroprotection is partially mediated through the inhibition of microglial activation. Dextromethorphan reduces the degeneration of dopaminergic neurons induced by lipopolysaccharide (LPS) or β-amyloid peptide (Aβ), and inhibits the production of tumor necrosis factor-α (TNF-α), prostaglandin E2, NO, superoxide free radicals and intracellular ROS in rat mesencephalic neuron-glia cultures, but not in neuron-enriched cultures [8], [10]. Dextromethorphan also reduces the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced loss of dopaminergic neurons in substantia nigra through suppression of ROS production [21].
Dextromethorphan is rapidly transformed through O-demethylation to its major metabolite, dextrorphan. Dextrorphan suppresses depolarization induced by N-methyl-d-aspartate (NMDA) and epileptiform activity elicited in Mg2+-free solution in rat hippocampus more potently than does dextromethorphan [2]. Concerning NMDA antagonism, dextrorphan, but not dextromethorphan, induces phencyclidine-like behavior in rats [18]. 3-Hydroxymorphinan, another metabolite of dextromethorphan, is neuroprotective against LPS- or MPTP-induced microglial activation and subsequent inflammatory neurotoxicity in dopaminergic neurons [22], [23]. 3-Hydroxymorphinan is even more potent than dextromethorphan due to its additional neurotrophic effect mediated through astroglia.
The effects of dextromethorphan are dependent on the presence of NADPH oxidase, the key enzymatic system responsible for ROS production in microglia [8], [21]. Thus, dextromethorphan reduces macrophage NADPH oxidase activity by decreasing membrane translocation of p47phox and p67phox through the inhibition of protein kinase C and extracellular signal-regulated kinase activation [9]. Dextromethorphan also inhibits the transcription of p47phox and gp91phox in microglia at subnanomolar concentrations [1]. Voltage-gated proton channels (Hv1) are predominantly distributed in the membrane of phagocytic cells, including microglia [5]. In these cells proton channels support NADPH oxidase activity and play an essential role in the production of ROS [6], [13], [14].
The role of proton channels in the neuroprotection of dextromethorphan has yet to be examined. To test the hypothesis that proton channel inhibition is related to the suppression of microglial production of ROS and other cytotoxic cytokines, we compared dextromethorphan with its metabolites for their inhibitory effects on the proton channels, and tested whether the proton channel inhibition was related to their well-documented differential effects on microglial activation. Here, we report that dextromethorphan, but not dextrorphan or 3-hydroxymorphinan, inhibits proton currents in BV2 microglial cells.
Section snippets
Materials and methods
Microglial BV2 cells were cultured in a 50:50 mixture of Dulbecco's Modified Eagle's Medium and Ham's F12 (Mediatech, Manassas, VA) along with 10% fetal bovine serum, 100 units/ml penicillin and 100 μg streptomycin/ml. Cells were harvested and plated in 6-well culture plates containing 12 mm glass coverslips that were previously coated with poly-l-lysine. These cells were allowed to grow for 2 days before using them for electrophysiological experiments.
The standard external solution contained (in
Results
Depolarizing pulses to +20 mV from a holding potential of −70 mV were employed to evoke voltage-gated proton currents in BV2 cells at an intracellular pH/extracellular pH = 5.5/7.3 (Fig. 1). The resultant outward currents were activated slowly during a 2-s depolarization and, upon repolarization to the holding potential, long tail currents developed, which are characteristics of proton currents.
Dextromethorphan at 100 μM slowly inhibited the proton currents, reaching a steady-state level of 28 ± 4.0%
Discussion
Dextromethorphan reversibly inhibited proton currents in microglial BV2 cells with an IC50 value of 51.7 μM. The inhibition was not due to intracellular pH change or altered gating voltage, as evidenced by the observations that dextromethorphan had no effect on the reversal potential or the conductance–voltage curve. Dextrorphan and 3-hydroxymorphinan, both O-demethylated metabolites of dextromethorphan, were ineffective. Thus, it appears that O-methyl at position 3 of dextromethorphan plays
Acknowledgments
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0022213).
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2022, Neurobiology of DiseaseCitation Excerpt :This voltage-gated proton channel compensates the cellular loss of electrons with protons, activating NADPH oxidase (Wu, 2014) (Fig. 2). Dextromethorphan that has anti-inflammatory properties was shown to reduce proton channels in BV2s (Song and Yeh, 2012) and primary rat microglia. Yang et al. demonstrated that dextromethorphan reduced LPS-induced TNF-α production and caspase-3 activation (Yang et al., 2020).
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