Elsevier

Nitric Oxide

Volumes 113–114, 1 September 2021, Pages 39-49
Nitric Oxide

Alcohol hangover induces nitric oxide metabolism changes by impairing NMDA receptor-PSD95-nNOS pathway

https://doi.org/10.1016/j.niox.2021.04.009Get rights and content

Highlights

  • Alcohol hangover reduces NO total content and nNOS activity and expression.

  • NO production by NMDAR stimulation is impaired due to hangover in synaptosomes.

  • NMDAR subunit GluN2B and PSD-95 protein expression are reduced by hangover.

  • Hangover alters NO metabolism by disrupting NMDAR/PSD-95/nNOS pathway.

Abstract

Alcohol hangover is defined as the combination of mental and physical symptoms experienced the day after a single episode of heavy drinking, starting when blood alcohol concentration approaches zero. We previously evidenced increments in free radical generation and an imbalance in antioxidant defences in non-synaptic mitochondria and synaptosomes during hangover. It is widely known that acute alcohol exposure induces changes in nitric oxide (NO) production and blocks the binding of glutamate to NMDAR in central nervous system. Our aim was to evaluate the residual effect of acute ethanol exposure (hangover) on NO metabolism and the role of NMDA receptor-PSD95-nNOS pathway in non-synaptic mitochondria and synaptosomes from mouse brain cortex. Results obtained for the synaptosomes fraction showed a 37% decrease in NO total content, a 36% decrease in NOS activity and a 19% decrease in nNOS protein expression. The in vitro addition of glutamate to synaptosomes produced a concentration-dependent enhancement of NO production which was significantly lower in samples from hangover mice than in controls for all the glutamate concentrations tested. A similar patter of response was observed for nNOS activity being decreased both in basal conditions and after glutamate addition. In addition, synaptosomes exhibited a 64% and 15% reduction in NMDA receptor subunit GluN2B and PSD-95 protein expression, respectively. Together with this, glutamate-induced calcium entry was significant decreased in synaptosomes from alcohol-treated mice. On the other hand, in non-synaptic mitochondria, no significant differences were observed in NO content, NOS activity or nNOS protein expression. The expression of iNOS remained unaltered in synaptosomes and non-synaptic mitochondria. Here we demonstrated that hangover effects on NO metabolism are strongly evidenced in synaptosomes probably due to a disruption in NMDAR/PSD-95/nNOS pathway.

Introduction

The abuse of alcohol consumption induces adverse effects on multi-organs, including the liver, pancreas, heart, and central nervous system. Alcohol hangover constitutes the main negative consequence after binge drinking. This state is defined as the combination of mental and physical symptoms experienced the day after a single episode of heavy drinking, starting when blood alcohol concentration approaches zero [1]. We previously established that alcohol hangover induced substantial negative changes in motor and affective behavior during at least 20 h since hangover onset [2,3]. Since synaptosomes constitute a suitable approach to study bioenergetics and mitochondrial function in the synapses [4], we previously demonstrated that oxidative stress is generated at the onset of alcohol hangover. The increment in free radicals generation and the imbalance in antioxidant defenses were mostly observed in the synaptosomal fration revealing that mitochondria at the synapse were deeply affected [5].

Other authors evidenced that adolescent binge alcohol exposure affects brain function through mitochondrial impairment [6,7] and we recently verified that mitochondrial dysfunction triggered by acute ethanol exposure could be persistent and evidenced at the onset of the hangover state mainly at synaptic terminals [8]. In addition, it is known that acute alcohol exposure can lead to changes in nitric oxide (NO) production, which is associated with different cognitive impairments such as memory and learning deficiencies [9].

Interestingly, we previously observed that ethanol hangover does not only impair mitochondrial function but also NO metabolism in crude mitochondrial fractions from brain cortex and cerebellum [10,11].

The activation of N-methyl-d-aspartate NMDA receptors (NMDARs) plays a central role in essential physiological processes [12]. It is widely known that ethanol blocks the binding of glutamate to NMDAR among other effects that compromise central nervous system [13]. NMDAR physiological activation results in the opening of its ion channel allowing calcium influx to the cell. Neuronal nitric oxide synthase (nNOS) is activated by calcium entry resulting in an increased NO production [14]; this activation pathway seems to be critically dependent on the postsynaptic density protein-95 (PSD-95), a scaffolding protein which binds simultaneously to the NMDAR and nNOS via its two N-terminal PDZ (PSD-95/Discs large/zona occludens-1) domains [15]. Thus, PSD-95 mediates a specific association between NMDAR activation, Ca2+ influx and NO production [15]. It was reported that PSD-95 changes could be associated with alcohol-induced behaviour changes, being also this protein postulated as a key mediator of the effects of multiple abuse drugs. For instance, it was demonstrated that PSD-95 knockout mice presented an exacerbated sensibility to acute ethanol exposure compared with wild-type animals [16]. Moreover, it was established that PSD95-nNOS interaction is critical for synaptic connections [17], and therefore, prolonged suppression of PSD95-nNOS signaling may lead to unknown risks [18].

There are no previous reports focusing on the study of NO metabolism associated to NMDAR-PSD-95 impairment due to alcohol after-effects (hangover). In line with this, and considering our previous research, it was of interest to study the mechanisms involved in the changes in NO production at the hangover state and to analyze if NO production alterations at the synapses could be associated to NMDAR-PSD-95 impairment.

Therefore, the aim of the work was to study alcohol residual effects on NO metabolism by detecting NO total content and NOS activity and expression in non-synaptic mitochondria and synaptosomes from mouse brain cortex. Moreover, the role of NMDA activation on NO content and production together with NMDAR GluN2B subunit and PSD-95 protein expression were evaluated in the same experimental model of alcohol hangover.

Section snippets

Materials

CaCl2, catalase, p-coumaric acid, dithiothreitol (DTT), EDTA, EGTA, FFA-BSA, Folin reagent, glutamic acid, glutathione, Hepes, H2O2, HRP, KH2PO4, KCl, K2HPO4, KCN, l-arginine, luminol, malic acid, mannitol, MgCl2, NADPH, NaH2PO4, Na2HPO4, NaN3, NO2, Nω-Nitro-l-arginine (L-NNA), oxyhemoglobin, sodium dodecilsulphate (SDS), superoxide dismutase (SOD), succinate, sucrose, Trizma base, Tween were obtained from Sigma Chemical Co. (Saint Louis, Missouri, United States). Acrylamide, APS,

Nitric oxide content

Nitric oxide total content was detected by flow cytometry using DAF-2DA and DAF-2 probes in synaptosomes and non-synaptic mitochondria respectively. Results of synaptosomes NO content are shown in Fig. 1A while the same for non-synaptic mitochondria are shown in Fig. 1B.

Dot plots of Forward Scatter-Height (FSC–H) versus Side Scatter-Height (SSC–H) indicating the gated synaptosomes or non-synaptic mitochondria populations from control samples are shown in Fig. 1A (i) and 1B (i), respectively.

Discussion

Alcohol hangover represents the condition that comprises the residual effects of acute alcohol exposure. Bae and colleagues (2005) have proposed that ethanol damage in CNS is provoked due to its site of action within the cell membrane at synapses [33]. We previously demonstrated the occurrence of mitochondrial dysfunction and oxidative stress mainly affecting mitochondria present at the synapse at the onset of alcohol hangover [5,8]. As we also reported alterations in NO metabolism in total

Conclusions

The present work demonstrated that ethanol induced impairments of NMDAR/PSD-95/nNOS pathway and NO synthesis observed in mouse brain cortex synaptosomes and non-synaptic mitochondria 6 h after acute ethanol exposure.

Results indicated that hangover induced a significant decrease in NO total content, nNOS activity and NO production due to NMDAR stimulation in synaptosomes. On the other hand, no changes were observed in NO content and nNOS activity in non-synaptic mitochondria. The expression of

Declaration of competing interest

None.

Acknowledgments

This research was supported by grants from Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET 112–20150100648) and Universidad de Buenos Aires (UBA, 20020170100516BA).

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      In the context of present findings, an impairment of NMDA receptor/PSD-95/nNOS complex integrity can be suggested. It is worthwhile to mention that in an experimental model of ethanol hangover, which likewise leads to brain NOS activity decrease (Karadayian et al., 2014) reduction in the expression of GluN2B, PSD-95 and nNOS proteins were recorded (Karadayian et al., 2021). Interestingly, in the present study, we observed a significant decrease in actin protein expression in synaptosomal membrane fractions by levocabastine treatment.

    1

    Note of authors: AK and SLA regret to communicate that unfortunately our dear colleague Juanita Bustamante passed away by the time we were finishing the manuscript. We will never forget her huge enthusiasm for scientific research and wish to express our profound recognition and admiration for her work.

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