Ethanol inhibition of NMDA-induced responses and acute tolerance to the inhibition in rat rostral ventrolateral medulla in vivo: Involvement of cAMP-dependent protein kinases
Introduction
The N-methyl-d-aspartate (NMDA) receptors are involved in the excitability, cognitive function, and motor coordination of the central nervous systems and are thought to mediate the intoxicating effects of ethanol (Kumari and Ticku, 2000). Ethanol inhibits NMDA receptor function at pharmacologically relevant concentrations in a variety of neuronal preparations including culture neurons, brain slices, and anesthetized animals (Lai et al., 2004, Lin et al., 2003, Lovinger et al., 1989, Woodward, 2000). Rostral ventrolateral medulla (RVLM) neurons play an important role in the regulation of the cardiovascular function. Our recent study showed that intravenous injection of ethanol selectively inhibited the pressor response induced by microinjection of NMDA into the RVLM leaving the effects of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), γ-aminobutyric acid A (GABA), or glycine on blood pressure unaltered, suggesting NMDA receptors as a major target of ethanol in the RVLM (Lai et al., 2004). The inhibitory effects of ethanol on the NMDA-induced pressor effect in the RVLM were reduced over time during continuous infusion of ethanol, indicating the development of acute tolerance or tachyphylaxis. We also observed acute tolerance to ethanol inhibition of NMDA receptors in rat sympathetic preganglionic neurons, another key neuron involved in the regulation of cardiovascular function (Lin et al., 2003). These studies provided in vivo evidence of acute tolerance to ethanol inhibition of neuronal NMDA receptor activation.
The precise mechanisms underlying acute ethanol tolerance have not been established. NMDA receptors are composed of 7 subunits including a NR1 subunit, a family of NR2 subunits (A, B, C, D), and two NR2 subunits (A and B) (Cull-Candy et al., 2001). NMDA receptor function is regulated by kinases and phosphatases. There are several serine residues on the NR1 subunit; serine residue 897 on the NR1 subunit is phosphorylated by cAMP-dependent protein kinases (PKA) (Ron, 2004). Activation of type 1 protein phosphatase (PP1) decreased NMDA receptor function whereas PKA activation overcame constitutive PP1 activity (Westphal et al., 1999). Ethanol affects several transduction pathways including the cAMP-PKA signaling pathway (Newton and Messing, 2006). Acute ethanol exposure to cortical neurons in vitro has been demonstrated to increase the levels of phosphoserine 897 on NR1 subunits by activating PKA (Ferrani-Kile et al., 2003). Since acute ethanol exposure has been found to activate the cAMP-PKA signaling pathway and NMDA receptor function is regulated by PKA, we hypothesized that activation of the PKA pathway may be involved in acute tolerance to ethanol inhibition of NMDA-induced responses in the RVLM in vivo. An in vitro study has already demonstrated that activation of the dopamine D1 receptor was able to counteract ethanol inhibition of NMDA receptor function through activation of cAMP-PKA signals in nucleus accumbens neurons (Maldve et al., 2002). The disinhibition of ethanol action by activation of cAMP-PKA signals has been suggested to be related to the development of acute tolerance to ethanol (Lovinger, 2002). The present study was undertaken to test the hypothesis that the PKA pathway is involved in acute ethanol tolerance in the RVLM in vivo. In addition, since protein phosphorylation has been suggested to play a role in ethanol actions (Newton and Messing, 2006), the relationship between PKA activity and ethanol sensitivity of NMDA receptors was also examined.
Section snippets
Animals
A breeding colony of Sprague–Dawley rats purchased from the National Laboratory Animal Breeding and Research Center (Taipei, Taiwan) was established at the Laboratory Animal Center, Tzu Chi University. Adult male rats weighing 320–350 g were selected from the colony for use in the present study. All experimental procedures were carried out in accordance with the guidelines of the Institutional Animal Care and Use Committee of Tzu Chi University.
Determination of blood ethanol levels
Under urethane anesthesia (1.2–1.5 g per kg, i.p.),
The levels of phosphoserine 897 on the NR1 subunit in the rostroventral medulla during prolonged application of ethanol
Intravenous injection of a bolus of 0.16 g ethanol followed by continuous infusion at a constant rate of 0.16 g/h gradually increased the blood ethanol levels; the blood ethanol concentrations were 72.2 ± 3.9 mg/dl, 84.8 ± 6.6 mg/dl, and 101.4 ± 6.1 mg/dl (n = 6) at 10 min, 40 min, and 70 min, respectively, following continuous infusion of ethanol. The protein content of the NR1 subunit and the level of phosphoserine 897 on the NR1 subunit in the rostroventral part of the medulla before and at 10 min, 40 min,
Discussion
Our previous study found that in RVLM, ethanol selectively inhibited NMDA receptor activation and the development of acute tolerance during prolonged ethanol exposure both in vitro and in vivo (Lai et al., 2004). In the present study, we demonstrated that prolonged ethanol treatment increased the levels of phosphoserine 897 on the NMDA NR1 subunit in the rostroventral medulla. We further showed that PKA inhibitors applied after administration of ethanol reversed the acute tolerance to ethanol
Acknowledgements
The authors would like to thank Dr Ted Chiu (Department of Pharmacology, Tzu Chi University) for his critical reading of the manuscript. This study was supported by grants from National Science Council (NSC 93-2320-B-320-026 and NSC 94-2745-B-320-001), Taiwan.
References (30)
Ethanol sensitivity of NMDA receptors
Neurochemistry International
(2002)- et al.
NMDA receptor subunits: diversity, development and disease
Current Opinion in Neurobiology
(2001) - et al.
Acute ethanol affects phosphorylation state of the NMDA receptor complex: implication of tyrosine phosphatases and protein kinase A
Brain Research. Molecular Brain Research
(2003) - et al.
Beyond the dopamine receptor: the DARPP-32/protein phosphatase-1 cascade
Neuron
(1999) - et al.
Intracellular signaling pathways that regulate behavioral responses to ethanol
Pharmacology and Therapeutics
(2006) - et al.
Alcohols inhibit N-methyl-d-aspartate receptors via a site exposed to the extracellular environment
Neuropharmacology
(2000) - et al.
Beta-adrenergic regulation of synaptic NMDA receptors by cAMP-dependent protein kinase
Neuron
(1996) - et al.
A site of alcohol action in the fourth membrane-associated domain of the N-methyl-d-aspartate receptor
Journal of Biological Chemistry
(2003) - et al.
Glutamate receptor phosphorylation and synaptic plasticity
Current Opinion in Neurobiology
(1994) - et al.
Ethanol inhibition of N-methyl-d-aspartate receptors is reduced by site-directed mutagenesis of a transmembrane domain phenylalanine residue
Journal of Biological Chemistry
(2001)
Reduced hypothermic and hypnotic responses to ethanol in PACAP-deficient mice
Regulatory Peptides
Importance of glycinergic and glutamatergic synapses within the rostral ventrolateral medulla for blood pressure regulation in conscious rats
Hypertension
cAMP-dependent protein kinase mediates activity-regulated synaptic targeting of NMDA receptors
Journal of Neuroscience
DARPP-32: regulator of the efficacy of dopaminergic neurotransmission
Science
Suppression of ethanol-reinforced behavior by naltrexone is associated with attenuation of the ethanol-induced increase in dialysate dopamine levels in the nucleus accumbens
Journal of Neuroscience
Cited by (24)
Ethanol induces persistent potentiation of 5-HT<inf>3</inf> receptor-stimulated GABA release at synapses on rat hippocampal CA1 neurons
2021, NeuropharmacologyCitation Excerpt :It seems very likely that factors downstream of the receptor contribute to this potentiation. Several studies have reported that EtOH activates cAMP-PKA signaling in the CNS (Asher et al., 2002; Asyyed et al., 2006; Lin et al., 2006). The observation that a cell permeable PKA blocker prevents EtOH potentiation of 5-HT3-mediated GABA release indicates that cAMP/PKA signaling plays a role in this plasticity.
NADPH oxidase contributes to oxidative damage and mitochondrial impairment induced by acute ethanol treatment in rat hippocampal neurons
2020, NeuropharmacologyCitation Excerpt :Ethanol not only induces an increase in the production of ROS but also affects the balance between oxidant and antioxidant pathways (Ramachandran et al., 2003), leading to an abnormal cellular redox state. Interestingly, previous studies have revealed that ethanol affects mitochondrial function (Almansa et al., 2009; Lin et al., 2006; Reddy et al., 2011, 2013; Tapia-Rojas et al., 2018, 2019). Mitochondria are the powerhouse of cells, and studies in several ethanol administration models have not only shown a decrease in ATP production and mitochondrial depolarization, but also an increase in mitochondrial ROS production and a detriment of mitochondrial antioxidant defense capacity (Lin et al., 2006; Reddy et al., 2011, 2013; Tapia-Rojas et al., 2018, 2019).
Immunoreactivity for the NMDA NR1 subunit in bulbospinal catecholamine and serotonin neurons of rat ventral medulla
2013, Autonomic Neuroscience: Basic and ClinicalCitation Excerpt :Because we did not stain retrogradely labeled TH or TpTH neurons for NR2 or NR3 subunits, we cannot exclude the possibility that a group of neurons in the ventral medulla express the NR1 subunit but do not produce functional NMDA channels, as is the case for Purkinje cells of the cerebellum (Petralia et al., 1994). Nevertheless, many physiological studies have demonstrated the functional importance of NMDA receptors in the ventral medulla (Mao and Abdel-Rahman, 1995; Sun and Reis, 1995; Lin et al., 2006; Bardgett et al., 2010); and we have shown here that virtually all of the bulbospinal catecholamine and serotonin neurons in the ventral medulla express the NR1 subunit. Although we used a very sensitive three-layer immunofluorescent procedure to assess the presence of NR1, it is possible that some neurons contained undetectable levels of this NMDA receptor subunit.