Elsevier

Neuropharmacology

Volume 51, Issue 4, September 2006, Pages 747-755
Neuropharmacology

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

https://doi.org/10.1016/j.neuropharm.2006.05.018Get rights and content

Abstract

Our recent study showed that intravenous ethanol selectively inhibited the pressor effects elicited by the microinjection of N-methyl-d-aspartate (NMDA) into rostral ventrolateral medulla (RVLM) and acute tolerance to the inhibition was observed during prolonged application of ethanol in anesthetized Sprague–Dawley rats. In this study, we examined the role of the cAMP-dependent protein kinase (PKA) signaling pathway in acute tolerance to ethanol inhibition of NMDA-induced responses in rat RVLM. A significant increase in the level of PKA-regulated phosphoserine 897 on the NMDA NR1 subunit was found in the rostroventral medulla during acute ethanol tolerance. Reduction of NMDA-induced pressor effects was observed at 10 min but disappeared at 40 min after continuous ethanol infusion. This effect was dose-dependently blocked by microinjection of KT5720 (0.04–4 pmol, a selective PKA inhibitor) or cAMPS-Rp (0.02, 0.2 pmol, a cAMP antagonist) into the RVLM 10 min post-injection of ethanol; KT 5720 or cAMPS-Rp alone at doses tested had no significant effects on NMDA-induced responses. Post-treatment with cAMPS-Sp (10 pmol, a cAMP activator) did not affect acute ethanol tolerance. Interestingly, administration of KT 5720 (0.4, 4 pmol) or cAMPS-Rp (2,10 pmol) into the RVLM 20 min before the injection of ethanol also reduced the inhibitory effects of ethanol on NMDA-induced pressor effects in a dose-dependent manner. Our results provide the first in vivo evidence that PKA signaling pathways participate in acute tolerance to ethanol inhibition of NMDA receptor function. Furthermore, PKA-mediated signaling pathways may also be involved in the interaction between ethanol and NMDA receptors.

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.

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