Abstract
Alcohol and tobacco use is highly correlated in humans, and studies with animal models suggest an interaction of alcohol with neuronal nicotinic acetylcholine receptors (nAChRs). The aim of the present study was to characterize the effect of acute ethanol treatment on different combinations of human nAChR (hnAChR) subunits expressed inXenopus oocytes. Ethanol (75 mM) potentiated ACh-induced currents in α2β4, α4β4, α2β2, and α4β2 receptors. This effect was due to an increase in E max, without a change in the EC50 or Hill coefficient. hnAChR α2β4 did not develop tolerance to repeated applications of ethanol or continuous exposure (10 min). The α3β2 and α3β4combinations were insensitive to ethanol. Low concentrations of ethanol (25 and 50 mM) significantly inhibited homomeric α7receptor function, but these receptors showed highly variable responses to ethanol. These results indicate that ethanol effects on hnAChRs depend on the receptor subunit composition. In light of recent evidence indicating that nAChRs mediate and modulate synaptic transmission in the central nervous system, we postulate that acute intoxication might involve ethanol-induced alterations in the function of these receptors.
There is a high correlation between alcohol and tobacco use in humans (reviewed in Istvan and Matarazzo, 1984). Alcoholics are more likely to use tobacco than nonalcoholics, and alcoholic smokers consume more cigarettes per day than nonalcoholic smokers (reviewed in Istvan and Matarazzo, 1984; DiFranza and Guerrera, 1990). In laboratory animals, nicotine treatment increases ethanol consumption (Blomqvist et al., 1996). Mouse and rat lines were selectively bred for differences in ethanol sensitivity, and it was found that animals that have high sensitivity to ethanol also have high sensitivity to nicotine (De Fiebre et al., 1987, 1990, 1991). Recently, Collins et al. (1996)suggested that cross-tolerance between nicotine and ethanol in laboratory animals might involve a change in neuronal nicotinic acetylcholine receptor (nAChR) function. The studies mentioned above suggest that common gene products are involved in the interaction between ethanol and nicotine, and there is considerable interest in determining whether this interaction is mediated, at least in part, by nAChRs.
nAChRs are members of a superfamily of ligand-gated ion channels. A gene family of 11 nAChR subunits (α2–9, β2–4) has been identified (McGehee and Role, 1995). Receptors containing α2–6 subunits are usually expressed as heteromers in combination with β2–4 subunits (reviewed in Role and Berg, 1996). The majority of heteromeric neuronal nicotinic binding sites expressed in the central nervous system possess the α4β2 or α4α5β2subunit combinations (Flores et al., 1992; Zoli et al., 1998). Recent experiments with β2 mutant mice suggest that α2β2 and α3β2 receptors are expressed in the interpeduncular nucleus and the hippocampus (Zoli et al., 1998). The α3β4 or α3α5β4subunit combinations are predominantly expressed in the medial habenula, interpeduncular nucleus, and dorsal medula (Zoli et al., 1998). The α4β4 or α2β4 subunit combinations are predominantly expressed in the lateral medial habenula and dorsal interpeduncular nucleus, respectively (Zoli et al., 1998). The α7, α8, and α9 subunits can form homomeric receptors (reviewed in Role and Berg, 1996). nAChRs receptors containing α7 subunits are the major binding sites for α-bungarotoxin (α-BGT) in the mammalian central nervous system and are predominantly expressed in the cortex and limbic areas (Orr-Urtreger et al., 1997; Zoli et al., 1998).
The acute effects of ethanol on nAChRs have been studied previously with conflicting results. It was recently reported that ethanol (5–100 mM) inhibits the function of α7 nAChRs expressed in Xenopus oocytes by a mechanism that involves the amino-terminal domain of the receptor (Yu et al., 1996). In rat pheochromocytoma (PC12) cells, continuous bath application of low concentrations of ethanol (0.1–10 mM) produced an increase in desensitization of nAChR responses, with variable changes in the current peak amplitude (Nagata et al., 1996). Recently, Covernton and Connolly (1997) showed that different combinations of rat nAChRs subunits expressed in Xenopus oocytes are sensitive to acute exposure to ethanol under some experimental conditions. The aim of the present study was to characterize the effect of acute ethanol treatment on the recently characterized human nAChRs (hnAChR; Chavez-Noriega et al., 1997) and to assess the effects of ethanol on receptors with different subunit compositions.
Materials and Methods
Materials.
Xenopus laevis female frogs were purchased from Xenopus I (Ann Arbor, MI) or Nasco (Fort Atkinson, WI). Acetylcholine chloride, atropine sulfate, collagenase type 1A, streptomycin/penicillin, gentamicin, and other reagents were purchased from Sigma Chemical Co. (St. Louis, MO). Ethanol was from Aaper Alcohol and Chemical (Shelbyville, KY). XL-1 Blue cells were from Stratagene (La Jolla, CA). The QIAFilter Maxi kit was from Qiagen (Chatworth, CA), and the mCAP mRNA capping kit was from Stratagene (La Jolla, CA).
cDNA and cRNA Preparation.
The hnAChR subunits α2, α3, α4, α7, β2, and β4 were cloned from cDNA libraries prepared from human brain and the human IMR32 neuroblastoma cell line and subcloned into different expression vectors, α2 and α3 in pCMV-T7 to 3; α4, α7, and β4 in pcDNA3; and β2 in pSP64T (Elliott et al., 1996). XL-1 Blue cells were transformed with the cDNAs, and amplified plasmid was purified with the QIAFilter Maxi kit. In vitro transcripts were prepared using the mRNA capping kit.
Eletrophysiological Recording of XenopusOocytes.
Isolation and injection of Xenopus laevisoocytes were performed as described previously by Mascia et al. (1996). Oocytes were injected with 40 nl of diethyl pyrocarbonate-treated water containing 20 to 100 ng of αxβy subunit combinations of cRNA in a 1:1 ratio or 50 ng of α7 cRNA.
For electrophysiological recording, oocytes were placed in a rectangular chamber (∼100 μl) and perfused (2 ml/min) with buffer ND96 (96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, and 10 mM HEPES, pH 7.4) containing 1 μM atropine sulfate. Oocytes were impaled with two glass electrodes (0.5–10 MΩ) filled with 3 M KCl and clamped at −70 mV using a Warner Instruments (Hamden, CT) oocyte clamp (model OC-725C).
Agonist (acetylcholine or nicotine) was applied for 20 s at 5-min intervals. Unless indicated otherwise, ethanol was preapplied for 2 min to allow complete equilibration in the bath and then immediately coapplied with agonist for 20 s. All solutions were prepared on the day of the experiment.
Statistical Analysis.
Results are expressed as percentages of change from control responses, which were measured before and after each ethanol application to take into account possible shifts in the control current throughout the experiment. The “n” values refer to number of oocytes studied. Each experiment was carried out with oocytes from at least two different frogs. Effects of ethanol were analyzed by either one-sample Student’s t test (against a theoretical mean of zero) or one-way ANOVA. Statistical analysis and curve fitting were performed using GraphPad Prism software (San Diego, CA). To analyze dose/response curves, a four-parameter logistic equation (sigmoid) with variable slope was used (Y = Bottom + (Top − Bottom)/(1+ 10((Log EC50-X) · (Hill Slope))), where X is the logarithm of concentration, and Y is the response). The best fit for the data was obtained without fixing any of the parameters in this equation. Data are presented as mean ± S.E.M.
Results
Effects of Ethanol on hnAChRs Are Reproducible and Reversible.
Our first goal was to evaluate whether the effects of ethanol were reproducible and reversible. Concentration-response curves for acetylcholine were obtained for the different hnAChR subunit combinations. Currents induced by an EC30 dose of ACh in α2β4 were equally potentiated after 2, 5, and 10 min of preincubation with 75 mM ethanol, and essentially the same level of potentiation was obtained after washout and re-exposure to ethanol (Fig.1; ANOVA, p > .05).
Effects of Ethanol Depend on the hnAChR Subunit Composition.
The effects of 75 mM ethanol on ACh concentration curves in oocytes expressing six different combinations of hnAChR subunits are shown in Figs.2–4 and Table 1. Ethanol potentiated ACh-gated currents in α2β2 and α4β2, and this was independent of the concentration of ACh (ANOVA, p > .05), and characterized by an increase in theE max without change in the EC50 or Hill coefficient. Ethanol also induced a significant increase in the E max of α2β4 and α4β4, and did not change the EC50 or Hill coefficient for ACh. The potentiation of agonist responses produced by ethanol in both of these receptor configurations was inversely proportional to ACh concentration (ANOVA, p ≤ .001 and p ≤ .05, respectively).
Receptors formed by the α3β2 combination of hnAChR subunits were insensitive to ethanol, as well as α3β4 receptors at submaximal agonist concentrations. A small but statistically significant decrease in E max induced by 75 mM ethanol (−13.8 ± 3.9%) was observed with α3β4.
Potentiation by Ethanol of α2β4 and α4β2 hnAChRs Is Concentration Dependent.
To study the ethanol concentration-response relationship, we chose α4β2 and α3β4 as nAChR subtype combinations that are thought to be abundantly expressed in the central nervous system and peripheral nervous system (McGehee and Role, 1995), and differ in ethanol sensitivity (present results). We also included the α2β4 combination, because it allowed us to determine whether the α or β subunit was critical for the alcohol resistance of the α3β4 receptor. To evaluate the concentration-response effect of ethanol on hnAChRs, we used an ACh concentration corresponding to the EC30 for each receptor, which was 23 μM for α2β4, 0.5 μM for α4β2, and 127 μM for α3β4. ACh-induced currents in α2β4 and α4β2 were potentiated in a concentration-dependent manner by ethanol. The lowest ethanol concentration that produced a statistically significant potentiation was 50 mM for both subunit combinations. In contrast, α3β4 receptors were insensitive to all concentrations of ethanol tested under these recording conditions (Fig. 5)
Low Concentrations of Ethanol Inhibit Homomeric α7hnAChRs.
We also tested different concentrations of ethanol (25–200 mM) on the nicotine-gated currents mediated by homomeric hnAChR α7. These receptors presented a highly variable response to ethanol, with changes ranging from 43% inhibition to 17% potentiation at 25 mM ethanol and from 75% inhibition to 40% potentiation at 200 mM ethanol. Despite the large variability in the responses, statistical analysis of average effects showed that low concentrations of ethanol significantly inhibited these receptors (25 mM: −17 ± 6% and 50 mM: −29 ± 6%; Student’st test, p ≤ .05), but higher concentrations did not alter its function (Fig. 6).
Discussion
Our results demonstrate that recombinant hnAChRs with distinct subunit configurations are differentially affected by pharmacologically relevant doses of ethanol (concentrations ≤ 100 mM; Deitrich and Harris, 1996). The α2β4and α4β2 combinations were the most sensitive receptors to potentiation by ethanol, and the α4β4 and α2β2 combinations were slightly less sensitive. Interestingly, receptors containing the α3 subunit plus either the β2 or β4 subunits were insensitive to ethanol. Homomeric α7 receptors displayed highly variable responses to ethanol, and the average effect of ethanol was inhibitory at low ethanol concentrations. From these results, we conclude that the sensitivity to ethanol of hnAChRs depends on the subunit composition of the receptors expressed.
The effects of ethanol on rat nAChR were previously suggested to be dependent on the subunit composition of the receptor (Covernton and Connolly, 1997). It was found that the α3β4 combination of rat nAChR had highly variable responses (ranging from potentiation to inhibition) to low concentrations of ethanol. The authors also reported that the α7, α3β2, α4β2, and α4β4 receptors were less sensitive to low concentrations of ethanol than α3β4 receptors. We obtained similar results in our initial studies (data not shown). However, variability was minimized, and results were different (see above) than those of Covernton and Connolly (1997) when ethanol was preapplied. We decided to preapply ethanol because, in vivo, the receptors are not only exposed to ethanol at the time they are activated by acetylcholine, but they would be expected to be in contact with ethanol before activation. Covernton and Connolly (1997) found that α3β4 receptors developed tolerance to repeated exposure to ethanol, which was not seen in our experiments. These differences might be due to either experimental protocol or species differences. Indeed, Chavez-Noriega et al. (1997) showed that human clones of nAChR have distinct pharmacological profiles compared with clones from other species. It should be noted that we obtained similar results as Covernton and Connolly (1997) with rat α7 receptors, which were modulated in a variable manner by ethanol. Interestingly, we found that the average effect of low concentrations of ethanol was inhibitory, which is in agreement with the findings of another study (Yu et al., 1996).
The differential sensitivity of hnAChRs with different subunit compositions to ethanol may be the basis for the observations that ethanol potentiates the excitatory responses to nicotine in the substantia nigra reticulata and ventral pallidum (Criswell et al., 1993) but inhibits them in the locus ceruleus (Fröhlich et al., 1994). The differential sensitivity of distinct nAChR configurations to ethanol might also explain why ethanol inhibits nAChR-dependent firing of Purkinje neurons (Freund and Palmer, 1997). Unfortunately, the precise subunit composition of functional nAChRs present in those regions has not yet been elucidated. Also, it is not clear whether alcohol acts on pre- or postsynaptic receptors, or both.
It is interesting to compare the magnitude of the acute effects of ethanol on nAChRs versus its effects on other members of this superfamily of ligand-gated ion channels studied previously inXenopus oocytes. Homomeric glycine α1 receptors were potentiated by 25 to 200 mM ethanol by 20 to 110% at glycine EC2concentrations (Mascia et al., 1996), whereas GABAA receptors with different subunit compositions were potentiated by 0 to 60% at submaximal agonist concentrations (Mihic et al., 1994, 1997; Harris et al., 1997). 5-HT3 receptors were potentiated by 25 to 200 mM ethanol by 25 to 40% at submaximal agonist concentrations (Machu and Harris, 1994). A striking difference is that ethanol alters the function of glycine, GABAA, and 5-HT3 receptors by changing the neurotransmitter EC50 with no change in theE max (Machu and Harris,1994; Mihic et al., 1994; Mascia et al., 1996). Thus, ethanol appears to have a rather unique action on hnAChRs dose/response curves when compared with other members of this family of ligand-gated ion channels.
Significance.
A number of recent studies found that α-BGT-sensitive as well as α-BGT-insensitive nAChRs modulate neurotransmitter release from presynaptic terminals (reviewed in Role and Berg, 1996). Therefore, it is possible that the effects of ethanol on presynaptic nAChRs result in alterations in the function of pathways involving a number of different neurotransmitters. For instance, nicotine enhances glutamatergic, cholinergic, and monoaminergic synaptic transmission by activation of presynaptic nAChRs that contain, at least in part, the α7 subunit (McGehee et al., 1995; Gray et al., 1996; Guo et al., 1998; Li et al., 1998). Experiments with α-conotoxin MII, which is a selective inhibitor of α3β2 nAChRs, suggest that these receptors mediate nicotine-stimulated dopamine release in rat striatal synaptosomes (Kulak et al., 1997). Experiments with α-conotoxin AuIB, which is a selective blocker of α3β4 nAChRs, suggest that these receptors mediate nicotine-induced release of norepinephrine in rat hippocampal synaptosomes (Luo et al., 1998). In our studies, the α3β2 and α3β4 receptor configurations were insensitive to ethanol, which suggests that ethanol does not act by modulating neurotransmitter release mediated by these receptors. However, ethanol could affect neurotransmitter release mediated by α7 receptors, which were inhibited by it at low concentrations. Ethanol could also enhance neurotransmitter release controlled by α4β2 nAChRs; Alkondon et al. (1997) recently postulated that activation of α4β2 receptors might induce GABA release from hippocampal interneurons. A similar finding was reported by Yang et al. (1996) in the rat medial septum.
Finally, it is interesting that most, if not all, drugs of abuse stimulate dopamine neurotransmission in the mesolimbic system, including nicotine and alcohol (Pontieri et al., 1996; Blomqvist et al., 1997). It has been suggested that the stimulation of dopaminergic transmission in the mesolimbic system by ethanol involves the activation of nAChRs in the ventral tegmental area; unfortunately, the subunit composition of mesolimbic nAChRs has not yet been addressed (Blomqvist et al., 1996, 1997). A better understanding of the regional distribution of different subunit combinations of nAChRs as well as of molecular interactions between ethanol and nAChRs might provide targets for the development of pharmacological tools to control alcohol and tobacco addictions.
Footnotes
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Send reprint requests to: R. Adron Harris, PhD, Institute for Cellular and Molecular Biology, University of Texas at Austin, 2500 Speedway, Austin, TX 78712-1095.
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↵1 Supported by NIH Grants K01-AA00227 (C.F.V.), AA06399 (R.A.H.), AA03527 (R.A.H.).
- Abbreviations:
- nAChR
- neuronal nicotinic acetylcholine receptor
- BGT
- bungarotoxin
- GABA
- γ-aminobutyric acid
- 5-HT
- 5-hydroxytryptamine
- ACh
- acetylcholine
- Nic
- nicotine
- HEPES
- 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid
- EtOH
- ethanol
- Received October 1, 1998.
- Accepted December 30, 1998.
- U.S. Government