Introduction

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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 (_2-9, _2-4) has been identified (McGehee and Role, 1995). Receptors containing _2-6 subunits are usually expressed as heteromers in combination with _2-4 subunits (reviewed in Role and Berg, 1996). The majority of heteromeric neuronal nicotinic binding sites expressed in the central nervous system possess the ₄₂ or ₄₅₂ subunit combinations (Flores et al., 1992; Zoli et al., 1998). Recent experiments with ₂ mutant mice suggest that ₂₂ and ₃₂ receptors are expressed in the interpeduncular nucleus and the hippocampus (Zoli et al., 1998). The ₃₄ or ₃₅₄ subunit combinations are predominantly expressed in the medial habenula, interpeduncular nucleus, and dorsal medula (Zoli et al., 1998). The ₄₄ or ₂₄ subunit combinations are predominantly expressed in the lateral medial habenula and dorsal interpeduncular nucleus, respectively (Zoli et al., 1998). The ₇, ₈, and ₉ subunits can form homomeric receptors (reviewed in Role and Berg, 1996). nAChRs receptors containing ₇ subunits are the major binding sites for -bungarotoxin (-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 ₇ 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

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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 ₂, ₃, _{4, 7}, ₂, and ₄ were cloned from cDNA libraries prepared from human brain and the human IMR32 neuroblastoma cell line and subcloned into different expression vectors, ₂ and ₃ in pCMV-T7 to 3; ₄, ₇, and ₄ in pcDNA3; and ₂ 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 Xenopus Oocytes. Isolation and injection of Xenopus laevis oocytes 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 _xy subunit combinations of cRNA in a 1:1 ratio or 50 ng of ₇ cRNA.

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

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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 ₂₄ 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).

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Currents induced by an EC30 ACh in 24 are equally potentiated after 2, 5, and 10 min of preincubation with 75 mM ethanol, and the same level of potentiation is obtained after washout and re-exposure to ethanol. Top panel, representative current tracing obtained from a single Xenopus oocyte. The bars represent the period of application of drugs. In the bottom panel, ethanol was preapplied for 2, 5, and 10 min before coapplication for 20 s with ACh (23 µM). Control responses to ACh were measured 5 min before and after ethanol. After that, oocytes were subjected to 5 min of washout and 2 min of preincubation with ethanol, and they were re-exposed to ACh+EtOH. After 5 min, a new ACh control response was determined. Values are mean ± S.E.M. from four to six oocytes.

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 ₂₂ and ₄₂, and this was independent of the concentration of ACh (ANOVA, p > .05), and characterized by an increase in the E_max without change in the EC₅₀ or Hill coefficient. Ethanol also induced a significant increase in the E_max of ₂₄ and _44, 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).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Ethanol selectively potentiates some hnAChR subunit combinations. Representative tracings of ACh-induced currents before and after preincubation with 75 mM ethanol. Oocytes were stimulated with ACh for 20 s, followed by 5 min of wash, then 2 min of preincubation with EtOH, which was immediately coapplied with ACh and once more washed for 5 min and re-exposed to ACh. Note that the effect of ethanol in all receptors was reversible. Bars represent period of treatment with drugs.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Effect of 75 mM ethanol in the ACh concentration-response curve in Xenopus oocytes expressing 22 (top), 32 (middle), and 42 (bottom). Drug application protocols are the same as in Fig. 2. ACh concentration-response curves were fitted using GraphPad Prism software, as described in Materials and Methods. Values are mean ± S.E.M. from five to seven oocytes. Shown in the inset is the percentage of potentiation (mean ± S.E.M.) produced by ethanol as a function of ACh concentration. Error bars not visible are smaller than symbols.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Effect of 75 mM ethanol on the ACh concentration-response curve in Xenopus oocytes expressing 24 (top), 34 (middle), and 44 (bottom). Drug application protocols are the same as in Fig. 2. ACh concentration-response curves were fitted using GraphPad Prism software, as described in Materials and Methods. Values are mean ± S.E.M. from 6 to 12 oocytes. Shown in the inset is the percentage of potentiation (mean ± S.E.M.) produced by ethanol as a function of ACh concentration. Error bars not visible are smaller than symbols.

Potentiation by Ethanol of ₂₄ and ₄₂ hnAChRs Is Concentration Dependent. To study the ethanol concentration-response relationship, we chose ₄₂ and ₃₄ 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 ₂₄ combination, because it allowed us to determine whether the  or  subunit was critical for the alcohol resistance of the ₃₄ 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 ₂₄, 0.5 µM for ₄₂, and 127 µM for ₃₄. ACh-induced currents in ₂₄ and ₄₂ 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, ₃₄ receptors were insensitive to all concentrations of ethanol tested under these recording conditions (Fig. 5)

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Effect of different concentrations of ethanol on ACh-induced currents in oocytes expressing 24, 34, and 42. Ethanol was applied for 2 min before coapplication with ACh for 20 s. An EC30 dose of ACh was used for each receptor: 23 µM for 24, 127 µM for 34, and 0.5 µM for 42. Values are mean ± S.E.M. from 7 to 11 oocytes. Error bars not visible are smaller than symbols. *, lowest concentration of ethanol with a significant (p  .05) effect on 24 and 42.

Low Concentrations of Ethanol Inhibit Homomeric ₇ hnAChRs. We also tested different concentrations of ethanol (25-200 mM) on the nicotine-gated currents mediated by homomeric hnAChR ₇. 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's t test, p  .05), but higher concentrations did not alter its function (Fig. 6).

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Effect of different concentrations of ethanol on nicotine-induced currents in oocytes expressing hnAChR 7. Nicotine (Nic) was applied for 20 s, and after 5 min of washout, oocytes were pre-exposed to 75 mM EtOH for 2 min before coapplication of Nic+EtOH. The nicotine concentration used was the EC30 (32 µM) for 7. Top, each point represents data from a single oocyte. Bottom, values are mean ± S.E.M. from 8 to 14 oocytes. *p  .05, Student's t test.

	Discussion

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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 ₂₄ and ₄₂ combinations were the most sensitive receptors to potentiation by ethanol, and the ₄₄ and ₂₂ combinations were slightly less sensitive. Interestingly, receptors containing the ₃ subunit plus either the ₂ or ₄ subunits were insensitive to ethanol. Homomeric ₇ 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 ₃₄ combination of rat nAChR had highly variable responses (ranging from potentiation to inhibition) to low concentrations of ethanol. The authors also reported that the ₇, ₃₂, ₄₂, and ₄₄ receptors were less sensitive to low concentrations of ethanol than ₃₄ 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 ₃₄ 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 ₇ 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 in Xenopus oocytes. Homomeric glycine ₁ receptors were potentiated by 25 to 200 mM ethanol by 20 to 110% at glycine EC₂ concentrations (Mascia et al., 1996), whereas GABA_A 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, GABA_A, and 5-HT₃ receptors by changing the neurotransmitter EC₅₀ with no change in the E_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 -BGT-sensitive as well as -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 ₇ subunit (McGehee et al., 1995; Gray et al., 1996; Guo et al., 1998; Li et al., 1998). Experiments with -conotoxin MII, which is a selective inhibitor of ₃₂ nAChRs, suggest that these receptors mediate nicotine-stimulated dopamine release in rat striatal synaptosomes (Kulak et al., 1997). Experiments with -conotoxin AuIB, which is a selective blocker of ₃₄ nAChRs, suggest that these receptors mediate nicotine-induced release of norepinephrine in rat hippocampal synaptosomes (Luo et al., 1998). In our studies, the ₃₂ and ₃₄ 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 ₇ receptors, which were inhibited by it at low concentrations. Ethanol could also enhance neurotransmitter release controlled by ₄₂ nAChRs; Alkondon et al. (1997) recently postulated that activation of ₄₂ receptors might induce GABA release from hippocampal interneurons. A similar finding was reported by Yang et al. (1996) in the rat medial septum.