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NEUROPHARMACOLOGY

Additive Effects of Endogenous Cannabinoid Anandamide and Ethanol on ₇-Nicotinic Acetylcholine Receptor-Mediated Responses in Xenopus Oocytes

National Institute on Drug Abuse, National Institutes of Health/Department of Health and Human Services, Intramural Research Program, Cellular Neurobiology Branch, Baltimore, Maryland (M.O., S.N.J., A.S.W., M.M.); and National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health/Department of Health and Human Services, Laboratory of Molecular and Cellular Neurobiology, Bethesda, Maryland (L.Z.)

Received November 23, 2004; accepted January 31, 2005.

	Abstract

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The interaction between the effects of the endogenous cannabinoid receptor agonist anandamide and ethanol on the function of homomeric ₇-nicotinic acetylcholine (nACh) receptors expressed in Xenopus oocytes were investigated using the two-electrode voltage-clamp technique. Anandamide and ethanol reversibly inhibited currents evoked with 100 µM acetylcholine in a concentration-dependent manner. Coapplication of anandamide and ethanol caused a significantly greater inhibition of ₇-nACh receptor function than anandamide or ethanol alone. The IC₅₀ value of 238 ± 34 nM for anandamide inhibition decreased significantly to 104 ± 23 nM in the presence of 30 mM ethanol. The inhibition of ₇-mediated currents by coapplication of anandamide and ethanol was not altered by phenylmethylsulfonyl fluoride, an inhibitor of anandamide hydrolyzing enzyme, or N-(4-hydroxyphenyl)-arachidonylamide, an anandamide transport inhibitor. Analysis of oocytes by matrix-assisted laser desorption/ionization technique indicated that ethanol treatment did not alter the lipid profile of oocytes, and there is negligible, if any, anandamide present in these cells. Results of studies with chimeric ₇-nACh-5-HT₃ receptors comprised of the amino-terminal domain of the ₇-nACh receptor and the transmembrane and carboxyl-terminal domains of 5-HT₃ receptors suggest that although ethanol inhibition of the ₇-nACh receptor is likely to involve the N-terminal region of the receptor, the site of action for anandamide is located in the transmembrane and carboxyl-terminal domains of the receptors. These data indicate that endocannabinoids and ethanol potentiate each other's inhibitory effects on ₇-nACh receptor function through distinct regions of the receptor.

However, increased levels of endocannabinoids by ethanol can also facilitate the interaction of these molecules with ethanol on common target proteins other than cannabinoid receptors. Several reports indicate that endocannabinoids produce effects that are not mediated by the activation of the cloned CB₁ and/or CB₂ receptors. For example, it was demonstrated that endocannabinoids such as anandamide can inhibit the function of voltage-dependent Ca²⁺ channels (Oz et al., 2000, 2004b; Chemin et al., 2001), Na⁺ channels (Nicholson et al., 2003), various types of K⁺ channels (Poling et al., 1996; Maingret et al., 2001), 5-HT₃ receptor function (Barann et al., 2002; Oz et al., 2002b; Godlewski et al., 2003), and nicotinic ACh receptors (Oz et al., 2003a, 2004c), suggesting that additional molecular targets for endocannabinoids exist in the central nervous system (for recent review, see Oz, 2005). Similar to endocannabinoids, ethanol also interacts directly with voltage-dependent Ca²⁺ channels (Oz et al., 2001, 2002a), Na⁺ channels (Oz and Frank, 1995; Shiraishi and Harris, 2004), K⁺ channels (Lewohl et al., 1999; Oz et al., 2003a), 5-HT₃ receptors (Lovinger and Zhou, 1998), and nicotinic ACh receptors (Yu et al., 1996; Cardoso et al., 1999).

Nicotinic acetylcholine (nACh) receptors containing the ₇ subunit belong to the ligand-gated ion channel super family (Lindstrom et al., 1996). The potential involvement of these receptors in pharmacological actions of ethanol has been reported in several earlier studies (for review, see Narahashi et al., 1999). Since both ethanol and endocannabinoid anandamide have been shown to modulate the function of ₇-nACh receptors, the present study was performed to investigate if there is an interaction between direct effects of anandamide and ethanol on the functional properties of ₇-nACh receptors. We found that anandamide and ethanol cause an additive inhibition on the function of ₇-nACh receptor by interacting with distinct regions of the receptor.

	Materials and Methods

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Mature female Xenopus laevis frogs were purchased from Xenopus I (Ann Arbor, MI) and were housed in dechlorinated tap water at 19 to 21°C with a 12-h light/dark cycle and fed with beef liver twice a week. Clusters of oocytes were removed surgically under tricaine (Sigma-Aldrich, St. Louis, MO) local anesthesia (0.15% w/v), and individual oocytes were dissected away manually in a solution containing 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO₃, 0.8 mM MgSO₄, and 10 mM HEPES, pH 7.5. Later, dissected oocytes were stored 2 to 7 days in modified Barth's solution (MBS) containing 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO₃, 0.3 mM Ca(NO₃)₂, 0.9 mM CaCl₂, 0.8 mM MgSO₄, and 10 mM HEPES, pH 7.5, supplemented with 2 mM sodium pyruvate, 10,000 IU/l penicillin, 10 mg/l streptomycin, 50 mg/l gentamicin, and 0.5 mM theophylline. The oocytes were placed in a 0.2-ml recording chamber and superfused at a constant rate of 3 to 5 ml/min. The bathing solution consisted of 95 mM NaCl, 2 mM KCl, 2 mM CaCl₂, and 5 mM HEPES, pH 7.5. The cells were impaled at the animal pole with two standard glass microelectrodes filled with 3 M KCl (1-10 M). The oocytes were voltage-clamped routinely at a holding potential of -70 mV using GeneClamp-500 amplifier (Axon Instruments Inc., Union City, CA), and current responses were recorded directly on a Gould 2400 rectilinear pen recorder (Gould Instrument Systems Inc., Cleveland, OH). Current-voltage curves were generated by holding each membrane potential in a series for 30 s, followed by a return to -70 mV for 10 min. Oocyte capacitance was measured by a paired-ramp method described earlier (Oz et al., 2004a). Briefly, voltage ramps were employed to elicit constant capacitive current (I_cap), and the charge associated with this current was calculated by the integration of I_cap. Ramps had slopes of 2 V/s and durations of 20 ms and started at a holding potential of -90 mV. A series of 10 paired ramps was delivered at 1-s intervals, and averaged traces were used for charge calculations. In each oocyte, the averages of five to six measurements were used to obtain values for membrane capacitance (C_m). Currents for I_cap recordings were filtered at 20 kHz and sampled at 50 kHz.

Compounds were applied externally by addition to the superfusate. All chemicals used in preparing the solutions were from Sigma-Aldrich. Anandamide, (-)-nicotine, AM404, and -bungarotoxin were from Sigma/RBI (Natick, MA). Procedures for the injections of BAPTA (50-100 nl, 100 mM) were performed as described previously (Oz et al., 1998). BAPTA was prepared in Cs₄-BAPTA. Injections were performed 1 h prior to recordings using an oil-driven ultra microsyringe pump (Micro4; WPI, Sarasota, FL). Stock solutions of anandamide were prepared in dimethylsulfoxide at a concentration of 100 mM. Dimethyl sulfoxide alone did not affect nicotinic receptors when added at concentrations up 0.3% (v/v) in MBS solutions, a concentration twice as high as that resulting from the most concentrated application of the agents used.

Synthesis of cRNA and Chimeric Construct. The cDNA clones of the chick nACh₇ subunit and 5-HT_3A subunit were provided by Dr. Jon Lindstrom (University of Pennsylvania, Philadelphia, PA) and Dr. David Julies (University of California, San Francisco, CA), respectively. Capped cRNA transcripts were synthesized in vitro using a mMESSAGE mMACHINE kit from Ambion (Austin, TX) and analyzed on 1.2% formaldehyde agarose gel to check the size and the quality of the transcripts. The chimeric ₇-nACh-5-HT_3A receptor was constructed as described previously (Eisele et al., 1993; Yu et al., 1996).

Anandamide Analysis. A voyager De-Pro matrix-assisted laser desorption/ionization (MALDI) time-of-flight instrument (Applied Biosystems, Foster City, CA) was used in this study for MALDI analysis of Xenopus oocytes. All mass spectra were acquired in positive ion mode and are the sum of 100 laser shots. Samples were prepared from eight to nine oocytes in controls (in MBS solution) or in 100 mM ethanol-containing MBS solutions. The individual oocytes were then fractured and spread across the target surface. The 2,5-dihydroxybenzoic acid matrix was deposited directly onto the sample prior to insertion into the mass spectrometer. 2,5-Dihydroxybenzoic acid was prepared in a 50:50 ethanol/water solution. Anandamide was used as a standard and was prepared a 3 mM in ethanol and was subsequently diluted in distilled water.

Data Analysis. Average values were calculated as mean ± S.E. Statistical significance was analyzed using Student's t test or ANOVA as indicated. Concentration-response curves were obtained by fitting the data to the logistic equation:

where x and y are concentration and response, respectively, E_max is the maximal response, EC₅₀ is the half-maximal concentration, and n is the slope factor (apparent Hill coefficient).

	Results

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Xenopus oocytes injected with distilled water (n = 4) did not demonstrate ion currents when 1 to 3 mM ACh in the presence of 1 µM atropine was applied. In oocytes injected with ₇-nAChR mRNA, a 4- to 5-s application of ACh activated a fast inward current that desensitized rapidly. These ACh-induced inward currents were elicited at 10-min intervals to avoid receptor desensitization and were irreversibly abolished by 10 nM -bungarotoxin (n = 3; data not shown), indicating that these responses were mediated by neuronal ₇-nACh receptor-ion channels.

In earlier studies, ethanol and anandamide were shown to release intracellular Ca²⁺ in endothelial cells and Xenopus oocytes (Wafford et al., 1989; Howlett and Mukhopadhyay, 2000). In the oocyte expression system, the increased level of intracellular Ca²⁺ can be detected by Ca²⁺-activated Cl^- channels and concomitant alterations in the membrane input resistance. For this reason, we examined the effects of ethanol and anandamide coapplication on membrane resistance, C_m, and resting membrane potential in oocytes injected with ₇-nACh receptor mRNA. In control and in the presence of anandamide together with ethanol, the values for the means of membrane resistance, C_m, and resting membrane potential values were 1.1 ± 0.3 and 1.0 ± 0.2 M, 194 ± 17 and 201 ± 18 nF, and -34.3 ± 2.9 and -31.7 ± 3.2 mV, respectively (n = 8-9; ANOVA, P > 0.05). Thus, the data indicate that coapplication of 300 nM anandamide and 100 mM ethanol did not cause a significant effect on passive membrane properties of oocytes.

In previous studies, we showed that anandamide alone and ethanol alone inhibited the function of ₇-nAChRs expressed in Xenopus oocytes with IC₅₀ values of 229 nM (Oz et al., 2003a) and 58 mM (Yu et al., 1996), respectively. In the present study, we investigated the interaction between the inhibitory effects of anandamide and ethanol by their coapplication to oocytes. Application of 30 mM ethanol for 10 min inhibited ACh (100 µM)-induced ion currents in oocytes expressing ₇-nACh receptors (first and second traces from left; Fig. 1A). Perfusion of oocytes for 20 min with extracellular solution containing 30 mM ethanol and 100 nM anandamide caused a further inhibition of ACh-induced currents (third trace from left; Fig. 1A). Following a 30-min recovery, 20-min applications of 100 nM anandamide alone caused a significant inhibition of ACh-induced currents in the same oocyte (fourth and fifth traces from left; Fig. 1A). Results of experiments demonstrating the time courses of the effects of anandamide, ethanol, and anandamide + ethanol applications on the mean amplitudes of the ACh-induced currents (normalized to current amplitudes induced by 100 µM ACh) from four to five oocytes are presented in Fig. 1B. Results summarizing the effects of ethanol, anandamide, and ethanol + anandamide on ACh-induced responses are demonstrated in Fig. 1C.

View larger version (18K): [in this window] [in a new window]   Fig. 1. The effects of anandamide, ethanol, and anandamide + ethanol on 7-nACh receptor-mediated ion currents. A, currents activated by 100 µM ACh and recorded from the same oocyte in control extracellular media (first trace from the left), at 10 min after bath application of 30 mM ethanol (second trace from the left), and 20 min after coapplication of 30 mM ethanol and 100 nM anandamide (third trace from the left), following 30 min of recovery (fourth trace from the left), and after a 20-min application of 100 nM anandamide alone (last trace from the left). B, time courses of the effects of anandamide, ethanol, and anandamide + ethanol on the peak ACh-induced currents. Each data point represents the normalized means ± S.E.M. of four to five experiments. The duration of the anandamide or anandamide + ethanol application is indicated by the horizontal bar. C, bar graph comparing the inhibitory effects of anandamide, ethanol, and anandamide + ethanol on the maximal amplitudes of ACh-induced currents. AEA and EtOH, anandamide and ethanol, respectively. *, statistical significance at the level of P < 0.05 (ANOVA).

View larger version (23K): [in this window] [in a new window]   Fig. 2. The effects of ethanol and anandamide on concentration-response curves for anandamide and ethanol-induced inhibition of 7-nACh receptor-mediated ion currents. A, concentration-response curve for anandamide in the absence and in the presence of 30 mM ethanol. Inset, analysis of data after the subtraction of tonic inhibition caused by 30 mM ethanol and normalizing to maximal inhibition for each data set. Data points represent the mean ± S.E.M. of four to six oocytes. B, concentration-response curve for ethanol in the absence and in the presence of 100 nM anandamide. Inset, analysis of data after subtraction of tonic inhibition caused by 100 nM anandamide and normalizing to maximal inhibition for each data set. Data points represent the mean ± S.E.M. of four to six oocytes. The curves are the best fit of the data to the logistic equation described in the methods. Currents were activated by applying 100 µM ACh.

Fatty acid ethyl esters are the major nonoxidative metabolites of ethanol in humans, representing the predominant ethanol metabolites in human brain after ethanol ingestion (Bora and Lange, 1993). Perfusion of tissue cultures by ethanol is known to cause production of fatty acid ethyl esters from arachidonic acid. These fatty acid ethyl esters, such as ethyl arachidonate, are reported to have pharmacological effects on ion channels (Gubitosi-Klug and Gross, 1996). Since the hydrolysis of anandamide by fatty acid amide hydrolase (FAAH) would produce arachidonic acid in the presence of ethanol in our extracellular solution, ethyl arachidonate produced from these molecules could mediate additive inhibitory effects of ethanol and anandamide on ₇-nACh receptor-mediated currents. To evaluate a role of anandamide hydrolysis by FAAH in our system, we compared the extent of anandamide + ethanol-induced inhibition of nACh responses in the presence and absence of phenylmethylsulfonyl fluoride (PMSF; 0.2 mM), an inhibitor for FAAH. The amount of the inhibition by anandamide + ethanol on ₇-nACh receptor-mediated currents was not altered significantly in the presence of PMSF (Fig. 3A).

View larger version (45K): [in this window] [in a new window]   Fig. 3. Additive effect of anandamide and ethanol on ACh-induced (100 µM) currents is not due to alterations in anandamide hydrolysis, anandamide membrane transport, endogenous Ca2+-activated Cl- channels, and membrane-potential. A, effects of the inhibitions of anandamide hydrolysis by PMSF (black bar) and anandamide transport by AM404 (gray bar) on the suppression of ACh-induced currents by coapplication of anandamide and ethanol. B, comparison of the effects of anandamide + ethanol on the amplitudes of ACh-induced currents in the absence or presence of factors contributing to the activation of Ca2+-dependent Cl- channels. Oocytes were either injected with 50 nl of distilled water and recorded in a 2 mM Ca2+-containing MBS solution (black bar) or injected with 50 nl of 100 mM BAPTA (gray bar) and recorded in a 2 mM Ba2+-containing MBS solution. Each data point represents the normalized mean ± S.E.M. of five to six experiments. C, current-voltage relationships of ACh-activated currents in controls, 30 mM ethanol, 100 nM anandamide, and anandamide + ethanol. Each data point represents the normalized means and S.E.M. of three to five experiments. D, effects of anandamide and anandamide + ethanol are presented as percentages of inhibition of ACh-activated currents at different voltages indicated in the figure.

Both anandamide and ethanol have been shown to enhance intracellular Ca²⁺ concentrations (Wafford et al., 1989; Howlett and Mukhopadhyay, 2000). Since activation of ₇-nACh receptors permit sufficient Ca²⁺ entry to activate endogenous Ca²⁺-dependent Cl^- channels in Xenopus oocytes (Sands et al., 1993), we determined whether or not coapplication of anandamide + ethanol can interact directly with endogenous Ca²⁺-dependent Cl^- channels or secondarily on other currents induced by Ca²⁺ entry. Thus, extracellular Ca²⁺ was replaced with Ba²⁺ since Ba²⁺ can pass through nACh ₇ receptors (Sands et al., 1993) but causes little, if any, activation of Ca²⁺-dependent Cl^- channels. In addition, because a small Ca²⁺-dependent Cl^- current remains, even in Ba²⁺, we injected oocytes with the Ca²⁺ chelator BAPTA (Sands et al., 1993). Under these conditions, coapplication of anandamide + ethanol produced the same level of inhibition (73 ± 7% in controls versus 76 ± 6% in BAPTA-injected group) of ACh-induced currents when compared with control oocytes (Fig. 3B).

Examination of the voltage-dependence of the inhibition by anandamide + ethanol coapplication indicated that the degree of inhibition of the ACh (100 µM)-induced currents did not vary with membrane potential (Fig. 3, C and D). In addition, there was no change on the reversal potential of the ACh-activated ion currents (4 ± 2 mV in controls versus 6 ± 3 mV in anandamide + ethanol), indicating that neither the ionic selectivity of the channel nor the driving force on Na⁺ and Ca²⁺ were affected by these molecules.

To study the possible effects of ethanol on endogenous anandamide levels, we looked for quantitative detection of anandamide in control and 100 mM ethanol-treated oocytes by MALDI analysis. To test the sensitivity of our technique, several standard solutions of different anandamide concentrations were detected. As shown in Fig. 4A, anandamide was easily detectable down to 150 fM. The two dominant mass spectrums corresponded to the protonated anandamide and its sodium adduct. In contrast, anandamide was not observed in mass spectra of either MBS (Fig. 4B) or 100 mM ethanol (Fig. 4C) containing MBS solution. Moreover, lipid distribution was not altered by ethanol treatment (Fig. 4, B and C).

View larger version (20K): [in this window] [in a new window]   Fig. 4. Lack of anandamide in Xenopus oocytes and the effect of ethanol on mass spectra of Xenopus oocytes measured with matrix-assisted laser desorption/ionization. A, anandamide standard (150 fM) was indicated in spectrum. Peaks correspond to the protonated anandamide and its sodium adduct. Mass spectra of oocytes in control (B) and in the presence of 100 mM ethanol (C). Cholesterol and phosphatidylcholine species were detectable in both control and ethanol-treated oocytes.

View larger version (19K): [in this window] [in a new window]   Fig. 5. Effect of anandamide, ethanol, and anandamide + ethanol on responses mediated by 7-nACh receptors, 5-HT3, and chimeric 7-nACh-5-HT3 receptors. Agonist concentration-response curves for 7-nACh receptors (A), 5-HT3 receptors (B), the chimeric receptors (C), and the effects of anandamide (100 nM) and anandamide + ethanol (30 mM) on the responses mediated by each of these receptors are shown. Each data point represents the average of three to five cells (mean ± S.E.M.). The error bars that are not visible are smaller than the size of the symbols. Paired concentration-response curves were constructed and responses normalized to the maximum response under control conditions. Current traces illustrating the effect of anandamide and anandamide + ethanol on currents mediated by 7-nACh receptors, 5-HT3 receptors, and chimeric receptors are presented as insets. The bar above each record indicates the period of agonist application. Vertical calibration bars for the 7-nACh receptors, the 5-HT3 receptors, and the chimeric receptors represent 100, 200, and 200 nA, respectively. Horizontal calibration bars indicate 2, 10, and 3 s, respectively. The 7-nACh receptor and chimeric receptor-mediated currents were activated by 100 µM ACh; the 5-HT3 receptor-mediated currents were activated by 1 µM 5-HT.

	Discussion

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In the present study, we provide evidence indicating that endogenous cannabinoid anandamide and ethanol have additive inhibitory effects on the function of neuronal ₇-nACh receptors expressed in Xenopus oocytes. Our results also suggest that sites of actions for ethanol and anandamide are different, and there is no allosteric cooperation between ethanol and anandamide on ₇-nACh receptors.

Alterations of intracellular Ca²⁺ levels by ethanol and anandamide have been reported in several cell types (Wafford et al., 1989; Howlett and Mukhopadhyay, 2000). In oocytes, Ca²⁺-activated Cl^- channels are highly sensitive to intracellular levels of Ca²⁺ (for review, see Dascal, 1987). Under voltage-clamp conditions, these alterations in intracellular Ca²⁺ levels can be detected by changes in holding currents. However, during our experiments, the coapplication of anandamide and ethanol to oocytes expressing ₇-nACh receptors did not cause a significant change in baseline holding currents, suggesting that the intracellular concentration of Ca²⁺ was not altered by the coapplication of ethanol and anandamide. In addition, passive membrane properties of oocytes were not significantly altered by anandamide and ethanol, suggesting that coapplication of these reagents to oocytes did not disrupt the integrity of the lipid membrane.

Endogenous cannabinoids, at the concentration range used in this study, have been shown to activate cannabinoid receptors (for review, see Howlett et al., 2002); however, binding studies conducted indicate that cannabinoid receptors are not expressed endogenously in Xenopus oocytes (Henry and Chavkin, 1995). In addition, our previous work has demonstrated that the CB₁ receptor antagonist SR-141716A and the CB₂ receptor antagonist SR144528 did not affect the anandamide-induced inhibition of ₇-nACh receptors expressed in oocytes, nor did pertussis toxin alter the inhibition by anandamide (Oz et al., 2003a), suggesting a direct effect of anandamide on the ₇-nACh receptors. Based on these observations, we propose that additive inhibitory effect of anandamide and ethanol on the function of neuronal ₇-nACh receptors expressed in oocytes is also independent of the participation of cannabinoid receptors.

Chronic application of ethanol to mammalian cells has been shown to increase extracellular concentration of anandamide by activating de novo synthesis of anandamide and inhibiting its intracellular transport (Basavarajappa and Hungund, 1999, 2002). Although in our ethanol experiments, oocytes were only acutely exposed to ethanol for 20 to 30 min of duration, we set up several experiments to evaluate if ethanol-induced changes in anandamide levels could account for the additive inhibitory effect of anandamide and ethanol on the function of neuronal ₇-nACh receptors expressed in oocytes. As a first step, we compared the spectra profiles of oocyte preparations under control conditions and after exposure to ethanol for 30 min and found that anandamide was not detectable in any of these preparations. Thus, ethanol-induced changes in anandamide levels do not account for the additive inhibitory effects of anandamide and ethanol on the function of neuronal ₇-nACh receptors expressed in oocytes. In additional studies, we demonstrated that the additive effects of ethanol and anandamide did not change when the anandamide transport inhibitor AM404 was present in bathing solution, suggesting that inhibition of anandamide transport does not mediate the additive inhibitory effect of anandamide and ethanol on the function of neuronal ₇-nACh receptors.

In our earlier experiments, we have demonstrated that inhibition of neuronal ₇-nACh receptors by anandamide is not mediated by its metabolic products (Oz et al., 2003a, 2004c). One of the products of anandamide hydrolysis is arachidonic acid (Cravatt and Lichtman, 2002), and the perfusion of tissue cultures by ethanol is known to cause production of pharmacologically active compounds such as ethyl arachidonate (fatty acid ethyl ester) from arachidonic acid (Gubitosi-Klug and Gross, 1996). However, inhibition of FAAH activity by PMSF did not prevent the additive actions of anandamide and ethanol on the function of neuronal ₇-nACh receptors, suggesting that the production of arachidonic acid ethyl esters from arachidonic acid and ethanol does not mediate the additive inhibitory effects of anandamide and ethanol in oocyte expression system.

Analysis of the inhibition of ₇-nACh receptors at different anandamide concentrations indicated that in the presence of ethanol, the IC₅₀ value for anandamide decreased significantly. In contrast, in the presence of anandamide, alteration of the IC₅₀ value for ethanol was not observed (Fig. 2). It is likely that in the presence of ethanol, this change in the IC₅₀ value for anandamide is due to the different efficacies of anandamide (complete inhibition) and ethanol (approximately 65% inhibition of ₇-nACh receptor-mediated currents). Thus, in the presence of ethanol, maximal inhibition of ACh-induced responses occurred at a lower anandamide concentration. As a result, the IC₅₀ shifted to lower values without a change in anandamide efficacy. On the other hand, only in the presence of anandamide was ethanol able to inhibit ACh-induced responses completely (increased efficacy without causing an alteration on IC₅₀ value). Both anandamide and ethanol are allosteric inhibitors of ₇-nACh receptors because these molecules bind to sites topographically distinct from the agonist binding sites for ligand-gated ion channels. Recently, allosteric interactions of ethanol with neurosteroids and membrane cholesterol on the function of ion channels have been investigated, and both positive and negative allosteric interaction between ethanol and these modulators have been reported (Akk and Steinbach, 2003; Crowley et al., 2003). Similarly, ethanol has been shown to potentiate anandamide activation of TRPV1 receptor-mediated currents in HEK293 cells (Trevisani et al., 2002).

Earlier studies have demonstrated positive or negative cooperation between different allosteric modulators for various ligand-gated ion channels (for review, see Changeux and Edelstein, 1998; Christopoulos, 2002). Here, we report for the first time that ethanol and anandamide have additive inhibitory effects on the function of ₇-nACh receptors. This additive effect did not show cooperativity between the binding sites for ethanol and cannabinoids on the ₇-nACh receptors. The lack of cooperativity between the binding sites for these allosteric modulators was confirmed in experiments employing the chimeric ₇-nACh-5-HT₃ receptor. The functional chimeric ₇-nACh-5-HT₃ receptor consisted of an N-terminal domain of the ₇-nACh receptor and the transmembrane and C-terminal domains of the 5-HT₃ receptor (Eisele et al., 1993; Zhang et al., 1997). We have demonstrated previously that anandamide and ethanol have allosteric binding sites on the ₇-nACh receptor (Yu et al., 1996; Oz et al., 2003a, 2004c).

In this study, we have examined if the chimeric ₇-nACh-5-HT₃ receptor uncouples a positive or negative linkage between the allosteric modulators ethanol and anandamide. Previous studies have demonstrated that anandamide inhibits the function of ₇-nACh receptors with a potency that was an order of magnitude higher than at 5-HT₃ receptors in Xenopus oocytes (Oz et al., 2002b, 2003a). In the present study, we found that anandamide did not inhibit the chimeric ₇-nACh-5-HT₃ receptor; however, coapplication of anandamide and ethanol caused a significant inhibition of the chimeric ₇-nACh-5-HT₃ receptor. Nevertheless, the extent of inhibition by the coapplication of anandamide and ethanol was not significantly different from the inhibition caused by ethanol alone. The lack of additive effects of anandamide and ethanol on the chimeric ₇-nACh-5-HT₃ receptor-mediated currents indicates that there is no cooperativity (positive or negative) inherent to topographically distinct binding sites between ethanol and anandamide and that these molecules act on different sites of the nicotinic ACh receptor.

In several earlier studies, ethanol has been shown to modulate the release and metabolism of arachidonic acid and prostaglandins (George and Collins, 1985; Westcott and Collins, 1985; Elmer and George, 1996; Basavarajappa et al., 1998). In addition to anandamide, fatty acids such as arachidonic acid and their oxygenated metabolites such as prostaglandins also are allosteric inhibitors of nicotinic ACh receptors (Vijayaraghavan et al., 1995; Tan et al., 1998; Du and Role, 2001; Oz et al., 2004c). Thus, by modulating fatty acid release and metabolism, ethanol may indirectly influence signaling via nicotinic ACh receptors.

To our knowledge, this is the first study investigating the interaction between two allosteric inhibitors on the function of ₇-nACh receptors; however, a similar study on nACh receptors from mouse skeletal muscle investigated the effects of arachidonic acid and prostaglandin D₂ on the desensitization of nACh receptor-mediated ion currents (Nojima et al., 2000). In this study, coapplication of arachidonic acid and prostaglandin D₂ caused a cooperative increase in their effects on desensitization of nACh receptor-mediated ion currents.

Because of the enhanced potency of the endocannabinoid modulation by ethanol and the presence of ₇-nACh receptors in these critical locations such as presynaptic terminals, it is possible that in the presence of ethanol, the activity of the ₇-nACh receptors may be further modulated by endocannabinoids released from postsynaptic sites in situ. Considering that chronic ethanol treatments enhance anandamide synthesis, it is likely that ethanol-induced inhibition of ₇-nACh receptors would be more relevant due to already suppressed function of ₇-nAChRs by enhanced endocannabinoid tone.

In conclusion, our results indicate that coadministration of ethanol and anandamide causes additive inhibition of ₇ nACh receptor-mediated currents in Xenopus oocytes. The results of studies with chimeric ₇-nACh-5-HT₃ receptor suggest that there are multiple allosteric modulatory sites for ethanol and anandamide. Collectively, these data suggest that ₇-nACh receptors may represent a novel link between endocannabinoid tone and ethanol in the intact nervous system.

	Acknowledgements

 
We thank Dr. Jon Lindstrom for kindly providing the cDNA clone of ₇ subunit of nACh receptors, David Julius for providing 5-HT₃-receptor cDNA, and Mary Pfeiffer for careful reading of the manuscript.

	Footnotes

 
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.104.081315.

ABBREVIATIONS: CB₁, cannabinoid receptor; ACh, acetylcholine; 5-HT, serotonin; nACh, nicotinic acetylcholine; MBS, modified Barth's solution; AM404, N-(4-hydroxyphenyl)-arachidonylamide; BAPTA, 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid; MALDI, matrix-assisted laser desorption/ionization; ANOVA, analysis of variance; FAAH, fatty acid amide hydrolase; PMSF, phenylmethylsulfonyl fluoride; SR-141716A, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride; SR144528, N-((1S)-endo-1,3,3-trimethyl bicyclo heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide).

Address correspondence to: Dr. Murat Oz, National Institute on Drug Abuse/Intramural Research Program, Cellular Neurobiology Branch, 5500 Nathan Shock Drive, Baltimore, MD 21224. E-mail: moz{at}intra.nida.nih.gov

	References

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