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NEUROPHARMACOLOGY

Differential Effects of Endogenous and Synthetic Cannabinoids 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 (M.O., C.R.L.), Behavioral Neurobiology Branch (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., A.R.)

Received for publication February 27, 2004
Accepted April 13, 2004.

	Abstract

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The effects of endogenous and synthetic cannabinoid receptor agonists, including 2-arachidonoylglycerol (2-AG), R-methanandamide, WIN55,212-2 [4,5-dihydro-2-methyl-4(4-morpholinylmethyl)-1-(1-naphthalenylcarbonyl)-6H-pyrrolo[3,2,1ij]quinolin-6-one], and CP 55,940 [1,2-(R)-5]-(-)-5-(1,1-dimethyl)-2-[5-hydroxy-2-(3-hydroxypropyl) cyclohexyl-phenol], and the psychoactive constituent of marijuana, ⁹-tetrahydrocannabinol (⁹-THC), on the function of homomeric ₇-nicotinic acetylcholine (nACh) receptors expressed in Xenopus oocytes was investigated using the two-electrode voltage-clamp technique. The endogenous cannabinoid receptor ligands 2-AG and the metabolically stable analog of anandamide (arachidonylethanolamide), R-methanandamide, reversibly inhibited currents evoked with ACh (100 µM) in a concentration-dependent manner (IC₅₀ values of 168 and 183 nM, respectively). In contrast, the synthetic cannabinoid receptor agonists CP 55,940, WIN55,212-2, and the phytochemical ⁹-THC did not alter ₇-nACh receptor function. The inhibition of ₇-mediated currents by 2-AG was found to be non-competitive and voltage-independent. Additional experiments using endocannabinoid metabolites suggested that arachidonic acid, but not ethanolamine or glycerol, could also inhibit the ₇-nACh receptor function. Whereas the effects of arachidonic acid were also noncompetitive and voltage-independent, its potency was much lower than 2-AG and anandamide. Results of studies with chimeric ₇-nACh-5-hydroxytryptamine (5-HT)₃ receptors comprised of the amino-terminal domain of the ₇-nACh receptor and the transmembrane and carboxyl-terminal domains of 5-HT₃ receptors indicated that the site of interaction of the endocannabinoids with the ₇-nAChR was not located on the N-terminal region of the receptor. These data indicate that cannabinoid receptor ligands that are produced in situ potently inhibit ₇-nACh receptor function, whereas the synthetic cannabinoid ligands, and ⁹-THC, are without effect, or are relatively ineffective at inhibiting these receptors.

Nicotinic acetylcholine (nACh) receptors containing the ₇ subunit belong to the ligand-gated ion channel superfamily and is found in many brain areas where cannabinoids are known to act (Lindstrom et al., 1996). In addition, the potential involvement of these receptors in pain transmission, neurodegenerative diseases, and drug abuse has been extensively reported (Damaj et al., 2000; Orr-Urteger et al., 2000; Picciotto et al., 2001). Biochemical and behavioral studies support the idea that functional interactions between nicotine receptors and cannabinoid receptor ligands exist (Pryor et al., 1978; Valjent et al., 2002). In addition, we have shown that the endogenous cannabinoid, anandamide, inhibits ion currents mediated by the activation of nACh ₇ receptors expressed in Xenopus oocytes (Oz et al., 2003). Another receptor that shares a high degree of homology with nACh ₇ receptors is the serotonin 5-HT₃ receptor (for reviews, see Jackson and Yakel, 1995; Reeves and Lummis, 2002). As might be expected, earlier studies demonstrated that both endogenous and synthetic cannabinoids could inhibit the function of 5-HT₃ receptors expressed in mammalian cell lines or in Xenopus oocytes (Barann et al., 2002; Oz et al., 2002), although the potency of these cannabinoids seems to be lower at 5-HT₃ receptors than at ₇-nACh receptors.

In addition to the phytochemical ⁹-THC, there are three different classes of cannabinoid receptor ligands that are currently used in pharmacological research. These include the nonclassical cannabinoids, typified by the agonist CP 55,940; the aminoalkylindoles, such as WIN55,212-2; and the arachidonic acid-derived eicosanoid molecules such as anandamide (arachidonylethanolamide) and 2-arachidonylglycerol (Pertwee, 1997). The present study was performed to compare the effects a range of cannabinoid receptor ligands representing each of these classes. We find that the endocannabinoids potently inhibit ₇-nACh receptor function, independently of cannabinoid receptor activation and that the synthetic cannabinoids and ⁹-THC do not seem to significantly disrupt ₇-nACh signaling.

	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 19-21°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. Ion currents were recorded as described previously (Oz et al., 2002b). Briefly, 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 a 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 directly recorded 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 previously (Schmitt and Koepsell, 2002). Briefly, voltageramps were used 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 were 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, R-(+)-methanandamide, 2-AG, (-)-nicotine, and -bungarotoxin were from Sigma-Aldrich. Procedures for the injections of pertussis toxin (50 nl, 50 µg/ml) or BAPTA (50-100 nl, 100 mM) were performed as described previously (Oz et al., 1998). Pertussis toxin was dissolved in distilled water, BAPTA was prepared in Cs₄-BAPTA. Injections were performed 1 h before recordings using oil-driven ultramicrosyringe pump (Micro4, WPI, Sarasota, FL). Stock solutions of anandamide were prepared in dimethyl sulfoxide 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. 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).

Data Analysis. Average values were calculated as mean ± S.E. Statistical significance was analyzed using Student's t test or analysis of variance 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 that were either not injected with ₇-nAChR mRNA (n = 6) or 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 rapidly desensitized. 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, endogenous cannabinoids such as anandamide and 2-AG were shown to release intracellular Ca²⁺ in endothelial and NG108-15 cells (Sugiura et al., 1996; Mombouli et al., 1999; for review, see Howlett and Mukhopadhyay, 2000). In the oocyte expression system, this increased level of intracellular Ca²⁺ would be detected by the activation of Ca²⁺-activated Cl^- channels and concomitant alterations in the membrane input resistance. For this reason, we examined the effects of 2-AG and R-methanandamide, a metabolically stable chiral analog of anandamide that is resistant to hydrolytic inactivation by fatty acid amide hydrolase (Abadji et al., 1994), on membrane resistance (R_m), membrane capacitance (C_m), and resting membrane potential (V_m) in uninjected oocytes. The results presented in Table 1 demonstrate that neither 2-AG nor R-methanandamide had a significant effect on the passive membrane properties of the oocytes.

In our earlier studies, we found that anandamide potently inhibited the function of ₇-nAChRs expressed in Xenopus oocytes (IC₅₀ = 229 nM; Oz et al., 2003). In the present study, the application of 10 nM to 3 µM of 2-AG also concentration dependently inhibited ACh (100 µM)-induced ion currents in ₇-nACh receptor-expressing oocytes (Fig. 1A). The ACh-induced currents were maximally inhibited by 2-AG within 4 to 5 min of its initial bath application, and it was applied for 30 to 40 min to ensure equilibrium concentrations (Fig. 1). In contrast, a high concentration of WIN55,212-2 (10 µM), a synthetic full agonist at CB receptors, did not affect the maximal amplitudes of the ACh-induced ion currents at application times up to 30 min (Fig. 1B). Results of experiments demonstrating the time course of the effects of 2-AG and WIN55,212-2 on the mean amplitudes of the ACh-induced currents from four to six oocytes are presented in Fig. 1C. In addition, in the absence of agonist neither 2-AG nor WIN55,212-2 (both at 30 µM) had effects on holding currents in oocytes voltage clamped at -70 mV (n = 3-5), suggesting that passive membrane properties of the oocytes were unaffected.

View larger version (14K): [in this window] [in a new window]   Fig. 1. Effects of 2-AG and WIN 55,212-2 on 7-nACh receptor-mediated ion currents. A, currents activated by ACh (100 µM) in control extracellular media (left), during bath application of 300 nM 2-AG (middle), and during a 10-min recovery (right). B, records of currents activated by ACh (10 µM) in control (left), during application of 10 µM WIN 55,212-2 (middle), and after a 10-min WIN 55,212-2 washout (right). C, time courses of the effects of 2-AG and WIN 55,212-2 on the peak ACh-induced currents. Each data point represents the normalized means and S.E.M. of four to six experiments. The duration of the 2-AG or WIN 55,212-2 application is indicated by the horizontal bar.

In the next series of experiments, we examined the concentration-response relationships of the effects 2-AG, R-methanandamide, CP 55,940, WIN55,212-2, and ⁹-THC on the function of nicotinic receptors (Fig. 2). Two arachidonoylglycerol and R-methanandamide inhibited the nicotinic receptor-mediated response with IC₅₀ values of 168 and 183 nM, respectively. These values were slightly lower than the IC₅₀ values of 229 nM described previously for anandamide (Oz et al., 2003). In contrast to the relatively potent inhibition by 2-AG and R-methanandamide, neither ⁹-THC nor WIN55,212-2 altered the maximal amplitudes of the ACh-induced inward currents. Furthermore, although the synthetic agonist CP 55,940 had no effect at concentrations up to 1 µM, at much higher concentrations it inhibited the nicotinic receptor response, demonstrating an IC₅₀ of 3.4 µM (slope = 2.6).

View larger version (21K): [in this window] [in a new window]   Fig. 2. Concentration dependence of the effect of cannabinoid receptor ligands on 7-nACh receptor-mediated ion currents. Data points represent the mean ± S.E.M. of four to seven oocytes. The curve is the best fit of the data to the logistic equation described under Materials and Methods. Currents were activated by applying ACh (100 µM). The IC50 and slope factors for 2-AG, R-methanandamide, and CP 55,940 were 118 nM and 0.92; 163 nM and 1.03; and 2.7 µM and 3.2, respectively.

Because activation of ₇-nACh receptors permit sufficient Ca²⁺ entry to activate endogenous Ca²⁺-dependent Cl^- channels in Xenopus oocytes (Sands et al., 1993; Séguéla et al., 1993), it was important to determine in the present study whether the effect of 2-AG was due to the inhibition of these currents or secondarily, on other currents induced by Ca²⁺ entry. Thus, extracellular Ca²⁺ was replaced with Ba²⁺, because 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, 2-AG (300 nM) produced the same level of inhibition (43 ± 4% of controls) of ACh-induced currents compared with control oocytes (Fig. 3A).

View larger version (29K): [in this window] [in a new window]   Fig. 3. Inhibition of ACh-induced currents by 2-AG is not due to an effect on endogenous Ca2+-activated Cl- channels, is independent of membrane potential, and is due to a noncompetitive interaction with the 7-nACh receptor. A, comparison of the effect of 2-AG (200 nM) on the amplitudes of ACh (100 µM)-induced currents in the absence or presence of factors contributing 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 column) or injected with 50 nl of BAPTA (100 mM, gray column) 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. B, current-voltage relationships of ACh-activated currents in the absence and presence of 2-AG or R-methanandamide. Normalized currents activated by 100 µM ACh. Each data point represents the normalized means and S.E.M. of four to six experiments. C, quantitative evaluation of the effects of 2-AG and R-methanandamide are presented as percentage of inhibition at different voltages indicated in the figure. D, concentration-response curves for peak ACh-induced currents in the presence and absence of 200 nM 2-AG or R-methanandamide. Oocytes were voltage clamped at -70 mV and currents were activated by applying ACh (0.01-3 mM). Paired concentration-response curves were constructed and responses normalized to the maximum response under control conditions. EC50 and slope values were determined by fitting the curves from four to six oocytes to the standard logistic equation as described under Materials and Methods.

Examination of the voltage dependence of the 2-AG and R-methanandamide inhibition indicated that the degree of inhibition of the ACh (100 µM)-induced currents by 2-AG (200 nM) or R-methanandamide (200 nM) did not vary with membrane potential (Fig. 3, B and C). 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 2-AG and 5 ± 4 in R-methanandamide), indicating that neither the ionic selectivity of the channel nor the driving force on Na⁺ and Ca²⁺ were affected by these molecules.

Another way 2-AG and R-methanandamide may alter ₇-nAChR channel activity might be through the competitive inhibition of ACh binding to the receptor. To test this possibility, the effects of 2-AG and R-methanandamide on ₇-nAChR function were examined at different concentrations of ACh. Neither 2-AG nor R-methanandamide altered the potency of ACh (Fig. 3D). Thus, in controls and in the presence of 2-AG or R-methanandamide, the EC₅₀ values for ACh were 142 ± 8 µM, 137 ± 7 µM, and 149 ± 8 (n = 4), and slope values were 1.4 ± 0.1, 1.6 ± 0.2, and 1.3 ± 0.2, respectively. However, both of these molecules inhibited the maximal response to ACh by approximately 45% (n = 4-7), suggesting that the inhibition of ₇-nAChR currents was noncompetitive. The effect of 2-AG (300 nM) on currents induced by a more potent agonist at ₇-nACh receptors, nicotine (10 and 100 µM), was also examined. The degree of inhibition of nicotine-induced currents was identical to ACh and did not vary with nicotine concentration [10 µM = 44 ± 5% (n = 4) and 100 µM = 41 ± 6% (n = 5) of controls, respectively].

The endocannabinoids tested in these experiments contain several major chemical moieties: arachidonic acid and ethanolamine are contained in anandamide, whereas glycerol and arachidonic acid are found in 2-AG. To determine whether these chemical moieties could by themselves alter ₇-nAChR function, we examined the effects of arachidonic acid, ethanolamine, and glycerol on the function of ₇-nACh receptor. Arachidonic acid (1 µM) reduced the maximal amplitudes of ₇-nACh receptor-mediated ion currents to 54 ± 7% of controls (n = 5). Conversely, 35-min incubation in ethanolamine or glycerol did not alter the maximal amplitudes of ACh-induced currents (Fig. 4A). We have shown that anandamide (Oz et al., 2003), R-methanandamide, and 2-AG (present study; Fig. 3C) inhibit the function of ₇-nACh receptors in voltage-independent and noncompetitive manners. If the endocannabinoid metabolite arachidonic acid mediated the effects of these molecules, it would be expected that the arachidonic acid inhibition of ₇-nACh receptor response would also be voltage-independent and noncompetitive. For this reason, we examined the voltage dependence of the inhibition of ₇-nACh receptor, by arachidonic acid. Similar to endocannabinoids, the inhibition of the ACh induced currents by arachidonic acid (1 µM) did not vary with membrane potential (Fig. 5, A and B), and the reversal potential of the ACh-activated ion currents was not changed (3 ± 2 mV in controls versus 5 ± 3 mV in arachidonic acid). In addition, we examined the inhibitory effect of arachidonic acid at increasing concentrations of ACh to determine whether its inhibitory effects on the ₇-nACh receptor-mediated currents were competitive (Fig. 5C). Similar to the endocannabinoid precursors, arachidonic acid did not cause any shift on the concentration-response curve, but it inhibited the maximal oocyte response to ACh to about 45 to 55% of controls (n = 4-7). In controls and in the presence of arachidonic acid, the EC₅₀ values for ACh were 138 ± 6 and 142 ± 7 µM (n = 5-6), and slope values were 1.4 ± 0.3 and 1.5 ± 0.3, respectively, suggesting that similar to anandamide, R-methanandamide, and 2-AG, arachidonic acid inhibited the function of the nicotinic receptors in a noncompetitive manner.

View larger version (18K): [in this window] [in a new window]   Fig. 4. Effects of endocannabinoid metabolites on 7-nACh receptor-mediated ion currents. A, effects of arachidonic acid (AA, 1 µM), ethanolamine (EA, 10 µM), and glycerol (GL, 10 µM) on the maximal amplitudes of 7-nACh receptor-mediated ion currents activated by 100 µM ACh. Each data point represents the normalized means ± S.E.M. of four to six experiments. The horizontal bar in the figure indicates duration of application. B, concentration dependence of the effect of endocannabinoid metabolites on 7-nACh receptor-mediated ion currents. Data points are the mean ± S.E.M. of four to five oocytes. The curve is the best fit of the data to the logistic equation described under Materials and Methods. The IC50 and slope factor for arachidonic acid were 1.2 µM and 0.9, respectively. The same data sets for 2-AG and R-methanandamide, shown in Fig. 2, are presented for comparison. The IC50 and slope factors for 2-AG and R-methanandamide were 118 nM and 0.9 and 163 nM and 1, respectively.

View larger version (14K): [in this window] [in a new window]   Fig. 5. Inhibition of 7-nACh receptor-mediated ion currents by arachidonic acid is independent of membrane potential and is noncompetitive. A, current-voltage relationships of 7-nACh receptor-mediated ion currents before (control; ) and during 30-min treatment with arachidonic acid (1 µM; ). Normalized currents were activated by 100 µM ACh. Each data point presents the normalized means ± S.E.M. of four to five experiments. B, quantitative evaluation of the effects of arachidonic acid is presented as the percentage of inhibition at different voltages indicated in the figure. C, concentration-response curves for peak ACh-induced currents in the presence () and absence of 1 µM arachidonic acid (). Oocytes were voltage clamped at -70 mV, and currents were activated by applying ACh (0.01-3 mM). Paired concentration-response curves were constructed and responses normalized to the maximum response under control conditions. EC50 and slope values were determined by fitting the curves from four to five oocytes to the standard logistic equation as described under Materials and Methods.

The preceding data suggested that the metabolite arachidonic acid might be responsible for the presumed endocannabinoid action on ₇-nACh receptor-mediated currents in the oocytes. However, a more critical test of this hypothesis would be achieved by comparing the potency of arachidonic acid with the endocannabinoid molecules in this system. Figure 4B demonstrates the effects of increasing concentrations of arachidonic acid, ethanolamine, and glycerol on the amplitudes of ACh (100 µM)-activated currents. Although, ethanolamine and glycerol were ineffective up to concentrations of 100 µM, arachidonic acid inhibited ACh-induced currents with an EC₅₀ value of 1.2 µM, indicating that the potency of arachidonic acid was lower than that of anandamide or 2-AG. Therefore, it is unlikely that the arachidonic acid metabolite was responsible for the inhibition of ₇-nACh receptors observed during anandamide application.

In earlier studies, we found that anandamide inhibited ₇-nACh receptor-mediated responses with a potency that was at least 1 order of magnitude higher than at 5-HT₃ receptors expressed in Xenopus oocytes (Oz et al., 2002, 2003). Thus, in these studies, the IC₅₀ values for anandamide were 229.0 nM and 3.7 µM at ₇-nACh and 5-HT₃ receptors, respectively. The development of chimeric ₇-nACh-5HT₃ receptors (Eisele et al., 1993; Zhang et al., 1997) and the differences in anandamide potency at these distinct receptor-operated ion channels provided an opportunity to evaluate the location of the anandamide interaction with the ₇-nACh receptor. Therefore, we used a functional chimeric receptorion channel constructed with the N-terminal domain of the ₇-nACh receptor and the C-terminal and transmembrane domains of the 5-HT₃ receptor (Eisele et al., 1993; Zhang et al., 1997). The properties of these chimeric receptors are similar to the native ₇-nACh receptor with regard to the potency and efficacy of ACh. However, they are different from the native ₇-nACh receptors in that they display slower activation and inactivation kinetics (Eisele et al., 1993). In agreement with our earlier results (Oz et al., 2003), application of 300 nM anandamide inhibited the ACh-induced currents mediated by ₇-nACh receptors in a noncompetitive manner (Fig. 6A). Thus, in the presence of anandamide, maximal ACh-induced currents were inhibited to 40 to 45% of control, and the EC₅₀ values of ACh were not significantly altered (122 ± 6 and 117 ± 5 µM; n = 4-6). In contrast, this same concentration of anandamide had no significant effect on 5-HT₃ receptor-mediated currents in oocytes injected with cRNA coding for this receptor (Fig. 6B). Similarly, when the ability of 300 nM anandamide to inhibit ACh-induced currents mediated by the ₇-nACh-5-HT₃ chimeric receptors was examined, it was found that the endocannabinoid did not significantly inhibit these currents (Fig. 6C). Thus, in the presence and absence of anandamide, the EC₅₀ values for ACh were 132 ± 5 and 127 ± 6 µM (n = 5-6), respectively. These data suggest that the N-terminal domain of the ₇-nAChRs was not the critical target for the anandamide inhibition of ₇-nAChRs and that this site must be located either at one of the transmembrane domains or near the carboxyl terminal of this receptor.

View larger version (19K): [in this window] [in a new window]   Fig. 6. Effect of anandamide on responses mediated by 7-nACh receptors, 5-HT3 receptors, and chimeric 7-nACh-5-HT3 receptors. Agonist concentration-response curves for 7-nACh receptors (A), 5-HT3 receptors (B), and chimeric receptors (C), and the effect of anandamide (300 nM) on the responses mediated by each of these receptors are shown. Each data point represents the average of four to six 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 300 nM anandamide (ANA) 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 200, 100, 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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The present results indicate that endogenous and synthetic cannabinoids have differential effects on the function of neuronal ₇-nACh receptors expressed in Xenopus oocytes. Whereas the endogenous cannabinoid receptor ligands such as 2-AG and R-methanandamide inhibited the function of ₇-nACh receptors at relatively low concentrations, the synthetic cannabinoid ligands, including WIN55,212-2 and CP 55,940, and the plant-derived ⁹-THC, were virtually ineffective in modulating ₇-nAChR-mediated ion currents. These results therefore suggest that whereas ₇-nACh receptors may represent molecular targets for endogenous cannabinoids in the mammalian brain, they are not likely to be affected by the psychoactive constituent in marijuana.

Endogenous cannabinoids, at the concentration range used in this study, have been shown to activate native cannabinoid receptors (for reviews, see Di Marzo et al., 2002; Sugiura et al., 2002). However, binding studies conducted in Xenopus oocytes indicate that the cloned cannabinoid receptors CB₁ and CB₂ are not expressed endogenously in these cells (Henry and Chavkin, 1995). In addition, our previous work has demonstrated that the CB₁ receptor antagonist SR141716A and the CB₂ receptor antagonist SR144528 did not affect the anandamide-induced inhibition of ₇-nACh receptors, nor did pertussis toxin alter the inhibitory effects of anandamide on the ₇-nACh receptors (Oz et al., 2003). Thus, it is unlikely that the observed effects of the endocannabinoids on ₇-nAChR function in the present study were due to the activation of cannabinoid receptors or to other G_i/G_o G protein-coupled receptors.

During our experiments, the application of endocannabinoids even at the highest concentrations did not cause a significant change in baseline holding currents, suggesting that the intracellular concentration of Ca²⁺ was not affected. Because Ca²⁺-activated Cl^- channels are highly sensitive to intracellular levels of Ca²⁺ (for review, see Dascal, 1987), its release would be reflected by changes in holding currents under voltage-clamp conditions. In addition, other passive membrane properties, such as membrane capacitance, were not significantly altered (Table 1), suggesting that endocannabinoids, at the concentrations used in this study, did not alternatively disrupt the integrity of the lipid membrane.

The present data strongly suggest that the effects of the endocannabinoids were due to a noncompetitive interaction with the ₇-nAChR. This is supported by data demonstrating that increases in the concentration of ACh could not overcome the endocannabinoid inhibition of the ACh-induced ion currents, and by the observation that the EC₅₀ for ACh was unchanged by these molecules. Although the noncompetitive nature of the inhibition by R-methanandamide (Fig. 3C) or anandamide (Oz et al., 2003) suggested that the ACh binding site itself was not targeted by the endocannabinoids, we used the chimeric ₇-nACh-5-HT₃ receptor to further examine the possible site of this interaction in more detail. The functional chimeric ₇-nACh-5-HT₃ receptor consisted of an N-terminal domain from the ₇-nACh receptor and the transmembrane and C-terminal domains from the 5-HT₃ receptor (Eisele et al., 1993; Zhang et al., 1997). 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., 2002a, 2003). In the present study, the observed resistance of the chimeric ₇-nACh-5-HT₃ receptor to inhibition by a concentration of anandamide (300 nM) that potently inhibited the ₇-nACh receptor-mediated response indicated that the site of interaction with the ₇-nACh receptor must be at the transmembrane domain, and/or the C-terminal receptor domains, but not at the ligand binding N-terminal domain. Thus, these data seem to confirm our previous observation that anandamide does not interfere with ₇-nACh receptor-mediated function by inhibiting the binding of ACh to this receptor, but rather, by disrupting some process in the plasma membrane, or within the cell at the C-terminal tail.

Earlier studies on 5-HT₃ receptors indicated that both endogenous and synthetic cannabinoids are effective inhibitors of this ligand-gated ion channel (Barann et al., 2002; Oz et al., 2002a; Godlewski et al., 2003). This is in contrast with the results of the present study where only the endogenous ligands were capable of inhibiting the function of ₇-nAChR, at reasonable concentrations. This suggests that despite the high degree of homology of ₇-nACh receptors and 5-HT₃ receptors (Reeves and Lummis, 2002), a distinct site for endocannabinoid versus synthetic cannabinoid interaction with these proteins must exist.

The endocannabinoid 2-AG is hydrolyzed in situ to form arachidonic acid and glycerol, whereas anandamide is metabolized to arachidonic acid and ethanolamine by fatty acid amide hydrolase (for reviews, see Piomelli et al., 1998; Piomelli, 2003). Also, earlier studies on nACh receptors at the neuromuscular junction demonstrated that many fatty acids, including arachidonic acid and prostaglandin E₂ could modulate the function of neuronal nicotinic ACh receptors via a direct action (Vijayaraghavan et al., 1995, Nishizaki et al., 1998; Tan et al., 1998; Du and Role, 2001; for review, see Arias, 1998). Because of these known effects, we wanted to determine whether the metabolites of the endocannabinoids could also alter the ₇-nAChR function. Under the present experimental conditions, we found that only arachidonic acid had significant effects on ₇-nACh receptor-mediated function in the oocytes. Thus, both the intact endocannabinoids and arachidonic acid shared similar time courses for the inhibition, as well as similar noncompetitive and voltage-independent modulation. However, the potencies of 2-AG or R-methanandamide were higher than that of arachidonic acid on ₇-nAChRs, suggesting that it was the intact endocannabinoid and not the metabolite, arachidonic acid that altered ₇-nACh receptor function. In addition, because glycerol and ethanolamine had virtually no effect at these receptors, it can also be concluded that the endocannabinoid, and not these other metabolites, mediated the inhibitory effect of anadamide and 2-AG on ₇-nACh receptor function.

The synthesis of endocannabinoids can be triggered in response to membrane depolarization and the subsequent influx of Ca²⁺ (Piomelli et al., 1998; Freund et al., 2003). Alternatively, postsynaptic activation of neurotransmitter receptors such group I metabotropic glutamate receptors (Maejima et al., 2001; Varma et al., 2001), M₁ and M₃ muscarinic receptors (Ohno-Shosaku et al., 2003), D₂ dopamine receptors (Giuffrida et al., 1999), or the coactivation of N-methyl-D-aspartate and ₇-nACh receptors (Stella and Piomelli, 2001; Freund et al., 2003) can induce endocannabinoid synthesis in neuronal preparations. Recent studies also indicate that endocannabinoids are synthesized postsynaptically and that they can modulate synaptic transmission in a retrograde manner, by occupying CB₁ receptors (for review, see Wilson and Nicoll, 2002). The evidence to date is also very strong that in the central nervous system, ₇-nACh receptors are located presynaptically and play an important modulatory role in synaptic transmission (McGehee et al., 1995; Girod et al., 2000; Dani, 2001). Because of the potency of the endocannabinoid modulation and the presence of ₇-nAChRs in these critical locations, it is possible that the activity of the ₇-nACh receptors may be modulated by endocannabinoids released from postsynaptic sites in situ.

In conclusion, our results indicate that endocannabinoids, but not synthetic or plant-derived cannabinoid ligands, inhibit the function of ₇ nicotinic receptors expressed in Xenopus oocytes. In addition, the results of studies with chimeric ₇-nACh-5-HT₃ receptor suggest that the site of this interaction is at the transmembrane or C-terminal domain of the ₇-nACh receptor. Collectively, these data suggest that ₇-nACh receptors may represent targets for the endocannabinoids in the intact nervous system.

	Acknowledgements

 
We thank Dr. Lindstrom for kindly providing the cDNA clone of ₇ subunit of nACh receptors and Dr. David Julius for providing 5-HT₃ receptor cDNA.

	Footnotes

 
doi:10.1124/jpet.104.067751.

ABBREVIATIONS: ⁹-THC, ⁹-tetrahydrocannabinol; CB, cannabinoid; 2-AG, 2-arachidonoylglycerol, 5-HT, 5-hydroxytryptamine; ACh, acetylcholine; nACh, nicotinic acetylcholine; MBS, modified Barth's solution; BAPTA, 1,2-bis (o-aminophenoxy) ethane-N,N, N',N'-tetraacetic acid; WIN55,212-2, 4,5-dihydro-2-methyl-4(4-morpholinylmethyl)-1-(1-naphthalenylcarbonyl)-6H-pyrrolo[3,2,1ij]quinolin-6-one; CP 55,940, 1,2-(R)-5]-(-)-5-(1,1-dimethyl)-2-[5-hydroxy-2-(3-hydroxypropyl) cyclohexyl-phenol; SR141716A, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride; SR144528, N-[(1S)-endo-1,3,3-trimethylbicyclo[2,2,1]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 Dr., Baltimore, MD 21224. E-mail: moz{at}intra.nida.nih.gov

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