Abstract
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 [1α,2β-(R)-5α]-(-)-5-(1,1-dimethyl)-2-[5-hydroxy-2-(3-hydroxypropyl) cyclohexyl-phenol], and the psychoactive constituent of marijuana, Δ9-tetrahydrocannabinol (Δ9-THC), on the function of homomeric α7-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 Δ9-THC did not alter α7-nACh receptor function. The inhibition of α7-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 α7-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 α7-nACh-5-hydroxytryptamine (5-HT)3 receptors comprised of the amino-terminal domain of the α7-nACh receptor and the transmembrane and carboxyl-terminal domains of 5-HT3 receptors indicated that the site of interaction of the endocannabinoids with the α7-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 α7-nACh receptor function, whereas the synthetic cannabinoid ligands, and Δ9-THC, are without effect, or are relatively ineffective at inhibiting these receptors.
Endogenous cannabinoids (endocannabinoids) are produced on demand from membrane-bound precursors in brain tissue via calcium and/or G protein-dependent processes (for a recent review, see Piomelli, 2003). After release, these molecules bind to cannabinoid CB1 and/or CB2 receptors and mimic the effects of synthetic cannabinoids in several in vitro preparations (for a recent review, see Freund et al., 2003). However, several reports also indicate that endocannabinoids and the psychoactive active ingredient of marijuana, Δ9-THC, can produce effects that are not mediated by the activation of the cloned CB1 and/or CB2 receptors. For example, it has been demonstrated that endocannabinoids such as anandamide and/or 2-AG can inhibit the function of gap junctions (Venance et al., 1995), voltage-dependent Ca2+ channels (Oz et al., 2000; Chemin et al., 2001), Na+ channels (Nicholson et al., 2003), various types of K+ channels (Poling et al., 1996; Van den Bossche and Vanheel, 2000; Maingret et al., 2001), 5-HT3 receptor function (Barann et al., 2002; Oz et al., 2002a; Godlewski et al., 2003), and nicotinic ACh receptors (Oz et al., 2003). This suggests that additional molecular targets for certain classes of cannabinoids exist in the central nervous system, and that these targets may represent important sites for cannabinoids to alter neuronal function.
Nicotinic acetylcholine (nACh) receptors containing the α7 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 α7 receptors expressed in Xenopus oocytes (Oz et al., 2003). Another receptor that shares a high degree of homology with nACh α7 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 α7-nACh receptors.
In addition to the phytochemical Δ9-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 α7-nACh receptor function, independently of cannabinoid receptor activation and that the synthetic cannabinoids and Δ9-THC do not seem to significantly disrupt α7-nACh signaling.
Materials and Methods
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 MΩ). 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, Icap, and the charge associated with this current was calculated by the integration of Icap. 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, Cm. Currents for Icap 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 α-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 nAChα7 subunit and 5-HT3A 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 α7-nACh-5-HT3A 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, Emax is the maximal response, EC50 is the half-maximal concentration, and n is the slope factor (apparent Hill coefficient).
Results
Xenopus oocytes that were either not injected with α7-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 α7-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 α-bungarotoxin (n = 3; data not shown), indicating that these responses were mediated by neuronal α7-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 (Rm), membrane capacitance (Cm), and resting membrane potential (Vm) 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 α7-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 α7-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.
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 Δ9-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 Δ9-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).
Because activation of α7-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 α7 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).
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 α7-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 α7-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 α7-nAChR currents was noncompetitive. The effect of 2-AG (300 nM) on currents induced by a more potent agonist at α7-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 α7-nAChR function, we examined the effects of arachidonic acid, ethanolamine, and glycerol on the function of α7-nACh receptor. Arachidonic acid (1 μM) reduced the maximal amplitudes of α7-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 α7-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 α7-nACh receptor response would also be voltage-independent and noncompetitive. For this reason, we examined the voltage dependence of the inhibition of α7-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 α7-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.
The preceding data suggested that the metabolite arachidonic acid might be responsible for the presumed endocannabinoid action on α7-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 α7-nACh receptors observed during anandamide application.
In earlier studies, we found that anandamide inhibited α7-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 α7-nACh and 5-HT3 receptors, respectively. The development of chimeric α7-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 α7-nACh receptor. Therefore, we used a functional chimeric receptorion channel constructed with the N-terminal domain of the α7-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 α7-nACh receptor with regard to the potency and efficacy of ACh. However, they are different from the native α7-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 α7-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 α7-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 α7-nAChRs was not the critical target for the anandamide inhibition of α7-nAChRs and that this site must be located either at one of the transmembrane domains or near the carboxyl terminal of this receptor.
Discussion
The present results indicate that endogenous and synthetic cannabinoids have differential effects on the function of neuronal α7-nACh receptors expressed in Xenopus oocytes. Whereas the endogenous cannabinoid receptor ligands such as 2-AG and R-methanandamide inhibited the function of α7-nACh receptors at relatively low concentrations, the synthetic cannabinoid ligands, including WIN55,212-2 and CP 55,940, and the plant-derived Δ9-THC, were virtually ineffective in modulating α7-nAChR-mediated ion currents. These results therefore suggest that whereas α7-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 α7-nACh receptors, nor did pertussis toxin alter the inhibitory effects of anandamide on the α7-nACh receptors (Oz et al., 2003). Thus, it is unlikely that the observed effects of the endocannabinoids on α7-nAChR function in the present study were due to the activation of cannabinoid receptors or to other Gi/Go 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 α7-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 α7-nACh-5-HT3 receptor to further examine the possible site of this interaction in more detail. The functional chimeric α7-nACh-5-HT3 receptor consisted of an N-terminal domain from the α7-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 α7-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 α7-nACh-5-HT3 receptor to inhibition by a concentration of anandamide (300 nM) that potently inhibited the α7-nACh receptor-mediated response indicated that the site of interaction with the α7-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 α7-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 α7-nAChR, at reasonable concentrations. This suggests that despite the high degree of homology of α7-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 α7-nAChR function. Under the present experimental conditions, we found that only arachidonic acid had significant effects on α7-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 α7-nAChRs, suggesting that it was the intact endocannabinoid and not the metabolite, arachidonic acid that altered α7-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 α7-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 α7-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, α7-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 α7-nAChRs in these critical locations, it is possible that the activity of the α7-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 α7 nicotinic receptors expressed in Xenopus oocytes. In addition, the results of studies with chimeric α7-nACh-5-HT3 receptor suggest that the site of this interaction is at the transmembrane or C-terminal domain of the α7-nACh receptor. Collectively, these data suggest that α7-nACh receptors may represent targets for the endocannabinoids in the intact nervous system.
Acknowledgments
We thank Dr. Lindstrom for kindly providing the cDNA clone of α7 subunit of nACh receptors and Dr. David Julius for providing 5-HT3 receptor cDNA.
Footnotes
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doi:10.1124/jpet.104.067751.
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ABBREVIATIONS: Δ9-THC, Δ9-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, 1α,2β-(R)-5α]-(-)-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.
- Received February 27, 2004.
- Accepted April 13, 2004.
- The American Society for Pharmacology and Experimental Therapeutics