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NEUROPHARMACOLOGY

The Endogenous Cannabinoid Anandamide Inhibits 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 Section (M.O.), Cellular Neurophysiology Section (O.D.R., M.M.), Baltimore, Maryland; National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health/Department of Health and Human Services, Laboratory of Molecular and Cellular Neurobiology (A.R., L.Z.), Bethesda, Maryland

Received February 4, 2003; accepted May 13, 2003.

	Abstract

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The effect of the endogenous cannabinoid ligand anandamide on the function of the cloned ₇ subunit of the nicotinic acetylcholine (ACh) receptor expressed in Xenopus oocytes was investigated by using the two-electrode voltage-clamp technique. Anandamide reversibly inhibited nicotine (10 µM) induced-currents in a concentration-dependent manner (10 nM to 30 µM), with an IC₅₀ value of 229.7 ± 20.4 nM. The effect of anandamide was neither dependent on the membrane potential nor meditated by endogenous Ca²⁺ dependent Cl^- channels since it was unaffected by intracellularly injected BAPTA and perfusion with Ca²⁺-free bathing solution containing 2 mM Ba²⁺. Anandamide decreased the maximal nicotine-induced responses without significantly affecting its potency, indicating that it acts as a noncompetitive antagonist on nicotinic acetylcholine (nACh) ₇ receptors. This effect was not mediated by CB₁ or CB₂ receptors, as neither the selective CB₁ receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride (SR 141716A) nor CB₂ receptor antagonist N-((1S)-endo-1,3,3-trimethyl-bicyclo-heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR 144528) reduced the inhibition by anandamide. In addition, inhibition of nicotinic responses by anandamide was not sensitive to either pertussis toxin treatment or to the membrane permeable cAMP analog 8-Br-cAMP (0.2 mM). Inhibitors of enzymes involved in anandamide metabolism including phenylmethylsulfonyl fluoride, superoxide dismutase, and indomethacin, or the anandamide transport inhibitor AM404 did not prevent anandamide inhibition of nicotinic responses, suggesting that anandamide itself acted on nicotinic receptors. In conclusion, these results demonstrate that the endogenous cannabinoid anandamide inhibits the function of nACh ₇ receptors expressed in Xenopus oocytes in a cannabinoid receptor-independent and noncompetitive manner.

Nicotinic acetylcholine (nACh) receptors containing the ₇ subunit are members of a ligand-gated ion channel family that has been proposed to mediate pre- and postsynaptic effects of nicotine in both the central and the peripheral nervous systems (McGehee et al., 1995; Role and Berg, 1996). The potential involvement of nACh ₇ receptors in pain transmission, neurodegenerative diseases, and drug abuse has been reported (Damaj et al., 2000; Orr-Urteger et al., 2000; Picciotto et al., 2001). Furthermore, biochemical and behavioral studies have demonstrated functional interactions between nicotine and cannabinoid receptor ligands (Pryor et al., 1978; Valjent et al., 2002). In the present study, we have tested the hypothesis that some of the actions of the endogenous cannabinoid anandamide may be mediated by nACh ₇ receptors. For this purpose, the cRNA encoding functional nACh ₇ receptor was homeometrically expressed in Xenopus oocytes, and the effect of anandamide on the function of nACh ₇ receptors was investigated. Preliminary results of this study were presented in abstract form (Oz et al., 1996).

	Materials and Methods

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Mature female Xenopus laevis frogs were purchased from Xenopus laevis I (Ann Arbor, MI) and were housed in dechlorinated tap water at 19-21°C with a 12:12-h light/dark cycle and fed with beef liver twice a week. Clusters of oocytes were surgically removed form the frogs under tricaine (Sigma-Aldrich, St. Louis, MO) local anesthesia (0.15% w/v). 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). Dissected oocytes were then 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. Oocytes were placed in a 0.2-ml recording chamber and superfused at a rate of 4 to 5 ml/min. The bathing solution consisted of 95 mM NaCl; 2 mM KCl, 2 mM CaCl₂, and 5 mM HEPES (pH 7.4). The cells were impaled at the animal pole with two glass microelectrodes filled with a 3 M KCl (1-10 M). The oocytes were routinely voltage clamped at a holding potential of -70 mV using a GeneClamp-500 amplifier (Axon Instruments, Inc., Burlingame, CA), and current responses were directly recorded on a Gould 2400 rectilinear pen recorder (Gould, Inc., Cleveland, OH). Agonists and antagonists were applied by gravity flow via a micropipette positioned about 2 mm from the oocyte. Some of the compounds were applied externally by addition to the superfusate. All chemicals used in preparing the solutions were from Sigma-Aldrich. Anandamide, R-(+)-methanandamide, (-)-nicotine, 8-Br-cAMP, and -bungarotoxin were from Sigma/RBI (St. Louis, MO). SR 141716A and SR 144528 were provided by NIDA Drug Supply System (NIH, Baltimore, MD). Both SR 141716A and SR 144528 were originally synthesized by Research Triangle Institute (Research Triangle Park, NC) on behalf of NIDA. AM404 was purchased from Tocris Cookson (St. Louis, MO). Procedures for the injections of pertussis toxin (PTX) (50 nl, 50 µg/ml) or BAPTA (50-100 nl, 100 mM) were performed as described previously (Oz et al., 1998). PTX was dissolved in distilled water; BAPTA was prepared in Cs₄-BAPTA. Injections were performed 1 h before recordings using oil-driven ultra microsyringe pump (Micro4; WPI, Inc. Sarasota, FL). Stock solutions of anandamide were prepared in dimethyl sulfoxide at a concentration of 100 mM. Dimethyl sulfoxide did not affect nicotinic responses when added at concentrations up to 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. The cDNA clone of the chick nACh₇ subunit was provided by Dr. Lindstrom (University of Pennsylvania, Philadelphia, PA). 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.

Data Analysis. Average values were calculated as the mean ± standard error (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: y = E_max/(1 + [x/EC₅₀]^-n), 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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At the highest concentration used in this study, nicotine (300 µM) did not cause detectable currents in uninjected oocytes (n = 5) or in oocytes injected with distilled water (n = 3). Application of 10 µM nicotine for 3 to 5 s activated fast inward currents that desensitized rapidly in oocytes injected with cRNA transcribed from cDNA encoding the ₇ subunit of the nACh receptor. Moreover, nicotine-induced inward currents were abolished completely with 10 nM -bungarotoxin (n = 3, data not shown), indicating that these responses are mediated by -bungarotoxin sensitive neuronal ₇ nACh receptor-ion channel complex.

Incubation of oocytes with 10 nM to 30 µM of anandamide for 20 to 30 min caused a gradually developing inhibition of nicotine-induced ion currents. The inhibition was partially reversible and concentration-dependent. The effect of the 30-min incubation with 300 nM anandamide on the nicotine-induced ion current is shown in Fig. 1A. The recovery was complete within 20 to 30 min. A time course of the effect of anandamide on the peak amplitudes of nicotine-induced currents is presented in Fig. 1B. Anandamide by itself (30 µM for 15 to 30 min) did not alter the magnitudes of holding-currents in oocytes voltage-clamped at -70 mV (n = 5).

View larger version (13K): [in this window] [in a new window]   Fig. 1. The effect of anandamide on nACh 7 receptor-mediated ion currents. A, records of currents activated by nicotine (10 µM) in control conditions (left), during coapplication of 300 nM anandamide and nicotine after a 30-min pretreatment with 300 nM anandamide (middle), and 40 min following anandamide washout (right). B, time course of the effect of 300 nM anandamide on the peaks of the nicotine induced currents. Each data point represents the normalized mean ± S.E. of four to six experiments. Duration of anandamide application is indicated by the horizontal bar in the figure.

We found that the threshold concentration for the inhibitory effect of anandamide on ₇ nACh receptor-mediated currents was 10 nM, and the maximum inhibitory effect was achieved at concentrations between 10 to 30 µM (Fig. 2). The inhibition of nicotine (10 µM)-induced current by anandamide was concentration-dependent, with an IC₅₀ value of 229.7 ± 20.4 nM and a slope value of 0.92 ± 0.05 (n = 4).

View larger version (13K): [in this window] [in a new window]   Fig. 2. Concentration-dependence of anandamide inhibition of nACh 7 receptor-mediated ion currents. Data points are the mean ± S.E. of four to seven oocytes. The curve is the best fit of the data to the logistic equation described under Materials and Materials. The IC50 value and slope factor derived from four oocytes were 229.7 ± 20.4 and 0.92 ± 0.05 nM (n = 4).

To investigate whether endogenous cannabinoid-like receptors in oocytes mediates the effects of anandamide, we tested the effect of SR 141716A, a selective antagonist of CB₁ receptors, on the maximal amplitudes of nicotine-induced currents. Application of 1 µM SR 141716A for 30 min did not significantly alter the amplitudes of peak currents in response to nicotine (Fig. 3A; P > 0.05, n = 6, paired t test). In another set of experiments, preincubation and continuous incubation of 1 µM SR 141716A for 30 min did not affect anandamide-inhibition of nicotine-induced currents, and the magnitudes of the inhibition by anandamide were not significantly different in the absence and presence of SR 141716A (Fig. 3A; P > 0.05, Student's t test, n = 6). The CB₂ receptor antagonist, SR 144528, was also tested to determine whether CB₂ receptors are involved in anandamide inhibition of nicotinic responses. Application of 1 µM SR 144528 for 30 min did not significantly alter the maximal amplitudes activated by nicotine (Fig. 3A; P > 0.05, n = 5, paired t test). Furthermore, the anandamide inhibition of nicotine-induced currents was not affected by 1 µM SR 144528, and the magnitudes of the inhibition 30 min following anandamide application were not significantly different in the absence and presence of SR 144528 (Fig. 3A; P > 0.05, Student's t test, n = 5).

View larger version (28K): [in this window] [in a new window]   Fig. 3. Effects of CB1 receptor antagonist SR 141716A, CB2 receptor antagonist SR 141528, cAMP regulating agents, and inhibitors of anandamide metabolism on the inhibition of nACh 7 receptor-mediated ion currents by anandamide. A, the effects of a 30-min application of CB1 receptor antagonist SR 141716A (1 µM) or CB2 receptor antagonist SR 144528 (1 µM) on nACh 7 receptor-mediated ion currents in the absence and presence of anandamide (300 nM). Bars represent the mean ± S.E. The numbers of experiments are presented on top of each bar. B, the effects of a 30-min application of 8-Br-cAMP (0.2 mM) and PTX injections on the time course of nACh 7 receptor-mediated ion currents in the absence and presence of anandamide. Bars represent the mean ± S.E. The numbers of experiments are presented on top of each bar. C, time course of the maximal amplitudes of nicotine induced currents during a 30-min application of 300 nM anandamide in oocytes injected with 50 nl distilled water and recorded in 2 mM Ca2+ containing MBS solution () or 50 nl of BAPTA (100 mM, ) injected and recorded in 2 mM Ba2+ containing MBS solution. Each data point represents the normalized mean ± S.E. of five to six experiments. D, effects of anandamide on nACh 7 receptor-mediated currents in the presence of PMSF, SOD, indomethacin, or AM404. Bars represent the means ± S.E. The numbers of experiments are presented on top of each bar. There was no statistically significant difference on the anandamide inhibition in the presence or the absence of agents applied for 30 min (P > 0.05, n = 5-8, ANOVA).

A functional cAMP pathway and its modulation by pharmacological agents including 8-Br-cAMP and forskolin have been reported in earlier studies in oocytes (Schorderet-Slatkine and Baulieu, 1982; Sadler and Maller, 1983; Oz et al., 2002b). Given the link between cannabinoids and the cAMP pathway (Martin et al., 1994), we investigated whether a membrane permeable analog of cAMP, 8-Br-cAMP (0.2 mM) was able to modulate the inhibitory effect of anandamide. As shown in Fig. 3B, 8-Br-cAMP had no effect on nicotinic responses under control conditions. In addition, 8-Br-cAMP did not alter the anandamide inhibition of nicotinic currents (Fig. 3B). As certain types of G-proteins involving the signaling of cannabinoid-like receptor mediated-events have been shown to be sensitive to PTX treatments (Martin et al., 1994; Di Marzo and Fontana, 1995), we tested the effect of anandamide in distilled-water and PTX injected oocytes expressing nACh ₇ receptors. At the end of the 30-min incubation periods, there was no significant difference in anandamide inhibition between controls and PTX injected cells (Fig. 3B; P > 0.05, Student's t test, n = 5).

Since activation of nACh ₇ receptors allows sufficient Ca²⁺ entry to activate endogenous Ca²⁺-dependent Cl^- channels in Xenopus oocytes (Séguéla et al., 1993; Sands et al., 1993), it was important to determine whether the effect of anandamide was exerted on nicotinic-receptor mediated currents or on 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 to Ba²⁺ replacement, a small contribution of remaining Ca²⁺-dependent Cl^- channel activity has been shown to be abolished by the injection of Ca²⁺ chelator, BAPTA (Sands et al., 1993). For this reason, we tested the effect of anandamide in a solution containing 2 mM Ba²⁺ in BAPTA injected oocytes. Anandamide (300 nM) produced the same level of inhibition (45 ± 4% of controls) on nicotine-induced currents when BAPTA injected oocytes were recorded in Ca²⁺-free, 2 mM Ba²⁺-containing solutions (Fig. 3C).

Anandamide may regulate the function of nACh ₇ receptors either directly or via breakdown products generated by hydrolytic and/or oxidative metabolism of anandamide. To distinguish between these possibilities, a series of compounds were included in anandamide-containing solutions: the amidohydrolase inhibitor phenylmethylsulfonyl fluoride (PMSF, 0.2 mM) was used to examine the possible hydrolysis of anandamide to arachidonic acid; the cyclooxygenase inhibitor indomethacin (5 µM) was used to investigate the possible involvement of endogenous prostaglandin synthesis from anandamide; superoxide dismutase (SOD, 25 U/ml) was used to examine the involvement of free-radical generation on anandamide hydrolysis. In addition, we tested the effect of AM404, anandamide membrane transport inhibitor (Beltramo et al., 1997) on the amplitudes of nicotine-induced currents. Application of 10 µM AM404 alone for 30 min did not affect the maximal amplitudes of nicotine-induced currents (n = 5, 102 ± 3% of controls). In the presence of AM404, anandamide inhibited the nicotine-induced responses to 45 ± 4% of controls (n = 4). The results of these experiments indicated that compared with anandamide alone, the presence of PMSF, indomethacin, SOD, or AM404 did not affect the inhibitory effects of anandamide on nACh ₇ receptors-mediated ion currents (n = 4-5, P > 0.05, ANOVA; Fig. 3D). We also tested the effect of R-methanandamide, a metabolically stable chiral analog of anandamide that is more resistant than anandamide to hydrolytic inactivation by fatty acid amide hydrolase (Abadji et al., 1994). Application of 300 nM R-methanandamide for 30 min inhibited the nicotine-induced responses to 38 ± 4% of controls (n = 3).

It has been reported that the effects of some compounds such as local anesthetics or polyamines on ligand-gated ion channels are sensitive to membrane potential (Hille, 2001). For this reason, voltage-dependence of the anandamide inhibition was examined. Each tested membrane potential was held for 30 s and then returned to -70 mV. As indicated in Fig. 4A, the inhibition of nicotine (10 µM)-induced currents by anandamide (300 nM) does not appear to be voltage-dependent. The extent of the anandamide inhibition was similar at all tested membrane potentials (from -80 to +20 mV). In the presence of anandamide, there was no change on the reversal potentials of nicotine-activated ion currents (6 ± 2 mV in controls versus 8 ± 3 mV in anandamide), indicating that the ionic selectivity of the channel was not affected by anandamide. Evaluation of data from current-voltage relationship indicated that the extent of the inhibitory effect of anandamide did not change significantly at different holding potentials (Fig. 4B; P > 0.05, n = 5, ANOVA). By definition, an open-channel blockade requires the opening of the channel by the binding of agonist to the receptor. In the absence of agonist, pretreatment with a blocker should not cause inhibition. In experiments conducted with this in mind, the extent of anandamide inhibition was compared in oocytes stimulated with 10 µM nicotine at 10-min intervals with those stimulated at 30-min intervals. During application of 300 nM anandamide for 30 min, anandamide was equally effective in inhibiting the currents activated at 10- and 30-min intervals. At 10- and 30-min intervals between nicotine applications, nicotine-induced currents were reduced to 44 ± 5 and 48 ± 7% of controls, respectively (Fig. 4C; P > 0.05; Student's t test, n = 6), indicating that the channel does not need to be opened by the agonist for anandamide to be effective.

View larger version (12K): [in this window] [in a new window]   Fig. 4. Anandamide inhibition of nicotine-induced currents is independent of membrane-potential. A, current-voltage relationships of nicotine-activated currents in the absence and presence of anandamide. Normalized currents activated by 10 µM nicotine in BAPTA-injected oocytes before (control, ) and after a 30-min treatment with anandamide (300 nM, ) in Ca2+-free 2 mM Ba2+ containing solutions. Each data point presents the normalized means and SE of four to six experiments. B, quantitative evaluation of the effects of anandamide is presented as percent inhibition at different voltages indicated in the figure. C, lack of effect of nicotine stimulation on anandamide inhibition of nACh 7 receptor-mediated responses. The percentage of anandamide inhibition on the nACh 7 receptor-mediated currents recorded at the end of a 30-min application period was not different between oocytes stimulated and not stimulated with nicotine application every 10 min (n = 5, P > 0.05, Student's t test). Duration of anandamide application is indicated by the horizontal bar in the figure.

Anandamide may decrease the binding of the agonist to the receptor by acting as a competitive antagonist. For this reason, the effect of anandamide was examined at different concentrations of nicotine. Concentration-response curves for nicotine in the absence and presence of 300 nM anandamide are presented in Fig. 5. Anandamide did not cause any shift on the concentration-response curve but inhibited the maximal response induced by nicotine to about 45% of controls (n = 4-7). In the presence and absence of anandamide, the EC₅₀ values were 10.8 ± 2.7 and 12.3 ± 3.2 µM (n = 4), and slope values were 1.4 ± 0.1 and 1.6 ± 0.2 (n = 4), respectively, suggesting that anandamide inhibits the nicotine responses in a noncompetitive manner. In addition, we have tested the effect of anandamide on currents induced by ACh (50 and 500 µM), endogenous activators of nACh ₇ receptors. Maximal amplitudes of currents induced by 50 and 500 µM ACh were inhibited to 46 ± 4 (n = 5) and 42 ± 5% (n = 5) of controls, respectively.

View larger version (17K): [in this window] [in a new window]   Fig. 5. Concentration-response curves for peak nicotine-induced currents in the presence and absence of 300 nM anandamide. Oocytes were voltage-clamped at -70 mV and currents were activated by applying nicotine (1-300 µM). Oocytes were exposed to anandamide for 30 min, and nicotine was reapplied. Paired concentration-response curves were constructed and responses normalized to maximal response under control conditions. EC50 and slope values were determined by fitting the curves from four oocytes to the standard logistic equation, as described under Materials and Methods.

	Discussion

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Our observations indicate that anandamide, at nanomolar concentrations, inhibits the function of neuronal nACh ₇ receptor expressed in Xenopus oocytes. The lowest concentration of anandamide producing inhibition of nicotine-induced currents was 30 nM, and inhibition reached maximal levels in concentration range of 10 to 30 µM. The effect of anandamide was concentration-dependent with an EC₅₀ value of 218 nM and a slope factor of 0.94. Increases in the concentration of nicotine did not overcome the anandamide inhibition of nicotine-induced ion currents. These results suggest that anandamide inhibited nicotine-induced responses in a noncompetitive manner.

Anandamide, at the concentration range used in this study, has been shown to bind cannabinoid receptors (Devane et al., 1992). On the other hand, binding studies conducted in Xenopus oocytes indicate that cannabinoid receptors are not expressed endogenously in these cells (Henry and Chavkin, 1995). Thus, in our experiments, it is unlikely that the observed effects of anandamide were due to the activation of cannabinoid receptors. In line with this finding, the CB₁ antagonist SR 141716A and CB₂ antagonist SR 144528 did not affect anandamide-induced inhibition of nACh ₇ responses. Furthermore, activation of cannabinoid-like receptors, if present, would be predicted to decrease intracellular cAMP concentrations in a PTX-sensitive manner. Neither the application of membrane permeable analog of cAMP, 8-Br-cAMP, nor preincubation with PTX affected the peak amplitudes of nACh ₇ receptor-mediated ion currents. The observation that 8-Br-cAMP and PTX treatments did not mimic the inhibitory effect of anandamide and that anandamide continues to inhibit the activation of nACh ₇ receptors following pretreatments with these agents suggests that the inhibition of nACh ₇ receptors is independent of CB₁ and/or CB₂ cannabinoid receptor-dependent second messengers.

In Xenopus oocytes, activation of nACh ₇ receptors, due to their high Ca²⁺ permeability, allows sufficient Ca²⁺ entry to activate endogenous Ca²⁺-dependent Cl^- channels (Sands et al., 1993; Séguéla et al., 1993). In oocytes injected with BAPTA and recorded in solution containing 2 mM Ba²⁺, anandamide continued to inhibit ₇ nACh receptor-mediated ion currents, suggesting that increases in intracellular Ca²⁺were not involved in anandamide inhibition of nicotinic responses. The inhibition of nACh ₇ receptor-mediated ion currents by anandamide was not affected by membrane potential, indicating that the effect of anandamide was voltage-independent. In addition, the reversal potential in solutions containing Ba²⁺ was not altered in the presence of anandamide, suggesting that the inhibitory effect of anandamide is not due to alterations in the Ba²⁺ permeability of the nACh ₇ receptor-channel complex.

During our experiments, application of anandamide, even at the highest concentration, did not cause any change in baseline holding currents, suggesting that the intracellular concentration of Ca²⁺ was not affected. Since the Ca²⁺-activated Cl^- channels are highly sensitive to intracellular levels of Ca²⁺ (for a review, see Dascal, 1987), the release of Ca²⁺ from internal stores of this ion would be reflected by the changes in the holding current under voltage-clamp conditions. In line with these observations, at the concentration range used in this study, anandamide does not affect intracellular Ca²⁺ levels in Chinese hamster ovary cell lines (Felder et al., 1993). Cannabinoid receptor-independent effects of anandamide have been reported in several earlier studies (Venance et al., 1995; Poling et al., 1996; Oz et al., 2000; Van den Bossche and Vanheel, 2000; Chemin et al., 2001; Maingret et al., 2001; Oz et al., 2002a).

So far, two different sets of physiological stimuli have been associated with anandamide synthesis (for a review, Wilson and Nicoll, 2002). Anandamide synthesis can be triggered in response to depolarization and subsequent influx of Ca²⁺ (Di Marzo et al., 1994; Piomelli et al., 1998). Alternatively, synthesis of anandamide and/or other endocannabinoids by postsynaptic activation of neurotransmitter receptors, such as group 1 metabotropic glutamate receptors (Varma et al., 2001; Maejima et al., 2001) and D₂ type dopamine (Giuffrida et al., 1999), or by coactivation of NMDA and nACh ₇ receptor (Stella and Piomelli, 2001) has been shown to occur in neuronal preparations. Recent studies indicate that anandamide is synthesized postsynaptically and modulates synaptic communication in a retrograde fashion regardless of the triggering stimuli (for a review, see Wilson and Nicoll, 2002). Several earlier studies have reported that in the central nervous system, nACh ₇ receptors are located presynaptically and play an important modulatory role in synaptic transmission (McGehee et al., 1995; Girod et al., 2000; Dani, 2001; Mang et al., 2001). Therefore, it is possible that the activity of nACh ₇ receptors can be modulated by anandamide released from postsynaptic sites that in turn, modulates the presynaptic release of neurotransmitters. Alternatively, increased anandamide synthesis triggered by the activation of NMDA and nACh ₇ receptors (Stella and Piomelli, 2001) may cause a feedback inhibition of nACh ₇ receptors.

Synthesis of anandamide has been linked to increase of intracellular Ca²⁺ levels (Di Marzo et al., 1994; Piomelli et al., 1998). We present several lines of evidence indicating that the endogenous formation of anandamide does not play a role in the observed effects of anandamide on nicotinic responses in oocytes, however. First, applications of nicotine (10 µM) every 10 min did not cause any inhibition of nicotinic responses, suggesting that the synthesis of endogenous cannabinoids due to increased intracellular Ca²⁺ levels during nicotinic receptor activation either did not occur or did not achieve levels sufficient to cause a significant effect on nicotinic responses. In line with this observation, in oocytes injected with BAPTA and recorded in solution containing 2 mM Ba²⁺, anandamide continued to inhibit ₇ nACh receptor-mediated ion currents, suggesting that increases in intracellular Ca²⁺ were not involved in anandamide inhibition of nicotinic responses. In addition, during repeated nicotine applications, holding current that would be affected by increased intracellular Ca²⁺ levels (Dascal, 1987) did not change significantly during the course of experiments. Second, inhibition of anandamide amide hydrolase by PMSF or the inhibition of anandamide transporter by AM404 did not alter anandamide inhibition of nicotinic responses. It is likely that sustained and/or global increases in intracellular Ca²⁺levels would be required for anandamide synthesis. In line with this hypothesis, a recent study in rat cortical neurons reports that although the ACh alone did not affect anandamide levels, coapplication of ACh and NMDA (N-methyl-D-aspartate) caused a 5-fold increase in anandamide formation (Stella and Piomelli, 2001). In another recent study, however, chronic nicotine treatments have been shown to cause an increase (in brain stem), a decrease (in hippocampus, limbic forebrain, and cerebral cortex), or no change (cerebellum, midbrain, and diencephalon) in anandamide levels measured in rat brain (Gonzalez et al., 2002).

Anandamide is structurally similar to other fatty acids such as arachidonic acid and prostaglandins. Many of these fatty acids have been shown to modulate the function of muscle-type nACh receptors in earlier studies (for a review Barrantes, 1993). In later studies, several fatty acids including arachidonic acid and prostaglandin E₂ have been shown to modulate the function of nACh ₇ receptors directly (Vijayaraghavan et al., 1995; Nishizaki et al., 1998; Tan et al., 1998; Du and Role, 2001). Thus, it is likely that the effects of anandamide and other fatty acids share some common mechanisms of action on nACh receptors.

	Acknowledgements

 
We thank Dr. Lindstrom (University of Pennsylvania, Philadelphia, PA) for kindly providing the cDNA clone of ₇ subunit of nACh receptors and Dr. Alex Hoffman of NIDA/NIH for helpful reading of the manuscript.

	Footnotes

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

DOI: 10.1124/jpet.103.049981.

ABBREVIATIONS: nACh, nicotinic acetylcholine; MBS, modified Barth's solution; SR 141716A, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride; SR 144528, N-((1S)-endo-1,3,3-trimethyl-bicyclo-heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide; PTX, pertussis toxin; BAPTA, 1,2-bis(o-aminophenoxy)ethane-N, N,N',N'-tetraacetic acid; ANOVA, analysis of variance; PMSF, phenylmethylsulfonyl fluoride; SOD, superoxide dismutase.

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

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