Address correspondence and reprint requests to Dr. B. L. Hungund at NYS Psychiatric Institute at NKI, Bldg. no. 39, 140 Old Orangeburg Road, Orangeburg, NY 10962, U.S.A.
Abstract : In an earlier study, we demonstrated that chronic ethanol (EtOH) exposure down-regulated the cannabinoid receptors (CB1) in mouse brain synaptic plasma membrane. In the present study, we investigated the effect of chronic EtOH on the formation of anandamide (AnNH), an endogenous cannabimimetic compound, and its precursor N-arachidonoylphosphatidylethanolamine (N-ArPE) in SK-N-SH cells that were prelabeled with [3H]arachidonic acid. The results indicate that exposure of SK-N-SH cells to EtOH (100 mM) for 72 h significantly increased levels of [3H]AnNH and [3H]N-ArPE (p < 0.05) (1.43-fold for [3H]AnNH and 1.65-fold for [3H]N-ArPE). Exposure of SK-N-SH cells to EtOH (100 mM, 24h) inhibited initially the formation of [3H]AnNH at 24 h, followed by a progressive increase, reaching a statistical significance level at 72 h (p < 0.05). [3H]N-ArPE increased gradually to a statistically significant level after 48 and 72 h (p < 0.05). Incubation with exogenous ethanolamine (7 mM) and EtOH (100 mM, 72 h) did not result in an additive increase in the formation of [3H]AnNH. The formation of [3H]AnNH and [3H]N-ArPE by EtOH was enhanced by the Ca2+ ionophore A23187 or by the depolarizing agent veratridine and the K+ channel blocker 4-aminopyridine. Further, the EtOH-induced formation of [3H]AnNH and [3H]N-ArPE was inhibited by exogenous AnNH, whereas only [3H]AnNH formation was inhibited by the CB1 receptor antagonist SR141716A and pertussis toxin, suggesting that the CB1 receptor and Gi/o protein mediated the regulation of AnNH levels. The observed increase in the levels of these lipids in SK-N-SH cells may be a mechanism for neuronal adaptation and may serve as a compensatory mechanism to counteract the continuous presence of EtOH. The present observation taken together with our previous results indicate the involvement of the endocannabinoid system in mediating some of the pharmacological actions of EtOH and may constitute part of a common brain pathway mediating reinforcement of drugs of abuse including EtOH.
Chronic EtOH has been shown to induce activation of phospholipase A2 (PLA2) in both in vivo and in vitro models (John et al., 1985 ; Stubbs et al., 1988 ; Hungund et al., 1994). Recently, we have demonstrated that chronic EtOH treatment leads to selective activation of arachidonic acid (AA)-specific PLA2 in human SK-N-SH cells and mouse brain (Basavarajappa et al., 1997, 1998b). Arachidonylethanolamide (AnNH), also known as anandamide, an endogenous cannabimimetic substance, has been identified as a naturally occurring brain constituent (Devane et al., 1992). AnNH has been shown to bind specifically to cannabinoid receptors (CB1) in the brain and to mimic many of the pharmacological and behavioral effects, including the reinforcing effect of ▵9-tetrahydrocannabinol, the active ingredient of marijuana and other synthetic agonists (Pertwee, 1995). CB1 receptors belong to the G protein-coupled receptors (Matsuda et al., 1990) and are known to share G proteins and/or effector molecules with other G protein-coupled receptors. We have documented that chronic EtOH administration results in down-regulation of CB1 receptors in mouse synaptic plasma membranes (Basavarajappa et al., 1998a). Furthermore, the CB1 receptor antagonist SR141716A has recently been shown to block voluntary EtOH intake in rats (EtOH-preferring) (Colombo et al., 1998) and mice (Arnone et al., 1997), suggesting the role of a common brain pathway mediating reinforcement of drugs of abuse including alcohol.
The present study was undertaken to examine whether chronic EtOH treatment has any effect on the formation of CB1 receptor agonist AnNH and its precursor N-arachidonoylphosphatidylethanolamine (N-ArPE) by using human neuroblastoma cells. The findings suggest that EtOH treatment increased the synthesis of AnNH and its precursor, N-ArPE, in SK-N-SH cells. The results also indicate that AnNH levels may be regulated by CB1 receptors and Gi/o proteins. AnNH and N-ArPE levels are stimulated by EtOH and may be regulated by Ca2+ ions.
MATERIALS AND METHODS
All plastic culture supplies were purchased from Falcon Labware (VWR Scientific, Piscataway, NJ, U.S.A.). Dulbecco's modified Eagle's medium (DMEM), heat-inactivated fetal bovine serum (FBS), and streptomycin and penicillin solutions were from GIBCO (Grand Island, NY, U.S.A.). Human neuroblastoma cells (SK-N-SH) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, U.S.A.). The cells used for the current studies were under passage 42. Liquid scintillation cocktail (Liquiscint) was purchased from National Diagnostics (Atlanta, GA, U.S.A.). AA, AnNH, and N-ArPE were obtained from Avanti Polar Lipids (Alabaster, AL, U.S.A.). [3H]AA (200 Ci/mmol) was purchased from American Radiolabeled Chemicals (St. Louis, MO, U.S.A.). All other chemicals were obtained from Sigma (St. Louis, MO, U.S.A.).
Neuroblastoma cell culture
Neuroblastoma cells (strain SK-N-SH) were cultured in DMEM supplemented with 10% FBS, penicillin (50 IU/ml), streptomycin (50 μg/ml), and 2 mM glutamine in a dish (Falcon, Meylan, France) maintained at 37°C in a humidified atmosphere of 5% CO2/95% air. Cultures were grown to 80% confluence, replaced with fresh DMEM supplemented with 10% FBS, penicillin (50 IU/ml), streptomycin (50 μg/ml), and 2 mM glutamine.
The cells were incubated with [3H]AA (1 μCi/ml) in 0.1% FBS/DMEM for 5 h. Media were monitored for uptake and 85% of the total AA was incorporated into the cells within 5 h of incubation. The cells were then washed three times with 0.1% bovine serum albumin/DMEM to remove essentially all of the free AA. The cells were then subjected to EtOH exposure. The culture conditions were similar to the ones described by us and others for chronic EtOH investigations (Hoffman et al., 1996 ; Basavarajappa et al., 1997), and EtOH levels (50, 100, and 150 mM) were maintained by adding appropriate concentrations of absolute alcohol. All dishes were flushed with 5% CO2/95% air and sealed with parafilm before incubation. The medium was then supplemented at 24 and 48 h with absolute EtOH (4 μl/5 ml for 50 mM ; 25 μl/5 ml for 100 mM ; and 30 μl/5 ml for 150 mM). EtOH level in the media at 72 h was determined by an enzymatic method (50 ± 3.0 mM ; 100 ± 8.0 mM ; and 150 ± 15.0 mM, n = 6). Appropriate control cultures were similarly maintained in a medium containing no EtOH. The cultures were incubated for various times in DMEM (5 ml) containing drugs at the indicated concentrations. Veratridine, A23187, phenylmethylsulfonyl fluoride (PMSF), and SR141716A were added from stock solutions in dimethyl sulfoxide. Final concentration of dimethyl sulfoxide never exceeded 0.2%, and this concentration had no effect on cell viability. For treatment with various agents, culture plates containing PMSF (50 μM) were cotreated with ethanolamine (7 mM), AnNH (100 μM), veratridine (20 μM), 4-aminopyridine (4-AP ; 3 mM), SR141716A (1 μM), or pertussis toxin (PTX ; 100 ng/ml), with or without EtOH. For Ca+ ionophore A23187 treatment, cells were first exposed to EtOH (100 mM, 72 h) and then to A23187 (10 μM) for 10 min. Cells were examined for viability by trypan blue exclusion and counted. PMSF (50 μM) was included in the incubation medium in all the experiments. The incubations were terminated by collecting the media. The cells were scraped and then suspended in 10 mM Tris-HCl buffer, pH 7.4, containing protease inhibitor cocktail (Sigma). Data (media or cells) are expressed as dpm per milligram of cellular protein (mean ± SEM values).
Extraction and chromatography
Lipids from cells and media were extracted by using a mixture of chloroform/methanol (2:1, vol/vol) (Bligh and Dyer, 1959) ; 2 μg of unlabeled anandamide was included as a carrier. Butylated hydroxytoluene (0.05%) was added to prevent lipid peroxidation. The dried extracts were redissolved in a mixture of chloroform/methanol (2:1, vol/vol), spotted onto TLC plates (silica gel 60, 250 μm thickness), and developed with one of the following solvent systems : chloroform/methanol/acetic acid (90:6:6, by volume) (solvent system A), which allows the separation of N-ArPE (Rf = 0.344) from AnNH (Rf = 0.566) and AA (Rf = 0.735) ; chloroform/methanol/acetic acid (85:15:1, by volume) (solvent system B), which allows separation of N-ArPE (Rf = 0.25) from AnNH (Rf = 0.63) and AA (Rf = 0.70) ; or the organic phase of a mixture of isooctane/ethylacetate/water/acetic acid (50:110:100:20, by volume) (solvent system C), which resolves AnNH (Rf = 0.372) from AA (Rf = 0.776). As initial analysis yielded identical results with all the three solvent systems, solvent system A was used for subsequent assays. All other phospholipids remained at the origin with all the solvent systems used. The band corresponding to authentic N-ArPE, AnNH, and AA were scraped off the plate and solubilized with tissue solubilizer (NCS-II, Amersham). The radioactivity was determined using 10 ml of scintillation liquid.
Protein concentration of the cell homogenates was determined by the procedure of Lowry et al. (1951), using bovine serum albumin as the standard.
Statistical analysis was performed using Student's t test and one-way ANOVA followed by Dunnett's test, using GraphPad Prism version 2.01 software program (GraphPad Software, San Diego, CA, U.S.A.). Differences were considered significant if p < 0.05. Data are presented as mean ± SEM values from at least three separate experiments run in triplicate, unless otherwise indicated.
Effect of EtOH on cell viability
Human neuroblastoma (SK-N-SH) cells were exposed to various concentrations of EtOH (50, 100, and 150 mM for 72 h) and also 100 mM EtOH for various times (24, 48, and 72 h). The cell viability was unaffected irrespective of the treatment conditions used as determined by the trypan blue exclusion method (data not shown).
Effect of EtOH on AnNH and N-ArPE formation
A higher basal level of [3H]AnNH (1.5-fold) but not [3H]N-ArPE was observed when cells were cotreated with PMSF (50 μM), an amidohydrolase inhibitor (data not shown). Incubation of cells with PMSF (50 μM) produced no cell death and no lifting of the cell monolayer. Hence, in all experiments PMSF (50 μM) was included in the incubation medium. We investigated the effect of EtOH on [3H]AnNH and [3H]N-ArPE formation, using SK-N-SH cells prelabeled with [3H]AA. Treatment with EtOH caused a concentration- and time-dependent increase in the formation of [3H]AnNH and its precursor [3H]N-ArPE (Figs. 1A and B and 2). The results also indicated that EtOH treatment of SK-N-SH cells did not have any significant effect on the levels of the two lipids at 50 mM for 72 h (p > 0.05) but produced a statistically significant increase at 100 mM for 72 h (p < 0.05). Exposure of SK-N-SH cells to EtOH (100 mM, 24 h) initially inhibited the formation of AnNH (Fig. 2). AnNH and N-ArPE increased gradually, reaching a statistical significance at 100 mM after 48 and 72 h (p < 0.05). Most of the synthesized [3H]AnNH was released from the cells and accumulated in the media (60-70%) at 72 h. As most [3H]N-ArPE remained in the cells (90%), data are shown as the total (media plus cells) (Figs. 1B and 2).
Effect of various agents on EtOH-induced formation of [3H]AnNH and [3H]N-ArPE
When SK-N-SH cells were incubated with ethanolamine (7 mM) plus EtOH (100 mM) for 72 h, the basal and EtOH-induced levels of [3H]AnNH remained unchanged, and only a higher level of [3H]N-ArPE was observed in EtOH-treated cells (p < 0.05). In a similar manner, when cells were cotreated with EtOH (100 mM) and cold AnNH (100 μM) for 72 h, exogenous AnNH inhibited significantly (p < 0.05) the EtOH-induced formation of both [3H]AnNH and [3H]N-ArPE without affecting the basal level (Fig. 3A and B).
To determine the possible mechanism of EtOH-induced formation of AnNH and N-ArPE, we treated cells with various agents that are known to influence the formation of AnNH and N-ArPE (Di Marzo et al., 1994 ; Cadas et al., 1996). When chronic EtOH-exposed cells were treated with A23187 (10 μM) for 10 min, in addition to its stimulatory action on control cells (2.0-fold for AnNH and 2.4-fold for N-ArPE), A23187 was potent at enhancing EtOH-induced formation of [3H]AnNH (3.3-fold) and [3H]N-ArPE (3.6-fold) (Fig. 4A and B). When cells were cotreated with a Na+ channel activator, veratridine (20 μM), or a K+ channel blocker (4-AP, 3 mM), a significant increase in [3H]AnNH (veratridine control, 1.3-fold ; veratridine + EtOH, 2.0-fold ; 4-AP control, 1.3-fold ; 4-AP + EtOH, 2.7-fold) and [3H]N-ArPE (veratridine control, 2.0-fold ; veratridine + EtOH, 2.6-fold ; 4-AP control, 1.7-fold ; 4-AP + EtOH, 2.4-fold) levels was observed in the presence or absence of EtOH (Fig. 4A and B).
Effect of PTX and CB1 receptor antagonist SR141716A on EtOH-induced formation of [3H]AnNH and [3H]N-ArPE
We studied the effect of PTX to determine further whether EtOH-induced formation of AnNH and N-ArPE in SK-N-SH cells is receptor/G protein mediated. Coexposure of cells to PTX (100 ng/ml) and EtOH (100 mM) for 72 h did not affect the cell viability. Our results show that PTX inhibited the EtOH-induced formation of [3H]AnNH (100%) without affecting the formation of [3H]N-ArPE. It is interesting that cotreatment of SK-N-SH cells with CB1 receptor antagonist SR141716A (1.0 μM) and EtOH (100 mM) for 72 h blocked (100%) the EtOH-induced formation of [3H]AnNH without affecting the cell viability and the synthesis of [3H]N-ArPE (Fig. 5A and B).
The SK-N-SH cell culture system used in the current studies has been used for investigating the cellular and molecular events that underlie EtOH intoxication, tolerance, and withdrawal (Charness et al., 1994 ; Basavarajappa et al., 1997 ; Ding et al., 1997). Although chronic EtOH treatment activates AA-specific PLA2 in SK-N-SH cells and mouse brain, the significance of this activation is not clear at the present time (Basavarajappa et al., 1997, 1998b). For the first time, we have presented here evidence that chronic EtOH exposure induces the synthesis of AnNH and its precursor, N-ArPE, in SK-N-SH cells. Recent findings by us that chronic EtOH down-regulated the CB1 receptors (Basavarajappa et al., 1998a) and the inhibition of EtOH intake by CB1 receptor antagonist SR141716A in rats (Colombo et al., 1998) and mice (Arnone et al., 1997) suggest a role for the endocannabinoid system in the pharmacology of EtOH actions.
Our results suggest that EtOH treatment leads to the enhanced formation of AnNH and its precursor, N-ArPE, in a dose- and time-dependent manner. The increased synthesis of AnNH may be an adaptation mechanism to the continuous presence of EtOH. Several N-acylethanolamines share the ability to bind to and activate cannabinoid receptors in the brain and in peripheral tissues. AnNH and other polyunsaturated N-acylethanolamines also activate CB1 receptors (for review, see Mechoulam et al., 1994). Although the levels of N-ArPE and AnNH are low in normal tissues, their levels increase significantly during cell injury, tissue degeneration, and the postmortem period (Schmid et al., 1990, 1995 ; Felder et al., 1996 ; Kempe et al., 1996). The exact mechanism by which AnNH is synthesized has not been clearly established. The available evidence suggests that AnNH could be synthesized by the following two pathways : (1) ATP-and CoA-independent condensation between AA and ethanolamine (Deutsch and Chin, 1993 ; Devane and Axelrod, 1994 ; Kruszka and Gross, 1994), and (2) calcium-dependent, phosphodiesterase-mediated hydrolytic cleavage of a phospholipid precursor such as N-ArPE (Di Marzo et al., 1994). The natural occurrence of N-ArPE has been shown in rat brain (Cadas et al., 1997) and its in vitro synthesis has been demonstrated in various tissues and cells (Cadas et al., 1996 ; Di Marzo et al., 1996b ; Sugiura et al., 1996). The occurrence of related N-acylphosphatidylethanolamines has been demonstrated (Matsumoto and Miwa, 1973 ; Epps et al., 1980). In the present study, when SK-N-SH cells were incubated with ethanolamine and EtOH (100 mM, 72 h) no significant additional synthesis of AnNH occurred. Under similar conditions, EtOH treatment has been shown to activate Ca2+-dependent, AA-selective PLA2, resulting in the release of AA in these cells (Basavarajappa et al., 1997). These results suggest that the biosynthesis of AnNH by these cells does not occur through the condensation between ethanolamine and AA. It has been shown that the biosynthesis of AnNH by N18TG2 mouse neuroblastoma cells and J774 mouse macrophages did not follow the condensation pathway (Di Marzo et al., 1996a). It is interesting that chronic EtOH-induced formation of AnNH was accompanied by increased formation of N-ArPE in SK-N-SH cells, suggesting the activation of one or more N-acyltransferase enzymes or intermolecular transfer of a fatty acyl group from glycerophospholipid to ethanolamine moiety of phosphatidylethanolamine (Natarajan et al., 1982 ; Schmid et al., 1990 ; Cadas et al., 1996 ; Di Marzo et al., 1996b ; Sugiura et al., 1996). Cellular localization and physiological regulation of N-ArPE remains unknown and such a mechanism of N-ArPE formation by chronic EtOH must be investigated further. However, EtOH has been shown to activate more than one acyltransferase as an adaptation to the continuous presence of EtOH (Le Petit-Thevenin et al., 1995 ; Zheng et al., 1996). The formation of N-ArPE was accompanied by a formation of AnNH, suggesting that N-ArPE-specific phospholipase D (PLD) may be constitutively active in chronic EtOH-exposed SK-N-SH cells. Although several studies have shown the activation of PLD in various EtOH models (Kiss, 1992 ; Lundqvist et al., 1994 ; Gustavsson, 1995), the involvement of an N-ArPE-specific PLD-like enzyme must be established. Stimulus-induced (glutamate-, ionophore-, and membrane-depolarizing agents) formation of AnNH and N-ArPE has been suggested (Di Marzo et al., 1994 ; Cadas et al., 1996 ; Hansen et al., 1997). Formation of N-acylphosphatidylethanolamine/N-acylethanolamine has been shown in response to cell injury (Schmid et al., 1990 ; Hansen et al., 1995).
Further, the substances that enhance the EtOH-induced formation of AnNH, including the Ca2+ ionophore A23187 and chemically unrelated membrane-depolarizing agents (veratridine and 4-AP), are also effective at increasing the biosynthesis of N-ArPE in SK-N-SH cells in culture (Fig. 4A and B). AnNH and N-ArPE biosynthesis, induced by a similar ionophore and membrane-depolarizing agents, occurs in various neuronal cells (Di Marzo et al., 1994 ; Hansen et al., 1995 ; Cadas et al., 1996) and in cell homogenate (Di Marzo et al., 1996a, b). It has been shown that chronic EtOH exposure leads to an increase in intracellular Ca2+ levels in mouse brain synaptosomes (Friedman et al., 1980), rat cerebellar macroneurons (Zou et al., 1995), PC12 cells (Belia et al., 1995), and cerebral vascular smooth muscle cells (Zhang et al., 1997, 1998). Based on these data, it is reasonable that EtOH might increase the formation of N-ArPE and AnNH in SK-N-SH cells in culture by increasing the intracellular Ca2+.
Our results with SR141716A and PTX suggest that the level of AnNH in chronic EtOH-treated SK-N-SH cells may be regulated by CB1 receptors and Gi/o proteins. These results, along with the data presented in Fig. 3, suggest the presence of a possible negative feedback mechanism for AnNH biosynthesis (Childers and Deadwyler, 1996), to protect neurons from insult such as the continuous presence of EtOH. These findings support a CB1 receptor-mediated stimulation of AnNH synthesis by tetrahydrocannabinol (Hunter and Burstein, 1997). It is noteworthy that chronic EtOH has been shown to decrease cyclic AMP levels (Gordon et al., 1986 ; Rabin et al., 1992), and cannabinoids also have been shown to decrease cyclic AMP levels by inhibiting the adenylate cyclase through Gi/o protein (Howlett and Fleming, 1984 ; Howlett et al., 1986 ; Bidaut-Russell et al., 1990). Moreover, the CB1 receptor antagonist SR141716A was shown to block voluntary EtOH intake in rats (EtOH-preferring) (Colombo et al., 1998) and mice (Arnone et al., 1997), suggesting that the endocannabinoid system may constitute part of a common brain pathway mediating reinforcement of drugs of abuse including alcohol. These results suggest a possible role for endogenously formed AnNH and its signal transduction system in alcohol tolerance and dependence. Further studies with in vivo models of the impact of the CB1 receptor agonist and antagonist on both pharmacological and behavioral effects will enhance the understanding of the mechanism involving the CB1 receptor-second messenger system in the development of tolerance to EtOH. Studies along these lines are currently in progress in our laboratory.
We thank Thomas B. Cooper, Chief, Department of Analytical Psychopharmacology, NYS Psychiatric Institute at NKI, for his continued support and encouragement throughout this study, Dr. Henry Sershen, Center for Neurochemistry, NKI, Orangeburg, NY, U.S.A., for helpful discussion during the preparation of the manuscript, and Dr. Mitsuo Saito, Division of Neurobiology, NKI, Orangeburg, NY, U.S.A., for providing the cell culture facility. This study was supported in part by a grant from New York State Psychiatric Institute.