Introduction

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Dopaminergic synaptic transmission in the mesolimbic and mesocortical system is considered a critical neurobiological component for behaviors caused by drugs of abuse (Robbins and Everitt, 1999). Over the past decade, it has been repeatedly shown in animals that acute administration of addicting drugs changes dopaminergic neurotransmission (Robinson and Berridge, 1993). Experiments with rodent models of alcohol-seeking behavior provide considerable evidence that dopamine D2 (D2) receptors are involved in ethanol self-administration and ethanol stimulation of locomotion (Hodge et al., 1997). Furthermore, recent studies show that alcohol preference and sensitivity are markedly reduced in mice lacking D2 receptors (Phillips et al., 1998). Despite the importance of dopamine in mediating responses to ethanol, little is known about the regulation of dopamine receptor responses after ethanol administration.

In many tissues, cell lines, and brain, activation of the D2 receptor inhibits adenylyl cyclase via the GTP-binding protein G_i (Obadiah et al., 1999). Activation of D2 receptors also produces other intracellular events, including decreases in Ca²⁺ (Lledo et al., 1992), activation of K⁺ channels (Waszczak et al., 1998), increases in inositol phosphate levels, release of arachidonic acid (Piomelli et al., 1991), and translocation of protein kinase C (Gordon et al., 2001).

Most investigators find that exposure to ethanol potentiates and then desensitizes receptor-stimulated cAMP production via G_s (Diamond and Gordon, 1997). It is possible that ethanol also modulates D2 receptor coupling to adenylyl cyclase. In this study, we used NG108-15 cells stably transfected with the long form of the D2 (D2L) receptor to investigate how ethanol alters D2 receptor coupling to adenylyl cyclase. We show here that ethanol added acutely prevents D2L receptor inhibition of cAMP production, whereas chronic exposure to ethanol diminishes ethanol sensitivity of D2L receptor coupling (tolerance), thereby enabling the D2 receptor agonist NPA to inhibit cAMP production. Acute ethanol does not change NPA binding to D2 receptor but abolishes GTP-induced changes toward a low-affinity state; chronic ethanol is without effect. Since ethanol also affects PKA localization and activity in these cells (Coe et al., 1996; Dohrman et al., 1996), we determined whether ethanol sensitivity of D2L receptor function involves PKA. We found that PKA appears to be required for acute ethanol inhibition of D2L receptor coupling to adenylyl cyclase. Activation of PKA reverses chronic ethanol-induced tolerance of D2L receptor coupling to adenylyl cyclase.

	Materials and Methods

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Materials. [³H]-L-()-N-propylnorapomorphine ([³H]NPA; PerkinElmer Life Science Products, Boston, MA), S-()-raclopride L (+)-tartrate salt (Sigma/RBI, Natick, MA), prostaglandin E1 (PGE₁; Sigma, St. Louis, MO), R-()-2,10,11-trihydroxy-N-propylnorapomorphine hydrobromide (NPA), carbachol, UK-14304, and enkephalin (D-Ala2, D-Leu5) (DADLE) were purchased from Sigma/RBI. Rp-cAMPS and Sp-cAMPS were purchased from Biolog (La Jolla, CA). Assay media consisted of Dulbecco's modified Eagle's medium/F-12 (3:1) containing 25 mM HEPES (pH 7.4) and 0.1 mM Ro 20-1724, a selective inhibitor of cAMP-specific phosphodiesterase (BIOMOL Research Laboratories, Plymouth Landing, PA).

Cell Culture. NG108-15 cells stably expressing the D2L receptor (NG108-15/D2L) (Asai et al., 1998) or cells stably transfected with vector alone (vector) were grown in 10% Nu-serum (Collaborative Research, Waltham, MA) and maintained for 3 days in complete defined medium as previously described (Asai et al., 1998). The cells were then seeded in six-well plates (Falcon, Franklin Lakes, NJ) at a density of 6 × 10⁴ cells/well and used for acute ethanol experiments on day 5. Media were replaced daily. For chronic ethanol experiments, cells seeded as above were treated on day 3 for an additional 3 days with or without 150 mM ethanol. The media were replaced daily.

Dopamine D2L Receptor Binding. Cells were subcultured in T175 flasks at 5.0 × 10⁶ cells/flask and grown for 3 days in complete defined medium. They were then re-seeded in T175 flasks at 3.0 × 10⁶ cells/flask for an additional 3 days in complete defined medium. For chronic ethanol experiments, cells were treated with or without 150 mM ethanol for 72 h. Crude membrane fractions were prepared and D2 receptor binding activity was measured as described by Monsma et al. (1989). Membranes (0.3 mg of protein/tube) were preincubated in 0.5 ml of binding buffer for 10 min at 37°C. After addition of an equal volume of the D2-specific agonist [³H]NPA dissolved in 50 mM Tris-HCl buffer (pH 7.4) containing 1 mM EDTA, 120 mM NaCl, 5 mM KCl, 4 mM MgCl₂, and 5.7 mM ascorbic acid, the membranes were incubated with or without 200 mM ethanol for 30 min at 37°C in the presence or absence of 100 µM GTPS. Nonspecific binding was measured in the presence of 10 µM raclopride. The reaction was terminated by filtration through glass fiber filters pretreated with 0.05% polyethyleneimine, followed by four washes with ice-cold 50 mM Tris-HCl buffer (pH 7.4).

Inhibition of Adenylyl Cyclase Activity by Dopamine D2L, M4 Muscarinic, Adrenergic 2_b, or -Opioid Receptor Agonists. Cells were incubated for 30 min at 37°C in 1 ml of assay medium/well containing 10 µM PGE₁ (Moylan and Brooker, 1981) in the presence or absence of 5.0 × 10⁸ M NPA (D2 receptor agonist), 1 × 10⁵ M carbachol (acetylcholine receptor agonist), 1 × 10⁵ M UK-14304 (2_b adrenergic receptor agonist), or 1 × 10⁶ M DADLE (-opioid receptor agonist). The concentrations of agonists were approximately 100 times the reported K_d values. The cells were then lysed with 100 µl of ice-cold 2% Nonidet P-40 in 1 N HCl. cAMP levels were measured by radioimmunoassay as previously described (Gordon et al., 1986). Acute experiments with ethanol were carried out by incubating with 1 ml of assay medium containing 10 µM PGE₁ with or without receptor agonists in the presence or absence of various concentrations of ethanol for 30 min. NPA concentration dependence was analyzed by incubating cells in 1 ml of assay medium containing 10 µM PGE₁ and increasing concentrations of NPA for 30 min in the presence or absence of 200 mM ethanol. The cells were lysed and adenylyl cyclase activity measured. For chronic ethanol experiments, cells were treated with or without 150 mM ethanol for 72 h, and adenylyl cyclase activity was measured as described above.

Protein Assay. Protein was measured with a Bio-Rad protein assay kit (Bio-Rad, Richmond, CA) with bovine gamma globulin as standard.

	Results

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D2L Receptors Inhibit Adenylyl Cyclase Activity. The D2 receptor has been shown to couple to G_i to inhibit adenylyl cyclase (Malek et al., 1993). To determine whether D2L receptors expressed in stably transfected NG108-15 cells are functionally coupled, the D2 receptor agonist NPA (5 × 10⁸ M) was used to activate D2L receptors and inhibit 10 µM PGE₁-stimulated adenylyl cyclase activity. Activation of D2L receptors reduced cAMP levels by 35 ± 2% (Fig. 1). NPA also decreased basal cAMP levels by 33 ± 4% in the absence of PGE₁ (Fig. 1). However, NPA had no effect on PGE₁-stimulated adenylyl cyclase activity or basal cAMP levels in stable clones isolated from NG108-15 cells transfected with vector alone.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   D2L receptors are functionally coupled to adenylyl cyclase. NG108-15/D2L cells were incubated for 30 min at 37°C with or without NPA for 30 min in the presence of 10 µM PGE1. Data shown are the means ± S.E. of seven experiments. *p < 0.05, **p < 0.01, compared with paired cells transfected with vector alone (Student's t test).

Ethanol Interaction with D2L and Other G_i-Coupled Receptors. To determine how acute ethanol exposure affects D2 receptor coupling to adenylyl cyclase, NPA inhibition of PGE₁-stimulated cAMP production was measured as described in the legend of Fig. 1 in the presence or absence of 25 to 200 mM ethanol. Ethanol inhibited D2L receptor coupling to adenylyl cyclase as a function of increasing ethanol concentration (Fig. 2A). Significant inhibition occurred at 25 mM ethanol. We have observed that acute ethanol also prevented D2 receptor inhibition of adenylyl cyclase (data not shown) in CHO cells overexpressing the D2L receptor (Fishburn et al., 1995). To determine whether ethanol competes with NPA for binding, PGE₁-stimulated cAMP production was assayed as a function of increasing NPA concentration in the presence or absence of ethanol. Increasing concentration of NPA did not overcome ethanol inhibition of D2L coupling to adenylyl cyclase (Fig. 2B).

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Acute ethanol inhibits coupling of D2L receptors to adenylyl cyclase. A, NPA inhibition of PGE1-stimulated cAMP production as a function of ethanol concentration. NG108-15/D2L cells exposed to increasing concentrations of ethanol for 30 min showed increasing inhibition of D2L receptor coupling to adenylyl cyclase. NPA was used as described in Fig. 1. Separate controls for ethanol-induced increases in PGE1-stimulated cAMP accumulation were subtracted from the results. Data shown are the means ± S.E. of six experiments. **p < 0.01, compared with control cells (one way analysis of variance and Dunnett's test). B, NPA inhibition of PGE1-stimulated cAMP production as a function of NPA concentration in the presence or absence of 200 mM ethanol. Data shown are the means ± S.E. of three experiments. *p < 0.05, **p < 0.01, compared with cells without NPA treatment (one way analysis of variance and Dunnett's test).

View this table: [in this window] [in a new window]   TABLE 1 Effect of acute ethanol exposure on Gi-coupled receptors NG108-15/D2L cells were incubated with or without agonists in the presence or absence of 200 mM ethanol for 30 min. The inhibition of PGE1-stimulated cAMP production was measured. Data shown are the means ± S.E.

Acute Ethanol Inhibition of D2L Receptor Coupling Is Abolished by Chronic Exposure to Ethanol (Tolerance). In many systems, chronic exposure to ethanol results in tolerance to rechallenge with ethanol, i.e., acute treatment with ethanol produces a diminished response when presented following prolonged exposure. We therefore determined whether D2L receptor coupling to G_i develops tolerance to ethanol inhibition. Cells were preincubated in the presence of 150 mM ethanol for 72 h, a blood alcohol concentration that can be encountered in chronic alcoholics (Messing and Diamond, 1997). Table 2 shows that acute treatment with ethanol inhibits D2L receptor coupling to adenylyl cyclase. After chronic exposure to ethanol, however, even rechallenge with high concentrations of ethanol no longer abolishes D2L receptor inhibition of PGE₁-stimulated cAMP production (Table 2). Therefore, it seems that D2L receptor coupling to adenylyl cyclase becomes tolerant to ethanol inhibition after prolonged exposure to ethanol.

Ethanol Alters D2L Receptor Coupling to G_i but not D2 Receptor Binding. The binding affinity of D2L receptors in membranes from stably transfected cells was assayed using [³H]NPA. Scatchard analysis of saturation binding data (Fig. 3A) revealed a K_d of 0.27 ± 0.01 nM and B_max of 23 ± 5 fmol/mg of protein (Table 3). Addition of 100 µM GTPS produced a lower-affinity state (K_d of 1.12 ± 0.06) but did not change B_max (Fig. 3A; Table 3). This is consistent with findings in CHO cell membranes expressing D2L receptors (Vanhauwe et al., 1999). GTP induces a shift in D2 receptor agonist affinity in CHO cell membranes from a high- to a low-affinity state.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   D2L receptor binding in stably transfected NG108-15 cells is unaffected by ethanol. Membranes prepared from NG108-15/D2L cells were incubated with [3H]NPA (0.1-5.0 nM) with or without 200 mM ethanol in the presence or absence of 100 µM GTPS. A, acute exposure to ethanol for 30 min at 37°C had no effect on the Kd (0.26 ± 0.03 nM) or the Bmax (23 ± 1 fmol/mg of protein). However, acute ethanol exposure abolished the shift toward lower affinity produced by GTPS (Kd, 1.12 ± 0.06; Bmax, 25 ± 2). Data are representative of three independent experiments. The Kd and Bmax values are listed in Table 3. B, membranes prepared from NG108-15/D2L cells exposed to 150 mM ethanol for 3 days were incubated with [3H]NPA (0.1-5.0 nM) with or without 200 mM ethanol in the presence or absence of 100 µM GTPS. Chronic ethanol exposure had no effect on the Kd or Bmax (Kd, 0.24 ± 0.01; Bmax, 23 ± 1) and no effect on the GTP-induced shift in D2 agonist affinity (Kd, 1.11 ± 0.06; Bmax 26 ± 2). Rechallenging the chronically treated cells with 200 mM ethanol did not change the shift toward lower affinity produced by GTPS (Kd, 1.00 ± 0.07; Bmax, 26 ± 3). Data are representative of three independent experiments. The Kd and Bmax values are listed in Table 3.

View this table: [in this window] [in a new window]   TABLE 3 D2L receptor binding kinetics Membranes prepared from stably transfected NG 108-15 cells were assayed for binding to [3H]NPA with or without 200 mM ethanol in the presence or absence of 100 µM GTPS for 30 min at 37°C. Chronic EtOH refers to cells incubated with 150 mM ethanol for 72 h. Values represent the mean ± S.E. of three experiments.

Ethanol Inhibition of D2L Receptor Coupling Requires PKA. PKA affects several membrane protein responses to ethanol, including the GABA_A receptor (Freund and Palmer, 1997) and the adenosine transporter (Coe et al., 1996). We used the PKA inhibitor, Rp-cAMPS, to determine whether PKA regulates acute ethanol sensitivity of D2L receptor coupling to adenylyl cyclase. Table 4 shows that Rp-cAMPS abolished acute ethanol sensitivity of D2L receptor coupling to adenylyl cyclase. This suggests that PKA may be required for acute ethanol inhibition of D2L receptor coupling to adenylyl cyclase.

View this table: [in this window] [in a new window]   TABLE 4 Ethanol inhibition of D2L receptor coupling requires PKA Cells were preincubated with or without the PKA inhibitor, Rp-cAMP (20 µM) (Coe et al., 1996) for 1 h and then incubated for an additional 30 min in the presence or absence of 200 mM ethanol and NPA as described in Fig. 1 and PGE1-stimulated cAMP production measured. Data shown are the means ± S.E. n = 4.

PKA Activation Reverses Tolerance of D2L Receptor Coupling to Ethanol Inhibition. We next determined whether activation of PKA in NG108-15/D2L cells chronically treated with ethanol could restore ethanol sensitivity of D2L receptor coupling after tolerance developed to ethanol. Cells were incubated with or without 150 mM ethanol for 72 h. Medium was then removed, and cells were incubated for 10 min in the presence or absence of Sp-cAMPS, a PKA agonist. The cells were then rechallenged with acute ethanol, and D2L receptor inhibition of adenylyl cyclase was measured. As shown in Table 2 and Fig. 4, chronic exposure to ethanol abolishes sensitivity of D2L receptor coupling to ethanol. However, when chronically exposed cells were treated with Sp-cAMPS for 10 min, sensitivity of D2L receptors to ethanol inhibition of receptor coupling to adenylyl cyclase was restored (Fig. 4). Sp-cAMPS had no effect on NPA inhibition of cAMP production (data not shown) or on acute ethanol sensitivity of D2L receptor coupling in control cells (Fig. 4). Rp-cAMPS did not affect chronically treated cells (data not shown).

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Sp-cAMPS reverses tolerance of D2L receptor coupling and restores sensitivity to acute ethanol inhibition. Cells were treated with or without 150 mM ethanol for 72 h and then incubated for 10 min in the presence or absence of 1 mM Sp-cAMPS (Coe et al., 1996), followed by incubation for 30 min with () or without () 200 mM ethanol and NPA as described in Fig. 2. Data shown are the means ± S.E. of three experiments. *p < 0.05, compared with NPA inhibition of PGE1-stimulated cAMP in control cells (two way analysis of variance and Newman-Keuls test).

	Discussion

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The major finding in this study is that acute exposure to ethanol prevents D2L receptor inhibition of cAMP production and that D2L receptor coupling to adenylyl cyclase becomes tolerant to ethanol inhibition after prolonged exposure.

We used a stable cell line of NG108-15 cells expressing dopamine D2L receptors at a density comparable with that found in central nervous system neurons (Joyce et al., 1991). G_i-coupled D2L receptors are functionally active because NPA activation significantly reduces cAMP production (Fig. 1). Inhibition of adenylyl cyclase by the transfected D2L receptors was comparable with inhibition produced by activation of endogenously expressed opioid receptors in the same cells (Table 1; Charness et al., 1983), suggesting that receptor responses were unaffected by transfection. Acute exposure to ethanol largely abolishes D2L receptor coupling so that NPA no longer inhibits adenylyl cyclase (Table 1). Parenthetically, ethanol added acutely to NG108-15 cells does increase PGE₁-stimulated accumulation of cAMP (Table 1). This is consistent with our earlier studies showing that ethanol increased receptor-stimulated cAMP accumulation (Gordon et al., 1986). Basal cAMP levels are also increased by ethanol in these experiments, but the effect is trivial when compared with PGE₁-stimulated cAMP production (Gordon et al., 1986). Ethanol inhibition is a function of increasing ethanol concentration (Fig. 2A) and does not appear to be due to changes in D2L receptor concentration (Fig. 3; Table 3) or competition for binding with NPA (Fig. 2B). However, ethanol does appear to prevent GTP-induced changes in D2L receptor binding toward a lower-affinity state (Vanhauwe et al., 1999) (Fig. 3A; Table 3). We propose the possibility, therefore, that ethanol may be acting at sites downstream from the D2L receptor to uncouple receptor activation from inhibition of adenylyl cyclase.

Receptor-mediated inhibition of adenylyl cyclase usually involves activation of a G_i subunit. O'Hara et al. (1996) found that the D2L receptor can couple effectively to G_i2 and G_i3 to inhibit adenylyl cyclase activity. The data of Senogles (1994) suggest that there is differential coupling of D2 receptor isoforms in the pituitary cell line GH₄Cl. She found that the short form of the D2 receptor is coupled to G_i2, whereas the D2L receptor interacts with G_i3. NG108-15 cells express G_i3, as well as G_i2 and G_o (Roerig et al., 1992). In this study using NG108-15 cells, therefore, it is possible that D2L receptors could couple to G_i3 or G_i2, since both are present in these cells. However, G_o is also present and might be involved.

The finding that acute ethanol can abolish dopamine receptor-dependent G_i coupling in cells stably transfected with the D2L receptor suggested that the coupling of other receptors to G_i might also be inhibited by ethanol. NG108-15/D2L cells endogenously express muscarinic M4, -opioid, and 2_b-adrenergic receptors, all linked to G_i (Graeser and Neubig, 1993). We found that acute ethanol inhibits D2L and -opioid receptor signaling but has no effect on 2_b-adrenergic or M4 muscarinic receptor function (Table 1). One possibility is that D2L and -opioid receptors are more vulnerable to ethanol because they are less potent inhibitors of adenylyl cyclase. Alternatively, as described for the D2L receptor, there is evidence that -opioid receptors also couple to G_i3 (Sanchez-Blazquez and Garzon, 1993). In this setting, it may be significant that 2_b-adrenergic receptors and M4 muscarinic receptors interact with G_i2 (McClue and Milligan, 1990; Migeon and Nathanson, 1994), not G_i3. Therefore, it is possible that G_i3, which can couple to D2L and -opioid receptors, confers vulnerability to inhibition of coupling by ethanol.

After NG108-15/D2L cells are exposed to ethanol for several days, ethanol no longer blocks D2L receptor coupling. Therefore, D2L receptor coupling via G_i/_o to adenylyl cyclase has become tolerant to ethanol, and D2L receptor inhibition of cAMP production is restored to levels encountered in naïve control cells. It is possible the development of tolerance is related, in part, to the failure of ethanol to inhibit the GTP shift to a lower D2 receptor binding affinity (Vanhauwe et al., 1999) (Fig. 3B; Table 3). Tolerance of D2L receptor coupling to ethanol inhibition may have relevance to drinking behaviors. Koob and Moal (1997) postulate a spiraling dysregulation of brain reward systems that progressively increases drug use by addicts. Since dopamine D2 receptors appear to reinforce responses to ethanol (Robbins and Everitt, 1999), ethanol-induced tolerance of D2 receptor coupling might be involved in this postulated phenomenon. Ethanol-induced tolerance of D2 receptor coupling restores D2 receptor responses to that found in naïve cells. Perhaps this somehow facilitates a desire for drinking in chronic alcoholics that leads to more alcohol consumption. In vivo studies will be needed to test this possibility in experimental models of ethanol self-administration. Note, however, that other neurotransmitter systems, including those for N-methyl-D-aspartate, GABA, serotonin, opioids, and adenosine, also play a major role in ethanol-seeking behavior (Diamond and Gordon, 1997).

Neurotransmitter-stimulated cAMP signaling and protein kinase activity appear to regulate neuronal responses to ethanol and alcohol-seeking behaviors (Moore et al., 1998; Thiele et al., 2000). PKA activation potentiates GABA_A sensitivity to ethanol-induced chloride flux (Freund and Palmer, 1997) and is required for ethanol inhibition of adenosine uptake in NG108-15 cells (Coe et al., 1996). Expression of D2L receptors in these cells allowed us to test whether PKA also regulates ethanol sensitivity of D2L receptor coupling to adenylyl cyclase. In the presence of the PKA inhibitor Rp-cAMPS, ethanol added acutely no longer inhibited D2L receptor coupling to adenylyl cyclase (Table 4). In these cells, prolonged exposure to ethanol induced tolerance of D2L receptor coupling to ethanol inhibition (Table 2). Rp-cAMPS had no effect on chronic ethanol-induced tolerance to ethanol (data not shown). However, after a 10-min incubation with Sp-cAMPS, a PKA agonist, the sensitivity of D2L receptor coupling to ethanol was restored (Fig. 4). Sp-cAMPS had no effect on acute ethanol sensitivity of D2L receptor coupling (Fig. 4). Consistent with the data in Fig. 4, we propose the possibility that PKA-mediated phosphorylation may be required for ethanol inhibition of D2L receptor coupling to adenylyl cyclase. We also propose that diminished PKA activity correlates with ethanol-induced tolerance of D2L receptors. We have observed a parallel requirement for PKA for ethanol inhibition of adenosine uptake and a reduction of PKA associated with the development of tolerance to ethanol inhibition (Coe et al., 1996). This could be explained by our findings in NG108-15 cells that chronic exposure to ethanol causes 75% of PKA to be redistributed into the nucleus (Dohrman et al., 1996) so that much less PKA is available to regulate plasma membrane protein functions. However, 25% of the PKA is still available to phosphorylate plasma membrane and other proteins when Sp-cAMPS is added (Fig. 4).

Abnormal cAMP/PKA signaling may be involved in several alcohol-induced changes in neural function and behavior. Sensitivity to alcohol intoxication is increased in Drosophila mutants that lack the gene "cheap date", an allele of amnesiac that regulates cAMP production (Moore et al., 1998). Sensitivity to ethanol sedation is reduced and ethanol intake increased in mice with reduced PKA in the striatum (Thiele et al., 2000). This may have genetic pathophysiologic significance since decreased sensitivity to intoxication appears to be a predictor for the development of alcoholism in normal young men with a positive family history for alcoholism (Raimo et al., 1999). This is consistent with the observation that blood cells from alcoholics show desensitization of cAMP production (Diamond et al., 1987; Menninger et al., 2000), just as in cultured cell lines chronically exposed to ethanol (Diamond et al., 1987). Taken together, these results suggest that ethanol-induced changes in cAMP levels and PKA activity may contribute significantly to the development of alcoholism. This may be particularly important in the nucleus accumbens, a region implicated in reinforcement and addiction (Self et al., 1998). Studies are underway to determine the role of ethanol-induced changes in cAMP signaling in nucleus accumbens neurons in animal models of alcoholism.