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Selective effects of ethanol on opiate receptor subtypes in brain
Neuropharmacology
Printed in Great
Vol. 26, Britain.
No. 10, pp. 1503-1507, All rights reserved
1987
Copyright 0
0028-3908/87 $3.00 + 0.00 1987 Pergamon Journals Ltd
SELECTIVE EFFECTS OF ETHANOL ON OPIATE RECEPTOR SUBTYPES IN BRAIN S. KHATAMI,’PAULA L. ‘Department of Biomedical IL 61107, U.S.A., ?%ction Studies, National Institute ‘Department
HOFFMAN?*T. SHIBUYA~and B. SALAFSKY’
Sciences, University of Illinois College of Medicine at Rockford, Rockford, on Receptor Mechanisms, Laboratory of Physiologic and Pharmacologic on Alcohol Abuse and Alcoholism, Bethesda, MD 20892, U.S.A. and of Pharmacology, Tokyo Medical College, Tokyo 160, Japan (Accepted 2 April 1987)
Snnunary-Large concentrations of ethanol in vitro decreased ligand binding to mu and delta opiate receptors in the frontal cortex of the C57BL mouse., but did not alter binding to kappa opiate receptors. Mu and delta receptors were equally sensitive to the inhibitory effect of ethanol. Since the effects of ethanol were significant only in large concentrations, ethanol may alter opiate binding through its membrane lipid-perturbing actions, and the selectivity of the effects of ethanol may reflect differences in the microenvironments of the opiate receptor subtypes in membranes. After chronic ingestion of ethanol by mice, in vim, there was a selective decrease in the number of mu receptors in the frontal cortex. This change may result from indirect effects of ethanol on the opiate receptor and may contribute to specific central effects of ethanol. Key words: ethanol, opiate receptors, kappa receptor, mu receptor, delta receptor.
Ethanol, in vitro, has been reported to alter opiate binding to receptors in brain tissue. In tissue from the striatum of the mouse ethanol had a biphasic effect on the binding of dihydromorphine (DHM): a small concentration of ethanol (50 mM) enhanced binding, while larger concentrations inhibited the binding of dihydromorphine. In contrast, ethanol only inhibited the binding of D-Ala*, D-Leu’-enkephalin (DADLE) to membranes from the striatum. The binding of DADLE was more sensitive to inhibition by ethanol than the binding of dihydromorphine (Tabakoff and Hoffman, 1983). Hiller, Angel and Simon (1981) also reported that ethanol selectively inhibited the binding of DADLE to opiate receptors in membranes of whole brain of the rat and had little effect on the binding of dihydromorphine. In the latter study, the binding of several ligands was examined and it was concluded that ethanol affected the properties of the binding site for enkephalin (delta receptor) rather than the ligands. In tissue from the striatum of mice that had ingested ethanol chronically, a decreased affinity of high-affinity binding sites for dihydromorphine and a decreased responsiveness of these receptors to the effects of sodium ions, was found (Hoffman, Urwyler and Tabakoff, 1982). No change in binding sites for enkephalin in the striatum was observed. In view of the greater sensitivity of delta opiate receptors in the striatum to ethanol in vitro, as compared to the mu receptors, this selective change in the properties of the mu receptor was unexpected. Gn the other hand, there have been some reports of changes in the ‘To whom all correspondence should be sent.
properties of delta receptors in membranes from the whole brain of rat (Gianoulakis, 1983) or from large areas of brain (Pfeiffer, Seizinger and Herz, 1981) after chronic ingestion of ethanol. Since subtypes of opiate receptors (Iwamoto and Martin, 1981; Wood, 1983; Chang and Cuatrecasas, 1979) are differentially distributed between regions of the brain (Chang, Cooper, Hazum and Cuatrecasas, 1979), the present authors wished to determine the acute and chronic effects of ethanol on the properties of opiate receptors in another area of the brain of the mouse. Because of interest in the effect of ethanol on regulation of dopaminergic activity by opiates (Hoffman et al., 1982; Urwyler and Tabakoff, 1981), the present study has investigated the effects of ethanol on opiate receptors in the frontal cortex that may modulate the activity of mesolimbic dopaminergic neurons (Pollard, Llorens, Bonnet, Costentin and Schwartz, 1977). METHODS Opiate binding
Mice were sacrificed by decapitation, the brains were removed and the frontal cortex was dissected. Tissue from 20 to 25 animals in each experiment was pooled and membranes from the brain were prepared as previously described (Tabakoff and Hoffman, 1983; Pasternak, Wilson and Snyder, 1975). Measurements of ligand binding were carried out in a total volume of 0.5 ml. Each assay contained, in 0.05 M Tris-HCl buffer (pH 7.7), 0.25 ml of homogenate (about 1 mg of protein/ml) (Lowry, Rosebrough, Farr and Randall, 195 1) and various concen-
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S. KHATAMI etal.
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trations of [‘HID-Ala2-MePhe4-GlyLol enkephalin PAGO) (New England Nuclear, Boston, Massachusetts, 30-60 Ci/mmol), [3H]r+Ala2-o-Leu5-enkephalin (DADLE) (New England Nuclear, 30-50 Ci/mmol) or [3H]bremazocine (BMZ) (New England Nuclear, 1S-30 Ci/mmol). For Scatchard analyses, the concentration range of [3H]DAG0 was 0.1-5 nM; of [3H]DADLE was 0.2-10 nM; and of [3H]bremazocine was OS-7 nM. Nonspecific binding was determined with 1 PM unlabelled DAGO, 1 PM unlabelled D-Se?-Leu5-enkephalin-Thr6 (DSLET), or 1 p M unlabelled [3H]bremazocine, respectively. The binding of [‘HIDADLE to delta receptors was assessed by performing binding assays in the presence of a IOO-fold excess of unlabelled DAGO, to mask mu receptor sites. Binding assays with [3H]bremazocine were performed in the presence of excess unlabelled DAGO and DSLET (Gillian, Kosterlitz and Paterson, 1980). Since the brain contains primarily mu, delta and kappa receptors (Wood, 1983), and [3H]bremazocine is a universal ligand (Simon, Hiller, Groth, Itzhak, Holland and Beck, 1982), the data obtained with [3H]bremazocine and the masking ligands are believed to reflect the binding of [3H]bremazocine to kappa receptors. The binding assays were carried out at 25°C for 75 min and were terminated by filtration of the tissue suspensions as previously described (Tabakoff and Hoffman, 1983; Pasternak et al., 1975). Bound radioactivity was quantified by scintillation counting. When the effect of ethanol in vitro was investigated, tubes were capped throughout the assay and binding in the presence and absence of ethanol was determined simultaneously. For dose-response studies of the effects of ethanol, ligand concentrations used were: [3H]DAG0, 0.2 nM; [3H]DADLE, 0.5 nM; (3H]bremazocine, 0.2nM. The binding of DADLE and [3H]bremazocine were assayed in the presence of excess unlabelled DSLET and/or DAGO, as described. The ligand concentrations were chosen to be
loo-
zQ-
Chronic ingestion of ethanol
Male C57BL mice (22-25 g) were housed 6 per cage in a laboratory environment (12-hr light cyctr , 22°C) for at least one week before use. In experiments to assess the chronic effects of ethanol, mice were housed individually and fed with ethanol in liquid diet, or control diet, for seven days, as previously described (Ritzmann and Tabakoff, 1976). The quantity of diet fed daily to the control animals was adjusted to equal the average daily consumption of the ethanol-consuming mice. On the morning of the eighth day, the diet containing ethanol was withdrawn and the mice were monitored for withdrawal symptoms (Ritzmann and Tabakoff, 1976). Mice were sacrified at 24 hr after withdrawal, when overt withdrawal symptoms had dissipated, but the mice were still tolerant to and physically dependent on ethanol (Ritzmann and Tabakoff, 1976). RESULTS
Scatchard plots of the binding of [3H]DAG0 and [3H]DADLE, the latter in the presence of an excess of unlabelled DAGO, appeared linear, while there was some indication of curvature in the Scatchard plot for the binding of [3H]bremazocine in the presence of excess DAGO and DSLET (Fig. 1). Analysis of the binding data using the LIGAND program (Munson and Rodbard, 1980) indicated that the data for the binding of [3H]DAG0 and [3H]DADLE were best fit by a one-site model. In two out of three cases, data for the binding of [‘Hlbremazocine were also best fit by a one-site model. In the instance where the binding data for [3H]bremazocine were better fit by a two-site model, there was a high degree of variability of the parameters for the low-affinity site (KD approximately 15 nM). For the purposes of these studies,
t”HIBMZ
C’HIDADLE
C3HlDAG0
g
3@-40% of the K, value, but still to allow easily detectable levels of ligand binding.
.5
loo
0
P
A 0
P .
0 .
&
50
SD
loo-
a
0 . 0
d d
0
‘5 A
\-x....-,, 50
SD Spectiic Swnd
50
100 IfmoWmg
100
150
protein1
Fig. 1. Scatchard plots of the binding of [3H]DAG0 (a), [‘HIDADLE (0) and [‘Hlbremaxocine ([3H]BMZ) (A) in tissue from the frontal cortex of C57BL mouse. Membrane preparation, binding assays and ligand concentrations used are described in the text. The binding of [3H]DADLE was assayed in the presence of high concentrations of unlabelled DAGO. The binding of [‘Hlbremazocine was assayed in the presence of large concentrations of unlabelled DAGO and DSLET. Values are from a single representative experiment; the data for all ligands were best fit by a one-site model.
Ethanol and opiate receptors
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Table I. Opiatebinding in the frontal cortexof C57BLmice Percentageof Bmm (fmol/mgprotein) total binding Ligand K. (nM) DAGO DADLE BMZ
0.63 + 0.03 1.15 f0.04 0.45 * 0.01
27% 38% 35%
87+4 12Ok8 116*2
Binding of [‘HIDAGO, [‘HIDADLE and [‘Hlbremazocine ([3H]BMZ) were assayed as described in the text. The binding of DADLE was assayed in the presence of unlabelled DAGO, to mask mu receptors, and the binding of [‘Hlbremazocine was assayed in the presence of DAGO and DSLET, to mask delta and mu receptors. Values represent mean f SEM from 3 experiments. tEthonol1
therefore, the binding data for [3H]bremazocine were assumed to be best fit by a one-site model. The kinetic parameters for the binding of [3H]DAG0, [3H]DADLE and [‘Hlbremazocine in frontal cortex of the C57BL mouse are shown in Table 1. The concentration of mu opiate receptors was smallest, while concentrations of delta and kappa receptors were larger (Table 1). Ethanol, added to binding assays in vitro, inhibited the binding of [3H]DAG0 and [3H]DADLE, but had no significant effect on the binding of [3H]bremazocine to the membranes from the frontal cortex (Fig. 2). The I&, value for the inhibition by ethanol of the binding of [3H]DAG0 was 592 (55&613) mM [95% confidence limits calculated by log-probit analysis of the average values from three inhibition curves in separate experiments; (Sokal and Rohlf, 1981)], and the I& for the inhibition by ethanol of the binding by DADLE was 789 (524-l 189) mM (also calculated from 3 experiments). Chronic treatment with ethanol resulted in tolerance to ethanol and physical dependence in the mice, as described earlier (Ritzmann and Tabakoff, 1976). The kinetic parameters of opiate binding to tissue of the frontal cortex of control mice and mice withdrawn from ethanol, at 24 hr after withdrawal, are shown in Table 2. The number of binding sites for [‘HIDAGO (mu binding sites) was significantly reduced in the mice withdrawn from ethanol, compared to controls. The number and KD of kappa binding sites in tissue of control animals were reduced in comparison to these parameters in naive, untreated animals (compare to Table 1). However, there was no statistically significant difference in the number of kappa or of delta binding sites and no significant
Table 2. Opiate binding
in the frontal
Ligand
K. (nM) 0.66 f 0.05 0.99 + 0.08 0.26 k 0.04
B_
of ethanol
difference in affinity for the ligand of any binding site, between the mice withdrawn from ethanol and control mice. The inhibition by ethanol of the binding of [3H]DAG0 and [3H]DADLE was unchanged after chronic ingestion of ethanol (DAGO: ICso = 478 mM in control animals and 458 mM in animals withdrawn from ethanol; DADLE: I&, = 534mM in control animals and 385 mM in animals withdrawn from ethanol; I&, values were calculated from log-probit analysis of mean values from three separate experiments and the 95% confidence limits (Sokal and Rohlf, 1981) showed no significant differences between control and animals withdrawn from ethanol). DISCUSSION
The binding data are consistent with the presence of at least three classes of opiate receptor in the frontal cortex of mouse, i.e. mu, delta and kappa opiate receptors. The percentage of each type of binding site was similar to that for membranes of whole brain of mouse (Wood, 1983) and was also similar to that found in the brain of rabbit (Ho, Hammonds and Li, 1985) in that the relative amount of mu receptors was less than that of delta or kappa
cortex of C57BL mice after chronic
ingestion
of ethanol
Ethanol-withdrawn
Control
DAGO DADLE BMZ
(mM)
on opiate binding to mu, delta and kappa receptors in the frontal cortex of mouse. Tissue was prepared and ligand binding was assayed in the presence of masking ligands, as described in the text. The concentrations of ligands used were: [3H]DAG0, 0.20 + 0.01 mM; [‘HIDADLE, 0.50 f 0.02 nM; and [‘Hlbremazocine ([3H]BMZ), 0.20 & 0.01 nM. Binding in the absence of ethanol was (in fmol/mg protein): [3H]DAG0, 19.1 k 1.1; [3H]DADLE 60.6 rf:9.4; [‘Hlbremazocine, 51.5 k 2.9 (mean +SEM from 3 experiments). Fig. 2. Effect
(fmol/mg
protein)
84 + 2 122 + 14 69 k 9
KD (nM) 0.69 k 0.07 0.88 f 0.03 0.39 * 0.05
B_
(fmol/mg
protein)
63f4* 112*21 71 *3
Binding assays were carried out as described in the text. Binding of [‘HIDADLE was assayed in the presence of excess unlabelled DACO, and binding of [‘Hlbremazocine ([‘HIBMZ) was assayed in the presence of excess unlabelled DAGO and DSLET. Mice were sacrificed at 24 hr after withdrawal from chronic ingestion of ethanol. Values represent mean * SEM from three experiments. *P < 0.01 compared to control (Student’s r-test).
1.506
S. KHATAMIet al.
receptors. The affinity of each ligand for its specific binding site was comparable to that reported in membranes of the brain of rat (Wood, 1983; Simon et al., 1982). The inhibitory effect of ethanol in vitro on the binding of DADLE to the delta receptor of the frontal cortex in the mouse was similar to its effects in striatal tissue in mouse (Tabakoff and Hoffman, 1983), membranes from the brain in rat (Hiller et af., 1981) and NG108-15 cells (Charness, Gordon and Diamond, 1983). The pattern of the effects of ethanol on the binding of DAGO to the mu opiate receptor in the frontal cortex of the mouse was somewhat different from results in the striatum of mouse, in that no stimulation of ligand binding to the mu receptors in the cortex was observed at small concentrations of ethanol. However, it had previously been suggested that the increase in the binding of [‘Hldihydromorphine (DHM) in the striatum induced by ethanol reflected an action on ligand bound to a low-affinity binding site for dihydromorphine (Hoffman, Chung and Tabakoff, 1984). Using [3H]DAG0, binding to a low-affinity site in cortical tissue was not observed. Larger concentrations of ethanol inhibited ligand binding to the mu receptor in cortex, similar to what was observed in striatal tissue (Tabakoff and Hoffman, 1983). However, in contrast to results obtained in striatal tissue in the mouse or whole brain membranes in the rat (Tabakoff and Hoffman, 1983; Hiller et al., 1981), ligand binding to the mu receptor in the frontal cortex was not less sensitive to the effect of ethanol than ligand binding to the delta receptor. In cortical tissue, there was no significant difference in sensitivity between the two receptors. Other investigators have reported that ligand binding to mu receptors in membranes of whole brain was not affected by ethanol in vitro (Hiller et al., 1981). As discussed in a previous report, (Tabakoff and Hoffman, 1983), this discrepancy could result from the use of large concentrations of ligand to assess the effects of ethanol (Hiller et al., 1981), since ethanol inhibits ligand binding to mu receptors in a “competitive” manner (Tabakoff and Hoffman, 1983). On the other hand, differences in the relative sensitivities of mu and delta opiate receptors in different areas of the brain may also contribute to the apparent insensitivity of mu opiate receptors to ethanol when membranes from whole brain are studied. In contrast to its effects on ligand binding to mu and delta opiate receptors, ethanol, in vitro, did not alter the binding of [3H]bremazocine to the kappa receptor in frontal cortex in the mouse. Similarly, Hiller, Angel and Simon (1984) reported that other alcohols did not alter ligand binding to kappa receptors in the brain of rat. The present results show that, under the conditions of these assays, opiate binding to receptors in brain was significantly affected only by large concentrations
of ethanol in vitro, consistent with the lack of effect reported when small concentrations of ethanol were tested (Jorgenson and Hole, 1981; 1986). The effects seen are most likely due to the membrane-perturbing properties of ethanol, which are also most pronounced in large concentrations (Harris and Schroeder, 1981). Therefore, the differential response of subtypes of opiate receptors and, in particular, the differential relative sensitivities of mu and delta receptors in different areas of the brain, may reflect different microenvironments of these proteins in membranes. The in vitro results indicate that acute inhibition of the binding of opiates by ethanol may not take place in viva [although, under more “physiological” in vitro conditions, smaller concentrations of ethanol did inhibit ligand binding to mu receptors in the striatum (Hoffman et al., 1984)]. However, the present results do indicate a selective effect of chronic ingestion of ethanol, in vivo, on ligand binding to mu receptors in the frontal cortex of mouse. The present authors had previously found a decrease in the affinity of the mu receptor for dihydromorphine in the striatum of mouse 24 hr after withdrawal from chronic ingestion of ethanol (Hoffman et al., 1982). In frontal cortex, a decrease in the number of receptors, rather than a change in affinity was observed, but in both areas of the brain there was a selective change in opiate binding to mu receptors. The decreased number and the increased affinity of kappa receptors in control animals (i.e. animals fed with control liquid diet), as compared to untreated (chow-fed) mice, could simply reflect experimental variability, since experiments using untreated mice were performed at a different time from those using control and ethanol-fed mice. On the other hand, a number of factors, including nutrition, stress or the novelty of the liquid diet regimen could contribute to differences between control and untreated mice. However, there appeared to be no significant change in the binding of opiates to kappa or delta receptors as a result of chronic ingestion of ethanol. There have been several previous reports of changes in the affinity (Pfeiffer et al., 1981; Hynes, Lockner, Bemis and Hymson, 1983) or number (Gianoulakis, 1983) of delta receptors after chronic ingestion of ethanol by rats or mice, or exposure of cells in culture to ethanol for several days (Chamess et al., 1983). In the latter studies, the NG108-15 cells were grown in the presence of a concentration of ethanol that was well outside the physiologicallyrelevant range and therefore the observed changes may not be comparable to the in uivo situation. The earlier in vivo studies measured opiate binding in whole brain or large areas of the brain. The possible differential responses to ethanol of opiate receptors in various areas of the brain makes it difficult to compare the earlier findings with the present results. As discussed above, the present authors wished to investigate alterations in opiate receptors that may be
Ethanol and opiate receptors associated with the terminals of dopaminergic neurons (Pollard et al., 1977) and therefore, regions of the brain with larger concentrations of delta receptors were not examined such as, for example, the periacqueductal gray. In such regions, changes in delta receptors might be more apparent. The present results indicate that, in the striatum and frontal cortex, the in oivo effect of ethanol did not appear to be related to the acute effects observed in vitro. Thus, although delta receptors were equally or more sensitive to ethanol than mu receptors in vitro, the mu receptors were selectively altered by chronic ingestion of ethanol. These findings suggest that chronic ingestion of ethanol may affect the characteristics of opiate receptors through indirect pathways. Gianoulakis (1983) reported resistance of opiate receptors in ethanol-fed rats to the in vitro effects of ethanol (i.e. “tolerance” to ethanol), but such a change was not observed in the present studies, in which mice were behaviorally tolerant to ethanol. The present data support the hypothesis that chronic ingestion of ethanol induces a selective and pathological alteration in opiate binding to a single subtype of receptor in the frontal cortex of the mouse. This change may contribute to certain specific effects of ethanol in the CNS, including possible alterations in regulation of mesolimbic dopaminergic activity, similar to those observed in the striatum (Hoffman ef al., 1982). Acknowledgement-This work was supported in part by a grant from ADAMHA, PHS (AA5372).
REFERENCES
Chang K.-J., Cooper B. R., Hazum E. and Cuatrecasas P. (1979) Multiple opiate receptors: Different regional distribution in the brain and differential binding of opiates and opioid peptides. Molec. Pharmac. 16: 91-104. Chang K.-J. and Cuatrecasas P. (1979) Multiple opiate receptors. J. biol. Chem. 254: 2610-2618. Charness M. E., Gordon A. S. and Diamond I. (1983) Ethanol modulation of opiate receptors in cultured neural cells. Science 222: 12461248. Gianoulakis C. (1983) Long term ethanol alters the binding of ‘H-opiates to brain membranes. Life Sci. 33: 725-733. Gillian M. G. C., Kosterlitz H. W. and Paterson S. J. (1980) Comparison of the binding characteristics of tritiated opioids and opioid peptides. Br. J. Pharmac. 70: 481490. Harris R. A. and Schroeder S. (1981) Ethanol and the physical properties of brain membranes: fluorescence studies. Molec. Pharmac. 20: 128-137. Hiller J. M., Angel L. M. and Simon E. J. (1981) Characterization of the selective inhibition of the delta subclass of opioid binding sites by alcohols. Science 214: 468469.
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Hiller J. M., Angel L. M. and Simon E. J. (1984) Multiple opiate receptors: alcohol selectively inhibits binding to delta receptors. Molec. Pharmac. 25: 249-255. Ho C. L., Hammonds R. G. Jr and Li C. H. (1985) Opiate receptor binding profile in the rabbit cerebellum and brain membranes. Biochem. Pharmac. 34: 925-93 1. Hoffman P. L., Chung C. T. and Tabakoff B. (1984) Effects of ethanol, temperature and endogenous regulatory factors on the characteristics of striatal opiate receptors. J. Neurochem. 43: 1003-1010.
Hoffman P. L., Urwyler S. and Tabakoff B. (1982) Alterations in opiate receptor function after chronic ethanol exposure. J. Pharmac. exp. Ther. 222: 182-189. Hynes M. D., Lockner M. A., Bemis K. G. and Hymson D. L. (1983) Chronic ethanol alters the receptor binding characteristics of the delta opioid receptor ligand, D-Ala2-D-Leu’ enkephalin in mouse brain. Life Sci. 33: 2331-2337.
Iwamoto E. T. and Martin N. R. (1981) Multiple opioid receptors. Med. Res. Rev. 1: 411-440. Jorgensen H. A. and Hole K. (1981) Does ethanol stimulate brain opiate receptors? Studies on receptor binding and naloxone inhibition of ethanol-induced effects. Eur. J. Pharmac. 75: 223-229.
Jorgenson H. A. and Hole K. (1986) Evidence from hehavioral and in vitro receptor binding studies that the enkephalinergic system does not mediate acute ethanol effects. Eur. J. Pharmac. 125: 249-256. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193: 265-275. Munson P. F. and Rodbard D. (1980) LIGAND: a versatile computerized approach for characterization of ligandbinding systems. Analyt. Biochem. 107: 22&239. Pastemak G. W., Wilson H. A. and Snyder S. H. (1975) Differential effects of protein-modifying reagents on receptor binding of opiate agonists and antagonists. Molec. Pharmac. 11: 34&35 1. Pfeiffer A., Seizinger B. R. and Herz A. (1981) Chronic ethanol imbibition interferes with 6-, but not with p-opiate receptors. Neuropharmacology 20: 1229-1232. Pollard H.. Llorens C.. Bonnet J. J.. Costentin J. and Schwartz’ J. C. (1977) Opiate receptors on mesolimbic dopaminergic neurons. Neurosci. Lett. 7: 295-299. Ritzmann R. F. and Tabakoff B. (1976) Body temperature in mice: a quantitative measure of alcohol tolerance and dependence. J. Pharmac. Exp. Ther. 199: 158-170. Simon E. J., Hiller J. M., Groth J., Itzhak Y., Holland M. J. and Beck S. G. (1982) The nature of opiate receptors in toad brain. Life Sci. 31: 1367-1370. Sokal R. R. and Rohlf F. J. (1981) Biomefry. 2nd edn, Chapt. 14. Freeman, San Francisco. Tabakoff B. and Hoffman P. L. (1983) Alcohol interactions with brain opiate receptors. Life Sci. 32: 197-204. Urwyler S. and Tabakoff B. (1981) Stimulation of dopamine synthesis and release by morphine and D-Ala*-D-Ledenkephalin in the mouse striatum in uiuo. Life Sci. 28: 2277-2286. Wood P. L. (1983) Opioid receptor affinities of kappa agonists, agonists/antagonists and antagonists in vitro and in vivo. Prog. Neuropsychopharmac. biol. Psychiat. 7: 657-662.
Printed in Great
Vol. 26, Britain.
No. 10, pp. 1503-1507, All rights reserved
1987
Copyright 0
0028-3908/87 $3.00 + 0.00 1987 Pergamon Journals Ltd
SELECTIVE EFFECTS OF ETHANOL ON OPIATE RECEPTOR SUBTYPES IN BRAIN S. KHATAMI,’PAULA L. ‘Department of Biomedical IL 61107, U.S.A., ?%ction Studies, National Institute ‘Department
HOFFMAN?*T. SHIBUYA~and B. SALAFSKY’
Sciences, University of Illinois College of Medicine at Rockford, Rockford, on Receptor Mechanisms, Laboratory of Physiologic and Pharmacologic on Alcohol Abuse and Alcoholism, Bethesda, MD 20892, U.S.A. and of Pharmacology, Tokyo Medical College, Tokyo 160, Japan (Accepted 2 April 1987)
Snnunary-Large concentrations of ethanol in vitro decreased ligand binding to mu and delta opiate receptors in the frontal cortex of the C57BL mouse., but did not alter binding to kappa opiate receptors. Mu and delta receptors were equally sensitive to the inhibitory effect of ethanol. Since the effects of ethanol were significant only in large concentrations, ethanol may alter opiate binding through its membrane lipid-perturbing actions, and the selectivity of the effects of ethanol may reflect differences in the microenvironments of the opiate receptor subtypes in membranes. After chronic ingestion of ethanol by mice, in vim, there was a selective decrease in the number of mu receptors in the frontal cortex. This change may result from indirect effects of ethanol on the opiate receptor and may contribute to specific central effects of ethanol. Key words: ethanol, opiate receptors, kappa receptor, mu receptor, delta receptor.
Ethanol, in vitro, has been reported to alter opiate binding to receptors in brain tissue. In tissue from the striatum of the mouse ethanol had a biphasic effect on the binding of dihydromorphine (DHM): a small concentration of ethanol (50 mM) enhanced binding, while larger concentrations inhibited the binding of dihydromorphine. In contrast, ethanol only inhibited the binding of D-Ala*, D-Leu’-enkephalin (DADLE) to membranes from the striatum. The binding of DADLE was more sensitive to inhibition by ethanol than the binding of dihydromorphine (Tabakoff and Hoffman, 1983). Hiller, Angel and Simon (1981) also reported that ethanol selectively inhibited the binding of DADLE to opiate receptors in membranes of whole brain of the rat and had little effect on the binding of dihydromorphine. In the latter study, the binding of several ligands was examined and it was concluded that ethanol affected the properties of the binding site for enkephalin (delta receptor) rather than the ligands. In tissue from the striatum of mice that had ingested ethanol chronically, a decreased affinity of high-affinity binding sites for dihydromorphine and a decreased responsiveness of these receptors to the effects of sodium ions, was found (Hoffman, Urwyler and Tabakoff, 1982). No change in binding sites for enkephalin in the striatum was observed. In view of the greater sensitivity of delta opiate receptors in the striatum to ethanol in vitro, as compared to the mu receptors, this selective change in the properties of the mu receptor was unexpected. Gn the other hand, there have been some reports of changes in the ‘To whom all correspondence should be sent.
properties of delta receptors in membranes from the whole brain of rat (Gianoulakis, 1983) or from large areas of brain (Pfeiffer, Seizinger and Herz, 1981) after chronic ingestion of ethanol. Since subtypes of opiate receptors (Iwamoto and Martin, 1981; Wood, 1983; Chang and Cuatrecasas, 1979) are differentially distributed between regions of the brain (Chang, Cooper, Hazum and Cuatrecasas, 1979), the present authors wished to determine the acute and chronic effects of ethanol on the properties of opiate receptors in another area of the brain of the mouse. Because of interest in the effect of ethanol on regulation of dopaminergic activity by opiates (Hoffman et al., 1982; Urwyler and Tabakoff, 1981), the present study has investigated the effects of ethanol on opiate receptors in the frontal cortex that may modulate the activity of mesolimbic dopaminergic neurons (Pollard, Llorens, Bonnet, Costentin and Schwartz, 1977). METHODS Opiate binding
Mice were sacrificed by decapitation, the brains were removed and the frontal cortex was dissected. Tissue from 20 to 25 animals in each experiment was pooled and membranes from the brain were prepared as previously described (Tabakoff and Hoffman, 1983; Pasternak, Wilson and Snyder, 1975). Measurements of ligand binding were carried out in a total volume of 0.5 ml. Each assay contained, in 0.05 M Tris-HCl buffer (pH 7.7), 0.25 ml of homogenate (about 1 mg of protein/ml) (Lowry, Rosebrough, Farr and Randall, 195 1) and various concen-
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S. KHATAMI etal.
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trations of [‘HID-Ala2-MePhe4-GlyLol enkephalin PAGO) (New England Nuclear, Boston, Massachusetts, 30-60 Ci/mmol), [3H]r+Ala2-o-Leu5-enkephalin (DADLE) (New England Nuclear, 30-50 Ci/mmol) or [3H]bremazocine (BMZ) (New England Nuclear, 1S-30 Ci/mmol). For Scatchard analyses, the concentration range of [3H]DAG0 was 0.1-5 nM; of [3H]DADLE was 0.2-10 nM; and of [3H]bremazocine was OS-7 nM. Nonspecific binding was determined with 1 PM unlabelled DAGO, 1 PM unlabelled D-Se?-Leu5-enkephalin-Thr6 (DSLET), or 1 p M unlabelled [3H]bremazocine, respectively. The binding of [‘HIDADLE to delta receptors was assessed by performing binding assays in the presence of a IOO-fold excess of unlabelled DAGO, to mask mu receptor sites. Binding assays with [3H]bremazocine were performed in the presence of excess unlabelled DAGO and DSLET (Gillian, Kosterlitz and Paterson, 1980). Since the brain contains primarily mu, delta and kappa receptors (Wood, 1983), and [3H]bremazocine is a universal ligand (Simon, Hiller, Groth, Itzhak, Holland and Beck, 1982), the data obtained with [3H]bremazocine and the masking ligands are believed to reflect the binding of [3H]bremazocine to kappa receptors. The binding assays were carried out at 25°C for 75 min and were terminated by filtration of the tissue suspensions as previously described (Tabakoff and Hoffman, 1983; Pasternak et al., 1975). Bound radioactivity was quantified by scintillation counting. When the effect of ethanol in vitro was investigated, tubes were capped throughout the assay and binding in the presence and absence of ethanol was determined simultaneously. For dose-response studies of the effects of ethanol, ligand concentrations used were: [3H]DAG0, 0.2 nM; [3H]DADLE, 0.5 nM; (3H]bremazocine, 0.2nM. The binding of DADLE and [3H]bremazocine were assayed in the presence of excess unlabelled DSLET and/or DAGO, as described. The ligand concentrations were chosen to be
loo-
zQ-
Chronic ingestion of ethanol
Male C57BL mice (22-25 g) were housed 6 per cage in a laboratory environment (12-hr light cyctr , 22°C) for at least one week before use. In experiments to assess the chronic effects of ethanol, mice were housed individually and fed with ethanol in liquid diet, or control diet, for seven days, as previously described (Ritzmann and Tabakoff, 1976). The quantity of diet fed daily to the control animals was adjusted to equal the average daily consumption of the ethanol-consuming mice. On the morning of the eighth day, the diet containing ethanol was withdrawn and the mice were monitored for withdrawal symptoms (Ritzmann and Tabakoff, 1976). Mice were sacrified at 24 hr after withdrawal, when overt withdrawal symptoms had dissipated, but the mice were still tolerant to and physically dependent on ethanol (Ritzmann and Tabakoff, 1976). RESULTS
Scatchard plots of the binding of [3H]DAG0 and [3H]DADLE, the latter in the presence of an excess of unlabelled DAGO, appeared linear, while there was some indication of curvature in the Scatchard plot for the binding of [3H]bremazocine in the presence of excess DAGO and DSLET (Fig. 1). Analysis of the binding data using the LIGAND program (Munson and Rodbard, 1980) indicated that the data for the binding of [3H]DAG0 and [3H]DADLE were best fit by a one-site model. In two out of three cases, data for the binding of [‘Hlbremazocine were also best fit by a one-site model. In the instance where the binding data for [3H]bremazocine were better fit by a two-site model, there was a high degree of variability of the parameters for the low-affinity site (KD approximately 15 nM). For the purposes of these studies,
t”HIBMZ
C’HIDADLE
C3HlDAG0
g
3@-40% of the K, value, but still to allow easily detectable levels of ligand binding.
.5
loo
0
P
A 0
P .
0 .
&
50
SD
loo-
a
0 . 0
d d
0
‘5 A
\-x....-,, 50
SD Spectiic Swnd
50
100 IfmoWmg
100
150
protein1
Fig. 1. Scatchard plots of the binding of [3H]DAG0 (a), [‘HIDADLE (0) and [‘Hlbremaxocine ([3H]BMZ) (A) in tissue from the frontal cortex of C57BL mouse. Membrane preparation, binding assays and ligand concentrations used are described in the text. The binding of [3H]DADLE was assayed in the presence of high concentrations of unlabelled DAGO. The binding of [‘Hlbremazocine was assayed in the presence of large concentrations of unlabelled DAGO and DSLET. Values are from a single representative experiment; the data for all ligands were best fit by a one-site model.
Ethanol and opiate receptors
1505
Table I. Opiatebinding in the frontal cortexof C57BLmice Percentageof Bmm (fmol/mgprotein) total binding Ligand K. (nM) DAGO DADLE BMZ
0.63 + 0.03 1.15 f0.04 0.45 * 0.01
27% 38% 35%
87+4 12Ok8 116*2
Binding of [‘HIDAGO, [‘HIDADLE and [‘Hlbremazocine ([3H]BMZ) were assayed as described in the text. The binding of DADLE was assayed in the presence of unlabelled DAGO, to mask mu receptors, and the binding of [‘Hlbremazocine was assayed in the presence of DAGO and DSLET, to mask delta and mu receptors. Values represent mean f SEM from 3 experiments. tEthonol1
therefore, the binding data for [3H]bremazocine were assumed to be best fit by a one-site model. The kinetic parameters for the binding of [3H]DAG0, [3H]DADLE and [‘Hlbremazocine in frontal cortex of the C57BL mouse are shown in Table 1. The concentration of mu opiate receptors was smallest, while concentrations of delta and kappa receptors were larger (Table 1). Ethanol, added to binding assays in vitro, inhibited the binding of [3H]DAG0 and [3H]DADLE, but had no significant effect on the binding of [3H]bremazocine to the membranes from the frontal cortex (Fig. 2). The I&, value for the inhibition by ethanol of the binding of [3H]DAG0 was 592 (55&613) mM [95% confidence limits calculated by log-probit analysis of the average values from three inhibition curves in separate experiments; (Sokal and Rohlf, 1981)], and the I& for the inhibition by ethanol of the binding by DADLE was 789 (524-l 189) mM (also calculated from 3 experiments). Chronic treatment with ethanol resulted in tolerance to ethanol and physical dependence in the mice, as described earlier (Ritzmann and Tabakoff, 1976). The kinetic parameters of opiate binding to tissue of the frontal cortex of control mice and mice withdrawn from ethanol, at 24 hr after withdrawal, are shown in Table 2. The number of binding sites for [‘HIDAGO (mu binding sites) was significantly reduced in the mice withdrawn from ethanol, compared to controls. The number and KD of kappa binding sites in tissue of control animals were reduced in comparison to these parameters in naive, untreated animals (compare to Table 1). However, there was no statistically significant difference in the number of kappa or of delta binding sites and no significant
Table 2. Opiate binding
in the frontal
Ligand
K. (nM) 0.66 f 0.05 0.99 + 0.08 0.26 k 0.04
B_
of ethanol
difference in affinity for the ligand of any binding site, between the mice withdrawn from ethanol and control mice. The inhibition by ethanol of the binding of [3H]DAG0 and [3H]DADLE was unchanged after chronic ingestion of ethanol (DAGO: ICso = 478 mM in control animals and 458 mM in animals withdrawn from ethanol; DADLE: I&, = 534mM in control animals and 385 mM in animals withdrawn from ethanol; I&, values were calculated from log-probit analysis of mean values from three separate experiments and the 95% confidence limits (Sokal and Rohlf, 1981) showed no significant differences between control and animals withdrawn from ethanol). DISCUSSION
The binding data are consistent with the presence of at least three classes of opiate receptor in the frontal cortex of mouse, i.e. mu, delta and kappa opiate receptors. The percentage of each type of binding site was similar to that for membranes of whole brain of mouse (Wood, 1983) and was also similar to that found in the brain of rabbit (Ho, Hammonds and Li, 1985) in that the relative amount of mu receptors was less than that of delta or kappa
cortex of C57BL mice after chronic
ingestion
of ethanol
Ethanol-withdrawn
Control
DAGO DADLE BMZ
(mM)
on opiate binding to mu, delta and kappa receptors in the frontal cortex of mouse. Tissue was prepared and ligand binding was assayed in the presence of masking ligands, as described in the text. The concentrations of ligands used were: [3H]DAG0, 0.20 + 0.01 mM; [‘HIDADLE, 0.50 f 0.02 nM; and [‘Hlbremazocine ([3H]BMZ), 0.20 & 0.01 nM. Binding in the absence of ethanol was (in fmol/mg protein): [3H]DAG0, 19.1 k 1.1; [3H]DADLE 60.6 rf:9.4; [‘Hlbremazocine, 51.5 k 2.9 (mean +SEM from 3 experiments). Fig. 2. Effect
(fmol/mg
protein)
84 + 2 122 + 14 69 k 9
KD (nM) 0.69 k 0.07 0.88 f 0.03 0.39 * 0.05
B_
(fmol/mg
protein)
63f4* 112*21 71 *3
Binding assays were carried out as described in the text. Binding of [‘HIDADLE was assayed in the presence of excess unlabelled DACO, and binding of [‘Hlbremazocine ([‘HIBMZ) was assayed in the presence of excess unlabelled DAGO and DSLET. Mice were sacrificed at 24 hr after withdrawal from chronic ingestion of ethanol. Values represent mean * SEM from three experiments. *P < 0.01 compared to control (Student’s r-test).
1.506
S. KHATAMIet al.
receptors. The affinity of each ligand for its specific binding site was comparable to that reported in membranes of the brain of rat (Wood, 1983; Simon et al., 1982). The inhibitory effect of ethanol in vitro on the binding of DADLE to the delta receptor of the frontal cortex in the mouse was similar to its effects in striatal tissue in mouse (Tabakoff and Hoffman, 1983), membranes from the brain in rat (Hiller et af., 1981) and NG108-15 cells (Charness, Gordon and Diamond, 1983). The pattern of the effects of ethanol on the binding of DAGO to the mu opiate receptor in the frontal cortex of the mouse was somewhat different from results in the striatum of mouse, in that no stimulation of ligand binding to the mu receptors in the cortex was observed at small concentrations of ethanol. However, it had previously been suggested that the increase in the binding of [‘Hldihydromorphine (DHM) in the striatum induced by ethanol reflected an action on ligand bound to a low-affinity binding site for dihydromorphine (Hoffman, Chung and Tabakoff, 1984). Using [3H]DAG0, binding to a low-affinity site in cortical tissue was not observed. Larger concentrations of ethanol inhibited ligand binding to the mu receptor in cortex, similar to what was observed in striatal tissue (Tabakoff and Hoffman, 1983). However, in contrast to results obtained in striatal tissue in the mouse or whole brain membranes in the rat (Tabakoff and Hoffman, 1983; Hiller et al., 1981), ligand binding to the mu receptor in the frontal cortex was not less sensitive to the effect of ethanol than ligand binding to the delta receptor. In cortical tissue, there was no significant difference in sensitivity between the two receptors. Other investigators have reported that ligand binding to mu receptors in membranes of whole brain was not affected by ethanol in vitro (Hiller et al., 1981). As discussed in a previous report, (Tabakoff and Hoffman, 1983), this discrepancy could result from the use of large concentrations of ligand to assess the effects of ethanol (Hiller et al., 1981), since ethanol inhibits ligand binding to mu receptors in a “competitive” manner (Tabakoff and Hoffman, 1983). On the other hand, differences in the relative sensitivities of mu and delta opiate receptors in different areas of the brain may also contribute to the apparent insensitivity of mu opiate receptors to ethanol when membranes from whole brain are studied. In contrast to its effects on ligand binding to mu and delta opiate receptors, ethanol, in vitro, did not alter the binding of [3H]bremazocine to the kappa receptor in frontal cortex in the mouse. Similarly, Hiller, Angel and Simon (1984) reported that other alcohols did not alter ligand binding to kappa receptors in the brain of rat. The present results show that, under the conditions of these assays, opiate binding to receptors in brain was significantly affected only by large concentrations
of ethanol in vitro, consistent with the lack of effect reported when small concentrations of ethanol were tested (Jorgenson and Hole, 1981; 1986). The effects seen are most likely due to the membrane-perturbing properties of ethanol, which are also most pronounced in large concentrations (Harris and Schroeder, 1981). Therefore, the differential response of subtypes of opiate receptors and, in particular, the differential relative sensitivities of mu and delta receptors in different areas of the brain, may reflect different microenvironments of these proteins in membranes. The in vitro results indicate that acute inhibition of the binding of opiates by ethanol may not take place in viva [although, under more “physiological” in vitro conditions, smaller concentrations of ethanol did inhibit ligand binding to mu receptors in the striatum (Hoffman et al., 1984)]. However, the present results do indicate a selective effect of chronic ingestion of ethanol, in vivo, on ligand binding to mu receptors in the frontal cortex of mouse. The present authors had previously found a decrease in the affinity of the mu receptor for dihydromorphine in the striatum of mouse 24 hr after withdrawal from chronic ingestion of ethanol (Hoffman et al., 1982). In frontal cortex, a decrease in the number of receptors, rather than a change in affinity was observed, but in both areas of the brain there was a selective change in opiate binding to mu receptors. The decreased number and the increased affinity of kappa receptors in control animals (i.e. animals fed with control liquid diet), as compared to untreated (chow-fed) mice, could simply reflect experimental variability, since experiments using untreated mice were performed at a different time from those using control and ethanol-fed mice. On the other hand, a number of factors, including nutrition, stress or the novelty of the liquid diet regimen could contribute to differences between control and untreated mice. However, there appeared to be no significant change in the binding of opiates to kappa or delta receptors as a result of chronic ingestion of ethanol. There have been several previous reports of changes in the affinity (Pfeiffer et al., 1981; Hynes, Lockner, Bemis and Hymson, 1983) or number (Gianoulakis, 1983) of delta receptors after chronic ingestion of ethanol by rats or mice, or exposure of cells in culture to ethanol for several days (Chamess et al., 1983). In the latter studies, the NG108-15 cells were grown in the presence of a concentration of ethanol that was well outside the physiologicallyrelevant range and therefore the observed changes may not be comparable to the in uivo situation. The earlier in vivo studies measured opiate binding in whole brain or large areas of the brain. The possible differential responses to ethanol of opiate receptors in various areas of the brain makes it difficult to compare the earlier findings with the present results. As discussed above, the present authors wished to investigate alterations in opiate receptors that may be
Ethanol and opiate receptors associated with the terminals of dopaminergic neurons (Pollard et al., 1977) and therefore, regions of the brain with larger concentrations of delta receptors were not examined such as, for example, the periacqueductal gray. In such regions, changes in delta receptors might be more apparent. The present results indicate that, in the striatum and frontal cortex, the in oivo effect of ethanol did not appear to be related to the acute effects observed in vitro. Thus, although delta receptors were equally or more sensitive to ethanol than mu receptors in vitro, the mu receptors were selectively altered by chronic ingestion of ethanol. These findings suggest that chronic ingestion of ethanol may affect the characteristics of opiate receptors through indirect pathways. Gianoulakis (1983) reported resistance of opiate receptors in ethanol-fed rats to the in vitro effects of ethanol (i.e. “tolerance” to ethanol), but such a change was not observed in the present studies, in which mice were behaviorally tolerant to ethanol. The present data support the hypothesis that chronic ingestion of ethanol induces a selective and pathological alteration in opiate binding to a single subtype of receptor in the frontal cortex of the mouse. This change may contribute to certain specific effects of ethanol in the CNS, including possible alterations in regulation of mesolimbic dopaminergic activity, similar to those observed in the striatum (Hoffman ef al., 1982). Acknowledgement-This work was supported in part by a grant from ADAMHA, PHS (AA5372).
REFERENCES
Chang K.-J., Cooper B. R., Hazum E. and Cuatrecasas P. (1979) Multiple opiate receptors: Different regional distribution in the brain and differential binding of opiates and opioid peptides. Molec. Pharmac. 16: 91-104. Chang K.-J. and Cuatrecasas P. (1979) Multiple opiate receptors. J. biol. Chem. 254: 2610-2618. Charness M. E., Gordon A. S. and Diamond I. (1983) Ethanol modulation of opiate receptors in cultured neural cells. Science 222: 12461248. Gianoulakis C. (1983) Long term ethanol alters the binding of ‘H-opiates to brain membranes. Life Sci. 33: 725-733. Gillian M. G. C., Kosterlitz H. W. and Paterson S. J. (1980) Comparison of the binding characteristics of tritiated opioids and opioid peptides. Br. J. Pharmac. 70: 481490. Harris R. A. and Schroeder S. (1981) Ethanol and the physical properties of brain membranes: fluorescence studies. Molec. Pharmac. 20: 128-137. Hiller J. M., Angel L. M. and Simon E. J. (1981) Characterization of the selective inhibition of the delta subclass of opioid binding sites by alcohols. Science 214: 468469.
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Hiller J. M., Angel L. M. and Simon E. J. (1984) Multiple opiate receptors: alcohol selectively inhibits binding to delta receptors. Molec. Pharmac. 25: 249-255. Ho C. L., Hammonds R. G. Jr and Li C. H. (1985) Opiate receptor binding profile in the rabbit cerebellum and brain membranes. Biochem. Pharmac. 34: 925-93 1. Hoffman P. L., Chung C. T. and Tabakoff B. (1984) Effects of ethanol, temperature and endogenous regulatory factors on the characteristics of striatal opiate receptors. J. Neurochem. 43: 1003-1010.
Hoffman P. L., Urwyler S. and Tabakoff B. (1982) Alterations in opiate receptor function after chronic ethanol exposure. J. Pharmac. exp. Ther. 222: 182-189. Hynes M. D., Lockner M. A., Bemis K. G. and Hymson D. L. (1983) Chronic ethanol alters the receptor binding characteristics of the delta opioid receptor ligand, D-Ala2-D-Leu’ enkephalin in mouse brain. Life Sci. 33: 2331-2337.
Iwamoto E. T. and Martin N. R. (1981) Multiple opioid receptors. Med. Res. Rev. 1: 411-440. Jorgensen H. A. and Hole K. (1981) Does ethanol stimulate brain opiate receptors? Studies on receptor binding and naloxone inhibition of ethanol-induced effects. Eur. J. Pharmac. 75: 223-229.
Jorgenson H. A. and Hole K. (1986) Evidence from hehavioral and in vitro receptor binding studies that the enkephalinergic system does not mediate acute ethanol effects. Eur. J. Pharmac. 125: 249-256. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193: 265-275. Munson P. F. and Rodbard D. (1980) LIGAND: a versatile computerized approach for characterization of ligandbinding systems. Analyt. Biochem. 107: 22&239. Pastemak G. W., Wilson H. A. and Snyder S. H. (1975) Differential effects of protein-modifying reagents on receptor binding of opiate agonists and antagonists. Molec. Pharmac. 11: 34&35 1. Pfeiffer A., Seizinger B. R. and Herz A. (1981) Chronic ethanol imbibition interferes with 6-, but not with p-opiate receptors. Neuropharmacology 20: 1229-1232. Pollard H.. Llorens C.. Bonnet J. J.. Costentin J. and Schwartz’ J. C. (1977) Opiate receptors on mesolimbic dopaminergic neurons. Neurosci. Lett. 7: 295-299. Ritzmann R. F. and Tabakoff B. (1976) Body temperature in mice: a quantitative measure of alcohol tolerance and dependence. J. Pharmac. Exp. Ther. 199: 158-170. Simon E. J., Hiller J. M., Groth J., Itzhak Y., Holland M. J. and Beck S. G. (1982) The nature of opiate receptors in toad brain. Life Sci. 31: 1367-1370. Sokal R. R. and Rohlf F. J. (1981) Biomefry. 2nd edn, Chapt. 14. Freeman, San Francisco. Tabakoff B. and Hoffman P. L. (1983) Alcohol interactions with brain opiate receptors. Life Sci. 32: 197-204. Urwyler S. and Tabakoff B. (1981) Stimulation of dopamine synthesis and release by morphine and D-Ala*-D-Ledenkephalin in the mouse striatum in uiuo. Life Sci. 28: 2277-2286. Wood P. L. (1983) Opioid receptor affinities of kappa agonists, agonists/antagonists and antagonists in vitro and in vivo. Prog. Neuropsychopharmac. biol. Psychiat. 7: 657-662.