Journal of Neurochemistry Raven Press, Ltd., New York 0 I992 International Society for Neurochemistry
Ethanol-Induced Increase in Endogenous Dopamine Release May Involve Endogenous Opiates Peter S. Widdowson and R. Bruce Holman Department of Biochemistry and Physiology. University of Reading, Whiteknights, Reading, Berkshire, England
Abstract: The effect of opiate peptides on basal and potassium-stimulated endogenous dopamine (DA) release from striatal slices was studied in vitro. Dual stimulation of the stnatal slices gave a reproducible increase in DA release that was calcium dependent. Addition of the &opiate receptor agonists Met5-enkephalin, [~-Ala*,~Leu~]enkephalin (DADLE), and [~-Se8]Leu-enkephalin-Thr(DSLET), increased the basal DA release without affecting potassiumstimulated release in a dose-dependent manner. The effect of DADLE was antagonized by the addition of naloxone. In contrast, the p-opioid receptor agonist [D-Ala2,NMePhe4,Gly-o15]enkephalin(DAGO) and the t-opioid agonist P-endorphin inhibited the stimulated DA release without changing the basal release. The inhibitory effect of DAGO on potassium-stimulated release was antagonized by naloxone. The addition of ethanol (75 mM) to the incu-
bation media produced a delayed increase of both the basal and stimulated DA release. There was no change in stimulated DA release when the change in basal release was subtracted, suggesting that ethanol produced a dose-dependent, selective increase in basal DA release. Naloxone and the selective d-opiate antagonist ICI 174864 inhibited the ethanol-induced increase in basal DA release. Naloxone and ICI 174864 added alone did not alter either basal or stimulated DA release. We therefore suggest that the ethanol-induced increase in basal DA release is an indirect effect involving an endogenous d-opiate agonist. Key Words: Dopamine-Opiate peptides-Ethanol-Enkephalins-Release. Widdowson P. S. and Holman R. B. Ethanol-induced increase in endogenous dopamine release may involve endogenous opiates. J. Neurochem. 59, 157-163 (1992).
Acute administration of ethanol has been shown to increase the turnover (Fadda et al., 1980; Reggiani et al., 1980; Yamanaka and Egashira, 1982) and release of endogenous dopamine (DA) (Holman and Snape, 1985; Imperato and DiChiara, 1986; DiChiara and Imperato, 1988). However, the intensity and the time course of these effects of ethanol appear to be different for the various DA systems in the brain (Holman and Snape, 1985;Imperato and DiChiara, 1986).Such observations imply a specificity of the action of ethanol on neurotransmission, especially DA. Attempts to delineate the mechanisms underlying the drug’s interactions with neurons has focused on changes in membrane fluidization (Goldstein, 1986)and ion channels (Little, 1991) as no “ethanol” receptor has been identified. However, whatever the neurochemical mechanism may be, it is still unclear whether changes in DA release are a direct effect of ethanol on DA neurons or
are mediated indirectly through the drug’s effects on another endogenous neuromodulator. The endogenous opioid peptides have been shown to modulate cerebral DA activity and there is increasing evidence for a role in the acute behavioral and neurochemical effects of ethanol. Both the p- and dopioid receptors and possible endogenous ligands for these receptors are present in the rat corpus striatum (Kuhar et al., 1973; Pollard et al., 1977; Fallon and Leslie, 1986; Mansour et al., 1988). Furthermore, 6hydroxydopamine lesion experiments have demonstrated that both p- and d-opioid receptors are located presynaptically on the DA terminals (Pollard et al., 1977; Murrin et al., 1980; Waksman et al., 1987). More direct evidence for opioid/DA interactions has come from experiments performed both in vitro and in vivo where d-opioid agonists have been shown to increase the synthesis (Biggio et al., 1978) and the re-
Received March 19, 1991; revised manuscript received November 10, 1991; accepted December 12, 1991. Address correspondence and reprint requests to Dr. R. B. Holman at Reckitt & Colman Psychopharmacology Unit, School of Medical Sciences, University Walk, Bristol, BS8 ITD, U.K. The present address of Dr. P. S. Widdowson is Department of
Psychiatry, MetroHealth Medical Center, 3395 Scranton Road, Cleveland, Ohio 44109, U.S.A. Abbreviations used: ANOVA, analysis of variance; DA, dopamine; DADLE, [~-Ala*,~-Leu~]enkephalin; DAGO, [D-Ala2,NMePhe4,Gly-o15]enkephalin;L-DOPA, L-dihydroxyphenylalanine; DSLET, [D-Se?]Leu-enkephalin-Thr.
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lease of DA (Lubetzki et al., 1982). Evidence for interactions of p-opioid receptors and the DA system are less clear. @-Endorphin,which has been suggested to act at r-type opiate receptors (Schultz et al., 1979),but also has some p-opiate agonist properties (Corbett et al., 1982), has been reported to be twice as potent as morphine to inhibit potassium-induced DA release in vitro (Loh et al., 1976). However, other work in vitro with morphine and other p-opiate agonists have reported no change in release (Chesselet et al., 1982, 1983; Mulder et al., 1984; Petit et al., 1986; Heijna et al., 1990). Finally, ethanol, as well as altering DA function, has been reported to increase the release of Met-enkephalin in striatum (Albertini et al., 1979) and to decrease the binding of d-opioid ligands as compared to p-opioid ligands (Hiller et al., 1981; Hoffman et al., 1984). In addition, Barbaccia et al. (1981) demonstrated naloxone-reversibleincreases in DA turnover following ethanol in C57 but not DBA 25 mouse strains. The DBA 25 strain has been shown to lack enkephalinergic modulation of striatal DA activity (Barbaccia et al., 1981). The selective 8-opioid receptor antagonist ICI 174864 has also been shown to reverse ethanol-induced hypothermia and narcosis in rats following microinjections into DA-rich brain regions such as the nucleus accumbens and medial septum (Widdowson, 1987). In man, naloxone has been reported to reverse acute alcoholic coma (Smensen and Mattisson, 1978; Ducobu, 1984). The present studies were undertaken to further our preliminary results which suggest that opioid modulation may be important for the ethanol-induced increase of endogenous DA release (Widdowson and Holman, 1986; Holman et al., 1987). MATERIALS AND METHODS Animals Adult (200-300 g) male Sprague-Dawley rats (Charles River, Margate, U.K.) were group housed in cages of between three and four animals and allowed free access to food and tap water. All animals were kept under a light/dark cycle of 13 h/ I 1 h (lights on at 0600 h) and killed by decapitation between 10:30 and 11:30 a.m.
(500 mm3)on a cooled glass surface with a razor blade. The tissue sliceswere transferred to incubation tubes containing 200 pl of incubation medium. The incubation tubes were I .5-ml polythene microfuge tubes into which had been inserted rubber syringe bungs to reduce the microfuge volumes to 0.75 ml and produce a large surface area for the tissue slices to come into contact with oxygenated incubation medium. The incubation medium was a modified Tyrode’s solution (Baron and Eyzaguirre, 1977) (NaCI 112 mM, KC1 4.7 mM, CaCI, 2.2 mhf, MgCI, 1.1 mM, sodium glutamate 42 mM, glucose 5.6 mM, HEPES 5 mM, pH 7.4) containing pargyline (350 p M ) and L-DOPA (4 p M ) which was continually gassed with 0, saturated water vapor at 37°C. The high potassium medium was the same as the normal medium except that the concentration of KCI was increased to 20 mM with a concomitant reduction in the concentration of NaCl to 82 mM. The tissue slices were incubated for 10 consecutive periods of I5 min each. For the first three incubation periods, the slices were exposed to normal media followed by one period of exposure to media containing 20 mMpotassium, then three periods in normal media, a second potassium stimulus, and two final periods in normal media. At the conclusion of each incubation period, all the media was transferred to a microcentifuge tube containing 50 p1 of 0.6 M perchloric acid with NaHSO, (1.2 mM) and EDTA (400 p M ) and fresh warm media put into the incubation tubes. All samples were kept on ice and analyzed for their DA content on the same day as the tissue slice incubation experiments. Drugs and peptides were added in 20-4 aliquots and the volume of incubation medium reduced to 180 p1 accordingly.
Quantitation of endogenous DA The concentration of DA in each incubation sample was determined by reverse phase, ion pair HPLC with electrochemical detection. Acetate buffer (0.1 M sodium acetate, 0.02 M citric acid, 100 mg/L sodium octyl sulphate, 50 mg/L EDTA, and 15% vol/vol methanol) was pumped at 1.0 ml/min with an LDC HPLC pump through a C18 column (Hichrom, 4.6 mm I.D. X 25 cm) at room temperature as described by Mefford (198 1). Endogenous DA was measured by injecting 50 pl of incubation buffer directly into the HPLC system and the DA concentration determined with the detector electrode set at +0.65 V. DA eluted from the column at 8 min and was quantitated by comparisons ofthe peak height with known standards.
Statistics
The results are expressed as means t SEM of between 8 and 12 replicates. Comparisons of drug- or peptide-induced Octyl sodium sulphate was obtained from Kodak, changes in basal DA release were performed by compariKirkby, Liverpool, U.K. Pargyline HCI and L-dihydroxysons of periods 3 and 7 whereas comparisons of peptide- or phenylalanine (L-DOPA) were from Sigma Chemical, drug-induced changes in the potassium-stimulated DA rePoole, U.K. The peptides Met5-enkephalin, [D-Ala2,Dlease were made by comparing sample periods 4 and 8. SigLe~~lenkephalin(DADLE), [D-Se?]Leu-enkephalin-Thr nificant differences between control and experimental sam(DSLET), P-endorphin, [~-Ala~,N-MePhe~,Gly-ol~]enples were assessed by Student’s t tests. Multiple cornpansons kephalin (DAGO), and dynorphin (1-8) were obtained were performed by analysis of variance (ANOVA) followed from Bachem, Saffron Walden, U.K. Naloxone was a gift by Duncan’s multiple range test. from DuPont Pharmaceuticals, U.K. ICI 174864 was a gift from ICI Pharmaceuticals, Macclesfield, Cheshire, U.K. All RESULTS other reagents were purchased from Fisons, U.K.
Reagents
Brain slice incubation Rats were killed by decapitation and the brains quickly removed. The striata were dissected out and cut in slices J. Neurochem., Vol. 59, No. 1. 1992
We have demonstrated that opiate peptides and ethanol have the ability to differentially alter basal and potassium-stimulated endogenous DA release
EFFECT OF OPIATES ON ENDOGENOUS DOPAMINE RELEASE from a striatal slices. To prevent a gradual reduction in basal DA release (basal 1 versus 2, n = 4; 0.45 f 0.04 1 versus0.25 k 0.036) and a reduction in stimulated DA release following a second stimulation period (stimulus l versus 2, n = 4; 1.532 f 0.066 versus 0.714 f 0.021), we found it necessary to add a DA precursor to the incubation medium. The addition of the DA precursor tyrosine (4 pA4) to the incubation medium increased to magnitude of DA release during a second potassium-induced stimulation (stimulus 1 versus 2, n = 4; 1.351 f 0.1 10 versus 0.956 k 0.06 1). However, when the DA release during the second stimulus was compared to the first stimulus, there was still a significant reduction in magnitude of DA release in the presence of tyrosine. The addition of the more immediate DA precursor, L-DOPA (4 p M ) , which bypasses the regulatory tyrosine hydroxylase stage of DA synthesis, was able to increase the amount of DA release during a second potassium stimulus. The increase in DA release during the second stimulus was sufficient that the second potassium stimulus produced an equivalent amount of DA release as compared to a first stimulation (stimulus 1 versus 2, n
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FIG. 2. Dose-response curves of opioid-induced changes in basal and potassium-stimulatedendogenous DA release. A Doseresponse effect of d-agonists on basal DA release. DADLE, 0; DSLET, 0; Met5-enkephalin,0; 5 pM DADLE 10 pM naloxone, m; 5 p M DADLE 25 pM naloxone, A. ' p < 0.05 (Duncan's multiple range test) as compared to control basal DA release. t p < 0.01 (Student's t test) as compared to 5 p M DADLE alone. 8: Dose-response effect of p-agonists on potassium-stimulated DA release. DAGO, 0; P-endorphin, 0; DAGO 5 pM naloxone 10 pM, 0. * p < 0.01 (Duncan's multiple range test) as compared to control stimulated DA release. t p < 0.01 (Student's t test) as compared to 5 pM DAGO alone. Mean of 8-1 2 replicates.
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= 4; 1.306 f 0.12 1 versus 1.26 1 f 0.144). The addition of L-DOPA did not, however, alter either basal release (basal 1 versus 2, n = 4; 0.499 0.05 1 versus 0.468 f 0.052) or the magnitude of the first potassium-stimulus as compared to experiments in which no DA precursors were present (see above). Therefore all further experiments were performed with the addition of 4 p M L-DOPA in the incubation medium. Figure 1A shows the results of a control experiment in which no drug was added to the preparation. Basal and potassium-induced release of endogenous DA was identical for both basal DA measurements and comparing a second potassium-stimulus with the first. In addition, when the tissue was incubated with calcium-free media, DA release was significantly reduced (Fig. 1B). When the opiate agonist ,&endorphin (Roemer et al., 1977; Kosterlitz and Paterson, 1981) was added to the incubation media, there was no change in the basal DA release (Fig. 1C). However, potassium-induced release was significantly inhibited at concentrations of P-endorphin from 0.5 to 5.0 pA4 [ANOVA (2,23) = 3.82; p < 0.051 (Figs. 1C and 2B). The same effect was seen with the p-opiate receptor agonist DAGO [ANOVA = (2,27) = 5 . 8 6 ; ~< 0.011 (Fig. 2B). The ability of DAGO (5.0 p M ) to reduce potassiumstimulated DA release was prevented by the addition of naloxone (10 pA4) to the incubation medium from sampling periods 6-10 (Fig. 2B). +_
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FIG. 1. Basal (open columns) and potassium-stimulated release (filled columns) of endogenous dopamine from striatal slices in vitro. Mean of 12 replicates. A: Effect of double stimulation on DA release. B Effect of calcium removal on basal and stimulated DA release. C: Effect of 5 pM 0-endorphin on basal and stimulated DA release. *p < 0.01 as compared to control stimulation (Student's t test). D: Effect of 5 f l DADLE on basal and stimulated DA release. p < 0.05 as compared to control basal release (Student's t test).
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The 6-agonist DADLE (Wei et al., 1977) had no effect on potassium-stimulated DA release (Fig. 1D). In contrast, the addition of DADLE caused a significant, dose-dependent (0.5- 10.0 p M ) increase in basal release [ANOVA (3,27) = 3.25; p < 0.051 (Figs. 1D and 2A). The 5 pMDADLE-induced increase in basal DA release was partially prevented by 10 pM naloxone and fully reversed with 25 pM naloxone (p < 0.01) (Fig. 2A). Another d-opiate agonist, DSLET, elicited an increase in basal DA release [ANOVA (2,23) = 3.73; p < 0.051 similar to that elicited by DADLE (Fig. 2A). Met5-enkephalin(5.0 pM), a third d-opiate agonist (Hughes et al., 1975), also significantly increased the basal DA release ( y < 0.05; Student’s t test) as compared to the control period, but only about half as much as the more stable enkephalin analogues (Fig. 2A).
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FIG. 3. Effect of ethanol and opiate antagonists on basal and stimulated DA release. A: Comparison between control DA release (open bars) and the presence of 75 mM ethanol throughout the experiment (filled columns). *p < 0.05 (Student’s t test) as compared to control basal release, ‘ p < 0.01 (Student’st test) as compared to control potassium-stimulated DA release. B Effect of calcium removal P.2 mM EDTA (striped columns) on ethanolinduced increase in DA release (filled columns). ‘ p < 0.01 (Student’st test) as compared to 75 mM ethanol alone. C: Effect of 50 pM naloxone (striped columns) and 10 pM ICI 174864 (filled columns) on ethanol-induced increased basal and stimulated DA release (open columns). Opiate antagonists added from periods 6-1 0. * p < 0.01 (Student’st test) as compared to alcohol alone. Mean of 8-1 2 replicates.
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When ethanol (75 mM) was added to the media throughout the experiment, there was a delayed increase in endogenous basal DA release and a significant increase in both potassium-stimulated DA release periods (Fig. 3A). Although potassium-stimulated DA release appeared to be increased, as compared to controls, it was not clear whether this was due to the enhanced basal release of DA. A comparison of the increase in the second period of potassium exposure for controls and ethanol-exposed tissue in which the basal release has been subtracted suggests that there are no significant effects on potassium-induced DA release. Within an hour of exposure to 75 mM ethanol, a significant increase in basal DA release occurred and was maintained for the remainder of an experiment (Fig. 3A). The effect of ethanol to increase DA release was calcium dependent (Fig. 3B), as removal of calcium from the buffer, coupled with the addition of 0.2 mM EDTA, abolished the ethanol-induced effect on DA release. Furthermore, the ethanol-induced increase in basal DA release was dose dependent (25-75 mM) (Fig. 4A). To determine if endogenous opioids have any role to play in the effects of ethanol on DA release, experiments were repeated in the presence of naloxone and the selective d-opiate antagonist ICI 174864 (Cotton et al., 1984). Naloxone (50 p M ) and ICI 174864 (10 p M ) significantly reduced ethanol-induced increase in basal DA release (Fig. 3C). The ICI 174864 was more potent than naloxone in reversing the ethanolinduced increased DA release and the effect was dose dependent [ANOVA, F(2,27) = 5 . 8 8 ; ~< 0.011. (Figs. 3C and 4B) Neither of the antagonists alone had any effect on basal or potassium-stimulated DA release [naloxone (20 p M ) versus control, n = 8; basal 1.15 k 0.0 13 versus I . 15 k 0.09; stimulated 2.12 +- 0.22 versus 2.33 & 0.241 [ICI174864 (10 pLM) versus control, n = 8; basal 1.15 k 0.06 versus0.97 k 0.08; stimulated 2.22 k 0.25 versus 2.45 & 0.181. Conclusions We have demonstrated that it is possible to study the effects of alcohol and opioid peptides on the release of endogenous DA from striatal slices in vitro. However, it was important to include the DA precursor L-DOPA in the incubation medium, to maintain consistent spontaneous and potassium-stimulated release of endogenous DA release. Although the conversion of tyrosine to DA is more tightly regulated by tyrosine hydroxylase in the striatal tissue, the addition of tyrosine to the incubation media failed to maintain a steady spontaneous DA release. In addition, tyrosine failed to produce a potassium-induced release of DA during a second stimulus which was equal in size to that following the first stimulus. Tyrosine may not have maintained DA release because its uptake into striatal tissue is slow or the conversion of the precursor to DA is slow. In addition, it did not appear that the L-DOPA was being taken up in nondopaminergic
EFFECT OF OPIATES ON ENDOGENOUS DOPAMINE RELEASE
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FIG. 4. A Dose-response effect of ethanol on basal DA release. *p < 0.01 (Duncan's multiple range test). Mean of 12 replicates. B Effect of increasing concentrations of ICI 174864 on ethanolinduced increasesin basal DA release. *p < 0.01 (Duncan's multiple range test) as compared to 75 mM alcohol alone. Mean of 4-1 2 replicates.
neurons and converted to DA. The magnitude of basal and potassium-stimulated release of DA in the presence of L-DOPA was not significantly different from basal and stimulated DA release without any DA precursor added. If L-DOPA was taken up into nondopaminergic tissues and converted to DA, one would expect a greater release of DA on potassium stimulation. Thus any conversion of L-DOPA to DA and its subsequent release from nondopaminergic terminals in this preparation was clearly minimal. Therefore, incubation of the striatal slices in the presence of L-DOPA in vitro provides a stable preparation with which to examine the effects of drugs on both basal and depolarization-induced release of endogenous DA. Both basal and potassium-induced DA release were shown to be calcium dependent. The significant reduction of basal DA release when the slices were incubated in calcium free media suggests that some calcium leakage into unstimulated striatal tissue does normally occur and contributes to the basal DA release. Removal of calcium from the media exposes the calcium-mediated component of the basal DA release. The p-opiate agonist DAGO (Roemer et al., 1977) and the 6-agonist P-endorphin (Schultz et al., 1979) did not alter basal DA release, but did inhibit potassium-induced DA release. Because these effects are inhibited by naloxone and are different from the effects of specific d-agonists, it suggests that both peptides are changing DA release via an action at p-opiate receptors. This agrees with a previous report in which they examined potassium-induced DA release in brain slices (Loh et al., 1976). However, with electrical stimulation of slices, Mulder and his colleagues have been unable to demonstrate a p-receptor-mediated effect on radiolabelled DA release from the striatum (Mulder et al., 1984; Heijna et al., 1990).
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The demonstration that p-agonists can inhibit stimulated DA release also does not agree with reports of DA release in vivo (Chesselet et al., 1982, 1983; Spanagel et al., 1990) in which p-agonists either had no effect or significantly enhanced DA release. We suggest these differences may be explained if the site of action of the p-agonists on the DA neurons in vivo versus in vitro is considered. In vitro the p-agonistsact on the DA terminals only, whereas in vivo studies allow the simultaneous activation of both preceptors on the DA terminals and on the DA cell bodies. Morphine, a potent p-agonist has been demonstrated to increase the firing rate of dopaminergic cells when administered to both the substantia nigra and the ventral tegmental area (Gysling and Wang, 1983). Thus, an inhibition of DA release through preceptor activation at the dopaminergic terminals could be balanced or overcome by a p-receptor-induced excitatory input at the dopaminergic cell somas. The d-opiate agonist DADLE elicited a naloxone reversible increase in basal DA release. These results agree with previous reports both in vitro and in vivo (Algeri et al., 1978; Biggio et al., 1978; Wood et al., 1980;Yonehara and Cloulet, 1984; Petit et al., 1986). In the present experiments other d-agonists, Met5-enkephalin and DSLET, also increased basal DA release without effecting potassium-stimulated release. The difference between these responses and those with p-agonists emphasize that these changes are not mediated via preceptors. Taken together our data suggest that DA release from the rat striatum in vitro can be regulated by different mechanisms utilizing p- and d-opiate receptors. Activation of preceptors results in the inhibition of potassium-induced release, whereas stimulation of dopiate receptors increases the basal release of DA. There does not, however, appear to be any endogenous opioid tone, because the administration of either of the opiate antagonists naloxone and ICI 174864 failed to alter either basal or stimulated DA release. This suggests that opioid neuromodulation of DA release must be driven or activated by unknown mechanisms. The failure of naloxone to alter potassiumstimulated DA release is surprising because the depolarizing stimulus would have been expected to release endogenous p-agonists which would inhibit DA release. The addition of naloxone would then be expected to cause an increase in stimulated DA release, as the inhibitory opiate component would be attenuated. It is possible that the high potassium-induced depolarization was not of sufficient strength to release the endogenousp ligand. On the other hand, the potassium stimulus could have been so great as to obliterate any small inhibitory opiate component produced by the release of an endogenous p ligand. The increased DA release following administration of ethanol to the striatal tissues agrees with previous reports both in vitro and in vivo (Barbaccia et al., 1981; Holman and Snape, 1985; Imperato and DiJ. Neurochem.. Vol. 59, No. 1. 1992
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Chiara, 1986). The effect of ethanol particularly on basal DA release was dose dependent, which argues against a nonspecific drug effect or the result of decreased viability of the tissue in our preparation. Further, the enhanced basal DA release was prevented by the addition of high concentration of naloxone or by the more specific %opiate antagonist, ICI 174864, which suggests pharmacological specificity of action of ethanol. The similarity of ethanol- and d-opiate-induced increases in endogenous DA release is interesting. The observation that ethanol-induced increased basal DA release can be reversed by opioid antagonists including ICI 174864 suggests that ethanol may indirectly alter DA release through release of an endogenous ligand as the enkephalins. The presence of enkephalins in the striatum and %receptors on DA terminals would support this hypothesis. Further, Albertini et al. (1 979) have demonstrated that acute administration of ethanol is capable of increasing the release of Met-enkephalin in the striatum. The proposal that some of the effects of ethanol may involve endogenous opiates is also supported by the demonstration that ethanol-induced narcosis and hypothermia in rats can be attenuated by intracerebrally administered ICI 174864 (Widdowson, 1987). These data provide further evidence for the involvement of endogenous opioids in the acute effects of ethanol. REFERENCES Albertini A., Saiai M., Cecchetin M., and Trabucchi M. (1979) Effect of acute and chronic elcohol intake on brain enkephalin neurons and other transmitter systems, in Radioimrnunoassay ofDrugs and Hormones in Cardiovascular Medicine (Albertini A., DaPrada M., and Peskar B. A., eds), pp. 176- 184. Elsevier/ North Holland Biomedical Press, Amsterdam. Algeri S., Brunello N., Calderini G., and Consolazione A. (1978) Effect of enkephalins on catechol metabolism in rat CNS. Adv. Biochem. Psychopharmacol. 18, 199-209. Barbaccia M. L., Reggiani A., Spano P. F., and Trabucchi M. ( I98 1) Ethanol-induced changes of dopaminergic function in three stains of mice characterized by a different population of opiate receptors. Psychopharmacology 74,260-262. Baron M. and EyzaguirreC. (1977) Effects of temperature on some membrane characteristics of carotid body cells. Am. J. Physiol. 233, C35-C46. Biggo C., Casa M., Corda M. G., DiBello C., and Gessa G. L. (1978) Stimulation of dopamine synthesis in caudate nucleus by intrastriatal enkephalins and antagonism by naloxone. Science 20, 552-554. Chang K.-J., Hazumn E., and Cuatrecasas P. (I98 I ) Novel opiate binding sites selective for benzomorphan drugs. Proc. Natl. Acad. Sci. USA 78,4141-4145. Chesselet M. F., Cheramy A., Reisine T. D., LubetzkiC., Glowinski J., Fournie-Zaluski M. C., and Roques B. (1982) Effects of various opiates including specific delta and mu agonists on dopaminergic neurons in vitro in the rat and in vivo in the cat. Life Sci. 31, 2291-2294. Chesselet M. R., Cheramy A,, Reisine T. D., Lubetzki C., Desban M., and Glowinski J. (1983) Local and distal effects induced by unilateral striatal application of opiates in the absence or in the presence of naloxone on the release of dopamine in both caudate nuclei and substantiae nigrae of the cat. Brain Res. 258, 229-242.
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Corbett A. D., Paterson S. J., McKnight A. T., Magnan J., and Kosterlitz H. W. (1982) Dynorphin,_, and dynorphinl-9 are ligands for the K-SUbtYPe ofopiate receptor. Nature 299.79-8 1. Cotton R., Giles M. G., Miller L., Shaw J. S., and Timms D. ( 1984) ICI 174864, a highly selective antagonist for the opioid d-receptor. Eur. J . Pharmacol. 97, 33 1-332. DiChiara G. and lmperato A. ( 1 988) Drugs abused by humans preferentially increase synaptic dopamine concentrations in the mesolimbic system of freely moving rats. Proc. Null. Acnd. Sci. USA 85,5274-5278. Ducobu J. (1984) Naloxone and alcohol intoxication. Ann Intern. Med. 100,6 17-6 18. Fadda F., Argiolas A,, Melis M. R., Serra G., and Gessa G. L. ( 1980) Differential effect of acute and chronic ethanol on dopamine metabolism in frontal cortex, caudate nucleus and substantia nigra. Lge Sci. 27, 979-986. Fallon J. H. and Leslie F. M. (1986) Distribution ofdynorphin and enkephalin peptides in the rat brain. J. Comp. Neurol. 249, 293-336. Goldstein D. B. (1986) The alcohol withdrawal syndrome. A view from the laboratory. Rec. Dev. Alc. 4,231-240. Gysling K. and Wang R. Y. (1983) Morphine-induced activation of A10 dopamine neurons in the rat. Brain Res. 277, 119-127. Heijna M. H., Padt M., Hogenboom G., Portoghese P. S., Mulder A. H., and Schoffelmeer A. N. (1990) Opioid receptor-mediated inhibition of dopamine and acetylcholine release from slices of rat nucleus accumbens, olfactory tubercle and frontal cortex. Eur. J. Pharmacol. 181,267-278. Hiller J. M., Angel L. M., and Simon E. J. (198 I ) Multiple opiate receptors: alcohol selectively inhibits binding to delta receptors. Science 214,468-469. 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. Holman R. B. and Snape B. M. (1985) Effects of ethanol in v i m and in vivo on the release of endogenous catecholamines from specific regions of rat brain. J . Neurochem. 44,357-363. Holman R. B., Snape B. M., and Widdowson P. S. (1987) Naloxone elicits dose-dependentchanges in alcohol-induced increases in endogenous dopamine release for rat striatum. In Advances in Biomedical Alcohol Research (Lindros K. O., Ylikahri R., and Kiianmaa K., eds), pp. 737-741. Pergamon Press, Oxford. Hughes J., Smith T. W., Kosterlitz H. W., Fothergill L. H., Morgan B. A., and Moms H. (1975) Identificationoftwo related pentapeptides from the brain with potent opiate agonist activity. Nature 258, 577-580. Imperato A. and DiChiara G. (1986) Preferential stimulation of dopamine release in the nucleus accumbens of freely moving rats by ethanol. J. Pharmacol. Exp. Ther. 239,219-228. Kosterlitz H. W. and Paterson S. J. (198 I ) Tyr-D-Ala-Gly-MePheNH(CH,),OH is a selectiveligand for the p-opiate binding site. Br. J. Pharmacol. 73,29913. Kuhar M. J., Pert C. B., and Snyder S. H. ( I 973) Regional distribution of opiate receptor binding in monkey and human brain. Nature 245,447-450. Little H. J. (1991) Mechanisms that may underlie the behavioural effects of ethanol. Prog. Neurobiol. 36, 17 1 - 194. Loh H. H., Brase D. A,, Sampath-Khanna S., Mar J. B., Way E. L., and Li C. H. (1976) P-Endorphin in vitro inhibition of striatal dopamine release. Nature 264, 567-568. Lubetzki C., Chesselet M. F., and Glowinski J. (1982) Modulation of dopamine release in rat striatal slices by %opiate agonists. J. Pharmacol. Exp. Ther. 222,435-440. Mansour A., Khachaturian H., Lewis M. E., Akil H., and Watson S. J. ( 1988) Anatomy of CNS opioid receptors. Trends Neurol. Sci. 11, 308-314. Mefford I. N. (1981) Application of HPLC with electrochemical detection to neurochemicalanalysis: measurement ofcatecholamines, serotonin and metabolites in rat brain. J. Neurosci. Meth. 3, 207-224. Mulder A. H., Wardeh G., Hogenboom F., and Frankhuyzen A. L.
EFFECT OF OPIATES ON ENDOGENOUS DOPAMINE RELEASE (1984) K- and a-opioid receptor agonists differentially inhibit stnatal dopamine and acetylcholine release. Nature 308,278280. Mumn L. C., Coyle J. T., and Kuhar M. J. (1980) Striatal opiate receptors: pre- and postsynaptic localization. Life Sci. 27, 1175-1 183. Petit F., Homen M., Fournie-Zaluski M. C., Roques B. P., and Glowinski J. (1986) Further evidence for a role of delta-opiate receptors in the presynaptic regulation of newly synthesized dopamine release. Eur. J. Pharmacol. 126, 1-9. Pollard H., Llorens-Cortes C., and Schwartz J. C. (1977) Enkephalin receptors on dopaminergic neurons in rat striatum. Nature 268,745-747. Reggiani A., Barbaccia M. L., Spano P. F., and Trabucchi M. (1980) Dopamine metabolism and receptor function after acute and chronic ethanol. J. Neurochem. 35,34-37, Roemer D., Buescher H. H., Hill R. L., Pless J., Bauer W., Cardinaux F., Close A,, Hauser D., and Huguenin R. (1 977) A synthetic enkephalin analogue with prolonged parented and oral analgesic activity. Nature 268, 547-550. Schultz R., Faase E., Wiister M., and Hen A. (1979) Selective receptors for beta-endorphin on the rat vas deferens. Life Sci. 24, 843-849. Ssrensen S . C. and Mattisson K. ( 1978) Naloxone as an antagonist in severe alcohol intoxication. Lancet 2, 688-689. Spanagel R., Herz A., and Shippenberg T. S. (1990) The effect of opioid peptides on dopamine release in the nucleus accum-
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bens: an in vivo microdialysis study. J. Neurochem. 55, 17341740. Waksman G., Hamel E., Delay-Goyet P., and Roques B. P. (1 987) Neutral endopeptidase-24.11, p and d opioid receptors after selective brain regions: an autoradiographic study. Brain Rex 436,205-2 16. Wei E. T., Tseng L. F., Loh H. H., and Li C. H. (1977) Comparison of the behavioural effects of beta-endorphin and enkephalin analogues. Life Sci. 21, 321-327. Widdowson P. S. (1987) The effect of neurotensin, TRH and the delta-opioid receptor antagonist ICI 174864 on alcohol-induced narcosis in rats. Brain Rex 424,28 1-289. Widdowson P. S. and Holman R. B. (1986) Effect of opiate receptor antagonists on alcohol-induced increase in endogenous dopamine release from rat corpus striatum in vitro. Br. J. Pharmacol. 87, 17 1P. Wood P. L., Stotland M., Richard J. W., and Rackham A. (1980) Actions of mu, kappa, sigma, delta and agonist/antagonist opiates on stnatal dopaminergic function. J . Pharmacol. Exp. Ther. 215,697-703. Yamanaka S. and Egashira T. (1982) Effects of ethanol on catecholamine levels and related enzyme activities in different brain regions of rats. Jpn. J. Pharmacol. 32, 599-606. Yonehara N. and Cloulet D. H. (1984) Effects of delta and mu opiopeptides on the turnover and release of dopamine in rat brain. J. Pharmacol. Exp. Ther. 231, 38-42.
J. Neurochem.. Vol. 59, No. I , 1992
Ethanol-Induced Increase in Endogenous Dopamine Release May Involve Endogenous Opiates Peter S. Widdowson and R. Bruce Holman Department of Biochemistry and Physiology. University of Reading, Whiteknights, Reading, Berkshire, England
Abstract: The effect of opiate peptides on basal and potassium-stimulated endogenous dopamine (DA) release from striatal slices was studied in vitro. Dual stimulation of the stnatal slices gave a reproducible increase in DA release that was calcium dependent. Addition of the &opiate receptor agonists Met5-enkephalin, [~-Ala*,~Leu~]enkephalin (DADLE), and [~-Se8]Leu-enkephalin-Thr(DSLET), increased the basal DA release without affecting potassiumstimulated release in a dose-dependent manner. The effect of DADLE was antagonized by the addition of naloxone. In contrast, the p-opioid receptor agonist [D-Ala2,NMePhe4,Gly-o15]enkephalin(DAGO) and the t-opioid agonist P-endorphin inhibited the stimulated DA release without changing the basal release. The inhibitory effect of DAGO on potassium-stimulated release was antagonized by naloxone. The addition of ethanol (75 mM) to the incu-
bation media produced a delayed increase of both the basal and stimulated DA release. There was no change in stimulated DA release when the change in basal release was subtracted, suggesting that ethanol produced a dose-dependent, selective increase in basal DA release. Naloxone and the selective d-opiate antagonist ICI 174864 inhibited the ethanol-induced increase in basal DA release. Naloxone and ICI 174864 added alone did not alter either basal or stimulated DA release. We therefore suggest that the ethanol-induced increase in basal DA release is an indirect effect involving an endogenous d-opiate agonist. Key Words: Dopamine-Opiate peptides-Ethanol-Enkephalins-Release. Widdowson P. S. and Holman R. B. Ethanol-induced increase in endogenous dopamine release may involve endogenous opiates. J. Neurochem. 59, 157-163 (1992).
Acute administration of ethanol has been shown to increase the turnover (Fadda et al., 1980; Reggiani et al., 1980; Yamanaka and Egashira, 1982) and release of endogenous dopamine (DA) (Holman and Snape, 1985; Imperato and DiChiara, 1986; DiChiara and Imperato, 1988). However, the intensity and the time course of these effects of ethanol appear to be different for the various DA systems in the brain (Holman and Snape, 1985;Imperato and DiChiara, 1986).Such observations imply a specificity of the action of ethanol on neurotransmission, especially DA. Attempts to delineate the mechanisms underlying the drug’s interactions with neurons has focused on changes in membrane fluidization (Goldstein, 1986)and ion channels (Little, 1991) as no “ethanol” receptor has been identified. However, whatever the neurochemical mechanism may be, it is still unclear whether changes in DA release are a direct effect of ethanol on DA neurons or
are mediated indirectly through the drug’s effects on another endogenous neuromodulator. The endogenous opioid peptides have been shown to modulate cerebral DA activity and there is increasing evidence for a role in the acute behavioral and neurochemical effects of ethanol. Both the p- and dopioid receptors and possible endogenous ligands for these receptors are present in the rat corpus striatum (Kuhar et al., 1973; Pollard et al., 1977; Fallon and Leslie, 1986; Mansour et al., 1988). Furthermore, 6hydroxydopamine lesion experiments have demonstrated that both p- and d-opioid receptors are located presynaptically on the DA terminals (Pollard et al., 1977; Murrin et al., 1980; Waksman et al., 1987). More direct evidence for opioid/DA interactions has come from experiments performed both in vitro and in vivo where d-opioid agonists have been shown to increase the synthesis (Biggio et al., 1978) and the re-
Received March 19, 1991; revised manuscript received November 10, 1991; accepted December 12, 1991. Address correspondence and reprint requests to Dr. R. B. Holman at Reckitt & Colman Psychopharmacology Unit, School of Medical Sciences, University Walk, Bristol, BS8 ITD, U.K. The present address of Dr. P. S. Widdowson is Department of
Psychiatry, MetroHealth Medical Center, 3395 Scranton Road, Cleveland, Ohio 44109, U.S.A. Abbreviations used: ANOVA, analysis of variance; DA, dopamine; DADLE, [~-Ala*,~-Leu~]enkephalin; DAGO, [D-Ala2,NMePhe4,Gly-o15]enkephalin;L-DOPA, L-dihydroxyphenylalanine; DSLET, [D-Se?]Leu-enkephalin-Thr.
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lease of DA (Lubetzki et al., 1982). Evidence for interactions of p-opioid receptors and the DA system are less clear. @-Endorphin,which has been suggested to act at r-type opiate receptors (Schultz et al., 1979),but also has some p-opiate agonist properties (Corbett et al., 1982), has been reported to be twice as potent as morphine to inhibit potassium-induced DA release in vitro (Loh et al., 1976). However, other work in vitro with morphine and other p-opiate agonists have reported no change in release (Chesselet et al., 1982, 1983; Mulder et al., 1984; Petit et al., 1986; Heijna et al., 1990). Finally, ethanol, as well as altering DA function, has been reported to increase the release of Met-enkephalin in striatum (Albertini et al., 1979) and to decrease the binding of d-opioid ligands as compared to p-opioid ligands (Hiller et al., 1981; Hoffman et al., 1984). In addition, Barbaccia et al. (1981) demonstrated naloxone-reversibleincreases in DA turnover following ethanol in C57 but not DBA 25 mouse strains. The DBA 25 strain has been shown to lack enkephalinergic modulation of striatal DA activity (Barbaccia et al., 1981). The selective 8-opioid receptor antagonist ICI 174864 has also been shown to reverse ethanol-induced hypothermia and narcosis in rats following microinjections into DA-rich brain regions such as the nucleus accumbens and medial septum (Widdowson, 1987). In man, naloxone has been reported to reverse acute alcoholic coma (Smensen and Mattisson, 1978; Ducobu, 1984). The present studies were undertaken to further our preliminary results which suggest that opioid modulation may be important for the ethanol-induced increase of endogenous DA release (Widdowson and Holman, 1986; Holman et al., 1987). MATERIALS AND METHODS Animals Adult (200-300 g) male Sprague-Dawley rats (Charles River, Margate, U.K.) were group housed in cages of between three and four animals and allowed free access to food and tap water. All animals were kept under a light/dark cycle of 13 h/ I 1 h (lights on at 0600 h) and killed by decapitation between 10:30 and 11:30 a.m.
(500 mm3)on a cooled glass surface with a razor blade. The tissue sliceswere transferred to incubation tubes containing 200 pl of incubation medium. The incubation tubes were I .5-ml polythene microfuge tubes into which had been inserted rubber syringe bungs to reduce the microfuge volumes to 0.75 ml and produce a large surface area for the tissue slices to come into contact with oxygenated incubation medium. The incubation medium was a modified Tyrode’s solution (Baron and Eyzaguirre, 1977) (NaCI 112 mM, KC1 4.7 mM, CaCI, 2.2 mhf, MgCI, 1.1 mM, sodium glutamate 42 mM, glucose 5.6 mM, HEPES 5 mM, pH 7.4) containing pargyline (350 p M ) and L-DOPA (4 p M ) which was continually gassed with 0, saturated water vapor at 37°C. The high potassium medium was the same as the normal medium except that the concentration of KCI was increased to 20 mM with a concomitant reduction in the concentration of NaCl to 82 mM. The tissue slices were incubated for 10 consecutive periods of I5 min each. For the first three incubation periods, the slices were exposed to normal media followed by one period of exposure to media containing 20 mMpotassium, then three periods in normal media, a second potassium stimulus, and two final periods in normal media. At the conclusion of each incubation period, all the media was transferred to a microcentifuge tube containing 50 p1 of 0.6 M perchloric acid with NaHSO, (1.2 mM) and EDTA (400 p M ) and fresh warm media put into the incubation tubes. All samples were kept on ice and analyzed for their DA content on the same day as the tissue slice incubation experiments. Drugs and peptides were added in 20-4 aliquots and the volume of incubation medium reduced to 180 p1 accordingly.
Quantitation of endogenous DA The concentration of DA in each incubation sample was determined by reverse phase, ion pair HPLC with electrochemical detection. Acetate buffer (0.1 M sodium acetate, 0.02 M citric acid, 100 mg/L sodium octyl sulphate, 50 mg/L EDTA, and 15% vol/vol methanol) was pumped at 1.0 ml/min with an LDC HPLC pump through a C18 column (Hichrom, 4.6 mm I.D. X 25 cm) at room temperature as described by Mefford (198 1). Endogenous DA was measured by injecting 50 pl of incubation buffer directly into the HPLC system and the DA concentration determined with the detector electrode set at +0.65 V. DA eluted from the column at 8 min and was quantitated by comparisons ofthe peak height with known standards.
Statistics
The results are expressed as means t SEM of between 8 and 12 replicates. Comparisons of drug- or peptide-induced Octyl sodium sulphate was obtained from Kodak, changes in basal DA release were performed by compariKirkby, Liverpool, U.K. Pargyline HCI and L-dihydroxysons of periods 3 and 7 whereas comparisons of peptide- or phenylalanine (L-DOPA) were from Sigma Chemical, drug-induced changes in the potassium-stimulated DA rePoole, U.K. The peptides Met5-enkephalin, [D-Ala2,Dlease were made by comparing sample periods 4 and 8. SigLe~~lenkephalin(DADLE), [D-Se?]Leu-enkephalin-Thr nificant differences between control and experimental sam(DSLET), P-endorphin, [~-Ala~,N-MePhe~,Gly-ol~]enples were assessed by Student’s t tests. Multiple cornpansons kephalin (DAGO), and dynorphin (1-8) were obtained were performed by analysis of variance (ANOVA) followed from Bachem, Saffron Walden, U.K. Naloxone was a gift by Duncan’s multiple range test. from DuPont Pharmaceuticals, U.K. ICI 174864 was a gift from ICI Pharmaceuticals, Macclesfield, Cheshire, U.K. All RESULTS other reagents were purchased from Fisons, U.K.
Reagents
Brain slice incubation Rats were killed by decapitation and the brains quickly removed. The striata were dissected out and cut in slices J. Neurochem., Vol. 59, No. 1. 1992
We have demonstrated that opiate peptides and ethanol have the ability to differentially alter basal and potassium-stimulated endogenous DA release
EFFECT OF OPIATES ON ENDOGENOUS DOPAMINE RELEASE from a striatal slices. To prevent a gradual reduction in basal DA release (basal 1 versus 2, n = 4; 0.45 f 0.04 1 versus0.25 k 0.036) and a reduction in stimulated DA release following a second stimulation period (stimulus l versus 2, n = 4; 1.532 f 0.066 versus 0.714 f 0.021), we found it necessary to add a DA precursor to the incubation medium. The addition of the DA precursor tyrosine (4 pA4) to the incubation medium increased to magnitude of DA release during a second potassium-induced stimulation (stimulus 1 versus 2, n = 4; 1.351 f 0.1 10 versus 0.956 k 0.06 1). However, when the DA release during the second stimulus was compared to the first stimulus, there was still a significant reduction in magnitude of DA release in the presence of tyrosine. The addition of the more immediate DA precursor, L-DOPA (4 p M ) , which bypasses the regulatory tyrosine hydroxylase stage of DA synthesis, was able to increase the amount of DA release during a second potassium stimulus. The increase in DA release during the second stimulus was sufficient that the second potassium stimulus produced an equivalent amount of DA release as compared to a first stimulation (stimulus 1 versus 2, n
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FIG. 2. Dose-response curves of opioid-induced changes in basal and potassium-stimulatedendogenous DA release. A Doseresponse effect of d-agonists on basal DA release. DADLE, 0; DSLET, 0; Met5-enkephalin,0; 5 pM DADLE 10 pM naloxone, m; 5 p M DADLE 25 pM naloxone, A. ' p < 0.05 (Duncan's multiple range test) as compared to control basal DA release. t p < 0.01 (Student's t test) as compared to 5 p M DADLE alone. 8: Dose-response effect of p-agonists on potassium-stimulated DA release. DAGO, 0; P-endorphin, 0; DAGO 5 pM naloxone 10 pM, 0. * p < 0.01 (Duncan's multiple range test) as compared to control stimulated DA release. t p < 0.01 (Student's t test) as compared to 5 pM DAGO alone. Mean of 8-1 2 replicates.
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= 4; 1.306 f 0.12 1 versus 1.26 1 f 0.144). The addition of L-DOPA did not, however, alter either basal release (basal 1 versus 2, n = 4; 0.499 0.05 1 versus 0.468 f 0.052) or the magnitude of the first potassium-stimulus as compared to experiments in which no DA precursors were present (see above). Therefore all further experiments were performed with the addition of 4 p M L-DOPA in the incubation medium. Figure 1A shows the results of a control experiment in which no drug was added to the preparation. Basal and potassium-induced release of endogenous DA was identical for both basal DA measurements and comparing a second potassium-stimulus with the first. In addition, when the tissue was incubated with calcium-free media, DA release was significantly reduced (Fig. 1B). When the opiate agonist ,&endorphin (Roemer et al., 1977; Kosterlitz and Paterson, 1981) was added to the incubation media, there was no change in the basal DA release (Fig. 1C). However, potassium-induced release was significantly inhibited at concentrations of P-endorphin from 0.5 to 5.0 pA4 [ANOVA (2,23) = 3.82; p < 0.051 (Figs. 1C and 2B). The same effect was seen with the p-opiate receptor agonist DAGO [ANOVA = (2,27) = 5 . 8 6 ; ~< 0.011 (Fig. 2B). The ability of DAGO (5.0 p M ) to reduce potassiumstimulated DA release was prevented by the addition of naloxone (10 pA4) to the incubation medium from sampling periods 6-10 (Fig. 2B). +_
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FIG. 1. Basal (open columns) and potassium-stimulated release (filled columns) of endogenous dopamine from striatal slices in vitro. Mean of 12 replicates. A: Effect of double stimulation on DA release. B Effect of calcium removal on basal and stimulated DA release. C: Effect of 5 pM 0-endorphin on basal and stimulated DA release. *p < 0.01 as compared to control stimulation (Student's t test). D: Effect of 5 f l DADLE on basal and stimulated DA release. p < 0.05 as compared to control basal release (Student's t test).
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The 6-agonist DADLE (Wei et al., 1977) had no effect on potassium-stimulated DA release (Fig. 1D). In contrast, the addition of DADLE caused a significant, dose-dependent (0.5- 10.0 p M ) increase in basal release [ANOVA (3,27) = 3.25; p < 0.051 (Figs. 1D and 2A). The 5 pMDADLE-induced increase in basal DA release was partially prevented by 10 pM naloxone and fully reversed with 25 pM naloxone (p < 0.01) (Fig. 2A). Another d-opiate agonist, DSLET, elicited an increase in basal DA release [ANOVA (2,23) = 3.73; p < 0.051 similar to that elicited by DADLE (Fig. 2A). Met5-enkephalin(5.0 pM), a third d-opiate agonist (Hughes et al., 1975), also significantly increased the basal DA release ( y < 0.05; Student’s t test) as compared to the control period, but only about half as much as the more stable enkephalin analogues (Fig. 2A).
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FIG. 3. Effect of ethanol and opiate antagonists on basal and stimulated DA release. A: Comparison between control DA release (open bars) and the presence of 75 mM ethanol throughout the experiment (filled columns). *p < 0.05 (Student’s t test) as compared to control basal release, ‘ p < 0.01 (Student’st test) as compared to control potassium-stimulated DA release. B Effect of calcium removal P.2 mM EDTA (striped columns) on ethanolinduced increase in DA release (filled columns). ‘ p < 0.01 (Student’st test) as compared to 75 mM ethanol alone. C: Effect of 50 pM naloxone (striped columns) and 10 pM ICI 174864 (filled columns) on ethanol-induced increased basal and stimulated DA release (open columns). Opiate antagonists added from periods 6-1 0. * p < 0.01 (Student’st test) as compared to alcohol alone. Mean of 8-1 2 replicates.
+
When ethanol (75 mM) was added to the media throughout the experiment, there was a delayed increase in endogenous basal DA release and a significant increase in both potassium-stimulated DA release periods (Fig. 3A). Although potassium-stimulated DA release appeared to be increased, as compared to controls, it was not clear whether this was due to the enhanced basal release of DA. A comparison of the increase in the second period of potassium exposure for controls and ethanol-exposed tissue in which the basal release has been subtracted suggests that there are no significant effects on potassium-induced DA release. Within an hour of exposure to 75 mM ethanol, a significant increase in basal DA release occurred and was maintained for the remainder of an experiment (Fig. 3A). The effect of ethanol to increase DA release was calcium dependent (Fig. 3B), as removal of calcium from the buffer, coupled with the addition of 0.2 mM EDTA, abolished the ethanol-induced effect on DA release. Furthermore, the ethanol-induced increase in basal DA release was dose dependent (25-75 mM) (Fig. 4A). To determine if endogenous opioids have any role to play in the effects of ethanol on DA release, experiments were repeated in the presence of naloxone and the selective d-opiate antagonist ICI 174864 (Cotton et al., 1984). Naloxone (50 p M ) and ICI 174864 (10 p M ) significantly reduced ethanol-induced increase in basal DA release (Fig. 3C). The ICI 174864 was more potent than naloxone in reversing the ethanolinduced increased DA release and the effect was dose dependent [ANOVA, F(2,27) = 5 . 8 8 ; ~< 0.011. (Figs. 3C and 4B) Neither of the antagonists alone had any effect on basal or potassium-stimulated DA release [naloxone (20 p M ) versus control, n = 8; basal 1.15 k 0.0 13 versus I . 15 k 0.09; stimulated 2.12 +- 0.22 versus 2.33 & 0.241 [ICI174864 (10 pLM) versus control, n = 8; basal 1.15 k 0.06 versus0.97 k 0.08; stimulated 2.22 k 0.25 versus 2.45 & 0.181. Conclusions We have demonstrated that it is possible to study the effects of alcohol and opioid peptides on the release of endogenous DA from striatal slices in vitro. However, it was important to include the DA precursor L-DOPA in the incubation medium, to maintain consistent spontaneous and potassium-stimulated release of endogenous DA release. Although the conversion of tyrosine to DA is more tightly regulated by tyrosine hydroxylase in the striatal tissue, the addition of tyrosine to the incubation media failed to maintain a steady spontaneous DA release. In addition, tyrosine failed to produce a potassium-induced release of DA during a second stimulus which was equal in size to that following the first stimulus. Tyrosine may not have maintained DA release because its uptake into striatal tissue is slow or the conversion of the precursor to DA is slow. In addition, it did not appear that the L-DOPA was being taken up in nondopaminergic
EFFECT OF OPIATES ON ENDOGENOUS DOPAMINE RELEASE
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%
120
2
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100 -
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Concentration
ICI 174864 I p M l
FIG. 4. A Dose-response effect of ethanol on basal DA release. *p < 0.01 (Duncan's multiple range test). Mean of 12 replicates. B Effect of increasing concentrations of ICI 174864 on ethanolinduced increasesin basal DA release. *p < 0.01 (Duncan's multiple range test) as compared to 75 mM alcohol alone. Mean of 4-1 2 replicates.
neurons and converted to DA. The magnitude of basal and potassium-stimulated release of DA in the presence of L-DOPA was not significantly different from basal and stimulated DA release without any DA precursor added. If L-DOPA was taken up into nondopaminergic tissues and converted to DA, one would expect a greater release of DA on potassium stimulation. Thus any conversion of L-DOPA to DA and its subsequent release from nondopaminergic terminals in this preparation was clearly minimal. Therefore, incubation of the striatal slices in the presence of L-DOPA in vitro provides a stable preparation with which to examine the effects of drugs on both basal and depolarization-induced release of endogenous DA. Both basal and potassium-induced DA release were shown to be calcium dependent. The significant reduction of basal DA release when the slices were incubated in calcium free media suggests that some calcium leakage into unstimulated striatal tissue does normally occur and contributes to the basal DA release. Removal of calcium from the media exposes the calcium-mediated component of the basal DA release. The p-opiate agonist DAGO (Roemer et al., 1977) and the 6-agonist P-endorphin (Schultz et al., 1979) did not alter basal DA release, but did inhibit potassium-induced DA release. Because these effects are inhibited by naloxone and are different from the effects of specific d-agonists, it suggests that both peptides are changing DA release via an action at p-opiate receptors. This agrees with a previous report in which they examined potassium-induced DA release in brain slices (Loh et al., 1976). However, with electrical stimulation of slices, Mulder and his colleagues have been unable to demonstrate a p-receptor-mediated effect on radiolabelled DA release from the striatum (Mulder et al., 1984; Heijna et al., 1990).
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The demonstration that p-agonists can inhibit stimulated DA release also does not agree with reports of DA release in vivo (Chesselet et al., 1982, 1983; Spanagel et al., 1990) in which p-agonists either had no effect or significantly enhanced DA release. We suggest these differences may be explained if the site of action of the p-agonists on the DA neurons in vivo versus in vitro is considered. In vitro the p-agonistsact on the DA terminals only, whereas in vivo studies allow the simultaneous activation of both preceptors on the DA terminals and on the DA cell bodies. Morphine, a potent p-agonist has been demonstrated to increase the firing rate of dopaminergic cells when administered to both the substantia nigra and the ventral tegmental area (Gysling and Wang, 1983). Thus, an inhibition of DA release through preceptor activation at the dopaminergic terminals could be balanced or overcome by a p-receptor-induced excitatory input at the dopaminergic cell somas. The d-opiate agonist DADLE elicited a naloxone reversible increase in basal DA release. These results agree with previous reports both in vitro and in vivo (Algeri et al., 1978; Biggio et al., 1978; Wood et al., 1980;Yonehara and Cloulet, 1984; Petit et al., 1986). In the present experiments other d-agonists, Met5-enkephalin and DSLET, also increased basal DA release without effecting potassium-stimulated release. The difference between these responses and those with p-agonists emphasize that these changes are not mediated via preceptors. Taken together our data suggest that DA release from the rat striatum in vitro can be regulated by different mechanisms utilizing p- and d-opiate receptors. Activation of preceptors results in the inhibition of potassium-induced release, whereas stimulation of dopiate receptors increases the basal release of DA. There does not, however, appear to be any endogenous opioid tone, because the administration of either of the opiate antagonists naloxone and ICI 174864 failed to alter either basal or stimulated DA release. This suggests that opioid neuromodulation of DA release must be driven or activated by unknown mechanisms. The failure of naloxone to alter potassiumstimulated DA release is surprising because the depolarizing stimulus would have been expected to release endogenous p-agonists which would inhibit DA release. The addition of naloxone would then be expected to cause an increase in stimulated DA release, as the inhibitory opiate component would be attenuated. It is possible that the high potassium-induced depolarization was not of sufficient strength to release the endogenousp ligand. On the other hand, the potassium stimulus could have been so great as to obliterate any small inhibitory opiate component produced by the release of an endogenous p ligand. The increased DA release following administration of ethanol to the striatal tissues agrees with previous reports both in vitro and in vivo (Barbaccia et al., 1981; Holman and Snape, 1985; Imperato and DiJ. Neurochem.. Vol. 59, No. 1. 1992
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Chiara, 1986). The effect of ethanol particularly on basal DA release was dose dependent, which argues against a nonspecific drug effect or the result of decreased viability of the tissue in our preparation. Further, the enhanced basal DA release was prevented by the addition of high concentration of naloxone or by the more specific %opiate antagonist, ICI 174864, which suggests pharmacological specificity of action of ethanol. The similarity of ethanol- and d-opiate-induced increases in endogenous DA release is interesting. The observation that ethanol-induced increased basal DA release can be reversed by opioid antagonists including ICI 174864 suggests that ethanol may indirectly alter DA release through release of an endogenous ligand as the enkephalins. The presence of enkephalins in the striatum and %receptors on DA terminals would support this hypothesis. Further, Albertini et al. (1 979) have demonstrated that acute administration of ethanol is capable of increasing the release of Met-enkephalin in the striatum. The proposal that some of the effects of ethanol may involve endogenous opiates is also supported by the demonstration that ethanol-induced narcosis and hypothermia in rats can be attenuated by intracerebrally administered ICI 174864 (Widdowson, 1987). These data provide further evidence for the involvement of endogenous opioids in the acute effects of ethanol. REFERENCES Albertini A., Saiai M., Cecchetin M., and Trabucchi M. (1979) Effect of acute and chronic elcohol intake on brain enkephalin neurons and other transmitter systems, in Radioimrnunoassay ofDrugs and Hormones in Cardiovascular Medicine (Albertini A., DaPrada M., and Peskar B. A., eds), pp. 176- 184. Elsevier/ North Holland Biomedical Press, Amsterdam. Algeri S., Brunello N., Calderini G., and Consolazione A. (1978) Effect of enkephalins on catechol metabolism in rat CNS. Adv. Biochem. Psychopharmacol. 18, 199-209. Barbaccia M. L., Reggiani A., Spano P. F., and Trabucchi M. ( I98 1) Ethanol-induced changes of dopaminergic function in three stains of mice characterized by a different population of opiate receptors. Psychopharmacology 74,260-262. Baron M. and EyzaguirreC. (1977) Effects of temperature on some membrane characteristics of carotid body cells. Am. J. Physiol. 233, C35-C46. Biggo C., Casa M., Corda M. G., DiBello C., and Gessa G. L. (1978) Stimulation of dopamine synthesis in caudate nucleus by intrastriatal enkephalins and antagonism by naloxone. Science 20, 552-554. Chang K.-J., Hazumn E., and Cuatrecasas P. (I98 I ) Novel opiate binding sites selective for benzomorphan drugs. Proc. Natl. Acad. Sci. USA 78,4141-4145. Chesselet M. F., Cheramy A., Reisine T. D., LubetzkiC., Glowinski J., Fournie-Zaluski M. C., and Roques B. (1982) Effects of various opiates including specific delta and mu agonists on dopaminergic neurons in vitro in the rat and in vivo in the cat. Life Sci. 31, 2291-2294. Chesselet M. R., Cheramy A,, Reisine T. D., Lubetzki C., Desban M., and Glowinski J. (1983) Local and distal effects induced by unilateral striatal application of opiates in the absence or in the presence of naloxone on the release of dopamine in both caudate nuclei and substantiae nigrae of the cat. Brain Res. 258, 229-242.
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