Release of endogenous dopamine and cholecystokinin from rat striatal slices: effects of amphetamine and dopamine antagonists

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BRE 21471

Short Communications

Release of endogenous dopamine and cholecystokinin from rat striatal slices: effects of amphetamine and dopamine antagonists JOHN B. HUTCHISON, JAMES STRUPISH and STEFAN R. NAHORSKI Department of Pharmacology and Therapeutics, Medical Sciences Building, UniversiO' of Leicester. University Road, Leicester LE1 7RH (U. K. ) (Accepted December 3rd. 1985) Key words. dopamine - - cholecystokinin - - striatum

The release of immunoreactive cholecystokinin (CCK) and dopamine was monitored simultaneously from superfused rat striatal slices. Exposure of the tissue to medium containing elevated K + or veratrine, induced a marked release of both substances. The addition of dopamine (10-7 and 10-6 M), the dopamine agonist pergolide (t0 -7 M), the D2-antagonist sulpiride (1 uM) or the Di-antagonist (SCH 23390) had no significant effect on basal overflow or on evoked release of CCK. On the other hand, preincubation of striatat slices with D-amphetamine (10-5 M) enhanced basal and veratrine-stimulated dopaminc release but markedly suppressed evoked CCK release. Sulpiride blocked this action of amphetamine whereas SCH 23390 was ineffective. The data suggests that whereas it is difficult to observe any effects of exogenous dopamine agonists or antagonists on evoked CCK release, endogenously released dopaminc appears to interact with D2-receptors to suppress evoked CCK release from rat striatal slices.

In a classical study, Hokfelt et al.S have provided evidence that cholecystokinin (CCK) may co-exist with d o p a m i n e in certain mesencephalic neurones that project primarily to mesolimbic areas, though CCK immunoreactivity was absent in the majority of dopamine nerve fibres in the rat caudate nucleus. These observations have p r o m p t e d a large n u m b e r of studies probing the possible interaction of these neurotransmitters 6. F o r e x a m p l e , depletion of dopamine modifies the C C K content of both the nucleus accumbens and striatum 5 and C C K can potentiate apomorphine-induced inhibition of d o p a m i n e neuronesg. F u r t h e r m o r e , striatal and mesolimbic C C K levels are elevated by subchronic neuroleptic treatment5 and C C K exhibits neuroleptic-like effects in a variety of behavioural models 19. H o w e v e r , perhaps the most striking interactions have been at a presynaptic level with very low concentrations of C C K suppressing both basal and electrically e v o k e d [3H]dopa-

mine release from the cat caudate nucleus L~. On the other hand, exogenous d o p a m i n e appears to enhance or suppress veratrine-evoked C C K release depending upon the concentration usedJ~, and it has been argued that these actions of d o p a m i n e are mediated via d o p a m i n e D,- and Dl-receptors, respectively1,13. In view of these observations generally, and in particular the surprising effects of low 'exogenous' dopamine levels on evoked C C K release 13 when extracellular endogenous d o p a m i n e might be expected to be relatively high, we have d e v e l o p e d a superfusion system that allows the simultaneous m e a s u r e m e n t of endogenous d o p a m i n e and C C K release from rat striatal slices. We have provided evidence that d o p a m i n e released by a m p h e t a m i n e may suppress depolarization-evoked C C K release by interacting with a dopamine D2-receptor. Superfusion ofstriatal slices. Entire brains were re-

Correspondence." S.R. Nahorski, Department of Pharmacology and Therapeutics, Medical Sciences Building, University of Leicester, University Road. Leicester LE1 7RH, U.K 0(X)6-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

311 moved from male Wistar rats (200-300 g) after cervical dislocation, and two coronal cuts were made anterior to the optic chiasmata. The ventral parts of the corpora striata were cleared of cortical tissue and 0.3 mm slices were made in two directions (at 90 °) on a Mcllwain tissue chopper. Striatal slices were suspended in 0.5-ml capacity perspex chambers and superfused with Krebs bicarbonate buffer containing 30/~M bacitracin and gassed with 95% 0 2 - 5 % CO2 (ref. 4). Generally, striata from two rats were pooled and suspended in two or 3 chambers which were superfused (0.5 ml/min) simultaneously. Each chamber contained around 30 mg (w/w) tissue. Samples of superfusate were collected at 2-min intervals (sample vol., 1 ml) after a 30-min equilibrium period. Samples were collected over 20 min and drugs were added to the superfusion system via a 'T'-tube sidearm. In experiments where amphetamine or dopamine agonists and antagonists were used, drugs were added 15 min prior to the start of the sample collection. Duplicate 200-/~1 aliquots were removed from the samples and stored at -20 °C in plastic tubes for CCK radioimmunoassay. The remaining sample was acidified to 0.1 M with perchloric acid for catecholamine estimation. Fixed volume incubations. In order to reproduce experimental conditions described by Meyer and Krauss 13, striatal slices were placed in scintillation vials containing 1 ml Krebs bicarbonate buffer. Tissue was gassed and preincubated for 30 min prior to veratrine (5/~M) stimulation when buffer medium was removed and replaced with test buffer. After 20 min, 2 x 100/~1 aliquots were removed for CCK assay and the remaining 3001zl acidified to 0.1 M with perchloric acid and assayed for catecholamines as below. Catecholamine estimation. Dopamine in the superfusate was estimated by electrochemical detection following high-performance liquid chromatography (HPLC-ED). Catechols were separated on a 3/~m Hypersil ODS column using a mobile phase of citrate-acetate buffer (pH 4.8), containing 5% methanol and 0.1 g/1 sodium octylsulphonate. The detector was a Bioanalytical Systems BAS LC4 using carbon paste or glassy carbon electrodes. The system was calibrated using dopamine, adrenaline, noradrenaline and dihydroxyphenylacetic acid, and was sensitive to 1 pmol/ml dopamine in the superfusate. Cholecystokinin estimation. CCK in the superfu-

sate was measured by radioimmunoassay using a 'COOH'-terminal-specific antiserum, which was a kind gift from Professor G.J. Dockray. This antiserum cross-reacts almost equally well with CCK-8 and Gastrin-17, but only 2% as well with the 'C'-terminal tetrapeptide fragment. The assays were conducted according to methods previously described 3. Briefly, monoiodinated 15Leu-gastrin-17 label was prepared by brief reaction with chloramine T and purified on diethylaminoethyl-cellulose columns in a gradient of ammonium carbonate (0.6-6%). Assays were incubated for 48 h at 4 °C in i ml of 0.02 M sodium barNtone buffer (pH 8.4), containing 1% bovine serum albumin and 0.02% sodium azide. Synthetic CCK-8 and 15Leu-gastrin-17 were used as standards. Assays were regularly able to detect 0.6 pmol/l CCK-8, with 50% inhibition of binding with 4.46 + 0.30 pmol/1 CCK-8 in 10 consecutive assays. Antibody boundand free-labelled antigen were separated by addition of 0.2 ml charcoal-dextran-plasma (10:1:10), and activity in both was counted in a LKB RIA gammacounter for one min. Result calculation. In most experiments, peptide or catecholamine release is expressed as femtomoles or picomoles per milligram protein per minute. Tissue protein was estimated by the Folin method l~. In some experiments, the release is expressed as a percentage of the maximum release, or as the 'total stimulation' release (i.e. stimulated release minus basal release). Chemicals and drugs. Drugs used and suppliers were as follows: dopamine, HCI and veratrine (Sigma), pergolide (Eli Lilly), sulpiride isomers (Ravizza), SCH 23390 (Schering), D-amphetamine sulphate (Smith, Kline & French). All other reagents were purchased from Sigma or Fisons (U.K.). Depolarizing stimuli. In preliminary experiments various depolarizing stimuli were examined for their ability to release CCK and dopamine. Increased extracellular K ÷ (15-65 mM) resulted in release of both substances in an entirely Ca2÷-dependent manner. However, more consistent results were obtained with the alkaloid veratrine (1-10 BM). Pulses of veratrine induced prompt release of both dopamine and CCK. A 4-rain pulse of 5 /~M veratrine increased CCK release 4-5-fold over basal whilst dopamine release from the same slices was enhanced 10-fold (Fig. 1). The time-courses of the release of CCK and dopamine were similar, and in two experiments perform-

312

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Fig. 1. Simultaneous release of dopamine (open circles) and CCK (closed circles) from superfused striatum in response to a 4-rain pulse of veratrine (VER) (5 ~uM) indicated as the horizontal bar, in the absence (top panel) and presence (lower panel) of 10 #M D-amphetamine (AMPHET). In these experiments two superfusion chambers containing pooled striata from two animals were run under identical conditions, with the exception that 10 ,uM D-amphetamine was added to the superfusate of the chamber represented by the lower panel 15 rain prior to veratrine. Dopamine release (right vertical axis) and CCK release (left vertical axis) are expressed as picomoles and femtomoles per milligram protein per minute. These results represent the means with standard errors of 3 experiments. Results using veratrine (10 #M) as a stimulus (not shown) were similar. ed in the absence of calcium, evoked release of both substances was totally abolished.

Effects of exogenous dopamine, pergolide and sulpiride. In view of reports 13-14 that exogenous dopamine can enhance CCK release from striatal slices, the effects of exogenous dopamine (10 -7 and 10 -6 M), the dopamine agonist pergolide (10 -7 M) and the D2-antagonist (-)-sulpiride (5 × 10-7 M) were examined on CCK release in superfused tissue. In no experiments, either under resting conditions or during evoked release, were any significant effects seen of the drugs above on peptide release (data not shown). In an attempt to reproduce data reported by Meyer and Krauss 13, we used a similar fixed-volume protocol for incubation and identical stimulation parameters. We were unable to observe any effects of exogenous dopamine (10 -s to 10-6 M) on veratrine-induced CCK release. Indeed, assay of endogenous dopamine in the incubation medium under these con-

ditions revealed levels (l.1 +_ 0.2 × 10-7 M; n = 6) that were higher than those reported by Meyer and Krauss 13 to be effective in enhancing CCK when added exogenously.

E[fects of amphetamine and dopamine receptor antagonists. These experiments were designed to expose striatal slices to endogenously released dopamine prior to a depolarizing concentration of veratrine. Preincubation (15 min) of the tissue with D-amphetamine (1(I-5 M) enhanced the basal dopamine release (0.26 + 0.04 to 1.42 _+ 0.13 pmol/mg protein/ rain; n = 3). The basal release of CCK was unaffected under these conditions (Fig. 1). In the presence of amphetamine, veratrine-stimulated dopamine release was approximately doubled but in contrast, evoked release of CCK was markedly reduced. Thus, in 3 experiments using 5 /~M veratrine, the peak CCK release was reduced from 4.25 + 0.89 to 1.96 _+ (/.05 fmol/mg protein/rain (54%) (Fig. 1). The possibility that dopamine released by amphetamine suppressed depolarization-evoked CCK release by interacting with cell-surface receptors was next examined. The selective D I- and D2-receptor antagonists SCH 23390 (1 #M) and (-)-sulpiride (l #M) had no significant effects on either basal or on veratrine-stimulated CCK release. However, (-)sulpiride effectively blocked the ability of amphetamine to suppress evoked CCK release (Fig. 2). On the other hand, the D~-antagonist SCH 23390 did not significantly alter this action of amphetamine. Discussion.In the present studies we have described a method for the simultaneous measurement of endogenous CCK and dopamine release from superfused rat striatal slices in vitro. We have recently established that estimation of endogenous dopamine release using H P L C - E D reveals a number of inadequacies in the more commonly used method of prelabelling stores with [3H]dopamine 7. In this study, depolarizing stimuli were shown to release both CCK and dopamine in a calcium-dependent manner and have allowed us to examine the possible interactions between these striatal transmitters. A recent series of studies by Conzelmann et al.~, Meyer and Krauss ~ and Meyer et al. j4 have suggested that exogenous dopamine can regulate veratrine or K+-evoked release of CCK from rat striatal slices. In a fixed incubation-volume system this group

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Fig. 2. This figure illustrates CCK release from superfused rat striata in response to 5 ~M veratrine (VER) in two series of experiments. In the first, CCK release was measured in the absence and presence of 10ktM D-amphetamine (AMPH), and in the absence and presence of amphetamine plus 1~M (-)-sulpiride (SULP). In the second series (on the right of the figure), (-)-sulpiride was replaced by 1 ,uM SCH 23390 (SCH). Total CCK release over a 4-min period with basal release subtracted is shown by the vertical axes, and is expressed in femtomoles per milligram protein. In these experiments, 3 superfusion chambers were employed, each containing an aliquot of pooled striatal tissue. Each chamber received a 4-min pulse of veratrine (VER). In addition, one received 10/~M amphetamine (VER + AMPH) 15 min prior to veratrine, whilst the third received the veratrine pulse plus amphetamine with either 1 ~M sulpiride (VER + AMPH + SULP) or 1 ~M SCH 23390 (VER + AMPH + SCH), also for 15 min prior to stimulation. Each histogram represents the mean plus standard errors of 3 experiments. Neither 1/~M sulpiride nor 1/~M SCH 23390 had significant effects on basal or veratrine-stimulated CCK release in the absence of amphetamine (results not shown).

reported that exogenous dopamine (10 -7 M) enhanced CCK release whereas at lower or higher concentrations, this effect was not observed~3. Pharmacological evidence was provided that dopamine D zreceptor stimulation enhances release I whereas D~receptor activation suppresses release of CCK 14, and that both effects can be observed with exogenous dopamine depending upon its concentration. In our own studies, using superfusion and various stimulation parameters, we have failed to observe any effects of exogenous dopamine on CCK release. Furthermore, the highly selective Dz-antagonist sulpiride or the Dl-antagonist SCH 23390 were also without effect on evoked CCK release. This suggests that the simultaneous release of endogenous dopamine with depolarizing stimuli does not influence ongoing CCK release by interacting with D1- or D2-receptor sites. In attempts to reconcile these differences in the effects of dopamine on striatal CCK release, we exam-

ined the fixed volume-incubation technique under identical conditions, as described by Meyer and Krausst3. During 20-min incubations with 5/~M veratrine, CCK release was similar to that previously reportedt3 but addition of exogenous dopamine (10-s to 10 -6 M) failed to significantly influence this release. Furthermore, we also observed that the concentration of extracellular endogenous dopamine released under these conditions rose to 10-7 M. If a similar release of dopamine occurred in the experiments of Meyer and Krauss ~3, then it is difficult to see how effects on CCK release were observed on addition of 10 .7 M exogenous dopamine when concentrations were already near to this level due to release of endogenous dopamine. Again, in contrast to our own studies, it should be recalled that in the initial study, Meyer and Krauss 13 reported an inhibitory effect of the D2-antagonist haloperidol on evoked CCK release which they ascribed to the effect of this drug antagonizing released endogenous dopamine at D 2 sites. However, in a subsequent study I these workers failed to observe an effect of the D2-antagonist domperidone on K*-evoked release of CCK. In contrast to the effect of exogenous dopamine, in this study preincubation of striatal slices with D-amphetamine markedly suppressed the ability of veratrine to evoke CCK release. Amphetamine (10-5 M) increased the basal dopamine release 5-fold from striatal slices and doubled the veratrine-induced release of this amine, so an effect of endogenously released dopamine on the peptide was suspected. Two strategies were considered to test such a hypothesis. Attempts could be made to prevent amphetamine's ability to release dopamine by using the synthesis inhibitor a-methyl-p-tyrosine. However, recent studies from this laboratory 7 using an identical superfusion system have established complex effects of this inhibitor on amphetamine's releasing effect and it was considered that interpretation of any results would be difficult. On the other hand, use of the selective dopamine antagonists sulpiride (D2) and SCH 23390 (DO provided results that strongly suggest the effect of endogenously released dopamine on C C K may be mediated by dopamine D2-receptors. Thus, concentrations of sulpiride that would be expected to selectively and totally block these sites]5,16 completely reversed the amphetamine effect. On the other hand, concentrations of SCH 23390 that would selectively

314 totally occupy Dl-receptors> were quite ineffective. Thus, on the basis that amphetamine itself possesses very low affinity for dopamine De-sites 15 and is therefore very unlikely to have direct effects, the most parsimonious explanation is that released dopamine sup-

provided that endogenously released dopamin¢ may be effective. Whether this relates to higher local concentrations of endogenous amine and/or that a finite pre-exposure of the D~-sites to dopamine is necessary, requires further investigation.

presses CCK release via De-receptors. Stimulation of De-receptors inhibits the release of several neurotransmitters ~6,w, including the neuropeptide fl-endorphin from hypothalamic slices Is. Unlike some of the latter studies, however, it has not

The authors would like to thank the M.R.C. and the Nuffield F o u n d a t i o n for financial support. J.B.H. was a Foulkes Fellow. Thanks are also due to Profes-

been possible here to establish effects of exogenous

sor G, Dockray for provision of the CCK antiserum and helpful advice with the assay, and to Jenny Bell

dopamine on CCK release though evidence has been

for manuscript preparation.

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