Characterisation of dopamine receptors in insect (Apis mellifera) brain

BRAIN RESEARCH ELSEVIER

Brain Research 706 (1996) 47-56

Research report

Characterisation of dopamine receptors in insect ( Apis mellifera) brain Ilona C. Kokay *, Alison R. Mercer Department of Zoology, University of Otago, Dunedin, New Zealand Accepted 5 September 1995

Abstract

In vitro binding experiments using the vertebrate D 1 dopamine receptor ligand [3H]SCH23390 and the vertebrate D 2 dopamme receptor ligand [3H]spiperone were conducted on membrane preparations of honey bee (Apis mellifera) brain. Specific binding of [3H]SCH23390 was saturable and reversible. Analysis of saturation data gave an apparent K d of 6.3 ___1.0 nM and Bmax of 1.9 + 0.2 pmol/mg protein for a single class of binding sites. The specificity of high affinity [3H]SCH23390 binding was confirmed in displacement experiments using a range o f dopaminergic antagonists and agonists. The rank order of potency for antagonists was: R(+)-SCH23390 > cis-(Z)-flupentixol > chlorpromazine > fluphenazine > S(+)-butaclamol > spiperone. R(+)-SKF38393 and dopamine were the most effective agonists tested. [3H]SCH23390 labels a site in bee brain that is similar, but not identical to the vertebrate D 1 dopamine receptor subtype. [3H]Spiperone also bound with high affinity to bee brain homogenates. Scatchard analysis of [3H]spiperone saturation data revealed a curvilinear plot suggesting binding site heterogeneity. The high affinity site had a apparent K a of 0.11 + 0.02 nM and Bmax of 9.2 + 0.5 fmol/mg protein. The calculated values for the low affinity site were a K a of 19.9 nM and Bmax of 862 fmol/mg protein. Kinetic analyses also indicated that [3H]spiperone recognises a heterogeneous population of sites in bee brain. Furthermore, agonist competition studies revealed a phenolaminergic as well as a dopaminergic component to [3H]spiperone binding in bee brain. The rank order of potency of dopaminergic antagonists in competing for [3H]spiperone binding was: spiperone > fluphenazine > S( + )-butaclamol > domperidone > R( + )-SCH23390 > S( - )-sulpiride. Keywords: Dopamine; D 1 receptor; [3H]SCH23390; [3H]Spiperone; Binding; Insect; Brain

1. Introduction

In contrast to the extensive literature on dopamine receptors in the vertebrate brain, surprisingly little is known about the receptors that mediate the actions of dopamine in the insect central nervous system. Dopamine has been detected in the nervous system of many insect species [8,20,34,46], and enzymes required for dopamine synthesis have also been identified [9,10,20,39,43]. Dopamine-sensitive adenylate cyclase activity in the insect brain indicates that at least some of the actions of dopamine are mediated t h r o u g h the elevation of intracellular c A M P [6,17,27,49,52,66], but the identity of the receptors involved has yet to be resolved. Dopamine receptors in the vertebrate brain have for some years, been classified into two major subtypes, D1 and D 2 receptors respectively [2,33,60,65]. With the advent of molecular cloning techniques, however, additional

* Corresponding author. Fax: [email protected]

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subtypes have been revealed [12,63,64]. To date, five pharmacologically distinct dopamine receptor subtypes have been recognized. Two of these cloned receptors (D 1 and Ds), exhibit functional and pharmacological properties that resemble classical D 1 dopamine receptors, whereas the other three (D 2, D 3 and D 4) are D2-1ike in their pharmacological characteristics [24,61,64]. Using radioligand binding studies with [3H]pifluthixol, Notman and Downer [48] identified a receptor in the brain of the cockroach, Periplaneta americana, with pharmacological properties similar to mammalian D1 receptors. D : like dopamine receptors have also been identified in the salivary glands of this insect [21,22] and in the locust [I]. However, a number of studies indicate that dopamine receptors in the insect central nervous system may be pharmacologically distinct from those found in vertebrates [14,49,56]. The existence of multiple dopamine receptor subtypes in the insect brain has yet to be clearly established. High affinity binding of the benzazepine antagonist SCH23390 is characteristic of the vertebrate D 1 dopamine

48

L C. Kokay, A.R. Mercer/Brain Research 706 (1996) 47-56

receptor subtype [31,32]. In contrast, the pharmacology of the D 2 receptor subtype is characterized by high affinity binding of the butyrophenone spiperone [13,38,60] and benzamides such as sulpiride and raclopride [2,24,61]. In the present study, [3H]SCH23390 and [3H]spiperone are used to determine whether receptors with characteristics similar to the vertebrate D 1 and D 2 dopamine receptor subtypes are present in insect brain. Here we show that both [3H]SCH23390 and [3H]spiperone bind reversibly and with high affinity to honey bee (Apis mellifera) brain homogenates. [3H]SCH23390 binds to a receptor that resembles the vertebrate D 1 dopamine receptor subtype. The binding of [3H]spiperone is more complex and is best described by a two-site model. While the pharmacological profile of [3H]spiperone binding is similar to that of a vertebrate D 2 dopamine receptor, the phenolamines tyramine and octopamine are more effective at displacing this ligand than dopamine. A preliminary report of some of the information in this paper has been presented elsewhere [35].

2. Materials and methods

2.1. Preparation of tissue Adult worker honey bees were collected from hives maintained at the University of Otago Department of Zoology and immobilised by chilling at 4°C. The brain (including optic lobes and suboesophageal ganglion) was removed from the head capsule of each bee, frozen in liquid nitrogen and stored ( < 3 weeks) at - 8 0 ° C until required. Frozen brain tissue was homogenized in a 50-fold volume of ice-cold Tris-HCl buffer (50 mM, pH 7.5) containing 120 mM NaC1, 1 mM MgC12, 5 mM KCI, and 200 /xM phenylmethylsulfonyl fluoride (PMSF) then centrifuged at 400 × g for 10 min at 4°C to remove retinal pigments and fragments of connective tissue. The homogenate was centrifuged twice at 20,000 × g for 45 min at 4°C. For the [3H]SCH23390 assays the final pellet was resuspended in fresh ice-cold Tris-HC1 buffer, homogenised and stored at - 8 0 ° C (for not more than 4 weeks), until use. For [3H]spiperone binding assays, icecold MOPS (3-[n-morpholino]propanesulfonic acid) buffer (20 mM adjusted to pH 7.5 with Tris-base) containing 120 mM NaCI, 20 mM MgC12, 2 mM CaC12, 10 mM KCI, 1 mM EDTA, and 200 /xM PMSF was substituted for the above Tris-HC1 buffer. Protein concentration of the homogenate was determined using a modification [51] of the method of Lowry et al. [40] using BSA as the standard and including a trichloroacetic acid precipitation step.

2.2. Radioligand binding assays Binding assays were performed in duplicate or triplicate and, unless stated otherwise, repeated three times. For

displacement and kinetic studies, aliquots of membrane preparation containing 100-200 /zg of protein were added to tubes containing vehicle or competing drug and either 2 nM [3H]SCH23390 or 0.5 nM [3H]spiperone. For saturation experiments a range of 8-12 radioligand concentrations was added. Final assay volumes were 1 ml ([3H]SCH23390) or 0.5 ml ([3H]spiperone). In [3H]spiperone agonist displacement experiments, NaCI was omitted from the buffer, 0.1% ascorbic acid was added and the incubations were carried out in a nitrogen-rich environment. A nitrogen-rich environment was necessary to prevent the oxidation of dopamine during the relatively long incubation period. After incubation for 45 min at 25°C ([3H]SCH23390), or 90 min at 20°C ([3H]spiperone), the samples were rapidly filtered under vacuum through Whatman G F / C filters presoaked in 0.5% polyethylenimine. Filters were washed three times in 3 ml of ice-cold Tris buffer ( 50 mM, pH 7.3), extracted overnight in 10 ml of scintillation fluid (26% Triton X-100, 0.4% 2,5-diphenyoxazole in toluene) and the radioactivity counted using a liquid scintillation analyser (Packard 1900CA) at 53-56% efficiency. Specific binding of [3H]SCH23390 was defined as the difference between total binding and binding in the presence of 5 × 10 -6 M cis-(Z)-flupentixol. [3H]Spiperone specific binding was defined as the difference between total binding and binding in the presence of 10-SM domperidone. Binding of [3H]SCH23390 to rat striatal tissue was included as a control in these experiments.

2.3. Drugs [3H]SCH23390 (specific activity 67-86 Ci/mmol) and [3H]spiperone (specific activity 96-107 Ci/mmol) were purchased from Amersham. The following compounds were purchased from Research Biochemicals, USA: R(+)-SCH23390 HCI, S(_)-SKF38393 HC1, cis-(Z)flupentixol 2HC1, fluphenazine HC1, S(+)-butaclamol HC1, domperidone, ketanserin. All other drugs were obtained from Sigma Chemical Co. All drugs were prepared just before use. Some drugs were dissolved initially in methanol (spiperone and domperidone), or ethanol (sulpiride and ketanserin), or acidified (butaclamol and serotonin) and then diluted in buffer before use. Dopamine octopamine, tyramine and histamine were dissolved in water through which N 2 had been bubbled.

2.4. Data analysis Saturation and competition results were analysed using the non linear least squares curve-fitting programme, LIGAND [45] modified for use on the microcomputer [41]. This programme (EBDA/LIGAND) was also used to calculate the inhibition constants ( g i s ) of the displacing drugs. Association and dissociation studies were analysed

LC. Kokay, A.R. Mercer~Brain Research 706 (1996) 47-56

using the iterative programme, KINETIC [41]. Data are expressed as means + S.E.M.

appeared to saturate within the range of concentrations used (Fig. 1B), close examination of the saturation binding isotherm suggested [3H]spiperone may be binding to more 0.08

3. Results

Saturable, specific binding of both the vertebrate D 1 ligand [3H]SCH23390 and the vertebrate D 2 ligand [3H]spiperone to honey bee brain membrane preparations was observed (Fig. 1). At 2 nM [3H]SCH23390, and 0.5 nM [3H]spiperone, specific binding represented 87 + 2.2% and 56 + 0.5% of total binding respectively.

3.1. Saturation of [3H]SCH23390 binding Preliminary experiments showed that the cations Na ÷ (10-100 mM) and Mg 2÷ (at 2 mM) tended to enhance [3H]SCH23390 binding, whereas the divalent cation Ca 2÷ inhibited the binding of this ligand. CaCI 2 was omitted from the buffer in all subsequent assays. Preliminary experiments also established that binding of 2 nM [3H]SCH23390 showed a linear relationship with protein concentration up to 300 p.g of protein. Specific binding was also found to be highest between pH 7.3-pH 8. For all subsequent experiments the buffer was adjusted to pH 7.5. Saturation studies were performed at equilibrium with 8 concentrations of [3H]SCH23390 ranging from 0.019 nM to 23 nM. A curve of the specific binding values of a representative experiment is shown in Fig. 1A. Scatchard transformation of these data (inset Fig. 1A) yields a straight line. Assuming a single class of binding sites the apparent K d for this experiment was calculated to be 6.9 nM. The Hill coefficient was 0.99 (data not shown), also suggesting no co-operatively or heterogeneity of sites in the concentration range tested. The density of [3H]SCH23390 binding sites was calculated as 2.2 pmol/mg protein. The apparent mean affinity constant for [3H]SCH23390 binding calculated from the results of three independent experiments was 6.3 _ 1.0 nM and the mean density of binding sites was 1.9 + 0.2 pmol/mg protein. [3H]SCH23390 bound with higher affinity to rat striatal tissue, included in these experiments as a control. The apparent K d at 25°C was 0.47 _ 0.05 nM, and Bmax was calculated as 1.01 _ 0.03 pmol/mg protein, values consistent with published data [4,7].

3.2. Saturation of [3H]spiperone binding Without EDTA in the homogenising buffer, specific binding of [3H]spiperone ranged from 0-15%. Inclusion of an EDTA wash in the receptor preparation however, increased specific binding levels to around 60% of total binding. EDTA (1 mM) was added routinely in all subsequent experiments. Saturation experiments were performed at equilibrium with 8 or 9 concentrations of radioligand ranging from 0.04 nM-5 nM. Although specific binding

49

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Fig. 1. A: saturation curve of specific [3H]SCH23390 binding to Dl-like binding sites in homogenates of honey bee brain. A single representative curve is shown which was repeated three times. Data points are the means of triplicate determinations. Membranes were incubated for 45 min at 25°C with 8 concentrations of [3H]SCH23390 ranging from 0.2-23 nM. Specific binding was calculated by subtracting nonspecific binding (binding in the presence of 5 × 10 -6 M cis-(Z)-flupentixol) from total binding. The curve was fitted using MINIM 2.0, a Macintosh application for non-linear parameter estimation. The inset shows a Scatchard plot of the saturation curve data. These data, analysed by LIGAND, are best fitted to a one site binding model. The binding parameters are K d = 6.9 nM, Bmax = 2.2 p m o l / m g membrane protein. The mean binding parameters calculated from the results of 3 independent experiments are K d = 6.3 + 1.0 nM, Bmax = 1.9+0.2 p m o l / m g membrane protein. B: saturation curve of specific [3H]spiperone binding to homogenates of honey bee brain. A single representative curve is shown which was repeated three times. Data points are the means of duplicate determinations. Membranes were incubated for 90 min at 20°C with 9 concentrations of [3H]spiperone ranging from 0.4 nM-4.6 nM. Specific binding was calculated by subtracting nonspecific binding (binding in the presence of 10 -s M domperidone) from total binding. The curve was fitted as in A above. The inset shows a Scatchard plot of the saturation curve data. These data are best fitted by LIGAND to a two site binding model. The calculated binding parameters are, K a = 0.1 nM, Bmax = 10 f m o l / m g membrane protein for site one and K a = 23.5 nM, Bmax = 559 f m o l / m g for site two. The mean binding parameters calculated from the results of 3 independent experiments are K d = 0 . 1 1 + 0 . 0 2 nM, B m a x = 9 . 2 + 0 . 5 f m o l / m g membrane protein for site one and K d = 19.9 + 3.0 nM, Bmax = 862+ 172 f m o l / m g for the second site.

I.C. Kokay, A.R. Mercer~Brain Research 706 (1996)47-56

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Fig. 2. A: association of specific [3H]SCH23390 binding to honey bee brain homogenates. Data points are the mean (+ S.E.M.) of three independent experiments, each done in triplicate. Aliquots of membrane preparations were incubated with 2 nM [3H]SCH23390 at 25°C for increasing periods of time before filtration. Non-specific binding was defined as binding in the presence of 5 × 10 -6 M cis-(Z)-flupentixol. B: dissociation of specific [3H]SCH23390 binding to honey bee brain homogenates. Data points are the mean (+ S.E.M.) of three independent experiments, each done in triplicate. Aliquots of membrane preparations were incubated with 2 nM [3H]SCH23390 at 25°C for 45 min. Cis-(Z)-flupentixol to a final concentration of 5 × 10-6 M was then added and specifically bound [3H]SCH23390, as a percentage of total specific binding, was measured at selected time intervals.

than one site. Scatchard transformation of the data also revealed a curvilinear plot indicating binding site heterogeneity (inset Fig. 1B). The high affinity site had an apparent K d of 0.1 nM and a Bmax of 10 f m o l / m g protein. A K d value of 23.5 nM and a Bmax of 559 f m o l / m g was calculated for the low affinity site. The mean affinity constants for [3H]spiperone binding calculated from the results of three independent experiments were, for the high affinity site, an apparent K d of 0.11 _ 0.02 nM and a Bmax of 9.2 + 0.5 f m o l / m g protein. Calculated mean values for the low affinity site were a K d of 19.9 _ 3.0 nM and a Bmax of 862 _ 172 nM.

3.31 Kinetics of [3H]SCH23390 binding [3H]SCH23390 (2 nM) bound rapidly and reversibly to brain homogenate. A t 25°C, specific binding of [3H]SCH23390 reached equilibrium in less than 40 min

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(Fig. 2A). Binding was stable for at least 120 min, indicating that degradation of neither the binding sites nor the radioligand was occurring under these conditions. Dissociation kinetics were examined by incubating 2 nM [3H]SCH23390 with brain homogenate at 25°C till equilibrium (45 min), then displacing the radioligand from the binding sites by the addition of 5 × 10 -6 M cis-(Z)flupentixol. As shown in Fig. 2B, the dissociation of [3H]SCH23390 occurred rapidly. Specific binding was fully reversed after approximately 10 min. The apparent equilibrium dissociation constant was calculated from the ratio of the association and dissociation constants ( k _ l / k 1) using the iterative analysis program, KINETIC. Data from the association and dissociation experiments were best fitted by a two site model, which gave a K d of 4.2 nM for the high affinity site and a K d of 24.8 nM for the low affinity site. The value for the high affinity site is in reasonable agreement with the dissociation constant calculated by

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Fig. 3. A: association of specific [3H]spiperone binding to honey bee brain homogenates. Data points are the mean (+ S.E.M.) of two independent experiments, each done in triplicate. Aliquots of membrane preparations were incubated with 0.5 nM [3H]spiperoneat 20°C for increasing periods of time before filtration. Non-specific binding was defined as binding in the presence of 10 -5 M domperidone. B: dissociation of specific [3H]spiperonebinding to honey bee brain homogenates. Data points are the mean (+ S.E.M.) of two independent experiments, each done in triplicate. Aliquots of membrane preparations were incubated with 0.5 nM [3H]spiperone at 20°C for 90 min. Domperidone to a final concentration of 10-5 M was then added and specifically bound [3H]spiperone, as a percentage of total specific binding, was measured at selected time intervals.

L C. Kokay, A.R. Mercer~Brain Research 706 (1996) 47-56

saturation studies (6.3 nM) for a one binding site model. The highest concentration of radiolabelled ligand tested in the saturation experiments was less than 25 nM. A low affinity site was not revealed in the saturation studies.

3.4. Kinetics of [3H]spiperone binding Binding of [3H]spiperone to honey bee brain homogenates was moderately rapid and was reversible. At room temperature, equilibrium was attained after approximately 60 min (Fig. 3A). Incubation for 90 min at room temperature was used routinely for all saturation and competition studies. Domperidone was used to examine the kinetics of [3H]spiperone binding. After incubation to equilibrium (90 min at RT), dissociation of [3H]spiperone from the binding sites was initiated by addition of domperidone at a final concentration of 10 -5 M. After 30 min, specific binding had decreased to around 35% (Fig. 3B). The kinetic data were best fitted by a two binding sites model, which yielded an apparent K d of 0.10 nM for site 1 and a K d of 0.08 nM for the second site. The K a values are in good agreement with the dissociation constant for a high affinity site identified by saturation studies (0.11 nM). The affinities of the two apparent sites identified by iterative analysis are so similar, they would be unlikely to be differentiated using saturation studies. Reliable detection of two binding sites by saturation analysis requires the radioligand to have at least 5-fold selectivity [11].

51

Table 1 Affinities of aminergic compounds for [3H]SCH23390 and [3H]spiperone labelled sites in honey bee brain [ 3 H]SCH23390 Ki

[ 3H]Spiperone Ki

Antagonists R( + )-SCH23390 9.5 + 1 nM 456 -I- 26 nM Spiperone 25354 + 6580 nM 0.17 + 0.02 nM * cis-(Z)-Flupentixol 218 + 39 nM Chlorpromazine 208 ___ 58 nM S(+)-Butaclamol 13810 -I- 2302 nM 55.5 -t- 20.9nM Fluphenazine 788 5256 nM 20.1 + 8.9 nM Domperidone 384 + 27 nM S( - )-Sulpiride 29690 5- 5325 nM Ketanserine 74982 -1-12500 nM

Agonists R( -I-)SKF38393 Dopamine Tyramine Serotonin Octopamine Histamine

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A series of competition experiments, using a range of aminergic antagonists and agonists was used to investigate the pharmacology of the high affinity [3H]SCH23390 binding site (Fig. 4, Table 1). Elf the dopaminergic antagonists examined, the most potent displacer was unlabelled SCH23390, whereas the least potent at displacing [3H]SCH23390 was the D 2 dopamine antagonist spiperone (Fig. 4A). The thioxanthene, cis-(Z)-flupentixol, and the phenothiazine, chlorpromazine, both inhibited [3H]SCH23390. binding with moderate potency but S( + )butaclamol, a dopamine antagonist that displaces [3H]SCH23390 binding with high affinity in vertebrate preparations, was a poor displacer of [3H]SCH23390 binding in the bee brain. The vertebrate 5-HT2/5HT~ antagonist ketanserin was largely ineffective at inhibiting [3H]SCH23390 binding (Table 1). The rank order of potency for antagonists was found to be: R( + )-SCH23390 > cis-(Z)-flupentixol > chlorpromazine > fluphenazine > S(+)-butaclamol > spiperone > ketanserin. Of the agonists tested, the vertebrate D~ dopamine receptor agonist R(+)SKF38393 was the most potent displacer of [ 3H]SCH23390 binding (Fig. 4B), with a K i of 3.2 /~M. Dopamine was also an effective inhibitor of [3H]SCH23390 binding, more effective than serotonin, octopamine, tyramine or histamine. Displacement data were analysed using the pro-

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L C. Kokay, A.R. Mercer~Brain Research 706 (1996) 47-56

52

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spiperone was the most potent antagonist at displacing [3H]spiperone binding, followed by fluphenazine (K i = 20.1 nM, high affinity site) and S(+)-butaclamol (K i = 55.5 nM). The butyrophenone-like compound domperidone was a moderately potent inhibitor of [3H]spiperone binding, whereas S(-)-sulpiride was poor at displacing this ligand (Table 1). The rank order of potency for dopaminergic antagonists was found to be: spiperone > fluphenazine > S( + )-butaclamol > domperidone > R( + )SCH23390 > S( - )-sulpiride. Of the biogenic amines tested, serotonin and histamine were less effective at displacing [3H]spiperone than dopamine, whereas the phenolamines, tyramine and octopamine, were more effective than dopamine at displacing this ligand (Fig. 5B, Table 1). The binding of octopamine to [3H]spiperone binding sites appeared to be unsaturable (Fig. 5B). Competition curves obtained using dopamine and octopamine are best fitted by a two site model suggesting that both high and low affinity sites for these amines are present in membrane preparations of honey bee brain. 4. Discussion

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Fig. 5. Displacement of [3H]spiperone binding to honey bee brain membranes by aminergic drugs. Curves shown are representative of three similar experiments each done in duplicate. Aliquots of membrane preparations were added to tubes containing increasing amounts of displacing drugs and [3H]spiperone at a final concentration of 2 nM. Mean K i values ( + S.E.M.) are shown in Table 1. A: displacement by antagonist drugs. Curves analysed using the program LIGAND were best fitted to a one site binding model except for spiperone and fluphenazine which were best fitted to a two site model. B: displacement by agonist drugs. In these experiments, NaC1 was omitted from the incubation buffer, 0.1% ascorbic acid was added and experiments were conducted in a N2-rich atmosphere. Displacement by octopamine and dopamine were best fitted by LIGAND to a two site binding model. Other agonists tested were best fitted to a one site model of binding.

gramme LIGAND, and were best fitted to a one site model. Data obtained using butaclamol could also be fitted to a two site model, but the fit was not significantly better than that obtained for a single site. Data obtained using chlorpromazine were in some experiments best fitted by a two site model but in others a one site model was appropriate.

3.6. Pharmacology of [3H]spiperone binding sites A range of compounds, including drugs known to be selective for D 2 dopaminergic receptors, was used to characterize the [3H]spiperone binding sites (Fig. 5, Table 1). With the exceptions of spiperone and fluphenazine, data from antagonist curves were best fitted to a one site binding model. Data from displacement with spiperone and fluphenazine were better fitted to a two site binding model. With a K i value of 0.17 nM (high affinity site), unlabelled

This paper clearly identifies a population of dopamine receptors in the brain of the honey bee with characteristics similar to those of the D 1 dopamine receptor subtype identified in the vertebrate brain. We have also demonstrated high affinity binding sites for the vertebrate D 2 ligand, [3H]spiperone. Although [3H]SCH23390 binds reversibly to a Dl-like site in the brain of the bee, the binding affinity of this ligand is lower than that reported for D 1 dopamine receptors in the vertebrate brain [4,7,23]. This difference in affinity was confirmed using rat striatal tissue processed in parallel with the bee brain homogenates. The dissociation constant recorded in this study for [3H]SCH23390 binding in rat striatum (K d = 0.46 nM) is consistent with published data [4,23] and is ca. 13-fold higher than the affinity of the [3H]SCH23390 binding site found in the brain of the bee (K d ---6.3 nM). A Hill coefficient of 0.99 suggests that under the experimental conditions used in the saturation experiments, [3H]SCH23390 is binding to a homogeneous class of sites in the bee brain. However, kinetic analyses suggest that this ligand may also bind to a low affinity site that has a dopaminergic component. It is possible that in insects Dl-like receptors exist in two interconverting affinity states. In the saturation experiments, however, the highest concentration of ligand used was less than that required to saturate and thus clearly detect the apparent low affinity site. The properties of this site therefore, have yet to be fully elucidated. Analysis of the high affinity [3H]SCH23390 binding site reveals a pharmacological profile remarkably similar to the vertebrate D 1 dopamine receptor. The most effective inhibitors of [3H]SCH23390 binding in honey bee brain homogenates were drugs reported to have higher affinity at

L C. Kokay, A.R. Mercer~Brain Research 706 (1996) 47-56

D 1 sites than at D 2 sites [23,24]. SCH23390 and cis-(Z)flupentixol for example, were considerably more potent at displacing [3H]SCH23390 than the D 2 antagonist spiperone, and the most effective agonist tested was the specific D 1 receptor agonist, S(+)-SKF38393. Compared to dopamine, relatively high concentrations of the biogenic amines serotonin, octopamine, tyramine and histamine were needed to displace the binding of [3H]SCH23390, supporting the view that this ligand is binding to a dopaminespecific receptor site in the brain of the bee. Fluphenazine and S( + )-butaclamol, which display high affinity for vertebrate [3H]SCH23390 binding sites [4,23,28,54] show a lower affinity for [3H]SCH23390 binding sites in bee brain. This suggests that the Dl-like receptor in the bee is similar, but not identical to the D 1 dopamine receptor subtype found in the vertebrate brain. Given that a single amino-acid difference in a receptor gene sequence can have profound effects on the pharmacological profile of the receptor [50], differences between the pharmacology of the D~ dopamine receptor subtype in the brains of vertebrates and invertebrates are not unexpected. Although widely used as a selective label for D~ receptors in the vertebrate brain [4,7,53,59], [3H]SCH23390 can also bind to serotonin receptors, particularly in areas of high 5-HTle receptor density [5,47]. For this reason, the serotonergic antagonist ketanserin is often included in [3H]SCH23390 binding assays to mask binding to serotonergic receptors. In bee brain, however, ketanserin was ineffective at displacing [-~'H]SCH23390 (K i of > 70,000 nM) and was not included in the assays. It has been shown that [3H]SCH23390 will selectively label D 1 receptors as long as sodium ions are present in the incubation buffer [54], conditions that were adopted in the present study. Differences between the pharmacological profiles of the D~-like dopamine receptors identified in the brain of the cockroach [48] and those found in the bee, are likely to reflect differences in the selectivity of ligands used to examine these sites. [3H]Pifluthixol, used to demonstrate the presence of Dl-like dopamine receptors in the cockroach brain, can bind with equal affinity to D 2 and D 1 dopamine receptors [30], and also interacts with serotonin and histamine receptors in the vertebrate brain (see [37]). Ligand selectivity may also explain why S( + )-butaclamol, which had a low affinity for [3H]SCH23390 binding sites in bee brain, was a relatively potent displacer of [3H]pifluthixol in the cockroach [48]. In the vertebrate brain, S(+)-butaclamol has been reported as having a similar affinity for D 2 and D 1 dopamine receptors [23,24,42], whereas in bee brain preparations, S(+)butaclamol showed a 250-fold greater affinity for [3H]spiperone binding sites than sites labelled with the D 1 dopamine receptor ligand [3H]SCH23390. At the concentrations used in this study, the vertebrate D 2 dopamine receptor ligand [3H]spiperone showed little specificity for D~-like dopamine receptor sites in the brain of the bee. Autoradiographical studies have also revealed

53

clear differences between the distribution of [3H]spiperone binding sites in the bee brain, and sites labelled with the D 1 dopamine receptor antagonist, [3H]SCH23390 [35]. The pharmacological profile of [3H]spiperone binding sites is markedly different from that of the Dl-like dopamine receptor in the bee but the calculated inhibition constants of specific [3H]spiperone binding for aminergic drugs should be interpreted with care as the non-specific binding component of this ligand was relatively high ( > 40%). Unlike [3H]SCH23390, [3H]spiperone binding is displaced more effectively by vertebrate dopamine receptor antagonists, such as spiperone and fluphenazine than by drugs that are selective for vertebrate D1 dopamine receptor sites. However, S(-)-sulpiride, which is a moderately potent displacer of [3H]spiperone binding in vertebrates [24,61] was relatively ineffective at displacing this ligand in the bee brain. S(-)-Sulpiride has also been reported to be a poor inhibitor of [3H]LSD binding to a dopamine-like site in the locust brain, a site at which spiperone is relatively effective (see [56]). S(-)-Sulpiride is also relatively ineffective at inhibiting dopamine stimulation of adenylate cyclase in the brain of the cockroach, although here also spiperone appears to be a moderately effective dopamine antagonist [49]. [3H]Spiperone bound with high affinity to bee brain homogenates. The K d value of 0.11 nM calculated from saturation experiments for the high affinity site is within the range of those calculated for the mammalian brain [60,69,70]. D 2 receptors in vertebrates are thought to exist in two agonist affinity states [15,26,62]. The binding profile for dopamine to [3H]spiperone sites in bee brain is also best described by a two site model, with binding sites of different affinities. The estimates of the binding parameters of the low affinity site must be viewed with some caution however, as they required extrapolation of the data well beyond the range of ligand concentrations used in the study. Although results from experiments using D 2 antagonists are not inconsistent with an interaction of [3H]spiperone at a D2-1ike site in tile bee brain, the identity of [3H]spiperone-binding sites in the brain of the bee remains uncertain. Kinetic analyses also reveal that binding of [3H]spiperone in bee brain homogenates is complex and is likely to involve more that one site. Furthermore, the agonist profile of this ligand indicates that [3H]spiperone recognizes phenolamine receptor sites in the brain of the bee. Tyramine is more effective at displacing [3H]spiperone than dopamine, and octopamine also displays high affinity for [3H]spiperone binding sites in bee brain. Dopamine and dopaminergic drugs such as apomorphine, S(+)-butaclamol, chlorpromazine and haloperidol have been found to be relatively ineffective at inhibiting the binding of ligands such as [3H]octopamine and [3H]mianserin used to label octopamine receptors in the insect brain [19,29,36,44]. Studies of amine stimulation of cyclic AMP also suggest that dopamine and octopamine

54

LC. Kokay, A.R. Mercer~Brain Research 706 (1996) 47-56

act via distinct receptor sites in insects [6,27,49,66]. Although there is evidence for both octopamine and tyramine receptors in the insect brain [29], interesting overlaps exist between the receptors that mediate the effects of these closely related amines [55,66]. Expression of the recently cloned Drosophila octopamine/tyramine receptor [3,55,57] in a mammalian cell line has shown, firstly, that both octopamine and tyramine are effective agonists at this cloned receptor site and secondly, that these closely related amines are capable of coupling this receptor to different second messenger systems [55]. These results illustrate the difficulties that exist in differentiating aminergic receptor subtypes in the insect brain. Although specific tyramine-mediated responses in insects have been reported [16,18,66], the lack of a definitive pharmacology for tyramine receptors makes it difficult to isolate the tyramine component of [3H]spiperone binding apparent in the brain of the bee. As dopaminergic drugs such as apomorphine and chlorpromazine are moderately effective inhibitors of [3H]tyramine binding in the locust brain [29], it is perhaps not surprising that binding of [3H]spiperone in honey bee brain homogenates displays a tyramine component. It would seem premature, however, to assume that [3H]spiperone is labelling a tyraminespecific site in the brain of the bee. In vertebrate brain, for example, [3H]para-tyraminebinds with high affinity (K d ca. 10 nM) to sites concentrated in membranes isolated from the corpus striatum and hypothalamus. Evidence suggests that rather than binding to a central tyramine receptor, [3H]para-tyramineassociates with a receptor that is functionally related to the transport of dopamine in these tissues [67,68]. Only when potent, selective antagonists for tyramine receptors are available will unequivocal characterization of this receptor subtype be possible. As in vertebrates, dopamine has a markedly higher affinity for [3H]spiperone binding sites in bee brain than for the Dl-like dopamine receptor site identified using [3H]SCH23390. In the bee, [3H]spiperone-binding sites are concentrated in areas of the brain such as the mushroom bodies ([35], and in preparation). The mushroom bodies, or corpora pedunculata, receive dense innervation from dopamine-immunoreactive neurons [58], but Di-like dopamine receptors are not found in the mushroom body neuropil [35]. These results are difficult to reconcile with there being only one dopamine receptor subtype in the bee brain. Although [3H]spiperone binding is complex and displays a phenolaminergic as well as a dopaminergic component, the results of this study, together with autoradiographic analyses presented elsewhere [35], suggest that in addition to a Dl-like dopamine receptor, a non Dl-like binding site with high affinity for dopamine is present in the brain of the bee. A receptor with significant homology to the human D I / D 5 receptor has recently been identified in Drosophila[25]. Identification of the putative dopamine/tyramine receptor subtype(s) revealed in this study using [3H]spiperone awaits molecular cloning and

characterization of amine receptor genes expressed in the mushroom bodies of the brain of the bee.

Acknowledgements We wish to thank Prof. Richard Laverty and Dr. Graham Young for encouragement and advice during this study. We also thank Dr. Robert Purves for help in data analysis and the use of his computer program MINIM 2.0. Rat brain tissue was kindly donated by Professor Laverty and Associate Professor W.C. Abraham. This research was supported by grants ORG MFZ B69-B77/MFZ B64-B72. I.C.K. was personally supported by a Blair scholarship from Otago University.

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