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Characterization of a dopamine-sensitive [3H]LSD binding site in honeybee (Apis mellifera) brain
Camp. Biochem. fhysiol. Vol. I IOC, No. 2, pp. 197-205, 1995 Copyright 0 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0742-8413/95 $9.50 + 0.00
Pergamon 0742-8413(94)00091-3
Characterization of a dopamine-sensitive binding site in honeybee (Apis mell@ra) W. Blenau,”
C3H]LSD brain
T. May? and J. Erber*
*Institut fiir Biologie, Technische Universitlt Berlin, Germany; and TInstitut fur Neuropsychopharmakologie, D-14050 Berlin, Germany
Franklinstr. 28/29, D-10587 Berlin, Freie Universitlt Berlin, Ulmenallee
30,
PHILysergic acid diethylamide (13H]LSD) binds on membrane homogenate of honeybee brain to both a dopamine-sensitive site (D-site) and a serotonin-sensitive site (S-site). Under suitable conditions the properties of the two sites can be studied separately. Specific binding of 13H]LSD to both the D-site and the S-site has high affinity and is saturable. The mean equilibrium dissociation constants (Ko) were 3.8 nM for the D- and 0.89 nM for the S-site. The densities (B,,,,, values) of both binding sites were 1.7 pmol/mg protein for the D-site and 0.79 pmol/mg protein for the S-site. 13H]LSD binding to the D-site was reversible and reached equilibrium in about 30 min. Pharmacological displacement studies display a high binding affinity of the putative natural agonist dopamine to the D-site (Ki = 22 nM). The most potent displacers of D-site binding were lisuride, (+)-bromocriptine, chlorpromazine, S( +)-butaclamol, and 6,7-ADTN. The 13H]LSD labelled D-site seems to be G-protein coupled, since addition of the stable GTP analogue GTPy S or NaCl to the incubation medium evoked a decrease of specific 13H]LSD binding to the D-site. Key words: Dopamine Neuromodulator. Comp. Biochem.
Physiol.
receptor;
5-HT
IIOC, 197-205,
receptor;
[3H]LSD
binding;
Honeybee;
Insect
brain;
1995.
Introduction demonstrated the immunoreactivity (IR) of neural elements for a large number of putative and established neurotransmitters and modulators [for a review see Erber et al. (1993)]. For amines, neuroanatomical descriptions exist for serotonin-, dopamineand octopamine-like IR in the bee brain (Rehder et al., 1987; Schafer and Bicker, 1986; Schiirmann and Klemm, 1984; Schafer and Rehder, 1989; Schiirmann et al., 1989; Kreissl et al., 1991). Our knowledge about the function of dopamine in the bee mainly comes from behavioural and electrophysiological analyses in the olfactory pathway of the brain. Dopamine injected globally through the ocellar tract can reduce the amount of sucrose uptake and can block the retrieval of a conditioned olfactory stimulus, while it does not interfere with the storage of an olfactory signal [for a review see Menzel et al. (1994)]. Local injection of dopamine into the
Biogenic amines in the bee brain play an important role in controlling and modulating different types of behaviour [for a review see Erber et al. (1993)]. The existence of different biogenic amines has been shown in the bee brain. The highest concentrations were found for dopamine (12-40 pmol/brain) and serotonin (6-21 pmol/brain), while octopamine had intermediate concentrations (approximately 8 pmol/brain) and noradrenaline showed the smallest concentration (l-4 pmol/brain) (Fuchs et al., 1989; Harris and Woodring, 1992; Mercer et al., 1983; Taylor et al., 1992). Immunocytological studies in the bee have Correspondence fo : W. Blenau, Institut fiir Biologie; FR I- 1, Technische Universitit Berlin, Franklinstrasse 28/29, D-10587 Berlin, Germany. Tel. (030) 314 73 344; Fax (030) 314 73 177. Received I1 May 1994; revised 29 September 1994; accepted 15 October 1994. 197
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W. Blenau et al
a-lobe of the mushroom bodies reduces the proboscis extension responses to water vapour (Blenau and Erber, 1993; Menzel et al., 1994). Application of dopamine onto the antenna1 lobes reduces the percentage of animals responding to a conditioned olfactory stimulus (Macmillan and Mercer, 1987). In many respects the response modifications after dopamine are similar to those after serotonin application. In the bee brain, as in other insect nervous tissues and glands, there is only very limited knowledge about the pharmacological characteristics of dopamine receptors (Evans and Green, 1990a,b; Roeder, 1994). In two studies, haloperidol, a vertebrate dopamine receptor antagonist, was used as a putative antagonist also in bees (Michelsen, 1988; Macmillan and Mercer, 1987). Recently a number of putative dopamine receptor antagonists were tested using the proboscis extension reflex in the bee (Blenau and Erber, 1993). This study showed that haloperidol had only weak antagonistic effects, while significant antagonistic effects were found for chlorpromazine, flupentixol, spiperone and buspirone. The pharmacological characterization of dopamine receptors in other invertebrates is also very incomplete. Electrophysiological studies indicate that the pharmacological profile of dopamine receptors on the soma of an inhibitory motoneuron of the cockroach does not conform well to mammalian D,- or D,-receptors (Pitman and Davis, 1988; Davis and Pitman, 1991). In homogenates of the ganglia of a mollusc, Helix porn&a, Drummond and coworkers (1978) demonstrated dopamine-sensitive binding sites for [3H]LSD. [3H]LSD labels also a dopamine receptor in the locust nervous tissue (Degen et al., 1992; Roeder, 1994). In membrane preparations of cockroach brain Notman and Downer (1987) demonstrated high affinity binding of [iH]pifluthixol, a vertebrate dopamine receptor antagonist. The bee offers the possibility to study the effects of dopamine at the level of different types of behaviour (Blenau and Erber, 1993; Menzel et al., 1994) and at the level of interneurons (Mercer and Erber, 1983). To understand the dopamine effects at these levels it is necessary to characterize the dopamine receptor(s) in this insect. As binding studies using [‘Hldopamine are not feasible in membrane homogenates of bee brain (see below), we studied the kinetics and the pharmacological profile of dopaminesensitive [jH]LSD binding to membrane homogenate of bee brain. It was the aim of this study in the bee to provide further data for a comparison of dopamine receptors in different invertebrate species.
Materials and Methods Insects
Foraging worker bees (Apis mellzjkra) were caught at the entrance of the hive and immobilized by chilling. They were mounted in small metal tubes as described by Erber and Menzel (1977) to dissect the brains. Tissue preparation
Brains (including optic lobes and suboesophageal ganglia) of worker bees were dissected under ice-cold ringer solution (268 mM NaCl, 3.19 mM KCl, 1.2 mM CaCI,, 10 mM sulfonic MgC&, 10 mM morpholinopropane acid, pH 7.4) as quickly as possible (about 4 min) and transferred into Eppendorf tubes. The tubes were then rapidly immersed in liquid nitrogen and kept at a temperature of -80°C until use. The tissue was homogenized using a glass/teflon homogenizer (10 strokes, 1000 rounds per minute) in ice-cold buffer (50 mM Tris-HCl, 5 mM MgSO,, 1OOpM phenylmethylsulforyl fluoride, pH 7.4). The homogenate was centrifuged (40,000 g, 10 min, 4°C) and the resulting pellet was rehomogenized in the buffer, incubated at 30°C for 10 min and centrifuged as before. The final pellet was rehomogenized in the buffer for use in the binding assay. Tissue aliquots (70&100 pg protein) were incubated at 22°C usually for 60 min in the buffer containing 100 PM ascorbic acid (final volume 1 ml, pH 7.4). The reaction was initiated by the addition of tissue and following incubation, bound and free ligand were separated by vacuum filtration of the samples through Whatman GF/B filters pre-soaked in 0.3% polyethyleneimine (Sigma) using a Brandell cell harvester. The filters were rapidly washed three times with 4 ml washing buffer (50mM Tris-HCI, pH 7.4). Bound radioactivity on the filters was determined by liquid scintillation spectrometry (counting efficiency about 60%). Radioligand binding to tissue samples was determined in duplicate. Dopamine-sensitive [3H]LSD binding (to the D-site) was estimated in the presence of 300 nM serotonin (5-HT). Non-specific binding was defined as binding in the presence of 10 PM dopamine, if not otherwise indicated. Specific binding was calculated as the difference between total and non-specific binding. The protein content of all samples was determined according to the method of Lowry and co-workers with bovine serum albumin (Sigma) as the standard (Lowry et al., 1951). Analyses of saturation, competition, and kinetic experiments were performed using EBDA/LIGAND and KINETIC computer software programs (G. A. McPherson,
Dopamine-sensitive
[‘HILSD
Elsevier-BIOSOFT, Cambridge, U.K.) which utilize non-linear least squares curve fitting techniques (Munson and Rodbard, 1980). The Ki values were calculated using the equation of Cheng and Prusoff for competitive inhibitors 1973): and Prusoff, Ki = Ic,,/ (Cheng ((1 + [L*])/K,), where IC,~ is the concentration of displacer, which inhibits 50% of specific binding, [L*] is the used concentration of radioligand and KD is the equilibrium dissociation constant. Drugs and other chemicals [3H]LSD (specific activity 72.4 Ci/mmol) and [3H]dopamine (specific activity 32 Ci/mmol) were purchased from DuPont NEN (Dreieich, Germany). [3H]Spiperone (specific activity 24 Ci/mmol) was from Amersham Buchler (Braunschweig, Germany). The following drugs were obtained from Research Biochemicals, Inc. (Ktiln, Germany): (+)-2-amino-6,7-dihydroxy- 1,2,3,4_tetrahydronaphtalene hydrobromide (6,7-ADTN HBr), (+)-bromocriptine HCl, methanesulfonate, S( +)-butaclamol R( -)-butaclamol HCl, cis(Z)-flupentixol dihydrochloride, trans(E)-flupentixol dihydrochloride, haloperidol, histamine dihydrochloride, (-)-quinpirole HCI, R( +)-SCH-23390 HCl, S( -)-SCH-23388 HCI, R( +)-SKF-38393, and spiperone HCI. Buspirone HCl, chlorpromazine HCl, dopamine HCI, GTPy S, 5-hydroxytryptamine HCI, fluphenazine dihydrochloride, metoclopramide HCl, (-)-norepinephrine bitartrate, (k)-octopamine HCL, phenylmethylsulforyl fluoride, S( -)-sulpiride, and tyramine HCl were purchased from Sigma Chemical Co. (Deisenhofen, Germany). Lisuride hydrogen maleate was a gift from Schering AG (Berlin, Germany). All other chemicals were of analytical grade.
binding
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Initial experiments using [3H]LSD as radioligand showed saturable binding. The specific binding (non-specific binding defined as binding in the presence of 1 mM dopamine) with a given concentration of [)H]LSD (2 nM) increased linearly with increasing amount of bee brain membrane homogenate (SO-250~s protein) in the assay mixture. Heating for 10 min at 100°C of the membrane homogenate decreased 98% of specific [3H]LSD binding in comparison with untreated tissue. In initial competition studies using a rH]LSD concentration of about 1 nM, various biogenic amines were tested for their ability to decrease specific [3H]LSD binding. At concentrations of 1 PM or less only dopamine and serotonin (5-HT) were efficacious displacers. (+)-Octopamine, (-)-norepinephrine, tyramine and histamine were much less active. Dopamine and 5-HT displaced L3H]LSD binding in a strongly biphasic manner (Fig. 1A). For the displacement experiments with serotonin, non-specific binding was defined as the amount of binding in
FL
e
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10-9
I
10"
I
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Competitor
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I
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I
104
!
10-Z
concentration (M)
0
Red ts First we tried to label dopamine receptors in the bee brain with [3H]dopamine, but the resulting binding was non-saturable within the range of l-50 nM and heat insensitive, arguing against receptor character of the binding site. The reasons for the unsuccessful labelling with [‘Hldopamine are unknown; similar observations were made in the locust (Roeder, personal communication). Furthermore we could not detect specific binding of another radioligand, [iH]spiperone, to the bee brain preparation. This ligand labels, with high affinity, dopamine Dz and serotonin 5-HT, receptors in mammalian brain. Also in this case the reasons for the unsuccessful labelling in the bee are unknown but similar to the findings in the mollusc Helix pomatia (Drummond et al., 1980).
I
lo-3
0
I ‘L..
0
5 [3H]LSD
10 concentratcin
15
20
(nM)
Fig. 1. Inhibition of specific [jH]LSD binding to membrane homogenate of bee brain by dopamine and 5-HT. (A): displacement of the specific [‘H]LSD binding (1.3 nM) by different concentrations of dopamine (0) and 5-HT (0). The definitions of specific binding are described in the text. (B): saturation curves of specific [‘H]LSD binding. (0): S-site; (0): D-site; (0): both sites experimentally determined; (A): both sites calculated as the sum of S-site + D-site. For experimental details see text.
200
W. Blenau
the presence of 1 mM 5-HT. For the experiments with dopamine, non-specific binding was defined in the presence of 3 mM dopamine. In both cases specific binding was approximately 95% of total binding. At low concentrations both amines displaced [3H]LSD probably from different binding sites. This hypothesis was tested by [3H]LSD binding saturation experiments. Saturation curves were determined in the absence of any displacer (total binding) and in the presence of the following displacers: 10pM dopamine, 1 PM 5-HT, and 10 PM dopamine + 1 PM 5-HT. Figure l(B) shows the resulting saturation curves of specific [3H]LSD binding which were determined by calculating the differences between total binding and binding in the presence of the displacers. At all used 13H]LSD concentrations the calculated sum of the specific [3H]LSD binding determined with 10 PM dopamine (D-site binding) and with 1 PM 5-HT (S-site binding) is in accordance with the experimentally determined specifically displaced [3H]LSD binding by 1 PM 5HT + 10 PM dopamine together (binding to both sites). With higher displacer concentrations (2 1 mM) specific binding to both sites (D- and S-site) was abolished by either amine acting alone, suggesting that the two sites exhibit specificity for dopamine and 5-HT only at lower concentrations. The estimated K, values of dopamine as [3H]LSD binding displacer were 22 nM for the dopamine-sensitive site (D-site) and 47 PM for the serotonin-sensitive site (Ssite), respectively. Serotonin as displacer revealed K, values for the S-site of 2.6 nM and for the D-site of 7 PM. The differences of dopamine and 5-HT in displacing [3H]LSD binding were used to derive conditions under which the radioligand was able to bind specifically to either the D- or the S-site. In the presence of 300 nM 5-HT, dopaminesensitive binding could be analysed, while 5-HTsensitive binding was analysed in the presence of 10 PM dopamine. Under the chosen conditions specific binding to both D-site (defined by 10 PM dopamine) and S-site (non-specific binding defined by 1 PM 5-HT) was higher than 80% of total binding when homogenate concentration was one brain/sample (70-100 pg protein/sample) and [3H]LSD concentration was 0.5 nM. The K,, for [3H]LSD binding to the D-site was 3.8 f 0.5 nM (Scatchard plot shown in Fig. 2), and for the S-site 0.89 f 0.03 nM (not shown). Figure l(B) also reveals that there were about twice as many saturable D-sites (B,,, = 1.7 f 0.1 pmol/mg protein) than S-sites (B,,, = 0.79 f 0.12 pmol/mg protein) in the bee brain preparation. Hill analysis of the D-site saturation binding data (Fig. 3) yielded a linear plot with a Hill
et al.
1t
0.6
J
0.0
!
I
I
05
0.0
I
1.0
1.5
(pmollmg protein)
Bound
Fig. 2. Scatchard plot of specific [‘HILSD binding on membrane homogenate of bee brain to the D-site, measured in the presence of 300 nM 5-HT. Membrane homogenate was incubated for 60min at 22°C with various concentrations of [‘H]LSD (0.1-10 nM). Non-specific binding was determined in the presence of 10nM dopamine. Points shown are from a typical experiment performed in duplicate which was replicated three times. K. = 3.8 + 0.5 nM and E max= 1.7+ 0.1 pmol/mg protein (mean & SD).
slope of 1.02 + 0.01. The linearity of the Scatchard plot and the Hill coefficient near unity suggest that under the chosen conditions a single class of dopamine-sensitive binding sites was labelled by [3H]LSD. Due to the limitations in the number of data points, we are unable to rule out the existence of multiple dopamine-sensitive [3H]LSD binding sites. The available data from our saturation experiments, however, support the existence of a homogenous [3H]LSD labelled D-site. Kinetic analysis of the dopamine-sensitive [3H]LSD binding (Fig. 4) was performed at a concentration of 0.5 nM [3H]LSD at 22°C. Under these experimental conditions, binding
0.75 nH = 1 02 0.50
1
t
-1 25 -1 50 -10.0
-9.5
I
I
I
1
-9.0
-0 5
-0 0
-7 5
log (M) free [3H]LSD
Fig. 3. Hill plot of specific [jH]LSD binding on membrane homogenate of bee brain (D-site). Data from Fig. 2. Hill plot analyses resulted in a Hill-coefficient (nu) of 1.02 f 0.01 (mean + SD).
Dopamine-sensitive
I
I
I
I
0
30
60
90 Time
I
120
I
150
[3H]LSD
I
160
(min)
Fig. 4. Kinetic analysis of specific [‘H]LSD binding to membrane homogenate of bee brain (D-site). Association (0) was stopped after the indicated times. Dissociation (0) was initiated after 60 min by the addition of lisuride to a final concentration of I pM. Non-specific binding was determined with 10pM dopamine. Values are the means of a single experiment performed in duplicate.
reached maximal values after about 30-60 min of incubation. A slight decline of specific binding was observed after longer incubations, probably due to receptor denaturation. At equilibrium (after 60 min association) dissociation was initiated by the addition of 1 PM lisuride which competes with reassociation of [3H]LSD binding. Dissociation of binding was found to occur slowly with an apparent half-life of about 20 min. Specific binding decreased to about 14% after 2 hr, indicating reversibility of the [‘H]LSD binding to the D-site. However, dissociation appears to be biphasic, with a rapid initial phase followed by a slow phase. The results of pharmacological studies by displacement of [3H]LSD binding to the D-site are summarized in Table 1. Table 1 shows the percentage of inhibition of the binding of 0.6 nM [jH]LSD by a variety of drugs at 1 PM. The K, values of selected drugs were determined by inhibition of [3H]LSD (0.6 nM) binding by coincubation with six different concentrations of the respective compounds (Figs 5 and 6 and Table 2). Among the biogenic amines dopamine and tyramine were the most potent displacers of dopamine-sensitive [3H]LSD binding. (+)-Octopamine, (-)-norepinephrine, 5-HT, and histamine were less efficacious. The compounds with highest affinity to the D-site were the ergot derivates, lisuride and (+)-bromocriptine. The vertebrate dopamine receptor agonist 6,7ADTN was also a potent displacer, whereas (-)-quinpirole and R( +)-SKF-38393 were much less effective. The vertebrate dopamine receptor antagonist with highest displacing potency was chlorpromazine. S( +)-Butaclamol and R(-)-butaclamol as well as cis(Z)-flupen-
binding
in Apis
201
tixol and trans(E)-flupentixol (Fig. 6) displayed stereoselective inhibition of [3H]LSD binding to the D-site. The enantiomers R( +)-SCH-23390 and S( -)-SCH-23388 were less stereoselective displacers. The rather low affinity of the butyrophenon group of compounds (spiperone and haloperidol) is remarkable. Metoclopramide and (-)-sulpiride were nearly ineffective in displacing [3H]LSD binding to the D-site. The dopamine-sensitive [3H]LSD binding site showed characteristics of G-protein coupled receptors. High concentrations of NaCl(lO0 mM) decreased the specific binding of 0.6 nM [3H]LSD at the D-site by 62%. The addition of the GTP stable analogue GTPyS also led to a reduction of specific [3H]LSD binding. Under the chosen conditions 100 PM GTPy S decreased specific binding to the D-site by 56%.
Discussion Radioligand binding studies were performed for a pharmacological in vitro characterization of a putative dopamine receptor in the honeybee brain. We demonstrated that [3H]LSD binds to both dopamine- and serotonin-sensitive sites in membrane homogenate of bee brain, and that conditions can be selected which allow the two sites to be studied separately. The K,, values calculated from the saturation data revealed that the affinity of [3H]LSD for the dopamine-sensitive site (K,, = 3.8 nM) is about four times lower than that for the serotonin-sensitive site (KD = 0.98 nM). The K,, value for the D-site in the bee is about 3.8 times higher than the respective K,, value for this site in desert locust brain preparation (Degen et al., 1992). It is nearly an order of magnitude higher than that calculated for dopamine receptors in the Roman snail Helix pomatiu (Drummond et al., 1978). The KD value for the S-site revealed that the affinity of [3H]LSD for this binding site in the bee brain is slightly higher than that reported for the S-site in locust nervous tissue (KD = 1.64 nM; Wedemeyer et al., 1992) and molluscan ganglia (KD = 1.2 nM; Drummond et al., 1980). A comparison of B,,,.,, values for the two binding sites revealed that the bee brain preparation contained about twice as many D-sites (B,,, = 1.7 pmol/mg protein) than Ssites (B,,, = 0.79 pmol/mg protein). The reverse situation has been found with the desert locust Schistocerca gregaria (Degen et al., 1992; Wedemeyer ef al., 1992). Here the B,,, values were found to be about 50 fmol/mg protein for the D-site and 79.8 fmol/mg protein for the S-site. The much higher density of both putative dopamine and serotonin receptor binding sites in the CNS of the honeybee compared with the locust preparation is remarkable.
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The pharmacological properties of dopaminesensitive [3H]LSD binding sites in the bee brain were assessed by competitive displacement experiments using a variety of unlabelled dopaminergic and serotonergic ligands. Ergot alkaloids and derivates such as lisuride and (+ )-bromocriptine, which are structurally related to the radioligand, were very potent displacers for D-site binding. D-site binding of [3H]LSD to membrane preparations of bee brain was stereoselective. Comparison of the Ki values displayed S( +)-butaclamol more than 1200 times more potent than R( -)-butaclamol and cis(Z)-flupentixol 24 times more potent than trans(E)-flupentixol as inhibitors of [3H]LSD binding to the D-site. The enantiomers R(+)SCH-23390 and S( -)-SCH-23388 were less selective. Stereospecificity is an important criterion for ligand receptor interaction (Laduron, 1984). The spectrum of drug action on the [3H]LSD-labelled dopamine-sensitive site in the
et al.
bee brain is clearly different from that of vertebrate dopamine D,- and D,-receptors labelled with [3H]SCH-23390 or [3H]spiperone (Seeman and Niznik, 1988). In the bee, D-site binding could be displaced with high affinity by the selective dopamine D, receptor ligands fluphenazine, cis(Z)-flupentixol and R( +)-SCH23390 or by the more selective dopamine Dz receptor ligands (+ )-bromocriptine and S( + )butaclamol. Conversely, some compounds such as metoclopramide (non-selective), SKF-38393 (D,), and S( -)-sulpiride (D,), which are potent dopaminergic agents in mammals, are nearly in displacing dopamine-sensitive inactive [‘H]LSD binding in the bee brain. Among the substances with highest affinity were substances without receptor subtype specificity in vertebrates, such as chlorpromazine and 6,7ADTN. The moderate affinity of spiperone (D2) may be related to the lack of specific [3H]spiperone binding in the bee brain
Table 1. Percentage inhibition of specific [‘H]LSD (0.6 nM) binding to the D-site by different compounds at 1 PM. Three hundred nM 5-HT were added to suppress binding to the S-site. Mean values, the range and the number (N) of independent experiments performed in duphcate are shown Compound Biogenic amines Dopamine Tyramine (+)-Octopamine (-)-Norepinephrine 5-Hydroxytryptamine Histamine Ergot alkaloids and derivates Lisuride (+ )-Bromocriptine Vertebrate dopamine receptor agonists 6,7-ADTN (-)-Quinpirole R( +)-SKF-38393 Vertebrate dopamine receptor antagonists Chlbrpromazke S( + )-Butaclamol Fhtphenazine cb(Z)-Flupentixol Spiperone R( +)-SCH-23390 Haloperidol trans(E)-Flupentixol S(-)-SCH-23388 Buspirone Metoclopramide S( -)-Sulpiride R( -)-Butaclamol
Percent inhibition (mean values)
Range
N
85 48 31 36 18 10
8487 4650 34-39 34-38 lo-27
2 2 2 2 2 1
108* 94
107-108 92-95
3 3
88
86-89 28-35
2 2
31 14
90 89 19 16 61 56 47 31 40 21 17 7
1
89-92 86-93 79-80 71-80 51-67 53-58 40-55 36-38 31-48 23-3 1 8-26 2-11 1-3
4 4 2 2 3 2 2 2 2 2 2 2 2
‘The calculated values of over 100% inhibition for lisuride are due to the fact, that 1 PM lisuride is more effective in displacing (‘H]LSD binding than 10 PM dopamine. This dopamine concentration was used to define non-specific binding.
Dopamine-sensitive
[3H]LSD
0
??.Y% 10"
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Competitor
I
1o-10
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104
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104
203
in Apis
Table 2. Inhibition of dopamine-sensitive [‘H]LSD binding to membrane homogenate of bee brain by various compounds. Inhibition of specific binding was determined with six different concentrations of the competing drugs (performed twice in duplicate). The K, values were determined using the LIGAND-program (Munson and Rodbard, 1980)
A
1 O-9
binding
w-5
(M)
Fig. 5. Displacement of specific D-site [3H]LSD binding by various agents. Non-specific binding was determined in the presence of 10 PM dopamine. (A): vertebrate dopamine receptor agonists: l , lisuride; 0, (+)-bromocriptine; 0, 6,7-ADTN. (B): vertebrate dopamine receptor antagonists: 0, chlorpromazine; 0, S( + )-butaclamol.
Because of the low affinity of haloperidol to the D-site it does not seem suitable to use this compound in future behavioural or electrophysiological experiments to block the action of dopamine in the honeybee brain. The pharmacological profile of [3H]LSD binding to the dopamine sensitive site in the preparation.
Fig. 6. Inhibition of specific [)H]LSD binding to the D-site by isomeric drugs. Non-specific binding was determined in the presence of 10 p M dopamine. Different concentrations cis(Z)-flupentixol (0) and [runs(E)-flupentixol (0) were added to displace specific [‘H]LSD binding.
Compound
Ki (nM)
Lisuride Dopamine Chlorpromazine 6,7-ADTN S( +)-Butaclamol cis(Z)-Flupentixol (+)-Bromocriptine frans(E)-Flupentixol 5Hydroxytryptamine R( -)-Butaclamol
4.1 22 48 78 89 150 160 3600 7000 > 100,000
locust Schistocerca gregaria is very similar to that of the bee (Roeder, 1994). Interestingly, the pharmacology of the bee D-site also resembles those of a [3H]LSD binding site in the nervous tissue of Helixpomatia (Drummond et al., 1978) and a [3H]pifluthixol binding site in the brain of Periplaneta americana (Notman and Downer, 1987). The pharmacological profile of the dopamine sensitive adenylate cyclase in the cockroach brain (Orr et al., 1987) and even of the dopamine receptor in the salivary gland acinar cells of the cockroach (Evans and Green, 1990a,b) is also similar to that of the dopamine sensitive [3H]LSD binding site in the bee nervous system. However correlation analyses of the pharmacological data from the putative dopamine receptor binding sites of the bee brain compared with those of other invertebrates are difficult to perform, since most calculated K, values were determined for different compounds in the different studies. The finding that dopamine-sensitive binding sites in the insect CNS have pharmacological properties distinct from those of mammalian dopamine receptors is supported by the observations on dopamine responses in insect neurones. Davis and Pitman (1991), studying depolarizing responses to dopamine in the soma of an inhibitory motoneurone of the cockroach Periplaneta americana, concluded that the receptors of this neurone, characterized on the basis of their pharmacological properties, do not fit the mammalian dopamine receptor classification scheme. The data of the in vitro binding study discussed here are in good agreement with the behavioural pharmacological experiments with honeybees using the unconditioned proboscis extension reflex to test the effects of putative dopamine receptor antagonists (Blenau and Erber, 1993). Chlorpromazine, cis(Z)-flupentixol and spiperone were efficacious both in the binding study and in the behavioural test.
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Haloperidol and S( -)-sulpirid, being less active in the behavioural test, were also less effective in displacing dopamine-sensitive [3H]LSD binding. Only buspirone, a very efficacious antagonist in the behavioural test, does not fit into this scheme. Buspirone does not displace D-site binding with high affinity in vitro. The dopamine-sensitive [3H]LSD binding site in the bee fulfils basic specificity criteria of receptor sites (Laduron, 1984) like displacement by agonists and antagonists belonging to different chemical and pharmacological classes, stereospecificity, saturability, reversibility, high affinity, and correlation between drug affinity in vitro and pharmacological potency in vivo. The significant reduction of the specific [3H]LSD binding to the D-site in the presence of GTPyS suggests that the dopamine-sensitive 13H]LSD binding site is coupled to a G-protein and therefore is likely to be a physiologically functional dopamine receptor. In mammals GTP and stable analogues are believed to convert high affinity dopamine receptors to a low affinity state for agonists (Leff et al., 1985; Grigoriadis and Seeman, 1985). Sodium chloride also apparently inhibited D-site binding which is an additional argument for a receptor binding site. Inhibitory effects of sodium have been reported for both D, and D, receptors in mammals, where sodium is believed to reduce agonist affinity (Grigoriadis and Seeman, 1985; Urwyler, 1987; Reader et al., 1992). The pharmacological characterization of a putative dopamine receptor in the honeybee brain indicates similarities to cockroach and molluscan receptors. There exist clear differences to dopamine receptors described in mammals. Thus, the pharmacological distinctiveness of invertebrate receptors, which has been demonstrated for other insect neuroreceptors (Eldefrawi et al., 1986), is also evident for dopamine receptors. Over the past few years five different mammalian dopamine receptors have been cloned and identified using molecular biological techniques (for a review see Civelli et al., 1993). These subtypes exhibit characteristic differences in their pharmacology and in their second messenger pathways through which they operate. The research on dopamine receptors in invertebrates is in its beginning, further research might reveal that dopamine receptors in invertebrates have a similar degree of complexity as found in mammals.
Acknvwl[~dnemenrs~This work was supported by Studienstiftung des deutschen Volkes and by-a grant- from the Deutsche Forschunasgemeinschaft (PF 128/6-3). We wish to thank Professor Dr-H. Coper of the Institut fur Neuropsychopharmakologie for his generous support which was essential for the experiments.
et al.
References Blenau W. and Erber J. (1993) The behavioural effects of dopamine and putative antagonists in the mushroom bodies of the honey bee Apis mellifera. In Proceedings of the 20th GBttingen Neurobiology Conference (Edited by Elmer N. and Heisenberg M.), p. 680. Thieme, Stuttgart. Cheng Y.-C. and Prusoff W. H. (1973) Relationship between the inhibition constant (K,) and the concentration of inhibitor which causes 50 per cent inhibition (I,,) of an enzymatic reaction. Biochem. Pharmac. 22, 3099-3
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Civelli O., Bunzow J. R. and Grandy D. K. (1993) Molecular diversity of the dopamine receptors. Ann. Rez>. Pharmacol.
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32, 28 l-307.
Davis J. P. L. and Pitman R. M. receptors mediating the actions ified inhibitory motoneurone Biol. 155, 2033217. Degen J., Roeder T. and Gewecke a dopamine receptor in the
(1991) Characterization of of dopamine on an identof the cockroach. J. e.~p. M. (1992) [‘HILSD labels locust nervous tissue. In
Proceedings qf the 20th Giittingen Neurobiology Conference (Edited by Elsner N. and Richter D. W.). p. 573.
Thieme, Stuttgart. Drummond A. H., Bucher F. and Levitan I. B. (1978) LSD labels a novel dopamine receptor in molluscan nervous system. Nature 272, 368-370. Drummond A. H., Bucher F. and Levitan 1. B. (1980) p-[‘HILysergic acid diethylamide binding to serotonin receptors in the molluscan nervous system. J. bivl. Chem. 255, 6679-6686.
Eldefrawi M. E., Abalis I. M., Sherby S. M. and Eldefrawi A. T. (1986) Neurotransmitter receptors of vertebrates and insects as targets for insecticides. In Neurophurmacology and Pesticide Action (Edited by Ford M. G., Lunt G. G., Reay R. C. and UsherwoodP. N. R.), pp. 154-173. Horwood, England. Erber J., Kloppenburg P. and Scheidler A. (1993) Neuromodulation by serotonin and octopamine in the honeybee: behaviour, neuroanatomy and electrophysioiogy. E.xperientia 49, 107331083. Erber J. and Menzel R. (1977) Visual interneurons in the median protocerebrum of the bee. J. cvmp. PhJ:Fio/. 121, 65-77.
Evans A. M. and Green K. L. (1990a) Characterization of the dopamine receptor mediating the hyperpolarization of cockroach salivary gland acinar cells in citro. Br. J. fharmac. 101, 1033108. Evans A. M. and Green K. L. (1990b) The action of dopamine receptor antagonists on the secretory response of the cockroach salivary gland in ritro. Comp. Biochem. Physiol. 97C,
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Fuchs E., Dustmann J. H., Stadler H. and Schiirmann F. W. (I 989) Neuroactive compounds in the brain of the honeybee during imaginal life. Camp. Biochem. Physiol. 92C, 337-342.
Grigoriadis D. and Seeman P. (1985) Complete conversion of brain Dz dopamine receptors from the high- to the lowaffinity state for dopamine agonists, using sodium ions and guanine nucleotide. J. Neurochem. 44, 5 12-520.
Harris J. W. and Woodring J. (1992) Effects of stress, age, season, and source colony on levels of octopamine, dopamine and serotonin in the honey bee (Apis meNifera L.) brain. J. Insect Physiol. 38, 29-35. Kreissl S., Eichmiiller S., Bicker G., Rapus J. and Eckert M. (1991) The distribution of octopamine-like immunoreactivity ‘in the brain of the honeybee. In Proceedings qf the 19th Gdttinaen Neurobiolopp Conference (Edited bv Elsner N. and Pet&in H.), p. 407. Thieme, Stuttgart. _ Laduron P. M. (1984) Criteria for receptor sites in binding studies. Biochem. Pharmac. 33, 833-839.
Dopamine-sensitive
[‘HILSD
Leff S. E., Hamblin M. W. and Creese I. (1985) Interactions of dopamine agonists with brain D, receptors labeled by [3H]-antagonis&: evidence for the presence of high and low affinity agonist-binding states. Molec. Pharmac. 27, 171-183. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. ‘biol. Chem. 193, 265-275. Macmillan C. S. and Mercer A. R. (1987) An investigation of the role of dopamine in the ‘antenna1 lobes of the honeybee, Apis mellifera. J. camp. Physiol. A 160,359-366. Menzel R., Durst C., Erber J., Eichmiiller S., Hammer M., Hildebrandt H., Mauelshagen J., Miiller I-J., Rosenboom H.. Rvbak J.. Schafer S. and Scheidler A. (1994) The mushroom bodies in the honeybee: From molecules to behaviour. In Neuronal Basis of Behavioural Adaptations. Fortschritte der Zoo1 (Edited by Schildberger K. and Elsner N.), Vol. 39, pp. 81-102. Fischer, Stuttgart. Mercer A. R. and Erber J. (1983) The effects of amines on evoked potentials recorded in the mushroom bodies of the bee brain. J. camp. Physiol. A 151, 469-476. Mercer A. R., Mobbs P. G., Davenport A. P. and Evans P. D. (1983) Biogenic amines in thebrain of the honeybee, Aois mellifera. Cell Tiss. Res. 234. 655-677. Michelsen D. B. (1988) Catecholamines affect storage and retrieval of conditioned odour stimuli in honey bees. Camp. Biochem. Physiol. 91C, 479-482. Munson P. J. and Rodbard D. (1980) LIGAND: a versatile computerized approach for characterization of ligandbinding systems. Analyt. Biochem. 107, 220-239. Notman H. J. and Downer R. G. H. (1987) Binding of [‘Hlpifluthixol, a dopamine antagonist, in the brain of the American cockroach, Periplaneta americana. Insect Biothem. 17, 587-590. Orr G. L., Gale, J. W. D., Notman H. J. and Downer R. G. H. (1987) Pharmacological characterisation of the dopamine-sensitive adenylate cyclase in cockroach brain: evidence for a distinct dopamine receptor. Life Sci. 41, 2705-2715. Pitman R. M. and Davis J. P. L. (1988) Pharmacology of dopamine responses recorded from an identified cock-
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roach motoneurone. In Progress in Catecholamine Research, Part A: Basic Aspects and Peripheral Mechanisms, pp. 307-31 I. Alan R. Liss. Reader T. A., Molina-Holgado E. and Dewar K. M. (1992) Comparative biochemical pharmacology of central nervous system dopamine D, and D, receptors. Molec. Neurobiol. 6, 425-450. Rehder V., Bicker G. and Hammer M. (1987) Serotoninimmunoreactive neurons in the antenna1 lobes and suboesophageal ganglion of the honeybee. Cell Tiss. Res. 247, 59-66. Roeder T. (1994) Biogenic amines and their receptors in insects. Camp. Biochem. Physiol. 107C, I-12. Schafer S. and Bicker G. (1986) Common .projection areas of 5-HT- and GABA-like immunoreactive fibers in the visual system of the honeybee. Brain Res. 380, 3688370. Schafer S. and Rehder V. (1989) Dopamine-like immunoreactivity in the brain and suboesophageal ganglion of the honeybee. J. camp. Neural. 280, $3-38. - _ Schiirmann F. W.. Elekes K. and Geffard M. (1989) Dooamine-like immunoreactivity in the bee brain. &II, Tk. Res. 256, 399-410. Schiirmann F. W. and Klemm N. (1984) Serotoninimmunoreactive neurons in the brain of the honeybee. J. camp. Neural. 225, 570-580. Seeman P. and Niznik H. B. (1988) Dopamine Dl receptor pharmacology. In ISI Atlas of Science: Pharmacology, pp. 161-170. Taylor D. J., Robinson G. E., Logan B. J., Laverty R. and Mercer A. R. (1992) Changes in brain amine levels associated with the morphological and behavioural development of the worker honeybee. J. camp. Physiol. A 170, 715-721. Urwyler S. (1987) Affinity shifts induced by cations do not reliably predict the agonistic or antagonistic nature of ligands at brain dopamine receptors. J. Neurochem. 49, 1415-1420. Wedemeyer S., Roeder T. and Gewecke M. (1992) Pharmacological characterization of a 5-HT receptor in locust nervous tissue. Eur. J. Pharmac. 223, 173-178.
Pergamon 0742-8413(94)00091-3
Characterization of a dopamine-sensitive binding site in honeybee (Apis mell@ra) W. Blenau,”
C3H]LSD brain
T. May? and J. Erber*
*Institut fiir Biologie, Technische Universitlt Berlin, Germany; and TInstitut fur Neuropsychopharmakologie, D-14050 Berlin, Germany
Franklinstr. 28/29, D-10587 Berlin, Freie Universitlt Berlin, Ulmenallee
30,
PHILysergic acid diethylamide (13H]LSD) binds on membrane homogenate of honeybee brain to both a dopamine-sensitive site (D-site) and a serotonin-sensitive site (S-site). Under suitable conditions the properties of the two sites can be studied separately. Specific binding of 13H]LSD to both the D-site and the S-site has high affinity and is saturable. The mean equilibrium dissociation constants (Ko) were 3.8 nM for the D- and 0.89 nM for the S-site. The densities (B,,,,, values) of both binding sites were 1.7 pmol/mg protein for the D-site and 0.79 pmol/mg protein for the S-site. 13H]LSD binding to the D-site was reversible and reached equilibrium in about 30 min. Pharmacological displacement studies display a high binding affinity of the putative natural agonist dopamine to the D-site (Ki = 22 nM). The most potent displacers of D-site binding were lisuride, (+)-bromocriptine, chlorpromazine, S( +)-butaclamol, and 6,7-ADTN. The 13H]LSD labelled D-site seems to be G-protein coupled, since addition of the stable GTP analogue GTPy S or NaCl to the incubation medium evoked a decrease of specific 13H]LSD binding to the D-site. Key words: Dopamine Neuromodulator. Comp. Biochem.
Physiol.
receptor;
5-HT
IIOC, 197-205,
receptor;
[3H]LSD
binding;
Honeybee;
Insect
brain;
1995.
Introduction demonstrated the immunoreactivity (IR) of neural elements for a large number of putative and established neurotransmitters and modulators [for a review see Erber et al. (1993)]. For amines, neuroanatomical descriptions exist for serotonin-, dopamineand octopamine-like IR in the bee brain (Rehder et al., 1987; Schafer and Bicker, 1986; Schiirmann and Klemm, 1984; Schafer and Rehder, 1989; Schiirmann et al., 1989; Kreissl et al., 1991). Our knowledge about the function of dopamine in the bee mainly comes from behavioural and electrophysiological analyses in the olfactory pathway of the brain. Dopamine injected globally through the ocellar tract can reduce the amount of sucrose uptake and can block the retrieval of a conditioned olfactory stimulus, while it does not interfere with the storage of an olfactory signal [for a review see Menzel et al. (1994)]. Local injection of dopamine into the
Biogenic amines in the bee brain play an important role in controlling and modulating different types of behaviour [for a review see Erber et al. (1993)]. The existence of different biogenic amines has been shown in the bee brain. The highest concentrations were found for dopamine (12-40 pmol/brain) and serotonin (6-21 pmol/brain), while octopamine had intermediate concentrations (approximately 8 pmol/brain) and noradrenaline showed the smallest concentration (l-4 pmol/brain) (Fuchs et al., 1989; Harris and Woodring, 1992; Mercer et al., 1983; Taylor et al., 1992). Immunocytological studies in the bee have Correspondence fo : W. Blenau, Institut fiir Biologie; FR I- 1, Technische Universitit Berlin, Franklinstrasse 28/29, D-10587 Berlin, Germany. Tel. (030) 314 73 344; Fax (030) 314 73 177. Received I1 May 1994; revised 29 September 1994; accepted 15 October 1994. 197
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a-lobe of the mushroom bodies reduces the proboscis extension responses to water vapour (Blenau and Erber, 1993; Menzel et al., 1994). Application of dopamine onto the antenna1 lobes reduces the percentage of animals responding to a conditioned olfactory stimulus (Macmillan and Mercer, 1987). In many respects the response modifications after dopamine are similar to those after serotonin application. In the bee brain, as in other insect nervous tissues and glands, there is only very limited knowledge about the pharmacological characteristics of dopamine receptors (Evans and Green, 1990a,b; Roeder, 1994). In two studies, haloperidol, a vertebrate dopamine receptor antagonist, was used as a putative antagonist also in bees (Michelsen, 1988; Macmillan and Mercer, 1987). Recently a number of putative dopamine receptor antagonists were tested using the proboscis extension reflex in the bee (Blenau and Erber, 1993). This study showed that haloperidol had only weak antagonistic effects, while significant antagonistic effects were found for chlorpromazine, flupentixol, spiperone and buspirone. The pharmacological characterization of dopamine receptors in other invertebrates is also very incomplete. Electrophysiological studies indicate that the pharmacological profile of dopamine receptors on the soma of an inhibitory motoneuron of the cockroach does not conform well to mammalian D,- or D,-receptors (Pitman and Davis, 1988; Davis and Pitman, 1991). In homogenates of the ganglia of a mollusc, Helix porn&a, Drummond and coworkers (1978) demonstrated dopamine-sensitive binding sites for [3H]LSD. [3H]LSD labels also a dopamine receptor in the locust nervous tissue (Degen et al., 1992; Roeder, 1994). In membrane preparations of cockroach brain Notman and Downer (1987) demonstrated high affinity binding of [iH]pifluthixol, a vertebrate dopamine receptor antagonist. The bee offers the possibility to study the effects of dopamine at the level of different types of behaviour (Blenau and Erber, 1993; Menzel et al., 1994) and at the level of interneurons (Mercer and Erber, 1983). To understand the dopamine effects at these levels it is necessary to characterize the dopamine receptor(s) in this insect. As binding studies using [‘Hldopamine are not feasible in membrane homogenates of bee brain (see below), we studied the kinetics and the pharmacological profile of dopaminesensitive [jH]LSD binding to membrane homogenate of bee brain. It was the aim of this study in the bee to provide further data for a comparison of dopamine receptors in different invertebrate species.
Materials and Methods Insects
Foraging worker bees (Apis mellzjkra) were caught at the entrance of the hive and immobilized by chilling. They were mounted in small metal tubes as described by Erber and Menzel (1977) to dissect the brains. Tissue preparation
Brains (including optic lobes and suboesophageal ganglia) of worker bees were dissected under ice-cold ringer solution (268 mM NaCl, 3.19 mM KCl, 1.2 mM CaCI,, 10 mM sulfonic MgC&, 10 mM morpholinopropane acid, pH 7.4) as quickly as possible (about 4 min) and transferred into Eppendorf tubes. The tubes were then rapidly immersed in liquid nitrogen and kept at a temperature of -80°C until use. The tissue was homogenized using a glass/teflon homogenizer (10 strokes, 1000 rounds per minute) in ice-cold buffer (50 mM Tris-HCl, 5 mM MgSO,, 1OOpM phenylmethylsulforyl fluoride, pH 7.4). The homogenate was centrifuged (40,000 g, 10 min, 4°C) and the resulting pellet was rehomogenized in the buffer, incubated at 30°C for 10 min and centrifuged as before. The final pellet was rehomogenized in the buffer for use in the binding assay. Tissue aliquots (70&100 pg protein) were incubated at 22°C usually for 60 min in the buffer containing 100 PM ascorbic acid (final volume 1 ml, pH 7.4). The reaction was initiated by the addition of tissue and following incubation, bound and free ligand were separated by vacuum filtration of the samples through Whatman GF/B filters pre-soaked in 0.3% polyethyleneimine (Sigma) using a Brandell cell harvester. The filters were rapidly washed three times with 4 ml washing buffer (50mM Tris-HCI, pH 7.4). Bound radioactivity on the filters was determined by liquid scintillation spectrometry (counting efficiency about 60%). Radioligand binding to tissue samples was determined in duplicate. Dopamine-sensitive [3H]LSD binding (to the D-site) was estimated in the presence of 300 nM serotonin (5-HT). Non-specific binding was defined as binding in the presence of 10 PM dopamine, if not otherwise indicated. Specific binding was calculated as the difference between total and non-specific binding. The protein content of all samples was determined according to the method of Lowry and co-workers with bovine serum albumin (Sigma) as the standard (Lowry et al., 1951). Analyses of saturation, competition, and kinetic experiments were performed using EBDA/LIGAND and KINETIC computer software programs (G. A. McPherson,
Dopamine-sensitive
[‘HILSD
Elsevier-BIOSOFT, Cambridge, U.K.) which utilize non-linear least squares curve fitting techniques (Munson and Rodbard, 1980). The Ki values were calculated using the equation of Cheng and Prusoff for competitive inhibitors 1973): and Prusoff, Ki = Ic,,/ (Cheng ((1 + [L*])/K,), where IC,~ is the concentration of displacer, which inhibits 50% of specific binding, [L*] is the used concentration of radioligand and KD is the equilibrium dissociation constant. Drugs and other chemicals [3H]LSD (specific activity 72.4 Ci/mmol) and [3H]dopamine (specific activity 32 Ci/mmol) were purchased from DuPont NEN (Dreieich, Germany). [3H]Spiperone (specific activity 24 Ci/mmol) was from Amersham Buchler (Braunschweig, Germany). The following drugs were obtained from Research Biochemicals, Inc. (Ktiln, Germany): (+)-2-amino-6,7-dihydroxy- 1,2,3,4_tetrahydronaphtalene hydrobromide (6,7-ADTN HBr), (+)-bromocriptine HCl, methanesulfonate, S( +)-butaclamol R( -)-butaclamol HCl, cis(Z)-flupentixol dihydrochloride, trans(E)-flupentixol dihydrochloride, haloperidol, histamine dihydrochloride, (-)-quinpirole HCI, R( +)-SCH-23390 HCl, S( -)-SCH-23388 HCI, R( +)-SKF-38393, and spiperone HCI. Buspirone HCl, chlorpromazine HCl, dopamine HCI, GTPy S, 5-hydroxytryptamine HCI, fluphenazine dihydrochloride, metoclopramide HCl, (-)-norepinephrine bitartrate, (k)-octopamine HCL, phenylmethylsulforyl fluoride, S( -)-sulpiride, and tyramine HCl were purchased from Sigma Chemical Co. (Deisenhofen, Germany). Lisuride hydrogen maleate was a gift from Schering AG (Berlin, Germany). All other chemicals were of analytical grade.
binding
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Initial experiments using [3H]LSD as radioligand showed saturable binding. The specific binding (non-specific binding defined as binding in the presence of 1 mM dopamine) with a given concentration of [)H]LSD (2 nM) increased linearly with increasing amount of bee brain membrane homogenate (SO-250~s protein) in the assay mixture. Heating for 10 min at 100°C of the membrane homogenate decreased 98% of specific [3H]LSD binding in comparison with untreated tissue. In initial competition studies using a rH]LSD concentration of about 1 nM, various biogenic amines were tested for their ability to decrease specific [3H]LSD binding. At concentrations of 1 PM or less only dopamine and serotonin (5-HT) were efficacious displacers. (+)-Octopamine, (-)-norepinephrine, tyramine and histamine were much less active. Dopamine and 5-HT displaced L3H]LSD binding in a strongly biphasic manner (Fig. 1A). For the displacement experiments with serotonin, non-specific binding was defined as the amount of binding in
FL
e
0 I
I
lo-'0
10-9
I
10"
I
IO-'
Competitor
I
I
lad
10-S
I
104
!
10-Z
concentration (M)
0
Red ts First we tried to label dopamine receptors in the bee brain with [3H]dopamine, but the resulting binding was non-saturable within the range of l-50 nM and heat insensitive, arguing against receptor character of the binding site. The reasons for the unsuccessful labelling with [‘Hldopamine are unknown; similar observations were made in the locust (Roeder, personal communication). Furthermore we could not detect specific binding of another radioligand, [iH]spiperone, to the bee brain preparation. This ligand labels, with high affinity, dopamine Dz and serotonin 5-HT, receptors in mammalian brain. Also in this case the reasons for the unsuccessful labelling in the bee are unknown but similar to the findings in the mollusc Helix pomatia (Drummond et al., 1980).
I
lo-3
0
I ‘L..
0
5 [3H]LSD
10 concentratcin
15
20
(nM)
Fig. 1. Inhibition of specific [jH]LSD binding to membrane homogenate of bee brain by dopamine and 5-HT. (A): displacement of the specific [‘H]LSD binding (1.3 nM) by different concentrations of dopamine (0) and 5-HT (0). The definitions of specific binding are described in the text. (B): saturation curves of specific [‘H]LSD binding. (0): S-site; (0): D-site; (0): both sites experimentally determined; (A): both sites calculated as the sum of S-site + D-site. For experimental details see text.
200
W. Blenau
the presence of 1 mM 5-HT. For the experiments with dopamine, non-specific binding was defined in the presence of 3 mM dopamine. In both cases specific binding was approximately 95% of total binding. At low concentrations both amines displaced [3H]LSD probably from different binding sites. This hypothesis was tested by [3H]LSD binding saturation experiments. Saturation curves were determined in the absence of any displacer (total binding) and in the presence of the following displacers: 10pM dopamine, 1 PM 5-HT, and 10 PM dopamine + 1 PM 5-HT. Figure l(B) shows the resulting saturation curves of specific [3H]LSD binding which were determined by calculating the differences between total binding and binding in the presence of the displacers. At all used 13H]LSD concentrations the calculated sum of the specific [3H]LSD binding determined with 10 PM dopamine (D-site binding) and with 1 PM 5-HT (S-site binding) is in accordance with the experimentally determined specifically displaced [3H]LSD binding by 1 PM 5HT + 10 PM dopamine together (binding to both sites). With higher displacer concentrations (2 1 mM) specific binding to both sites (D- and S-site) was abolished by either amine acting alone, suggesting that the two sites exhibit specificity for dopamine and 5-HT only at lower concentrations. The estimated K, values of dopamine as [3H]LSD binding displacer were 22 nM for the dopamine-sensitive site (D-site) and 47 PM for the serotonin-sensitive site (Ssite), respectively. Serotonin as displacer revealed K, values for the S-site of 2.6 nM and for the D-site of 7 PM. The differences of dopamine and 5-HT in displacing [3H]LSD binding were used to derive conditions under which the radioligand was able to bind specifically to either the D- or the S-site. In the presence of 300 nM 5-HT, dopaminesensitive binding could be analysed, while 5-HTsensitive binding was analysed in the presence of 10 PM dopamine. Under the chosen conditions specific binding to both D-site (defined by 10 PM dopamine) and S-site (non-specific binding defined by 1 PM 5-HT) was higher than 80% of total binding when homogenate concentration was one brain/sample (70-100 pg protein/sample) and [3H]LSD concentration was 0.5 nM. The K,, for [3H]LSD binding to the D-site was 3.8 f 0.5 nM (Scatchard plot shown in Fig. 2), and for the S-site 0.89 f 0.03 nM (not shown). Figure l(B) also reveals that there were about twice as many saturable D-sites (B,,, = 1.7 f 0.1 pmol/mg protein) than S-sites (B,,, = 0.79 f 0.12 pmol/mg protein) in the bee brain preparation. Hill analysis of the D-site saturation binding data (Fig. 3) yielded a linear plot with a Hill
et al.
1t
0.6
J
0.0
!
I
I
05
0.0
I
1.0
1.5
(pmollmg protein)
Bound
Fig. 2. Scatchard plot of specific [‘HILSD binding on membrane homogenate of bee brain to the D-site, measured in the presence of 300 nM 5-HT. Membrane homogenate was incubated for 60min at 22°C with various concentrations of [‘H]LSD (0.1-10 nM). Non-specific binding was determined in the presence of 10nM dopamine. Points shown are from a typical experiment performed in duplicate which was replicated three times. K. = 3.8 + 0.5 nM and E max= 1.7+ 0.1 pmol/mg protein (mean & SD).
slope of 1.02 + 0.01. The linearity of the Scatchard plot and the Hill coefficient near unity suggest that under the chosen conditions a single class of dopamine-sensitive binding sites was labelled by [3H]LSD. Due to the limitations in the number of data points, we are unable to rule out the existence of multiple dopamine-sensitive [3H]LSD binding sites. The available data from our saturation experiments, however, support the existence of a homogenous [3H]LSD labelled D-site. Kinetic analysis of the dopamine-sensitive [3H]LSD binding (Fig. 4) was performed at a concentration of 0.5 nM [3H]LSD at 22°C. Under these experimental conditions, binding
0.75 nH = 1 02 0.50
1
t
-1 25 -1 50 -10.0
-9.5
I
I
I
1
-9.0
-0 5
-0 0
-7 5
log (M) free [3H]LSD
Fig. 3. Hill plot of specific [jH]LSD binding on membrane homogenate of bee brain (D-site). Data from Fig. 2. Hill plot analyses resulted in a Hill-coefficient (nu) of 1.02 f 0.01 (mean + SD).
Dopamine-sensitive
I
I
I
I
0
30
60
90 Time
I
120
I
150
[3H]LSD
I
160
(min)
Fig. 4. Kinetic analysis of specific [‘H]LSD binding to membrane homogenate of bee brain (D-site). Association (0) was stopped after the indicated times. Dissociation (0) was initiated after 60 min by the addition of lisuride to a final concentration of I pM. Non-specific binding was determined with 10pM dopamine. Values are the means of a single experiment performed in duplicate.
reached maximal values after about 30-60 min of incubation. A slight decline of specific binding was observed after longer incubations, probably due to receptor denaturation. At equilibrium (after 60 min association) dissociation was initiated by the addition of 1 PM lisuride which competes with reassociation of [3H]LSD binding. Dissociation of binding was found to occur slowly with an apparent half-life of about 20 min. Specific binding decreased to about 14% after 2 hr, indicating reversibility of the [‘H]LSD binding to the D-site. However, dissociation appears to be biphasic, with a rapid initial phase followed by a slow phase. The results of pharmacological studies by displacement of [3H]LSD binding to the D-site are summarized in Table 1. Table 1 shows the percentage of inhibition of the binding of 0.6 nM [jH]LSD by a variety of drugs at 1 PM. The K, values of selected drugs were determined by inhibition of [3H]LSD (0.6 nM) binding by coincubation with six different concentrations of the respective compounds (Figs 5 and 6 and Table 2). Among the biogenic amines dopamine and tyramine were the most potent displacers of dopamine-sensitive [3H]LSD binding. (+)-Octopamine, (-)-norepinephrine, 5-HT, and histamine were less efficacious. The compounds with highest affinity to the D-site were the ergot derivates, lisuride and (+)-bromocriptine. The vertebrate dopamine receptor agonist 6,7ADTN was also a potent displacer, whereas (-)-quinpirole and R( +)-SKF-38393 were much less effective. The vertebrate dopamine receptor antagonist with highest displacing potency was chlorpromazine. S( +)-Butaclamol and R(-)-butaclamol as well as cis(Z)-flupen-
binding
in Apis
201
tixol and trans(E)-flupentixol (Fig. 6) displayed stereoselective inhibition of [3H]LSD binding to the D-site. The enantiomers R( +)-SCH-23390 and S( -)-SCH-23388 were less stereoselective displacers. The rather low affinity of the butyrophenon group of compounds (spiperone and haloperidol) is remarkable. Metoclopramide and (-)-sulpiride were nearly ineffective in displacing [3H]LSD binding to the D-site. The dopamine-sensitive [3H]LSD binding site showed characteristics of G-protein coupled receptors. High concentrations of NaCl(lO0 mM) decreased the specific binding of 0.6 nM [3H]LSD at the D-site by 62%. The addition of the GTP stable analogue GTPyS also led to a reduction of specific [3H]LSD binding. Under the chosen conditions 100 PM GTPy S decreased specific binding to the D-site by 56%.
Discussion Radioligand binding studies were performed for a pharmacological in vitro characterization of a putative dopamine receptor in the honeybee brain. We demonstrated that [3H]LSD binds to both dopamine- and serotonin-sensitive sites in membrane homogenate of bee brain, and that conditions can be selected which allow the two sites to be studied separately. The K,, values calculated from the saturation data revealed that the affinity of [3H]LSD for the dopamine-sensitive site (K,, = 3.8 nM) is about four times lower than that for the serotonin-sensitive site (KD = 0.98 nM). The K,, value for the D-site in the bee is about 3.8 times higher than the respective K,, value for this site in desert locust brain preparation (Degen et al., 1992). It is nearly an order of magnitude higher than that calculated for dopamine receptors in the Roman snail Helix pomatiu (Drummond et al., 1978). The KD value for the S-site revealed that the affinity of [3H]LSD for this binding site in the bee brain is slightly higher than that reported for the S-site in locust nervous tissue (KD = 1.64 nM; Wedemeyer et al., 1992) and molluscan ganglia (KD = 1.2 nM; Drummond et al., 1980). A comparison of B,,,.,, values for the two binding sites revealed that the bee brain preparation contained about twice as many D-sites (B,,, = 1.7 pmol/mg protein) than Ssites (B,,, = 0.79 pmol/mg protein). The reverse situation has been found with the desert locust Schistocerca gregaria (Degen et al., 1992; Wedemeyer ef al., 1992). Here the B,,, values were found to be about 50 fmol/mg protein for the D-site and 79.8 fmol/mg protein for the S-site. The much higher density of both putative dopamine and serotonin receptor binding sites in the CNS of the honeybee compared with the locust preparation is remarkable.
W. Blenau
202
The pharmacological properties of dopaminesensitive [3H]LSD binding sites in the bee brain were assessed by competitive displacement experiments using a variety of unlabelled dopaminergic and serotonergic ligands. Ergot alkaloids and derivates such as lisuride and (+ )-bromocriptine, which are structurally related to the radioligand, were very potent displacers for D-site binding. D-site binding of [3H]LSD to membrane preparations of bee brain was stereoselective. Comparison of the Ki values displayed S( +)-butaclamol more than 1200 times more potent than R( -)-butaclamol and cis(Z)-flupentixol 24 times more potent than trans(E)-flupentixol as inhibitors of [3H]LSD binding to the D-site. The enantiomers R(+)SCH-23390 and S( -)-SCH-23388 were less selective. Stereospecificity is an important criterion for ligand receptor interaction (Laduron, 1984). The spectrum of drug action on the [3H]LSD-labelled dopamine-sensitive site in the
et al.
bee brain is clearly different from that of vertebrate dopamine D,- and D,-receptors labelled with [3H]SCH-23390 or [3H]spiperone (Seeman and Niznik, 1988). In the bee, D-site binding could be displaced with high affinity by the selective dopamine D, receptor ligands fluphenazine, cis(Z)-flupentixol and R( +)-SCH23390 or by the more selective dopamine Dz receptor ligands (+ )-bromocriptine and S( + )butaclamol. Conversely, some compounds such as metoclopramide (non-selective), SKF-38393 (D,), and S( -)-sulpiride (D,), which are potent dopaminergic agents in mammals, are nearly in displacing dopamine-sensitive inactive [‘H]LSD binding in the bee brain. Among the substances with highest affinity were substances without receptor subtype specificity in vertebrates, such as chlorpromazine and 6,7ADTN. The moderate affinity of spiperone (D2) may be related to the lack of specific [3H]spiperone binding in the bee brain
Table 1. Percentage inhibition of specific [‘H]LSD (0.6 nM) binding to the D-site by different compounds at 1 PM. Three hundred nM 5-HT were added to suppress binding to the S-site. Mean values, the range and the number (N) of independent experiments performed in duphcate are shown Compound Biogenic amines Dopamine Tyramine (+)-Octopamine (-)-Norepinephrine 5-Hydroxytryptamine Histamine Ergot alkaloids and derivates Lisuride (+ )-Bromocriptine Vertebrate dopamine receptor agonists 6,7-ADTN (-)-Quinpirole R( +)-SKF-38393 Vertebrate dopamine receptor antagonists Chlbrpromazke S( + )-Butaclamol Fhtphenazine cb(Z)-Flupentixol Spiperone R( +)-SCH-23390 Haloperidol trans(E)-Flupentixol S(-)-SCH-23388 Buspirone Metoclopramide S( -)-Sulpiride R( -)-Butaclamol
Percent inhibition (mean values)
Range
N
85 48 31 36 18 10
8487 4650 34-39 34-38 lo-27
2 2 2 2 2 1
108* 94
107-108 92-95
3 3
88
86-89 28-35
2 2
31 14
90 89 19 16 61 56 47 31 40 21 17 7
1
89-92 86-93 79-80 71-80 51-67 53-58 40-55 36-38 31-48 23-3 1 8-26 2-11 1-3
4 4 2 2 3 2 2 2 2 2 2 2 2
‘The calculated values of over 100% inhibition for lisuride are due to the fact, that 1 PM lisuride is more effective in displacing (‘H]LSD binding than 10 PM dopamine. This dopamine concentration was used to define non-specific binding.
Dopamine-sensitive
[3H]LSD
0
??.Y% 10"
IO-’
Competitor
I
1o-10
“,““‘,
““‘“‘A
10-s
104
concentration
“““‘I
IO-8
Competitor
1 o-5 (M)
----“I
““““I
IO-7
concentration
104
203
in Apis
Table 2. Inhibition of dopamine-sensitive [‘H]LSD binding to membrane homogenate of bee brain by various compounds. Inhibition of specific binding was determined with six different concentrations of the competing drugs (performed twice in duplicate). The K, values were determined using the LIGAND-program (Munson and Rodbard, 1980)
A
1 O-9
binding
w-5
(M)
Fig. 5. Displacement of specific D-site [3H]LSD binding by various agents. Non-specific binding was determined in the presence of 10 PM dopamine. (A): vertebrate dopamine receptor agonists: l , lisuride; 0, (+)-bromocriptine; 0, 6,7-ADTN. (B): vertebrate dopamine receptor antagonists: 0, chlorpromazine; 0, S( + )-butaclamol.
Because of the low affinity of haloperidol to the D-site it does not seem suitable to use this compound in future behavioural or electrophysiological experiments to block the action of dopamine in the honeybee brain. The pharmacological profile of [3H]LSD binding to the dopamine sensitive site in the preparation.
Fig. 6. Inhibition of specific [)H]LSD binding to the D-site by isomeric drugs. Non-specific binding was determined in the presence of 10 p M dopamine. Different concentrations cis(Z)-flupentixol (0) and [runs(E)-flupentixol (0) were added to displace specific [‘H]LSD binding.
Compound
Ki (nM)
Lisuride Dopamine Chlorpromazine 6,7-ADTN S( +)-Butaclamol cis(Z)-Flupentixol (+)-Bromocriptine frans(E)-Flupentixol 5Hydroxytryptamine R( -)-Butaclamol
4.1 22 48 78 89 150 160 3600 7000 > 100,000
locust Schistocerca gregaria is very similar to that of the bee (Roeder, 1994). Interestingly, the pharmacology of the bee D-site also resembles those of a [3H]LSD binding site in the nervous tissue of Helixpomatia (Drummond et al., 1978) and a [3H]pifluthixol binding site in the brain of Periplaneta americana (Notman and Downer, 1987). The pharmacological profile of the dopamine sensitive adenylate cyclase in the cockroach brain (Orr et al., 1987) and even of the dopamine receptor in the salivary gland acinar cells of the cockroach (Evans and Green, 1990a,b) is also similar to that of the dopamine sensitive [3H]LSD binding site in the bee nervous system. However correlation analyses of the pharmacological data from the putative dopamine receptor binding sites of the bee brain compared with those of other invertebrates are difficult to perform, since most calculated K, values were determined for different compounds in the different studies. The finding that dopamine-sensitive binding sites in the insect CNS have pharmacological properties distinct from those of mammalian dopamine receptors is supported by the observations on dopamine responses in insect neurones. Davis and Pitman (1991), studying depolarizing responses to dopamine in the soma of an inhibitory motoneurone of the cockroach Periplaneta americana, concluded that the receptors of this neurone, characterized on the basis of their pharmacological properties, do not fit the mammalian dopamine receptor classification scheme. The data of the in vitro binding study discussed here are in good agreement with the behavioural pharmacological experiments with honeybees using the unconditioned proboscis extension reflex to test the effects of putative dopamine receptor antagonists (Blenau and Erber, 1993). Chlorpromazine, cis(Z)-flupentixol and spiperone were efficacious both in the binding study and in the behavioural test.
204
W. Blenau
Haloperidol and S( -)-sulpirid, being less active in the behavioural test, were also less effective in displacing dopamine-sensitive [3H]LSD binding. Only buspirone, a very efficacious antagonist in the behavioural test, does not fit into this scheme. Buspirone does not displace D-site binding with high affinity in vitro. The dopamine-sensitive [3H]LSD binding site in the bee fulfils basic specificity criteria of receptor sites (Laduron, 1984) like displacement by agonists and antagonists belonging to different chemical and pharmacological classes, stereospecificity, saturability, reversibility, high affinity, and correlation between drug affinity in vitro and pharmacological potency in vivo. The significant reduction of the specific [3H]LSD binding to the D-site in the presence of GTPyS suggests that the dopamine-sensitive 13H]LSD binding site is coupled to a G-protein and therefore is likely to be a physiologically functional dopamine receptor. In mammals GTP and stable analogues are believed to convert high affinity dopamine receptors to a low affinity state for agonists (Leff et al., 1985; Grigoriadis and Seeman, 1985). Sodium chloride also apparently inhibited D-site binding which is an additional argument for a receptor binding site. Inhibitory effects of sodium have been reported for both D, and D, receptors in mammals, where sodium is believed to reduce agonist affinity (Grigoriadis and Seeman, 1985; Urwyler, 1987; Reader et al., 1992). The pharmacological characterization of a putative dopamine receptor in the honeybee brain indicates similarities to cockroach and molluscan receptors. There exist clear differences to dopamine receptors described in mammals. Thus, the pharmacological distinctiveness of invertebrate receptors, which has been demonstrated for other insect neuroreceptors (Eldefrawi et al., 1986), is also evident for dopamine receptors. Over the past few years five different mammalian dopamine receptors have been cloned and identified using molecular biological techniques (for a review see Civelli et al., 1993). These subtypes exhibit characteristic differences in their pharmacology and in their second messenger pathways through which they operate. The research on dopamine receptors in invertebrates is in its beginning, further research might reveal that dopamine receptors in invertebrates have a similar degree of complexity as found in mammals.
Acknvwl[~dnemenrs~This work was supported by Studienstiftung des deutschen Volkes and by-a grant- from the Deutsche Forschunasgemeinschaft (PF 128/6-3). We wish to thank Professor Dr-H. Coper of the Institut fur Neuropsychopharmakologie for his generous support which was essential for the experiments.
et al.
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