Dopamine receptors in the goldfish retina: 3H-spiroperidol and 3H-domperidone binding; and dopamine-stimulated adenylate cyclase activity

Life Sciences, Vol. 27, Printed in the U.S.A.

pp.

Pergamon Press

23-31

DOPAMINE RECEPTORS IN THE GOLDFISH RETINA: ‘H-SPIROPERIDOL AND ‘h-DOMPERIDONE BINDING; AND DOPAMINE-STIMULATED ADENYLATE CYCLASE ACTIVITY

Dianna A. Redburnl,

Yvonne Clement-Cormier

12

’ , and Dominic M.K. Lam

3

Departments of Neurobiology and Anatomy 1,2 , and Pharmacology’, The University of Texcy Medical School at Houston and Cullen Eye Institute , Baylor College of Medicine, Houston, Texas 77030 (Recieved

in final

form April

29,

1980)

Summary Dopamine reyp tars. in the goldf+h retina have been examined by binding studies using H-splroperidol and H-domperidone as specific ligands, and by measuring retinal adenylate cyclase activities in the presence and absence of dopamine. Our results indicate that washed membranes from goldfish retinal homogenate bind a variety of dopamine agonists and antagonists with high affinities and with characteristics similar to those reported for the brain, with the exception that in this jetina there is virtually no binding of the H-domperidone. In addition, there-is a very specific D2 rqcyptor antagonist, low basal actlvlty of adenylate cyclase which can be greatly stimulated by dopamine, possibly reflecting a high degree of coupling between this enzyme and the dopamine receptor. Taken together, our findings indicate that the goldfish retina contains a high density of D, type dopamine receptors and few, if any, D2 type receptors. There is considerable evidence that dopamine is a neurotransmitter in the vertebrate retina (l-5). In most retinas examined, dopamine has been localized to some amacrine cells (6). Dopaminergic neurons of certain teleost and primate retinas, however, appear to be a class of interplexiform cells which make synaptic contacts in both outer and inner plexiform layers (1,4). For instance in the goldfish retina, electron microscopic and autoradiographic studies indicate that most presumptive dopaminergic neurons are interplexiform cells which contact some cone horizontal cells and bipolar cell dendrites in the outer plexiform layer, and amacrine cells in the inner plexiform layer (1,4). Biochemical analyses of dopaminergic systems in the retina have concentrated on pre-synaptic mechanisms, such as synthesis, uptake and release of the transmitter, rather than on the post-synaptic events (4,5). Because the morphology, electrophysiology and synaptic chemistry of dopaminergic neurons have been studied most extensively in the goldfish retina, we have begun to characterize the post-synaptic dopamine receptors in this retina. Two different biochemical techniques were selected for this study because they have been used successfully to characterize dopamine receptors in the central nervous system. One technique entails the analysis of dopamine stimulation of adenylate The other technique involves the3 determination of receptor binding cyclase (7). characteristics using various radioligands such as H-spiroperidol which is a dopamine receptor antagonist with very high affinity for dopamine receptor and which blocks dopamine stimulation of adenylate cyclase (8,9). However, it is noteworthy that studies using these two receptor analyys have not always yielded consistent results (7,8,10,11). In fact, it now appears that H-spiroperidol can label two populations of dopamine receptors (presently designated D and D ) only one of which (D ) is associated with dopamine-stimulated cyclase activ 1ty (lO,l’i). Domperidone is alsb a very potent and 0024-3205/80/270023-09$02.00/O Copyright (c) 1980 Pergamon Press

Ltd

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Dopamine Receptors in Goldfish Retina

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selective dopamine receptor antagonist, however, unlike 3H-spiroperidol, this compound has l i t t l e effect~n the activity of dopamine sensitive adenylate cyclase in brain or retina (12,13). Thus, H-domperidone binding can be used as a measure of the number of D 2 dopamine receptors, i.e., those which are not associated with adenylat~ cyclase. In studies r~,ported herein, goldfish retinas and brains were analyzed for H-spiroperidol binding, H-domperidone binding and adenylate cyclase activity in order to characterize the pharmacological and kinetic properties of D ! and D7 dopamine receptors associated with the dopaminergic neurons which are a class o'f interpl'exiform cells in this retina. Methods and Materials Goldfish (Carassius auratus, 6 to 7 inches long) were obtained from Ozark Fisheries (Stouland, MO). After at least 2 hours of dark adaptation, brains were removed; the eyes were enucleated and retinas isolated. The vitreous humor was carefully removed from each retina. All further procedures were carried out at 4Oc by methods previously described for rabbit (14). Tubes containing isolated retinas in 10 mls of 0.32 M sucrose were gently vortexed to remove most rod outer segments which were then decanted. The remaining retinas, with most rod outer segments removed, were then hand homogenized using a glass-Teflon tissue grinder. Homogenates were centrifuged at 150 xg for 10 min to remove a pre-P / fraction consisting of cell debris and nuclei. For mammalian retinal samples, the superfiatant was decanted and centrifuged at 800 xg for 10 minutes to obtain a Pl or outer plexiform layer fraction which was shown by EM analysis to be highly enriched in photoreceptor cell, outer plexiform layer, synaptosomes. Goldfish retinal tissue proved unsuitable for isolation of i n t a c t photoreceptor cell synaptosomes; therefore, the P/ or outer plexiform layer fraction from goldfish was not analysed. A P~ or inner plexifor~n layer fraction was obtained by centrifuging the supernatant from th~ Pl pellet at 15,000 xg for 12 rain. Em analysis showed this fraction from mammalial and goldfish retinas, contained conventional sized synaptosomes from the numerous amacrine terminals in the inner plexiform layer as well as small endings from the outer plexiform layer.

The binding assay used was based on the techniques developed by Fields, et al, (9) for brain tissue. Synaptosomal fractions were resuspended in 0.05 M Na-K phosphate buffer pH 7.# and homogenized at setting No. 5 on a polytron ([3rinkmann Instruments, Westbury, N.Y.) with three 15 sec bursts. The homogenate was diluted up to 30 mls and centrifuged at lg,000rpm for 20 min in a Sorvall RC 2-[3 centrifuge. The resulting pellet was washed once by resuspension and centrifugation as before. The final pellet was ~esuspended in buffer. Tissue samples containing 0.3-0.6 mg protein were incubated with H-spiroperidol, 23.6 Ci/m Mole sp. act., (New England Nuclear, Boston, Mass.) at 37°C with agitation for 30 min in the presence or absence of specific drugs as indicated in the Results section. Incubation was t e r m i n a t e d by rapid filtration through GF/C glass fiber filters (Whatman, England) followed by three 5 ml washes of buffer. Filters were placed in counting vials containing 1 ml 95% alcohol for 30 minutes. Counting solution containing Triton X-100 was added and samples w~re counted on a Searle Mark III liquid scintillation counter, at 45% efficiency. Specific H-spiroperidol binding was experimentally determined as the difference between binding in the absence and in the presence of 0.1 }aM (+) butaclamol. The dopamine agonist, (+) butaclamol exhibits absolute stereospecificity for dopamine receptor binding and was used to displace ( H)-spiroperidol bound specifically to the receptor. Thus, the amount of (+) butaclamol-displaceable IHspiroperidol was used as a measure of the amount of specific (i.e., receptor) binding. The binaing assay for 3H-domperidone used was previously describe~ by Laduron and Leysen (12). The incubation volume was 2.2 mls and contained 1 nM H-domperidone, varying concentrations of tissue and Tris-HCl buffer 0.05M; pH 7.6 with 120 mM NaCI, 5ram KCI, 2 mM CaCI?, 1 mM MgClo, 10 p M pargyline and 0.1% ascorbic acid. The tissue was incubated for-10 min at 37°~. The reaction was stopped by rapid filtration on GF/[3 glass fiber filters, which were washed twice with 5 mls ice-cold buffer.

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Dopamine Receptors in Goldfish Retina

25

For determination of adenylate cyclase activity, retinas and brains were homogenized in 2 mM Tris-2 mM EGTA. The standard assay mixture for measurement of adenylate cyclase a c t i v i t y had a final volume of 0.5 ml and contained (in mM) Tris (hydroxmethyl) aminomethanemaleate, 80.2, pH 7.4; ATP, 1.5; GTP, 0.003; MgSO4, 6.0; theophylline, 10; EGTA, 0.6j 0.05 ml of tissue homogenate i and test substances as indicated. The enzyme was preincubated with all components of the standard assay system, except ATP and GTP which were added at the beginning of the 10 minute incubation period at 300 . The reaction was terminated by boiling, and cyclic AMP was measured, as described previously (7). The data on adenylate cyclase activity was expressed as pmoles cyclic AMP formed/rag protein/rain and represent mean values and ranges for three separate experiments. The standard errors for the data were less than i0 percent. Protein was determined by the mothod of Lowry, et ai (15). The sources of drugs were as follows: Fluphenazine, Squibb, (Princeton, N.3.); (+) and (-) butaclamol, Ayerst, (Montreal, Canada); dopamine, Calbiochem, (San Diego, CA); haloperidol, Janssen Pharmaceutical, (Beerse, Belgium); ATP, cyclic AMP and ethylene glycolbis-(B-aminoethylether)-N, N' tetraacetic acid, EGTA, Sigma Co. (St. Louis, MO); chlorpromazine, Smith, Kline and French (Philadelphia, PA); sulpiride, Delagrange International (Paris, France); 2-amino-B, 7-dihydroxy-l, 2, 3, 4-tetrahydronaphtalene (ADTN) was a gift from ~ r . 3ohn McDermed, Wellcome Laboratories, Research Triangle Park, North Carolina. 3 H-spiroperidol (23.6 Ci/mMole) was obtained from New England Nuclear (Boston, Mass.); H-domperidone (24.6 Ci/mMole) was a gift from Dr. Sam Enna. Results and Discussion The binding of 3H-spiroperidol in washed membrane fractio3r}s from goldfish retinal homogenates is shown in Figure I. At low concentrations of H-spiroperidol (0.5 nM) approximately t h i r t y percent of the total bound ligand was displaced by 0.I IJM (+) butaclamol (Figure IA; inset). This represents forty percent of total binding to the tissue. Although this represents a somewhat low percentage compared to the other systems, the assay reliably detected binding at this level. Binding in the presence of (+) butaclamol was statistically different from3tota| binding, i . e , binding in the absence of this drug (P < 0.01) at all concentrations of H-spiroperidol > 0.05 nM. The difference between total binding and binding in the presence of 0 . l - p ~ l (+) butaclamoJ was defined as specific binding to the dopamine receptc~,s. As shown in Figure A (inset), specific binding was saturable at concentrations of H-spiroperidol > 0.5 nM. Scatchard analysis (Fig. IA) revealed only a single dissociation constant of ap-proximately 0.23 nM which is similar to 0.3 nM reported for bovine retinal synaptosomes (13) and for homogenates from the highly dopaminergic brain region, the striatum (9). Receptor density was also calculated by Scatchard analysis and equalled approximately 37 pmol/gm protein which is considerably less than values of 300-500 pmole/gm protein reported for human, bovine and rat caudate homogenates (9). Based on specific binding a c t i v i t y at 0.5 nM 3H-spiroperidol, the relative concentrations of dopamine receptors in goldfish, rabbit and bovine retinal homogenates are remarkable similar 37,57~and 48 pmol/gm protein respectively, (16). Washed membrane fractions from goldfish brain also demonstrated a high a f f i n i t y binding site for "Hspiroperidol which was displaced by 0.I p M (+) butaclamol. The apparent K D was 0.85 nM and the Bmax was 6.6 p tool/gin protein as compared to a K D of 0.8 nM and a Bmax of 152 p/mol/gm protein in rat brain (9). The ability of a variety of dopamine receptor antagonists to displace 3H-spiroperidol binding indicate a high degree of pharmacological specificity associated with the receptor (Fig. 2A). Potent antipsychotic drugs such as fluphenazine and haloperidol which are also potent dopamine antagonists were able to compete for receptor binding sites at low concentrations. Agonists such as dopamine and the inactive (-) stereoisomer of the antagonist (+) butaclamol were far less potent displacers (See Table I). The calculated inhibition constants (Ki) for selected dopamine agonists and antagonists in the goldfish retina were somewhat higher than those reported for caudate and anterior pituitary from rat brain (8).

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Dopamine Receptors in Goldfish Retina

Vol. 27, No. i, 1980

I

A

5O

I

0.3

I

I

I)

I ~ "e~

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FIG IA. Scatchard plot ol specilic binding of 3H- spiroperidol to washed membranes from goldfish retinal homogenates. Insert shows binding curves for total binding (dashed li~e) and specific binding (solid lines) in pmoles/gm protein (Y axis) at concentrations of ~Hspiroperidol from 0.05 to InM (X axis). Specific binding is defined as the difference between binding in the presence and absence of 0.I 12M (+) butaclamol. Data are means of triplicate determinations of each of three different preparations) SEM < I0%, The slope of the Scatchard plot was determined by linear regression analysis) r=0,95. FIG. IB, Effect of increasing concentrations of dopamine on adenylate cyclase activity in a homogenate of the goldfish retina, The tissue was prepared as described in the methods, The data are expressed as pmol/mg protein/rain and represent the mean and range; n=3.

Vol. 27, No. i, 1980

Dopamine Receptors in Goldfish Retina

27

TABLE I

Inhibition of dopamine stimulated adenylate cyclase and H-splroperldol binding m the goldfish r e t i n a by n e u r o l e p t l c drugs. Drug

ADTN (+) B u t a c l a m o l Fluphenazine Haloperidol Dopamine (-) Butaclamol Sulpiride

Ki* (M) A d e n y l a t e C y c l a s e Inhibition -- _9, t~ x 10..~ 2 x 10 "'~--10 -5 10 -9

Ki + (M) 3H-Spiroperidol Binding 3,2 x 10-~ 1.2 X lO: 9 x 10-~7 1.3 x l ~ 1010 -4 --

Reported Ki values for adenylate cyclase were calculated from the relationship ICso=Ki (I + S/KIn) where S is the concentration of dopamine (I00 IJM) and Km is the cofi~entration of dopamine required for half-maximal stimulation of adenylate cyclase a c t i v i t y (2 la M). + Reported Ki values for binding were calculated from equation ICsn=Ki (l+C/Kd) where C is the concentration of H- spiroperidol (0.5nM) and Kd is t11~ dissociation constant (0.2nM).

Although butyrophenones such as spiroperidol and (+) butaclamol have a very high a f f i n i t y for the dopamine receptor) there are reports that at higher concentrations they also bind to serotonin receptors (17,18). In3the experiments reported here, low concentrations were used for both the ligand, H-spiroperidol) and the displacer, (+) butaclamoI, which should assure that most of the displaceable binding measured was associated with the dopamine receptor. As a further test, an additional compound ADTN, which binds3only to dopamine receptors, (19,20) was used as a displacer in the retina. The amount of H-spiroperidoI displaced by maximal (101aM) concentrations o£ ADTN and 0.I ~HM butaclamol were similar (Fig. 2A). These data suggest that most of the displaceable -spiropericlol binding measured under our conditions was associated with the dopamine receptor. In order to distinguish between the two proposed types of dopamine receptors) binding characteristics of a specific ~.2 receptor Iigand H-domperidone was studied. As reported by Laduron and Leysen(J2) ~-domperidone binding sites are only detectable in homogenates of dopaminergic brain areas in ratr3 the most prominent of which is the striatum. We also found significant amounts3of H-domperidone binding a c t i v i t y in rat striatai synaptosomes. F i f t y percent of the H-domperidone binding at InM concentrations was inhibited by I0 IJM ADTN; (filter bIanks a~counte~ for I0% of the total ~inding). In contrast, ADTN, at concentrations from 10-° to I0 , displaced none of the H-domperidone binding in goldfish retinal homogenate or synaptosome fractions indicating no specific binding of this D 2 receptor ~gand. This lack of specific Hdomperidon binding was unique to goldfish retina. H-domperidone binding in goldfish brain areas was displaced by ADTN: tectal-striataI area > cerebellum = brainstem. ( See Table If). Retinal synaptoso3me fractions from a mammalian species (cow) also demonstrated ADTN displaceable H-domperidone binding. These data suggest that D2-type (domperidone-sensitive) dopamine receptors are not measureable in goldfish retina under

28

Dopamine Receptors in Goldfish Retina

Vol. 27, No. i, 1980

conditions which d e m o n s t r a t e significant binding in other dopaminergic areas of goldlish brain and in retinas of other species.

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FIG. 2A. Inhibition ol 3H-spiroperidol binding by selected c~rugs in washed membranes Irom goldlish retina homogenate. The concentration of -H-spiroperidol was 0.5nM. Results are expressed as the percent displacement of total binding by the drug. Data are means ol triplicate determinations of each ol t h r ee separate preparations; SEM < 1096. FIG, 2B. Inhibition of dopamine-sensitive adenylate cyclase activity by selected drugs in a homogenate of the goldsfish retina prepared as described in the methods. The concentration of dopamine was 2 ]aM; inhibitory drugs were added to the incubation mixture simultaneously with the dopamine. The data are expressed as pmol/mg protein/rain and are the mean from t r i p l i c a t e determinations from a r e p r e s e n t a t i v e experiment. The range for mean values was less that ten percent.

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Dopamine Receptors in Goldfish Retina

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TABLE II Specific Binding of 3H-Domperidone* Tissue Source

pmole/g protein

Rat striatal synaptosomes

70.0

Bovine retina homogenate

0

Pl synaptosomes

0.6

P2 synaptosomes

1.2

Goldfish brain homogenate tectal-striatal area cerebellum-brainstem frontal and inferior lobes

6.0 1.6 1.5

Goldfish retina homogenate P2 synaptomes

0 0

C o n c e n t r a t i o n of 3 H - d o m p e r i d o n e was lnM. 10 tJ M ADTN was used as t h e d i s p l a c e r . D a t a a r e m e a n s , n=3, SEM < I0%.

Dopamine-sensitive adenylate cyclases isolated from brain and retina have provided a successful in vitro model for the study of dopamine receptors and for investigation of the mechanism of antipsychotic drug action (21-24). In the mammalian retina, dopamine produces a dose dependent increase in cyclic AMP formation (5,25,26). A fifty-percent increase in enzyme a c t i v i t y is observed with I ]a M dopamine which is very similar to that seen in brain (21). Pharmacological studies with both intact and broken cell preparations reveal that the response to dopamine can be competitively inhibited by neuroleptic drugs, with the phenothiazines being more potent thatn the butyrophenones (7,24,25). Dopamine-sensitive adenylate cyclase in homogenates of the goldfish retina demonstrate pharmacological specificity similar to that reported for brain and mammalian retina. The dose response curve for dopamine-stimulated adenylate cyclase activity in homogenates of the goldfish retina is shown in Figure IB. The maximal increase in enzyme a c t i v i t y was observed with I0 )J M dopamine~ half maximal response was obtained with 2 ~M dopamine. The absolute number of picomoles of cyclic AMP formed above basal levels in response to dopamine stimulation, 25 pmol/mg/protein/min, is comparable to that seen in rabbit retina (34 pmol/mg/protein/min, ref. 5). Based on this calculation, i t appears that goldfish and rabbit retinas have roughly the same number of receptorcyclase complexes, i . e , D I dopamine receptor sites. The inhibition of the goldfish retinal dopamine-sensitive adenyl~te cyclase by selected antipsychotic drugs is shown in Fig. 2B. The phenothiazine, fluphenazine, was the most potent inhibitor of dopamine activationqof adenylate cyclase in homogenates of the goldfish retina with a calculated Ki of 2 x 10-'M (Table If). The enzyme a c t i v i t y exhibited stereoselectivity to (-) and (+) butaclamol with the (÷) isomer being the active inhibitor. As observed with homogenates of mammalian brain, no dopamine stimulation of cyclase was found in the goldfish brain homogenates with concentrations of dopamine up to ImM. One major finding in these studies was the unusually low basal a c t i v i t y of adenylate cyclase in the goldfish retina (2.5 -+ 0.5 pmol/gm/min) as compared to goldfish brain (15 -+ 1.0 pmol/gm/min) or to our previous reports in the rabbit retina (39.1 -+ 3.9 pmol/gr/min)

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Dopamine Receptors in Goldfish Retina

Vol. 27, No. i, 1980

(5). Because the basal a c t i v i t y is so low, the rather modest level of dopamine stimulated enzyme activity observed actually represents a tenfold increase over basal levels. This is the highest percent increase in dopamine-stimulated enzyme a c t i v i t y reported for any dopaminergic system to-date in a broken cell preparation. Thus, under our experimental conditions most of the adenylate cyclase in this system is functionally coupled to dopamine binding sites.

In summary, washed membranes from goldfish retinal homogenates bind to a variety of dopamine agonists and antagonists with high affinities and with characteristics similar to those reported for the brain. It is especially interesting that in this retina, there is a very low basal activity of adenylate cyclase which can be greatly stimulated by dopamine, possibly reflecting a high degree3of coupling between this enzyme and the dopamine receptor. In addition, the lack of H-domperidone binding in goldfish retina homogenate and synaptosomes suggests that few if any of the retinal receptors are of the D9 type. Thus, the goldfish retina demonstrates a unique combination of properties: most-of the measureable adenylate cyclase is coupled to dopamine receptors, and smilarly most of the rneasureable dopamine receptors are associated with adeny~ate cyclase. In a recent publication by Watling et. al. (13), no dopamine displaceable H-domperidone binding was found in guinea pig retina homogenates. In our studies, the low levels of specific binding detected in bovne retinal synaptosomal fractions were not demonstratable in homogenates from the same retina. It would be of i~terest to determine if guinea pig retina synaptosomal fractions demonstrate specific H-domperidone (in D2 receptor) binding. The functional significance of the tightly coupled receptor-cyclase system in goldfish retina is, at this point, unclear. Nonetheless, this property, together with the well characterized dopaminergic pathways in the goldfish retina, makes this retina a unique model system to study the synaptic interactions and regulation of dopaminergic neurons in the central nervous system. Acknowledl~ements We thank Ms. Cheryl K. Mitchell for excellent technical assistance, Dr. C.A. Chin and Mr. S.C. Fung for skillful dissections, Dr. William Gordon for illustrations, Ms. Patricia Cloud and Mrs. Lillian Puccio for typing this manuscript. This research was supported by NIH grants EY-01655 (to Dianna A. Redburn), EY-02423 (to Dominic M.K. Lain), NSF grants BNS-7gI6003 (to Yvonne C. Clement-Cormier) and the Retina Research Foundation, Houston (to Dominic M.K. Lain). Yvonne C. Clement-Cormier is a recipient of a Faculty Development Award from the Pharmaceutical Manufacturer's Association, Dianna A. Redburn and Dominic M.K. Lain are recipients of Research Career Development Awards from the United States National Institutes of Health.

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