Characterization of Binding Sites for H-Spiroperidol in Human Retina

Characterization of Binding Sites For 3H-Spiroperidol in Human Retina

Paul McGonigle,* Martin B. Wax,f and Perry B. Molinoff*

Binding sites for the D-2-selective antagonist (3H)-spiroperidol were characterized in human retina. Nonspecific binding, measured in the presence of 2 nM (+)-butaclamol, made up 20% of total binding. Scatchard analysis of the binding of (3H)-spiroperidol resulted in linear plots and yielded a Kd value of 87 pM and a Bmax value of 1500 fmol/mg protein. In studies of the inhibition of the binding of (3H)-spiroperidol, (+)-butaclamol was approximately 1000-fold more potent than the (-)-stereoisomer. The inhibition curve for dopamine was shifted to the right and the Hill coefficient was increased by the addition of 300 ixM GTP. This effect was agonist-specific and suggests that some of the receptors are coupled to stimulation or inhibition of the enzyme adenylate cyclase. The inhibition curves for most of the antagonists had Hill coefficients between 0.6 and 0.8. Hill coefficients were also consistently less than 1.0 for agonists even in the presence of GTP. Nonlinear regression analysis of untransformed data revealed that these shallow inhibition curves were best explained by the presence of two populations of binding sites, 40% of the sites having a high affinity for dopamine in the presence of GTP and domperidone and the remaining 60% having a lower affinity for these ligands. The larger population of sites had a higher affinity for sulpiride, fluphenazine, and N-propylnorapomorphine in the presence of GTP. The possibility that either of these classes of sites consisted of serotonin receptors was ruled out by the finding that the 5-HT2 antagonist ketanserin had a low affinity for both classes of sites. Invest Ophthalmol Vis Sci 29:687-694, 1988

The neurotransmitter dopamine, acting in the cen- diated inhibition of the secretion of prolactin and tral nervous system, is involved in the regulation of a a-melanocyte-stimulating hormone secretion have variety of behaviors including the coordination and been shown not to involve an increase in adenylate control of motor activity. Results reported over the cyclase activity.4 Stimulation of D-2 receptors in both last several years have led to the conclusion that do- the anterior and intermediate lobes of the pituitary pamine interacts with multiple subtypes of dopamine has since been shown to result in the inhibition of receptors.1 In 1979, Kebabian and Calne2 formulated adenylate cyclase activity.4'5 the most widely accepted classification scheme for The application of radioligand binding techniques subtypes of dopamine receptors. Those receptors that has permitted a direct examination of the interactions stimulated the enzyme adenylate cyclase were termed of agonists and antagonists with subtypes of dopa- D-l receptors. Prototypic D-l receptors are found in mine receptors. Results of studies carried out using the parathyroid gland, where dopamine causes re- the butyrophenone (3H)-spiroperidol were consistent lease of parathyroid hormone through an increase in with the existence of binding sites in the striatum that 3 cyclic AMP levels. Conversely, those receptors that had a high affinity for antipsychotic drugs.6 A good did not mediate their response through stimulation of correlation was observed between the affinity of these adenylate cyclase were termed D-2 receptors. Proto- binding sites for various antipsychotic agents and the typic D-2 receptors exist in the anterior and interme- clinical potencies of these compounds.7 The proper- diate lobes of the pituitary, where dopamine-me- ties and distribution of the binding sites labeled with (3H)-spiroperidol differed from those observed in studies of dopamine-stimulated adenylate cyclase ac- From the Departments of *Pharmacology and "("Ophthalmology, tivity,8 suggesting that this radioligand does not label University of Pennsylvania, School of Medicine, Philadelphia, D-l receptors. Furthermore, the pharmacological Pennsylvania. 3 Supported by grants from the Public Health Service (NS-18591, specificity of the binding sites for ( H)-spiroperidol in GM-34781, NS-07272, MH-14654). the anterior pituitary was similar to that determined Submitted for publication: March 25, 1987; accepted December in studies of the prototypical D-2 receptor-mediated 8, 1987. response, inhibition of prolactin secretion.9 More re- Reprint requests: Dr. Paul McGonigle, Department of Pharma- cology, University of Pennsylvania, School of Medicine, Philadel- cently, spiroperidol has been shown to be 500-fold phia, PA 19104-6084. selective for D-2 receptors on the basis of results of

687

Downloaded from iovs.arvojournals.org on 09/29/2021 688 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / May 1988 Vol. 29

binding assays carried out with the nonselective an- ml. (3H)-Spiroperidol (24 Ci/mmol), drugs, and GTP tagonist (3H)-flupenthixol.10 were diluted in 2.6 mM ascorbic acid containing 20 Dopamine appears to be the predominant cate- Mg/ml of bovine serum albumin. A typical assay con- cholamine in the mammalian retina" where it acti- sisted of 50 /A radioligand, 50 n\ drug, 100 /A tissue vates adenylate cyclase and increases the concentra- homogenate and 2.8 ml of Hepes buffer containing tion of cyclic AMP.12 This suggests that D-l receptors 154 mM NaCl. Saturation experiments were carried exist in this tissue. Although it has been suggested out at concentrations of (3H)-spiroperidol from that mammalian retina does not contain D-2 recep- 0.01-2 nM. In competition experiments, the concen- tors,13 binding sites for the D-2-selective antagonist tration of (3H)-spiroperidol was between 0.2 and 0.3 (3H)-spiroperidol are present in homogenates of ret- nM, and bound radioligand was always less than 10% ina from several mammalian species including calf, of the total amount of radioligand in the assay. A rat, rabbit and monkey.14"16 Moreover, stimulation- concentration of GTP of 300 or 500 nM was included evoked release of (3H)-dopamine appears to be mod- in all experiments performed with agonists. ulated by an autoreceptor that exhibits the pharma- Homogenates of human retina were incubated for cological profile of a D-2 receptor.17 Thus, mamma- 30 min at 37°C, which provided sufficient time for lian retina may contain both the D-l and D-2 the binding of (3H)-spiroperidol to reach equilibrium. subtypes of the dopamine receptor. Reactions were terminated by the addition of 10 ml On the basis of in vitro binding assays with (3H)- of ice-cold Tris buffer (pH 7.5) containing 154 mM spiroperidol in the rat striatum, Huff and Molinoff18 NaCl. Samples were then filtered through glass-fiber suggested that the D-2 subtype of the dopamine re- filters (Schleicher and Schuell, No. 30, Keene, NH). ceptor may be further subdivided. Since antipsy- Each filter was washed with an additional 10 ml of chotic and antiparkinsonian drugs mediate their ef- Tris buffer. The amount of radioactivity remaining fects through inhibition of dopamine receptors, it was on the filter was determined by liquid scintillation of considerable interest to determine whether sub- spectroscopy with a counting efficiency for tritium of types of D-2 receptors exist in human tissue. One of 32%. Specific binding was defined as the difference the difficulties with using (3H)-spiroperidol to charac- between the amount of radioligand bound in the terize dopamine receptors is that this ligand also has a presence and absence of 2 /uM (+)-butaclamol. Pro- high affinity for serotonin receptors.19 Moreover, re- tein content was determined by the method of Brad- cent evidence suggests that there may be substantial ford,23 using bovine serum albumin as the standard. numbers of serotonin receptors in mammalian stria- (3H)-Spiroperidol was obtained from New England tum.20-21 In contrast, the mammalian retina contains Nuclear Corp. (Boston, MA); (+)- and (-)-butacla- little or no detectable serotonin.22 Since it has been mol and N-propylnorapomorphine were purchased shown to contain a moderately high density of bind- from Research Biochemicals Inc. (Wayland, MA); ing sites for (3H)-spiroperidol, the retina may be a dopamine, serotonin, yohimbine, and GTP were ob- promising tissue in which to look for subtypes of D-2 tained from Sigma Chemical Co. (St. Louis, MO). receptors. The following drugs were kindly provided as gifts by the company indicated: domperidone, ketanserin, Materials and Methods and R-5573, Janssen Pharmaceutica (Beerse, Bel- Freshly enucleated eyes from human donors (aged gium); sulpiride, Ravizza (Milan, Italy); fluphena- 14 to 91) were obtained within 24 hr of death (pri- zine, Squibb and Sons (New York, NY); prazosin, marily due to cardiorespiratory failure) and were dis- Pfizer Inc. (Groton, CT); phentolamine, Ciba-Geigy sected on ice in 20 mM Hepes buffer (pH 7.5) con- Corp. (Summit, NJ); clonidine, Boehringer Ingel- taining 154 mM NaCl. The pars plana was incised 4 heim Ltd. (Ridgefield, CT). Other reagents were pur- mm posterior to the correoscleral limbus. The retina chased from standard commercial sources. was removed from the underlying retinal pigment epithelium by blunt dissection and was detached sur- Data Analysis gically from its adherence at the optic nerve. Retinal Saturation isotherms were transformed using the tissue was homogenized in 20 mM Hepes buffer (pH 24 method of Scatchard. Estimates of the K^ and B 7.5) containing 154 mM NaCl and 5 mM EDTA. max values were obtained using unweighted linear regres- Following centrifugation (20,000 g for 10 min at sion analysis of the transformed data. Competition 4°C), pellets were resuspended in Hepes buffer con- curves were initially modeled using the following taining 154 mM NaCl, incubated at 37°C for 30 min, one-site equation: and subjected to centrifugation as above. The pellet was resuspended in 20 mM Hepes buffer containing B 154 mM NaCl at a concentration of 0.5 mg of tissue/ B, = n + NS (1) (L/IC50)

Downloaded from iovs.arvojournals.org on 09/29/2021 No. 5 BINDING SITES FOR (°H)-SPIROPERIDOL IN HUMAN RETINA / McGonigle er ol. 689

5009 In this equation BL is the amount of radioligand 016 bound to the tissue, B is the total number of binding sites labeled in the absence of competing ligand, L is 4000. the concentration of competing ligand, IC50 is the concentration of competing ligand that inhibits 50% 3000. •• of total specific binding, n is the Hill coefficient and NS is the amount of radioligand not specifically 2000. • bound to the tissue. Nonspecific binding was rou- tinely constrained to the value determined when 1000. •• assays were carried out in the presence of 2 /uM (+)- butaclamol. This value was determined in each ex- periment. Curve-fitting was done by nonlinear least squares regression analysis according to the method cSPIROPERIDOLD 3 of Marquardt and Levenberg.25 The analysis was per- Fig. 1. Binding of ( H)-spiroperidol in human retina. Mem- branes were incubated for 30 min at 37°C with varying concentra- formed using the mathematical modeling program 3 26 tions of ( H)-spiroperidol (10 pM to 2 nM) in the absence (O) and NEWFITSITES available on the NIH-sponsored presence (A) of 1 nM (+)-butaclamol. Specific binding (•) was PROPHET system. If the estimate of the Hill coeffi- saturable and of high affinity. Each point represents the mean of cient was significantly different from 1, the competi- triplicate determinations. Inset: Scatchard transformation of the tion data were fit to the following multiple-site specific binding data. model: determine the rate constant of dissociation, (3H)- B = (2) L 1 + (L/ICJOI) + spiroperidol was allowed to equilibrate with retinal tissue for 30 min, at which time 2 /xM (+)-butaclamol In this equation n is the number of independent was added to initiate dissociation. Specific binding of classes of binding sites, Bi is the amount of radioli- (3H)-spiroperidol was determined at various times gand bound to site "i" in the absence of competing after the addition of butaclamol (Fig. 2B). The rate of ligand and IC i is the IC value of the competing 5O 50 dissociation was rapid, with a t of approximately 15 ligand for site "i." The curve-fitting was performed by 1/2 min. A first-order plot of the data was linear, indicat- the NEWFITSITES program as described above. Im- ing that dissociation was monophasic (Fig. 2B, inset). provement in fit was determined by comparing the The rate constant of dissociation calculated from the residual sum of squares of the respective fits using a first-order plot was 0.04 min"1. The K value of 100 partial F-test.27 IC values were transformed to K; d 50 pM calculated from the ratio of the kinetic rate con- values using the method of Cheng and Prusoff.28 stants (k_i/ki) was in good agreement with the Kd value of 87 pM determined from the equilibrium sat- Results uration analysis. The monophasic rates of association The specific binding of (3H)-spiroperidol was satu- and dissociation provide further evidence that all of 3 rable and Scatchard transformation resulted in a lin- the binding sites have the same affinity for ( H)-spi- ear plot (Fig. 1). The linear Scatchard plot indicates roperidol. that (3H)-spiroperidol is either labeling a single class Studies of the inhibition of the binding of (3H)- of receptors or is nonselectively labeling multiple spiroperidol were performed with a number of com- classes of receptors with the same affinity for the ra- peting ligands. At concentrations of radioligand of dioligand. The Kd value from the Scatchard analysis 200 to 300 pM, specific binding represented 80% of was 87 pM and the Bmax value was 1650 fmol/mg of total binding. The potent serotonin antagonist ketan- protein. Both of these values were in good agreement serin exhibited a low, almost micromolar affinity for with measurements made in rat striatum.1826 These the binding sites labeled with (3H)-spiroperidol. This results indicate that human retina contains a high is in contrast to the nanomolar affinity of serotonin density of high-affinity, saturable binding sites for the receptors for ketanserin.29 A Hill coefficient close to 1 D-2 selective antagonist spiroperidol. indicated that ketanserin has a uniformly low affinity Experiments were carried out to determine the rate for all of the binding sites for (3H)-spiroperidol. This constants of association and dissociation for (3H)- result supports the contention that there are no de- spiroperidol. The time course of association was tectable 5-HT2 receptors in the human retina. rapid and monophasic (Fig. 2), yielding a linear Experiments were carried out to determine the ef- pseudo-first-order rate plot (Fig. 2A, inset). The fect of GTP (300 MM) on the inhibition of the binding pseudo-first-order rate constant of association de- of (3H)-spiroperidol by the agonist dopamine. GTP rived from this plot was 3.99 X 108 min"1 M"1. To increased the Hill coefficient from 0.62 to 0.82 and

Downloaded from iovs.arvojournals.org on 09/29/2021 690 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / Moy 1988 Vol. 29

Sites labeled with (3H)-spiroperidol had a signifi- A. cantly higher affinity for the active stereoisomer (+)- h 1200.• butaclamol than for the inactive stereoisomer (-)- butaclamol (Fig. 4, upper panel). In addition, the competition curve for (+)-butaclamol was markedly 809 • 2. T biphasic, suggesting that (+)-butaclamol exhibited selectivity for a subclass of binding sites for (3H)-spiroperidol. Nonlinear regression analysis was used to fit 400. multiple-site models to the competition data. The data were best fit by a two-site model in which (+)- 0. 5. 10. butaclamol had a high affinity for 60% of the sites and TIME CMIN5 a 100-fold lower affinity for the remaining 40% of the 0. 10. 20. 30. 40. 50. 60. sites. Fitting the data to a three-site model did not TIME CMIN5 result in a statistically significant improvement of fit. 1600 Inhibition studies with other antagonists yielded similar results. The competition curves for domperidone and sulpiride were also fit better by a two-site model 1200 than a simple one-site model (Fig. 4B). According to this analysis, domperidone exhibited a high affinity for 40% of the sites and a 15-fold lower affinity for the 800 remaining 60% of the sites. Conversely, sulpiride demonstrated a high affinity for 60% of the sites and a 20-fold lower affinity for 40% of the sites. Thus, it 400. appears that there are two distinct subclasses of binding sites labeled by (3H)-spiroperidol. Experiments carried out with several antagonists and two agonists in the presence of GTP revealed that 10. 15. 20. 25. 30. 35. 40. 45. 3 TIME CMIhO the binding sites for ( H)-spiroperidol could be subdivided into two populations (Table 1). The results Fig. 2. Kinetics of the binding of (3H)-spiroperidol. (A) Mem- branes were prewarmed and incubated at 37°C as described in were consistent with the existence of two classes of Methods. The binding reaction was terminated by the addition of sites present in a ratio of approximately 3:2. Two ice-cold buffer followed by rapid nitration through glass-fiber drugs, spiroperidol and ketanserin, were not selective filters. The resulting association curve was monophasic. Inset: A for either class of sites. The small class of sites had a pseudo-first-order rate plot was derived by transformation of the higher affinity for the antagonist domperidone and association binding data. X is equal to Be/^-B) where Be is the amount of radioligand bound at equilibrium and B is the amount the agonist dopamine, whereas the large class of sites of radioligand bound at time t. (B) After the retinal membranes had been incubated for 30 min at 37°C, dissociation was initiated by II 1 I 1 1 I I mil addition of 2 uM (+)-butaclamol. The resulting dissociation curve O Contr ol | f was monophasic. Inset: Transformation of the dissociation binding 100 ( • + 300 uM GTP ' A + 500 uM GTP- data resulted in a linear first-order rate plot. Each point represents the mean of triplicate determinations. 80 ••

>l nH = 0-82 60 • ••

: \ lowered the affinity by approximately one order of 40 • nH = 0.62\ ••

magnitude (Fig. 3). A concentration of GTP of 300 20 • juM had a maximal effect and no additional change in

the shape of the competition curve was detected when 0 • u | 1 I I ii - assays were carried out in the presence of 500 ^M 10 -8 10-6 ,0-4 a-2 GTP. The sensitivity to guanine nucleotides suggests :DOPAMINE: CMJ that some of the D-2 receptors in human retina are Fig. 3. Effect of GTP on binding of dopamine to D-2 receptors in linked to the enzyme adenylate cyclase. A Hill coeffi- human retina. Homogenates were incubated as described in cient less than 1 in the presence of a maximally effec- Methods. In the absence of GTP (O), the EC50 value was 2 /uM and the Hill coefficient was 0.62. In the presence of 300 uM GTP (•), tive concentration of GTP may be explained by the the EC50 value increased by six-fold and the Hill coefficient in- coexistence of a heterogeneous population of binding creased to 0.82. Each point represents the mean of triplicate deter- sites. minations.

Downloaded from iovs.arvojournals.org on 09/29/2021 No. 5 DINDING SITES FOR (3H)-SPIROPERIDOL IN HUMAN RETINA / McGonigle er ol. 691

I mini ml 1 i iiinn i— i— ...i— i had a higher affinity for the antagonists sulpiride, SITE 100 • • . — ' (+)-butaclamol and fluphenazine, and the agonist N- SITES propylnorapomorphine. Z 80 - The affinities of the large class of sites correlated well with the affinities of the D-2 receptor in rat stria- S 60 •• tum for these drugs (Fig. 5), indicating that the large class of sites represents D-2 receptors. The affinities fc 40 • \ o P of the small class of sites did not correlate well with UJ (o 20 the affinities of the D-2 receptor in the rat striatum • C-OBUTACLAMOL for these ligands. • C-3BUTACLAM0L Additional studies were performed to try to deter- 1 SITE mine the identity of the small class of binding sites. 100 SITES Competition experiments with yohimbine, prazosin, o clonidine, and phentolamine revealed that these 2 80 + drugs, which exhibit selectivity for alpha-adrenergic H receptors, had a low (micromolar) affinity for both m 60 • > classes of binding sites labeled by (3H)-spiroperidol (Table 2). This result ruled out the possibility that any 40 ••• • of the binding sites specifically labeled by (3H)-spiro- 20 • • DOMPERIDONE w peridol were alpha or alpha -adrenergic receptors. r 2 • SULPIRIDE Competition experiments carried out with R-5573 • •"•"•—• ' ""'1—• • •••••• demonstrated that all of the (3H)-spiroperidol binding 10-" 10 -9 10-7 10- sites had a low affinity for this spirodecanone-specific :DRUG: CMJ ligand, indicating that neither subclass of sites was a Fig. 4. Inhibition curves for antagonists at dopamine receptors. spirodecanone site. Homogenates of retina were incubated as described in Methods. Upper panel: The inhibition curve for (-)-butaclamol was best fit by a one-site equation (dashed line) whereas the active stereoisomer Discussion (+)-butaclamol was best fit by a two-site equation (solid line) as The results of these in vitro binding assays indicate determined by nonlinear regression analysis. According to the two-site model, (+)-butaclamol was 100-fold selective fora subclass that the human retina contains D-2 receptors that can 3 3 of ( H)-spiroperidol binding sites that represent 60% of the total be characterized with the radioligand ( H)-spiroperi- number of sites labeled. Lower panel: Inhibition data for the dopa- dol. The properties of these receptors were similar to minergic antagonists domperidone and sulpiride were also better fit by the two-site equation than the one-site equation. Domperidone those of D-2 receptors in rat pituitary. The receptors 3 have a uniformly high affinity for the radioligand as was 15-fold selective for 40% of the sites labeled by ( H)-spiroperidol whereas sulpiride was 20-fold selective for 60% of the sites. Each evidenced by the linear Scatchard plot and monopha- curve represents data pooled from three experiments in which each sic rates of association and dissociation. The close point was measured in duplicate. The improvement in fito f the agreement between the dissociation constants deter- two-site over the one-site model was P < 0.0001 in each case.

Table 1. Subclasses of binding sites for (3H)-spiroperidol Small site* Large sile

K, (nM)t % Kj (nM) %

Selective for small sile Dopamine + GTP (3)t 548 ± 440§ 34 ±3.1 10,500 ± 3,700 66 ±3.1 Domperidone (4) 0.62 ± 0.08 42 ± 1.7 9.5 ± 1.3 58 ± 1.7 Selective for large site NPA + GTP(3) 8.0 ± 1.1 39 ±2.5 0.42 ± 0.23 61 ±2.5 Sulpiride (4) 912 ± 50 39 ± 2.4 44 ±9 61 ±2.4 Fluphenazine (3) 15 ±4.5 38 ± 1.8 0.3 ±0.12 62 ± 18 Butaclamol (3) 34 ±7 40 ± 3.5 0.33 ±0.10 60 ± 3.9 Nonselective Spiroperidol (3) 0.087 ± 26 Ketanserin (3) 200 ± 74

* Displacement curves were fitt o a two-site model using the NEWFIT- Prusoft'. SITES program which provides IC50 values and relative proportions for each i Number of individual experiments performed in duplicate. class of sites. 5 Mean ± SEM. t IC50 values were corrected to K, values by the method of Cheng and

Downloaded from iovs.arvojournals.org on 09/29/2021 692 INVESTIGATIVE OPHTHALMOLOGY G VISUAL SCIENCE / May 1988 Vol. 29

11. peridol overestimated the density of D-2 receptors spiroper idol. because labeling of 5-HT2 receptors by this radioli- 10. bijtoc lomol. A"fluphen. gand was not accounted for or prevented. In this

9. study we have detected the existence of binding sites with high affinity for domperidone in human retina. / 14 8. •*» nper idone However, Watling and Iversen obtained no specific y 3 ^/•sulpir ide binding with ( H)-domperidone in retina from other 7. kelonserin-/' mammals. They suggested that the lack of specific y^ binding was due to the technical limitations asso- 6. • ciated with the use of (3H)-domperidone including y^ dopomi ne 5. • high levels of nonspecific binding. Furthermore, the studies were performed with bovine and guinea pig -log K. CSTRIRTUM) retina which could have different proportions Fig. 5. Comparison of Ki values obtained in membranes pre- of high-affinity sites for domperidone than human pared from human retina and rat striatum. The slope of the corre- retina. lation between results obtained with the retina and with striatal D-2 receptors was 0.92 with a correlation coefficient of 0.91. The values In many receptor systems, inhibition of the binding for retina represent the mean of at least three experiments in which of a radiolabeled antagonist by an agonist results in each point was measured in duplicate. The values for rat striatum displacement curves with a shallow slope.30 The low were reported by Lin et al.38 Hill coefficients of such curves may be erroneously interpreted as reflecting the existence of multiple mined by kinetic and by equilibrium analyses also subtypes of receptors.31 It is unlikely that the hetero- supports the contention that (3H)-spiroperidol is la- geneous binding sites described in this paper reflect beling a homogeneous population of receptors. De- agonist-specific phenomena. First, inhibition of the spite this apparent homogeneity, several drugs dis- binding of (3H)-spiroperidol by the agonists dopa- criminated two populations of binding sites, present mine and N-propylnorapomorphine results in com- in a ratio of 3:2. Competition experiments revealed plex displacement curves even in the presence of the existence of both agonists and antagonists that maximally effective concentrations of GTP. Second, were selective for each population of sites. in the presence of guanine nucleotides the agonists It has been reported that the density of sites labeled are selective for different populations of binding sites. with (3H)-spiroperidol in mammalian retina is rela- Third, and most convincing, is the fact that several tively low compared to other dopamine-containing antagonists also exhibit this apparent selectivity. brain regions such as the striatum.14 In the present (3H)-Spiroperidol has been shown to specifically study, the density of (3H)-spiroperidol sites in human label other classes of binding sites in addition to D-2 retina were found to be approximately equal to the receptors. Leysen et al19 reported that 5-HT2 recep- density of D-2 receptors in the rat striatum.1826 It is tors in the frontal cortex have a high affinity for (3H)- likely that earlier studies performed with (3H)-spiro- spiroperidol. Neither population of binding sites in the human retina appears to be 5-HT2 receptors since Table 2. Interaction of drugs with binding sites both populations of sites have the same low micro- labeled with (3H)-spiroperidol molar affinity for the potent 5-HT2 antagonist ketanserin. Moreover, the 5-HT2 agonist serotonin has Ki (nM)*-\ a Kd value greater than 10 nM for both classes of sites. 3 Serotonergic ( H)-Spiroperidol has also been reported to label Serotonin (2)% > 10,000 alpha-adrenergic receptors in the mouse frontal cor- Ketanserin (3) 200 ± 74§ tex.32 Since norepinephrine- and epinephrine-con- a-Adrenergic taining neurons have been shown to exist in the ret- 33 34 Yohimbine (2) 1,800 ± 840 ina, ' it was necessary to rule out the possibility that Prazosin (2) > 10,000 one of the subclasses of sites labeled by (3H)-spiroper- Phentolamine (2) > 10,000 Clonidine (2) > 10,000 idol was an alpha-adrenergic receptor. Based on the very low affinity exhibited by both classes of binding Spirodecanone sites for several potent antagonists at alpha-adrener- R-5573 (2) 250 ± 122 gic receptors, neither subclass of (3H)-spiroperidol-la- * Displacement curves were fitt o a single-site model using the NEWFIT- SITES program. beled sites in the retina appears to be alphar or t IQo values were corrected to Kj values by the method of Cheng and alpha2-adrenergic receptors. Spirodecanone sites are Prusoff. 3 % Number of individual experiments performed in duplicate. also specifically labeled by ( H)-spiroperidol. These § Mean ± SEM. sites are characterized by a high affinity for dopa-

Downloaded from iovs.arvojournals.org on 09/29/2021 No. 5 BINDING SITES FOR (°H)-SPIROPERIDOL IN HUMAN RETINA / McGonigle er ol. 690

mine, spiroperidol and related butyrophenones but a rat striatum.40 It has also been reported that activa- low affinity for such selective antagonists as sulpiride, tion of D-2 receptors results in inhibition of the activ- butaclamol and flupenthixol.35 Spirodecanone bind- ity of the enzyme adenylate cyclase in rat anterior ing sites have been localized to regions of the hippo- pituitary4 and in rat striatum.41 Thus, it is likely that campus, nucleus accumbens, olfactory tubercle and the D-2 receptors in the retina also mediate their ef- cerebral cortex using autoradiographic techniques.36 fects through the inhibition of adenylate cyclase. The possibility that one of these populations of bind- The small class of sites labeled by (3H)-spiroperidol ing sites is a spirodecanone site was ruled out by the in the human retina does not correspond to any of the finding that the potent spirodecanone-specific ligand specific sites that are known to be labeled by this R-5573 had a uniformly low affinity for both popula- radioligand. These binding sites have properties that tions of binding sites. are characteristic of specific recognition sites, since The two subclasses of binding sites identified by they are saturable, reversible and of high affinity. It antagonists at dopamine receptors in the retina do remains to be established whether they represent sig- not correspond to the subtypes of D-2 receptor hy- nificant neurochemical receptors that mediate mea- pothesized to exist in the rat striatum.18 This may surable biochemical or physiological responses. A result from the complexity of the binding profile of pharmacological profile of any functional response (3H)-spiroperidol in the rat striatum which contains mediated by these sites must be obtained before 5-HT2 receptors,20 alpha-adrenergic receptors37 and they can be considered a new subtype of dopamine spirodecanone sites35 in addition to high- and low-af- receptor. finity states of the D-2 receptor. These additional fac- 3 tors complicate studies of putative subtypes of dopa- Key words: retina, human, dopamine receptors, ( H)-spiroperidol, D-2 receptors mine receptors in the striatum. In contrast, the bind- 3 ing profile of ( H)-spiroperidol in the human retina is References simplified by the lack of detectable 5-HT2 receptors, 1. Cools AR and Van Rossum JM: Excitation-mediating and alpha-adrenergic receptors or spirodecanone binding inhibition-mediating dopamine-receptors: A new concept to- sites in this tissue. wards a better understanding of electrophysiological, biochem- The pharmacological specificity of the large class of ical, pharmacological, functional, and clinical data. Psycho- 3 pharmacologia (Berlin) 45:243. 1976. sites labeled by ( H)-spiroperidol in the retina corre- 2. Kebabian J W and Calne DB: Multiple receptors for dopamine. sponds to the D-2 receptors described in the striatum Nature 277:93, 1979. of the rat. The order of potency of the compounds 3. Brown EM, Caroll RJ, and Aurbach GD: Dopaminergic stim- tested was identical in the rat striatum and human ulation of cyclic AMP accumulation and parathyroid hormone retina, and the affinities of the large class of sites release from dispersed bovine parathyroid cells. Proc Natl correlated well with the affinities of the D-2 receptor AcadSci USA 74:4210, 1977. 4. Munemura M, Cote TE, Tsuruta K, Eskay RL, and Kebabian in rat striatum for these compounds.38 It appears 3 JK: The dopamine receptor in the intermediate lobe of the rat likely that the large class of sites labeled by ( H)-spi- pituitary gland: Pharmacological characterization. Endocri- roperidol in the retina is linked to the enzyme adenyl- nology 107:1676, 1980. ate cyclase. In systems whose function is mediated by 5. Onali P, Schwartz JP. and Costa E: Dopaminergic modulation increases or decreases in the activity of adenylate cy- of adenylate cyclase stimulation of vasoactive intestinal pep- tide (VIP) in anterior pituitary. Proc Natl Acad Sci USA clase, the formation of a ternary complex is thought 78:6531, 1981. to precede the interaction of the receptor with the 6. Seeman P, Chau-Wong M. Tedesco J. and Wong K: Brain enzyme. In this reaction sequence, the agonist binds receptors for antipsychotic drugs and dopamine: Direct bind- to the receptor in the first step. The agonist-bound ing assays. Proc Natl Acad Sci USA 72:4376. 1975. receptor then binds to a guanine nucleotide-binding 7. Seeman P: Anti-schizophrenic drugs—membrane receptor protein, forming a ternary complex. In the presence sites of action. Biochem Pharmacol 26:1741, 1977. 8. Clement-Cormier YC and George RJ: Multiple dopamine of a guanine nucleotide, the ternary complex is de- binding sites: Subcellular localization and biochemical charac- stabilized so that only the first step in the reaction terization. J Neurochem 32:1061, 1979. sequence is detected. When inhibition of the binding 9. Caron MG, Beaulieu M, Raymond V, Gagne B. Drouin J. Lefkowitz RJ, and Labrie F: Dopaminergic receptors in the of a radiolabeled antagonist by an agonist is studied 3 in the presence of GTP, the affinity of the receptor for anterior pituitary gland: Correlation of H-dihydroergocryp- tine binding with the dopaminergic control of prolactin re- the agonist is decreased, and the competition curve 30 lease. J Biol Chem 253:2244, 1978. becomes steeper. This is the effect that GTP had on 10. Huff RM and Molinoff PB: Assay of dopamine receptors with the inhibition of the binding of (3H)-spiroperidol by (3H)-a-flupenthixol. J Pharmacol Exp Ther 232:57. 1985. the agonist dopamine in homogenates from human 11. Haggendal J and Malmfors T: Identification and cellular local- retina. This effect of GTP has been demonstrated for ization of the catecholamine in the retina and the choroid of 39 the rabbit. Acta Physiol Scand 64:58. 1965. the D-2 receptor in rat intermediate pituitary and in 12. Bucher MB and Schorderet M: Dopamine- and apomorphine-

Downloaded from iovs.arvojournals.org on 09/29/2021 694 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / Moy 1988 Vol. 29

sensitive adenylate cyclase in homogenates of rabbit retina. 27. Snedecor GW and Cochran WG: Statistical Methods. Ames, Naunyn Schmiedebergs Arch Pharmacol 288:103, 1975. Iowa, Iowa State University Press, 1967, pp. 447-471. 13. Watling KJ, Dowling JE, and Iversen LL: Dopamine receptors 28. Cheng YC and Prusoff WH: Relationship between the inhibi- in the retina may all be linked to adenylate cyclase. Nature tion constant (K;) and the concentration of inhibitor which 281:578, 1979. causes 50 percent inhibition (I50) of an enzymatic reaction. 14. Watling KJ and Iversen LL: Comparison of the binding of Biochem Pharmacol 22:3099, 1973. (3H)-spiroperidol and (3H)-domperidone in homogenates of 29. Leysen JE, Niemegeers CJE, Van Nueten JM, and Laduron mammalian retina and caudate nucleus. J Neurochem PM: (3H)-Ketanserin (R41 468), a selective 3H-ligand for sero- 37:1130, 1981. tonin2 receptor binding sites. Mol Pharmacol 21:301, 1982. 15. Magistretti PJ and Schorderet M: Dopamine receptors in bo- 30. Maguire ME, Ross EM, and Gilman AG: /?-Adrenergic recep- vine retina: Characterization of the 3H-spiroperidol binding tor: Ligand binding properties and the interaction with ade- and its use for screening dopamine receptor affinity of drugs. nylyl cyclase. Adv Cyclic Nucleotide Res 8:1, 1977. Life Sci 25:1675, 1979. 31. Molinoff PB, Wolfe BB, and Weiland GA: Quantitative analy- 16. Makman MH, Dvorkin B, Horowitz SG, and Thai LJ: Proper- sis of drug-receptor interactions: II. Determination of the prop- ties of dopamine agonist and antagonist binding sites in mam- erties of receptor subtypes. Life Sci 29:427, 1981. malian retina. Brain Res 194:403, 1980. 32. Morgan DG, Marcuso JO, and Finch CE: Contamination of 17. Dubocovich ML and Weiner N: Modulation of the stimula- serotonin-2 binding sites by an alpha-1 adrenergic component tion-evoked release of (3H)-dopamine in the rabbit retina. J in assays with 3H-spiperone. Life Sci 34:2507, 1984. Pharmacol ExpTher 219:701, 1981. 33. Osborne NW: Noradrenaline, a transmitter candidate in the 18. HuffRM and Molinoff PB: Quantitative determination of do- retina. J Neurochem 36:17, 1981. pamine receptor subtypes not linked to activation of adenylate 34. Hadjiconstantinous M, Cohen J, and Neff NH: Epinephrine: A cyclase in rat striatum. Proc Natl Acad Sci USA 79:7561, 1982. potential neurotransmitter in retina. J Neurochem 41:1440, 19. Leysen JE, Niemegeers CJE, Tolenaere JP, and Laduron PM: 1983. Serotonergic component of neuroleptic receptors. Nature 35. Howlett DR, Morris H, and Nahorski SR: Anomalous proper- 272:168, 1978. ties of (3H)-spiroperidol binding sites in various areas of the rat 20. Hamblin MW, Leff SE, and Creese 1: Interactions of agonists limbic system. Mol Pharmacol 15:506, 1979. with D-2 dopamine receptors: Evidence for a single receptor 3 36. Palacios JM, Niehoff DL, and Kuhar MJ: ( H)-Spiroperidol population existing in multiple agonist-states in rat striatal binding sites in brain: Autoradiographic localization of multi- membranes. Biochem Pharmacol 33:877, 1984. ple receptors. Brain Res 213:277, 1981. 21. Withy RM, Mayer RJ, and Strange PG: Use of (3H)-spiperone for labelling dopaminergic and serotonergic receptors in bo- 37. U'Prichard DC, Bechtel WB, Rouot B, and Snyder SH: Multi- vine caudate nucleus. J Neurochem 37:1144, 1981. ple alpha-noradrenergic receptor binding sites in rat brain: Ef- 22. Ehinger B: Biogenic amines as transmitters in the retina. In fect of 6-hydroxydopamine. Mol Pharmacol 16:47, 1979. Transmitters in the Visual Process, Bontig SL, editor. Oxford, 38. Lin C, McGonigle P, and Molinoff PB: Characterization of Pergammon Press, 1976, pp. 145-167. D-2 dopamine receptors in a tumor of the rat anterior pituitary 23. Bradford MM: A rapid and sensitive method for the quantita- gland. J Pharmacol Exp Ther 242:950, 1987. 3 tion of microgram quantities of protein utilizing the principle 39. Frey EA, Cote TE, Grewe CW, and Kebabian JW: ( H)-Spiro- of protein-dye binding. Anal Biochem 72:248, 1976. peridol identifies a D-2 dopamine receptor inhibiting adenyl- 24. Scatchard G: The attractions of proteins for small molecules ate cyclase activity in the intermediate lobe of the rat pituitary and ions. Ann NY Acad Sci 51:660, 1949. gland. Endocrinology 110:1897, 1982. 25. Magar ME: Data Analysis in Biochemistry and Biophysics. 40. Zahniser NR and Molinoff PB: Effect of guanine nucleotides New York, Academic Press, 1972, pp. 93-166. on striatal dopamine receptors. Nature (Lond) 275:452, 1978. 26. McGonigle P, HuffRM, and Molinoff PB: A comprehensive 41. Onali P, Olinanas MC, and Gessa GL: Characterization of method for the quantitative determination of dopamine re- dopamine receptors mediating inhibition of adenylate cyclase ceptor subtypes. Ann NY Acad Sci 430:77, 1984. activity in rat striatum. Mol Pharmacol 28:138, 1985.

Downloaded from iovs.arvojournals.org on 09/29/2021