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

Investigative Ophthalmology & Visual Science, Vol. 29, No. 5, May 1988 Copyright Association for Research in Vision and Ophthalmology Characterization of Binding Sites For 3 H-Spiroperidol in Human Retina Paul McGonigle,* Martin B. Wax,f and Perry B. Molinoff* Binding sites for the D-2-selective antagonist ( 3 H)-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 ( 3 H)-spiroperidol resulted in linear plots and yielded a Kd value of 87 pm and a B max value of 1500 fmol/mg protein. In studies of the inhibition of the binding of ( 3 H)-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 central nervous system, is involved in the regulation of a variety of behaviors including the coordination and control of motor activity. Results reported over the last several years have led to the conclusion that dopamine interacts with multiple subtypes of dopamine receptors. 1 In 1979, Kebabian and Calne 2 formulated the most widely accepted classification scheme for subtypes of dopamine receptors. Those receptors that stimulated the enzyme adenylate cyclase were termed D-l receptors. Prototypic D-l receptors are found in the parathyroid gland, where dopamine causes release of parathyroid hormone through an increase in cyclic AMP levels. 3 Conversely, those receptors that did not mediate their response through stimulation of adenylate cyclase were termed D-2 receptors. Prototypic D-2 receptors exist in the anterior and intermediate lobes of the pituitary, where dopamine-me- From the Departments of *Pharmacology and "("Ophthalmology, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania. Supported by grants from the Public Health Service (NS-18591, GM-34781, NS-07272, MH-14654). Submitted for publication: March 25, 1987; accepted December 8, 1987. Reprint requests: Dr. Paul McGonigle, Department of Pharmacology, University of Pennsylvania, School of Medicine, Philadelphia, PA 19104-6084. diated inhibition of the secretion of prolactin and a-melanocyte-stimulating hormone secretion have been shown not to involve an increase in adenylate cyclase activity. 4 Stimulation of D-2 receptors in both the anterior and intermediate lobes of the pituitary has since been shown to result in the inhibition of adenylate cyclase activity. 4 ' 5 The application of radioligand binding techniques has permitted a direct examination of the interactions of agonists and antagonists with subtypes of dopamine receptors. Results of studies carried out using the butyrophenone ( 3 H)-spiroperidol were consistent with the existence of binding sites in the striatum that had a high affinity for antipsychotic drugs. 6 A good correlation was observed between the affinity of these binding sites for various antipsychotic agents and the clinical potencies of these compounds. 7 The properties and distribution of the binding sites labeled with ( 3 H)-spiroperidol differed from those observed in studies of dopamine-stimulated adenylate cyclase activity, 8 suggesting that this radioligand does not label D-l receptors. Furthermore, the pharmacological specificity of the binding sites for ( 3 H)-spiroperidol in the anterior pituitary was similar to that determined in studies of the prototypical D-2 receptor-mediated response, inhibition of prolactin secretion. 9 More recently, spiroperidol has been shown to be 500-fold selective for D-2 receptors on the basis of results of 687

688 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / May 1988 Vol. 29 binding assays carried out with the nonselective antagonist ( 3 H)-flupenthixol. 10 Dopamine appears to be the predominant catecholamine in the mammalian retina" where it activates adenylate cyclase and increases the concentration of cyclic AMP. 12 This suggests that D-l receptors exist in this tissue. Although it has been suggested that mammalian retina does not contain D-2 receptors, 13 binding sites for the D-2-selective antagonist ( 3 H)-spiroperidol are present in homogenates of retina from several mammalian species including calf, rat, rabbit and monkey. 14 " 16 Moreover, stimulationevoked release of ( 3 H)-dopamine appears to be modulated by an autoreceptor that exhibits the pharmacological profile of a D-2 receptor. 17 Thus, mammalian retina may contain both the D-l and D-2 subtypes of the dopamine receptor. On the basis of in vitro binding assays with ( 3 H)- spiroperidol in the rat striatum, Huff and Molinoff 18 suggested that the D-2 subtype of the dopamine receptor may be further subdivided. Since antipsychotic and antiparkinsonian drugs mediate their effects through inhibition of dopamine receptors, it was of considerable interest to determine whether subtypes of D-2 receptors exist in human tissue. One of the difficulties with using ( 3 H)-spiroperidol to characterize dopamine receptors is that this ligand also has a high affinity for serotonin receptors. 19 Moreover, recent evidence suggests that there may be substantial numbers of serotonin receptors in mammalian striatum. 20-21 In contrast, the mammalian retina contains little or no detectable serotonin. 22 Since it has been shown to contain a moderately high density of binding sites for ( 3 H)-spiroperidol, the retina may be a promising tissue in which to look for subtypes of D-2 receptors. Materials and Methods Freshly enucleated eyes from human donors (aged 14 to 91) were obtained within 24 hr of death (primarily due to cardiorespiratory failure) and were dissected on ice in 20 mm Hepes buffer (ph 7.5) containing 154 mm NaCl. The pars plana was incised 4 mm posterior to the correoscleral limbus. The retina was removed from the underlying retinal pigment epithelium by blunt dissection and was detached surgically from its adherence at the optic nerve. Retinal tissue was homogenized in 20 mm Hepes buffer (ph 7.5) containing 154 mm NaCl and 5 mm EDTA. Following centrifugation (20,000 g for 10 min at 4 C), pellets were resuspended in Hepes buffer containing 154 mm NaCl, incubated at 37 C for 30 min, and subjected to centrifugation as above. The pellet was resuspended in 20 mm Hepes buffer containing 154 mm NaCl at a concentration of 0.5 mg of tissue/ ml. ( 3 H)-Spiroperidol (24 Ci/mmol), drugs, and GTP were diluted in 2.6 mm ascorbic acid containing 20 Mg/ml of bovine serum albumin. A typical assay consisted of 50 /A radioligand, 50 n\ drug, 100 /A tissue homogenate and 2.8 ml of Hepes buffer containing 154 mm NaCl. Saturation experiments were carried out at concentrations of ( 3 H)-spiroperidol from 0.01-2 nm. In competition experiments, the concentration of ( 3 H)-spiroperidol was between 0.2 and 0.3 nm, and bound radioligand was always less than 10% of the total amount of radioligand in the assay. A concentration of GTP of 300 or 500 nm was included in all experiments performed with agonists. Homogenates of human retina were incubated for 30 min at 37 C, which provided sufficient time for the binding of ( 3 H)-spiroperidol to reach equilibrium. Reactions were terminated by the addition of 10 ml of ice-cold Tris buffer (ph 7.5) containing 154 mm NaCl. Samples were then filtered through glass-fiber filters (Schleicher and Schuell, No. 30, Keene, NH). Each filter was washed with an additional 10 ml of Tris buffer. The amount of radioactivity remaining on the filter was determined by liquid scintillation spectroscopy with a counting efficiency for tritium of 32%. Specific binding was defined as the difference between the amount of radioligand bound in the presence and absence of 2 /um (+)-butaclamol. Protein content was determined by the method of Bradford, 23 using bovine serum albumin as the standard. ( 3 H)-Spiroperidol was obtained from New England Nuclear Corp. (Boston, MA); (+)- and (-)-butaclamol and N-propylnorapomorphine were purchased from Research Biochemicals Inc. (Wayland, MA); dopamine, serotonin, yohimbine, and GTP were obtained from Sigma Chemical Co. (St. Louis, MO). The following drugs were kindly provided as gifts by the company indicated: domperidone, ketanserin, and R-5573, Janssen Pharmaceutica (Beerse, Belgium); sulpiride, Ravizza (Milan, Italy); fluphenazine, Squibb and Sons (New York, NY); prazosin, Pfizer Inc. (Groton, CT); phentolamine, Ciba-Geigy Corp. (Summit, NJ); clonidine, Boehringer Ingelheim Ltd. (Ridgefield, CT). Other reagents were purchased from standard commercial sources. Data Analysis Saturation isotherms were transformed using the method of Scatchard. 24 Estimates of the K^ and B max values were obtained using unweighted linear regression analysis of the transformed data. Competition curves were initially modeled using the following one-site equation: B, = B (L/IC 50 ) n + NS (1)

No. 5 BINDING SITES FOR ( H)-SPIROPERIDOL IN HUMAN RETINA / McGonigle er ol. 689 In this equation B L is the amount of radioligand bound to the tissue, B is the total number of binding sites labeled in the absence of competing ligand, L is the concentration of competing ligand, IC 50 is the concentration of competing ligand that inhibits 50% of total specific binding, n is the Hill coefficient and NS is the amount of radioligand not specifically bound to the tissue. Nonspecific binding was routinely constrained to the value determined when assays were carried out in the presence of 2 /um (+)- butaclamol. This value was determined in each experiment. Curve-fitting was done by nonlinear least squares regression analysis according to the method of Marquardt and Levenberg. 25 The analysis was performed using the mathematical modeling program NEWFITSITES 26 available on the NIH-sponsored PROPHET system. If the estimate of the Hill coefficient was significantly different from 1, the competition data were fit to the following multiple-site model: B L = 1 + (L/ICJOI) + (2) In this equation n is the number of independent classes of binding sites, Bi is the amount of radioligand bound to site "i" in the absence of competing ligand and IC 5O i is the IC 50 value of the competing ligand for site "i." The curve-fitting was performed by the NEWFITSITES program as described above. Improvement in fit was determined by comparing the residual sum of squares of the respective fits using a partial F-test. 27 IC 50 values were transformed to K; values using the method of Cheng and Prusoff. 28 Results The specific binding of ( 3 H)-spiroperidol was saturable and Scatchard transformation resulted in a linear plot (Fig. 1). The linear Scatchard plot indicates that ( 3 H)-spiroperidol is either labeling a single class of receptors or is nonselectively labeling multiple classes of receptors with the same affinity for the radioligand. The Kd value from the Scatchard analysis was 87 pm and the B max value was 1650 fmol/mg of protein. Both of these values were in good agreement with measurements made in rat striatum. 1826 These results indicate that human retina contains a high density of high-affinity, saturable binding sites for the D-2 selective antagonist spiroperidol. Experiments were carried out to determine the rate constants of association and dissociation for ( 3 H)- spiroperidol. The time course of association was rapid and monophasic (Fig. 2), yielding a linear pseudo-first-order rate plot (Fig. 2A, inset). The pseudo-first-order rate constant of association derived from this plot was 3.99 X 10 8 min" 1 M" 1. To 5009 4000. 3000. 2000. 1000. 016 cspiroperidold Fig. 1. Binding of ( 3 H)-spiroperidol in human retina. Membranes were incubated for 30 min at 37 C with varying concentrations of ( 3 H)-spiroperidol (10 pm to 2 nm) in the absence (O) and presence (A) of 1 nm (+)-butaclamol. Specific binding () was saturable and of high affinity. Each point represents the mean of triplicate determinations. Inset: Scatchard transformation of the specific binding data. determine the rate constant of dissociation, ( 3 H)- spiroperidol was allowed to equilibrate with retinal tissue for 30 min, at which time 2 /xm (+)-butaclamol was added to initiate dissociation. Specific binding of ( 3 H)-spiroperidol was determined at various times after the addition of butaclamol (Fig. 2B). The rate of dissociation was rapid, with a t 1/2 of approximately 15 min. A first-order plot of the data was linear, indicating that dissociation was monophasic (Fig. 2B, inset). The rate constant of dissociation calculated from the first-order plot was 0.04 min" 1. The K d value of 100 pm calculated from the ratio of the kinetic rate constants (k_i/ki) was in good agreement with the K d value of 87 pm determined from the equilibrium saturation analysis. The monophasic rates of association and dissociation provide further evidence that all of the binding sites have the same affinity for ( 3 H)-spiroperidol. Studies of the inhibition of the binding of ( 3 H)- spiroperidol were performed with a number of competing ligands. At concentrations of radioligand of 200 to 300 pm, specific binding represented 80% of total binding. The potent serotonin antagonist ketanserin exhibited a low, almost micromolar affinity for the binding sites labeled with ( 3 H)-spiroperidol. This is in contrast to the nanomolar affinity of serotonin receptors for ketanserin. 29 A Hill coefficient close to 1 indicated that ketanserin has a uniformly low affinity for all of the binding sites for ( 3 H)-spiroperidol. This result supports the contention that there are no detectable 5-HT2 receptors in the human retina. Experiments were carried out to determine the effect of GTP (300 MM) on the inhibition of the binding of ( 3 H)-spiroperidol by the agonist dopamine. GTP increased the Hill coefficient from 0.62 to 0.82 and

- 690 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / Moy 1988 Vol. 29 h 1200. 809 400. 1600 1200 800 400. A. 2. T 0. 5. 10. TIME CMIN5 0. 10. 20. 30. 40. TIME CMIN5 10. 15. 20. 25. 30. 35. TIME CMIhO 50. 60. 40. 45. Fig. 2. Kinetics of the binding of ( 3 H)-spiroperidol. (A) Membranes were prewarmed and incubated at 37 C as described in Methods. The binding reaction was terminated by the addition of ice-cold buffer followed by rapid nitration through glass-fiber filters. The resulting association curve was monophasic. Inset: A pseudo-first-order rate plot was derived by transformation of the association binding data. X is equal to Be/^-B) where B e is the amount of radioligand bound at equilibrium and B is the amount of radioligand bound at time t. (B) After the retinal membranes had been incubated for 30 min at 37 C, dissociation was initiated by addition of 2 um (+)-butaclamol. The resulting dissociation curve was monophasic. Inset: Transformation of the dissociation binding data resulted in a linearfirst-orderrate plot. Each point represents the mean of triplicate determinations. Sites labeled with ( 3 H)-spiroperidol had a significantly higher affinity for the active stereoisomer (+)- butaclamol than for the inactive stereoisomer (-)- butaclamol (Fig. 4, upper panel). In addition, the competition curve for (+)-butaclamol was markedly biphasic, suggesting that (+)-butaclamol exhibited selectivity for a subclass of binding sites for ( 3 H)-spiroperidol. Nonlinear regression analysis was used to fit multiple-site models to the competition data. The data were best fit by a two-site model in which (+)- butaclamol had a high affinity for 60% of the sites and a 100-fold lower affinity for the remaining 40% of the sites. Fitting the data to a three-site model did not result in a statistically significant improvement of fit. Inhibition studies with other antagonists yielded similar results. The competition curves for domperidone and sulpiride were also fit better by a two-site model 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 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 appears that there are two distinct subclasses of binding sites labeled by ( 3 H)-spiroperidol. Experiments carried out with several antagonists and two agonists in the presence of GTP revealed that the binding sites for ( 3 H)-spiroperidol could be subdivided into two populations (Table 1). The results were consistent with the existence of two classes of sites present in a ratio of approximately 3:2. Two drugs, spiroperidol and ketanserin, were not selective for either class of sites. The small class of sites had a higher affinity for the antagonist domperidone and the agonist dopamine, whereas the large class of sites 100 ( f 80 II 1 I 1 1 I I mil O Contr ol + 300 um GTP ' A + 500 um GTP- 60 >l nh = 0-82 lowered the affinity by approximately one order of magnitude (Fig. 3). A concentration of GTP of 300 jum had a maximal effect and no additional change in the shape of the competition curve was detected when assays were carried out in the presence of 500 ^M GTP. The sensitivity to guanine nucleotides suggests that some of the D-2 receptors in human retina are linked to the enzyme adenylate cyclase. A Hill coefficient less than 1 in the presence of a maximally effective concentration of GTP may be explained by the coexistence of a heterogeneous population of binding sites. 40 20 0 : \ n H = 0.62\ u 1 I I i i 10-8 10-6, 0-4 :DOPAMINE: CMJ Fig. 3. Effect of GTP on binding of dopamine to D-2 receptors in human retina. Homogenates were incubated as described in Methods. In the absence of GTP (O), the EC 50 value was 2 /um and the Hill coefficient was 0.62. In the presence of 300 um GTP (), the EC 50 value increased by six-fold and the Hill coefficient increased to 0.82. Each point represents the mean of triplicate determinations. a-2

No. 5 DINDING SITES FOR ( 3 H)-SPIROPERIDOL IN HUMAN RETINA / McGonigle er ol. 691 had a higher affinity for the antagonists sulpiride, (+)-butaclamol and fluphenazine, and the agonist N- propylnorapomorphine. The affinities of the large class of sites correlated well with the affinities of the D-2 receptor in rat striatum for these drugs (Fig. 5), indicating that the large class of sites represents D-2 receptors. The affinities of the small class of sites did not correlate well with the affinities of the D-2 receptor in the rat striatum for these ligands. Additional studies were performed to try to determine the identity of the small class of binding sites. Competition experiments with yohimbine, prazosin, clonidine, and phentolamine revealed that these drugs, which exhibit selectivity for alpha-adrenergic receptors, had a low (micromolar) affinity for both classes of binding sites labeled by ( 3 H)-spiroperidol (Table 2). This result ruled out the possibility that any of the binding sites specifically labeled by ( 3 H)-spiroperidol were alpha r or alpha 2 -adrenergic receptors. Competition experiments carried out with R-5573 demonstrated that all of the ( 3 H)-spiroperidol binding sites had a low affinity for this spirodecanone-specific ligand, indicating that neither subclass of sites was a spirodecanone site. Discussion 100 Z 80 S 60 fc 40 o UJ (o 20 100 o 2 80 + H m 60 40 20 - I mini ml 1 i iiinn C-OBUTACLAMOL C-3BUTACLAM0L DOMPERIDONE SULPIRIDE "" ' " " ' 1 \ i i...i i. ' w > 10-" 10-9 10-7 :DRUG: CMJ 1 P SITE SITES SITE SITES Fig. 4. Inhibition curves for antagonists at dopamine receptors. 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 (+)-butaclamol was best fit by a two-site equation (solid line) as determined by nonlinear regression analysis. According to the two-site model, (+)-butaclamol was 100-fold selective fora subclass of ( 3 H)-spiroperidol binding sites that represent 60% of the total number of sites labeled. Lower panel: Inhibition data for the dopaminergic antagonists domperidone and sulpiride were also better fit by the two-site equation than the one-site equation. Domperidone was 15-fold selective for 40% of the sites labeled by ( 3 H)-spiroperidol whereas sulpiride was 20-fold selective for 60% of the sites. Each curve represents data pooled from three experiments in which each point was measured in duplicate. The improvement infitof the two-site over the one-site model was P < 0.0001 in each case. The results of these in vitro binding assays indicate that the human retina contains D-2 receptors that can be characterized with the radioligand ( 3 H)-spiroperidol. The properties of these receptors were similar to those of D-2 receptors in rat pituitary. The receptors have a uniformly high affinity for the radioligand as evidenced by the linear Scatchard plot and monophasic rates of association and dissociation. The close agreement between the dissociation constants deter- 10- Table 1. Subclasses of binding sites for ( 3 H)-spiroperidol Small site* Large sile K, (nm)t % Kj (nm) % Selective for small sile Dopamine + GTP (3)t Domperidone (4) Selective for large site NPA + GTP(3) Sulpiride (4) Fluphenazine (3) Butaclamol (3) Nonselective Spiroperidol (3) Ketanserin (3) 548 ± 440 0.62 ± 0.08 8.0 ± 1.1 912 ± 50 15 ±4.5 34 ±7 0.087 ± 26 200 ± 74 * Displacement curves werefit to a two-site model using the NEWFIT- SITES program which provides IC50 values and relative proportions for each class of sites. t IC50 values were corrected to K, values by the method of Cheng and 34 ±3.1 42 ± 1.7 39 ±2.5 39 ± 2.4 38 ± 1.8 40 ± 3.5 10,500 ± 3,700 9.5 ± 1.3 0.42 ± 0.23 44 ±9 0.3 ±0.12 0.33 ±0.10 66 ±3.1 58 ± 1.7 61 ±2.5 61 ±2.4 62 ± 18 60 ± 3.9 Prusoft'. i Number of individual experiments performed in duplicate. 5 Mean ± SEM.

692 INVESTIGATIVE OPHTHALMOLOGY G VISUAL SCIENCE / May 1988 Vol. 29 11. 10. 9. 8. 7. 6. 5. kelonserin-/' y ^ y^ dopomi ne y ^/sulpir ide -log K. / CSTRIRTUM) spiroper idol. bijtoc lomol. A"fluphen. *» nper idone Fig. 5. Comparison of Ki values obtained in membranes prepared from human retina and rat striatum. The slope of the correlation between results obtained with the retina and with striatal D-2 receptors was 0.92 with a correlation coefficient of 0.91. The values for retina represent the mean of at least three experiments in which each point was measured in duplicate. The values for rat striatum were reported by Lin et al. 38 mined by kinetic and by equilibrium analyses also supports the contention that ( 3 H)-spiroperidol is labeling a homogeneous population of receptors. Despite this apparent homogeneity, several drugs discriminated two populations of binding sites, present in a ratio of 3:2. Competition experiments revealed the existence of both agonists and antagonists that were selective for each population of sites. It has been reported that the density of sites labeled with ( 3 H)-spiroperidol in mammalian retina is relatively low compared to other dopamine-containing brain regions such as the striatum. 14 In the present study, the density of ( 3 H)-spiroperidol sites in human retina were found to be approximately equal to the density of D-2 receptors in the rat striatum. 1826 It is likely that earlier studies performed with ( 3 H)-spiro- Table 2. Interaction of drugs with binding sites labeled with ( 3 H)-spiroperidol Serotonergic Serotonin (2)% Ketanserin (3) a-adrenergic Yohimbine (2) Prazosin (2) Phentolamine (2) Clonidine (2) Spirodecanone R-5573 (2) Ki (nm)*-\ > 10,000 200 ± 74 1,800 ± 840 > 10,000 > 10,000 > 10,000 250 ± 122 * Displacement curves werefit to a single-site model using the NEWFIT- SITES program. t IQo values were corrected to Kj values by the method of Cheng and Prusoff. % Number of individual experiments performed in duplicate. Mean ± SEM. peridol overestimated the density of D-2 receptors because labeling of 5-HT2 receptors by this radioligand was not accounted for or prevented. In this study we have detected the existence of binding sites with high affinity for domperidone in human retina. However, Watling and Iversen 14 obtained no specific binding with ( 3 H)-domperidone in retina from other mammals. They suggested that the lack of specific binding was due to the technical limitations associated with the use of ( 3 H)-domperidone including high levels of nonspecific binding. Furthermore, the studies were performed with bovine and guinea pig retina which could have different proportions of high-affinity sites for domperidone than human retina. In many receptor systems, inhibition of the binding of a radiolabeled antagonist by an agonist results in displacement curves with a shallow slope. 30 The low Hill coefficients of such curves may be erroneously interpreted as reflecting the existence of multiple subtypes of receptors. 31 It is unlikely that the heterogeneous binding sites described in this paper reflect agonist-specific phenomena. First, inhibition of the binding of ( 3 H)-spiroperidol by the agonists dopamine and N-propylnorapomorphine results in complex displacement curves even in the presence of maximally effective concentrations of GTP. Second, in the presence of guanine nucleotides the agonists are selective for different populations of binding sites. Third, and most convincing, is the fact that several antagonists also exhibit this apparent selectivity. ( 3 H)-Spiroperidol has been shown to specifically label other classes of binding sites in addition to D-2 receptors. Leysen et al 19 reported that 5-HT2 receptors in the frontal cortex have a high affinity for ( 3 H)- spiroperidol. Neither population of binding sites in the human retina appears to be 5-HT2 receptors since both populations of sites have the same low micromolar affinity for the potent 5-HT2 antagonist ketanserin. Moreover, the 5-HT2 agonist serotonin has a Kd value greater than 10 nm for both classes of sites. ( 3 H)-Spiroperidol has also been reported to label alpha-adrenergic receptors in the mouse frontal cortex. 32 Since norepinephrine- and epinephrine-containing neurons have been shown to exist in the retina, 33 ' 34 it was necessary to rule out the possibility that one of the subclasses of sites labeled by ( 3 H)-spiroperidol was an alpha-adrenergic receptor. Based on the very low affinity exhibited by both classes of binding sites for several potent antagonists at alpha-adrenergic receptors, neither subclass of ( 3 H)-spiroperidol-labeled sites in the retina appears to be alpha r or alpha 2 -adrenergic receptors. Spirodecanone sites are also specifically labeled by ( 3 H)-spiroperidol. These sites are characterized by a high affinity for dopa-

No. 5 BINDING SITES FOR ( H)-SPIROPERIDOL IN HUMAN RETINA / McGonigle er ol. 690 mine, spiroperidol and related butyrophenones but a low affinity for such selective antagonists as sulpiride, butaclamol and flupenthixol. 35 Spirodecanone binding sites have been localized to regions of the hippocampus, nucleus accumbens, olfactory tubercle and cerebral cortex using autoradiographic techniques. 36 The possibility that one of these populations of binding sites is a spirodecanone site was ruled out by the finding that the potent spirodecanone-specific ligand R-5573 had a uniformly low affinity for both populations of binding sites. The two subclasses of binding sites identified by antagonists at dopamine receptors in the retina do not correspond to the subtypes of D-2 receptor hypothesized to exist in the rat striatum. 18 This may result from the complexity of the binding profile of ( 3 H)-spiroperidol in the rat striatum which contains 5-HT2 receptors, 20 alpha-adrenergic receptors 37 and spirodecanone sites 35 in addition to high- and low-affinity states of the D-2 receptor. These additional factors complicate studies of putative subtypes of dopamine receptors in the striatum. In contrast, the binding profile of ( 3 H)-spiroperidol in the human retina is simplified by the lack of detectable 5-HT2 receptors, alpha-adrenergic receptors or spirodecanone binding sites in this tissue. The pharmacological specificity of the large class of sites labeled by ( 3 H)-spiroperidol in the retina corresponds to the D-2 receptors described in the striatum of the rat. The order of potency of the compounds tested was identical in the rat striatum and human retina, and the affinities of the large class of sites correlated well with the affinities of the D-2 receptor in rat striatum for these compounds. 38 It appears likely that the large class of sites labeled by ( 3 H)-spiroperidol in the retina is linked to the enzyme adenylate cyclase. In systems whose function is mediated by increases or decreases in the activity of adenylate cyclase, the formation of a ternary complex is thought to precede the interaction of the receptor with the enzyme. In this reaction sequence, the agonist binds to the receptor in the first step. The agonist-bound receptor then binds to a guanine nucleotide-binding protein, forming a ternary complex. In the presence of a guanine nucleotide, the ternary complex is destabilized so that only the first step in the reaction sequence is detected. When inhibition of the binding of a radiolabeled antagonist by an agonist is studied in the presence of GTP, the affinity of the receptor for the agonist is decreased, and the competition curve becomes steeper. 30 This is the effect that GTP had on the inhibition of the binding of ( 3 H)-spiroperidol by the agonist dopamine in homogenates from human retina. This effect of GTP has been demonstrated for the D-2 receptor in rat intermediate pituitary 39 and in rat striatum. 40 It has also been reported that activation of D-2 receptors results in inhibition of the activity of the enzyme adenylate cyclase in rat anterior pituitary 4 and in rat striatum. 41 Thus, it is likely that the D-2 receptors in the retina also mediate their effects through the inhibition of adenylate cyclase. The small class of sites labeled by ( 3 H)-spiroperidol in the human retina does not correspond to any of the specific sites that are known to be labeled by this radioligand. These binding sites have properties that are characteristic of specific recognition sites, since they are saturable, reversible and of high affinity. It remains to be established whether they represent significant neurochemical receptors that mediate measurable biochemical or physiological responses. A pharmacological profile of any functional response mediated by these sites must be obtained before they can be considered a new subtype of dopamine receptor. Key words: retina, human, dopamine receptors, ( 3 H)-spiroperidol, D-2 receptors References 1. Cools AR and Van Rossum JM: Excitation-mediating and inhibition-mediating dopamine-receptors: A new concept towards a better understanding of electrophysiological, biochemical, pharmacological, functional, and clinical data. Psychopharmacologia (Berlin) 45:243. 1976. 2. Kebabian J W and Calne DB: Multiple receptors for dopamine. Nature 277:93, 1979. 3. 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