Investigative Ophthalmology & Visual Science, Vol. 29, No. 5, May 1988 Copyright © Association for Research in Vision and Ophthalmology 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 (-)-stereo- isomer. 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.