Characteristics of agonist displacement of [3H]ketanserin binding to platelet 5-HT2A receptors: implications for psychiatric research1

Psychiatry Research 80 Ž1998. 227]238

Characteristics of agonist displacement of w 3 Hxketanserin binding to platelet 5-HT2A receptors: implications for psychiatric research 1 George N.M. Gurguis a,b,U , Stephanie P. Phan a , Jaishri E. Blakeley b a

b

Department of Psychiatry, Uni¨ ersity of Texas at Southwestern Medical Center, Dallas, TX 72535, USA Mental Health Ser¨ ice, Dallas Veterans Affairs Medical Center, Box 116A, 4500 S. Lancaster Road, Dallas, TX 75216, USA Received 1 April 1998; received in revised form 8 June 1998; accepted 21 June 1998

Abstract Brain 5-HT2A receptors exist in two agonist affinity states as a function of their coupling to Gq protein. This has not yet been shown in platelets. We examined w 3 Hxketanserin’s saturable binding to platelet 5-HT2A receptors and characteristics of agonist displacement curves of w 3 Hxketanserin binding in healthy control subjects. w 3 Hxketanserin saturation curves showed a trend for a two-site model, reflecting two independent binding sites. At low w 3 Hxketanserin concentrations, agonist displacement curves were flat and best fit a two-site model, indicating the existence of two agonist affinity states. Guanylyl 59-imidotriphosphate wGppŽNH.px induced a significant rightward shift in agonist displacement curves to fit a one-site model. Platelet membrane 5-HT2A receptors exist in two agonist affinity states that are regulated by Gq protein. Platelet 5-HT2A receptors provide an accessible model for examining possible dysregulation in the agonist affinity or coupling efficiency to the phosphoinositide system in psychiatric disorders and their modulation by psychotropic medications. Published by Elsevier Science Ireland Ltd. Keywords: w 3 Hxketanserin; Human platelets; 5-HT2A receptors; Agonist affinity; Coupling efficiency; Gq protein

U 1

Corresponding author. Tel.: q1 214 3765451; fax: q1 214 3727987; e-mail: [email protected] This data was presented in part at the annual meeting of the Society of Biological Psychiatry, May 1996.

0165-1781r98r$ - see front matter Published by Elsevier Science Ireland Ltd. PII S0165-1781Ž98.00077-8

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1. Introduction 5-HT2A receptors are coupled to Gq protein ŽSmrcka et al., 1991.. Agonist stimulation of 5HT2A receptors induces coupling to Gq and activates phospholipase C, resulting in the hydrolysis of phosphatidylinositol 4,5-bisphosphate into the second messengers inositol 1,4,5-triphosphate and 1,2-diacylglycerol ŽConn and Sanders-Bush, 1985; Doyle et al., 1986; Ivins and Molinoff, 1990.. Agonist displacement studies show that brain 5-HT2A receptors display two agonist affinity states and that guanylyl 59-imidotriphosphate wGppŽNH.px causes a significant rightward shift in displacement curves, suggesting that these affinity states are regulated by G protein ŽBattaglia et al., 1983, 1984; Shannon et al., 1984; Pazos et al., 1985; Shearman and Strange, 1988; Branchek et al., 1990; Ivins and Molinoff, 1990; Teitler et al., 1990; Waeber and Palacios, 1994.. Upon agonist binding, G protein-coupled receptors form a transitory high-affinity state composed of the agonist]receptor]G protein ternary complex. According to this ternary model, the percentage of receptors in the high-affinity state Ž%R H . and the ratio of the agonist dissociation constant from the receptor in the low-rhigh-affinity state Ž K L rK H . correlate with the agonist’s intrinsic activity and have been proposed as indices of coupling efficiency of the receptor to G protein ŽDeLean et al., 1980, 1982; Kent et al., 1980; Stadel et al., 1980; Marsh and Smith, 1985; Lee et al., 1986.. Agonist affinity and coupling efficiency are important measures of receptor function and signal transduction. The significance of these binding measures is underscored by studies showing that changes in receptor sensitivity, but not density, correlate more accurately with behavior following Mianserin treatment ŽSmith et al., 1990.. 5-HT2A receptor binding studies have used w 3 Hxketanserin, a 5-HT2A antagonist, as a ligand over wide concentration ranges Ž0.01]16 nM.. Estimates of receptor density and ketanserin’s dissociation constant were consequently variable ŽLeysen et al., 1982, 1983; Shearman and Strange, 1988; Pierce and Peroutka, 1989; Sanders-Bush et al., 1989; Branchek et al., 1990; Dewar et al., 1990; Gross-Isseroff et al., 1990; Smith et al.,

1990; Tsuchihashi et al., 1991; Burris and Sanders-Bush, 1992; Andres et al., 1993; Javaid et al., 1993; Steckler et al., 1993; Ohsuka et al., 1995.. This variability may be due, in part, to differences in concentration ranges. Ketanserin’s varied dissociation constant, receptor density and characteristics of antagonist saturation experiments also suggested that ketanserin may bind to a heterogeneous 5-HT2A receptor population, different affinity states of the same receptor, or a non-5-HT2A site ŽCross, 1982; Dewar et al., 1990; Apud, 1991; Oliva et al., 1992; Peroutka, 1994; Waeber and Palacios, 1994.. 5-HT2A receptors are distributed in the frontal cortex, hippocampus and other areas of the limbic system and subcortical extrapyramidal nuclei ŽMartin and Humphrey, 1994; Saudou and Hen, 1994.. These brain regions and 5-HT2A receptors play important roles in the neurobiology and pathophysiology of psychiatric disorders: stress and anxiety disorders ŽTorda et al., 1990; Morinobu et al., 1992; Pandey et al., 1993a., depression ŽBiegon et al., 1987; Cheetham et al., 1988; Pandey et al., 1990; Biegon et al., 1990a; McBride et al., 1993; Hrdina et al., 1995; Bakish et al., 1997., alcoholism ŽOverstreet et al., 1994; Pandey and Pandey, 1996., suicide and aggression ŽMann et al., 1986; Arora and Meltzer, 1989; Arango et al., 1990; Gross-Isseroff et al., 1990; Blumensohn et al., 1995., psychoses ŽArora and Meltzer, 1991; Pandey et al., 1993b., dementia ŽCrow et al., 1984. and responses to psychostimulantsrhallucinogens ŽJavaid et al., 1993.. Also, changes in 5-HT2A receptor density or receptor-mediated signal transduction are implicated in the mechanisms of action of antidepressants, electroconvulsive therapy and antipsychotic medications ŽBlackshear et al., 1986; Conn and Sanders-Bush, 1986; Cowen et al., 1986; Hotta et al., 1986; Stockmeier and Kellar, 1986; Sanders-Bush et al., 1987; Eison et al., 1989; Sanders-Bush et al., 1989; Biegon et al., 1990b; Smith et al., 1990; Pandey et al., 1992; Gurevich et al., 1993; Klimek et al., 1994; Ohsuka et al., 1995.. Platelet and brain 5-HT2A receptors are of the same subtype Žde Chaffoy de Courcelles et al., 1984; Hoyer et al., 1994; Qi et al., 1996.. 5-HT2A receptors are coupled to Gq protein and the

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phosphoinositide ŽPI. second messenger system Žde Chaffoy de Courcelles et al., 1984; Doyle et al., 1986; Conn and Sanders-Bush, 1987; Ivins and Molinoff, 1990; Sanders-Bush, 1990.. Although it was shown that brain 5-HT2A receptors display two agonist affinity states, similar investigations on platelet 5-HT2A receptors have not been conducted in healthy control subjects or psychiatric patients. If platelet 5-HT2A receptors, similar to brain 5-HT2A receptors, display two agonist affinity states, agonist]displacement studies of platelet 5-HT2A receptors can provide important measures of 5-HT2A receptor function, such as agonist affinity and coupling efficiency to Gq protein and the PI second messenger system. This study examined characteristics of agonist displacement of w 3 Hxketanserin to determine if platelet 5-HT2A receptors exist in two affinity states and the effect of GppŽNH.p on agonist displacement curves to determine if agonist affinity is regulated by G protein. We also examined w 3 Hxketanserin binding over a wide concentration range and its displacement with unlabeled ketanserin in order to address conflicting reports regarding the existence of two binding sites. 2. Materials and methods 2.1. Subjects Radioreceptor binding studies were conducted on platelet membranes obtained from 18 healthy control subjects Žnine males and nine females. with a mean age of 41.2" 13.8 years. Control subjects had no current or previous significant history of medical or psychiatric illness. All subjects were free of medication for at least 2 weeks prior to participating in the study. Two of the nine females were on birth control pills; however, a blood sample was obtained on day 5 of their menstrual cycle before the resumption of birth control pills. A third female was post-menopausal. The study was approved by the Human Subjects Committee at the Dallas VA Medical Center, and all subjects signed an informed written consent form before participating in the study. The blood

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samples Ž60]90 ml. were collected from the antecubital vein under fasting conditions. 2.2. Membrane preparation The blood sample was collected on acidcitrate-dextrose. Platelet-rich plasma ŽPRP. was obtained by centrifugation at 200 g for 10 min at room temperature. The PRP was then centrifuged at 1100 g for 20 min at room temperature. The platelet pellet was resuspended in Tyrodes buffer Žsodium chloride 137 mM, potassium chloride 2.7 mM, sodium monobasic phosphate 0.36 mM, magnesium chloride 0.10 mM, sodium bicarbonate 12.0 mM, dextrose 0.56 mM, pH 8.0. and incubated at 378C for 10 min to hydrolyze intraplatelet serotonin. The platelet pellet was recentrifuged at 1100 g for 20 min at room temperature. Platelets were then resuspended, washed and recentrifuged twice before being resuspended in a hypotonic Tris]EDTA buffer Ž5 mM Tris, 5 mM EDTA, pH 7.5. and homogenized using a Teflon pestle glass homogenizer. Platelet membranes were obtained by centrifugation at 40 000 g for 10 min at 48C. Platelet membranes were suspended in the incubation buffer Ž50 mM Tris]HCl, pH 7.4. to achieve a final protein concentration of 25]45 m gr100 m l. 2.3. Binding experiments Receptor binding experiments were conducted according to Leysen et al. Ž1982, 1983., with minor modifications using w 3 Hxketanserin Žspecific activity 60]70 Cirmmol. as a ligand. Fresh membrane preparations were used in all experiments. Platelet membranes Ž200 m l. were incubated in a total incubation volume of 1000 m l. Seven to 16 w 3 Hxketanserin concentrations Ž20 pM]9.0 nM. were used in saturation experiments. Non-specific binding was measured in various saturation experiments in the presence of either 1 mM unlabeled ketanserin, 1 mM serotonin or 1 mM quipazine dissolved in 0.1 mM ascorbic acid. Non-specific binding was also determined mathematically using LIGAND, which estimates nonspecific binding as a function of total amount of labeled ligand added.

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Displacement experiments were conducted using two w 3 Hxketanserin concentrations, 0.4 nM and 2.0 nM. w 3 Hxketanserin was displaced using 16]19 concentrations of serotonin, quipazine or ketanserin. Membranes were allowed to equilibrate at 378C for 15 min before the addition of w 3 Hxketanserin. Incubation lasted for 20 min at 378C after the addition of w 3 Hxketanserin. The reaction was terminated by the addition of 5 ml of incubation buffer at room temperature Ž50 mM Tris-HCl, pH 7.4. and quick filtration over Schleicher and Schuell glass fiber filters a30, presoaked in 0.5% polyethyleneimine using a Brandel M-24 cell harvester ŽBrandel, Gaithersburg, MD, USA.. The tubes were washed three times using 5 ml of incubation buffer. Filters were air-dried and radioactivity was counted using a scintillation counter Ž2200 Tri-Carb, Packard. with 60% counting efficiency. 2.4. Data analysis Binding data from saturation and displacement experiments were analyzed using LIGAND ŽMunson and Rodbard, 1983., an iterative]curve]linear curve fitting program using weighted averages. Data from saturation and displacement binding curves were tested for the presence of a two-site model. Data from a two-site model were accepted only if the residual sum of squares was significantly lower in a two-site model compared to a one-site model Ž F-test, P- 0.05.. From saturation experiments, ketanserin’s dissociation constant Ž K d . from one or two sites and the maximum binding capacity of each site were measured. From displacement experiments we measured receptor density in the high- Ž R H . and low-affinity Ž R L . states and the agonist Žserotonin or quipazine. dissociation constants from the receptor in the two affinity states Ž K H and K L , respectively.. Total agonist-measured receptor density Ž R T , where R T s R H q R L ., %R H and K L rK H ratio were calculated. Non-specific binding was defined by LIGAND in deriving final binding estimates. Data are reported as mean " S.D. of the mean.

2.5. Chemicals w 3 Hxketanserin Žspecific activity 61.9 Cirmmol. was obtained from DuPont NEN: Life Sciences ŽBoston, MA, USA.. Serotonin hydrochloride or bitartarate, ascorbic acid, quipazine and GppŽNH.p were obtained from Sigma ŽSt. Louis, MO, USA.. Ketanserin was obtained from Research Products International ŽMount Prospect, IL, USA.. Glass fiber filters Ža30. were obtained from Schleicher and Schuell ŽKene, ME, USA.. 3. Results Two sets of saturation experiments were condu cte d. A t th e 0.04 ] 1 .5 n M ra n ge , w 3 Hxketanserin’s K d was 0.52" 0.27 nM and maximum binding capacity Ž Bmax . was 42.46" 10.75 fmolrmg protein Ž n s 6.. In saturation experiments using w 3 Hxketanserin concentrations up to 5.0 nM, K d was 4.87" 1.42 nM Ž n s 6. and Bmax was 143.33" 35.87 fmolrmg protein. Saturation experim ents Ž n s 3 . using w 3 Hxketanserin concentration ranges from 20 pM to 9 nM showed a trend toward statistical significance for a two-site model Ž P- 0.07. ŽFig. 1.. In this analysis, at the site for which ketanserin had high affinity, the dissociation constant Ž K dH . was 0.33 " 0.29 nM and BmaxH was 11.04 " 5.98 fmolrmg, whereas at the site for which ketanserin had low affinity, the dissociation constant Ž K dL . was 8.48" 1.78 nM and BmaxL was 182.61 " 40.24 fmolrmg protein. Estimates from this analysis underestimated binding parameters of the high-affinity site and overestimated binding parameters of the low-affinity site. Thus, in the latter analysis, the site for which ketanserin had a low affinity appeared ill-defined. In addition, at extreme concentrations of the saturation curve Žhigh or low., the percentage of non-specific binding as a function of the total was higher than 50%. Unlabeled ketanserin displacement experiments Ž n s 4. of 0.4 nM w 3 Hxketanserin best fit a two-site model with an antagonist R H of 36.23" 7.40 fmolrmg protein, antagonist R L of 15.16"

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Fig. 1. Scatchard transformation of w 3 Hxketanserin binding data obtained from three saturation experiments measured in duplicate in human platelet membranes. At both ends of the curve non-specific binding Ž0.02]9.1 nM. was higher than 50% of the total binding. Hence, they were assigned less weight in the curve-fitting process. Cumulative modeling of binding data best fit a two-site model with only a trend toward statistical significance Ž P- 0.07.. In this two-site model, binding estimates of the site for which ketanserin has high affinity were underestimated compared to data obtained from w 3 Hxketanserin saturation experiments using low concentration ranges, while binding estimates of the site for which ketanserin had low affinity were overestimated compared to binding estimates derived from saturation experiments using high w 3 Hxketanserin concentrations.

3.74 fmolrmg protein, total density of 51.39" 8.93 fmolrmg protein, %R H of 70.42" 6.65, K dH of 0.59" 0.21 nM and K dL of 68.96" 45.55 nM. Again, the low-affinity site in this model had a very high K dL . The site for which ketanserin had low affinity represented 30% of w 3 Hxketanserin binding at a concentration of 0.4 nM ŽFig. 2.. Se roton in displa ce m e n t of 2.0 n M w 3 Hxketanserin Ž n s 3. showed a steep curve with a Hill coefficient close to unity. Binding data best fit a one-site model, with agonist-measured R T of 198.84" 52.00 fmolrmg protein and serotonin dissociation constant K H of 37.75" 37.77 m M ŽFig. 3.. In contrast, serotonin displacement curves of 0.4 nM w 3 Hxketanserin Ž n s 7. were flat with a Hill coefficient less than unity. LIGAND analysis of these displacement data best fit a two-site model ŽFig. 3.. Quipazine displacement curves Ž n s 2. also best fit a two-site model. Characteris-

tics of displacement curves using both agonists Žserotonin and quipazine. were similar. Binding parameters derived from displacement curves of 0.4 nM w 3 Hxketanserin by either serotonin or quipazine are summarized in Table 1. Binding parameters derived from displacement experiments in nine males and six females were tested for gender-related differences. There were no statistically significant differences in any receptor binding parameters. Serotonin displacement of w 3 Hxketanserin Ž0.4 nM. was conducted in the presence of GppŽNH.p. In four of eight experiments, serotonin displacement curves best fit a one-site model. K H increased significantly to 813.07" 548.77 nM Ž Ps 0.004. compared to a two-site model ŽFig. 3.. In four other experiments, a two-site model was still observed in the presence of GppŽNH.p; however, K H increased Ži.e. affinity decreased. to 249.18"

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Fig. 2. A representative curve of unlabeled ketanserin displacement of w 3 Hxketanserin Ž0.4 nM. binding in human platelet membranes. Binding data best fit a two-site model, with the site for which ketanserin has low affinity representing approx. 30% of total binding.

35.19 nM Ž Ps 0.02. and the K L rK H ratio decreased to 32.48" 20.21 Ž Ps 0.07, Welch’s t-test.. In saturation experiments, non-specific binding, defined as the percent of total binding in the presence of serotonin Ž1 mM., was 17.55" 17.83% at 0.4 nM w 3 Hxketanserin Ž n s 9.. In displacement experiments, serotonin’s Ž n s 9. or quipazine’s Ž n s 2. Ž1 mM. non-specific binding after maximum displacement of w 3 Hxketanserin Ž0.4 nM. was 35.69" 5.74% of total binding. The maximum displacement of w 3 Hxketanserin Ž2.0 nM. by serotonin was only 45% of total binding. When non-specific binding measured in the presence of unlabeled serotonin was defined mathematically by LIGAND as a function of the concentration of total w 3 Hxketanserin added, it averaged 0.91" 0.53% of total labeled ligand Ž n s 9. in experiments using excess unlabeled serotonin. This was not different from LIGANDdefined non-specific binding measured in experiments using excess unlabeled ketanserin Ž3.24" 8.56%.. Maximum displacement of w 3 Hxketanserin Ž0.4

nM. by unlabeled ketanserin Ž1 mM. was 35.08" 1.67 Ž n s 4.. Thus, there was no significant difference between agonist- Žserotonin, quipazine. and antagonist- Žketanserin . defined non-specific binding. 4. Discussion This investigation demonstrates that ketanserin binds to platelet membrane receptors with nanomolar affinity and a maximum binding capacity of approx. 50 fmolrmg protein. Displacement of w 3 Hxketanserin binding to 5-HT2A receptors by two agonists Žserotonin and quipazine. yielded flat curves that best fit a two-site model, indicating that platelet 5-HT2A receptors exist in two affinity states, with dissociation constants in nanomolar and micromolar ranges. Agonist interaction with G protein-coupled receptors induces conformational changes in the receptor and the formation of a high-affinity state as a result of coupling to G protein. Biphasic agonist interaction indicates binding to a single receptor popula-

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Fig. 3. A representative graph of serotonin displacement of w 3 Hxketanserin Ž2.0 nM. binding to human platelet membranes Žopen triangles .. Data best fit a one-site model, with the serotonin dissociation constant in the micromolar range. Serotonin displacement of w 3 Hxketanserin Ž0.4 nM. best fit a two-site model Žopen circles. with two agonist affinity states in the nanomolar and micromolar range. Fifty-five percent of the receptors were in the high-affinity state. In the presence of GppŽNH.p, serotonin displacement of w 3 Hxketanserin Ž0.4 nM. showed a significant rightward shift which best fit a one-site model with the serotonin dissociation constant in the micromolar range Žsolid circles..

tion which exists in two affinity states ŽDeLean et al., 1980; Kent et al., 1980; DeLean et al., 1982.. The rightward shift in agonist displacement curves by GppŽNH.p in this study indicates that the affinity states are regulated by G protein, consistent with predictions of the ternary model. To our

knowledge, this is the first report to demonstrate that platelet 5-HT2A receptors exist in two affinity states that are regulated by G protein. Our findings are also consistent with studies reporting similar observations in transfected cell cultures and in rat, pig, calf and human brain ŽBattaglia et

Table 1 5-HT2A receptor binding parameters derived from serotonin and quipazine displacement curves of w 3 Hxketanserin Ž0.4 nM. binding to platelet membrane 5-HT2A receptors RH Žfmolrmg protein.

RL Žfmolrmg protein.

RT Žfmolrmg protein.

%RH

Serotonin Ž n s 7.

33.43" 10.31

26.76" 8.26

60.19" 14.48

55.36" 8.48

Quipazine Ž n s 2.

44.54" 5.51

23.78" 4.71

68.32" 10.32

65.32" 1.70

Data are mean " S.D.

KL Ž m M.

KL rKH

57.80" 51.36

27.32" 23.68

337.97" 376.01

165.24" 90.86

23.58" 2.30

172.64" 108.80

KH ŽnM.

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al., 1983, 1984; Shannon et al., 1984; Pazos et al., 1985; Shearman and Strange, 1988; Branchek et al., 1990; Ivins and Molinoff, 1990; Teitler et al., 1990; Waeber and Palacios, 1994.. Although the ternary model was first applied to b-adrenergic receptors as a prototypic G proteincoupled receptor, it may be applied to other G protein-coupled receptors, including 5-HT2A receptors. Observations from the present investigation demonstrate that the coupling efficiency of 5-HT2A receptors can be examined using platelet membranes. To date, studies on 5-HT2A receptors in psychiatric disorders have focused primarily on measuring antagonist affinity and receptor density. Antagonist binding does not induce second messenger responses. Hence, physiological significance of the antagonist affinity remains unclear. Furthermore, changes in receptor coupling or agonist affinity and, consequently, in signal transduction, could occur in the absence of changes in receptor density. Indeed, changes in 5-HT2A receptor sensitivity, but not density, correlate closely with behavior ŽSmith et al., 1990.. Changes in coupling efficiency were also implicated in the mechanism of action of antidepressants ŽSanders-Bush et al., 1989.. It is important, therefore, to investigate coupling efficiency as an index of receptor function. Platelets may be a useful peripheral model of monoaminergic neurons ŽStahl, 1985; Pletscher, 1987; Wirz-Justice, 1988.. a 2-Adrenergic receptors, b 2-adrenergic receptors, 5-HT2A receptors and serotonin reuptake sites have been characterized on platelet membranes ŽGarcıa-Sevilla et al., ´ 1981; Kerry and Scrutton, 1983; Winther et al., 1985.. Platelet a 2-adrenergic receptors are of the a 2A subtype, which exists in the brain and are coupled to Gi protein. Therefore they provide a useful Gi protein-coupled adenylyl cyclase system ŽKim and Neubig, 1987; Gurguis, unpublished data.. Additional similarities between platelet and brain 5-HT2A receptors have been reported ŽMcBride et al., 1983; De Clerck et al., 1984; Geaney et al., 1984; Grahame-Smith et al., 1988; Tsuchihashi et al., 1991; Andres et al., 1993; Ostrowitzki et al., 1993; Ohsuka et al., 1995.. Platelets have a

monoamine oxidase enzyme system and share a common neuroectodermal origin with neurons. Our finding that platelet membrane 5-HT2A receptors exist in two-affinity states, like brain 5HT2A receptors, supports the use of the Gq protein-coupled phospholipase C system in platelets as an accessible peripheral model. w 3 HxKetanserin saturation experiments revealed two sites with two dissociation constants, a high-affinity site with a K dH of approx. 0.5 nM and a low-affinity site with a K dL of approx. 2]4 nM. The density of the high-affinity site was onethird that of the low-affinity site. Dewar et al. Ž1990. found similar relative densities in rabbit frontal cortex. Our data regarding ketanserin’s dissociation constant from the high-affinity site are also consistent with Dewar and colleagues’ results. In addition, unlabeled ketanserin displacement experiments in our laboratory showed that the dissociation constant of ketanserin from the low-affinity site was approx. 68 nM, close to the 55 nM figure reported by Dewar et al. Ž1990.. At extreme w 3 Hxketanserin concentrations, non-specific binding was more than 50% of total binding. This may have contributed to the fact that the goodness-of-fit of the two-site modeling of w 3 Hxketanserin saturation curves had only a trend toward statistical significance. In this twosite model, the low-affinity site was ill-defined, with a high dissociation constant and density. Since antagonist binding follows simple kinetics and does not involve formation of a transitory affinity state, the observed two-site model for antagonist binding suggests binding to two independent binding sites rather than to two affinity states of the same receptor population. Thus, ketanserin may be labeling another site, from which it is not displaceable by serotonin. LSD and ketanserin binding, among other 5HT2A antagonists, suggested the presence of two binding sites ŽCross, 1982; Dewar et al., 1990.. In the present study, serotonin’s maximum displacement of 2.0 nM w 3 Hxketanserin was 45% of total binding. Serotonin’s maximum displacement of 0.4 nM w 3 Hxketanserin was 65%. These observations suggest a dose effect and indicate that the higher the ketanserin concentration, the more

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ketanserin binds to the site for which it has low affinity and from which it is not displaced by serotonin. Conflicting data exist regarding the nature of the ketanserin binding site. Cell culture studies support a two-affinity state of the 5-HT2A receptor ŽBranchek et al., 1990; Teitler et al., 1990., whereas results by others in mammalian brain and human platelets ŽPierce and Peroutka, 1989; Dewar et al., 1990; Apud, 1991; Oliva et al., 1992; Waeber and Palacios, 1994. suggest binding to either a heterogeneous 5-HT2A receptor population or to a site allosterically coupled to the 5-HT2A receptor. Our data provide preliminary support for the latter. Thus, it is prudent to use low w 3 Hxketanserin concentrations in investigating platelet 5-HT2A receptors. In summary, results of the present investigation demonstrate that platelet 5-HT2A receptors display two agonist affinity states that exist as a function of their coupling to Gq protein upon agonist stimulation. The results also suggest that w 3 Hxketanserin binds to a non-5-HT2A site with low affinity. Binding to this site was non-displaceable by serotonin or quipazine. Future investigations into the role of 5-HT2A receptors in psychiatric disorders may employ agonist displacement studies to investigate 5-HT2A receptor coupling efficiency to Gq protein and the PI second messenger system as a likely site for dysregulation and modulation by psychotropics. Acknowledgements This project was generously supported, in part, by the Seay Foundation. The authors would like to thank A. John Rush, M.D., Betty Jo Hay Distinguished Chair in Mental Health and ViceChairman for Research, for his commitment, guidance and scientific mentoring. The authors would also like to thank Kenneth Z. Altschuler, M.D., Stanton Sharp Distinguished Chair and Chairman of the Department of Psychiatry, for his valuable administrative support. References Andres, A.H., Rao, M.L., Ostrowitzki, S., Entzian, W., 1993. Human brain cortex and platelet serotonin 2 receptor bind-

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