Domperidone from High-Affinity Sites of the Dopamine D2 Receptor, But

SYNAPSE 49:209–215 (2003)

Dopamine Displaces [3H]Domperidone From High-Afﬁnity Sites of the Dopamine D2 Receptor, But Not [3H]Raclopride or [3H]Spiperone in Isotonic Medium: Implications for Human Positron Emission Tomography

PHILIP SEEMAN,1,2* TERESA TALLERICO,2 AND FRANC¸ OISE KO1 1Department of Pharmacology, University of Toronto, Toronto, Ontario, Canada M5S JA8 2Department of Psychiatry, University of Toronto, Toronto, Ontario, Canada M5S JA8

KEY WORDS high-affinity state of D2; antipsychotics; brain imaging; amphetamine ABSTRACT Because the high-affinity state of the dopamine D2 receptor, D2High, is the functional state of the receptor, has a role in demarcating typical from atypical antipsychotics, and is markedly elevated in amphetamine-sensitized rats, it is important to have a method for the convenient detection of this state by a ligand. The present data show that, in contrast to [3H]spiperone or [3H]raclopride, [3H]domperidone labels D2High sites in the presence of isotonic NaCl in either striatum or cloned D2Long receptors, yielding a dopamine dissociation constant (1.75 nM) in agreement with that found with [3H]dopamine. Increased labeling of D2High sites occurred with [3H]domperidone after severe disruption of the cells, suggesting that [3H]domperidone has better access to the D2 receptor from the cytoplasmic aspect of the cell membrane. The density of the [3H]domperidone-labeled D2 receptors was the same as that of the [3H]raclopride- labeled D2 receptors, but twice the density of [3H]spiperone sites for human cloned D2Long receptors, compatible with the monomer-dimer concept of the D2 receptor. [3H]domperidone readily labels the D2High sites in postmortem human brain homoge- nates. Although [3H]spiperone or [3H]raclopride can occupy D2High sites, the inability of 1–10 nM dopamine to displace these ligands under isotonic conditions suggests that these ligands may not be suitable for monitoring the physiological high-affinity state of the dopamine D2 receptor by means of [11C]methylspiperone or [11C]raclopride in humans. Synapse 49:209–215, 2003. © 2003 Wiley-Liss, Inc.

INTRODUCTION predominantly (ϳ85–90% of the total population) in The dopamine D2 receptor exists in two states. The the low-affinity state (Seeman et al., 1985; Huff and high-affinity state of the D2 receptor is sensitive to Molinoff, 1982). However, using striatal slices and 3 dopamine concentrations in the 1–10 nM range (al- [ H]spiperone, Richfield et al. (1986) found that 90% of though values of up to 100 nM have been reported). The low-affinity state of the D2 receptor is sensitive to high dopamine concentrations between 100–2,000 nM Contract grant sponsors: NARSAD (the National Alliance for Research on Schizophrenia and Depression), the CIHR (Canadian Institutes of Health Re- (Hamblyn et al., 1984; Watanabe et al., 1985; Grigori- search), NIDA (the National Institute on Drug Abuse), the Stanley Medical adis and Seeman, 1986). Research Institute, the Stanley Scholars Program of the Stanley Medical Re- search Institute, and by donations from Dr. Karolina Jus and the Medland and Although the high-affinity state of the dopamine D2 O’Rorke families, the Grace Lumsden/Margaret Nicholds Graduate Scholarship (to FK). receptor (or D2High) is the functional state of the re- *Correspondence to: Philip Seeman, Department of Psychiatry, 1 King’s Col- ceptor in the anterior pituitary (George et al., 1985), lege Circle, Medical Sciences Building, Room 4344, Toronto, Ontario, Canada D2High has not been widely studied in nervous tissue. M5S JA8. E-mail: [email protected] This is because dopamine competition with the com- Received 30 March 2003; Accepted 21 April 2003 DOI 10.1002/syn.10232 monly used ligands ([3H]spiperone or [3H]raclopride) on homogenized tissues reveal that D2 receptors are

© 2003 WILEY-LISS, INC. 210 P. SEEMAN ET AL. the D2-labeled sites were in the high-affinity state, the binding of [3H]spiperone, [3H]raclopride, and with a dopamine dissociation constant of 8.2 nM. [3H]domperidone (Martres et al., 1978; Baudry et al., The clinical relevance of D2High in the nervous sys- 1979; Grigoriadis and Seeman, 1986) to cloned dopa- tem has recently become apparent in several different mine D2 receptors. types of studies. First, the striata from rats sensitized to amphetamine had a normal density of D2 receptors, MATERIALS AND METHODS but revealed a 360% increase in the density of the Frozen rat brains were purchased (Pel-Freez Biologi- D2High sites, compatible with a marked supersensitiv- cals, Rogers, AR) and stored at –70° until used. The ity to dopamine-mimetics (Seeman et al., 2002). brain was partly thawed and the striata removed. The Second, the absolute value of the dopamine dissoci- striata were homogenized in buffer (4 mg frozen tissue ation constant at D2High, 1.75 nM, separates antipsy- per ml buffer) using a Teflon-glass homogenizer with chotic drugs into traditional ones (which typically elicit the piston rotating at 500 rpm and 10 up-and-down parkinsonism) and atypical ones (which elicit little or strokes of the glass container. The buffer contained 50 no parkinsonism) (Seeman, 2002). While this value of mM Tris-HCl (pH 7.4 at 20°), 1 mM EDTA, 5 mM KCl, 1.75 nM was obtained on human cloned D2 receptors, 1.5 mM CaCl2, 4 mM MgCl2, and either 10 mM or 120 the dopamine dissociation constant reported in the lit- mM NaCl (see Results). The homogenate was not 3 erature for D2High ranges from 2 nM (using [ H]raclo- washed, centrifuged, or preincubated because previous pride; Malmberg et al., 1998) to 4.5–229 nM (using work found that more than 30% of the D2 receptors 3 [ H]spiperone; Gru¨ newald et al., 1996; Chio et al., were lost by these procedures (Seeman et al., 1984). 1994; Wiens et al., 1998; Payne et al., 2002) to 22–56 Postmortem human frozen tissues were prepared in 125 nM (using [ I]iodosulpride; Sokoloff et al., 1992; Pera- the same manner as that for the rat tissues. The frozen chon et al., 1999). postmortem human striata were obtained from the Na- Third, although D2High is the physiologically functional Neurological Research Specimen Bank (Los An- tional state, it has not been possible to measure the geles, CA). The diagnosis of schizophrenia was taken density or proportion of D2High states by either from the case notes as recorded by the physicians in 11 11 [ C]raclopride or [ C]methylspiperone by means of charge. The frozen human tissues were dissected free positron emission tomography in humans (Aalto et al., of gross myelin tracts, homogenized, and further pro- 2002). cessed as described above for the rat striata. One of the reasons for different dopamine dissocia- We used the human cloned dopamine D2Long recep- tion constants at D2High is that the competition curves tor (in Chinese hamster ovary cells; Liu et al., 2000). 3 3 between dopamine and [ H]spiperone, [ H]raclopride, The cells were harvested by gently scraping the cells off 125 or [ I]iodosulpride do not reveal a clear, unambiguous the bottom of the Petri dish, centrifuged, resuspended high-affinity component which is distinct or separate in phosphate-buffered saline (0.9% NaCl), recentri- from the low-affinity component. That is, the high- and fuged, and the pellet frozen at –70°. When used, the low-affinity components blend into one another and one frozen pellet was thawed and the cells suspended at must use computer-assisted separation of the compo- 200 ␮g protein/ml. The suspension was homogenized nents, with attendant assumptions and limitations, to for 5 sec (Polytron, setting 5), without any further determine the dopamine dissociation constant at washing or centrifugation. D2High (Watanabe et al., 1985). Another reason is that [3H]Raclopride (60–80 Ci/mmol; final concentration the dissociation constant can depend on the ligand of 2 nM in the incubation tube), [3H]spiperone (101 used and on the final concentration of NaCl, despite all Ci/mmol; final concentration of 250 pM), were pur- the appropriate corrections (Watanabe et al., 1985; chased from PerkinElmer Life Sciences (Boston, MA). Seeman and Van Tol, 1995). [3H]Domperidone was custom-synthesized as [phenyl- In addition to the difficulty in determining the true 3H(N)]domperidone (42 Ci/mmol) by PerkinElmer Life dopamine dissociation constant at D2High, there is Sciences and used at a final concentration of 1.2 nM. lack of agreement on the density of dopamine D2 re- The competition between a drug and a [3H]ligand for ceptors as labeled by the various dopaminergic ligands. binding at the receptors was done as follows. Each In particular, the density of [3H]spiperone-labeled D2 incubation tube (12 ϫ 75 mm, glass) received, in the sites has been reported as either equal to the density of following order, 0.5 ml. buffer, 0.25 ml [3H]ligand, and [3H]domperidone-labeled D2 sites (Huff and Molinoff, 0.25 ml of tissue homogenate. The tubes, containing a 1982; Lazareno and Nahorski, 1982) or higher than the total volume of 1 ml, were incubated for2hatroom density of [3H]domperidone-labeled D2 sites (Hall and temperature (20°C), after which the incubates were Wedel, 1986). filtered using a 12-well cell harvester (Titertek, Ska- Therefore, in order to obtain a reliable method for tron, Lier, Norway) and buffer-presoaked glass fiber measuring the dopamine dissociation constant at filter mats (No. 7034, Skatron, Sterling, VA). After D2High and to compare the densities of the various filtering the incubate the filter mat was rinsed with ligands on cloned human D2 receptors, we compared buffer for 15 sec (7.5 ml buffer). The filters were pushed DOPAMINE DISPLACES [3H]DOMPERIDONE 211 out and placed in scintillation minivials (Packard In- struments, Chicago, IL). The minivials received 4 ml each of scintillant (Ready Solve, Beckman, Palo Alto, CA) and were monitored 6 h later for tritium in a Packard 4660 scintillation spectrometer at 55% effi- ciency. The competition data were analyzed as previ- ously described (Seeman et al., 1985); the program provided two statistical criteria to judge whether a two-site fit was better than a one-site fit, or whether a three-site fit was better than a two-site fit.

RESULTS Dopamine dissociation constant at D2High in striatum In order to determine the dopamine dissociation constant at D2High, it was necessary to have a procedure which revealed a substantial number of receptors to be in the high-affinity state. Isotonic levels of NaCl (100– 120 mM) induce D2High receptors into their D2Low state, especially at 37°C (Watanabe et al., 1985), ob- scuring the dissociation constant of dopamine at D2High and increasing the error in the computer-assisted resolution of the dissociation constant at the high-affinity state. In fact, earlier studies deliberately omitted NaCl in order to determine the affinities of dopamine agonists at the high- and low-affinity states of D2 (Seeman et al., 1985). However, because the binding of antipsychotic benzamides (raclopride, sulpiride) requires sodium ions (Seeman et al., 1985; Jarvie et al., 1987), the present study used a low con- Fig. 1. Rat striatum: competition between dopamine and [3H]spip- 3 centration of NaCl (10 mM) to permit a clear demarca- erone, or between dopamine and [ H]raclopride, revealed less than 4% of the dopamine D2 receptors to be in the high-affinity state when tion of the D2High state. This is shown in Figure 1, measured in isotonic NaCl. However, when measured in hypotonic where [3H]spiperone and [3H]raclopride, although re- NaCl (10 mM), the competition data revealed between 10% and 40% vealing less than 4% of the D2 receptors in the high- of D2 sites to be in the high-affinity state with a dopamine Ki of approximately 1.8 nM. The high-affinity states were entirely removed affinity state in the presence of 120 mM NaCl, detected by 200 ␮M guanilylimidodiphosphate (not shown). Nonspecific bind- 30 Ϯ 5% (SE, n ϭ 10) of the receptors in the high- ing defined by 10 ␮M S-sulpiride. affinity state in the presence of 10 mM NaCl. The addition of 200 ␮M guanilylimidodiphosphate tion constant, Ki, was 1.75 Ϯ 0.2 nM (SE, n ϭ 8). This (Gpp[NH]p) eliminated the high-affinity component, agreed with a direct determination of 1.5 Ϯ 0.2 nM (SE, but had no effect on the low-affinity site (data not n ϭ 6) for the dopamine dissociation constant, Kd, shown). using [3H]dopamine on the cloned D2 receptor (Fig. 3). The observation that the Kd for dopamine is lower than Dopamine dissociation constant at D2High for the Ki for dopamine is consistent with earlier work cloned D2 receptors indicating that the apparent dissociation constant, Ki, While the proportion of D2High sites was less than of a compound varies in practice with the affinity of the 4% (0–4%, n ϭ 8) in the rat striatum, using [3H]spip- competing radioligand; in general, for any particular erone or [3H]raclopride (Fig. 1), the proportion of drug it has been repeatedly noted in the literature that D2High sites was hardly detectable with these ligands the Kd is consistently lower than the Ki (Seeman and when using human cloned D2 receptors in isotonic Van Tol, 1995). NaCl (Fig. 2A,B). 3 [3H]Domperidone, however, in contrast to [3H]spip- Characteristics of [ H]domperidone erone or [3H]raclopride, readily revealed 40 Ϯ 5% (SE, binding to D2 receptors n ϭ 8) of the D2 receptors to be in the high-affinity Using a range of [3H]domperidone concentrations state in isotonic NaCl (Fig. 2C) or in hypotonic NaCl (0.08–4.5 nM), the Kd value was 0.42 Ϯ 0.05 nM (SE, (data not shown). In a series of such dopamine/ n ϭ 4) on the cloned D2 receptor and 0.47 Ϯ 0.05 nM [3H]domperidone experiments, the dopamine dissocia- (SE, n ϭ 3) on the rat striatal homogenate, in agree- 212 P. SEEMAN ET AL.

TABLE I. Antipsychotic Ki dissociation constants (D2Long in CHO cells)

Using [3H]raclopride Using [3H]domperidone (1.5 nM; Kd ϭ 1.7 nM) (0.9 nM, Kd ϭ 0.41 nM) in 120 mM NaCl in 120 mM NaCl (average of n ϭ 3–25) (average of n ϭ 2) (Seeman, 2002) nM nM

Quetiapine 101 122 Clozapine 58 63 Amoxapine 21 21 Loxapine 10 9.6 Olanzapine 6.5 5.1 Prochlorperazine 1.5 1.7 Pimozide 1.3 1.4 Raclopride 1.3 1.7 Haloperidol 1.2 0.6 Risperidone 1 1.1 Chlorpromazine 0.8 0.99 Spiperone 0.07 0.04

peridone was 50 fmol/filter, compared to 57 fmol/filter for [3H]raclopride and 22.6 fmol/filter for [3H]spiperone, using 50 ␮g protein per filter. The inhibition constants of antipsychotics, when competed vs. [3H]domperidone on cloned D2 receptors, were similar to those found when competing vs. [3H]raclopride (Seeman, 2002), as shown in Table I. These Fig. 2. Human cloned dopamine D2Long receptors in CHO cells: although competition between dopamine and [3H]spiperone (250 pM; data indicate that the nature of the dopamine D2 re- 3 60 pM Kd), or dopamine and [3H]raclopride (2 nM; 1.9 nM Kd), ceptors labeled by [ H]domperidone is similar to that revealed no obvious high-affinity component for dopamine at dopa- labeled by [3H]raclopride, except that [3H]domperidone mine D2 receptors in isotonic NaCl, competition between dopamine 3 and [3H]domperidone (1.2 nM; 0.41 nM Kd) in isotonic NaCl revealed is far more sensitive than [ H]raclopride to being dis- a clear high-affinity component (31% of specific binding) for dopamine placed by dopamine. with a Ki of 1.9 nM. Representative experiments. The high-affinity states were entirely removed in the presence of 200 ␮M guanilylimidodiphosphate. Nonspecific binding defined by 10 ␮M S-sulpiride. Role of Na؉ ions for D2High Thus, raising the NaCl concentration from 10 to 120 mM prevented the ready detection of D2High by dopamine/[3H]spiperone or dopamine/[3H]raclopride competition experiments (Figs. 1, 2). In order to see whether raising the NaCl could also eliminate the detection of D2High by [3H]domperidone, dopamine/[3H]domperidone competition experiments were done in 200 mM NaCl. Figure 4 shows that 200 mM NaCl either abolished the D2High state or masked the labeling of D2High by [3H]domperidone. These data suggest that sodium ions may either alter the conformation of the dopamine D2 differentially altering the binding of the three ligands, or that the three ligands may have different potencies in competing with sodium ions for binding to the D2 receptor, as discussed in Jarvie et al. Fig. 3. Human cloned dopamine D2Long receptors in CHO cells. (1987). Saturation of the D2 receptors with 0.02–4 nM [3H]dopamine revealed a Kd of 1.5 nM, in agreement with the dopamine Ki in Figure 2. Representative experiment. Nonspecific binding defined by 200 nM Access of D2High to [3H]domperidone haloperidol. Because [3H]domperidone is a positively charged molecule at biological pH, it has low permeability ment with earlier values (Grigoriadis and Seeman, across biological membranes. Therefore, the effect of 1986). membrane disruption was tested. We found that the Using the same homogenate of cloned D2 receptors highest proportion of D2High sites were detected by in CHO cells, the density of D2 receptors for [3H]dom- [3H]domperidone when the thawed CHO cells were DOPAMINE DISPLACES [3H]DOMPERIDONE 213

Fig. 6. Postmortem human putamen from a control individual (L2508; 77 years, male, prostate cancer; 10 h postmortem) revealed 32% of the D2 sites to be in the high-affinity state, as detected by [3H]domperidone in isotonic NaCl, while a postmortem putamen from an individual with schizophrenia (L2211; 73 years, male, 2 years of haloperidol premortem, 14 h postmortem) revealed 19% of the D2 sites to be in the high-affinity state. Representative experiments. Nonspecific binding defined by 10 ␮M S-sulpiride. tissues. For example, a series of human control striata Ϯ ϭ Fig. 4. Human cloned dopamine D2Long receptors in CHO cells. (putamen) revealed that D2High was 35 5% (SE, n Although isotonic NaCl (120 mM) permitted the detection of a high- 5) of the D2 population, while that for schizophrenia affinity component for dopamine (15.4% of specific binding) by means tissues was 19 Ϯ 5% (SE, n ϭ 3), as illustrated in the of dopamine/[3H]domperidone experiments (with a dopamine Ki of 1.5 nM in this experiment), hypertonic NaCl (200 mM) abolished the examples in Figure 6. However, because of the large D2High state or prevented the labeling of the D2High state by number of uncontrolled premortem variables in such [3H]domperidone. Representative experiment. Nonspecific binding de- postmortem human tissues, these results only serve to fined by 10 ␮M S-sulpiride. report that a more extensive set of such experiments to measure D2High is feasible. Such experiments should also be done in hypotonic medium.

DISCUSSION The main finding is that, in contrast to [3H]spiperone or [3H]raclopride, [3H]domperidone labels D2High sites in the presence of isotonic NaCl (Figs. 1, 2, 4). Second, dopamine/[3H]domperidone competition experiments yielded a dopamine dissociation constant, Ki, of 1.75 Ϯ 0.2 nM, in reasonable agreement with the dissociation constant, Kd, found directly with [3H]dopamine for D2High on cloned D2 receptors (Fig. 3). These two findings, therefore, justify using [3H]domperidone to measure D2High sites in human postmortem tissue, as exemplified in Figure 6. In brain tissues, Fig. 5. Increased disruption of the CHO cells (containing the human cloned dopamine D2Long receptors) by means of Polytron ho- however, the endogenous dopamine, especially in non- mogenization, compared to suspending the cells by gentle vortex washed brain tissues, can be expected to occupy a pro- mixing, increased the number of D2High sites detected by [3H]dom- 3 portion of D2High sites and could thereby alter the peridone, suggesting that [ H]domperidone may approach the D2High 3 sites more readily from the cytoplasmic aspect of the cell membrane. dopamine dissociation constant detected by [ H]dom- Representative experiment. Nonspecific binding defined by 10 ␮M peridone. For example, in the case of postmortem S-sulpiride. schizophrenia tissues (Fig. 6), the endogenous dopamine can be higher than normal, thus reducing the broken by Polytron homogenization (setting 5; 5 sec), apparent density of the D2High sites. Furthermore, compared to just vortexing the cells for 5 sec (Fig. 5). endogenous dopamine could account for the high Ki value of 8.2 nM dopamine found for the high-affinity D2High in human striatum component of dopamine D2 receptors labeled by The present use of [3H]domperidone to detect [3H]spiperone in brain slices (Richfield et al., 1986). D2High in rat striatum or cloned D2 receptors in CHO The increased labeling of D2High sites by [3H]dom- cells in isotonic NaCl is readily applicable to human peridone after severe disruption of the cells (Fig. 5) 214 P. SEEMAN ET AL. suggests that [3H]domperidone may actually have bet- these ligands are insensitive to 1–10 nM dopamine in ter access to the D2 receptor from the cytoplasmic isotonic medium. aspect of the cell membrane. Although it is known that Furthermore, although [3H]domperidone detected the aspartic acid binding site on the dopamine D2 40% of the dopamine D2 receptors to be in the high- receptor is in the middle of the hydrophobic transmem- affinity state in homogenized striata, an even higher brane region, the mode of access of the antipsychotic to proportion of 90% for the high-affinity component of this site has not yet been elucidated. the D2 receptors occurs in brain slices (Richfield et al., Using the same homogenate of cloned D2 receptors 1986). It is possible that factors which promote the in CHO cells, the density of D2 receptors for [3H]dom- high-affinity state have been disrupted or removed in peridone was 1 fmol/␮g protein, compared to 1.1 the homogenized tissue. Considering that the high- fmol/␮g protein for [3H]raclopride and 0.45 fmol/␮g affinity state of the dopamine D2 receptor is the func- protein for [3H]spiperone. These values are consistent tional physiological state (George et al., 1985), and with previous data showing that the density of [3H]ben- considering that the proportion of the high-affinity zamide sites (i.e., [3H]nemonapride) was twice the den- component is higher in the more intact tissue slice, it is sity of [3H]spiperone sites for human cloned D2Long possible or likely that more than 90% of the D2 recep- receptors (Seeman et al., 1992), and compatible with tors in vivo are in the functional high-affinity state. the idea that butyrophenones label the monomer of D2, These considerations provide further justification for using radioligands which are more reflective of the D2 while benzamides label the D2 dimer (Zawarynski et high-affinity state in health and disease. al., 1998). Equally important, the density of the [3H]domperidone-labeled D2 receptors was essentially the same as ACKNOWLEDGMENTS that of the [3H]raclopride-labeled D2 receptors, indicat- We thank Dr. H.-C. Guan for excellent assistance ing that the higher density of D2High sites detected by and Dr. Ivy Liu for preparing the human cloned D2 [3H]domperidone cannot be attributed to a higher den- receptor in CHO cells. For providing postmortem hu- sity of D2 receptors labeled by [3H]raclopride. man tissues, we thank Dr. W.W. Tourtellotte, J.S. The present data only hold for the labeling of dopa- Riehl, and D. Kamrava of the National Neurological mine D2High receptors by [3H]domperidone in vitro, Research Bank ( sponsored by the NINDS/NIMH, the because [3H]domperidone does not permeate the blood– National Multiple Sclerosis Society, the Hereditary brain barrier in vivo. Disease Foundation, the Comprehensive Epilepsy Pro- The present data are relevant to physiological program, the Tourette Syndrome Association, the Dysto- cesses in the dopamine system examined by means of nia Medical Research Foundation, and the Veterans human PET. 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