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[3H]-YM-09151-2 binding sites in human brain postmortem
Neurochemistry International 55 (2009) 643–647
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[3H]-YM-09151-2 binding sites in human brain postmortem Donatella Marazziti a,*, Stefano Baroni a, Irene Masala a, Gino Giannaccini a, Laura Betti a, Lionella Palego a, Mario Catena Dell’Osso a, Giorgio Consoli a, Maura Castagna b, Antonio Lucacchini a a b
Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, University of Pisa, via Roma, 67, I-56100 Pisa, Italy Dipartimento di Chirurgia, University of Pisa, Italy
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1 June 2009 Received in revised form 9 June 2009 Accepted 10 June 2009 Available online 18 June 2009
The controversial and limited data on the distribution of dopamine (DA) receptors of type 4 (D4) in the human brain prompted us to explore their density and pharmacological characteristics in the prefrontal cortex, striatum and hippocampus, through a series of binding assays. Brain samples were taken during autopsy from seven subjects. Tissue homogenates were incubated with increasing concentration of [3H]YM-09151-2, a D2-like receptor antagonist, and L-745,870 and/or sulpiride to define the non-specific binding, while PPAP was used to block sigma receptors. The results showed a low density of D4 receptors in the hippocampus only, with a preponderance of D2/D3 and sigma receptors in the prefrontal cortex and striatum. In conclusion, these findings underline that it is possible to label D4 receptors by means of [3H]-YM-09151-2, provided that D2, D3 and sigma receptors are blocked. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: Dopamine D2-like receptors D4 receptors [3H]-YM-09151-2 Human brain Prefrontal cortex Striatum Hippocampus
1. Introduction Dopamine (DA) receptors are classified into two major classes called D1- and D2-like, on the basis, respectively, of the high and low affinity for the neurotransmitter. The D1-like class includes D1 and D5 receptors, while the D2, D3, D4 subtypes belong to the D2-like class (Civelli et al., 1991). Much interest has been directed towards the D4 subtype after the demonstration that clozapine, the prototype of atypical antipsychotics, shows a high affinity for it (Meltzer, 1994). Both the clinical effectiveness and the low risk of extrapyramidal side effects of clozapine have been related to its preferential activity on D4 receptors which, have been hypothesized to be involved not only in the pathophysiology of different psychotic disorders, but also of Parkinson’s disease, mood disorders and hypercynetic syndrome (Tarazi and Baldessarini, 1999; Tarazi et al., 2004). However, data on D4 receptors in these conditions are few and indirect (Davis et al., 1991; MacQueen et al., 2003; Kempf et al., 2005; Maletic et al., 2007). In fact, D2 and D4 receptor had been evaluated by different authors in the prefrontal cortex of schizophrenic patients through the messenger ribonucleic acid (mRNA) levels, and
* Corresponding author. Tel.: +39 050 835412; fax: +39 050 21581. E-mail address: [email protected] (D. Marazziti). 0197-0186/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2009.06.005
the ensuing findings were controversial, as both increased or similar levels of expression were reported, in comparison with those of healthy subjects (Mulcrone and Kerwin, 1996; Roberts et al., 1996; Stefanis et al., 1998). Others employed an indirect binding assay, while subtracting the binding of [3H]-raclopride, which labels only the D2 and D3 receptors, from that of [3H]-YM09151-2, which labels all D2-like receptors, while reporting a sixfold increase of D4 receptors density in the striatum of schizophrenic patients (Seeman et al., 1993a). However, the density of D2-like receptors, evaluated by means of [11C]methylspiperone, resulted higher in schizophrenic than in healthy subjects, but similar in the two groups when [11C]raclopride was employed (Hagberg et al., 1998). Given that methylspiperone labels all D2-like receptors, these results seemed to support the presence of D4 receptors in the striatum. The interpretation of these data is, however, complicated by the evidence that [11C]-methylspiperone binds only to the D2-like receptors in monomeric configuration, at variance with raclopride that binds to both monomeric and oligomeric configurations, and [3H]-YM-09151-2 labels also the sigma and serotonin receptors of type 1a and 2a (5HT1A and 5HT2A) (Helmeste et al., 1996, 1997; Ujike et al., 1996; Tang et al., 1997). These controversies may also be related to the fact that little is known on the distribution of D2-like receptors, and particularly of D4 ones, in the human brain in normal conditions.
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D. Marazziti et al. / Neurochemistry International 55 (2009) 643–647
D4 receptors have been described at high density in the prefrontal cortex by means of a binding method employing [3H]NGD 94–1 (Primus et al., 1997). However, in a recent study exploring D4 receptors in human prefrontal cortex by means of the binding of [3H]-YM-09151-2, their density resulted inconsistent (Marazziti et al., 2007). Therefore, given the paucity of available information, the aim of the present study, which can be considered an extension of the previous, was to explore and characterize the distribution and density of D4 receptors in the prefrontal cortex, striatum and hippocampus of human brain postmortem, by comparing and/or subtracting the values of binding parameters obtained with [3H]-YM-09151-2 and different compounds to define the non-specific binding. As a consequence, the presence of sigma sites in human prefrontal cortex and striatum was evaluated as well. 2. Experimental procedure 2.1. Subjects Prefrontal cortex, striatum and hippocampus samples were taken postmortem, during autoptic sessions, from seven subjects (four men and three women; age, mean SD: 61 5 years). They were immediately frozen in dry ice and rapidly stored to 808. Autolysis time (i.e., the time between death and freezing the samples) ranged between 7 h and 25 h, an interval which does not interfere with binding assays, as demonstrated in preliminary experiments (data not shown). All subjects had died for causes not involving primarily the brain (four from myocardial infarction, two from pulmonary embolism and one from respiratory failure), had not suffered from any psychiatric or neurological disorders and were not administered psychotropic drugs, as recorded by their medical charts. The study was approved by the Ethics Committee at Pisa University. 2.2. Preparation of brain tissue homogenates Brain tissue samples were defrosted and separated by white substance. Tissues were re-suspended in D4 buffer (120 mM NaCl, 1.5 mM CaCl2, 4 mM MgCl2, EDTA 1 mM, pH 7.4) to yield 4 mg original wet weight per ml, according to the method of Seeman et al. (1993a,b), and then homogenized with an Ultraturrax in D4 buffer. The homogenates were not washed and centrifuged, because a consistent loss of receptors ranging between 15% and 60% may occur during these steps. 2.3. Binding of [3H]-YM-09151-2 Five hundred microliters of cortex, striatum and hippocampus homogenates were incubated with 10 increasing concentration of [3H]-YM-09151-2 (PerkinElmer, Milan, Italy, specific activity: 85.5 Ci/mmol) ranging between 0.015 nM and 10 nM, at a final volume of 1.5 ml. The non-specific binding was carried out by using L-745,870 30 mM and/or sulpiride 10 mM (Helmeste et al., 1996, 1997; Seeman et al., 1993; Tang et al., 1997; Tarazi et al., 1998). Given that [3H]-YM-09151-2 may bind to sigma receptors (Ujike et al., 1996), 1-phenyl-2-propylaminopentane (PPAP) 100 nM was used to block them in some assays. To determine the amount of sigma sites bound by [3H]-YM-09151-2, we performed several binding assays with increasing radioligand concentrations (0.01–10 nM) and PPAP 500 nM to define the non-specific binding. Furthermore, to evaluate the different potency of [3H]-YM09151-2 binding to D2/D3 and D4 receptors, we carried out some assays with this ligand (concentration range: 0.015–10 nM) and raclopride 300 nM or L-745,870 300 nM. In these cases sulpiride 10 mM was used in these cases to define the nonspecific binding and PPAP 100 nM to block sigma receptors. The incubation with [3H]-YM-09151-2 was performed for 2 h at 22 8C. The separation of bound and free ligand was carried out by vacuum filtration through GF/C fiber filters (Whatman, UK), which were pre-soaked in polyethyleneimine (2%) to minimize the non-specific binding to filters. Radioactivity was counted by a liquid scintillation counter (Packard LS-1600). For the pharmacological characterization of the binding, the homogenates were incubated with increasing concentrations of L-745,870, raclopride, PPAP and 0.5 nM of the radioligand. 2.4. Data analysis and statistics Equilibrium-saturation binding data, the maximum binding capacity (Bmax, fmol/mg tissue) and the dissociation constant (Kd, nM) were analyzed by means of iterative curve-fitting computer programmes EBDA (McPherson, 1985). The potency of the different compounds in displacing the [3H]-YM-09151-2 binding was expressed as inhibition constant (Ki) values, calculated from the IC50 (concentration of drug causing 50% inhibition of the specific binding), using the Cheng and Prusoff equation (1973). Each determination was performed in triplicate, but not in all areas, for the lack of sufficient brain tissue.
Fig. 1. Example of Scatchard analysis of the prefrontal cortex.
3
H-YM-09151-2 binding to the
3. Results 3.1. [3H]-YM-09151-2 binding to the prefrontal cortex and striatum with L-745,870 to define the non-specific binding The Scatchard analysis of the [3H]-YM-09151-2 binding to the prefrontal cortex showed the presence of high and low affinity binding sites (Fig. 1). The Bmax (mean SD, fmol/mg tissue) and Kd (mean SD, nM) values of the two sites were, respectively, 12 5 and 0.091 0.0099, and 710 241 and 6.51 1.42 (Table 1). The pharmacological competition studies with L-745,870 and raclopride did not lead to the expected diphasic curves, but rather to a monophasic curve with high Ki values (236 33 nM for L-745,870 and 1590 170 nM for raclopride): this effect might be attributed to the interference of receptors other than the D2-like ones. The displacement curve of PPAP was also monophasic, with a very low Ki (10 3 nM). The situation in the striatum was similar, as high and low affinity binding sites were detected in this area (the Bmax and Kd values of both sites at this level are reported in Table 1). 3.2. [3H]-YM-09151-2 binding to the prefrontal cortex and striatum with sulpiride to define the non-specific binding and PPAP to block sigma receptors In this case, in both areas, the Scatchard analysis revealed that the binding was specific and saturable, but only a single population of binding sites was detected (Fig. 2, panel a). The Bmax was 9.4 3 and the Kd 0.21 0.08 in the first area, and 97 15 and 0.07 0.005 in the striatum (Table 2A). The pharmacological competition studies with raclopride and L-745,870 led to no curve in the prefrontal cortex, probably for the low density of DA receptors in this area. On the contrary, in the striatum a one phase-displacement curve was obtained with both L-745,870 (Ki = 925 140 nM) and raclopride (Ki = 11.4 3 nM) (Fig. 3, panel a and b). Table 1 Bmax (mean SD, fmol/mg tissue) and Kd (mean SD, nM) values of the 3H-YM09151-2 binding to the prefrontal cortex and striatum with L-745,870 to define the non-specific binding. Bmax
Prefrontal cortex Striatum
Kd
Hi
Lo
Hi
Lo
12 5 103 21
710 241 645 30
0.091 0.009 0.06 0.008
6.51 1.42 7.11 1.12
Hi: high-affinity site; Lo: low affinity site.
D. Marazziti et al. / Neurochemistry International 55 (2009) 643–647 Table 2A Bmax (mean SD, fmol/mg tissue) and Kd (mean SD, nM) values of the 3H-YM09151-2 binding to the prefrontal cortex and striatum with sulpiride to define the nonspecific binding and PPAP to block sigma receptors.
Prefrontal cortex Striatum
Bmax
Kd
9.4 3 97 15
0.21 0.08 0.07 0.005
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Table 3 Bmax (mean SD, fmol/mg tissue) and Kd (mean SD, nM) values of the 3H-YM09151-2 binding to the prefrontal cortex, striatum and hippocampus with raclopride to block D2/D3 receptors, sulpiride to define the non-specific binding and PPAP to block sigma receptors.
Prefrontal cortex Striatum Hippocampus
Bmax
Kd
ND ND 8.9 1.1
ND ND 0.32 0.04
ND: not detectable.
3.4. [3H]-YM-09151-2 binding to the prefrontal cortex, striatum and hippocampus with raclopride to block D2/D3 receptors, sulpiride to define the non-specific binding and PPAP to block sigma receptors In this case, it was not possible to carry out a Scatchard analysis in the prefrontal cortex and striatum. In the hippocampus, the binding was specific and saturable and the Scatchard analysis led to a Bmax of 8.9 1.1 and a Kd of 0.32 0.04 (Table 3). 3.5. [3H]-YM-09151-2 binding to the prefrontal cortex, striatum and hippocampus with L-745,870 to block D4 receptors, sulpiride to define the non-specific binding and PPAP to block sigma receptors In the prefrontal cortex and in the striatum the Scatchard analysis revealed that the binding was specific and saturable, and it showed the presence of a single population of binding sites. The Bmax values in the prefrontal cortex and the striatum were, respectively, 6.8 0.9 and 66 8, and the Kd values 0.31 0.02 and
Fig. 2. Examples of saturation curves and Scatchard analysis in the prefrontal cortex (panel a) and striatum (panel b) with blocked sigma sites.
3.3. [3H]-YM-09151-2 binding to the prefrontal cortex and striatum with PPAP to define the non-specific binding The Scatchard analysis revealed that the binding was specific and saturable, as well as it showed the presence of a single population of binding sites (Fig. 2, panel b). In the prefrontal cortex, the Bmax was 795 82 and the Kd 4.42 1.32; in the striatum, the Bmax was 666 78 and the Kd 6.33 1.54 (Table 2B). Table 2B Bmax (mean SD, fmol/mg tissue) and Kd (mean SD, nM) values of the 3H-YM09151-2 binding to the prefrontal cortex and striatum with PPAP to define the nonspecific binding.
Prefrontal cortex Striatum
Bmax
Kd
795 82 666 78
4.42 1.32 6.33 1.54
Fig. 3. Displacement curve with L-745,870 (panel a) and raclopride (panel b) in the striatum.
D. Marazziti et al. / Neurochemistry International 55 (2009) 643–647
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Table 4 Bmax (mean SD, fmol/mg tissue) and Kd (mean SD, nM) values of the 3H-YM09151-2 to the prefrontal cortex, striatum and hippocampus with L-745,870 to block D4 receptors, sulpiride to define the non-specific binding and PPAP to block sigma receptors.
Prefrontal cortex Striatum Hippocampus
Bmax
Kd
6.8 0.9 66 8 ND
0.31 0.02 0.08 0.007 ND
ND: not detectable.
0.08 0.007. On the contrary, it was not possible to perform a Scatchard analysis in the hippocampus (Table 4). 4. Discussion Given the potential relevance of D4 receptors in the pathophysiology of different neuropsychiatric disorders and the few information available (Helmeste and Tang, 2000; Muglia et al., 2002; Oak et al., 2000; Tarazi and Baldessarini, 1999; Tarazi et al., 2004; Engelborghs et al., 2008), we explored their distribution in some human brain areas, in particular prefrontal cortex, striatum and hippocampus obtained postmortem by means of binding techniques. The present study may be considered an extension of a previously carried out by our group in human prefrontal cortex in a smaller sample (Marazziti et al., 2007). Given the lack of specific radioligands for labelling D4 receptors, we compared and subtracted a series of binding assays carried out with [3H]-YM09151-2, which is a D2-like receptor antagonist, and L-745,870, raclopride, sulpiride and PPAP as displacers. Although other compounds, such as [3H]-NGD-94-1 or [3H]-PNU-101958 have been proposed as more selective for D4 receptors, it should noted that the selectivity observed in preliminary studies on cloned receptors may not match with that observed in vivo: for this reason, perhaps, the few data obtained with them have been disappointing (Primus et al., 1997; Lahti et al., 1998; De La Garza and Madras, 2000). Our results showed that we could detect D4 receptors only in the hippocampus, and not in the striatum and prefrontal cortex where the D2/D3 subtypes resulted to be prevalent. Our findings in the hippocampus are consistent with those of one study employing monoclonal antibody for D4 receptors (Lanau et al., 1997), but at variance with others that evaluated the mRNAs for D2, D3 or D4, receptors and reported a differential distribution of each subtype in distinct hippocampal areas (Primus et al., 1997; Khan et al., 1998). The majority of data on D4 receptors in prefrontal cortex and striatum have been gathered by means of expression studies and have shown a relevant presence of the mRNA for D4 receptors in both areas (Matsumoto et al., 1996; Meador-Woodruff et al., 1996; De La Garza and Madras, 2000), in disagreement with our observations. This is not surprising, as the mRNA presence does not necessarily correspond to the receptor expression or density, because different mechanisms, such as re-arrangement and dimerization (or etherodimerization), might change the pharmacological features of the monomeric configuration of the receptor. Interestingly, no expression of D4 receptors has been also reported in the prefrontal cortex and striatum (Mulcrone and Kerwin, 1996; Stefanis et al., 1998). However, further studies are needed to investigate the occurrence of these re-arrangement mechanism for D4 receptors, as up to now the presence of an oligomeric configuration has been demonstrated only for the D2 subtype (Lee et al., 2000; Franekova et al., 2008). As far as the pharmacological competition studies are concerned, we obtained one phase-displacement curve, while
indicating that [3H]-YM-09151-2 could not distinguish between the D2/D3 and D4 receptors. The Ki values were different from those reported in the literature, while corresponding to an intermediate level of affinity, however, the different studies are not easily comparable, as most of them have been carried out in animals. In the binding assay employing L-745,870 to define the non-specific binding, the Scatchard analysis resulted in a curvilinear plot, suggestive of the presence of two different sites. It is possible that the concentration of L-745,870 used (30 mM) might displace not only the D2-like receptors, but also other receptors (Bristow et al., 1997); however, since [3H]-YM-09151-2 binds to sigma receptors with a high affinity, these are the main candidates for explaining the interference with the above-mentioned binding assay (Helmeste et al., 1996, 1997; Tang et al., 1997; Ujike et al., 1996). When we added the 1-phenyl-2-propylaminopentane (PPAP), a selective drug to block sigma receptors, in the prefrontal cortex and in the striatum, we obtained a monophasic Scatchard plot with a low Ki, corresponding to a high-affinity site: this might hence suggest that sigma receptors labelled by [3H]-YM-09151-2 were significantly higher than D2-like receptors. This observation was confirmed by the high [3H]-YM-09151-2 binding performed by using PPAP to define the non-specific binding which can be also attributed to sigma receptors (Florijn et al., 1997; Assie et al., 2005). These data are consistent with those resulting from the assay in which the non-specific binding was defined by L-745,870: in this case, we obtained a non-straight Scatchard curve that could be divided in two populations of binding sites. The binding sites with the lower affinity probably correspond to sigma receptors whose interference with the [3H]-YM-09151-2 binding was directly related to their density and to the displacer used to define the non-specific binding, when were not blocked. According to the present findings, therefore, previous data on the [3H]-YM-09151-2 binding to D2like receptors should be reconsidered critically. Regarding the D2like component, the competition studies did not permit to evaluate separately the D2/D3 and D4 receptors; in agreement with previous studies (Tarazi et al., 1998), we carried out the binding while masking the D2/D3 component by adding raclopride 300 nM as displacer. It was not possible to evaluate the binding in the prefrontal cortex or striatum, while in the hippocampus a very low density was found which probably corresponds to D4 receptors. Similarly, the experiments performed to evaluate the D2/D3 receptors, while blocking the D4 ones with L-745,870, revealed that the binding occurred only in the prefrontal cortex and striatum. In conclusion, our findings suggest that D4 receptors are mainly distributed in the hippocampus, while in the striatum or prefrontal cortex, the high density of sigma receptors interfere significantly with their labelling. Although the detection of D4 receptors in the human brain with the available methodologies appears still difficult, our study supports the notion that the [3H]-YM-091512 may label the D4 receptors, provided that D2, D3 and sigma receptors are appropriately blocked. References Assie, M.B., Consul-Denjean, N., Koek, W., Newman-Tancredi, A., 2005. Differential in vivo inhibition of [3H]nemonapride binding by atypical antipsychotics in rat striatum, olfactory lobes, and frontal cortex. Pharmacology 75, 63–68. Bristow, L.J., Patel, S., Ragan, C.I., Ridgill, M., Saywell, K.L., Tricklebank, M.D., 1997. L745,870, a subtype selective dopamine D4 receptor antagonist, does not exhibit a neuroleptic-like profile in rodent behavioral tests. J. Pharmacol. Exp. Ther. 283 (December (3)), 1256–1263. Cheng, Y., Prusoff, W.H., 1973. Relationship between inhibition constant (Ki) and concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic-reaction. Biochem. Pharmacol. 22, 3099–3108. Civelli, O., Bunzow, J.R., Grandy, D.K., Zhou, Q.Y., Van Tol, H.H., 1991. Molecular biology of the dopamine receptors. Eur. J. Pharmacol. 207, 277–286. Davis, K.L., Kahn, R.S., Ko, G., Davidson, M., 1991. Dopamine in schizophrenia: a review and reconceptualization. Am. J. Psychiatry 148, 1474–1486.
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Neurochemistry International journal homepage: www.elsevier.com/locate/neuint
[3H]-YM-09151-2 binding sites in human brain postmortem Donatella Marazziti a,*, Stefano Baroni a, Irene Masala a, Gino Giannaccini a, Laura Betti a, Lionella Palego a, Mario Catena Dell’Osso a, Giorgio Consoli a, Maura Castagna b, Antonio Lucacchini a a b
Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, University of Pisa, via Roma, 67, I-56100 Pisa, Italy Dipartimento di Chirurgia, University of Pisa, Italy
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1 June 2009 Received in revised form 9 June 2009 Accepted 10 June 2009 Available online 18 June 2009
The controversial and limited data on the distribution of dopamine (DA) receptors of type 4 (D4) in the human brain prompted us to explore their density and pharmacological characteristics in the prefrontal cortex, striatum and hippocampus, through a series of binding assays. Brain samples were taken during autopsy from seven subjects. Tissue homogenates were incubated with increasing concentration of [3H]YM-09151-2, a D2-like receptor antagonist, and L-745,870 and/or sulpiride to define the non-specific binding, while PPAP was used to block sigma receptors. The results showed a low density of D4 receptors in the hippocampus only, with a preponderance of D2/D3 and sigma receptors in the prefrontal cortex and striatum. In conclusion, these findings underline that it is possible to label D4 receptors by means of [3H]-YM-09151-2, provided that D2, D3 and sigma receptors are blocked. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: Dopamine D2-like receptors D4 receptors [3H]-YM-09151-2 Human brain Prefrontal cortex Striatum Hippocampus
1. Introduction Dopamine (DA) receptors are classified into two major classes called D1- and D2-like, on the basis, respectively, of the high and low affinity for the neurotransmitter. The D1-like class includes D1 and D5 receptors, while the D2, D3, D4 subtypes belong to the D2-like class (Civelli et al., 1991). Much interest has been directed towards the D4 subtype after the demonstration that clozapine, the prototype of atypical antipsychotics, shows a high affinity for it (Meltzer, 1994). Both the clinical effectiveness and the low risk of extrapyramidal side effects of clozapine have been related to its preferential activity on D4 receptors which, have been hypothesized to be involved not only in the pathophysiology of different psychotic disorders, but also of Parkinson’s disease, mood disorders and hypercynetic syndrome (Tarazi and Baldessarini, 1999; Tarazi et al., 2004). However, data on D4 receptors in these conditions are few and indirect (Davis et al., 1991; MacQueen et al., 2003; Kempf et al., 2005; Maletic et al., 2007). In fact, D2 and D4 receptor had been evaluated by different authors in the prefrontal cortex of schizophrenic patients through the messenger ribonucleic acid (mRNA) levels, and
* Corresponding author. Tel.: +39 050 835412; fax: +39 050 21581. E-mail address: [email protected] (D. Marazziti). 0197-0186/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2009.06.005
the ensuing findings were controversial, as both increased or similar levels of expression were reported, in comparison with those of healthy subjects (Mulcrone and Kerwin, 1996; Roberts et al., 1996; Stefanis et al., 1998). Others employed an indirect binding assay, while subtracting the binding of [3H]-raclopride, which labels only the D2 and D3 receptors, from that of [3H]-YM09151-2, which labels all D2-like receptors, while reporting a sixfold increase of D4 receptors density in the striatum of schizophrenic patients (Seeman et al., 1993a). However, the density of D2-like receptors, evaluated by means of [11C]methylspiperone, resulted higher in schizophrenic than in healthy subjects, but similar in the two groups when [11C]raclopride was employed (Hagberg et al., 1998). Given that methylspiperone labels all D2-like receptors, these results seemed to support the presence of D4 receptors in the striatum. The interpretation of these data is, however, complicated by the evidence that [11C]-methylspiperone binds only to the D2-like receptors in monomeric configuration, at variance with raclopride that binds to both monomeric and oligomeric configurations, and [3H]-YM-09151-2 labels also the sigma and serotonin receptors of type 1a and 2a (5HT1A and 5HT2A) (Helmeste et al., 1996, 1997; Ujike et al., 1996; Tang et al., 1997). These controversies may also be related to the fact that little is known on the distribution of D2-like receptors, and particularly of D4 ones, in the human brain in normal conditions.
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D4 receptors have been described at high density in the prefrontal cortex by means of a binding method employing [3H]NGD 94–1 (Primus et al., 1997). However, in a recent study exploring D4 receptors in human prefrontal cortex by means of the binding of [3H]-YM-09151-2, their density resulted inconsistent (Marazziti et al., 2007). Therefore, given the paucity of available information, the aim of the present study, which can be considered an extension of the previous, was to explore and characterize the distribution and density of D4 receptors in the prefrontal cortex, striatum and hippocampus of human brain postmortem, by comparing and/or subtracting the values of binding parameters obtained with [3H]-YM-09151-2 and different compounds to define the non-specific binding. As a consequence, the presence of sigma sites in human prefrontal cortex and striatum was evaluated as well. 2. Experimental procedure 2.1. Subjects Prefrontal cortex, striatum and hippocampus samples were taken postmortem, during autoptic sessions, from seven subjects (four men and three women; age, mean SD: 61 5 years). They were immediately frozen in dry ice and rapidly stored to 808. Autolysis time (i.e., the time between death and freezing the samples) ranged between 7 h and 25 h, an interval which does not interfere with binding assays, as demonstrated in preliminary experiments (data not shown). All subjects had died for causes not involving primarily the brain (four from myocardial infarction, two from pulmonary embolism and one from respiratory failure), had not suffered from any psychiatric or neurological disorders and were not administered psychotropic drugs, as recorded by their medical charts. The study was approved by the Ethics Committee at Pisa University. 2.2. Preparation of brain tissue homogenates Brain tissue samples were defrosted and separated by white substance. Tissues were re-suspended in D4 buffer (120 mM NaCl, 1.5 mM CaCl2, 4 mM MgCl2, EDTA 1 mM, pH 7.4) to yield 4 mg original wet weight per ml, according to the method of Seeman et al. (1993a,b), and then homogenized with an Ultraturrax in D4 buffer. The homogenates were not washed and centrifuged, because a consistent loss of receptors ranging between 15% and 60% may occur during these steps. 2.3. Binding of [3H]-YM-09151-2 Five hundred microliters of cortex, striatum and hippocampus homogenates were incubated with 10 increasing concentration of [3H]-YM-09151-2 (PerkinElmer, Milan, Italy, specific activity: 85.5 Ci/mmol) ranging between 0.015 nM and 10 nM, at a final volume of 1.5 ml. The non-specific binding was carried out by using L-745,870 30 mM and/or sulpiride 10 mM (Helmeste et al., 1996, 1997; Seeman et al., 1993; Tang et al., 1997; Tarazi et al., 1998). Given that [3H]-YM-09151-2 may bind to sigma receptors (Ujike et al., 1996), 1-phenyl-2-propylaminopentane (PPAP) 100 nM was used to block them in some assays. To determine the amount of sigma sites bound by [3H]-YM-09151-2, we performed several binding assays with increasing radioligand concentrations (0.01–10 nM) and PPAP 500 nM to define the non-specific binding. Furthermore, to evaluate the different potency of [3H]-YM09151-2 binding to D2/D3 and D4 receptors, we carried out some assays with this ligand (concentration range: 0.015–10 nM) and raclopride 300 nM or L-745,870 300 nM. In these cases sulpiride 10 mM was used in these cases to define the nonspecific binding and PPAP 100 nM to block sigma receptors. The incubation with [3H]-YM-09151-2 was performed for 2 h at 22 8C. The separation of bound and free ligand was carried out by vacuum filtration through GF/C fiber filters (Whatman, UK), which were pre-soaked in polyethyleneimine (2%) to minimize the non-specific binding to filters. Radioactivity was counted by a liquid scintillation counter (Packard LS-1600). For the pharmacological characterization of the binding, the homogenates were incubated with increasing concentrations of L-745,870, raclopride, PPAP and 0.5 nM of the radioligand. 2.4. Data analysis and statistics Equilibrium-saturation binding data, the maximum binding capacity (Bmax, fmol/mg tissue) and the dissociation constant (Kd, nM) were analyzed by means of iterative curve-fitting computer programmes EBDA (McPherson, 1985). The potency of the different compounds in displacing the [3H]-YM-09151-2 binding was expressed as inhibition constant (Ki) values, calculated from the IC50 (concentration of drug causing 50% inhibition of the specific binding), using the Cheng and Prusoff equation (1973). Each determination was performed in triplicate, but not in all areas, for the lack of sufficient brain tissue.
Fig. 1. Example of Scatchard analysis of the prefrontal cortex.
3
H-YM-09151-2 binding to the
3. Results 3.1. [3H]-YM-09151-2 binding to the prefrontal cortex and striatum with L-745,870 to define the non-specific binding The Scatchard analysis of the [3H]-YM-09151-2 binding to the prefrontal cortex showed the presence of high and low affinity binding sites (Fig. 1). The Bmax (mean SD, fmol/mg tissue) and Kd (mean SD, nM) values of the two sites were, respectively, 12 5 and 0.091 0.0099, and 710 241 and 6.51 1.42 (Table 1). The pharmacological competition studies with L-745,870 and raclopride did not lead to the expected diphasic curves, but rather to a monophasic curve with high Ki values (236 33 nM for L-745,870 and 1590 170 nM for raclopride): this effect might be attributed to the interference of receptors other than the D2-like ones. The displacement curve of PPAP was also monophasic, with a very low Ki (10 3 nM). The situation in the striatum was similar, as high and low affinity binding sites were detected in this area (the Bmax and Kd values of both sites at this level are reported in Table 1). 3.2. [3H]-YM-09151-2 binding to the prefrontal cortex and striatum with sulpiride to define the non-specific binding and PPAP to block sigma receptors In this case, in both areas, the Scatchard analysis revealed that the binding was specific and saturable, but only a single population of binding sites was detected (Fig. 2, panel a). The Bmax was 9.4 3 and the Kd 0.21 0.08 in the first area, and 97 15 and 0.07 0.005 in the striatum (Table 2A). The pharmacological competition studies with raclopride and L-745,870 led to no curve in the prefrontal cortex, probably for the low density of DA receptors in this area. On the contrary, in the striatum a one phase-displacement curve was obtained with both L-745,870 (Ki = 925 140 nM) and raclopride (Ki = 11.4 3 nM) (Fig. 3, panel a and b). Table 1 Bmax (mean SD, fmol/mg tissue) and Kd (mean SD, nM) values of the 3H-YM09151-2 binding to the prefrontal cortex and striatum with L-745,870 to define the non-specific binding. Bmax
Prefrontal cortex Striatum
Kd
Hi
Lo
Hi
Lo
12 5 103 21
710 241 645 30
0.091 0.009 0.06 0.008
6.51 1.42 7.11 1.12
Hi: high-affinity site; Lo: low affinity site.
D. Marazziti et al. / Neurochemistry International 55 (2009) 643–647 Table 2A Bmax (mean SD, fmol/mg tissue) and Kd (mean SD, nM) values of the 3H-YM09151-2 binding to the prefrontal cortex and striatum with sulpiride to define the nonspecific binding and PPAP to block sigma receptors.
Prefrontal cortex Striatum
Bmax
Kd
9.4 3 97 15
0.21 0.08 0.07 0.005
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Table 3 Bmax (mean SD, fmol/mg tissue) and Kd (mean SD, nM) values of the 3H-YM09151-2 binding to the prefrontal cortex, striatum and hippocampus with raclopride to block D2/D3 receptors, sulpiride to define the non-specific binding and PPAP to block sigma receptors.
Prefrontal cortex Striatum Hippocampus
Bmax
Kd
ND ND 8.9 1.1
ND ND 0.32 0.04
ND: not detectable.
3.4. [3H]-YM-09151-2 binding to the prefrontal cortex, striatum and hippocampus with raclopride to block D2/D3 receptors, sulpiride to define the non-specific binding and PPAP to block sigma receptors In this case, it was not possible to carry out a Scatchard analysis in the prefrontal cortex and striatum. In the hippocampus, the binding was specific and saturable and the Scatchard analysis led to a Bmax of 8.9 1.1 and a Kd of 0.32 0.04 (Table 3). 3.5. [3H]-YM-09151-2 binding to the prefrontal cortex, striatum and hippocampus with L-745,870 to block D4 receptors, sulpiride to define the non-specific binding and PPAP to block sigma receptors In the prefrontal cortex and in the striatum the Scatchard analysis revealed that the binding was specific and saturable, and it showed the presence of a single population of binding sites. The Bmax values in the prefrontal cortex and the striatum were, respectively, 6.8 0.9 and 66 8, and the Kd values 0.31 0.02 and
Fig. 2. Examples of saturation curves and Scatchard analysis in the prefrontal cortex (panel a) and striatum (panel b) with blocked sigma sites.
3.3. [3H]-YM-09151-2 binding to the prefrontal cortex and striatum with PPAP to define the non-specific binding The Scatchard analysis revealed that the binding was specific and saturable, as well as it showed the presence of a single population of binding sites (Fig. 2, panel b). In the prefrontal cortex, the Bmax was 795 82 and the Kd 4.42 1.32; in the striatum, the Bmax was 666 78 and the Kd 6.33 1.54 (Table 2B). Table 2B Bmax (mean SD, fmol/mg tissue) and Kd (mean SD, nM) values of the 3H-YM09151-2 binding to the prefrontal cortex and striatum with PPAP to define the nonspecific binding.
Prefrontal cortex Striatum
Bmax
Kd
795 82 666 78
4.42 1.32 6.33 1.54
Fig. 3. Displacement curve with L-745,870 (panel a) and raclopride (panel b) in the striatum.
D. Marazziti et al. / Neurochemistry International 55 (2009) 643–647
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Table 4 Bmax (mean SD, fmol/mg tissue) and Kd (mean SD, nM) values of the 3H-YM09151-2 to the prefrontal cortex, striatum and hippocampus with L-745,870 to block D4 receptors, sulpiride to define the non-specific binding and PPAP to block sigma receptors.
Prefrontal cortex Striatum Hippocampus
Bmax
Kd
6.8 0.9 66 8 ND
0.31 0.02 0.08 0.007 ND
ND: not detectable.
0.08 0.007. On the contrary, it was not possible to perform a Scatchard analysis in the hippocampus (Table 4). 4. Discussion Given the potential relevance of D4 receptors in the pathophysiology of different neuropsychiatric disorders and the few information available (Helmeste and Tang, 2000; Muglia et al., 2002; Oak et al., 2000; Tarazi and Baldessarini, 1999; Tarazi et al., 2004; Engelborghs et al., 2008), we explored their distribution in some human brain areas, in particular prefrontal cortex, striatum and hippocampus obtained postmortem by means of binding techniques. The present study may be considered an extension of a previously carried out by our group in human prefrontal cortex in a smaller sample (Marazziti et al., 2007). Given the lack of specific radioligands for labelling D4 receptors, we compared and subtracted a series of binding assays carried out with [3H]-YM09151-2, which is a D2-like receptor antagonist, and L-745,870, raclopride, sulpiride and PPAP as displacers. Although other compounds, such as [3H]-NGD-94-1 or [3H]-PNU-101958 have been proposed as more selective for D4 receptors, it should noted that the selectivity observed in preliminary studies on cloned receptors may not match with that observed in vivo: for this reason, perhaps, the few data obtained with them have been disappointing (Primus et al., 1997; Lahti et al., 1998; De La Garza and Madras, 2000). Our results showed that we could detect D4 receptors only in the hippocampus, and not in the striatum and prefrontal cortex where the D2/D3 subtypes resulted to be prevalent. Our findings in the hippocampus are consistent with those of one study employing monoclonal antibody for D4 receptors (Lanau et al., 1997), but at variance with others that evaluated the mRNAs for D2, D3 or D4, receptors and reported a differential distribution of each subtype in distinct hippocampal areas (Primus et al., 1997; Khan et al., 1998). The majority of data on D4 receptors in prefrontal cortex and striatum have been gathered by means of expression studies and have shown a relevant presence of the mRNA for D4 receptors in both areas (Matsumoto et al., 1996; Meador-Woodruff et al., 1996; De La Garza and Madras, 2000), in disagreement with our observations. This is not surprising, as the mRNA presence does not necessarily correspond to the receptor expression or density, because different mechanisms, such as re-arrangement and dimerization (or etherodimerization), might change the pharmacological features of the monomeric configuration of the receptor. Interestingly, no expression of D4 receptors has been also reported in the prefrontal cortex and striatum (Mulcrone and Kerwin, 1996; Stefanis et al., 1998). However, further studies are needed to investigate the occurrence of these re-arrangement mechanism for D4 receptors, as up to now the presence of an oligomeric configuration has been demonstrated only for the D2 subtype (Lee et al., 2000; Franekova et al., 2008). As far as the pharmacological competition studies are concerned, we obtained one phase-displacement curve, while
indicating that [3H]-YM-09151-2 could not distinguish between the D2/D3 and D4 receptors. The Ki values were different from those reported in the literature, while corresponding to an intermediate level of affinity, however, the different studies are not easily comparable, as most of them have been carried out in animals. In the binding assay employing L-745,870 to define the non-specific binding, the Scatchard analysis resulted in a curvilinear plot, suggestive of the presence of two different sites. It is possible that the concentration of L-745,870 used (30 mM) might displace not only the D2-like receptors, but also other receptors (Bristow et al., 1997); however, since [3H]-YM-09151-2 binds to sigma receptors with a high affinity, these are the main candidates for explaining the interference with the above-mentioned binding assay (Helmeste et al., 1996, 1997; Tang et al., 1997; Ujike et al., 1996). When we added the 1-phenyl-2-propylaminopentane (PPAP), a selective drug to block sigma receptors, in the prefrontal cortex and in the striatum, we obtained a monophasic Scatchard plot with a low Ki, corresponding to a high-affinity site: this might hence suggest that sigma receptors labelled by [3H]-YM-09151-2 were significantly higher than D2-like receptors. This observation was confirmed by the high [3H]-YM-09151-2 binding performed by using PPAP to define the non-specific binding which can be also attributed to sigma receptors (Florijn et al., 1997; Assie et al., 2005). These data are consistent with those resulting from the assay in which the non-specific binding was defined by L-745,870: in this case, we obtained a non-straight Scatchard curve that could be divided in two populations of binding sites. The binding sites with the lower affinity probably correspond to sigma receptors whose interference with the [3H]-YM-09151-2 binding was directly related to their density and to the displacer used to define the non-specific binding, when were not blocked. According to the present findings, therefore, previous data on the [3H]-YM-09151-2 binding to D2like receptors should be reconsidered critically. Regarding the D2like component, the competition studies did not permit to evaluate separately the D2/D3 and D4 receptors; in agreement with previous studies (Tarazi et al., 1998), we carried out the binding while masking the D2/D3 component by adding raclopride 300 nM as displacer. It was not possible to evaluate the binding in the prefrontal cortex or striatum, while in the hippocampus a very low density was found which probably corresponds to D4 receptors. Similarly, the experiments performed to evaluate the D2/D3 receptors, while blocking the D4 ones with L-745,870, revealed that the binding occurred only in the prefrontal cortex and striatum. In conclusion, our findings suggest that D4 receptors are mainly distributed in the hippocampus, while in the striatum or prefrontal cortex, the high density of sigma receptors interfere significantly with their labelling. Although the detection of D4 receptors in the human brain with the available methodologies appears still difficult, our study supports the notion that the [3H]-YM-091512 may label the D4 receptors, provided that D2, D3 and sigma receptors are appropriately blocked. References Assie, M.B., Consul-Denjean, N., Koek, W., Newman-Tancredi, A., 2005. Differential in vivo inhibition of [3H]nemonapride binding by atypical antipsychotics in rat striatum, olfactory lobes, and frontal cortex. Pharmacology 75, 63–68. Bristow, L.J., Patel, S., Ragan, C.I., Ridgill, M., Saywell, K.L., Tricklebank, M.D., 1997. L745,870, a subtype selective dopamine D4 receptor antagonist, does not exhibit a neuroleptic-like profile in rodent behavioral tests. J. Pharmacol. Exp. Ther. 283 (December (3)), 1256–1263. Cheng, Y., Prusoff, W.H., 1973. Relationship between inhibition constant (Ki) and concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic-reaction. Biochem. Pharmacol. 22, 3099–3108. Civelli, O., Bunzow, J.R., Grandy, D.K., Zhou, Q.Y., Van Tol, H.H., 1991. Molecular biology of the dopamine receptors. Eur. J. Pharmacol. 207, 277–286. Davis, K.L., Kahn, R.S., Ko, G., Davidson, M., 1991. Dopamine in schizophrenia: a review and reconceptualization. Am. J. Psychiatry 148, 1474–1486.
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