Dopamine receptors in human lymphocytes: radioligand binding and quantitative RT-PCR assays

Abstract

Analysis of dopamine receptors (DR) in lymphocytes of the human peripheral blood mononuclear cell (PBMC) fraction is an attractive tool for evaluation of functional properties of dopaminergic function underlying variation in complex psychological/psychopathological traits. Receptor binding assays (RBAs) with selective radioligands, which are widely used in CNS studies, have not produced consistent results when applied to isolated PBMC. We tested the assay conditions that could be essential for detection of DR in human PBMC and their membrane preparations. Using [(3)H]SCH23390, a dopamine D1-like receptor antagonist, we demonstrated the presence of two binding sites in PBMC-derived membrane fraction. One of them is characterized by the K(d) value consistent with that reported for D5 dopamine receptors in human lymphocytes, whereas the other K(d) value possibly corresponds to serotonin receptor(s). Although D5 receptor binding sites in PBMC membranes could be characterized by binding assays, the low protein expression and the large volume of blood needed for membrane preparation render the binding method impracticable for individual phenotyping. In contrast, real-time RT-PCR may be used for this purpose, contingent on the relationship between DR expression in the brain and in lymphocytes. The expression of the DRD2-DRD5 genes, as detected by this method, varied widely among samples, whereas the DRD1 expression was not detected. The expression levels were comparable with those in the brain for DRD3 and DRD4, and were significantly lower for DRD2 and DRD5.

Figure 1

Binding of [³H]SCH 23390 with isolated PBMC. Cells (1.5×10⁶) were incubated with 0.8 nM of [³H]SCH 23390 for 60 min at 23°C in 170 mM Tris-HCl buffer with 1 µM (+)-butaclamol (nonspecific binding, NB) or without it (total binding, TB). Binding was terminated by centrifugation and aspiration of unbound ligand. Specific binding (SB) was determined as a difference between TB and NB values. Data represent mean±SEM values of triplicate measurements. Binding is expressed in absolute units (disintegrations per minute, dpm; the left ordinate), and as percent of total binding in intact cells. Grey bars – intact cells; black bars – cells preincubated in the boiling water bath for 8 min prior binding assay.

Figure 2

Dissociation of [³H]SCH 23390 from PBMC during washing procedure. Binding was conducted in five parallel sets of reaction tubes, each in triplicate. Reaction mixtures with 1.5×10⁶ cells (TB) or without cells (background, BKG) were incubated in the presence of 0.8 nM of [³H]SCH 23390 for 60 min at 23°C in 170 mM Tris-HCl buffer (TB). Binding was terminated by centrifugation and aspiration of unbound ligand. One, two or three washing steps were made by addition of ice-cold assay buffer to the cell pellets, which was removed by centrifugation and aspiration. The scales are the same as in Figure 1. Data represent mean± SEM values of triplicate measurement. BKG is the background radioactivity absorbed by the Eppendorf microfuge tubes used for assay.

Figure 3

Binding of [³H]SCH 23390 with PBMC in the assay buffers of different composition. Cells (3.8×10⁶) were incubated with 0.8 nM of [³H]SCH 23390 for 60 min at 23°C in Buffer 1 (170 mM Tris-HCl), Buffer 2 (50 mM Tris-HCL), or Buffer 3 (HBSS/HEPES). Binding was terminated by centrifugation and aspiration of unbound ligand. NB was determined in the presence of 1 µM (+)-butaclamol. A. Binding data are presented in absolute values (dpm) and relative (%) to the highest TB values in Buffer 3. B. Ratios of binding parameters determined in buffers 1, 2 and 3: SB/TB is fraction of the ligand bound to cells specifically, and TB/total ligand is the fraction of the ligand associated with cells at the end of incubation with 0.8 nM of the ligand. Data represent mean± SEM values of triplicate measurement.

Figure 3

Binding of [³H]SCH 23390 with PBMC in the assay buffers of different composition. Cells (3.8×10⁶) were incubated with 0.8 nM of [³H]SCH 23390 for 60 min at 23°C in Buffer 1 (170 mM Tris-HCl), Buffer 2 (50 mM Tris-HCL), or Buffer 3 (HBSS/HEPES). Binding was terminated by centrifugation and aspiration of unbound ligand. NB was determined in the presence of 1 µM (+)-butaclamol. A. Binding data are presented in absolute values (dpm) and relative (%) to the highest TB values in Buffer 3. B. Ratios of binding parameters determined in buffers 1, 2 and 3: SB/TB is fraction of the ligand bound to cells specifically, and TB/total ligand is the fraction of the ligand associated with cells at the end of incubation with 0.8 nM of the ligand. Data represent mean± SEM values of triplicate measurement.

Figure 4

Time- and temperature-dependent dynamics of the specific [³H]SCH23390 binding with PBMC. Isolated cells were incubated in three parallel sets of tubes (1.6×10⁶ cells/tube) with HBSS/HEPES buffer (Buffer 3) containing 0.6 nM of [³H]SCH23390. Binding was terminated by centrifugation followed by ligand aspiration. Specific binding expressed in absolute values (dpm) was determined from TB and NB in the presence of 1 µM (+)-butaclamol. Data represent mean± SEM values of triplicate measurements.

Figure 5

Concentration-dependent association of [³H]SCH23390 with PBMC. Cells were incubated in the presence of increasing concentrations of [³H]SCH23390 in HBSS/HEPES buffer at 37°C for 60 minutes with (NB) or without (TB) 1 µM (+)-butaclamol (the concentration scale is truncated to depict binding at low ligand concentrations). Binding was terminated by centrifugation followed by ligand aspiration. Data are expressed as the amount of ligand (fmol) bound to 2.4×10⁶ cells in reaction tubes. Data represent mean± SEM values of triplicate measurement.

Figure 6

Time- and temperature-dependent dynamics of specific [³H]SCH23390 binding with PBMC membranes. Membranes (135 µg) were incubated with 0.6 nM of [³H]SCH 23390. Reaction was terminated by vacuum filtration. Specific binding was calculated by subtraction of non-specific binding in the presence of 10 µM (+)-butaclamol from total binding‥ Data represent mean values (± SEM) of triplicate measurements.

Figure 7

Concentration-dependent binding of [³H]SCH 23390 with PBMC membranes. Membranes (260 µg) were incubated in the presence of increasing concentrations of [³H]SCH23390 for 30 minutes at 23°C (the concentration scale is truncated to depict binding at low ligand concentrations). Reaction was terminated by vacuum filtration. Non-specific binding was determined in the presence of 10 µM (+)-butaclamol. Data are expressed as the amount of ligand (fmol) bound to 260 µg of membrane protein. Data represent mean values (± SEM) of triplicate measurements.