Methodology of 3H-spiperone binding to lymphocytes

Bondy, Brigitta; Ackenheil, Manfred; Engel, Rolf R.

doi:10.1016/0022-3956(90)90026-m

Cited by 19 publications

(17 citation statements)

References 22 publications

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“…The total number of PBMC isolated from 60 ml of six specimens of buffy coat varied from 350×10 6 to 800×10 6 cells. All techniques utilizing blood samples met the criteria recommended for PBMC isolation to conserve the integrity of the cell membrane (Bondy et al, 1990), and did not exceed 2 hr. Preparation of PBMC from the buffy coat was more time-consuming (2.5–3 hr) due to the larger sample volume.…”

Section: Resultsmentioning

confidence: 99%

“…Using a single ligand concentration, we evaluated binding of [ 3 H]SCH23390 to PBMC under assay conditions previously described in other studies (Ricci and Amenta, 1994; Bondy et al, 1990). The methods of separation of the unbound ligand (centrifugation or filtration procedures), the buffer composition, and the temperature and duration of the binding steps were among the tested conditions.…”

Section: Resultsmentioning

confidence: 99%

“…The blood was diluted two-fold with NaCl physiological solution (Ricci and Amenta, 1994), or HBSS/HEPES buffer (136 mM NaCl, 4.16 mM NaHCO 3 , 0.34 mM Na 2 HPO 4 , 5.36 mM KCl, 0.44 mM KH 2 PO 4 , 1.25 mM CaCl 2 , 0.81 mM MgSO 4 , 22.3 mM HEPES, 1 mM EDTA, pH 7.4) (Bondy et al, 1990), or PBS (phosphate buffered saline, pH 7.4). The diluted blood (15 ml) was layered on Histopaque-1077 reagent (15 ml) in 50-ml polypropylene centrifuge tubes and centrifuged at 22–23°C for 40 min at 400×g.…”

Section: Methodsmentioning

confidence: 99%

“…Human peripheral blood lymphocytes have attracted attention as a convenient candidate for evaluation of the functional properties of DR. Molecular biological studies have detected D3, D4 and D5 receptors mRNA in human lymphocytes (Takahashi et al, 1992; Nagai et al, 1993; Bondy et al, 1996). However, DR detection in lymphocytes by the radioligand binding method (Bondy et al, 1990) produced controversial results (Fleminger et al, 1982; Maloteaux et al, 1982, 1983; Rao et al, 1990; Vile and Strange, 1995, 1996;). Nevertheless, one group has consistently reported quantitative detection of D3, D4 and D5, but not D1 or D2 receptors on human lymphocytes (Ricci and Amenta, 1994; Ricci et al, 1995, 1997–1999; Amenta et al, 1999).…”

Section: Introductionmentioning

confidence: 99%

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

Kirillova

Hrutkay

Shurin

et al. 2008

Journal of Neuroscience Methods

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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 (RBA) 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 [3H]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 Kd value consistent with that reported for D5 dopamine receptors in human lymphocytes, whereas the other Kd 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.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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

Kirillova

Hrutkay

Shurin

et al. 2008

Journal of Neuroscience Methods

View full text Add to dashboard Cite

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“…The exact methodology of the assay has been published in detail previously (Bondy et al, 1990a). Briefly, 50ml venous blood was drawn using 200mg EDTA as anticoagulant.…”

Section: Binding Of ~H-spiperonementioning

confidence: 99%

3H-spiperone binding to lymphocytes fails in the differential diagnosis of de novo Parkinson syndromes

Arnold

Bondy

Bandmann

et al. 1993

J Neural Transm Gen Sect

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In order to investigate the diagnostic value of 3H-spiperone binding capacity to lymphocytes in the differential diagnosis of de novo Parkinson's disease (idiopathic Parkinson syndrome, PD), we performed a double blind prospective study of spiperone binding capacity of 123 patients and 23 healthy control persons, belonging to different diagnostic groups (PD, Parkinsonian syndrome due to vascular lesions, multiple system atrophy [MSA], essential tremor). Diagnoses were based on medical history, clinical examination, CT or MRI scan, acute response to dopamimetic drugs, one year follow up, and long term response to L-DOPA treatment. Spiperone binding was assayed using ten different concentrations (0.03-3 nmol) in absence or presence of 1 mumol (+)-butaclamol to determine nonspecific binding. There was no significant difference in spiperone binding between patients with PD not treated with L-DOPA, and patients with other basal ganglia disorders including parkinsonian syndrome due to vascular lesions, multiple system atrophy, or progressive supranuclear palsy, and age matched controls. Binding was significantly higher in parkinsonian patients with PD treated with L-DOPA and patients with essential tremor. It is concluded that at present 3H-spiperone binding gives no further information in the differential diagnosis of de novo Parkinson's disease.

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