Dopamine D2-like receptors on human peripheral blood lymphocytes: a radioligand binding assay and immunocytochemical study.
1. Peripheral blood lymphocytes express dopamine D1-like and D2-like receptors which were investigated using radioligand binding assay and molecular biology techniques. Analysis of dopamine D2-like receptors expressed by human peripheral blood lymphocytes with radioligand binding assay may offer a rapid technique for assessing receptor changes in disorders characterized by involvement of the dopaminergic system. However, the suitability of radioligand binding assay techniques to measure dopamine D2-like receptors is questioned. 2. In view of the discrepancy between data of dopamine D2-like receptor determination with molecular biology and radioligand binding assay techniques, we have assayed dopamine D2-like receptors expressed by human peripheral blood lymphocytes using as radioligands the dopamine receptor agonist 7-[3H]-hydroxy-N,N-di-n-propyl-2-aminotetraline ([3H]-7-OH-DPAT) and two antagonists ([3H]-spiperone and [3H]-nemonapride). 3. Analysis of saturation curves revealed a concentration-dependent binding of all compounds to human peripheral blood lymphocytes. Dissociation constant (Kd) values averaged between 0.15 and 0.40 nM for different radioligands. The maximum density of binding sites (Bmax) was low, ranging from 4.15 +/- 0.05 fmol/10(6) cells with [3H]-spiperone and 8.66 +/- 0.04 fmol/10(6) cells with [3H]-7-OH-DPAT. 4. Displacement curves of [3H]-7-OH-DPAT, [3H]-spiperone and [3H]-nemonapride binding to human peripheral blood lymphocytes revealed, using radioligand concentrations giving the highest specific:non-specific binding ratio, a pharmacological profile consistent with the labelling of dopamine D2-like receptors. The use of higher radioligand concentrations resulted in a poorly displaceable and characterizable binding. 5. Detection of dopamine D2, D3 and D4 receptor immunoreactivity in cytospin centrifuged peripheral blood lymphocytes revealed dopamine D3 and D4 but not D2 receptor immunostaining. 6. The above findings indicate in agreement with molecular biology studies, that dopamine D2-like receptors expressed by human peripheral blood lymphocytes belong to the D3 and D4 receptor subtypes. These receptors are detectable using either dopamine D2-like receptor agonists and antagonists as radioligands if controlled experimental conditions are followed. The standardisation of immunocytochemical techniques for detecting human peripheral blood lymphocyte dopamine receptors may contribute to clarify their role in lymphocyte function or as a peripheral marker of the status of the dopaminergic system.
Amenta
F
F
Dipartimento di Scienze Farmacologiche e Medicina Sperimentale, Università di Camerino, Italy.
Bronzetti
E
E
Felici
L
L
Ricci
A
A
Tayebati
S K
SK
D001549:Benzamides / Q000494:pharmacology*; D001667:Binding, Competitive; D018491:Dopamine Agonists / Q000494:pharmacology; D018492:Dopamine Antagonists / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007106:Immune Sera; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D008214:Lymphocytes / Q000276:immunology / Q000502:physiology*; D011485:Protein Binding; D011869:Radioligand Assay; D017448:Receptors, Dopamine D2 / Q000502:physiology*; D012995:Solubility; D013134:Spiperone / Q000494:pharmacology*; D013764:Tetrahydronaphthalenes / Q000494:pharmacology*
D001549:Benzamides; D018491:Dopamine Agonists; D018492:Dopamine Antagonists; D007106:Immune Sera; D017448:Receptors, Dopamine D2; D013764:Tetrahydronaphthalenes; D013134:Spiperone; C030265:nemonapride; C031349:7-hydroxy-2-N,N-dipropylaminotetralin