Characterisation of nicotine receptors on human peripheral blood mononuclear cells (PBMC)

The aim of the work was to characterise the nAChRs on human PBMC. PBMC were isolated from human blood buffy coats provided by the blood transfusion service and were used for radioligand binding studies with [H-3]-nicotine. RT-PCR experiments were used to determine nAChR subunit expression while immunoblotting experiments were used to confirm that nAChR subunits identified by RT-PCR were translated into protein. Binding studies suggested the presence of one binding site for (-)- nicotine on human peripheral blood lymphocytes. Competition studies showed that only (-)- nicotine, epibatidine and alpha-bungarotoxin, displaced radiolabelled nicotine from cells. RT-PCR studies demonstrated mRNA for alpha 4, alpha 5, alpha 7, beta 1 and beta 2 nAChRs subunits in PBMC. Expression of mRNA for the a5 subunit of nAChR was observed in all lymphocyte samples tested. In contrast, the expression pattern of mRNAs for alpha 4, alpha 7, beta 1, and beta 2 mRNAs subunits of nAChRs, varied between samples. Western blot analysis showed that protein for alpha 4, alpha 5, and alpha 7 and beta 2 nAChR subunits was expressed in most, but not all of the PBMC samples tested but some of the bands obtained were faint. The results obtained suggest that human PBMC contain nAChRs containing alpha 4 beta 2, alpha 4 beta 2 alpha 5, and/or alpha 7 subunits.