Characterisation of nicotine receptors on human peripheral blood mononuclear cells (PBMC)
Aim and objective: The aim of the work was to characterise the nAChRs on human PBMC. Method: PBMC were isolated from human blood buffy coats provided by the blood transfusion service and were used for radioligand binding studies with [3H]-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. Results: Binding studies suggested the presence of one binding site for (-)- nicotine on human peripheral blood lymphocytes. Competition studies showed that only (-)- nicotine, epibatidine and α-bungarotoxin, displaced radiolabelled nicotine from cells. RT-PCR studies demonstrated mRNA for α4, α5, α7, β1 and β2 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 α4, α7, β1, and β2 mRNAs subunits of nAChRs, varied between samples. Western blot analysis showed that protein for α4, α5, and α7 and β2 nAChR subunits was expressed in most, but not all of the PBMC samples tested but some of the bands obtained were faint. Conclusion: The results obtained suggest that human PBMC contain nAChRs containing α4β2, α4β2α5, and/or α7 subunits.