Investigation of nicotine binding to THP-1 cells: evidence for a non-cholinergic binding site

Nicotine is known to modulate immune function, but reports have produced conflicting evidence as to whether nicotinic acetylcholine (nACh) receptors are responsible for these effects. This study was designed to examine the identity of nicotine-binding sites on immune cells using a human leukaemic monocytic cell line, THP-1, that is known to have functions that are modulated by nicotine. Binding studies were performed on THP-1 whole cells using [3H]nicotine as a probe to analyse any possible nicotine-binding sites on these cells. Saturation analysis of THP-1 cells revealed the presence of 2 distinct binding sites; one with a Kd1 of 3.5 ± 2.1 × 10−9 M and a Bmax1 of 4100 ± 560 sites/cell (designated the high-affinity site) and the other with a Kd2 of 27 ± 9.2 × 10−9 M and a Bmax2 of 11,600 ± 630 sites/cell (low-affinity site). Competition analysis revealed that one site had an affinity to a range of cholinergic ligands including epibatidine and cytisine. When saturation analysis of [3H](−)-nicotine to THP-1 cells was performed in the presence of 1 × 10−6 M epibatidine, only one binding site was detected. Comparisons of Kd and Bmax values showed that the high-affinity site was not occluded by epibatidine. No drugs tested displayed any affinity for the high-affinity site except the two enantiomers of nicotine. The high-affinity site was shown to be stereoselective for the (+)-enantiomer of nicotine as shown by Ki values produced by competition analysis in the presence of 1 × 10−6 M epibatidine. These values were 5.7 ± 0.32 × 10−11 M and 1.9 ± 4.9 × 10−9 M for (+)-nicotine and (−)-nicotine, respectively. This study presents evidence for a possible non-cholinergic binding site that may play a role in the mechanism of immunomodulation by nicotine.