Long‐term exposure to the new nicotinic antagonist 1,2‐bisN‐cytisinylethane upregulates nicotinic receptor subtypes of SH‐SY5Y human neuroblastoma cells

Abstract

1 Nicotinic drug treatment can affect the expression of neuronal nicotinic acetylcholine receptors (nAChR) both in vivo and in vitro through molecular mechanisms not fully understood. The present study investigated the effect of the novel cytisine dimer 1,2-bisN-cytisinylethane (CC4) on nAChR natively expressed by SH-SY5Y neuroblastoma cells in culture. 2 CC4 lacked the agonist properties of cytisine and was a potent antagonist (IC 50 ¼ 220 nM) on nAChRs. Chronic treatment of SH-SY5Y cells with 1 mM CC4 for 48 h increased the expression of 3 H-epibatidine ( 3 H-Epi; 3-4-fold) or 125 I-a-bungarotoxin ( 125 I-aBgtx; 1.2-fold) sensitive receptors present on the cell membrane and in the intracellular pool. Comparable data were obtained with nicotine or cytisine, but not with carbamylcholine, d-tubocurarine, di-hydro-b-erythroidine or hexametonium. 3 Immunoprecipitation and immunopurification studies showed that the increase in 3 H-Epi-binding receptors was due to the enhanced expression of a3b2 and a3b2b4 subtypes without changes in subunit mRNA transcription or receptor half-life. The upregulation was not dependent on agonist/antagonist properties of the drugs, and did not concern muscarinic or serotonin receptors. 4 Whole-cell patch clamp analysis of CC4-treated cells demonstrated larger nicotine-evoked inward currents with augmented sensitivity to the blockers a-conotoxin MII or methyllycaconitine. 5 In conclusion, chronic treatment with CC4 increased the number of nAChRs containing b2 and a7 subunits on the plasma membrane, where they were functionally active. In the case of b2-containing receptors, we propose that CC4, by binding to intracellular receptors, triggered a conformational reorganisation of intracellular subunits that stimulated preferential assembly and membrane-directed trafficking of b2-containing receptor subtypes.

A Comparative Study of the Effects of the Intravenous Self-Administration or Subcutaneous Minipump Infusion of Nicotine on the Expression of Brain Neuronal Nicotinic Receptor Subtypes

“…In line with previous experiments (Gotti et al, 2005b), we found that approximately 10 to 15% of SC nAChRs contained the ␣3␤2* subtype, which was shown to be strongly up-regulated by nicotine in vitro (Sallette et al, 2004;Kuryatov et al, 2005;Riganti et al, 2005). In the present in vivo experiment, however, this subtype was not up-regulated in rats self-administering nicotine or treated with nicotine via MP.…”

α9‐ and α7‐containing receptors mediate the pro‐proliferative effects of nicotine in the A549 adenocarcinoma cell line

“…In order to confirm the expression of nAChR subunits in the NSCLC cell lines at protein level, we carried out immunoprecipitation and Western blotting studies, using Abs against various nAChR subunits whose specificity was checked and is shown in Supporting Information Figure S1. The positive control was the SH‐SY5Y neuroblastoma cell line, which has high levels of receptors containing the α3, α5, α7, β2 and β4 subunits (Riganti et al, ).…”

The effects of lobeline on α4β2* nicotinic acetylcholine receptor binding and uptake of [18F]nifene in rats

“…Lobeline has also been found to be roughly 10 times less potent at the α7* nAChR compared to nicotine and cytisine (Houlihan et al, 2001; Miller et al, 2004), giving it advantages over nicotine and cytisine in selectivity for the α4β2 subtype. Additionally, chronic exposure of both nicotine and cytisine has been found to upregulate nAChR expression, while this effect is not observed with lobeline (Bhat et al, 1991; Riganti et al 2005). As a result, measurement of binding levels with PET experiments in subjects with long-term cytisine or nicotine exposure would be increased relative to subjects naïve to these drugs.…”