Neuronal nicotinic acetylcholine receptors: functional properties in lung cancer cell lines and response to tobacco-specific nitrosamines

The neuronal nicotinic acetylcholine receptors are ligand-gated ion channels permeable to cations. Several α and β subunits can associate to form homo- or heteropentamers, which exhibit distinct kinetics, ion permeability and pharmacological properties. The nAChRs are also expressed in non-neuronal tissues and recent evidence indicates that they can regulate cell proliferation, apoptosis and angiogenesis in a variety of neoplastic cells, including small (SCLC) and non-small (NSCLC) cell lung cancer. These observations are suggestive because smoking is an established risk factor for cancer, especially in the lung. We studied nAChRs in SCLC (U2020) and NSCLC (A549) cell lines. These were voltage-clamped in the whole-cell configuration of the patch-clamp methods, at room temperature. During the experiments, cells were maintained in physiological saline solutions. In both cell lines, Vrest was usually between -17 and -19 mV. At -60 mV, application of 100 μM nicotine consistently elicited inward desensitizing currents in both cell types, although the average current density was generally higher in NSCLC. In SCLC and NSCLC the nicotinic currents was significantly blocked by 10 nM methyllycaconitine and 1 μM dihydro-β-erythroidine. Several tobacco-derived carcinogens structurally similar to nicotine, such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) bind to nAChRs with high affinity. Moreover, modulation of nAChRs is thought to be implicated in cerebral tobacco addiction. However, very little is known about the mechanism of action of these drugs on nAChRs. We studied the effect of NNK on human α4β2 nAChRs stably expressed in human embryonic kidney cells, with patch-clamp methods. Our results suggest that NNK is a partial agonist with high affinity for α4β2 nAChRs. Accordingly, it produced nAChR potentiation in the presence of low concentrations of the full agonists (with a threshold around 0.1 μM), and nAChR competitive inhibition at higher concentrations of the full agonist. In keeping with a competitive action on the ligand binding site, the effect of NNK showed negligible voltage-dependence. We conclude that NNK can stimulate or inhibit heteromeric nAChRs, depending on the concomitant concentration of the full agonists.