Chronic Nicotine Treatment Up-regulates Human α3β2 but Not α3β4 Acetylcholine Receptors Stably Transfected in Human Embryonic Kidney Cells

Abstract

Human nicotinic acetylcholine receptor (AChR) subtypes ␣3␤2, ␣3␤2␣5, ␣3␤4, and ␣3␤4␣5 were stably expressed in cells derived from the human embryonic kidney cell line 293. ␣3␤4 AChRs were found in prominent 2-m patches on the cell surface, whereas most ␣3␤2 AChRs were more diffusely distributed. The functional properties of the ␣3 AChRs in tsA201 cells were characterized by whole cell patch clamp using both acetylcholine and nicotine as agonists. Nicotine was a partial agonist on ␣3␤4 AChRs and nearly a full agonist on ␣3␤2␣5 AChRs. Chronic exposure of cells expressing ␣3␤2 AChRs or ␣3␤2␣5 AChRs to nicotine or carbamylcholine increased their amount up to 24-fold but had no effect on the amount of ␣3␤4 or ␣3␤4␣5 AChRs, i.e. the up-regulation of ␣3 AChRs depended on the presence of ␤2 but not ␤4 subunits in the AChRs. This was also found to be true of ␣3 AChRs in the human neuroblastoma SH-SY5Y. In the absence of nicotine, ␣3␤2 AChRs were expressed at much lower levels than ␣3␤4 AChRs, but in the presence of nicotine, the amount of ␣3␤2 AChRs exceeded that of ␣3␤4 AChRs. Up-regulation was seen for both total AChRs and surface AChRs. Up-regulated ␣3␤2 AChRs were functional. The nicotinic antagonists curare and dihydro-␤-erythroidine also up-regulated ␣3␤2 AChRs, but only by 3-5-fold. The channel blocker mecamylamine did not cause up-regulation of ␣3␤2 AChRs and inhibited up-regulation by nicotine. Our data suggest that up-regulation of ␣3␤2 AChRs in these lines by nicotine results from both increased subunit assembly and decreased AChR turnover.

Desensitization of neuronal nicotinic receptors

“…2). Likewise, it is probable that desensitization of the lower affinity putative ␣3␤4*-nAChRs, as observed in various cells representative of both the autonomic nervous system and CNS (Higgins and Berg, 1988;Oortigiesen and Vijverberg, 1989;Mathie et al, 1990;Khiroug et al, 1997Khiroug et al, , 1998Boyd, 1987;Ifune and Steinbach, 1993;Lester and Dani, 1995), in addition to systems designed to specifically express ␣3␤4 receptors (Cachelin and Jaggi, 1991;Hsu et al, 1996;Fenster et al, 1997;Wang et al, 1998;Xiao et al, 1998), can also be explained in terms of the two-state model, although due to the slower time course of desensitization for ␤4 subunit-containing receptors (see below), the biphasic nature may only be seen clearly during longer agonist applications (e.g., Lester and Dani, 1995). Concentration-dependent analyses of desensitization of presumed native ␣3␤4* nAChRs have estimated the affinity of the desensitized state for nicotine to be Ϸ20 -300 nM (Higgins and Berg, 1988;Lester and Dani, 1995).…”

“…Following on from the work of Cachelin and Jaggi (1991), several studies have suggested that the ␤ subunit (␤4/␤2) strongly influences the rate of onset of desensitization: receptors containing ␤2 subunits desensitize faster than those containing ␤4 subunits (Hsu et al, 1995;Fenster et al, 1997;Wang et al, 1998;Bohler et al, 2001). The difference is effectively due to an altered balance of the fast and slow desensitization components: Ͼ90% desensitization for ␤4*nAChRs is slow; Ͼ90% desensitization for ␤2*nAChRs is fast ( Fig.…”

SSR180711, a Novel Selective α7 Nicotinic Receptor Partial Agonist: (1) Binding and Functional Profile

“…SH-SY5Y cells were grown in Ham's F12/MEM (v/v) supplemented with 10% fetal bovine serum, 1% antibiotics (penicillin, streptomycin) and 1% non-essential amino acids. Except for ha7-GH 4 C 1 , nicotine (100 mM-1 mM) was added into the culture medium and the cells were incubated for 24-48 h before harvesting in order to upregulate n-AChR expression (Wang et al, 1998). All cell lines were grown at 371C in a humidified atmosphere containing 5% CO 2 .…”

Cellular and synaptic mechanisms of nicotine addiction

“…Upregulation of other receptor subtypes can occur with higher nicotine concentrations in some cells (e.g., Schwartz and Kenneth, 1985;Rogers and Wonnacott, 1997;Molinari et al, 1998). Nicotinic receptor upregulation has previously been reported to involve an increase in the number of receptors, but this is not associated with changes in mRNA and is thought to reflect increased assembly (Olale et al, 1997;Wang et al, 1998). Recent studies suggest that the increased binding following upregulation may reflect a change in receptor state rather than receptor number (W. Green personal communication).…”