Regulation of Human α4β2 Neuronal Nicotinic Acetylcholine Receptors by Cholinergic Channel Ligands and Second Messenger Pathways

Abstract

The alpha4beta2 nicotinic acetylcholine receptors (nAChRs), a major subtype in the brain, have been shown to be modulated by chronic treatment with nicotine. In this study, the regulation of recombinant human alpha4beta2 nAChR subtype by (-)-nicotine and other cholinergic channel modulators was studied using human embryonic kidney 293 cells stably expressing this subunit combination. The treatment of transfected cells with (-)-nicotine and other activator ligands, including (-)-cytisine, 1,1-dimethyl-4-phenylpiperazinium, (S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole, and (+/-)-epibatidine, resulted in concentration-dependent increases in the levels of alpha4beta2 nAChRs. The increase in [3H]cytisine binding sites was initiated by low concentrations of (-)-nicotine (<100 nM); was maximal at 10 microM (15-fold), rapid (t0.5 = 4.0 +/- 0.5 hr), and totally reversible (t0.5 = 11.7 +/- 0.1 hr); and occurred with no change in ligand binding affinity. Antagonists, including dihydro-beta-erythroidine, d-tubocurarine, and methyllycaconitine, also elicited significant increases in receptor levels. A good correlation was observed between the Ki values for binding inhibition and the EC50 values for receptor up-regulation. Treatment of cells with mecamylamine, a noncompetitive antagonist, did not change receptor levels or alter (-)-nicotine-evoked up-regulation. (-)-Nicotine-evoked up-regulation was blocked by cycloheximide, suggesting a role for protein synthesis. Treatment of cells with (-)-nicotine or dihydro-beta-erythroidine differentially modulated the efficacy of acetylcholine to activate cation efflux. Both 6-beta-[beta'(piperidino)propionyl]forskolin and phorbol-12-myristate-13-acetate increased [3H]cytisine binding sites and nAChR function and enhanced the effects of chronic (-)-nicotine treatment in a synergistic manner. These results collectively demonstrate that human alpha4beta2 nAChRs can be differentially up-regulated by chronic treatment with nAChR ligands and activation of protein kinase A- and protein kinase C-dependent mechanisms.

Up-regulation of L-type Voltage-dependent Calcium Channels after Long Term Exposure to Nicotine in Cerebral Cortical Neurons

“…The Western blot analysis demonstrated the up-regulation of ␣ 4 /␤ 2 nnAChRs after the long term exposure of the neurons to 0.1 M nicotine. These data are in good agreement with the previous reports using human embryonic kidney cells (14) and Xenopus oocytes (15), although in the latter study the induction of up-regulation of ␣ 3 /␤ 4 nnAChRs required a 10-fold higher nicotine concentration for up-regulating ␣ 4 /␤ 2 nnAChRs (15).…”

“…Among various regions of the central nervous system, the ␣ 3 , ␣ 4 , ␤ 2 , and ␤ 4 subunits of nnAChRs are unequally distributed in the different layers of the cerebral cortex (60), and several investigations have reported that the up-regulation of the receptors is due to the increased numbers of ␣ 4 /␤ 2 , ␣ 3 /␤ 4 , and ␣ 7 nnAChR subunits in neurons and nonneuronal expression systems (13,17,61). In addition to these data, nnAChRs composed with ␣ 4 /␤ 2 are adequately activated by nicotine at concentrations less than 10 - 100 nM (13) and also have been reported to show up-regulation after long term nicotine treatment (13)(14)(15)18). Based on these data, we attempted to examine which of the ␣ 3 , ␣ 4 , and ␤ 2 subunits showed up-regulation under the conditions used in the present study.…”

“…Chronic exposure to low doses of nicotine induces the up-regulation of high affinity ␣ 4 /␤ 2 subunit of nnAChRs possessing [ 3 H]nicotine binding sites in the neurons (13)(14)(15), although chronic nicotine exposure also leads to association with long lasting down-regulation in responsiveness to nicotine (16 -18). This up-regulation of nnAChRs is supposed to be a compensatory reaction against agonist-induced desensitization (16,19).…”

An Extracellular Protein Microdomain Controls Up-regulation of Neuronal Nicotinic Acetylcholine Receptors by Nicotine

“…Thus, a common pharmacological spectrum for up-regulation and high affinity binding is observed. Moreover, a correlation between the ligand binding concentrations and those required to upregulate has been observed (16,17,46,48). In our opinion, this suggests that the binding site for up-regulation corresponds to a low affinity conformation of the classical binding site that bridges the interface between the ␣ and ␤ subunits.…”

“…A wide range of agonists specific for the binding site, such as nicotine, epibatidine, carbamylcholine, cytisine, dimethyl phenyl piperazinium (12,15,16,46,47), and even in some cases antagonists such as d-tubocurarine and dihydro-␤-erythroidine (14,46) have been shown to promote up-regulation of heteromeric receptors expressed in cell lines. Thus, a common pharmacological spectrum for up-regulation and high affinity binding is observed.…”