title: Long-term exposure to the new nicotinic antagonist 1,2-bisN-cytisinylethane upregulates nicotinic receptor subtypes of SH-SY5Y human neuroblastoma cells.
abstract: 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. CC4 lacked the agonist properties of cytisine and was a potent antagonist (IC50=220 nM) on nAChRs. Chronic treatment of SH-SY5Y cells with 1 mM CC4 for 48 h increased the expression of 3H-epibatidine (3H-Epi; 3-4-fold) or 125I-alpha-bungarotoxin (125I-alphaBgtx; 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-beta-erythroidine or hexametonium. Immunoprecipitation and immunopurification studies showed that the increase in 3H-Epi-binding receptors was due to the enhanced expression of alpha3beta2 and alpha3beta2beta4 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. Whole-cell patch clamp analysis of CC4-treated cells demonstrated larger nicotine-evoked inward currents with augmented sensitivity to the blockers alpha-conotoxin MII or methyllycaconitine. In conclusion, chronic treatment with CC4 increased the number of nAChRs containing beta2 and alpha7 subunits on the plasma membrane, where they were functionally active. In the case of beta2-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 beta2-containing receptor subtypes..
authors.affiliation: CNR, Institute of Neuroscience, Cellular and Molecular Pharmacology, Department of Medical Pharmacology and Center of Excellence on Neurodegenerative Diseases, University of Milan, Via Vanvitelli 32, 20129 Milan, Italy.
authors.forename: Loredana
authors.initials: L
authors.lastname: Riganti
authors.forename: Cosetta
authors.initials: C
authors.lastname: Matteoni
authors.forename: Silvia
authors.initials: S
authors.lastname: Di Angelantonio
authors.forename: Andrea
authors.initials: A
authors.lastname: Nistri
authors.forename: Annalisa
authors.initials: A
authors.lastname: Gaimarri
authors.forename: Fabio
authors.initials: F
authors.lastname: Sparatore
authors.forename: Caterina
authors.initials: C
authors.lastname: Canu-Boido
authors.forename: Francesco
authors.initials: F
authors.lastname: Clementi
authors.forename: Cecilia
authors.initials: C
authors.lastname: Gotti
mesh_terms: D000470:Alkaloids / Q000494:pharmacology; D001392:Azocines / Q000494:pharmacology*; D001665:Binding Sites; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002038:Bungarotoxins / Q000494:pharmacology; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D020128:Inhibitory Concentration 50; D008564:Membrane Potentials / Q000187:drug effects; D009419:Nerve Tissue Proteins / Q000187:drug effects / Q000378:metabolism*; D009447:Neuroblastoma; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D009538:Nicotine / Q000494:pharmacology; D018722:Nicotinic Agonists / Q000494:pharmacology; D018733:Nicotinic Antagonists / Q000494:pharmacology*; D020836:Protein Structure, Quaternary / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D011725:Pyridines / Q000494:pharmacology; D011807:Quinolizines / Q000494:pharmacology*; D011978:Receptors, Nicotinic / Q000187:drug effects / Q000378:metabolism*; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D013997:Time Factors; D015854:Up-Regulation; D064569:alpha7 Nicotinic Acetylcholine Receptor; D000093843:Quinolizidine Alkaloids
chemical_list: D000470:Alkaloids; D001392:Azocines; D019086:Bridged Bicyclo Compounds, Heterocyclic; D002038:Bungarotoxins; D009419:Nerve Tissue Proteins; D009538:Nicotine; D018722:Nicotinic Agonists; D018733:Nicotinic Antagonists; D011725:Pyridines; D011807:Quinolizines; D011978:Receptors, Nicotinic; D064569:alpha7 Nicotinic Acetylcholine Receptor; C482518:1,2-bis-N-cytisinylethane; C447159:CHRNB4 protein, human; C579167:Chrna7 protein, human; C419362:nicotinic receptor alpha3beta2; C004712:cytisine; D000093843:Quinolizidine Alkaloids; C082748:epibatidine