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Chronic nicotine treatment increases the number of neuronal nicotinic acetylcholine receptors (nAChRs). Localization of nAChRs at a cellular level determines their functional role. However, changes in the localization of nAChRs caused by chronic nicotine treatment are not well known. In this study, we have examined the effects of chronic nicotine treatment on α7 and β2 nAChR subunits in vitro in cell lines and in vivo in mouse striatum. In vitro, two different cell lines were used, SH-SY5Y cells endogenously expressing several nAChR subtypes and SH-EP1-hα7 cells, transfected with the human α7 nAChR subunit gene. Effects of chronic nicotine treatment (10 μM, 3 days) were studied in vitro by using confocal and electron microscopy and calcium fluorometry. In vitro in SH-SY5Y cells, α7 and β2 subunits formed groups, unlike α7 subunits in SH-EP1-hα7 cells, which were partially localized on endoplastic reticulum. Chronic nicotine treatment did not change the localization of nAChRs in endosomes, but caused clustering of α7 subunits in SH-EP1-hα7 cells. In vivo, nicotine was given to mice in their drinking water for 7 weeks. Results showed that α7 and β2 subunits formed groups, and that chronic nicotine treatment increased the size of the clusters. As a conclusion, our data show that there are large intracellular pools of nAChR subunits, which are partially localized on endoplastic reticulum. Chronic nicotine treatment does not change endocytotic trafficking of nAChRs. Chronic nicotine treatment increased clustering of nAChRs, which could have a role in the release of dopamine (DA) evoked by nicotine. Synapse 59:383-393, 2006. © 2006 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

(–)-Methadone acts as an agonist at opioid receptors. Both (+)- and (–)-enantiomers of methadone have been suggested to be potent non-competitive antagonists of α3β4 neuronal nicotinic acetylcholine receptors (nAChRs). In the present study, we have examined interactions of methadone with nAChRs by using receptor binding assays, patch-clamp recording and calcium fluorometry imaging with SH-SY5Y cells naturally expressing α7 and α3* nAChR subtypes and SH-EP1-hα7 cells heterologously expressing human α7 nAChRs. Methadone potently inhibited binding of [3H]methyllycaconitine to α7 nAChRs and that of [3H]epibatidine to α3* nAChRs. Methadone pretreatment induced up-regulation of epibatidine binding sites in SH-SY5Y cells. Using whole-cell patch-clamp recording, both isomers of methadone activated cation currents via mecamylamine-sensitive nAChRs in SH-SY5Y cells. Nicotine and both (+)- and (–)-methadone evoked increases in [Ca2+]i in both fluo-3AM loaded cell lines, and these effects were blocked by mecamylamine and by the α7 selective antagonist methyllycaconitine, suggesting effects of methadone as α7-nAChR agonist. Sensitivity of sustained nicotine and methadone effects to blockade by CdCl2, ryanodine and xestospongin-c implicates voltage-operated Ca2+ channels and intracellular Ca2+ stores as downstream modulators of elevated [Ca2+]i. Collectively, our results suggest that methadone engages in complex and potentially pharmacologically significant interactions with nAChRs. [ABSTRACT FROM AUTHOR]

Nicotine releases dopamine in the brain by activating neuronal nicotinic acetylcholine receptors (nAChRs). Chronic nicotine treatment increases the number of nAChRs, which represents plasticity of the brain. Together these phenomena have been suggested to have a role in the development of nicotine addiction. In the brain nAChRs can be localized synaptically, extrasynaptically or intracellularly. The purpose of these studies was to clarify the effects of chronic nicotine treatment on the localization ofβ2 andα7 nAChR subunits in brain areas involved in nicotine addiction. Nicotine was administered orally in drinking water to male NMRI mice for 7 weeks. At the end of chronic nicotine treatment the localization of the nAChR subunits was studied in the dorsal striatum and in the ventral tegmental area (VTA) by using electron microscopy. In the brain areas studiedβ2 andα7 subunits were localized presynaptically and postsynaptically in axon endings and in dendrites. In both areas the majority of theβ2 andα7 subunits were localized at extrasynaptic sites. In response to chronic nicotine treatment theβ2 andα7 nAChR subunit labelling was increased at synaptic and extrasynaptic sites as well as intracellularly. This suggests that the trafficking of nAChR subunits is increased as a result of chronic nicotine treatment and nAChRs in all parts of neurons could have functional roles in the formation of nicotine addiction. [ABSTRACT FROM AUTHOR]

SH-SY5Y cells express α7 and α3[sup *] subtypes of nicotinic acetylcholine receptors (AChR). Numbers of these receptors are upregulated by chronic treatment with nicotinic agonists or KC1. In this study we have examined the functional consequences of these drug treatments on nicotine- or KCl-evoked increases in [Ca[sup 2+]][sub i], in SH-SY5Y cells. 2 In untreated cells, nicotine increased [Ca[sup 2+]][sub i] (EC[sub 50] 7.5 μm). Responses to 10 μM nicotine were abolished by the non-selective nicotinic antagonist mecamylamine and were partially blocked by α7selective antagonists, the α3β2[sup *]-selective antagonist y-conotoxin-MII, and by cadmium and verapamil. 3 After treatment for 4 days with nicotinic agonists, nicotine-evoked increases in [Ca[sup 2+]][sub i] were significantly decreased by about 25%. Nicotine-evoked responses were paradoxically increased in the presence of acute methyllycaconitine (MLA; an α7-selective antagonist) although other α7-selective antagonists were without effect, while α-conotoxin-MII gave a partial inhibition. The increase observed with MLA was abolished by mecamylamine but not by α-conotoxin-MII and was still observed 24 h after chronic nicotine treatment. 4 After treatment for 4 days with KC1, nicotine-evoked increases in [Ca[sup 2+]][sub i] were also decreased by 25%, but acute MLA was without effect. Responses to 20 mM KC1 were unchanged by prior treatment with nicotine or KC1. Treatment for 4 days with 5 μM verapamil reduced responses to both nicotine and KC1 by about 50%. 5 Multiple nicotinic AChR subtypes contribute to nicotine-evoked increases in [Ca[sup 2+]][sub i] in SH-SY5Y cells. Responses to acute nicotine are reduced after chronic nicotine or KC1 treatment, with loss of the component attributed to the α7 subtype. However, in nicotine-treated cells this effect is reversed when nicotine stimulation is applied in the presence of acute MLA. The... [ABSTRACT FROM AUTHOR]