Nicotine-induced upregulation of human neuronal nicotinic α7-receptors is potentiated by modulation of cAMP and PKC in SH-EP1-hα7 cells

Abstract

Chronic nicotine exposure induces upregulation of nicotinic receptors, but the mechanisms underlying this phenomenon are not well understood. The aim of this study was to examine the role of different second messenger systems in the nicotine-induced upregulation of α7-nicotinic receptors in SH-EP1-hα7 human epithelial cells. We show here that chronic exposure to nicotine results in accumulation of cAMP. Furthermore, an enhanced cAMP signalling potentiates nicotine-induced upregulation of α7-nicotinic receptors measured by [³H]methyllycaconitine ([³H]MLA) binding suggesting that cAMP is involved in the α7-nicotinic receptor upregulation. Down-regulation of protein kinase C (PKC) with a phorbol ester abolishes the nicotine-induced upregulation of α7-nicotinic receptors. Furthermore, overexpression of PKCα in SH-EP1-hα7 cells results in potentiation of nicotine-evoked upregulation indicating that PKC has a role in regulation of α7-nicotinic receptor number. The Ca²⁺-calmodulin kinase II (CaMKII) and extracellular signal regulated kinase 1/2 (ERK1/2) appear not to participate in α7-nicotinic receptor upregulation since the specific inhibitors of these kinases did not have an effect on the nicotine-induced upregulation. Taken together this study provides evidence that nicotine induces accumulation of cAMP and that the upregulation mechanisms of α7-nicotinic receptors are potentiated both by cAMP and PKC. As nicotine-evoked upregulation of heteromeric nicotinic receptors in SH-SY5Y cells was unaffected by the treatment with drugs affecting cAMP signalling or PKC activity, our results suggest that the upregulation mechanisms of homomeric α7-nicotinic receptors and heteromeric nicotinic receptors differ from each other.

Introduction

Nicotine is the compound of cigarette smoke responsible for the development of tobacco addiction. The effects of nicotine are mediated through neuronal nicotinic acetylcholine receptors that are widely expressed in the central nervous system. Several different subtypes of nicotinic receptors occur in mammalian brain, including the two major subtypes of homomeric α7-and heteromeric α4β2-nicotinic receptors (Lukas et al., 1999).

Homomeric α7-nicotinic receptors have received particular attention as possible candidates in mediation of nicotine's effects on cognition (Levin, 2002) and neuroprotection (Jonnala et al., 2003). A role in nicotine dependence has also been shown for α7-nicotinic receptors (Grabus et al., 2005, Mansvelder and McGehee, 2000). The α7-nicotinic receptors are a unique class of receptors that differ from the heteromeric nicotinic receptors in many aspects. They bind α-bungarotoxin with high affinity and desensitize rapidly but reversibly following acute exposure to agonist (McGehee and Role, 1995). Due to their high Ca²⁺-permeability opening of α7-receptor channel is able to activate both directly and indirectly many Ca²⁺-dependent pathways, such as adenylate cyclase (AC), protein kinase C (PKC), Ca²⁺-calmodulin-dependent protein kinase (CaMK), phosphoinositide 3-kinase (PI3K) and extracellular signal-regulated kinase 1/2 (ERK1/2) (Dajas-Bailador and Wonnacott, 2004). In turn, activation of these pathways can result in expression of genes important in neuronal adaptation to chronic nicotine exposure (Berg and Conroy, 2002, Dajas-Bailador and Wonnacott, 2004).

Chronic nicotine exposure increases the number of nicotinic receptor radioligand binding sites in smoker's brain, in rodent brain as well as in cell lines in culture (for a review, see Gentry and Lukas, 2002). In rodent brain the number of all the subtypes of nicotinic receptors is increased by chronic nicotine treatment but the doses and the duration of nicotine exposure required to induce this vary across different nicotinic receptors. As compared with the heteromeric α4β2-nicotinic receptors the upregulation of α7-nicotinic receptors is usually less robust and may require longer exposure or higher concentration of nicotine to develop (Marks et al., 1986, Nuutinen et al., 2005, Pauly et al., 1991, Pietilä et al., 1998, Sparks and Pauly, 1999). Interestingly, differing from earlier studies (Gentry and Lukas, 2002) Kawai and Berg (2001) showed that upregulated α7-nicotinic receptors remain functional after chronic nicotine treatment confirming the role of these receptors in the mediation of long-term effects of nicotine.

Recent studies have concentrated on examining the mechanisms underlying the nicotine-induced upregulation of heteromeric nicotinic receptors (Darsow et al., 2005, Nashmi et al., 2003, Sallette et al., 2004, Sallette et al., 2005, Vallejo et al., 2005) whereas the pathways involved in the upregulation of homomeric α7-nicotinic receptors are still poorly understood. However, some studies suggest that the mechanisms involved in the upregulation of α7-nicotinic receptors may differ from those of heteromeric nicotinic receptors. For example, a study in rat cortical neurons showed that in contrast to heteromeric nicotinic receptors nicotine-evoked upregulation of α7-nicotinic receptors is dependent on newly synthesized receptors (Kawai and Berg, 2001). In addition, Ridley et al. (2001) found that nicotine-evoked α7-upregulation is independent from CaMK II activity whereas for the upregulation of α3 containing nicotinic receptors it is essential. Recent studies have also shown a role for tyrosine kinase in the regulation of α7-nicotinic receptors although it is still unclear whether activation or inactivation of tyrosine kinase results in increased number α7-nicotinic receptors (Charpantier et al., 2005, Cho et al., 2005, Kawai et al., 2002, Liu et al., 2001, Zhou et al., 2004). In addition, the possible involvement of tyrosine kinase in nicotine-induced upregulation remains to be examined.

In the present study we have examined the role of cAMP, PKC, CaMK II and ERK1/2 in the nicotine-induced upregulation of α7-nicotinic receptors in SH-EP1-hα7 cells. The SH-EP1-hα7 cell line was chosen because it stably expresses human α7-nicotinic receptors and lacks the other subtypes of nicotinic receptors. This cell line has been earlier characterised and shown to express functional α7-nicotinic receptors (Zhao et al., 2003). In order to examine possible differences in regulation of nicotine-evoked upregulation between homomeric and heteromeric nicotinic receptors, we extended this study to neuroblastoma SH-SY5Y cell line expressing α3, α5, α7, β2 and β4 nicotinic receptor subunits (Peng et al., 1994).