Central role of fibroblast alpha3 nicotinic acetylcholine receptor in mediating cutaneous effects of nicotine

Smoking is associated with aberrant cutaneous tissue remodeling, such as precocious skin aging and impaired wound healing. The mechanism is not fully understood. Dermal fibroblasts (DF) are the primary cellular component of the dermis and may provide a target for pathobiologic effects of tobacco products. The purpose of this study was to characterize a mechanism of nicotine (Nic) effects on the growth and tissue remodeling function of DF. We hypothesized that the effects of Nic on DF result from its binding to specific nicotinic acetylcholine receptors (nAChRs) expressed by these cells and that downstream signaling from the receptors alters normal cell functioning, leading to changes in skin homeostasis. Using RT-PCR and Western blotting, we found that a 24-hour exposure of human DF to 10 micro M Nic causes a 1.9- to 28-fold increase of the mRNA and protein levels of the cell cycle regulators p21, cyclin D1, Ki-67, and PCNA and a 1.7- to 2-fold increase of the apoptosis regulators Bcl-2 and caspase 3. Nic exposure also up-regulated expression of the dermal matrix proteins collagen type Ialpha1 and elastin as well as matrix metalloproteinase-1. Mecamylamine (Mec), the specific antagonist of nAChRs, abolished Nic-induced alterations, indicating that they resulted from a pharmacologic stimulation of nAChRs expressed by DF. To establish the relevance of these findings to a specific nicotinergic pathway, we studied human DF transfected with anti-alpha3 antisense oligonucleotides and murine DF from alpha3 nAChR knockout mice. In both cases, lack of alpha3 was associated with alterations in fibroblast growth and function that were opposite to those observed in DF treated with Nic, suggesting that the nicotinic effects on DF were mostly mediated by alpha3 nAChR. In addition to alpha3, the nAChR subunits detected in human DF were alpha5, alpha7, beta2, and beta4. The exposure of DF to Nic altered the relative amounts of each of these subunits, leading to reciprocal changes in [(3)H]epibatidine-binding kinetics. Thus, some of the pathobiologic effects of tobacco products on extracellular matrix turnover in the skin may stem from Nic-induced alterations in the physiologic control of the unfolding of the genetically determined program of growth and the tissue remodeling function of DF as well as alterations in the structure and function of fibroblast nAChRs.     

Nicotine stimulates human lung cancer cell growth by inducing fibronectin expression

The mechanisms by which tobacco promotes lung cancer remain incompletely understood. Herein, we report that nicotine, a major component of tobacco, promotes the proliferation of cultured non-small cell lung carcinoma (NSCLC) cells; this effect was most noticeable at 5 days. However, nicotine had no effect on apoptosis of NSCLC cells. In experiments designed to unveil the mechanisms for this effect, we found that nicotine also stimulated mRNA and protein expression of fibronectin. Fibronectin is a matrix glycoprotein that regulates important cellular processes (e.g., adhesion, proliferation, and differentiation) and is highly expressed in tobacco-related lung disorders. Of note, reagents against the integrin alpha5beta1 (antibodies, RGD peptides, alpha5 shRNA) blocked the mitogenic effects of nicotine. Thus, nicotine stimulated NSCLC cell proliferation indirectly via fibronectin induction. We then focused on the mechanisms responsible for nicotine-induced fibronectin expression in NSCLC cells and found that nicotine stimulated the surface expression of alpha7 nicotinic acetylcholine receptor (alpha7 nAChR), and that alpha-bungarotoxin, an inhibitor of alpha7 nAChR, abolished the nicotine-induced fibronectin response. The fibronectin-inducing effects of nicotine were associated with activation of extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3-K)/mammalian target of rapamycin (mTOR) signaling pathways, and were abrogated by inhibitors of ERK (PD98059), PI3-K (LY294002), and mTOR (rapamycin), but not by inhibitors of protein kinase (PK)C (calphostin C) and PKA (H89). These observations suggest that nicotine stimulates NSCLC proliferation through induction of fibronectin, and that these events are mediated through nAChR-mediated signals that include ERK and PI3-K/mTOR pathways. This work highlights the role of fibronectin and alpha5beta1 integrins as potential targets for anti-lung cancer therapies.     

Modulation of nicotine but not ethanol preference by the mouse Chrna4 A529T polymorphism

Available evidence indicates that common genes influence alcohol and tobacco abuse in humans. The studies reported here used mouse models to evaluate the hypothesis that genetically determined variability in the alpha4beta2* nicotinic receptor modulates genetically determined variability in the intake of both nicotine and alcohol. Data obtained with inbred mouse strains suggested an association between a polymorphism in the mouse alpha4 nAChR subunit gene, Chrna4, and variability in nicotine and ethanol preference. These associations were assessed in F2 animals derived by crossing C57BL/6-super(beta2-/-) mice and A/J mice. The results obtained by the authors indicate that the polymorphism in Chrna4 plays an important role in modulating variability in oral nicotine intake but is linked to a gene that regulates alcohol intake.     

Smoking upregulates alpha4beta2* nicotinic acetylcholine receptors in the human brain

The role of the alpha4beta2* nicotinic acetylcholine receptors (nAChR) in tobacco addiction in humans is largely unresolved. We visualized brain alpha4beta2* nicotinic acetylcholine receptors of smokers and non-smokers with positron emission tomography using 2-[(18)F]fluoro-3-(2(S)azetidinylmethoxy)pyridine, commonly known as 2-[(18)F]F-A-85380. The total brain distribution volume of 2-[(18)F]F-A-85380 was significantly increased in smokers. Statistical parametric mapping revealed that the most prominent regional differences of distribution volumes (DV) were found in cerebellum and brainstem with an increased uptake in smokers. The up-regulation of alpha4beta2* nAChR upon chronic nicotine exposure via tobacco smoking incorporates subcortical brain regions which may play an important role in nicotine addiction.     

Mouse strain-specific changes in nicotinic receptor expression with age

The onset and severity of age-related loss of neuronal nicotinic acetylcholine receptor (nAChR) expression in the mammalian hippocampus can vary considerably between individuals. We have examined the expression of four nAChR subunits (nAChR alpha4, nAChR alpha5, nAChR alpha7 and nAChR beta4) in the dorsal hippocampus of adult (12-14 months) and aged (24-28 month) animals from two-mouse strains (CBA/J and C57BL/6). The expression of nAChR alpha4 was selectively diminished with age in both strains, and there was a significant loss of nAChR alpha7 in CA1 of aged CBA/J, but not C57BL/6. There was no change in nAChR alpha5 expression with age whereas nAChR beta4 preferentially diminished in the C57BL/6 CA1 region and remained the same or slightly increased in the aged CBA/J. Coincident with the loss of neuronal nAChR alpha4 in the CBA/J strain was a significant age-related increase of nAChR alpha4 staining of astrocytes, most notably in the stratum radiatum. These results suggest that mouse strains of different genetic backgrounds undergo dissimilar age-related changes in the expression of nAChRs.     

Effects of chronic neonatal nicotine exposure on nicotinic acetylcholine receptor binding, cell death and morphology in hippocampus and cerebellum

Nicotine, the major psychoactive ingredient in tobacco interacting with nicotinic acetylcholine receptors (nAChR), is believed to have neuroprotective and neurotoxic effects on the developing brain. Neurotoxicity has been attributed to activation of homomeric alpha7 nAChRs, neuroprotection to heteromeric alpha4beta2 nAChRs. Thus, developmental nicotine could have opposite effects in different brain regions, depending on nAChR subtype expression. Here, we determined if chronic neonatal nicotine exposure (CNN), during a period of brain growth corresponding to the third human trimester, differentially regulates nAChR expression, cell death, and morphological properties in hippocampus and cerebellum, two structures maturing postnatally. Rat pups were orally treated with 6 mg/kg/day nicotine from postnatal day (P)1 to P7. On P8, expression for alpha4, alpha7 and beta2 mRNA was determined by in situ hybridization; nAChR binding sites by receptor autoradiography, dying neurons by TUNEL and Fluoro-Jade staining and morphological properties by analysis of Cresyl Violet-stained sections. In control cerebellum, strong expression of alpha4, beta2 mRNA and heteromeric nAChRs labeled with [125I]-epibatidine was found in granule cells, and alpha7 mRNA and homomeric nAChRs labeled with [125I]-alpha-bungarotoxin were in the external germinal layer. In control hippocampus, low expression of alpha4 mRNA and heteromeric nAChRs and high expression of alpha7 mRNA and homomeric nAChRs were detected. CNN increased heteromeric nAChR binding in hippocampus but not cerebellum and significantly decreased neuronal soma size and increased packing density in hippocampal principal cells but not in cerebellum. CNN did not increase the number of dying cells in any area, but significantly fewer TUNEL-labeled cells were found in CA3 strata oriens and radiatum and cerebellar granule layer. Thus, the hippocampus seems to be more sensitive than the cerebellum to CNN which could result from different nAChR subtype expression and might explain long-lasting altered cognitive functions correlated with gestational nicotine exposure due to changes in hippocampal cell morphology.     

Postnatal upregulation of alpha4 and alpha3 nicotinic receptor subunits in the brain of alpha7 nicotinic receptor-deficient mice

The nicotinic receptor subtypes are important for several physiological functions in brain and may therefore play a critical role in brain development. The alpha7 nicotinic receptors which have high Ca2+ permeability are important for cognitive, neuroprotective and trophic functions. In this study, the brain development and the expression of alpha4, alpha3, alpha7, alpha5 and beta2 nicotinic receptors were investigated in the brains of alpha7 deficient (alpha7 -/-), alpha7 heterozygous null (alpha7 +/-) and alpha7 wild-type (alpha7 +/+) mice from postnatal days (P) 7-84. The specific binding of [3H] cytisine and [3H] epibatidine, as well as the expressions of alpha4 and alpha3 nicotinic receptor subunits at mRNA and protein levels, were significantly increased in the cortex and hippocampus of alpha7 -/- and alpha7 +/- mice compared with alpha7 +/+ mice. Furthermore, the alpha4 and alpha3 nicotinic acetylcholine receptor (nAChR) subunits appeared to co-assemble with the alpha5 nAChR subunit in these above brain regions of these mice. No significant change in synaptophysin level was observed. These data suggest that increased levels of alpha4, alpha3-containing nAChRs, co-assembled with the alpha5 nAChR subunit, may contribute to the normal brain development of alpha7 -/- and alpha7 +/- mice.     

α7神经型尼古丁受体的神经保护作用及其与阿尔茨海默病发病的关系

目的探讨α7神经型尼古丁受体（nAChR）表达改变与淀粉样蛋白前体蛋白（APP）代谢、细胞存活率及脂质过氧化水平的关系,以了解α7 nAChR的神经保护作用,以及该受体水平与阿尔茨海默病发病的关系。方法设计并合成α7 nAChR基因特异性小分子干扰RNA（siRNA）,转染SH-SY5Y细胞;用20μmol／LDMXB处理细胞;培养48h后收集细胞,分别应用逆转录聚合酶链反应（RT-PCR）和Western blot方法检测转染细胞及DMXB处理后的细胞α7 nAChR mRNA及蛋白表达水平的变化;并用1μmol／L β-淀粉样肽25-35（Aβ25-35处理细胞,测定分泌型APP、总APP蛋白表达的变化;比色法测定脂质过氧化产物含量;MTT方法测定细胞活力。结果转染siRNA后,与对照组相比,α7 nAChR mRNA及蛋白表达量降低（抑制率分别为80％和69％）、脂质过氧化产物丙二醛含量明显增加、分泌型APP表达下降、细胞活力明显低于对照组,且能增强Aβ的细胞毒性作用;用DMXB处理细胞后能使α7 nAChR的表达增加23％、增加分泌型APP的表达、并能对抗Aβ引起的细胞活力下降及脂质过氧化水平的增强。结论α7 nAChR可能通过增强α-分泌酶对APP的切割、增强细胞抗氧化能力及对抗Aβ的神经毒性作用来发挥神经保护作用,其表达减少与阿尔茨海默病的发病机制有密切的关系。     

Liquid chromatographic studies with immobilized neuronal nicotinic acetylcholine receptor stationary phases: effects of receptor subtypes, pH and ionic strength on drug-receptor interactions

Nicotinic acetylcholine receptor (nAChR) alpha3-subunits, beta4-subunits, alpha3/beta4-subunit combination and alpha4/beta2-subunit combination were immobilized on chromatographic stationary phases and the binding affinities of the different nAChR subtypes were chromatographically evaluated. The observed relative binding affinities of epibatidine were alpha4/beta2>alpha3/beta4 and epibatidine did not bind at alpha3-subunits and beta4-subunits. No significant difference in binding affinities was observed on the alpha4/beta2 nAChRs immobilized in immobilized artificial membrane (IAM) particles and those sterically immobilized on Superdex 200 beads. The effects of mobile phase pH and ionic strength on the binding affinities of the alpha3/beta4 nAChRs support were also investigated. The results are consistent with the proposed ligand-nAChR binding model in which a cationic center exists at the binding site.     

Attenuation of ethanol-induced ataxia by alpha(4)beta(2) nicotinic acetylcholine receptor subtype in mouse cerebellum: a functional interaction

Many epidemiological studies support the notion that people who drink alcohol also smoke cigarettes and vice versa thereby suggesting a possible functional interaction between these two most widely used psychoactive substances. We have earlier demonstrated that direct intracerebellar (ICB) microinfusion of nicotine dose-dependently antagonizes ethanol-induced ataxia and further that this antagonism occurs in a glutamate-nitric oxide-cyclic guanylyl monophosphate (cGMP) sensitive manner. The present study was designed to determine the possible involvement of specific nicotinic acetylcholine receptor (nAChR) subtype alpha(4)beta(2) in nicotine-induced attenuation of ethanol ataxia. Using the Rotorod test and direct ICB microinfusion technique in stereotaxically cannulated CD-1 male mice, we performed the Rotorod test following ICB administration of the alpha(4)beta(2)-selective agonist, (E)-N-methyl-4-(3-pyridinyl)-3-buten-1-amine (RJR-2403; 31.25, 62.5, 125 ng) on ethanol (2 g/kg; i.p.) ataxia at 15, 30, 45, 60 min post-ethanol injection. RJR-2403 dose-dependently attenuated ethanol ataxia suggesting a role of alpha(4)beta(2) subtype in ameliorating ethanol-induced ataxia. Pretreatment with ICB dihydro-beta-erythroidine (DHbetaE: 125, 250, 500, 750 ng), a potent alpha(4)beta(2)-selective antagonist, significantly reduced RJR-2403's effect further supporting the alpha(4)beta(2) involvement. DHbetaE (ICB) also antagonized ICB nicotine-induced attenuation of ethanol ataxia again reinforcing the role of alpha(4)beta(2) subtype. Additional evidence for the role of alpha(4)beta(2) subtype was provided when ICB alpha(4)beta(2) antisense oligodeoxynucleotide treatment markedly antagonized RJR 2403-induced attenuation of ethanol ataxia compared with missense-treated animals. This was confirmed with an associated decrease in the expression of alpha(4)beta(2) subtypes indicated by immunoblot experiments. In conclusion, the results of the present investigation support an important role of alpha(4)beta(2) nAChR subtype in the expression of nicotine-induced attenuation of ethanol ataxia.     

Nicotinic AChR in subclassified capsaicin-sensitive and -insensitive nociceptors of the rat DRG

Nociceptive cells of the dorsal root ganglion (DRG) were subclassified, in vitro, according to patterns of voltage-activated currents. The distribution and form of nicotinic ACh receptors (nAChRs) were determined. nAChRs were present on both capsaicin-sensitive and -insensitive nociceptors but were not universally present in unmyelinated nociceptors. In contrast, all A delta nociceptors (types 4, 6, and 9) expressed slowly decaying nAChR. Three major forms of nicotinic currents were identified. Specific agonists and antagonists were used to demonstrate the presence of alpha7 in two classes of capsaicin-sensitive, unmyelinated nociceptors (types 2 and 8). In type 2 cells, alpha7-mediated currents were found in isolation. Whereas alpha7 was co-expressed with other nAChR in type 8 cells. These were the only classes in which alpha7 was identified. Other nociceptive classes expressed slowly decaying currents with beta4 pharmacology. Based on concentration response curves formed by nicotinic agonists [ACh, nicotine, dimethyl phenyl piperazinium (DMPP), cytisine] evidence emerged of two distinct nAChR differentially expressed in type 4 (alpha3beta4) and types 5 and 8 (alpha3beta4 alpha5). Although identification could not be made with absolute certainty, patterns of potency (type 4: DMPP > cytisine > nicotine = ACh; type 5 and type 8: DMPP = cytisine > nicotine = ACh) and efficacy provided strong support for the presence of two distinct channels based on an alpha3beta4 platform. Studies conducted on one nonnociceptive class (type 3) failed to reveal any nAChR. After multiple injections of Di-I (1,1'-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) into the hairy skin of the hindlimb, we identified cell types 2, 4, 6, 8, and 9 as skin nociceptors that expressed nicotinic receptors. We conclude that at least three nicotinic AChR are diversely distributed into discrete subclasses of nociceptors that innervate hairy skin.     

Strain-specific nicotinic modulation of glutamatergic transmission in the CA1 field of the rat hippocampus: August Copenhagen Irish versus Sprague-Dawley

Prepulse inhibition (PPI), a measure of sensorimotor gating impaired in patients with schizophrenia, is more sensitive to disruption by apomorphine in prepubertal August Copenhagen Irish (ACI) than Sprague-Dawley (SD) rats. In brain regions including the hippocampus, PPI is modulated by alpha7* nicotinic receptors (nAChRs) and kynurenic acid (KYNA), a kynurenine metabolite that blocks alpha7 nAChRs. Here, KYNA levels and nAChR activities were measured in the hippocampi of 10- to 23-day-old ACI and SD rats of both sexes. Hippocampal KYNA levels were not different between ACI and SD rats. In hippocampal slices from both rat strains, choline (10 mM) evoked alpha7* nAChR-mediated type IA currents in CA1 stratum radiatum (SR) interneurons. In the presence of alpha7 nAChR antagonists, acetylcholine (ACh, 1 mM) evoked alpha4beta2* nAChR-mediated type II currents. ACh also triggered excitatory postsynaptic currents (EPSCs) that resulted from alpha3beta4* nAChR activation in glutamatergic neurons/axons synapsing onto the interneurons. The magnitude of the nicotinic responses did not differ significantly between male and female rats. Only the magnitude of alpha3beta4* nAChR responses and the frequency of spontaneous EPSCs recorded from CA1 SR interneurons differed between the rat strains, being significantly larger in ACI than SD rats. These results indicate that the alpha3beta4* nAChR activity in glutamatergic neurons/axons and the number of glutamatergic terminals synapsing onto CA1 SR interneurons are larger in prepubertal ACI than SD rats. The differential sensitivity of these rats to PPI disruption by apomorphine may result from strain-specific levels of glutamatergic activity and its strain-specific modulation by alpha3beta4* nAChRs in the hippocampus.     

Postnatal expression of alpha2 nicotinic acetylcholine receptor subunit mRNA in developing cortex and hippocampus

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated cation channels composed of alpha and beta subunits. nAChR subunit expression is highly regulated during development. Previous studies have revealed increased expression of alpha3, alpha5, alpha7, and beta4 subunit mRNAs and alpha7 binding sites during hippocampal and cortical development. Here, we examined the expression of alpha2 subunit mRNA in rat cortex and hippocampus using highly sensitive radioactive in situ hybridization. alpha2 Subunit mRNA expression was first detected at P3 in cortex and hippocampus. During postnatal development the distribution of alpha2 subunit mRNA expression was spatially similar to the one found in adult, exhibiting highly restricted expression in scattered cells mostly in cortical layer V and retrosplenial cortex, and in scattered cells in CA1/CA3 stratum oriens and CA3 stratum radiatum. However, the expression intensity and number of alpha2 positive cells strongly increased to reach peak levels in both cortex and hippocampus at P7 and decreased thereafter to moderate to low to levels. Double in situ hybridization revealed that most, but not all, alpha2 mRNA expression was located in non-pyramidal GAD-positive cortical and hippocampal interneurons. Thus, similar to other nAChR subunits, alpha2 mRNA expression is transiently upregulated during postnatal development and nAChRs containing alpha2 subunits could regulate GABAergic activity during a critical period of network formation.     

Activation of nicotinic acetylcholine receptors increases the frequency of spontaneous GABAergic IPSCs in rat basolateral amygdala neurons

The basolateral amygdala (BLA) is a critical component of the amygdaloid circuit, which is thought to be involved in fear conditioned responses. Using whole cell patch-clamp recording, we found that activation of nicotinic acetylcholine receptors (nAChRs) leads to an action potential-dependent increase in the frequency of spontaneous GABAergic currents in principal neurons in the BLA. These spontaneous GABAergic currents were abolished by a low-Ca2+/high-Mg2+ bathing solution, suggesting that they are spontaneous inhibitory postsynaptic currents (sIPSCs). Blockade of ionotropic glutamate receptors did not prevent this increased frequency of sIPSCs nor did blockade of alpha7 nAChRs. Among the nAChR agonists tested, cystisine was more effective at increasing the frequency of the sIPSCs than nicotine or 1,1-dimethyl-4-phenyl piperazinium iodide, consistent with a major contribution of beta4 nAChR subunits. The nicotinic antagonist, dihydro-beta-erythroidine, was less effective than d-tubocurarine in blocking the increased sIPSC frequency induced by ACh, suggesting that alpha4-containing nAChR subunits do not play a major role in the ACh-induced increased sIPSC frequency. Although alpha2/3/4/7 and beta2/4 nAChR subunits were found in the BLA by RT-PCR, the agonist and antagonist profiles suggest that the ACh-induced increase in sIPSC frequency involves predominantly alpha3beta4-containing nAChR subunits. Consistent with this, alpha-conotoxin-AuIB, a nAChR antagonist selective for the alpha3beta4 subunit combination, inhibited the ACh-induced increase in the frequency of sIPSCs. The observations suggest that nicotinic activation increases the frequency of sIPSCs in the BLA by acting mainly on alpha3beta4-containing nicotinic receptors on GABAergic neurons and may play an important role in the modulation of synaptic transmission in the amygdala.     

The role of neuronal nicotinic acetylcholine receptor subunits in autonomic ganglia: lessons from knockout mice

Neuronal nicotinic acetylcholine receptors (nAChR), composed of 12 subunits (alpha2-alpha10, beta2-beta4), are expressed in autonomic ganglia, playing a central role in autonomic transmission. The repertoire of nicotinic subunits in autonomic ganglia includes alpha3, alpha5, alpha7, beta2 and beta4 subunits. In the last 10 years, heterologous expression studies have revealed much about the nature of neuronal nAChRs. However, there is only limited understanding of subunit actions in autonomic system. Functional deletions of subunit by gene knockout in animals could overcome these limitations. We review recent studies on nAChRs on autonomic ganglia for physiological and pharmacological properties and potential locations of the subunits.