Nicotinic receptors in airway disease

Abstract

With continued smoking of tobacco products and expanded use of nicotine delivery devices worldwide, understanding the impact of smoking and vaping on respiratory health remains a major global unmet need. Although multiple studies have shown a strong association between smoking and asthma, there is a relative paucity of mechanistic understanding of how elements in cigarette smoke impact the airway. Recognizing that nicotine is a major component in both smoking and vaping products, it is critical to understand the mechanisms by which nicotine impacts airways and promotes lung diseases such as asthma. There is now increasing evidence that α7 nicotinic acetylcholine receptors (α7nAChRs) are critical players in nicotine effects on airways, but the mechanisms by which α7nAChR influences different airway cell types have not been widely explored. In this review, we highlight and integrate the current state of knowledge regarding nicotine and α7nAChR in the context of asthma and identify potential approaches to alleviate the impact of smoking and vaping on the lungs.

NICOTINE CHEMISTRY AND PHARMACOLOGY

Although both tobacco and cigarette smoke contain many different components, nicotine is recognized as the addictive substance present in both (1). Since the additional components pose substantial health risks, particularly for cancer, there has been a large gravitation toward the use of nicotine-delivery devices. Therefore, understanding the role of nicotine per se in lung disease is of substantial impact.

Nicotine is an alkaloid compound extracted from plants belonging to the Solanaceae family, particularly tobacco (Nicotiana tabacum) (2). Nicotine in tobacco or cigarette smoke occurs in the particulate phase along with a mix of other alkaloids such as nornicotine, anatabine, and anabasine (3). Chemically, nicotine is a tertiary amine consisting of a pyridine and pyrrolidine-infused ring structure. The S-isomer of nicotine is the common isomer found in tobacco or cigarette smoke and binds to the nicotinic cholinergic receptors (nAChRs) to elicit functional activity. The R-nicotine, on the other hand, is a weak isomer and a weak agonist of the cholinergic receptors (3).

Being extremely lipophilic in nature, nicotine is rapidly absorbed from the linings of the gastrointestinal (GI) tract, skin, and mucosal membranes (4, 5). However, the absorption capacity of nicotine depends on the methods of preparation (3, 6). Since nicotine is also a weak base, its movement across cell membrane barriers is pH-dependent (3, 7). In addition, nicotine readily crosses the placenta from circulation and is, therefore, absorbed rapidly via maternal skin and can access the fetus (8, 9). Upon inhalation, nicotine is well absorbed from oral mucus membranes as well as by the airways and alveoli (10, 11). Absorption is facilitated by rapid buffering from an initial alkaline pH to physiological pH. Following absorption, nicotine enters the circulation and rapidly distributes to different organs and tissue systems, including the brain. Postabsorption and distribution, nicotine is metabolized to cotinine in the liver (12, 13). Previous studies suggest that the brain, liver, kidney, spleen, and lungs have the highest tissue affinity for nicotine, whereas adipose tissue has the lowest tissue affinity (14, 15). The resulting distribution of nicotine in these organs is dependent on the expression of the nAChRs in the various cell types that engender the tissue, indicating that adipose tissues have the least expression of the nAChRs (14, 16, 17). Importantly, the lung is particularly susceptible to nicotine effects because of two modes of contact: directly via inhalation and indirectly via circulation.

The endogenous ligand ACh activates two categories of receptors: ionotropic nAChRs and metabotropic muscarinic acetylcholine receptors (mAChRs) (18–20). nAChRs have two subclasses: muscular and neuronal (21). Muscle type nAChRs are well-known for their presence at the postsynaptic neuromuscular junctions, and for their role in neuromuscular transmission that generates skeletal muscle endplate potentials and subsequent muscle contraction. Neuronal nAChRs are found in the brain and autonomic ganglia, and are well-recognized for their preganglionic roles in both sympathetic and parasympathetic systems (22). However, recent studies suggest that nAChRs may also be expressed in other peripheral organs such as the lungs, which is the conceptual basis explored in this review (23, 24). nAChRs are pentameric structures originating from a complex protein of 270 kD and consisting of an extensive family of subunits ranging from α1 to α10, β1 to β4, γ, δ, and ε (25, 26). Each subunit consists of a large extracellular domain, four transmembrane domains, and a cytoplasmic domain. The extracellular domain is involved in binding acetylcholine, whereas the transmembrane domain spans the cell membrane and regulates the flow of ions through the pore. The cytoplasmic domain is responsible for intracellular signaling and regulation of receptor activity. The binding sites and variations in subunit structures explain the diverse effects of nicotine (3). Nicotine binding leads to allosteric changes in the receptor subunits leading to an activated state with an open ionic channel (27, 28).

Recent studies have demonstrated that the α7 subunit of the nAChR (α7nAChR) is particularly involved in mediating cellular response to nicotine (29–32). α7nAChRs have been investigated for their roles in multiple pathologies ranging from cognitive disorders such as Alzheimer’s disease, schizophrenia, and depression to cancer, infectious diseases, and allergic diseases (33–35). The rationale for focusing on α7nAChR rather than other subunits is due to 1) its heterotrimeric structure resulting in a higher probability of channel opening upon sensitization and desensitization; 2) high calcium permeability, and 3) various orthosteric binding sites (36). The role of α7nAChR is also extensively studied in the central nervous system and shown to regulate processes such as cellular excitability (27, 36, 37). In addition to its functional ability as an ion channel, α7nAChR is also involved in the cholinergic anti-inflammatory pathway (38–42). Although other subunits of the nAChR family of receptors have also been implicated in the anti-inflammatory pathway, much attention has been focused on the α7nAChR for its crucial role in regulating immune responses of multiple cell types. Accordingly, in the context of potential nicotine effects on the airways and its contribution to asthma and other lung diseases, the focus on α7nAChR in this review is justified.

UPSTREAM REGULATION OF nAChRs

nAChR expression and functionality at the plasma membrane is unique as it can be upregulated in response to agonist binding through both genomic and nongenomic mechanisms. Several complementary pathways are responsible such as 1) increasing migration of nAChRs to the cell surface; 2) conformational changes in nAChRs isoforms thereby affecting function; 3) increased intracellular trafficking of nAChRs with a reduced turnover on the cell surface; 4) increased exocytic trafficking of nAChRs; 5) cross talk or aberrant activations of secondary messenger pathways; 6) increased assembly in the endoplasmic reticulum (ER), most likely through intracellular activation of chaperones (RIC-3, NACHO, and others), and 7) potential receptor recycling mechanisms (43). Therefore, it is important to determine how these mechanisms cross talk and/or function independently upon exposure to nicotine (44). Moreover, recent reports suggest that the age of an individual may impact nAChR expression on the cell surface, ultimately influencing the individual’s predisposition to nicotine responses, including dependency (45). In addition, it is likely that the portfolio of nAChR subunits is organ- and cell type-specific, and further modulated by disease (46). nAChR expression and function may be differentially regulated during various developmental stages or under specific conditions of nicotine exposure, dose, duration, and/or by genetic differences (Table 1). More recently, several groups have used next-generation sequencing approaches to comprehensively estimate the binding regions on nAChR genes, although this is still in its infancy. Overall, these data suggest that nicotine effects in asthma may be substantially influenced by factors that drive cell- and context-dependent nAChR expression, even before any downstream signaling via nicotine and nAChRs may occur (Fig. 1).

Table 1.

Transcription factor binding site analyses of the nAChR gene reveal binding sites for the promoter sequences as shown below

Transcriptional Factor	nAChR Subunit	Agonist	Mechanism	Cell Lines	References
AP-2 α	α7	Nicotine	Involved in inducing EP4 expression via α7-PI3K/JNK and PKC signaling	A549 and H1838	(47)
NF-κB	α7	Nicotine	Upregulates α7 and induces activation of the JAK-STAT pathway	PC12	(48)
	α7	Nicotine	Attenuates MMP-9 production and inhibits NF-κB translocation into the nucleus via the PI3K/Akt pathway	RAW264.7	(49)
		Nicotine	Accelerates autophagic process via the ROS/ NF-κB signaling pathway	Apoe−/− mice	(50)
	α7	Acetylcholine	Activation of α7nAChR inhibits the nuclear translocation via JAK/STAT pathway	MDMs	(51)
	α4β2	Nicotine	Inhibits nuclear translocation of NF-κB	hα4β2 SH-EP1	(52)
	α7	Nicotine	Increases NF-κB translocation via NGF promoter	Lung fibroblasts	(53–55)
ER	α9	Nicotine	Activation of α7nAChR via the PI3K/Akt or the MAPK pathway	MCF-7 and MDA-MB-231	(56)
Ubx	α3	UBXN2A cofactor	Receptor ubiquitination and degradation	PC12 and HEK293T	(57)
c-Jun		Nicotine	Upregulates transcriptional activity of c-fos, c-jun, and JunB	Male SD rats	(58)
c-Fos	α4β2	Nicotine	Induction of nAChR via PI3K/ERK/AKT pathway	Male Wistar rats	(59)
		Nicotine and quinpirole	Induction of nAChR via p70S6K	D3R−/− mice	(60)
AP-1	α7	Nicotine	Downregulates ERK1/2 signaling pathway	RAW264.7 and MOVAS	(61)

α7nAChR, α7 nicotinic acetylcholine receptor.

Figure 1.

Upstream regulation of α7nAChR. In response to triggers or external stimuli, including nicotinic activation of α7nAChR, Ca²⁺, inflammation, and ROS, a number of transcriptional factors can be activated to induce α7nAChR gene expression. Following protein production of α7nAChR, the assembled receptor is chaperoned to the plasma membrane via RIC-3 and NACHO. Nicotine can accelerate ER stress by unfolded protein response (UPR) pathways. α7nAChR, α7 nicotinic acetylcholine receptor; ER, endoplasmic reticulum; ROS, reactive oxygen species. [Images created with a licensed version of BioRender.com.]

MECHANISMS OF α7nAChR EFFECTS

Nicotine per se is known to have a multitude of cellular effects such as contraction, proliferation and apoptosis, migration and adhesion, and angiogenesis (42, 62–66). Whether all nicotine effects involve α7nAChR is not known.

As an ionotropic receptor, α7nAChR activation leads to an influx of calcium across the plasma membrane, which can independently lead to downstream activation of signaling pathways. Nicotine via α7nAChR signaling mobilizes calcium from ryanodine-sensitive intracellular calcium stores leading to an increase in cellular cAMP levels (67, 68). The increase in intracellular calcium activates downstream signaling pathways, including calcium/calmodulin-dependent protein kinase (CaMK), PKC, and the cAMP response element-binding protein (CREB). However, calcium signaling via the α7nAChR is still a subject of investigation, particularly whether the effects occur in the context of acute versus chronic effects of nicotine. Recently, our group has shown that chronic nicotine exposure increases intracellular calcium influx in human airway smooth muscle cells, an effect that is abrogated in the absence of α7nAChR protein, or by channel blockers such as α-bungarotoxin and MLA (69, 70).

Nicotine via α7nAChR has indirect effects by activating other GPCR signaling pathways via direct binding to large heterotrimeric GTP-binding proteins (G proteins) (71, 72). Studies in nonlung cell types show that α7nAChR signaling via Rho GTPases enables mechanical plasticity during wound healing, thereby impacting cytoskeleton growth and formation (71, 72).

Nicotine via α7nAChR activates PI3K via Src family of proteins (Fig. 1), subsequently activating antiapoptotic AKT signaling pathway (73, 74). The antiapoptotic effects of nicotine are regulated by the MAPK pathway, which includes mitogen-activated protein kinase (MEK), extracellular signal-regulated-kinase (ERK), c-Jun, N-terminal kinase (JNK), and p38 kinase (75). These effects are mainly due to the action of nicotine on the Ser112, Ser136, or Ser155 residues of Bcl-2 antagonist of cell death (BAD) (76). Nicotine has also been shown to induce antiapoptotic signaling through phosphorylation of PKC, PKA, and NF-κB, further triggering downstream activation of tumor suppressor gene p53 (77, 78). Nicotine-induced NF-κB phosphorylation is mainly attributed to the inactivation of the apoptotic protein, BAD (79, 80). With respect to its direct effect on the Bcl class of proteins, nicotine can induce phosphorylation of Bcl-2 and its related proteins resulting in cisplatin-induced apoptosis in human small-cell lung carcinoma cells (81–83). Furthermore, administration of the nonspecific nAChR blocker, hexamethonium chloride counteracts the effects of nicotine on the ratio of proapoptotic Bax and antiapoptotic Bcl-2 proteins. These findings suggest that alterations in the levels of Bax/Bcl-2 proteins may underlie nicotine-induced apoptosis (84). Interestingly, nicotine induces the secretion of growth factors such as VEGF, bFGF, BDNF, TGFα, and PDGF in vitro (85–87), thus indirectly influencing cell proliferation. Such effects may involve α7nAChR-mediated recruitment of the β-arrestin-Ras-Raf kinase pathway leading to cell cycle entry (88), and conversely, controlling the progress from G2 to M phase of the cell cycle via modulation of NF-κB signaling pathway (84).

NICOTINE EFFECTS ON INFLAMMATION

α7nAChR is expressed in immune cells, including macrophages, dendritic cells, and T cells (89). The consensus is that nicotine via α7nAChR exerts a proinflammatory response, although there are some reports of anti-inflammatory effects (42, 74, 90, 91). These contradictory findings may reflect differences in concentration/dose, time of exposure, model system, and whether nicotine per se is responsible for α7nAChR activation. The proinflammatory effects of nicotine involve enhanced production of proinflammatory cytokines such as IL-1β, IL-6, and TNFα. Nicotine via α7nAChR also activates immune cells such as macrophages and dendritic cells leading to the production of proinflammatory cytokines and chemokines. α7nAChR activation can further enhance oxidative stress, thereby promoting inflammation. Notably, vagal modulation of immune responses is interlinked with lung function, and recent work suggests that vagal activity and α7nAChR signaling are important mechanisms for controlling inflammation (92). Recent reports have advanced our understanding of the intracellular signaling pathways involved in the pro- or anti-inflammatory potential of α7nAChR via nicotine, but more details remain to be established.

Conversely, to the proinflammatory effects, nicotine via α7nAChR has been shown to reduce the production of TNFα, IL-1β, and IL-6, and to inhibit the activation of immune cells. For example, in macrophages and dendritic cells, nicotine via α7nAChR reduces cell surface expression of costimulatory molecules: CD80 and CD86 (93). Mice deficient in α7nAChR exhibit increased TNFα production, but this effect remains independent of vagal stimulation (94). Similarly, Tracey et al. (95) showed that disruption of the α7nAChR in vivo significantly increased endotoxin-induced TNFα release. Here, ablation of the α7 subunit in the vagus nerve rendered the vagus nerve inefficient at inhibiting TNFα, indicating the importance of α7nAChR in vagal nerve inhibition of TNFα during systemic inflammation (95). As described previously, nicotine via α7nAChR inhibits the NF-κB signaling pathway, which is a key regulator of inflammation (96). This is supported by studies in human airway epithelial cells through α7nAChR-dependent inhibition of NF-κB signaling activity (97). Nicotine can induce the production of regulatory T cells to indirectly suppress inflammation and immune responses (98). In addition, α7nAChR enhances the activity of suppressor of cytokine signaling 3 (SOCS3) to regulate inflammation (99). α7nAChR activation initiates signal transduction and activation of the signal transducer and activator of transcription 3 (STAT3), which is phosphorylated by Janus kinase 2 (JAK2) and thus negatively controls NF-κB binding to the DNA (99). There are reports demonstrating that α7nAChR activation significantly inhibits inflammasome complex binding and activation, thereby preventing mitochondrial DNA release (100). Thus, nicotine through α7nAChR has the potential to modulate the immune system and defense functions, subsequently affecting growth and cell function and homeostasis. The plethora of nicotine effects is clearly prominent downstream. Collectively, α7nAChR has a pivotal role in mediating the inflammatory milieu, and importantly, α7nAChR may be differentially activated based on cell type and organ systems. Most likely, α7nAChR activation may be interfering with other signaling pathways and transcriptional factors (101, 102). Therefore, special emphasis should be placed on developing innovative therapeutic strategies to restore the equilibrium between α7nAChR-mediated stimulatory and inhibitory pathways. Overall, mechanistic research is needed to elucidate the context(s) under which this receptor exerts protective versus detrimental effects on the immune system. Numerous questions remain unanswered in understanding the role of α7nAChR in lung inflammation.

β-Arrestins belong to a family of scaffold proteins that recruit a broad spectrum of signaling molecules to membrane-bound receptors in an extremely coordinated manner (103, 104). Although only a handful of studies have examined the role of β-arrestin in nicotine or α7nAChR agonist responses, they have emerged as critical for nicotine-mediated activation of nAChRs to mediate cell proliferation and signaling (105, 106). Since nAChRs do not have any intrinsic tyrosine kinase activity, the molecular mechanisms underlying nicotine-mediated mitogenic activity are unclear (106, 107). A few elegant studies demonstrate that nicotine-mediated induction of cell proliferation involves the recruitment of β-arrestin to the nAChR, followed by facilitation of the Src for subsequent binding of Raf-1 kinase to Rb, and activation of the MEK pathway, leading to cell cycle entry (104–106). These signaling events promote the recruitment of transcription factors involved in cell proliferation, such as E2F genes belonging to the family promoting G1/S cell cycle transitions and others (108). Some studies investigated the association of β-arrestin polymorphisms in nicotine-dependent individuals in the nervous system (109, 110). Further research is needed to elucidate the biological mechanisms of this β-arrestin polymorphism in the increased risks associated with nicotine dependence in certain populations. In a separate study, adolescent β-arrestin2 knockout mice had impaired locomotor activity, whereas repeated administration of nicotine rescued locomotion in the same mouse model. This study suggests that β-arrestin-dependent changes are reflected in perceived nicotine reward (111).

NICOTINE EFFECTS ON OXIDATIVE STRESS AND MITOCHONDRIA

The balance between cell cycle progression and cell death is regulated by managing oxidative stress (112–114). Physiologically, ROS and other signaling molecules are involved in maintaining the normal homeostatic functions of the cell. The link between oxidative stress and apoptosis is well established (115–118) and nicotine promotes the expression of several apoptotic markers such as Bax/Bcl-2 (82, 119). Nicotine induction of apoptosis has been studied in neuronal and non-neuronal cells, but the mechanisms of nicotine-induced apoptosis and modulation of ROS generation in the context of e-cigs and vapes is a relatively new field under investigation. However, pilot studies using various lung cell types in vitro and studies investigating nicotinic effects in other tissues may help us glean potential mechanisms of nicotine on oxidative stress in the lung.

Cigarette smoke is well known to induce oxidative stress. The role of nicotine per se in inducing ROS is documented within a variety of tissues in the context of oxidative stress (120–122). For example, nicotine increases oxidative stress in rat mesencephalic cells (120). Specifically in the case of nicotine-containing aerosols, flavoring agents used as additives in many e-cigs also promote ROS, as shown in mouse lung epithelial cells (123). ROS generated from e-cigs is predominantly dependent on the flavor of the e-liquid used (e.g., fruity vs. menthol) (124). Separately, chronic nicotine exposure upregulates ROS generation in murine and lung epithelial cells, further triggering ER stress and UPR activation (Fig. 2) coincident with dysregulated p53 function (125). Although not well explored in the context of receptor subunit effects on ROS, one study showed that nicotine exposure reduced ROS production and enhanced cell proliferation via the α4nAChR subunit in human-induced pluripotent stem cells (126).

Figure 2.

Nicotine via α7nAChR influences ER stress and cellular metabolism. Nicotine, through α7nAChR, triggers endoplasmic reticulum (ER) stress target genes such as eIF2α and XBP1 via phosphorylation of the PERK, ATF6, and IREIα activation on the ER. Activation of ER stress response elements leads to transcriptional activation of genes related to inflammation, proliferation, and migration signaling pathways. α7nAChR, α7 nicotinic acetylcholine receptor. [Images created with a licensed version of BioRender.com.]

There is increasing interest in understanding the potential direct role of nAChRs localized at the mitochondrial membrane and their role in ROS generation when exposed to nicotine (Fig. 3) (100, 127–132). One comprehensive study showed that α7nAChR is the most abundant subtype expressed at the mitochondrial membrane (128). Given this subtype is most permeable to calcium, α7nAChR may indeed influence mitochondrial calcium buffering (133). Whether such functional effects occur broadly in the context of mitochondrial calcium regulation is yet to be determined.

Figure 3.

Nicotine via α7nAChR influences mitochondrial structure and function. Nicotine causes mitochondrial fusion in response to chronic exposure by increasing the accumulation of Mfn1/2 and Opa1 thereby leading to a reduced oxygen consumption rate and increasing extracellular acidification rate (ECAR). The inverse holds true under pathologically healthy conditions. Mitochondrial fission occurs in response to increased ROS resulting in a downregulation of the fusion genes such as Mfn1/2 and Opa1 and an activation of the fission genes such as Drp1 and Fis1. These subsequently leads to mitochondrial DNA mutations, thereby mitochondrial dysfunction and mitophagy.α7nAChR, α7 nicotinic acetylcholine receptor; ROS, reactive oxygen species. [Images created with a licensed version of BioRender.com.]

NICOTINE AND α7nAChR IN ASTHMA

Asthma poses a significant health challenge to people of all age groups and is influenced by predisposing factors such as sex, comorbidities, and likely lifestyle habits (134–137). Inflammation plays a crucial role in regulating the pathophysiology of asthma, and therefore compounds that inflame or irritate the immune system or cells of the airway (e.g., cigarette smoke and/or nicotine) are risk factors for asthma induction or exacerbation (138–141). In this regard, although a WHO survey in the early 2000s showed no significant difference in the proportion of smokers suffering from asthma versus individuals with asthma in the general population, the detrimental impact of both cigarette smoke and second-hand smoke exposure including in children and in asthmatic young adults was not in question (142). Furthermore, given observed sex differences in asthma, although postpubertal males have a significantly higher prevalence of smoking than postpubertal females (26% and 5%, respectively), the incidence and severity of asthma due to cigarette-smoking or nicotine-induced asthma are increasingly more prevalent in postpubertal females (143, 144). These observations highlight the importance of considering sex differences in studies. Beyond inflammation, factors such as oxidative stress and ROS induced by cigarette smoke and/or nicotine can further lead to worsening asthma and respiratory health outcomes.

The influence of maternal smoke exposure on asthma risk for the fetus represents a unique morbidity. Unfortunately, this concern is heightened knowing that there are targeted advertisements of nicotine-containing products aimed toward females, including those who are pregnant (145, 146). Although most published data to date have focused exclusively on the effects of nicotine exposure in adult mice, it has been demonstrated fairly well that secondhand smoke exposure or maternal cigarette smoking/smoke exposure adversely affects the developing fetus and neonates. This leads to pulmonary distress, including developmental defects and loss of weight (147). Here, infants born to mothers exposed to smoke are reported to have an increased risk of bronchial hyperresponsiveness and altered pulmonary function tests (145). To determine sex-specific effects of perinatal nicotine exposure, a study on male and female rats showed male offspring being functionally more susceptible to asthma after in-utero nicotine exposure (148). Moreover, the same study also showed worsened airway resistance and compliance in male offspring as compared with their female counterparts. Multiple studies have explored potential mechanisms underlying the postnatal occurrence of asthma following smoke or nicotine exposure (148). One group showed that maternal exposure to nicotine activates the PI3K-Akt pathway and upregulates β-catenin levels, thereby dysregulating the Th2-Th17 biases and checkpoints in the offspring, a process that persists after birth (149).

In youth and adults, it is often observed that nicotine dependence develops faster with symptoms of asthma (150). Asthma is commonly associated with a Th2-skewed inflammatory response (151, 152). Recently, studies have shown an anti-inflammatory role of vagal α7nAChR signaling in regulating ILC2 recruitment in the lung (153). α7nAChR^−/− mice exhibit exaggerated lung inflammation and there appears to be a pivotal role via S1P-dependent signaling from Th2 lymphocytes to dampen the recruitment of ILC2 to the lungs. Although this might support an anti-inflammatory role for α7nAChR signaling in the context of asthma, multiple other studies, including our own suggest a proinflammatory role. For example, using human airway smooth muscle cells, we show an increase in expression of α7nAChR in individuals with asthma, with higher expression in smoking asthmatics compared with nonasthmatics and nonsmokers, respectively (154, 155). α7nAChR is further increased by exposure to nicotine, proinflammatory cytokines, and cigarette smoke extract. Inflammation promotes NF-κB, AP-1, and CREB signaling in enhancing α7nAChR expression and signaling in human airway cells (154). In a lung injury model, lung endothelial cells show a dose-related loss of barrier function, involving enhanced oxidative stress (156). Other studies note a role for other nAChR subunits such as α3, α5, and β4 in regulating cell proliferation and behavior in bronchial epithelial cells (157).

Although the focus has previously been on studying nicotine in the context of cigarette smoke exposure, there is increasing interest in understanding nicotine in the asthmatic airway in the context of e-cigarettes (e-cigs) (158–161), with the recognition that e-cigs can also cause respiratory harm similar to and/or different from traditional cigarette smoking (162–164). This is particularly an issue given the greater societal and familial leniency toward vaping as opposed to smoking, especially in teenagers and young adults. Here, a further complicating factor is the potential detrimental effects of flavored liquids within e-cigs, which can exacerbate nicotine effects or independently lead to lung injury. One study investigated the effect of chronic e-liquid exposure containing propylene glycol alone and in combination with nicotine on wild-type and α7nAChR knockout mice (131). To our knowledge, this study was the first to reveal α7nAChR-dependent cardiopulmonary dysfunction in vivo: mice deficient in α7nAChR exhibited heightened inflammatory response to nicotine compared with wild-type counterparts (131). A probable explanation for the lack of studies on first- and second-hand vaping/vapor exposure on pulmonary physiology can be attributed to the lack of animal models that accurately reflect findings from cross-sectional studies in humans. Therefore, more comprehensive investigations are warranted to clearly demonstrate potentially detrimental effects of vaping or e-cigs on the lung and asthma pathogenesis.

NICOTINE AND nAChRs IN AIRWAY SMOOTH MUSCLE CELLS

Decreased lung function is associated with increased airway smooth muscle (ASM) mass, which is often the most important contributing factor responsible for airway remodeling-associated changes (165, 166). However, the mechanisms underlying these changes in ASM are unclear. Early studies explored the effects of nicotine via α7nAChR in rat ASM cells that express α2–7, β2, and β3-nAChR subunits, with α7nAChR most abundantly expressed and playing a major role in nicotine effects on Ca²⁺ regulation and cell proliferation via the Akt pathway (167, 168). Another study suggested that nicotine induces ASM cell proliferation via activation of α7nAChR and transient receptor potential protein 6 (TRPC6) (169). Conversely, inhibition of α7nAChR attenuated PI3K/Akt phosphorylation, thereby regulating calcium entry and proliferation. A role for α5nAChR in conjugation with TRPC3 in exaggerating ASM cell proliferation and calcium increase has also been shown (170). In terms of human airways, nicotinic receptors were previously assumed to be present only in airway neurons; however, we first showed that human ASM also expresses these receptors, specifically α7nAChR (154). We further showed that expression of α7nAChR is exaggerated in individuals with asthma and by proinflammatory cytokines, cigarette smoke, or nicotine itself (154). These initial findings support the idea that α7nAChR may play a role in asthma pathophysiology, particularly in the context of nicotine exposure. More recently, we demonstrated that functional α7nAChRs are indeed expressed in human ASM and that α7nAChR activation leads to an increase in ASM [Ca²⁺]_i and proliferation (155). Thus, exploring the role of nicotine and ASM nAChR, especially α7nAChR, in asthma could lead to new therapeutic strategies, but more research is needed to fully understand the different effects of α7nAChR in the context of ASM and asthma.

NICOTINE AND nAChRs IN EPITHELIUM

Initial studies showed that nicotine induces ASM contraction via the airway epithelium (171). Using tracheal rings with/without epithelium, dose-response curves in the presence of ACh and nicotine showed that ASM-induced contraction is lost by denudation of the epithelial layer. Furthermore, removal of nerves close to the epithelium attenuates nicotine-induced bronchoconstriction (171). Interestingly, unlike ASM cells, there is a wealth of evidence investigating the effects of nicotine via nAChRs in epithelial cells of the lung (172, 173). Not only limited to the lung, epithelial cell lines from other origins such as the retina, colon, and GI have also shown that nicotine via α7nAChR has effects in modulating systemic inflammatory response, thus contributing to processes such as cell development, death, migration, and angiogenesis (174, 175). With respect to the lung, nicotine via α7nAChR modulates multiple inflammatory responses. In a mouse model harboring an α7nAChR-specific point mutation that selectively uncouples the receptor from α7nAChR-associated calcium signaling mechanisms, LPS responses are blunted and mediated through the epithelium. However, the precise effects of nicotine in this model are not known (176). Another study in cultured bronchial epithelial cells (BECs) of the rhesus monkey found that chronic nicotine exposure significantly upregulates the expression of α3nAChR, α4nAChR, α7nAChR, α9nAChR, α10nAChR, β2nAChR, and β4nAChR (177). Moreover, chronic nicotine exposure increases nicotine-induced currents in voltage-clamped BECs via protein phosphorylation and dephosphorylation of tyrosine kinase. In human and murine lung epithelial cells, increases in expression of α3nAChR, α4nAChR, α5nAChR, and α7nAChR subunits following stimulation with nicotine have been noted. Interestingly, increasing the concentration of nicotine results in receptor desensitization, implicating that pathobiological effects of nicotine may result from both activation of the nAChR as well as nAChR desensitization (178). In contrast, a study in HT29 epithelial cell lines (relevant to ulcerative colitis) observed that increasing concentrations of nicotine inhibited release of IL-8 indicating the beneficial effect of nicotine (168). Separately, a study in two sets of primary human airway epithelial cell cultures, normal human bronchial epithelial cells (NHBEs) and small airway epithelial cells (SAECs) demonstrated that nicotinic activation of nAChRs is regulated by Akt/PI3K (73).

NICOTINE AND nAChRs IN LUNG FIBROBLASTS

Chronic lung diseases involve tissue remodeling processes such as fibrosis, which is mediated in large part by fibroblasts. Injured lungs, resulting from vaping, for example, have alterations in the basement membrane that are thought to support migration of surface epithelial cells. Furthermore, this can promote ASM-derived extracellular matrix deposition and further remodeling in the airways. Diseases that are driven by smoking, such as chronic obstructive pulmonary disease (COPD) are well known to involve fibrosis. Nicotine via α7nAChR has been shown to increase collagen and fibronectin content in murine lung fibroblasts (179). Even in utero nicotine exposure potentiates the expression of collagen in isolated pulmonary fibroblasts of progeny and this effect is mediated by α7nAChR (180). Moreover, studies have shown that among the numerous glycoproteins expressed by lung fibroblasts, fibronectin is the most common glycoprotein that is stimulated by α7nAChR via activation of protein kinase C and mitogen-activated protein kinases (181). Interestingly, airway fibroblasts express all the different nAChRs, although only α7nAChR is highly expressed in human airway fibroblasts (182). However, the mechanisms of α7nAChR action in airway fibroblasts have not been extensively studied (182, 183). Interestingly, in mouse models, α7nAChR appears to induce nerve growth factor (NGF) release via NF-κB by lung fibroblasts (53). This aspect of α7nAChR activity also appears to be important for idiopathic pulmonary fibrosis (IPF) (184). Nicotine activation of α3nAChR and α7nAChR is accompanied by activation of the PKC and Wnt signaling pathways resulting in a fibroblast to myofibroblast transdifferentiation (185).

NICOTINE AND nAChRs IN NERVE CELLS

It is well known that nAChRs are expressed in airway-related vagal preganglionic neurons (AVPNs) and that nicotine acts centrally to increase bronchomotor tone and airway constriction (186) (Fig. 4). Among the numerous subunits of the nAChRs, α7nAChR is the most abundant and functionally characterized (187, 188). Beyond effects on neurotransmission that lead to bronchoconstriction, α7nAChR activates the PI3K/Akt pathway through activation of a member of the Src family in neuronal cells from an Alzheimer's disease model (189). In the developing lung, nicotine causes significant increases in the number of neuroepithelial cells and neuroendocrine cells via binding to α7nAChR (190). Nicotine also increases α7nAChR expression in other developing organ systems (191–193). Cigarette smoke-induced airway cough and irritation are triggered by the binding of nicotine to neuronal nAChRs and increases in [Ca²⁺]_i (194). In guinea pigs, nicotine induces bronchoconstriction through the cholinergic reflex pathway by activating nAChRs in bronchopulmonary sensory nerves (195).

Figure 4.

Nicotinic receptors after activation in the airway. Nicotine, inflammatory stimuli, and other exogenous triggers such as cigarette smoke exposure leads to conformation changes in nAChRs receptors expressed in airway epithelial cells, airway smooth muscle cells, and fibroblasts. These triggers are usually resultant of ACh release from the nerves that further stimulate the postganglionic nerve fibers, subsequently stimulating nAChRs present on the effector organ, in this case, the lung resident cells. nAChRs, nicotinic acetylcholine receptors. [Images created with a licensed version of BioRender.com.]

NICOTINE AND nAChRs AS POTENTIAL THERAPEUTIC TARGETS

Given the substantial detrimental effects of cigarette smoke and the increased introduction of e-cigs as a “safer” option for individuals with asthma, there is a perception that nicotine use via e-cigs might be beneficial. However, there is still a focus on the use of medications to promote smoking cessation per se. Varenicline and bupropion are both Food and Drug Administration (FDA)-approved therapeutics for smoking cessation (196, 197). Varenicline is a partial α4β2 nAChR agonist while also having less potent effects on α7 and α3β4 nAChRs and is the drug of choice for smoking cessation. The dual action of varenicline functions by partially blocking nAChRs without creating the full effect of nicotine while simultaneously blocking the nAChR as an antagonist. Bupropion, originally discovered as an antidepressant, works as a weak antagonist of α4β2 and α3β4 nAChRs (198). Dianicline (SSR591813) is another partial agonist of the α4β2 nAChR providing reduced withdrawal symptoms during smoking cessation (199, 200). The rationale behind the use of partial agonists for smoking cessation is that binding to central α4β2 nAChRs in the reward pathway results in a modicum of dopamine release from mesolimbic dopamine neurons. This then, provides some relief from withdrawal symptoms, without a strong level of stimulation sufficient to evoke the reinforcing effect. There is considerable progress in understanding the mechanism of nicotine addiction and their translation into potential drugs or targets that interfere with nicotine signaling. Newer compounds such as the cytisine, which is a structural analog of nicotine and a partial α4β2 nAChR agonist, have been used for smoking cessation in parts of Europe (201–203). Cytisine also has a high affinity for other nAChR subtypes, the mechanisms of which are still unclear. Although these medications have clear central nervous system and systemic effects toward smoking cessation and addiction, their role in blunting the effects of smoking or nicotine in asthma has not been explored. Undoubtedly, interventions such 77as smoking cessation strategies with/without pharmacology will continue, especially due to the increased use of e-cigs, and the (mis)perception that e-cigs might be a safer option. Ultimately, it is challenging to simply pursue a cessation strategy and implement a simple “one size fits all” approach per se. Therefore, further research is necessary to understand whether and how nAChRs, particularly a7nAChRs within airways could be modulated in a beneficial manner to reduce asthma burden in e-cig users and/or smokers.

PERCEPTIONS, LIMITATIONS, AND PROSPECTS

Many research questions remain to be answered regarding the interactions between nicotine, the nicotinic acetylcholine receptor, and asthma, such as 1) Are there any confounding effects of corticosteroids on nicotine abstinence or addiction in individuals with asthma? 2) What are the characteristic phenotypic features in asthmatic airways of chronic smokers? 3) Does smoking cessation improve asthma therapy or response to corticosteroids? 4) While the focus has been on understanding the specific effects of α7nAChR in the airways, are there any specific subunit interactions playing a greater role in potentiating asthma or asthma in smokers? 5) Whether α7nAChR localization in airway resident cells plays a role in mediating downstream signaling cascades is a matter of current investigation: what are the dose/concentration, timing of administration, and duration factors to be considered when proposing a therapy? 6) How do we build experimental studies to obtain consistent and reliable results since such confounding cross talk between the receptors (including several subunits and ligands) exists? These questions, along with others, remain to be identified and answered, as long as smoking persists.

GRANTS

This work was supported by the National Institutes of Health (NIH) Grants R01 HL142061 (to C.M.P. and Y.S.P.) and R01 HL088029 (to Y.S.P.).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

N.A.B. conceived and designed research; N.A.B. prepared figures; N.A.B., M.A.T., C.M.B., and L.K. drafted manuscript; N.A.B., M.A.T., C.M.B., L.K., S.S., V.S., Y.S.P., and C.M.P. edited and revised manuscript; N.A.B., M.A.T., C.M.B., L.K., S.S., V.S., Y.S.P., and C.M.P. approved final version of manuscript.