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Endocrinology Vol. 145, No. 5 2498-2506
Copyright © 2004 by The Endocrine Society

Acetylcholine Is an Autocrine or Paracrine Hormone Synthesized and Secreted by Airway Bronchial Epithelial Cells

Becky J. Proskocil, Harmanjatinder S. Sekhon, Yibing Jia, Valentina Savchenko, Randy D. Blakely, Jon Lindstrom and Eliot R. Spindel

Division of Neuroscience (B.J.P., H.S.S., Y.J., E.R.S.), Oregon National Primate Research Center, Beaverton, Oregon 97006; Department of Pathology (H.S.S.), Oregon Health & Science University, Portland, Oregon 97201; Department of Pharmacology (V.S., R.D.B.), Vanderbilt University School of Medicine, Nashville, Tennessee 37232; and Department of Neuroscience (J.L.), University of Pennsylvania, Philadelphia, Pennsylvania 19104

Address all correspondence and requests for reprints to: Dr. Eliot Spindel, Division of Neuroscience, Oregon National Primate Research Center, 505 Northwest 185th Avenue, Beaverton, Oregon 97006. E-mail: Spindele{at}ohsu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The role of acetylcholine (ACh) as a key neurotransmitter in the central and peripheral nervous system is well established. However, the role of ACh may be broader because ACh may also function as an autocrine or paracrine signaling molecule in a variety of nonneuronal tissues. To begin to establish ACh of nonneuronal origin as a paracrine hormone in lung, we have examined neonatal and adult monkey bronchial epithelium for the components involved in nicotinic cholinergic signaling. Using immunohistochemistry and RT-PCR, we have demonstrated in lung bronchial epithelial cells (BECs) expression of choline acetyltransferase, the vesicular ACh transporter, the choline high-affinity transporter, {alpha}7, {alpha}4, and ß2 nicotinic ACh receptor (nAChR) subunits, and the nAChR accessory protein lynx1. Confocal microscopy demonstrates that these factors are expressed in epithelial cells and are clearly distinct from neighboring nerve fibers. Confirmation of RNA identity has been confirmed by partial sequence analysis of PCR products and by cDNA cloning. Primary culture of BECs confirms the synthesis and secretion of ACh and the activity of cholinesterases. Thus, ACh meets all the criteria for an autocrine/paracrine hormone in lung bronchial epithelium. The nonneuronal cholinergic signaling pathway in lung provides a potentially important target for cholinergic drugs. This pathway may also explain some of the effects of nicotine on fetal development and also provides additional mechanisms by which smoking affects lung cancer growth and development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE IMPORTANCE OF acetylcholine (ACh) as a neurotransmitter in both the central and peripheral nervous systems is well established. As a neurotransmitter, ACh plays essential roles in learning, memory, pain perception, and autonomic regulation. ACh may not only be a neurotransmitter, but it may also be a widely expressed paracrine factor synthesized in nonneuronal cells. Yet, despite more than 25 yr of isolated reports, the role of ACh as an autocrine signaling molecule expressed by a variety of nonneuronal cell types is not yet generally accepted. This likely reflects the lack of systematic studies demonstrating all aspects of cholinergic signaling in a single cell type and the lack of a clear function for nonneuronal ACh.

The initial description of nonneuronal ACh synthesis was by Morris (1), who in 1966 reported that ACh was synthesized in the placenta. Subsequent to the report by Morris, there have been multiple reports of the expression of ACh, acetylcholinesterase, and cholinergic receptors in the placenta (for reviews, see Refs.2 ,3). Parnavelas et al. (4) reported choline acetyltransferase (ChAT) expression in vascular endothelial cells in brain and suggested it plays a role in regulating vasodilation. ChAT has also been reported in glia (5, 6), white blood cells (7), keratinocytes (8), and esophageal epithelium (9). The case for nonneuronal autocrine signaling may be strongest in lung bronchial epithelial cells (BECs). Reports by Klapproth et al. (10) and Reinheimer et al. (11) have described ACh synthesis by BECs, and multiple reports have shown that BECs express active nicotinic receptors (12, 13, 14). Importantly, these components of cholinergic signaling have also been reported in lung cancers, and nicotinic activation appears to stimulate lung cancer cell growth (15, 16, 17).

As well as affecting cell growth, autocrine cholinergic signaling likely plays a role in lung development and may explain some of the effects of maternal smoking during pregnancy on lung development. Offspring of women who smoked during pregnancy show diminished lung function at birth (18) and increased incidence of respiratory illnesses (19). In monkeys, prenatal nicotine exposure similarly alters pulmonary function at birth (20). Because nicotine freely passes the placenta and is detected in the amniotic fluid of smoking mothers (21), the simplest explanation for this sensitivity would be the expression of nicotinic ACh receptors (nAChR) in developing lung. Consistent with this hypothesis, examination of airway epithelium in fetal monkey lung showed abundant expression of nAChR (13, 22, 23).

A key question yet to be addressed is which components of neuronal cholinergic signaling are present in nonneuronal systems. In neural systems, choline is transported into neurons by the recently characterized choline high-affinity transporter (CHT) (24, 25), synthesized into ACh by ChAT, packaged into vesicles by the vesicular ACh transporter (VAChT) (26), and released to interact with cholinergic receptors. In this article, we demonstrate that all the components for a nonneuronal autocrine/paracrine cholinergic loop are present in monkey airway BECs. The data presented here demonstrate that ACh must be regarded not only as a neurotransmitter but also as an autocrine hormone synthesized by nonneuronal cells and that this finding provides a mechanism to explain the effects of nicotine on both lung growth and lung development.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Immunohistochemistry (IHC)
Whole lungs were dissected from fetal to adult rhesus macaques. Lungs were either inflated to 20 cm H2O with zinc formalin (Fisher Scientific, Hampton, NH) or immersion fixed in Bouin’s fixative (Sigma Chemical Co., St. Louis, MO) or 4% paraformaldehyde. Zinc formalin and Bouin’s fixed tissue were processed for paraffin sectioning (5 µm), and paraformaldehyde fixed tissue was processed for cryostat sectioning (10 µm). All animal procedures were approved by the Oregon National Primate Research Center Institutional Animal Care and Utilization Committee. Sections were processed for IHC as previously described (22). Antibodies used were as follows: rat antihuman {alpha}4 nAChR [monoclonal antibody (mAB) 299, 1:250] (27), rabbit antihuman ß2 nAChR (antibody 3724, 1:1000) (28), rat anti-{alpha}7 nAChR (mAB 319, 1:250) (28), mouse anti-ChAT (mAB 305, 1:400; Chemicon International, Inc., Temecula, CA), rabbit antihuman VAChT (1:500; Phoenix Pharmaceuticals, Inc., Belmont, CA) (29), rabbit anti-CHT (polyclonal antibodies and mABs as previously described in Refs. 25 and 30), and rabbit anti-PGP9.5 (1:1000; Research Diagnostics, Flanders, NJ). Antibodies to lynx1 were custom-made as described previously (31). Lynx1 antibody LR4 was used at a dilution of 1:600. All antibody reactions were also tested with nonimmune serum to test for nonspecific staining.

Dual fluorescence IHC
Fluorescent IHC on tissue fixed in 4% paraformaldehyde was processed similarly to standard IHC with the following exceptions. Blocking and primary antibody solutions contained both sera and antibodies, respectively, required for the dual fluorescence. Rabbit PGP9.5 was used to visualize neuronal fibers. Fluorescence for proteins of low abundance (ChAT and VAChT) was amplified using the Tyramide Signal Amplification System (PerkinElmer Life Sciences, Wellesley, MA). The slides were mounted with Vectashield mounting media for fluorescence containing 4',6-diamidino-2-phenylindole (DAPI) for nuclear labeling (Vector Laboratories, Burlingame, CA). Specificity of staining was tested by inclusion of nonimmune sera in both single- and dual-antibody reactions. Slides were viewed on a confocal microscope, and images were captured as stacks and projections.

RT-PCR and real-time PCR
RNA was prepared from adult monkey hypothalamus, whole monkey lung of the ages shown in Fig. 3Go, and monkey lung BEC scrapings. For BEC scrapings, the lungs were dissected out and then carefully sliced along the major airways to expose the internal lumen. The left and right bronchi and the primary branches were gently scraped with a plastic inoculation loop. RNA from tissue was prepared with guanidine thiocyanate and cesium chloride for whole tissues (32), and TriReagent (Molecular Research Center, Inc., Cincinnati, OH) was used isolate RNA from the BEC scrapings. PCR was performed as described previously (22) using specific primers for monkey {alpha}4 nAChR, ß2 nAChR, {alpha}7 nAChR, and lynx (see Table 1Go for sequences). All amplified bands were of the correct size consistent with the chosen primers. Primers designed to amplify single exons of ChAT (N, S, 5, 10, and 12, as denoted in Ref.33) were used to characterize the expression of ChAT in newborn monkey lung. Expression was confirmed by probing blotted gels with 32P-labeled internal primers. CHT expression was examined with primer sets corresponding to the N terminus and C terminus of the coding region of CHT (Table 1Go). Real-time PCR for ChAT and {alpha}7 and ß2 nAChR was performed as described previously (23). All samples were standardized to 18S RNA concentrations measured simultaneously in duplex reactions.



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  		FIG. 3. Levels of ChAT, {alpha}7 nAChR, and ß2 nAChR mRNAs in monkey whole lung as a function of age. Levels of RNA were measured by real-time PCR as described in Materials and Methods. Age is shown as days postconception (term = 165) and then in years of age. Slope of lines fitted to RNA levels was not significantly different from 1.0 for any of the RNAs, indicating no significant change with age.

 

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  		TABLE 1. RT-PCR and real-time PCR primers

 
cDNA library construction and {alpha}7 nAChR cDNA sequencing
A cDNA library was prepared from newborn monkey lung in the vector {lambda}-Zap with methods as previously described (34). The library was screened with the amplified {alpha}7 nAChR fragment. Hybridizing clones were excised into the plasmid bluescript and sequenced in both directions using an ABI 373 (Applied Biosystems, Foster City, CA) automated sequencer. The {alpha}7 nAChR cDNA sequence has been deposited in GenBank with accession no. AF486623.

Epithelial cell culture
BEC cultures were established as described by Wu et al. (35). In brief, monkey lungs were obtained at necropsy and immersed in ice cold MEM (MEM + 15 mM HEPES + 50 µg/ml gentamicin + penicillin-streptomycin). The major bronchi were crudely dissected and then incubated overnight at 4 C in 0.1% Protease type 14 (Sigma) in MEM. The cells were washed from the mucosal surface of the airways using MEM with 5% fetal calf serum, centrifuged, resuspended, and plated in bronchial epithelium culture medium (50% Ham’s nutrient mixture F12 + 50% DMEM + 1.8 mM calcium chloride, 5.0 µg/ml insulin, 5.0 µg/ml transferrin, 20 ng/ml epidermal growth factor, 0.1 µM dexamethasone, 20 ng/ml cholera toxin, 30 µg/ml bovine hypothalamic extract, and 1.0 µM retinol) containing 2% fetal calf serum. The following day, the cells were changed to serum-free bronchial epithelium culture media. To measure ACh released from the cells, media samples were spun at 1000 rpm for 5 min to remove cellular debris and immediately frozen on dry ice. For ACh assay, 20 µl medium was injected directly into a HPLC using enzyme-coupled electrochemical detection as previously described (17). Neostigmine (50 µM) was added to some cells for 3 d to prevent ACh degradation.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Lungs were collected from newborn rhesus monkeys delivered by cesarean section at d 160 of gestation (5 d before natural birth). IHC showed expression of ChAT, VAChT, and the {alpha}4, ß2, and {alpha}7 nAChR subunits in airway BECs (Fig. 1Go). Expression was primarily found throughout BECs (see Figs. 1Go and 5Go), except {alpha}4 was occasionally observed concentrated in the basal region of the BEC layer (Fig. 1aGo). Control experiments in which nonimmune serum was added in substitute for primary antibody showed no specific binding (Fig. 1Go, a and e, inserts).



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  		FIG. 1. a, {alpha}4 nAChR; b, ß2 nAChR; c, {alpha}7 nAChR; d, ChAT; and e, VAChT are detected by IHC in newborn monkey lung airway bronchial epithelium (original magnification, a and e, x100; b–d, x200). The picture insetsin a and e show the control IHC for {alpha}4 and VAChT, respectively, using nonimmune serum in replacement of the primary antibody. The cell nuclei are counterstained with hematoxylin (blue). A, Airway; E, epithelium; C, cartilage. Chromogen = AEC.

 


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  		FIG. 5. RT-PCR analysis showing expression of ChAT in RNA from BEC scrapings. RNA was made from BECs scraped from the inside of main bronchi and then amplified with primers corresponding to the human ChAT exons as shown. The same-sized PCR products were amplified from RNA from monkey hypothalamus (brain) and whole monkey lung (lung). Primers used are described in Materials and Methods. Primers from exons 6 and 18 also amplified the expected sized bands (data not shown).

 
Miwa et al. (36) and Ibanez-Tallon et al. (37) have recently described the protein lynx1, which has a similar folding structure to {alpha}-bungarotoxin. Lynx1 is a cell surface glycosylphosphatidylinositol-linked protein that is highly expressed in mouse brain in neurons that express {alpha}7 and {alpha}4ß2 nAChR, and lynx1 appears to interact with nAChR to modulate ACh-evoked responses (36, 37). IHC showed strong lynx1 staining in newborn monkey lung bronchial epithelium, similar to {alpha}4, {alpha}7, ß2, ChAT, and VAChT (Fig. 2Go). Rabbit preimmune serum, in place of lynx1 primary antibody, showed no staining (data not shown).



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  		FIG. 2. IHC using lynx1 antibody LR4 showed strong staining in airway epithelium. a, Magnification, x200. b, Higher power view of boxed regionin a; magnification, x630. Lynx1 immunoreactivity is evenly distributed throughout cells. The cell nuclei are counterstained with hematoxylin (blue). A, Airway; E, epithelium. Chromogen = AEC. Note: antibody LR1 prepared to a different portion of lynx1 showed a similar pattern of staining (data not shown). No staining was seen with nonimmune serum.

 
The expression of these proteins is not just a developmental phenomenon. Lung tissue was collected from differently aged monkeys. IHC showed an identical pattern of ChAT and nAChR expression in 1- and 7-yr-old monkeys as was seen in newborn monkeys (data not shown). This was further quantified by measuring {alpha}7, ß2, and ChAT mRNA levels by real-time PCR in monkey lung from fetal to adult-aged animals (Fig. 3Go). As shown in Fig. 3Go, no clear developmental pattern of expression is evident.

Because the lung epithelium is innervated with fine nerve fibers, we tested the possibility that the observed lynx1 and the {alpha}4, ß2, and {alpha}7 nAChR were actually expressed in those fibers. PGP9.5 was used as a marker for peripheral nerve fibers. As shown in Fig. 4Go, {alpha}4, ß2, VAChT, and ChAT were clearly expressed in cells distinct from the PGP9.5-containing nerve fibers by confocal imaging ({alpha}7 and lynx1 were similarly not expressed in the bronchial epithelial nerve fibers, data not shown). Thus, nAChR and the enzymes necessary for ACh synthesis and secretion are primarily expressed in airway BECs and are not apparent in nerve fibers.



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  		FIG. 4. Confocal microscopy showing that expression of nicotinic receptors and VAChT is apparent in airway BECs and not in nerve fibers. PGP9.5 was used to stain nerve fibers (green). Confocal images show {alpha}4 (a, red), ß2 (b, red), and VAChT (c, red) are expressed in bronchial epithelial cells and do not colocalize with nerve fibers in airway bronchial epithelium. d, Colocalization (yellow) of {alpha}4 (green) and ChAT (red) in bronchial epithelium. Original magnification, a, x2040; b and c, x1280; and d, x600. Cell nuclei are counterstained with 4',6-diamidino-2-phenylindole (blue). A, Airway; E, epithelium.

 
Because Gault et al. (38) has reported expression of an {alpha}7 nAChR pseudogene, we felt it important to confirm that authentic {alpha}7 nAChR was expressed in lung. A cDNA library from neonatal monkey lung was prepared and screened for {alpha}7 nAChR. Multiple positive clones were identified, and six clones were sequenced in full. The cDNA sequence obtained from all clones was highly homologous to human neuronal {alpha}7, and the overall identity level was 96% identical to human {alpha}7. No clones encoding the pseudogene exons reported by Gault et al. (38) were identified. The complete amino acid sequence of monkey lung {alpha}7 had only five differences from the human neuronal {alpha}7 amino acid sequence. Thus, authentic neuronal {alpha}7 nAChR is clearly expressed in lung. This sequence has been deposited in GenBank with accession no. AF486623.

Next, RT-PCR was performed to prove the specificity of the IHC and to confirm the expression of cholinergic RNA in the BECs. Taking advantage of the high homology between monkey and human sequences that typically allows primers complementary to human DNA sequences to amplify the corresponding monkey sequences, primers were prepared for exons N, S, 5, 10, and 12 of ChAT (33), and RNA was amplified from adult monkey hypothalamus, newborn lung, and BEC scrapings from monkey lung. All ChAT exons examined were present in airway epithelium, confirming that the form of ChAT expressed in bronchial epithelium is highly similar to neuronal ChAT (Fig. 5Go). All bands gave the correct size PCR product from the designed primers and hybridized to internal 32P-labeled oligo probes. Similarly, primer sets for {alpha}4, ß2, {alpha}7, and lynx1 were prepared, and the cholinergic RNAs were shown to be present in the BEC scrapings (Fig. 6Go). The amplified PCR fragments from lung for {alpha}4, ß2, lynx1, and ChAT were sequenced to further confirm the identity of the amplified products.



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  		FIG. 6. RT-PCR analysis showing expression of {alpha}4 nAChR, {alpha}7 nAChR, ß2 nAChR, and lynx1 in RNA from BEC scrapings, whole lung, and hypothalamus. All bands were of expected size, and PCR products were blotted and probed with an internal 32P-labeled probe to confirm expression. Primers used are described in Materials and Methods.

 
Because the presence of mRNAs and proteins does not prove actual ACh synthesis and secretion, BEC cultures were established (35), and ACh secretion was measured by HPLC. As shown in Fig. 7Go, BEC cultures actively synthesize and secrete ACh. Levels of ACh were 4 nM/ml culture medium. In the absence of the cholinesterase inhibitor neostigmine, ACh could not be detected. This both further proves the specificity of the ACh assay and suggests that BECs in culture express cholinesterases to inactivate ACh.



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  		FIG. 7. Epithelial cell cultures prepared from the bronchi of a 1-yr-old monkey. a, Picture of cells showing typical rounded appearance of epithelial cells. b, HPLC tracing demonstrating that, in the presence of 50 µM neostigmine to prevent ACh degradation, airway BECs secrete ACh. c, HPLC tracing showing that endogenous cholinesterase activity is present and degrades ACh in the absence of neostigmine. d, Spiked sample showing retention time of ACh.

 
A key component in cholinergic neurons is CHT, which has been recently cloned (24, 25, 39). As determined by IHC, CHT protein was expressed in BECs (Fig. 8Go). As determined by RT-PCR of RNA prepared from BEC scrapings, CHT RNA was similarly expressed in BECs (Fig. 8Go). The identity of CHT was further confirmed because two different CHT antibodies (25), both a mAB and a polyclonal antibody, gave identical staining patterns (data not shown). CHT expression was also uniformly distributed throughout the BECs (Fig. 8Go).



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  		FIG. 8. Expression of CHT in airway bronchial epithelium. IHC using a CHT polyclonal antibody shows presence of CHT in airway bronchial epithelium. CHT immunoreactivity is evenly distributed throughout BECs (magnification: a, x100; b, x630 of boxed regionin a). The cell nuclei are counterstained with hematoxylin (blue). Similar staining was seen with a mAB to CHT (data not shown). c, RT-PCR showing the presence of CHT mRNA in lung BEC scrapings, whole lung, and hypothalamus (brain). RNA was amplified with primers corresponding to the N terminus or C terminus of CHT and then blotted to an internal 32P-labeled probe. Primers used are described in Materials and Methods. A, Airway; E, epithelium. Chromogen = AEC.

 
Thus, the data shown here clearly demonstrates that all the components for a nonneuronal ACh autocrine signaling loop are present in airway bronchial epithelium.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The concept that ACh is not only a neurotransmitter but is also a local signaling molecule synthesized by nonneuronal cells significantly broadens the role of ACh. For ACh to be a locally acting hormone in airway bronchial epithelium, ACh must be synthesized, secreted, and released and have an affect on a target cell. In this article, we present data that demonstrates that ACh meets all the criteria to be an autocrine and/or paracrine hormone in airway bronchial epithelium.

ACh synthesis has been documented in diverse nonneuronal tissues. Initial reports of ACh synthesis were in placenta (1, 2, 40, 41), followed by reports in brain vascular endothelium (4, 42), pituitary cells (43), glia (5, 6), and lung and gut epithelium (9, 10, 11). Synthesis has been documented both by demonstrating the presence of ChAT and by directly measuring ACh by HPLC. Thus, ACh appears to be widely expressed by nonneuronal cells, and its functions are likely just as diverse. How nonneuronal ACh synthesis is regulated and the components needed for ACh local signaling remain to be fully characterized.

The first step in ACh synthesis is transport of choline into the cells. Cholinergic neurons use a sodium-dependent CHT with a Michaelis-Menten constant for choline of approximately 1.2 µM (39, 44). This transporter has been cloned and designated CHT (24). As shown in Fig. 8Go, airway bronchial epithelium expresses CHT as determined by immunoreactivity using two different antibodies and by RT-PCR using two different primer sets. Further confirming the expression and potential role of CHT in lung, we have observed that CHT is present in bronchial epithelium of wild-type mice but not present in CHT knockout mice (data not shown) (45). Our finding of CHT in bronchial epithelium is consistent with reports by Pfeil et al. (46) showing CHT expression in rat trachea and functional studies showing that the airway epithelial cell line A549 expresses both a sodium-dependent CHT with the characteristics of CHT and a lower affinity sodium-independent choline transporter (47). The expression of CHT by BECs will allow highly efficient uptake of choline for the synthesis of ACh.

The next steps required for cholinergic signaling in neurons is synthesis of ACh by ChAT and packaging into vesicles by VAChT. By IHC, ChAT and VAChT were shown to be expressed primarily in lung epithelium (Fig. 1Go, d and e, and Fig. 4cGo). The peripheral fiber marker PGP9.5 (48) was used to show that the expression of ChAT, VAChT, and the nAChRs was not evident in the nerve fibers intertwined among the bronchial epithelium (Fig. 4cGo). Rather, the expression of these proteins is primarily in the nonneuronal cells of the lung bronchial epithelium. The pharmacology of the fine nerve fibers of the lung bronchial epithelium appears to be highly species specific. Jeffery (49) and El Bermani et al. (50) reported evidence that cholinergic nerves innervate the mouse lung and rabbit lung, respectively, but Reinheimer et al. (11) suggested that cholinergic nerves do not innervate the human lung bronchial epithelium. Canning and Fischer (51) similarly showed no ChAT-positive nerve fibers innervate guinea pig lung bronchial epithelium. In all species, it appears that the majority of nerve fibers innervating lung bronchial epithelium are peptidergic (52). Thus, although in some species there may be some ChAT expression in nerves innervating bronchial epithelium, the majority of ChAT expression is likely in the epithelial cells themselves.

The data shown in Fig. 5Go demonstrate that the isoform(s) of ChAT expressed in airway bronchial epithelium are highly similar to the isoforms of ChAT expressed in neurons. As shown in Fig. 5Go, epithelial ChAT amplifies and hybridizes to primers corresponding to exons N, S, 5, 10, and 12 of neuronal ChAT. There are more than six different isoforms of ChAT described in humans (33), differing primarily in their 5'-untranslated region, although minor forms with different coding regions also exist (53). Exons N and S are in the 5'-untranslated regions, and there presence defines the N and S forms of ChAT. Exons 5, 10, and 12 are in the coding region. Given that the N and S exons amplify bands from epithelial RNA, this suggests that at least the N and S forms of ChAT are likely present in epithelium, whereas additional forms may also be present.

VAChT is also expressed in airway bronchial epithelium (Figs. 1eGo and 4Go). However, because VAChT is expressed from the same genomic locus as ChAT, VAChT may be present but is not required for ACh secretion. In placenta, Wessler et al. (54) reported that the VAChT inhibitor vesamicol does not affect ACh secretion, so therefore, VAChT may not be needed for ACh secretion, and they suggested that, instead, the organic cation transporters Oct1 and Oct3 (55) may mediate ACh secretion in nonneuronal cells. Conversely, however, Song et al. (17) have shown that vesamicol reduces ACh secretion from lung cancer cells. Thus, although VAChT is clearly present in airway bronchial epithelium, its role in ACh secretion from airway BECs remains to be established.

By IHC (Fig. 1Go, a–c, and Fig. 4Go, a and b) and RT-PCR (Fig. 6Go), {alpha}4, ß2, and {alpha}7 nAChR are expressed in airway bronchial epithelium in both neonatal and adult monkeys. {alpha}3 and {alpha}5, but not {alpha}6, ß3, or ß4, were also shown to be expressed in BEC by RT-PCR (data not shown). Several reports (12, 13, 14) have also reported the expression of nicotinic receptors in airway bronchial epithelium and demonstrated by electrophysiology that the receptors form active channels. Thus, nAChRs are clearly expressed in BEC and are physiologically active. There is some disagreement over the expression of {alpha}4 in lung epithelium. We and Zia et al. (12) have detected {alpha}4 in monkey and human airway bronchial epithelium, whereas Maus et al. (13) did not detect {alpha}4 RNA transcript in human bronchial cell cultures and in rat trachea (by RT-PCR and in situ hybridization). These differences likely reflect species differences and differences in levels of bronchi sampled and the sensitivity of primers used for low gene expression. Although the focus of this article is on nAChR, muscarinic receptors, particularly the M3 subtype, are also present in airway bronchial epithelium (56) and will also be targets of the cholinergic autocrine loop. Characterization of the exact muscarinic subtypes expressed in bronchial epithelium and their role in autocrine cholinergic signaling awaits further description.

The function of the nicotinic cholinergic signaling pathway in airway bronchial epithelium is highly likely to be affected by nicotine in smokers. In smokers, plasma nicotine levels peak around 200 nM during the day and drop to 5–10 nM during sleep (57, 58). Nicotine levels in lung airways directly exposed to smoke may be 5- to 10-fold higher, and peaks and troughs are much sharper (57, 58). These levels are high enough to activate {alpha}4ß2 nAChR (59, 60) and may either inhibit (59) or activate (61) {alpha}7 nAChR. It is also interesting that the expression of nAChR appears highly expressed at the apical regions of cells, where they are more exposed to airway nicotine.

The cholinergic signaling pathway in airway BECs is clearly linked to cell growth. Nicotine has been shown to stimulate the growth of lung cancer cell lines that express {alpha}7 nAChR (62, 63, 64), an effect that is blocked by {alpha}-bungarotoxin (63). West et al. (15) has shown that nicotine stimulates the growth of airway BEC lines through activation of the Akt pathway. Our laboratory has also reported that the ACh synthesized by small-cell lung carcinoma cell lines acts as an autocrine growth factor to stimulate growth through both nicotinic and muscarinic pathways (16, 17). The potential of epithelial ACh to modulate lung development is also seen by the profound effects of fetal nicotine exposure on lung morphology and subsequent function (20, 22, 23). Similarly, Arredondo et al. (65) have recently shown that {alpha}7 nAChR expressed in stratified squamous epithelium plays a key role in keratinocyte differentiation.

In monkey lung bronchial epithelium, ACh may not be the sole ligand for cholinergic signaling. Choline has been demonstrated to be an agonist for {alpha}7 nAChR (66) and to potentiate responses of {alpha}4ß2 receptors to ACh (67). This is potentially significant because, uniquely in lung, choline can come from degradation and recycling of surfactant as well as from serum and membrane recycling (68). Thus, available choline from surfactant-coated airways may be able to modulate epithelial cell responses to released ACh. Thus lung, like brain, may be highly sensitive to alterations in choline availability both by effects on ACh synthesis and effects on nicotinic signaling (69, 70). The expression of CHT in bronchial epithelium as well as providing choline for the synthesis of ACh can, thus, also provide a mechanism to terminate nicotinic signaling by choline.

Another potential modulator of cholinergic function in airway bronchial epithelium is lynx1. This nicotinic receptor accessory protein, described by Miwa et al. (36) and Ibanez-Tallon et al. (37), appears to modulate responses to ACh by {alpha}7 and {alpha}4ß2 nAChR and to enhance receptor desensitization by nicotine. Because bronchial epithelium is exposed to some of the highest levels of nicotine in the body, it is likely that lynx1 will play a key role in modulating the effect of nicotine on autocrine cholinergic signaling. Thus, in airway bronchial epithelium lynx1, high levels of nicotine and high extracellular levels of choline can all combine to significantly modulate BEC responses to released ACh.

The data presented here clearly demonstrate that all the components needed for an autocrine cholinergic signaling pathway are expressed in airway BECs. The function of this pathway remains to be determined, although evidence strongly suggests it affects lung development and cell proliferation. In addition, the epithelial cholinergic autocrine pathway is a target for nicotine and may explain some of the effects of smoking on fetal development and on the development of lung cancer. This additional role and source of ACh suggests new approaches to developing therapeutics for diseases affected by cholinergic signaling ranging from Alzheimer’s disease to asthma, chronic obstructive pulmonary disease, and lung cancer.


   Acknowledgments
 
We thank Dr. Anda Cornea for assistance with confocal microscopy and Ellen Martin for assistance in sequencing the {alpha}7 nAChR. We also thank Reen Wu for assistance in establishing bronchial epithelial cell cultures.


   Footnotes
 
This work was supported by National Institute of Health Grants RR-00163, HD/HL-37131, and NS-11323.

Abbreviations: Ach, Acetylcholine; BEC, bronchial epithelial cell; ChAT, choline acetyltransferase; CHT, choline high-affinity transporter; IHC, immunohistochemistry; mAB, monoclonal antibody; nAChR, nicotinic acetylcholine receptor; VAChT, vesicular acetylcholine transporter.

Received December 22, 2003.

Accepted for publication January 26, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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