Available online at www.sciencedirect.com

ScienceDirect Non-neuronal cholinergic airway epithelium biology Wolfgang Kummer and Gabriela Krasteva-Christ Acetylcholine, a major regulator of airway function, is not solely produced by neurons but also by a variety of non-neurons cells including various airway epithelial cells utilizing a molecular machinery of acetylcholine synthesis and release that differs from that of neurons. While canonical neuronal cholinergic signaling pathways, for example, nerve driven bronchoconstriction, are still valid, new pathways of cholinergic communication have emerged which center around innate immunity. These include cholinergic luminal signaling via the airway lining fluid to reach receptors at the apical side of epithelial cells and on macrophages patrolling on the surface, and preneuronal cholinergic signaling where sensory neurons are the target of ACh released from a recently identified chemosensory cell that monitors the airway lining fluid for the presence of potentially hazardous compounds utilizing the classical taste transduction cascade. Addresses Institute of Anatomy and Cell Biology, Universities of Giessen and Marburg Lung Center, Excellence Cluster Cardiopulmonary System, Members of the German Center for Lung Research, Justus-LiebigUniversity Giessen, Aulweg 123, 35385 Giessen, Germany Corresponding author: Kummer, Wolfgang ([email protected])

Current Opinion in Pharmacology 2014, 16:43–49

previously unexpected low grade of reliability and suitability of antibodies against proteins involved in cholinergic (and other) signaling [2–4], so that data based solely on immunohistochemistry at least should be revisited and supplemented with additional techniques. In the case of organs which entirely lack nerve fibers, for example, the placenta, it is obvious that all intrinsic cholinergic effects are non-neuronal. Lung cancer cells in culture, derived from both small cell and non-small cell cancer, synthesize and secrete acetylcholine, and this promotes cell growth via muscarinic and nicotinic receptors in an autocrine fashion independent from neuronal influence [5,6,7,8]. In case of the intact airways, however, classical parasympathetic cholinergic nerve fibers and non-neuronal cholinergic cells coexist, and it is difficult to dissect which of the known multiple cholinergic effects operating in airways are primarily driven by neurons and which by other cells, or by both systems. Recent advances have been made particularly in addressing the epithelial mechanisms of cholinergic signaling and their contribution to airway physiology, and this will be the focus of the present review. Of course, this is only one facet of continuously emerging additional roles of ACh in airway biology, for example, triggering remodeling and inflammatory processes in obstructive diseases. This subject has been covered by a recent excellent review in this series [9].

This review comes from a themed issue on Respiratory Edited by John T Fisher and Julia K Walker

1471-4892/$ – see front matter, # 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.coph.2014.03.001

Introduction Acetylcholine (ACh) is a major regulator of airway function, and its potent bronchoconstrictor and secretagogue actions are targeted by inhibitors of cholinergic muscarinic receptors in common diseases such as COPD. It was the first neurotransmitter to be discovered, and this seminal finding not only boosted development of neurosciences enormously, it also dominated the view on this small molecule so strongly that its additional occurrence outside the nervous system (‘non-neuronal ACh’) was neglected for decades. It is now well established that a plethora of nonneuronal cell types synthesize and release ACh, especially certain epithelial cells and cells of the immune system, but this by far does not cover the entire spectrum [1]. Caution has to be applied, however, not to overestimate the extent of the non-neuronal system as recent years have shown a www.sciencedirect.com

Airway epithelium handles ACh synthesis and release different from neurons A very efficient way of quick ACh synthesis (via choline acetyltransferase = ChAT), vesicular storage in high concentration (via vesicular ACh transporter = VAChT), stimulus-evoked exocytotic release in high quantities and reuptake of the essential precursor choline (via high-affinity choline transporter-1 = CHT1) has evolved in neurons (Figure 1), and allows for precise, spatially and temporally controlled mode of action such as in regulation of airway muscle tone. Although expression of all of these proteins has been detected in the airway epithelial layer, they are not common to all cell types, and alternative mechanisms of ACh handling prevail [10]. Specifically, most epithelial cell types appear not to concentrate ACh in vesicles but release it directly from the cytoplasm through membrane transporters. Polyspecific organic cation transporters 1 and 2 (OCT1, OCT2; members of solute carrier family SLC22) have directly been shown to transport ACh in both directions (in and out of cells), are expressed by airway epithelial cells (ciliated and others), and epithelial ACh content is elevated in OCT1/OCT2 double-knockout mice, indicating that these transporters are indeed involved in epithelial ACh release in vivo [11]. Notably, various glucocorticoids used in pharmacotherapy Current Opinion in Pharmacology 2014, 16:43–49

44 Respiratory

Figure 1

AcetylCoA Nucleus

CHT1 CTLs

ACh

Choline

ChAT CarAT

VAChT OCT(N)s

CTLs?

Choline

Choline ACh BChE Acetate

Acetate N N

Target cell

N X β-Ar JAK

M

Current Opinion in Pharmacology

Recycling pathways of acetylcholine synthesis, release, action and breakdown, the neuronal pathway is indicated by blue arrows. ACh, acetylcholine; AChE, acetylcholineesterase (blue line indicates anchoring at cell membrane in neurons, but not in non-neuronal cells); b-Ar, b-arrestin; BChE, butyrylcholineesterase; CarAT, carnitine acetyltransferase, suggested to synthesize ACh in some non-neuronal cells; ChAT, choline acteyltransferase; CHT1, high-affinity choline transporter-1; CTLs, choline transporter-like proteins; JAK, Janus kinase; M, muscarinic acetylcholine receptor; N, nicotinic acetylcholine receptor OCT(N)s, organic action (carnitine) transporters; VAChT, vesicular acetylcholine transporter; X, postulated unknown binding partner for nicotinic receptor subunits in atypical receptors found outside the nervous system.

of asthma inhibit ACh release by directly binding to OCT1 and 2 so that part of the acute action of these drugs might be due to interference with this epithelial cholinergic system [11]. In general, transport by OCTs depends on concentration gradient and on membrane potential, but evidence for a stimulated epithelial release of ACh via OCTs has not been provided yet. On this background, airway epithelial ACh did not appear as a candidate for short-term or midterm signaling but rather as a more or less constitutively released trophic signal. Very elegant studies on another SLC22 family member, SLC22A4 or OCTN1, however, have recently put more dynamics on the scene. This transporter is also expressed by nasal and lower airway epithelia [12–14,15,16,17]. Human OCTN1 reconstituted in liposomes catalyzes ACh transport, and this is stimulated by internal, but not external, ATP, and is inhibited by various polyamines, external K+ and microbial derived substances, for example, g-butyrobetaine [18,19]. This is the first identified system allowing for regulated ACh release from epithelia. Importantly, ACh Current Opinion in Pharmacology 2014, 16:43–49

efflux is defective in the OCTN1 mutant L503F [18] which is associated with Crohn’s disease [20,21] providing a possible link between epithelial ACh and inflammatory diseases. As for the airways, associations of OCTN1 polymorphisms to certain diseases have not been reported yet and OCTN1 gene deficient mice do not show obvious phenotypic alterations [22]. Variants of the closely related isoform OCTN2 (=SLC22A5), however, are linked to asthma as determined by a genomewide association study, leading the authors to assume that ‘asthma and Crohn’s disease may therefore have shared mechanisms, perhaps involving a modulation of microbial interactions with the mucosa’ [23]. Among the members of the OCT and OCTN families, OCTN2 mRNA expression has been reported to be highest in human bronchial epithelium and cell lines derived from it [12,24], and OCTN2 protein was localized to airway epithelial cell membranes by immunohistochemistry [15,16,12,24]. On the other hand, it was undetectable in human lung and cultured airway epithelia by quantitative liquid chromatography-tandem www.sciencedirect.com

Non-neuronal cholinergic airway epithelium biology Kummer and Krasteva-Christ 45

mass spectrometry which revealed OCTN1 as the dominating transporter [17]. Both OCTN1 and 2 transport the clinically relevant muscarinic receptor inhibitors ipratropium and tiotropium [25], and inhibitor studies suggest that OCTN2 is mainly responsible for tissue accumulation of ipratropium applied to the mouse trachea lumen [15]. These very recent studies clearly demonstrate an important role of OCTNs in cholinergic, anti-muscarinic airway pharmacotherapy, and it appears to be an intriguing task to directly address the ACh transport capacity of OCTN2 and its relevance for pathogenesis, for example, of asthma. In neurons, re-uptake of choline through the plasma membrane via the Na+-dependent, high-affinity choline transporter CHT1 is absolutely required for sufficient ACh synthesis to maintain synaptic function [26–28]. CHT1 is also found in some non-neuronal cells, including airway ciliated cells [29] and some small cell lung carcinoma (SCLC) cell lines [30], but non-neuronal cells are capable of ACh synthesis also in the absence of CHT1 [5,31]. Recent studies have shown a widespread tissue distribution of intermediate-affinity choline transporters termed choline transporter-like proteins 1–5 (CTL1-5) that constitute the SLC44 family [32,33]. All of them are expressed by SCLC cell lines, and siRNA knockdown of CTL1, 2 and 5 diminishes choline uptake in cholinergic H82 SCLC cells, albeit not reducing ACh secretion into the medium [30]. Knockdown of the less abundantly expressed CTL4, on the other hand, had no significant impact upon choline uptake but reduced ACh secretion to about 50% of control. This might be explained by compartmentalization of choline transport and synthesis, as speculated by the authors [30], but it also might be worth investigating whether CTL4 by itself is capable of transporting ACh directly across the plasma membrane. In any case, these data highlight the difference between neuronal and airway epithelial ACh synthesis and release, and point towards CTL4 as a novel protein that is a crucial and potentially pharmacologically targeted mechanism.

From the top: luminal cholinergic signaling in the airway lining fluid In airway ciliated epithelial cells, immunoreactivity for the ACh synthesizing enzyme, ChAT, is enriched in the apical cell portion [10,34,35], and CHT1 [29], OCT1-3 and OCTN1 and 2 have been localized to the luminal membrane of ciliated cells [11,15,16,12] leading to the suggestion of luminal release of ACh [11]. Direct proof for the presence of ACh in the airway lining fluid could now be provided by sampling from the mucosal surface of the mouse trachea by desorption electrospray ionization and mass spectrometric analysis [36]. In Ussing chamber experiments, membrane impermeable agonists of muscarinic and nicotinic receptors activate apical chloride secretion in the mouse tracheal epithelium demonstrating it is also responsive to cholinergic agonists coming from the luminal side [36,37]. Thus, the thin layer of airway www.sciencedirect.com

lining fluid lying on top of the epithelial cells may serve as a transport medium for ACh in autocrine/paracrine signaling. It also has to be considered that, in contrast to other body surfaces, cells of the immune system, in particular macrophages, regularly patrol on the surface. Alveolar macrophages are equipped with both, nicotinic and muscarinic receptors that mainly exert anti-inflammatory and pro-inflammatory effects, respectively [38– 41], so that they also might be a target of ACh released by epithelial cells into the lining fluid. While these all are plausible scenarios based on well supported, solid evidence, it should not be overlooked that it still needs to be finally validated whether there are indeed causal links between luminal ACh of epithelial origin and regulation of ion transport, mucociliary clearance or immune functions.

‘Bad taste’ — a cholinergic link between hazardous luminal content and defense reactions Utilizing mice expressing enhanced green fluorescent protein (eGFP) driven by the ChAT promoter, slender, solitary cholinergic epithelial cells were noted in the airway epithelium from the nose and pharynx/auditory tube down to the main bronchi [42,43,44,45,46]. In the trachea, they represent the majority (85%) of so-called ‘brush cells’ which have received this name due to the presence of a characteristic apical tuft of microvilli and whose function remained unclear until very recently [47,48]. In the upper airways, cells with very similar, but not identical morphology, are termed ‘microvillous cells’ in the olfactory epithelium and ‘solitary chemosensory cells’ elsewhere [44,49,50,51]. These cells express the elements of the canonical taste transduction cascade for bitter, umami and sweet detection as known from oropharyngeal taste buds, that is G-protein coupled taste receptors of the Tas1R and Tas2R families, the taste specific G-protein a-gustducin, phospholipase Cb2, and the transient receptor potential cation channel, subfamily M, member 5 (TRPM5) [5,43,44,45,46,49,53]. In series of exciting discoveries, these cells have been shown to ‘taste’ the presence of potentially hazardous substances, including bacterial products such as Pseudomonas aeruginosa quorum sensing molecules, on the mucosal surface and initiate protective local responses and neural reflexes [43,45,52,54]. In the trachea it was demonstrated that ACh is the effector molecule utilized by these sentinel cells. Since they exhibit VAChT-immunoreactivity [43,45], vesicular ACh storage and exocytotic release appear to be likely. A considerable percentage, albeit not all tracheal cholinergic brush cells, are approached by sensory nerve fibers expressing the nicotinic ACh receptor subunit a3, and intratracheal application of the bitter substance cycloheximide evokes a reduction in respiratory rate which is sensitive to nicotinic receptor blockade and to epithelium removal [45]. Hence, this newly recognized protective system operates Current Opinion in Pharmacology 2014, 16:43–49

46 Respiratory

with ‘preneuronal’ ACh in that a non-neuronal epithelial cell utilizes ACh to excite a sensory neuron, which is opposite to the classical view of ACh being a neuronal transmitter controlling contractile and secretory responses of non-neuronal cells.

How deep do we get — does epithelial ACh regulate airway smooth muscle tone? Since cholinergic bronchoconstriction is a hallmark feature of both COPD and asthma, it is highly relevant to determine whether ACh originating from the epithelium also reaches the smooth muscle in sufficient amounts to evoke constriction. In the mouse trachea, serotonin evokes a constriction that is sensitive to epithelium removal and to atropine, suggesting release of ACh from the epithelium acting on smooth muscle [55]. A similar experiment in mouse bronchi, however, revealed that serotonin induced bronchoconstriction in mice lacking muscarinic receptors M2 and M3, which no longer show cholinergic bronchoconstriction, and this effect was still blocked by atropine [56]. Thus, at least in mouse bronchi, atropine effects are not due to muscarinic receptor inhibition and there is no evidence for cholinergic bronchoconstriction originating from the epithelium. Of course, the situation in the trachea might be different, and one striking difference is the presence of cholinergic chemosensory cells only in the tracheal but not in intrapulmonary bronchial mucosa [45,53]. Notably, however, these cells are concentrated in the fibrocartilagenous part and are nearly lacking in the paries membranaceus of the

trachea, that is that part of the mucosa overlying the tracheal muscle [45,53], so that there is no spatial relationship between these sentinel cells and airway smooth muscle. Thus, currently available data do not exclude the possibility that epithelial ACh might exert bronchoconstrictor effects under certain conditions, but there is insufficient evidence to postulate such an effect. Much against expectation, even an opposite effect of epithelial ACh on muscle tone has been proposed very recently. Brain natriuretic peptide (BNP) exerts relaxant activity on and evokes ACh release from human bronchi which is dependent on epithelial integrity [57]. Further analysis strongly suggested that epithelial ACh acts in very low dose (10 9–10 12 M) upon muscular M2 receptors to stimulate inducible NO synthase (preferably in sensitized muscle), thereby leading to NO-mediated relaxation [58]. Since neuronal cholinergic activity can also be expected in vivo in sensitized airways, it remains to be clarified how additional low amounts of ACh derived from the epithelium can counteract cholinergic bronchoconstriction operating at the same time.

Conclusions Epithelial cells are a source of ACh in the airways but the molecular machinery of ACh synthesis and release is neither identical to that of cholinergic neurons, nor is it the same among all epithelial cell types. An increasing number of transporter systems for uptake of the essential precursor choline and for release of ACh have been

Figure 2

auto-/paracrine luminal cholinergic signaling

ACh

MΦ

ACh

ACh

sensory neuron

“pre-neuronal” cholinergic signaling

“non-neuronal” cholinergic signaling

non-neuronal cell → neuron

cholinergic neurotransmission

cholinergic efferent neuron

ACh

smooth muscle cells Current Opinion in Pharmacology

Summary of cholinergic signaling pathways in the airways. Cells of the immune system are depicted as circles. Orange: cholinergic cells, blue: noncholinergic cells, brown: bacteria on the mucosal surface. MF, macrophage. Current Opinion in Pharmacology 2014, 16:43–49

www.sciencedirect.com

Non-neuronal cholinergic airway epithelium biology Kummer and Krasteva-Christ 47

identified over the last few years, and more are likely to be identified. Notably, the very same transporter systems are involved in accumulating commonly used anti-muscarinic drugs in the tissue and are directly inhibited by clinically used glucocorticoids indicating these therapeutic approaches might also interfere with epithelial ACh release. While this newly recognized multiplicity of molecular pathways adds another level of complexity and does not simplify our view on cholinergic signaling in airway physiology and pathophysiology, its cell type specificity at least implies the possibility to address certain pathways with more specificity than with the currently used general receptor blockers. The coexistence of cholinergic parasympathetic nerve fibers and of non-neuronal cholinergic cells such as epithelia in the very same organ raises the question for what different purposes they are designed for and how their effects can be discriminated. On the basis of our current state of knowledge, there is no unequivocal evidence that non-neuronal ACh contributes to a significant extent to what has classically been considered a parasympathetic nerve driven cholinergic effect, that is airway constriction and stimulation of glandular secretion. Instead, new areas of cholinergic signaling have emerged (Figure 2). One of them is cholinergic luminal signaling via the airway lining fluid which is suited to reach receptors at the apical side of epithelial cells and on macrophages patrolling on the surface; the functional relevance of this pathway still needs to be explored. Another emerging area is preneuronal cholinergic signaling where sensory neurons are the target of epithelial-derived ACh, as is the case for cholinergic chemosensory cells of the upper airways and trachea being innervated by sensory nerve fibers carrying nicotinic receptors. This particular system represents a newly recognized mechanism of detection of potentially hazardous material, including bacteria, on the mucosal surface. Collectively, the airway epithelial cholinergic system can be taken as part of our mucosal innate defense system.

Acknowledgements Our work in this area was funded by the German Research Foundation (KR 4338/1-1), the German Center for Lung Research (ALI-1.1 WK) and the LOEWE Research Focus Non-neuronal Cholinergic Systems.

References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest 1.

Wessler I, Kirkpatrick CJ: Acetylcholine beyond neurons: the non-neuronal cholinergic system in humans. Br J Pharmacol 2008, 154:1558-1571.

2.

Moser N, Mechawar N, Jones I, Gochberg-Sarver A, OrrUrtreger A, Plomann M, Salas R, Molles B, Marubio L, Roth U et al.: Evaluating the suitability of nicotinic acetylcholine receptor antibodies for standard immunodetection procedures. J Neurochem 2007, 102:479-492.

www.sciencedirect.com

3.

Jositsch G, Papadakis T, Haberberger RV, Wolff M, Wess J, Kummer W: Suitability of muscarinic acetylcholine receptor antibodies for immunohistochemistry evaluated on tissue sections of receptor gene-deficient mice. Naunyn Schmiedebergs Arch Pharmacol 2009, 379:389-395.

4.

Pradidarcheep W, Stallen J, Labruye`re WT, Dabhoiwala NF, Michel MC, Lamers WH: Lack of specificity of commercially available antisera against muscarinergic and adrenergic receptors. Naunyn Schmiedebergs Arch Pharmacol 2009, 379:397-402.

5. 

Song P, Sekhon HS, Jia Y, Keller JA, Blusztajn JK, Mark GP, Spindel ER: Acetylcholine is synthesized by and acts as an autocrine growth factor for small cell lung carcinoma. Cancer Res 2003, 63:214-221. First demonstration of an autocrine cholinergic loop in small cell lung carcinomas with ACh acting as a growth factor. 6.

Song P, Sekhon HS, Lu A, Arredondo J, Sauer D, Gravett C, Mark GP, Grando SA, Spindel ER: M3 muscarinic receptor antagonists inhibit small cell lung carcinoma growth and mitogen-activated protein kinase phosphorylation induced by acetylcholine secretion. Cancer Res 2007, 67:3936-3944.

7.

Song P, Sekhon HS, Fu XW, Maier M, Jia Y, Duan J, Proskosil BJ, Gravett C, Lindstrom J, Mark GP et al.: Activated cholinergic signaling provides a target in squamous cell lung carcinoma. Cancer Res 2008, 68:4693-4700.

Zovko A, Viktorsson K, Lewensohn R, Kolosˇa K, Filipicˇ M, Xing H, Kem WR, Paleari L, Turk T: APS8, a polymeric alkylpyridinium salt blocks a7 nAChR and induces apoptosis in non-small cell lung carcinoma. Mar Drugs 2013, 11:2574-2594. This study shows that a naturally occurring polymer from a marine sponge is cytotoxic to non-small cell lung carcinoma cells by interfering with autocrine cholinergic signaling.

8. 

9. 

Meurs H, Dekkers BG, Maarsingh H, Halayko AJ, Zaagsma J, Gosens R: Muscarinic receptors on airway mesenchymal cells: novel findings for an ancient target. Pulm Pharmacol Ther 2013, 26:145-155. An outstanding review of muscarinic cholinergic effects upon remodeling and inflammation in pulmonary diseases and therapeutic implications.

10. Kummer W, Lips KS, Pfeil U: The epithelial cholinergic system of the airways. Histochem Cell Biol 2008, 130:219-234. 11. Lips KS, Bru¨ggmann D, Pfeil U, Vollerthun R, Grando SA, Kummer W: Nicotinic acetylcholine receptors in rat and human placenta. Placenta 2005, 26:735-746. 12. Horvath G, Schmid N, Fragoso MA, Schmid A, Conner GE, Salathe M, Wanner A: Epithelial organic cation transporters ensure pH-dependent drug absorption in the airway. Am J Respir Cell Mol Biol 2007, 36:53-60. 13. Salomon JJ, Endter S, Tachon G, Falson F, Buckley ST, Ehrhardt C: Transport of the fluorescent organic cation 4-(4(dimethylamino)styryl)-N-methylpyridinium iodide (ASP+) in human respiratory epithelial cells. Eur J Pharm Biopharm 2012, 81:351-359. 14. Mukherjee M, Latif ML, Pritchard DI, Bosquillon C: In-cell WesternTM detection of organic cation transporters in bronchial epithelial cell layers cultured at an air–liquid W interface on Transwell( ) inserts. J Pharmacol Toxicol Methods 2013, 68:184-189. 15. Nakanishi T, Hasegawa Y, Haruta T, Wakayama T, Tamai I: In vivo  evidence of organic cation transporter-mediated tracheal accumulation of the anticholinergic agent. J Pharm Sci 2013, 102:3373-3381. Provides strong evidence for OCTN2 located at the apical side of the airway epithelium to mediated tissue uptake of the muscarinic inhibitor iprattropium. 16. Shao D, Massoud E, Anand U, Parikh A, Cowley E, Clarke D, Agu RU: Organic cation transporters in human nasal primary culture: expression and functional activity. Ther Deliv 2013, 4:439-451. 17. Sakamoto A, Matsumaru T, Yamamura N, Uchida Y, Tachikawa M,  Ohtsuki S, Terasaki T: Quantitative expression of human drug transporter proteins in lung tissues: analysis of regional, gender, and interindividual differences by liquid Current Opinion in Pharmacology 2014, 16:43–49

48 Respiratory

chromatography–tandem mass spectrometry. J Pharm Sci 2013, 102:3395-3406. Provides the currently broadest comparison of drug transporter expression in human lung and airways at protein level.

32. Traiffort E, Ruat M, O’Regan S, Meunier FM: Molecular characterization of the family of choline transporter-like proteins and their splice variants. J Neurochem 2005, 92:11161125.

18. Pochini L, Scalise M, Galluccio M, Pani G, Siminovitch KA,  Indiveri C: The human OCTN1 (SLC22A4) reconstituted in liposomes catalyzes acetylcholine transport which is defective in the mutant L503F associated to the Crohn’s disease. Biochim Biophys Acta 2012, 1818:559-565. The first direct demonstration that OCTN1 transports ACh and that this process can be acutely regulated.

33. Traiffort E, O’Regan S, Ruat M: The choline transporter-like  family SLC44: properties and roles in human diseases. Mol Aspects Med 2013, 34:646-654. Comprehensive overview on the choline transporter-like proteins.

19. Pochini L, Scalise M, Galluccio M, Indiveri C: Regulation by  physiological cations of acetylcholine transport mediated by human OCTN1 (SLC22A4). Implications in the non-neuronal cholinergic system. Life Sci 2012, 91:1013-1016. Shows regulation of OCTN1-mediated ACh transport by extracellular sodium and intracellular potassium ions. 20. Lin Z, Nelson L, Franke A, Poritz L, Li TY, Wu R, Wang Y, MacNeill C, Thomas NJ, Schreiber S, Koltun WA: OCTN1 variant L503F is associated with familial and sporadic inflammatory bowel disease. J Crohns Colitis 2010, 4:132-138. 21. Tamai I: Pharmacological and pathophysiological roles of  carnitine/organic cation transporters (OCTNs: SLC22A4, SLC22A5 and Slc22a21). Biopharm Drug Dispos 2013, 34:29-44. Provides an overview on the general biological function of OCTNs including association of single nucleotide polymorphisms with diseases. 22. Kato Y, Kubo Y, Iwata D, Kato S, Sudo T, Sugiura T, Kagaya T, Wakayama T, Hirayama A, Sugimoto M et al.: Gene knockout and metabolome analysis of carnitine/organic cation transporter OCTN1. Pharm Res 2010, 27:832-840. 23. Moffatt MF, Gut IG, Demenais F, Strachan DP, Bouzigon E, Heath S, von Mutius E, Farrall M, Lathrop M, Cookson WO:  GABRIEL Consortium: a large-scale, consortium-based genomewide association study of asthma. N Engl J Med 2010, 363:1211-1221. Demonstrates an association of variants of OCTN2 with asthma. 24. Macdonald C, Shao D, Oli A, Agu RU: Characterization of Calu-3 cell monolayers as a model of bronchial epithelial transport: organic cation interaction studies. J Drug Target 2013, 21:97106. 25. Nakamura T, Nakanishi T, Haruta T, Shirasaka Y, Keogh JP, Tamai I: Transport of ipratropium, an anti-chronic obstructive pulmonary disease drug, is mediated by organic cation/ carnitine transporters in human bronchial epithelial cells: implications for carrier-mediated pulmonary absorption. Mol Pharm 2010, 7:187-195. 26. Apparsundaram S, Ferguson SM, George AL Jr, Blakely RD: Molecular cloning of a human, hemicholinium-3-sensitive choline transporter. Biochem Biophys Res Commun 2000, 276:862-867. 27. Okuda T, Haga T, Kanai Y, Endou H, Ishihara T, Katsura I: Identification and characterization of the high-affinity choline transporter. Nat Neurosci 2000, 3:120-125. 28. Ferguson SM, Bazalakova M, Savchenko V, Tapia JC, Wright J, Blakely RD: Lethal impairment of cholinergic neurotransmission in hemicholinium-3-sensitive choline transporter knockout mice. Proc Natl Acad Sci U S A 2004, 101:8762-8767. 29. Pfeil U, Lips KS, Eberling L, Grau V, Haberberger RV, Kummer W: Expression of the high-affinity choline transporter, CHT1, in the rat trachea. Am J Respir Cell Mol Biol 2003, 28:473-477. 30. Song P, Rekow SS, Singleton CA, Sekhon HS, Dissen GA, Zhou M,  Campling B, Lindstrom J, Spindel ER: Choline transporter-like protein 4 (CTL4) links to non-neuronal acetylcholine synthesis. J Neurochem 2013, 126:451-461. Clear proof by siRNA approach that CTL1, CTL2 and CTL5 are essential for choline uptake by small cell lung carcinoma cells, and CTL4 for increase in ACh released into the medium. 31. Yajima T, Inoue R, Matsumoto M, Yajima M: Non-neuronal release of ACh plays a key role in secretory response to luminal propionate in rat colon. J Physiol 2011, 589:953-962. Current Opinion in Pharmacology 2014, 16:43–49

34. Klapproth H, Reinheimer T, Metzen J, Mu¨nch M, Bittinger F, Kirkpatrick CJ, Ho¨hle KD, Schemann M, Racke´ K, Wessler I: Nonneuronal acetylcholine, a signalling molecule synthezised by surface cells of rat and man. Naunyn Schmiedebergs Arch Pharmacol 1997, 355:515-523. 35. Proskocil BJ, Sekhon HS, Jia Y, Savchenko V, Blakely RD, Lindstrom J, Spindel ER: Acetylcholine is an autocrine or paracrine hormone synthesized and secreted by airway bronchial epithelial cells. Endocrinology 2004, 145:2498-2506. 36. Hollenhorst MI, Lips KS, Wolff M, Wess J, Gerbig S, Takats Z,  Kummer W, Fronius M: Luminal cholinergic signalling in airway lining fluid: a novel mechanism for activating chloride secretion via Ca2+ dependent ClS and K+ channels. Br J Pharmacol 2012, 166:1388-1402. First direct demonstration of ACh on the mucosal surface of the trachea utilizing desorption electrospray ionization and mass spectrometric analysis. 37. Hollenhorst M, Lips K, Weitz A, Krasteva G, Kummer W, Fronius M: Evidence for functional atypical nicotinic receptors that activate K+ dependent ClS secretion in mouse tracheal epithelium. Am J Resp Cell Mol Biol 2012, 46:106-114. 38. Profita M, Di Giorgi R, Sala A, Bonanno A, Riccobono L, Mirabella F, Gjomarkaj M, Bonsignore G, Bousquet J, Vignola AM: Muscarinic receptors, leukotriene B-4 production and neutrophilic inflammation in COPD patients. Allergy 2005, 60:1361-1369. 39. Giebelen IA, van Westerloo DJ, LaRosa GJ, de Vos AF, van der Poll T: Local stimulation of alpha7 cholinergic receptors inhibits LPS-induced TNF-alpha release in the mouse lung. Shock 2007, 28:700-703. 40. Mikulski Z, Hartmann P, Jositsch G, Zasłona Z, Lips KS, Pfeil U, Kurzen H, Lohmeyer J, Clauss WG, Grau V et al.: Nicotinic receptors on rat alveolar macrophages dampen ATP-induced increase in cytosolic calcium concentration. Resp Res 2010, 11:133. 41. Koarai A, Traves SL, Fenwick PS, Brown SM, Chana KK, Russell RE, Nicholson AG, Barnes PJ, Donnelly LE: Expression of muscarinic receptors by human macrophages. Eur Respir J 2012, 39:698-704. 42. Tallini YN, Shui B, Greene KS, Deng KY, Doran R, Fisher PJ, Zipfel W, Kotlikoff MI: BAC transgenic mice express enhanced green fluorescent protein in central and peripheral cholinergic neurons. Physiol Genomics 2006, 27:391-397. 43. Ogura T, Krosnowski K, Zhang L, Bekkerman M, Lin W:  Chemoreception regulates chemical access to mouse vomeronasal organ: role of solitary chemosensory cells. PLoS ONE 2010, 5:e11924. First demonstration that solitary chemosensory cells in the upper airways — entrance to the vomeronasal duct — are cholinergic. 44. Ogura T, Szebenyi SA, Krosnowski K, Sathyanesan A, Jackson J, Lin W: Cholinergic microvillous cells in the mouse main olfactory epithelium and effect of acetylcholine on olfactory sensory neurons and supporting cells. J Neurophysiol 2011, 106:1274-1287. 45. Krasteva G, Canning BJ, Hartmann P, Veres TZ, Papadakis T,  Mu¨hlfeld C, Schliecker K, Tallini YN, Braun A, Hackstein H et al.: Cholinergic chemosensory cells in the trachea regulate breathing. Proc Natl Acad Sci U S A 2011, 108:9478-9483. First demonstration that tracheal brush cells are cholinergic chemosensory cells and elicit respiratory reflexes. 46. Krasteva G, Hartmann P, Papadakis T, Bodenbenner M, Wessels L, Weihe E, Schu¨tz B, Langheinrich AC, Chubanov V, Gudermann T et al.: Cholinergic chemosensory cells in the auditory tube. Histochem Cell Biol 2012, 137:483-497. www.sciencedirect.com

Non-neuronal cholinergic airway epithelium biology Kummer and Krasteva-Christ 49

47. Sbarbati A, Osculati F: A new fate for old cells: brush cells and related elements. J Anat 2005, 4:349-358. 48. Krasteva G, Kummer W: ‘‘Tasting’’ the airway lining fluid. Histochem Cell Biol 2012, 138:365-383. 49. Finger TE, Bo¨ttger B, Hansen A, Anderson KT, Alimohammadi H,  Silver WL: Solitary chemoreceptor cells in the nasal cavity serve as sentinels of respiration. Proc Natl Acad Sci U S A 2003, 100:8981-8986. Established the presence of chemosensory cells in the nasal respiratory epithelium. 50. Gulbransen BD, Clapp TR, Finger TE, Kinnamon SC: Nasal solitary chemoreceptor cell responses to bitter and trigeminal stimulants in vitro. J Neurophysiol 2008, 99:2929-2937. 51. Hansen A, Finger TE: Is TrpM5 a reliable marker for chemosensory cells? Multiple types of microvillous cells in the main olfactory epithelium of mice. BMC Neurosci 2008, 9:115. 52. Tizzano M, Gulbransen BD, Vandenbeuch A, Clapp TR,  Herman JP, Sibhatu HM, Churchill ME, Silver WL, Kinnamon SC, Finger TE: Nasal chemosensory cells use bitter taste signaling to detect irritants and bacterial signals. Proc Natl Acad Sci U S A 2010, 107:3210-3215. This study shows that bacterial products can be detected by chemosensory cells of the nose and induce respiratory reflexes. 53. Tizzano M, Cristofoletti M, Sbarbati A, Finger TE: Expression of taste receptors in solitary chemosensory cells of rodent airways. BMC Pulm Med 2011, 11:3.

www.sciencedirect.com

54. Krasteva G, Canning BJ, Papadakis T, Kummer W: Cholinergic brush cells in the trachea mediate respiratory  responses to quorum sensing molecules. Life Sci 2012, 91:992-996. This study reveals that cholinergic airway epithelial cells sense bacterial quorum sensing molecules in the airway lining fluid and communicate this to the CNS via ACh release and nicotinic stimulation of sensory neurons. 55. Moffatt JD, Cocks TM, Page CP: Role of the epithelium and acetylcholine in mediating the contraction to 5hydroxytryptamine in the mouse isolated trachea. Br J Pharmacol 2004, 141:1159-1166. 56. Kummer W, Wiegand S, Akinci S, Wessler I, Schinkel AH, Wess J, Koepsell H, Haberberger RV, Lips KS: Role of acetylcholine and polyspecific cation transporters in serotonin-induced bronchoconstriction in the mouse. Respir Res 2006, 7:65. 57. Matera MG, Calzetta L, Passeri D, Facciolo F, Rendina EA, Page C, Cazzola M, Orlandi A: Epithelium integrity is crucial for the relaxant activity of brain natriuretic peptide in human isolated bronchi. Br J Pharmacol 2011, 163:1740-1754. 58. Calzetta L, Passeri D, Kanabar V, Rogliani P, Page C, Cazzola M,  Matera MG, Orlandi A: Brain natriuretic peptide protects against hyperreactivity of human asthmatic airway smooth muscle via an epithelial cell dependent mechanism. Am J Resp Cell Mol Biol 2013 September 27. [Epub ahead of print]. This study demonstrates that very low doses of ACh can relax sensitized airway smooth muscle cells via M2 receptor-mediated activation of inducible NO synthase.

Current Opinion in Pharmacology 2014, 16:43–49