Physiological Parameters Affecting the Modulatory Role of Airway Epithelium on Airway Smooth Muscle Responsiveness

Apostolia Hatziefthimiou, MD

Associate Professor, Laboratory of Physiology, Department of Medicine, School of Health Sciences, University of Thessaly, 3 Panepistimiou Str  41500, BIOPOLIS Larissa, Greece, Tel. +30 2410 685561; Fax: +30 2410 685555; E-mail: axatzi@med.uth.gr

INTRODUCTION

Respiratory epithelium belongs to the class of ciliated pseudostratified columnar epithelium due to the arrangement of the columnar epithelial cells. Airway epithelium functions as a barrier to potential pathogens and foreign objects and prevents infection by the action of the ciliary escalator. It acts as a non-specific upper airways defence mechanism, that entraps particles and other inhaled material in mucus, and transports them away from the lungs. Efficient mucociliary transport is the result of the co-ordination of three airway epithelial functions, i.e. mucus secretion, ciliary beat and ion and fluid transport. Another important function of airway epithelium is its ability to produce endogenous biologically active substances like Nitric Oxide (NO), prostanoids, and endothelin.^1,2,3,4 It is also worth mentioning that the function of airway epithelium is influenced by physiological parameters, like age, gender, load, and bronchi size.

Airway Smooth Muscles (ASM), whether contracted or relaxed, affect airway diameter and thus air flow to alveoli where gas exchange occurs. Excessive responsiveness of ASM to contractile agents is often characteristic of chronic respiratory diseases, with asthma being a typical example. This over-responsiveness results in airway obstruction and decline of airway flow. Remarkably, a common finding in asthma is epithelium damage, inflammation and in some cases airway remodelling. Moreover, epidemiological data suggest that the incidence of asthma becomes higher in females than males with the onset of puberty, and that this tendency prevails throughout the reproductive years.⁵

These observations triggered research interest toward the modulatory role of airway epithelium on ASM, in connection with the action of NO, prostanoid, cholinergic agents, mediators of inflammation and growth factors, but also in connection with airway size, animal age or gender and the initial load applied on airway smooth muscle.

In the following paragraphs, we discuss the impact of the above factors on the modulatory role of airway epithelium on ASM, as well as the possible implications on the pathophysiology of airway diseases like asthma and Chronic Obstructive Pulmonary Disease (COPD) (Table 1).

Endogenous factors: insulin, histamine, acetylcholine

Endogenous factors like insulin, histamine or acetylcholine cause airway contraction. On the other hand, they act on epithelial cells and cause the release of biologically active mediators, mainly NO, that relax ASM and as a result limit excessive airway contraction.

Insulin is the major modulator of blood glucose levels exhibiting also growth factor activity on many cell types including ASM cells. Namely, it promotes ASM cell proliferation, via activation of the Phosphatidylinositol 3-kinase (PI3K) pathway,⁶ switches the ASM phenotype to contractile,^7,8,9 induces the release of contractile prostanoids from sources other than epithelium10 and increases the responsiveness of ASM to contractile agents.⁷ Moreover, available data suggest that in airways, insulin promotes the survival of epithelial cells¹¹ and participates in epithelium restoration integrity¹² after its damage. Additionally, insulin may stimulate NO release from epithelial cells and thus cause relaxation of precontracted airways.¹³

Histamine, a classical inflammatory agent, induces airway contraction.¹⁴ Histamine further affects airway epithelium as it promotes H₂O₂ production from epithelial cells of bronchi,¹⁵ mucus release and swelling of mucosa.¹⁶ In fact, the effect of histamine on ASM contraction is mediated by the release of biologically active molecules like NO from epithelial cells and thus, depends on epithelium integrity.^14,17,18 Studies on vessels demonstrate that histamine stimulates endothelial cell H₃ receptors19 by increasing epithelial NO synthase phosphorylation and activity.²⁰

Acetylcholine, the neurotransmitter released from postganglionic parasympathetic vagus nerves, induces airways contraction via Ca²⁺ release from intracellular stores and Ca²⁺ entry from extracellular space.^21,22 Additionally, acetylcholine is also released from epithelial cells²³ and can promote the chemeotaxis of monocytes and neutrophils^24,25 via, at least in part, the release of interleukin-8.²⁶ ASM express mainly M₂ and M₃ muscarinic receptors.²⁷ Acetylcholine, as well as other muscarinic agonists, may induce proliferation of ASM cells that depends on the activity of the MAPK and PI3K pathways, either alone²⁸ or in combination with growth factors.²⁹ In vitro studies demonstrate that the airway epithelium stimulates the breakdown of acetylcholine.³⁰ In addition, acetylcholine seems to stimulate NO release from the epithelium and its mechanical removal increases airway responsiveness to acetylcholine.³¹

Age

Animal studies suggest that increased age decreases airway responsiveness to contractile agents,³² and increases their capability to relax.^33,34 These age-dependent alterations in airway responsiveness are attributed to changes in airway architecture and organization,^35,36 to the maturation of the non-adrenergic non cholinergic system³⁷ and to increased activity of acetylcholinesterase.³⁸

Evidence suggests that age may affect the release of biologically active factors, mainly NO, from the airway epithelium.The three NO synthase isoforms are expressed in airways, but their levels remain unchanged during life.⁴⁰ However, the capacity of airway epithelium to produce NO seems to increase with age. Namely, in rabbits (Figure 1, our unpublished results) and pigs³⁹ age affects the acetylcholine-induced NO production. Specifically, contractility studies were performed on tracheal strips obtained from young (four weeks old) or adult (eight weeks old) rabbits in the presence of 10^-9 to 10^-3 M of acetylcholine. In adult rabbits epithelium damage increases acetylcholine-induced contractions.²² On the contrary, these experiments revealed that in young rabbits the mechanical removal of epithelium (Figure 1A, our unpublished results) as well as the treatment of preparations with the inhibitor of NO synthase NG-nitro-L-arginine methyl ester (L-NAME), the precursor of NO formation L-arginine or the inhibitor of cyclooxygenase indomethacin had no effect on ACh-induced contractions (Figure 1B, our unpublished results).

Figure 1: The effect of epithelium removal (A) or the presence of NOS inhibitor, NG-nitro-Larginine methyl ester (L-NAME), NO precursor, L-arginine and cyclooxygenase inhibitor, indomethacin (B) on acetylcholine (ACh)-induced contractions of tracheal strips obtained from young rabbits (<4 weeks).
Data are means, vertical lines show SE. N refers to the number of animals studied.

Histological studies reveal that the expression of Μ₃ receptors, which are involved in acetylcholine mediated NO release from the epithelium, increase with age.⁴⁰

Gender

Epidemiological data indicate a role of sex hormones in the etiology of some chronic airway diseases, in particular, asthma. Gender differences in the incidence of asthma are attributed mainly to differences in the immune response, as testosterone is considered to be immunosuppressant while female sex steroids proinflammatory. Moreover, studies on blood vessels have provided evidence that testosterone may exert direct effects on smooth muscle.

Immunohistochemistry studies show that ASM obtained from male rabbits express classical androgen⁴¹ and estrogens receptors.⁴² Similarly, immunofluorescence experiments performed in our laboratory revealed that rabbit ASM cells express Androgen Receptors (ARs). In the majority of cells ARs are cytoplasmic. However, in a few ASM cells ARs are also present in the cell nucleus (Figure 2, our unpublished results).

Figure 2: Airway smooth muscle cells express classical Androgen Receptors (ARs). Airway smooth muscle cells (passages 3-6) were analyzed by immunofluorescence using an antihuman classical Androgen Receptor (AR) mouse monoclonal antibody (G122-25, 1:200, BD Transduction laboratories). ARs are located mainly in the cytoplasm (magnification 20 X) (A). However, in a few ASM cells ARs are also present in the cell nucleus (magnification 40 X) (B). The smooth muscle origin of cells was confirmed by immunofluorescence with the anti SM α-actin mouse monoclonal antibody A104 (1:400, Sigma (magnification 20 X) (C). The position of the cell nuclei was visualized by DAPI. The fluorescent signal was analyzed with an Optiphot-2 microscope and UFX-DX camera system (Nikon) at the indicated magnification.

During embryonic life, sex hormones contribute to growth and maturation of the respiratory system.^43,44,45,46 Androgens seem to delay the maturation of embryonic lungs⁴⁷ and might be involved in the pathogenesis of the Acute Respiratory Distress Syndrome (ARDS) that has increased incidence in male newborn. In vitro studies revealed that both androgens and estrogens may affect, via classical receptors, the proliferation of ASM.^48,49 Also, sex hormones modulate directly the responsiveness of airway smooth muscle to contractile agents via a non-genomic pathway. Namely, in rabbit trachea 17β-estradiol relaxes precontracted airways in an epithelium independent way.^41,50 On the other hand, testosterone may increase vagal activity and thus contract rabbit⁴¹ or guinea pig airways.⁵¹ This effect of testosterone depends on animal gender as it is present only in male animals.⁴¹ On the contrary, testosterone relaxes precontracted ASM^52,53 in an epithelium-dependent way.⁵² Also, androgens seem to act synergistically with salbutamol.⁵⁴ Although these studies are limited, they suggest that in guinea pigs testosterone acts directly on airway smooth muscle and inhibits Ca²⁺ entry via voltage dependent channels, while in rabbits its action requires epithelium integrity and is, at least in part, mediated via NO release from epithelial cells.

Airway size

Several studies demonstrated that the responsiveness of smooth muscle to contractile agents varies in different parts of the bronchial tree and is influenced by airway innervation, receptor density, mechanical properties,^55,56 airway wall anatomy⁵⁷ and the distribution of acetylcholinesterase.^58,59 In addition, structural and functional regional differences in airway epithelium have also been described.^60,61,62,63 Regional differences in airway smooth muscle contractility are of physiological importance because smooth muscle contraction in central airways determines the resistance to airflow and gas distribution, whereas in peripheral airways it mainly controls the regional ratio of perfusion to ventilation. The regional differences in smooth muscle responsiveness to contractile agents may also be the basis of the non-homogeneous distribution of bronchoconstriction observed in pathological conditions such as Chronic Obstructive Pulmonary Disease (COPD) and asthma.

There are a few studies investigating the impact of airway size on the modulatory role of airway epithelium. Studies on 2^nd and 3^rd order of canine airways revealed that epithelium mainly modulate the responsiveness of the large airways.⁶⁴ On the contrary, we demonstrated that on sheep airways, epithelium integrity affects mainly the responsiveness of small airways.⁶⁵ Specifically, acetylcholine or KCl-induced contraction of epithelium intact airways is independent of airway size. In contrast, the mechanical removal of epithelium affects mainly the responsiveness of 3^rd and 4^th order airways to acetylcholine. This difference seems to be attributed to the capability of epithelial cells to produce NO along the tracheo-bronchial tree.

Load

During the tidal action of breathing load fluctuations are imposed continuously on ASM that undergo shortening and lengthening. Stress and strain can both be the mechanical signals involved in mechanosensitive modulation of ASM activation^66,67 depended on the applied contractile stimuli.^68,69,70 The mechanisms involved comprise ASM stiffness and extensibility, alterations in intracellular Ca²⁺ concentration and regulation of molecules involved in contractile protein activation. Despite the above involved mechanisms, the epithelium may also have a modulatory role in the mechanosensitive modulation of ASM responsiveness. To be precise, studies suggest that airway epithelium modulates ASM responsiveness to acetylcholine at increased load. This effect is mediated at least in part via NO release from epithelial cells.⁶⁸ The involved pathway comprises the calcium dependent activation of epithelial NO synthase.²² As epithelium responds to stretch by modulating epithelial NO synthase activity, and thus NO production with a consequent reduction of airway responsiveness, this protective mechanism could be impaired in epithelium damage seen in airways diseases in particular asthma.⁴

Conclusion

In conclusion, airway epithelium has a significant modulatory role in ASM responsiveness to contractile agents. This role depends on age, gender, bronchi size and load. Also, endogenous released substances like hormones (insulin, sex hormones), inflammatory factors (histamine) and neurotransmitters (acetylcholine) act on airway epithelial cells, induce NO release and limit airway contraction. These factors may contribute to the pathophysiology of some airway diseases like asthma and Chronic Obstructive Pulmonary Disease (COPD).

1. Ricciardolo FL. Multiple roles of nitric oxide in the airways. Thorax. 2003; 58(2): 175-182. doi: 10.1136/thorax.58.2.175

2. Holgate ST. The airway epithelium is central to the pathogenesis of asthma. Allergol Int. 2008; 57(1): 1-10. doi: 10.2332/allergolint.R-07-154

3. Knight DA, Stewart GA, Thompson PJ. Histamine tachyphylaxis in human airway smooth muscle. The role of H2-receptors and the bronchial epithelium. Am Rev Respir Dis. 1992; 146(1): 137-140. doi: 10.1164/ajrccm/146.1.137

4. Knight DA, Holgate ST. The airway epithelium: structural and functional properties in health and disease. Respirology 2003; 8(4): 432-446. doi: 10.1046/j.1440-1843.2003.00493.x

5. de Marco R, Locatelli F, Sunyer J, Burney P. Differences in incidence of reported asthma related to age in men and women. A retrospective analysis of the data of the European Respiratory Health Survey. Am J Respir Crit Care Med. 2000; 162(1): 68-74. doi: 10.1164/ajrccm.162.1.9907008

6. Papagianni M, Hatziefthimiou A, Chachami G, Gourgoulianis K, Molyvdas PA, Paraskeva E. Insulin causes a transient induction of proliferation via activation of the PI3-kinase pathway in airway smooth muscle cells. Exp Clin Endocrinol Diabetes. 2007; 115(2): 118-123. doi: 10.1055/s-2007-967094

7. Gosens R, Nelemans SA, Hiemstra M, Grootte Bromhaar MM, Meurs H, Zaagsma J. Insulin induces a hypercontractile airway smooth muscle phenotype. Eur J Pharmacol. 2003; 481(1): 125-131. doi: 10.1016/j.ejphar.2003.08.081

8. Dekkers BG, Schaafsma D, Tran T, Zaagsma J, Meurs H. Insulin-induced laminin expression promotes a hypercontractile airway smooth muscle phenotype. Am J Respir Cell Mol Biol. 2009; 41(4): 494-504. doi: 10.1165/rcmb.2008-0251OC

9. Schaafsma D, McNeill KD, Stelmack GL, et al. Insulin increases the expression of contractile phenotypic markers in airway smooth muscle. Am J Physiol Cell Physiol. 2007; 293(1): C429- C439. doi: 10.1152/ajpcell.00502.2006

10. Schaafsma D, Gosens R, Ris JM, Zaagsma J, Meurs H, Nelemans SA. Insulin induces airway smooth muscle contraction. Br J Pharmacol. 2007; 150(2): 136-142. doi: 10.1038/sj.bjp.0706985

11. Numata T, Araya J, Fujii S, et al. Insulin-dependent phosphatidylinositol 3-kinase/Akt and ERK signaling pathways inhibit TLR3-mediated human bronchial epithelial cell apoptosis. J Immunol. 2011; 187(1): 510-519. doi: 10.4049/jimmunol.1004218

12. Romberger DJ, Pladsen P, Claassen L, Yoshida M, Beckmann JD, Rennard SI. Insulin modulation of bronchial epithelial cell fibronectin in vitro. Am J Physiol. 1995; 268(2 Pt 1): L230-L238. doi: 10.1152/ajplung.1995.268.2.l230

13. Papayianni M, Gourgoulianis KI, Molyvdas PA. Insulin NO-dependent action on airways smooth muscles. Nitric Oxide. 2001; 5(1): 72-76. doi: 10.1006/niox.2000.0317

14. Sadeghi-Hashjin G, Folkerts G, Henricks PA, et al. Induction of guinea pig airway hyperresponsiveness by inactivation of guanylate cyclase. Eur J Pharmacol. 1996; 302(1-3): 109-115. doi: 10.1016/0014-2999(96)00081-7

15. Rada B, Boudreau HE, Park JJ, Leto TL. Histamine stimu-lates hydrogen peroxide production by bronchial epithelial cells via histamine H1 receptor and dual oxidase. Am J Respir Cell Mol Biol. 2014; 50(1): 125-134. doi: 10.1165/rcmb.2013-0254OC

16. Huang HT, Guo JJ, Huang YH, Fu YS. Histamine-induced changes in rat tracheal goblet cell mucin store and mucosal edema. Histochem Cell Biol. 2013; 139(5): 717-726. doi: 10.1007/s00418-012-1060-y

17. Spicuzza L, Basile L, Belvisi MG, et al. The protective role of epithelium-derived nitric oxide in isolated bovine trachea. Pulm Pharmacol Ther. 2002; 15(4): 357-362. doi: 10.1006/pupt.2002.0377

18. Gourgoulianis K, Iliodromitis Z, Hatziefthimiou A, Molyvdas PA. Epithelium-dependent regulation of airways smooth muscle function. A histamine-nitric oxide pathway. Mediators Inflamm. 1998; 7(6): 409-411. doi: http://dx.doi.org/10.1248/bpb.33.58

19. Sun P, Jin X, Koyama T, Li S, Kitamura Y, Kawasaki H. R-(-)-alpha-methylhistamine, a histamine H3 receptor agonist, induces endothelium-dependent vasodilation in rat mesenteric resistance arteries. Biol Pharm Bull. 2010; 33(1): 58-63. doi: http://dx.doi.org/10.1248/bpb.33.58

20. Thors B, Halldórsson H, Thorgeirsson G. eNOS activation mediated by AMPK after stimulation of endothelial cells with histamine or thrombin is dependent on LKB1. Biochim Biophys Acta. 2011; 1813(2): 322-331. doi: 10.1016/j.bbamcr.2010.12.001

21. Gourgoulianis KI, Chatziparasidis G, Chatziefthimiou A, Molyvdas PA. Magnesium as a relaxing factor of airway smooth muscles. J Aerosol Med. 2001; 14(3): 301-307. doi: 10.1089/089426801316970259

22. Kitsiopoulou E, Hatziefthimiou AA, Gourgoulianis KI, Molyvdas PA. Resting tension affects eNOS activity in a calcium-dependent way in airways. Mediators Inflamm. 2007; 24174. doi: 10.1155/2007/24174

23. Proskocil BJ, Sekhon HS, Jia Y et al. Acetylcholine is an autocrine or paracrine hormone synthesized and secreted by airway bronchial epithelial cells. Endocrinology. 2004; 145(5): 2498-2506. doi: http://dx.doi.org/10.1210/en.2003-1728

24. Koyama S, Rennard SI, Robbins RA. Acetylcholine stimulates bronchial epithelial cells to release neutrophil and monocyte chemotactic activity. Am J Physiol. 1992; 262 (4 Pt 1): L466-L471. doi: 10.1152/ajplung.1992.262.4.L466

25. Koyama S, Sato E, Nomura H, Kubo K, Nagai S, Izumi T. Acetylcholine and substance P stimulate bronchial epithelial cells to release eosinophil chemotactic activity. J Appl Physiol (1985). 1998; 84(5): 1528-1534. doi: 10.1152/jappl.1998.84.5.1528

26. Profita M, Bonanno A, Siena L, et al. Acetylcholine mediates the release of IL-8 in human bronchial epithelial cells by a NFkB/ERK-dependent mechanism Eur J Pharmacol. 2008; 582(1-3): 145-153. doi: 10.1016/j.ejphar.2007.12.029

27. Barnes PJ. Muscarinic receptors subtypes in airways. Eur Respir J. 1993; 6(3): 328-331. doi: 10.1016/0024-3205(93)90310-y

28. Stamatiou R, Paraskeva E, Vasilaki A, Mylonis I, Molyvdas PA, Gourgoulianis K, Hatziefthimiou A. Long-term exposure to muscarinic agonists decreases expression of contractile proteins and responsiveness of rabbit tracheal smooth muscle cells. BMC Pulm Med. 2014;14: 39.

29. Gosens R, Zaagsma J, Meurs H, Halayko AJ. Muscarinic receptor signaling in the pathophysiology of asthma and COPD. Respir Res. 2006; 7: e73. doi: 10.1186/1465-9921-7-73

30. Folkerts G, Kloek J, Geppetti P, Van der Linde HJ, Nijkamp FP. Factors that determine acetylcholine responsiveness of guinea pig tracheal tubes. Eur J Pharmacol. 2001; 420(2-3): 151-157. doi: 10.1016/S0014-2999(01)01016-0

31. Spicuzza L, Basile L, Belvisi MG, Bellofiore S, Matera MG, Cazzola M, Di Maria GU. The protective role of epithelium-derived nitric oxide in isolated bovine trachea. Pulm Pharmacol Ther. 2002; 15(4): 357-362. doi: 10.1006/pupt.2002.0377

32. Sparrow MP, Mitchell HW. Contraction of smooth muscle of pig airway tissues from before birth to maturity. J Appl Physiol (1985). 1990; 68(2): 468-477. doi: 10.1152/jappl.1990.68.2.468

33. Chitano P, Cox CM, Murphy TM. Relaxation of guinea pig trachealis during electrical field stimulation increases with age. J Appl Physiol (1985). 2002; 92(5): 1835-1842. doi: 10.1152/japplphysiol.00688.2001

34. Grunstein MM, Rosenberg SM, Schramm CM, Pawlowski NA. Mechanisms of action of endothelin 1 in maturing rabbit airway smooth muscle. Am J Physiol. 1991; 260(6 Pt 1): L434-443. doi: 10.1152/ajplung.1991.260.6.l434

35. Deoras KS, Wolfson MR, Searls RL, Hifler SR, Shaffer TH. Developmental changes in tracheal structure. Pediatr Res. 1991; 30(2): 170-175.doi: 10.1203/00006450-199108000-00010

36. Ramchandani R, Shen X, Elmsley CL, Ambrosius WT, Gunst SJ, Tepper RS. Differences in airway structure in immature and mature rabbits. J Appl Physiol (1985). 2000; 89(4): 1310-1316. doi: 10.1152/jappl.2000.89.4.1310

37. Colasurdo GN, Loader JE, Graves JP, Larsen GL. Maturation of nonadrenergic noncholinergic inhibitory system in normal and allergen-sensitized rabbits. Am J Physiol. 1994; 267(6 Pt 1): L739-L744. doi: 10.1152/ajplung.1994.267.6.l739

38. Murphy TM, Mitchell RW, Phillips IJ, Leff AR. Ontogenic expression of acetylcholinesterase activity in trachealis of young swine. Am J Physiol. 1991; 261(4 Pt 1): L322-L326. doi: 10.1152/ajplung.1991.261.4.L322

39. Jakupaj M, Martin RG, Dreshaj IA, Potter CF, Haxhiu MA, Ernsberger P. Role of endogenous NO in modulating airway contraction mediated by muscarinic receptors during development. Am J Physiol. 1997; 273(3 Pt 1): L531-L536. doi: 10.1152/ajplung.1997.273.3.l531

40. Sherman TS, Chen Z, Yuahanna IS, Lau KS, Margraf LR, Shaul PW. Nitric oxide synthase isoform expression in the developing lung epithelium. Am J Physiol.1999; 276(2 Pt 1): L383-L390. doi: 10.1152/ajplung.1999.276.2.l383

41. Kouloumenta V, Hatziefthimiou A, Paraskeva E, Gourgoulianis K, Molyvdas PA. Sexual dimorphism in airway responsiveness to sex hormones in rabbits Am J Physiol Lung Cell Mol Physiol. 2007; 293(2): L516. doi: 10.1152/ajplung.00192.2007

42. Dahlman-Wright K, Cavailles V, Fuqua SA, et al. International Union of Pharmacology. LXIV. Estrogen receptors. Pharmacol Rev. 2006; 58(4): 773-781. doi: 10.1124/pr.58.4.8

43. Hibbert M, Lannigan A, Raven J, Landau L, Phelan P. Gender differences in lung growth. Pediatr Pulmonol. 1995; 19(2): 129-134. doi: 10.1002/ppul.1950190208

44. Torday JS, Nielsen HC, Fencl Mde M, Avery ME. Sex differences in fetal lung maturation. Am Rev Respir Dis. 1981; 123(2): 205-208. doi: 10.1164/arrd.1981.123.2.205

45. Osman M. Therapeutic implications of sex differences in asthma and atopy. Arch Dis Child. 2003; 88(7): 587-590. doi: 10.1136/adc.88.7.587

46. Levesque BM, Vosatka RJ, Nielsen HC. Dihydrotestosterone stimulates branching morphogenesis, cell proliferation and programmed cell death in mouse embryonic lung explants. Pediatr Res. 2000; 47(4 Pt 1): 481-491.

47. Wilson CM, McPhaul MJ. A and B forms of androgen receptor are expressed in a variety of human tissues. Mol Cell Endocrinol. 1996; 120(1): 51-57. doi: 10.1016/0303-7207(96)03819-1

48. Dashtaki R, Whorton AR, Murphy TM, Chitano P, Reed W, Kennedy TP. Dehydroepiandrosterone and analogs inhibit DNA binding of AP-1 and airway smooth muscle proliferation. J Pharmacol Exp Ther. 1998; 285(2): 876-883. doi: 10.1203/00006450-200004000-00012

49. Stamatiou R, Paraskeva E, Papagianni M, Molyvdas PA, Hatziefthimiou A. The mitogenic effect of testosterone and 17β-estradiol on airway smooth muscle cells. Steroids. 2011; 76(4): 400-408. doi: 10.1016/j.steroids.2010.12.010

50. Pang JJ, Xu XB, Li HF, Zhang XY, Zheng TZ, Qu SY. Inhibition of beta-estradiol on trachea smooth muscle contraction in vitro and in vivo. Acta Pharmacol Sin. 2002; 23(3): 273-277.

51. Card JW, Voltz JW, Ferguson CD, et al. Male sex hormones promote vagally mediated reflex airway responsiveness to cholinergic stimulation. Am J Physiol Lung Cell Mol Physiol. 2007; 292(4): L908-L914. doi: 10.1152/ajplung.00407.2006

52. Kouloumenta V, Hatziefthimiou A, Paraskeva E, Gourgoulianis K, Molyvdas PA. Non-genomic effect of testosterone on airway smooth muscle. Br J Pharmacol. 2006; 149(8): 1083-1091. doi: 10.1038/sj.bjp.0706936

53. Bordallo J, de Boto MJ, Meana C, et al. Modulatory role of endogenous androgens on airway smooth muscle tone in isolated guinea-pig and bovine trachea; involvement of β2-adrenoceptors, the polyamine system and external calcium. Eur J Pharmacol. 2008; 601(1-3): 154-162. doi: 10.1016/j.ejphar.2008.10.039

54. Suárez L, Pipa M, Granda J, et al. Sex hormones modulate salbutamol-elicited long-term relaxation in isolated bovine tracheal strips. Pharmacology. 2011; 87(5-6): 249-256. doi: 10.1159/000324315

55. Ma X, Li W, Stephens NL. Detection of two clusters of mechanical properties of smooth muscle along the airway tree. J Appl Physiol (1985). 1996; 80(3): 857-861. doi: 10.1152/jappl.1996.80.3.857

56. Bai Y, Zhang M, Sanderson MJ. Contractility and Ca2+ signaling of smooth muscle cells in different generations of mouse airways. Am J Respir Cell Mol Biol. 2007; 36(1): 122-130. doi: 10.1165/rcmb.2006-0036OC

57. McKay KO, Wiggs BR, Paré PD, Kamm RD. Zero-stress state of intra- and extraparenchymal airways from human, pig, rabbit, and sheep lung. J Appl Physiol (1985). 2002; 92(3): 1261-1266. doi: 10.1152/japplphysiol.00131.2001

58. Goroumaru-Shinkai M, Yamamoto R, Funayama N, Takayanagi I. Regional differences of the contractile responses to acetylcholine and neurokinin A in rabbit airway: heterogeneous distribution of the metabolic enzymes. Gen Pharmacol. 1992; 23(6): 1057-1061. doi: 10.1016/0306-3623(92)90286-S

59. Maize DF, Fedan JS, Dey RD. Characterization of neural control and contractile function in airway smooth muscle of the ferret. Pulm Pharmacol Ther. 1998; 11(1): 57-64. doi: 10.1006/pupt.1998.0117

60. Boucher RC Jr, Bromberg PA, Gatzy JT. Airway transepithelial electric potential in vivo: species and regional differences. J Appl Physiol Respir Environ Exerc Physiol. 1980; 48(l): 169-176. doi: 10.1152/jappl.1980.48.1.169

61. Mariassy AT, Plopper CG. Tracheobronchial epithelium of the sheep: I. Quantitative light-microscopic study of epithelial cell abundance, and distribution. Anat Rec. 1983; 205(3): 263-275. doi: 10.1002/ar.1092050304

62. Ballard ST, Schepens SM, Falcone JC, Meininger GA, Taylor AE. Regional bioelectric properties of porcine airway epithelium. J Appl Physiol (1985). 1992; 73(5): 2021-2027. doi: 10.1152/jappl.1992.73.5.2021

63. Al-Bazzaz FJ, Hafez N, Tyagi S, et al. Detection of Cl–HCO3- and Na+-H+ exchangers in human airways epithelium. JOP. 2001; 2(4 Suppl): 285-290.

64. Hay DW, Raeburn D, Fedan JS. Regional differences in reactivity and in the influence of the epithelium on canine intrapulmonary bronchial smooth muscle responsiveness. Eur J Pharmacol. 1987; 141(3): 363-370. doi: 10.1016/0014-2999(87)90553-x

65. Hatziefthimiou A, Kiritsi M, Kiropoulou C, Vasilaki A, Sakellaridis N, Molyvdas PA. Regional differences in the modulatory role of the epithelium in sheep airway. Clin Exp Pharmacol Physiol. 2009; 36(7): 668-674. doi: 10.1111/j.1440-1681.2008.05131.x

66.Pratusevich VR, Seow CY, Ford LE. Plasticity in canine airway smooth muscle. J Gen Physiol. 1995; 105(1): 73-94. doi: 10.1085%2Fjgp.105.1.73

67. Kuo KH, Herrera AM, Wang L, Paré PD, Ford LE, Stephens NL, Seow CY. Structure-function correlation in airway smooth muscle adapted to different lengths. Am J Physiol Cell Physiol. 2003; 285(2): C384-C390. doi: 10.1152/ajpcell.00095.2003

68. Hatziefthimiou AA, Karetsi E, Pratzoudis E, Gourgoulianis KI, Molyvdas PA. Resting tension effect on airway smooth muscle: the involvement of epithelium. Respir Physiol Neurobiol. 2005; 145(2-3): 201-208. doi: 10.1016/j.resp.2004.06.004

69. Mehta D, Wu MF, Gunst SJ. Role of contractile protein activation in the lenght-dependent modulation of tracheal smooth muscle force. Am J Physiol. 1996; 270(1 Pt 1): C243-C252. doi:  10.1152/ajpcell.1996.270.1.c243

70. Gilbert EK, Weaver BA, Rembold CM. Depolarisation decreaces the [Ca2+]i sensitivity of myocin light-chain kinase in arterial smooth muscle: comparison of aequorin and fura 2 [Ca2+]i estimates. FASEB J. 1991; 5(11): 2593-2599. doi: 10.1096/fasebj.5.11.1868983

71. Mandal TK. Inhaled insulin for diabetes mellitus. Am J Health Syst Pharm. 2005; 62(13): 1359-1364. doi: 10.1002/14651858.CD003890

72. Szczeklik A, Pieton R, Sieradzki J. Alternation in both insulin release and its hypoglycemic effects in atopic bronchial asthma. J Allergy Clin Immunol. 1980; 66(5): 424-427. doi: 10.1016/0091-6749(80)90123-2

73. Gosens R, Zaagsma J, Meurs H, Halayko AJ. Muscarinic receptor signaling in the pathophysiology of asthma and COPD. Respir Res. 2006; 7: 73. doi: 10.1186%2F1465-9921-7-73

74. Sherman TS, Chen Z, Yuahanna IS, Lau KS, Margraf LR, Shaul PW. Nitric oxide synthase isoform expression in the developing lung epithelium. Am J Physiol. 1999; 276 (2 Pt 1): L383-L390. doi: 10.1152/ajplung.1999.276.2.l383

75. Mhanna MJ, Dreshaj IA, Haxhiu MA, Martin RJ. Mechanism for subsance induced relaxation of precontracted airway smooth muscle during development. Am J Physiol. 1999; 276 (1 Pt 1): L51-L56. doi: 10.1203/00006450-199804001-01992

76. Semple PD, Beastall GH, Watson WS, Hume R. Serum testosterone depression associated with hypoxia in respiratory failure. Clin Sci (Lond). 1980; 58(1): 105-106. doi: 10.1042/cs0580105

77. Leifke E, Friemert M, Heilmann M, Puvogel N, Smaczny C, von zur Muhlen A, Brabant G. Sex steroids and body composition in men with cystic fibrosis. Eur J Endocrinol. 2003; 148(5): 551-557. doi: 10.1530/eje.0.1480551

78. Semple PD, Beastall GH, Brown TM, Stirling KW, Mills RJ, Watson WS. Sex hormone suppression and sexual impotence in hypoxic pulmonary fibrosis. Thorax. 1984; 39(1): 46-51. doi: 10.1136/thx.39.1.46

79. Creutzberg EC, Casaburi R. Endocrinological disturbances in chronic obstructive pulmonary disease. Eur Respir J Suppl. 2003; 46: 76s-80s. doi: 10.1183/09031936.03.00004610

80. Kapsali T, Permutt S, Laube B, Scichilone N, Togias A. Potent bronchoprotective effect of deep inspiration and its absence in asthma. J Appl Physiol (1985). 2000; 89(2): 711-720. doi: 10.1152/jappl.2000.89.2.711

81. Scichilone N, Kapsali T, Permutt S, Togias A. Deep inspiration-induced bronchoprotection is stronger than bronchodilation. Am J Respir Crit Care Med. 2000; 162(3 Pt 1): 910-916. doi: 10.1164/ajrccm.162.3.9907048

82. Scichilone N, Permutt S, Togias A. The lack of the bronchoprotective and not the bronchodilatory ability of deep inspiration is associated with airway hyperresponsiveness. Am J Respir Crit Care Med. 2001; 163(2): 413-419. doi: 10.1164/ajrccm.163.2.2003119

83. Scichilone N, Marchese R, Catalano F, Vignola AM, Togias A, Bellia V. Bronchodilatory effect of deep inspiration is absent in subjects with mild COPD. Chest. 2004; 125(6): 2029-2035. doi: 10.1378/chest.125.6.2029