N-acetylcysteine effects on sinonasal cilia function

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N-acetylcysteine effects on sinonasal cilia function
Zeliha Kapusuz Gencer1, Levent Saydam1, Noam A. Cohen2,3, Cemal Cingi4
1Department of Otorhinolaryngology, Head and Neck Surgery, Bozok University, Yozgat, Turkey
2Department of Otorhinolaryngology, Head and Neck Surgery, University of Pennsylvania, Philadelphia, PA, USA
3Surgical Services, Philadelphia Veterans Affairs Medical Center, Philadelphia, PA, USA
4Department of Otorhinolaryngology, Head and Neck Surgery, Osmangazi University, Eskiflehir, Turkey
The mucociliary transport system plays an important role in
the clearance of excessive mucus and inhaled foreign mate-
rials from the respiratory tract. Airway epithelium’s ciliary
structure is a part of the natural defense system that protects
the respiratory system by propelling debris laden mucus.
The mucus flow is generated by the vigorous asymmetric
beating of the cilia, which can be named as effective strokes
and recovery strokes.[1] During normal mucus clearance,
inhaled pathogens and particles become trapped in the
mucus layer and are then expelled before they colonize the
airways.[2] The airway secretions which line the respiratory
tract form a biphasic layer composed of an aqueous ‘sol’
layer and a more superficial ‘gel’ layer. The sol layer is also
described as the ‘periciliary’ layer or ‘airway surface fluid’,
where the cilia beat and relax. The lubricant sol layer
enables the gel mucus present at the tips of the cilia to be
Experimental Study
ENT Updates 2015;5(3):87–92
doi:10.2399/jmu.2015003002
Correspondence: Zeliha Kapusuz Gencer, MD. Department of Otorhinolaryngology, Head and Neck Surgery,
University of Bozok, Yozgat, Turkey.
e-mail: drzeliha19@hotmail.com
Received: October 13, 2015; Accepted: November 5, 2015
©2015 Continuous Education and Scientific Research Association (CESRA)
Online available at:
www.entupdates.org
doi:10.2399/jmu.2015003002
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Özet: N-asetil sisteinin sinonazal siliyer at›m
frekans›na etkileri
Amaç: N-asetil sisteinin (NAC) insan respiratuvar epitelyal hücre
kültüründe, baflta elektrolit transportu ve siliyer at›m frekans› olmak
üzere farmakolojik etkilerinin de¤erlendirilmesi.
Yöntem: Hava-s›v› arayüzünde üretilmifl, iyi diferansiye edilmifl insan
bronfliyal epitelyal hücre kültüründe farkl› konsantrasyonlarda NAC
uygulamas› yap›ld›. ‹deal siliyer at›m frekans›n› sa¤layan NAC konsan-
trasyonu saptand›. N-asetil sisteinin at›m h›z›na etkisi saptand›ktan son-
ra, etkinli¤i ATP veya IBMX ile araflt›r›larak etki mekanizmas› ortaya
konmaya çal›fl›ld›. Siliyer at›m frekans› Sissons-Ammons video analiz
sistemi ile saptand›.
Bulgular: Siliyer fonksiyonlarda maksimum stimülasyonun 10 mg/ml
NAC konsantrasyonunda oldu¤u saptand›. Y›kama sonras› siliyer ha-
reketler dramatik olarak artt›. Bu art›fl NAC art› IBMX ve NAC art›
ATP y›kamalar›ndan sonra daha da art›fl gösterdi.
Sonuç: N-asetil sisteinin apikal kullan›m›, siliyer at›m frekans›n› be-
lirgin düzeyde art›rmaktad›r. N-asetil sistein sonras› PBS ile y›kamak
ile klinik etkinlik elde edilebilir.
Anahtar sözcükler: N-asetil sistein, sinonazal, siliya.
Abstract
Objective: To evaluate the pharmacological effects of the N-acetylcys-
teine (NAC) on human respiratory epithelial cultures specifically
addressing electrolyte transport and cilia beat frequency.
Methods: Well-differentiated human bronchial epithelial cultures
grown at an air liquid interface were treated on the apical or basolateral
surface with varying concentrations of NAC. The best NAC concentra-
tion for ideal cilia beat frequency was found. The effects of NAC were
evaluated on cilia beat frequency. After the effect of N-acetylcysteine on
beat rate was found, its efficiency was investigated by ATP or IBMX to
understand its mechanism of action. Changes in ciliary beat frequency
were determined using the Sissons-Ammons video analysis system.
Results: Maximal stimulatory effect on cilia function was evident at 10
mg/ml NAC concentration. After wash up, cilia movement were
increased very dramatically. This increase of cilia beat frequency was
even higher after NAC plus IBMX and NAC plus ATP washings.
Conclusion: Apical application of NAC prominently stimulates cilia
beat frequency and after wash up, cilia movement was increased very
dramatically. After NAC use by washing with PBS in clinical efficacy
can be enhanced.
Keywords: N-acetylcysteine, sinonasal, cilia.

transported by the ciliary beating of the ciliated cells. The
mucus layer has a variable range of thickness, and mucus is
kept away from ciliated airway epithelia by the presence of
a ~7 μm periciliary liquid layer which, as the name suggests,
surrounds the beating cilia and acts as a lubricant to keep
mucus away from the epithelial cell surface. All layers con-
sisting mucus layer make up the airway surface liquid
(ASL).[3] The rate of mucociliary clearance is strongly influ-
enced by the degree of hydration of the ASL and mucus lay-
ers.[4] Mucus hydration is regulated by epithelial ion trans-
port processes from both the superficial epithelium and the
submucosal glands.[3,4]
Many techniques have been used to improve mucociliary
clearance in patients with a variety of chronic mucus hyper-
secretory conditions. The goal is to hydrate airway surfaces
by stimulating secretion and/or inhibiting absorption.
N-acetylcysteine (NAC) is a widely used mucolytic
agent, with known anti-oxidative and anti-apoptotic proper-
ties (references). However, some clinical and pharmacolog-
ical effects of NAC are still unclear. In addition to thinning
the mucus, animal studies suggest that NAC also stimulates
ciliary beating frequency at low concentrations, while it
inhibits the same function at higher concentrations (need
references here). Thus, in this study we investigated the in
vitro effect of NAC on human sinonasal ciliary activity.
Ciliary beating frequency increased arithmetically at the
best concentration of NAC and decreased after washout.
NAC has an effect on some ligands and receptors and by this
way it can change mucociliary clearance.
Materials and Methods
Surgical technique
Air-liquid interface cultures
Human sinonasal epithelium from patients who under-
went chronic rhino sinusitis operation was cultured
according to the described protocol below.
Briefly, tissue was harvested which grown on 6.5-mm
diameter permeable filter supports (Corning Life Sciences,
Lowell, MA, USA) submerged in culture medium. The
media was removed from the surface at the 4th day after
reaching confluence of the cells which was fed via the basal
chamber. Differentiation and ciliogenesis occurred in all
cultures within 10 days to 14 days.
Ciliary beat frequency analysis
Images were visualized using a 20×lens on an inverted
scope (Leica Microsystems, Inc., Bannockburn, IL, USA).
Image data was captured using a Model A602f-2 Basler area
scan high-speed monochromatic digital video camera
(Basler AG, Ahrensburg, Germany) at a sampling rate of
100 frames per second with a resolution of 640x480 pixels.
The video images were analyzed using the Sisson-Ammons
video analysis (SAVA) system version 2.1 (Ammons
Engineering, Mt. Morris, MI, USA).18
For each experiment, a large area of beating cilia was
detected with the inverted microscope. The digital image
signal was then routed from the camera directly into a dig-
ital image acquisition board (National Instruments Corp.,
Austin, TX, USA) within a Dell XPS 710 Workstation
(Dell, Roundrock, TX, USA) running Windows XP
Professional (Microsoft, Redmond, WA, USA) operating
system. Images were captured, compressed, and stored to
disk. Files were reloaded and analyzed with virtual instru-
mentation software highly customized to perform ciliary
beat frequency (CBF) analysis. All of the recordings in the
present experiments were made at 200×magnification.
Experiments were all performed at ambient temperature
(22 °C).
Whole field analysis was performed with each point
measured representing one cilium.
For each sample, the reported frequencies represent the
arithmetic means of these values, followed by standard devi-
ations. As our data includes thousands of individual points
(cilia), very small changes in CBF result in a statistical sig-
nificance due to the high power of the study. Thus, statisti-
cal analysis of the arithmetic means derived from each cul-
ture was performed using two-tailed unpaired t-tests to
ensure reproducibility.
Experimental paradigm
Well-differentiated human bronchial epithelial cultures
grown at an air liquid interface were treated on the apical
or basolateral surface with varying concentrations N-
acetylcysteine. First step was to estimate the best concen-
tration for cilia beat frequency. Cell cultures of four
chronic rhino sinusitis patients were used to find the best
NAC concentration in this study. In the second step, the
effect of NAC were evaluated on cilia beat frequency. The
effects which occurred at cilia during 20 minutes after
wash up the cell with DPS using best concentration of
NAC were studied. After wash up, beat frequency of cilia
was increased dramatically and decreased gradually in 10
minutes.
In the third step, four different chronic rhinosinusitis
cell cultures (transwells) were used. The efficiency of
NAC was investigated with adenosine triphosphate (ATP)
ENT Updates
Kapusuz Gencer Z et al.
88

or 3’-isobutyl-1-methylxanthine (IBMX) using four tran-
swells for better understanding on NAC mechanism. First
transwell was used for showing ATP effect, second tran-
swell was used for showing “ATP + NAC” effect, third
transwell was used for showing IBMX effect and fourth
transwell was used for showing “IBMX + NAC” effect.
Four transwells were prepared from each patient’s mate-
rial. Two transwells were used to demonstrate the effects of
NAC and ATP, while two transwells were used to demon-
strate the effects of NAC and IBMX. IBMX effects on cilia
were examined in 10 minutes in two transwells. After ten
minutes with IBMX, NAC was added to the first transwell,
while Dulbecco's phosphate buffered saline (DPBS) was
added to the second transwell. Following next ten minutes,
both transwells were washed up. ATP effects on cilia were
examined in 10 minutes in two transwells. After ten minutes
with ATP, NAC was added to the third transwell, while
DPBS was added to the fourth transwell. Following next ten
minutes, both transwells were washed up. These mentioned
steps were repeated for all four patients.
Changes in ciliary beat frequency were determined using
the Sissons-Ammons Video Analysis system. Images were
captured, compressed, and stored to disk. Files were
reloaded and analyzed with virtual instrumentation software
highly customized to perform CBF analysis. After CBF was
analyzed, arithmetic mean and standard deviation were cal-
culated.
Results
N-acetylcysteine concentration was titrated to identify the
optimal concentration for our experimental paradigm for
effects on cilia function. CBF was analyzed following
application of NAC at 0.5 mg/ml, 5 mg/ml, 7.5 mg/ml, 10
mg/ml, 15 mg/ml, and 50 mg/ml. We found at 50 mg/ml
that NAC was ciliotoxic with the cessation of cilia beating
2 minutes following application. Maximal stimulatory
effect on cilia function was evident at 10 mg/ml NAC con-
centration. At first step, we found 10 mg/ml for maximal
stimulatory effect. Afterwards, CBF was analyzed at 5
mg/ml, 10 mg/ml, and 15 mg/ml NAC concentrations for
five minutes, and then cilia were washed up two times with
DPBS. After wash up, cilia movement increased very dra-
matically (Fig. 1).
NAC was ciliotoxic with the cessation of cilia beating 2
minutes following application. Maximal stimulatory effect
on cilia function was evident at 10 mg/ml NAC concentra-
tion. At first step, we found 10 mg/ml for maximal stimu-
latory effect. Afterwards, CBF was analyzed at 5 mg/ml, 10
mg/ml, and 15 mg/ml NAC concentrations for five min-
utes, and then cilia were washed up two times with DPBS.
After wash up, cilia movement increased very dramatically
(Fig. 1).
CBFs measured after IBMX, after IBMX+NAC, and
after washout were shown in Fig. 2. CBFs measured after
IBMX, after IBMX+ DPBS, and after washout were shown
in Fig. 3. CBF measured after ATP, after ATP+ NAC, and
after washout were shown in Fig. 4. CBF measured after
ATP, after ATP+ DPBS, and after washout were shown in
Fig. 5.
Comparison of ATP-NAC and ATP-DPBS effects on
CBF was shown in Table 1. Comparison of IBMX-NAC
and IBMX-DPBS effects on CBF was shown in Table 2.
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N-acetylcysteine effects on sinonasal cilia function
89
Fig. 2. IBMX and NAC effects on sinonasal cilia function.Fig. 1. N-acetylcysteine effects on sinonasal cilia function.

Discussion
Inhaled pathogens in the nose and paranasal sinuses are
cleared by the sinonasal respiratory epithelium. Proper cil-
iary beating and the biological properties of ASL, which con-
sists of cross-linked glycoproteins, are the main properties of
respiratory epithelial mucociliary clearance (MCC).[5] The
mucus flow is mediated by the ciliary beat frequency. Mucus
transportability by the cilia is impaired when it becomes
more rigid and more viscous.[5]
N-acetylcysteine has been frequently used as supplemen-
tary medication for various diseases i.e. chronic obstructive
pulmonary disease, respiratory distress syndrome, and CF.[6]
Cysteine, the main glutathione precursor, is the reduced
form of NAC.[7] The wide use of NAC depends on its capa-
bility to interrupt disulfide bonds and to decrease mucus vis-
cosity; however, recent literature emphasizes the anti-oxi-
dant properties of NAC. A few studies emphasized the ion
transport ability of NAC in the respiratory epithelium.[8,9]
Furthermore, GSNO (S-nitrosoglutathione), a deriva-
tive of glutathione, is shown to improve Cl2(chloride)
efflux from CF airway epithelial cells via CFTR.[10–12]
Consequently, it seemed rational to examine the effect of
NAC, which is an indirect glutathione precursor, on Cl2
efflux from epithelial cells of the respiratory tract.[13]
N-acetylcysteine initiated a straight dose- and time-
depended reduction in ciliary beat frequency. This decrease
ENT Updates
Kapusuz Gencer Z et al.
90
Fig. 3. IBMX and DPBS effects on sinonasal cilia function. Fig. 4. ATP and NAC effects on sinonasal cilia function.
Fig. 5. ATP and DPBS effects on sinonasal cilia function.
ATP-NAC/ATP-DPBS 0.047828
Entry ATP-NAC wo/ATP-DPBS wo 0.044302
Exit ATP-NAC wo/ATP-DPBS wo 0.106019
Table 1. Comparison of ATP-NAC and ATP-DPBS effects on sinonasal
cilia function.
IBMX-NAC/IBMX-DPBS 0.066444
Entry IBMX-NAC wo/IBMX-DPBS wo 0.401604
Exit IBMX-NAC wo/IBMX-DPBS wo 0.000623
Table 2. Comparison of IBMX-NAC/IBMX-DPBS effects on sinonasal
cilia function.

was first shown at 2 mg/ml and reached significant levels at
20 mg/ml. Total cessation of ciliary movement was initiat-
ed within 15 s and at 200 mg/ml; however, this effect was
completely reversible within 15 min of perfusion with medi-
um alone. Although NAC had no effect on the ciliary beat-
ing pattern, it appears to have a completely reversible
inhibitory effect on the ciliary beating frequency of human
epithelium.[14]
Chloride efflux measured with the fluorescent probe
MQAE revealed that treatment with NAC increased Cl2
efflux from CFBE cells in a dose-dependent manner, with
the concentration of 10 mM producing the highest effect.
Anions have an important role in the regulation of ASL
volume, viscosity and pH.
In this experiment, the effect of NAC on human nasal
cilia in tissue was studied. Sample of human sinonasal
epithelium of patients who underwent chronic rhino
sinusitis operation was cultured according to previously
described protocol. Cell cultures of four chronic rhino
sinusitis patients were used to find the best NAC concen-
tration. Maximum effect on cilia was determined at 10
mg/ml NAC concentration. Cilia were examined at differ-
ent NAC concentrations for five minutes, and later
washed up two times with DPBS at second step. After
wash up, ciliary movement increased dramatically. After
this increase, ciliary movement decreased gradually in ten
minutes. The reason for this increase on CBF was ques-
tioned. It was postulated that some cell cycles might be
activated after the wash up procedure.
In the third step, ATP and IBMX were added to NAC
transwell. IBMX is a calcium channel agonist. Extracellular
nucleotides not only ATP, but also ADP and AMP, are key
components of the signaling network regulating airway
clearance. They are released by the epithelium into ASL to
stimulate cilia beating activity, mucus secretion and airway
hydration.[15] The reaction 2ADP <--> ATP + AMP, provid-
ing energy for the beating of cilia.[16]
Dynamic regulation of respiratory CBF is regulated by
fluxes in intracellular calcium Ca2+. P2X receptors (P2XR)
are extracellular ATP-gated, Ca2+-permeable, nonselective
cation channels. CBF significantly increased four times
over baseline from 5.99±3.16 Hz to 22.4 ± 4.33 Hz in the
presence of zinc chloride (50 micromoles) and calcium
chloride (3 mM).[17]
In the third step of this experiment both ATP and
IBMX increased CBF after wash up returned to normal. If
NAC was added to these transwell mediums, dramatic
increase was noted after wash up. This can be attributed to
the positive effect of NAC on Cl2efflux by a direct effect
on cystic fibrosis transmembrane conductance regulator
protein and/or alternative chloride channels.[18] IBMX ago-
nist of NAC enhancing Cl2efflux from CFBE cells indi-
cates that not only CFTR, but also alternative Cl2chan-
nels are responsible for the increase in Cl2efflux observed
after treatment with NAC.[18] Then, intracellular Cl-con-
centration in bronchial epithelial cell was significantly
decreased. It was concluded that further studies were
needed to show the exact reason of this dramatic increase
which probably was caused by the blockage of Ca++ and Cl-
channel activity cells.
Conflict of Interest: No conflicts declared.
References
1. Gibbons IR. The relationship between the fine structure and
direction of beat in gill cilia of a lamellibranch mollusc. J Biophys
Biochem Cytol 1961;11:179–205.
2. Chmiel JF, Davis PB. State of the art: why do the lungs of patients
with cystic fibrosis become infected and why can’t they clear the
infection? Respir Res 2003;4:8.
3. Boucher RC. Regulation of airway surface liquid volume by
human airway epithelia. Pflugers Arch 2003;445:495–8.
4. Tarran R, Button B, Boucher RC. Regulation of normal and cys-
tic fibrosis airway surface liquid volume by phasic shear stress.
Annu Rev Physiol 2006;68:543–61.
5. King M. Mucus, mucociliary clearance and coughing. In: Bates
DV, editor. Respiratory function in disease. 3rd ed. Philadelphia,
PA: Saunders; 1989. p. 69–78.
6. Boogaard R, de Jongste JC, Merkus PJ. Pharmacotherapy of
impaired mucociliary clearance in non-CF pediatric lung disease.
A review of the literature. Pediatr Pulmonol 2007;42:989–1001.
7. Gillissen A, Nowak D. Characterization of N-acetylcysteine and
ambroxol in anti-oxidant therapy. Respir Med 1998;92:609–23.
8. Köttgen M, Busch AE, Hug MJ, Greger R, Kunzelmann K. N-
Acetyl-L-cysteine and its derivatives activate a Cl-conductance in
epithelial cells. Pflugers Arch 1996;431:549–55.
9. Rochat T, Lacroix JS, Jornot L. N-acetylcysteine inhibits Na+
absorption across human nasal epithelial cells. J Cell Physiol 2004;
201:106–16.
10. Andersson C, Gaston B, Roomans GM. S-Nitrosoglutathione
induces functional DeltaF508-CFTR in airway epithelial cells.
Biochem Biophys Res Commun 2002;297:552–7.
11. Zaman K, Palmer LA, Doctor A, Hunt JF, Gaston B.
Concentration-dependent effects of endogenous S-nitrosoglu-
tathione on gene regulation by specificity proteins Sp3 and Sp1.
Biochem J 2004;380(Pt 1):67–74.
12. Servetnyk Z, Krjukova J, Gaston B, et al. Activation of chloride
transport in CF airway epithelial cell lines and primary CF nasal
epithelial cells by S-nitrosoglutathione. Respir Res 2006;7:124.
Volume 5|Issue 3|December 2015
N-acetylcysteine effects on sinonasal cilia function
91

13. Ventresca GP, Cicchetti V, Ferrari V. Acetylcysteine. In: Braga
PC, Allegra L, editors. Drugs in bronchial mucology. New York,
NY: Raven Press; 1989. p. 77–102.
14. Stafanger G, Bisgaard H, Pedersen M, Mørkassel E, Koch C.
Effect of N-acetylcysteine on the human nasal ciliary activity in
vitro. Eur J Respir Dis 1987;70:157–62.
15. Garcia GJ, Picher M, Zuo P, et al. Computational model for the
regulation of extracellular ATP and adenosine in airway epithelia.
Subcell Biochem 2011;55:51–74.
16. Milara J, Armengot M, Mata M, Morcillo EJ, Cortijo J. Role of
adenylate kinase type 7 expression on cilia motility: possible link in
primary ciliary dyskinesia. Am J Rhinol Allergy 2010;24:181–5.
17. Woodworth BA, Zhang S, Tamashiro E, Bhargave G, Palmer JN,
Cohen NA. Zinc increases ciliary beat frequency in a calcium-
dependent manner. Am J Rhinol Allergy 2010;24:6–10.
18. Varelogianni1 G. Oliynyk I, Roomans G. M, Johannesson M. The
effect of N-acetylcysteine on chloride efflux from airway epithelial
cells. Cell Biol Int 2010;34:245–52.
ENT Updates
Kapusuz Gencer Z et al.
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Please cite this article as: Kapusuz Gencer Z, Saydam L, Cohen NA, Cingi C. N-acetylcysteine effects on sinonasal cilia function. ENT Updates 2015;
5(3):87–92.