High Nitric Oxide Production in Human Paranasal Sinuses

J.O.N. Lundberg, T. Farkas-Szallasi, E. Weitzberg, J. Rinder et al
Summarized by Jeong Hoon Oh, MD
Nature Medicine, Vol. 1, No. 4, April 1995

Physiological role of the human paranasal sinuses ; several theories, but enigma
Paranasal sinuses - generally sterile in healthy subjects
* mech. of sterility ; not fully understood
- ciliary activity & secretory Ig with intact ostium : cleansing of the sinus ostium
0.5% of all common colds - complicated by sinusitis
Nitric Oxide (NO) : produced in mammalian cells by NO synthase (NOS)
* substrate ; amino acid L-arginine
* 3 isoforms of human NOSs cloned : neuronal, endothelial & inducible NOS
- neuronal & endothelial NOS ; constitutively expressed & produce low level of NO
( activity is dependent on Ca influx )
- inducible NOS ; expressed only after induction by certain cytokines or by bacterial lipopolysaccharide
( not dependent on Ca influx )
( susceptible to glucocorticosteroid - suppressed )
- All isoforms are blocked by L-arginine analogues (ex. N-nitro-L-arginine methyl ester (L-NAME))
Role of NO in host defence
- implied when produced in large quantities by an inducible NOS
- involved in mouse macrophage-mediated killing of a variety of pathogens
- antiviral properties
* The origin & role of NO of healthy subjects ; not known
In nasal airways, continuous production of NO
- indicated by presence of gas in nasally derived air
- In Kartagener's syndrome, pts lack NO in nasal air & have severe problems with recurrent airway infection
In this study, we identify the epithelium in paranasal sinuses as a major site of NO production and suggest a role for airway-derived NO in primary host defence.

Methods
Measurement of NO in sinus & nasal air
in 5 healthy subjects (ages 29-41, 4 males)
; after topical anesth.
-> max. antrum puncture with an autoinjector (Sinoject ) through the inferior meatus
-> a syringe was connected to a catheter placed in the sinus
-> 20ml of air aspirated over a period of 15 sec
-> repeated every minutes for 5 minutes
-> NO measurement performed before & after intrasinus instillation of NOS inhibitor L-NAME (3.7mM in saline) & as a control D-NAME (3.7 mM in saline)
-> entire sinus was filled with the solution & emptied again after a 10-min incubation period
; additional experiment
nasal air was aspirated (20ml over a period of 15s) using an occlusive nasal olive (connected to a syringe & introduced into the vestibulum of the nose)
-> asked to hold breath with mouth closed while the contralateral nostril was left open
- ( air was forced from one nostril to the other via the nasopharynx )
-> before & after measurement of intranasal administration of a total of 20mlg L-NAME (14.8mM in saline 5ml) - inhaled as an aerosol through the nose over 10-min period
in 4 pts (56-77 yrs, 3 males) undergoing routine surgery to alleviate orbital compression due to proptosis
; air was aspirated repeatedly from one max. sinus
in all experiments ; the aspirated air was immediatelt injected into an NO chemiluminescence analyzer
; NO values remained stable in the syringe for longer than 2 min at widely varied conc. of NO in air
in 49 healthy non-smoking subjects (age 0-62, 22 male)
measurement of nasal NO concentrations continuously by sampling air (0.7 l /min) form one nostril
-> nasal olive was connected directly to the sampling tube of chemiluminescence analyzer and introduced into one nostril
-> asked to breath through the mouth (contrlateral nostril was left open)
: NO levels in ambient air were less than 4 ppm in all
Immunohistochemical & in situ hybridization studies
Mucosal Bx from maxillary, frontal, or sphenoid sinus in 8 pts after surgical opening
Nasal Bx from inferior or middle turbinate in all patients
-> specimens incubated with
- monoclonal & polyclonal antibodies raised against fragments of mouse macrophage NOS, and
- monoclonal antibodies against human neuronal & endothelial NOS
(the mouse macrophage NOS shows 77% identity to human hepatocyte inducible NOS)
-> sections were incubated with fluorescein isothiocyanate-conjugated anti-mouse IgG & anti-rabbit IgG antibodies
-> analyzed in microscope
* Control : macrophage NOS antibodies were preincubated with mouse macrophage lysate for 2 hrs

Results
Sinus and nasal NO concentrations
High concentrarions of NO ; found in sinus air on first aspiration
- in all healthy subjects & in the pts undergoing surgery
Mean sinus NO concentration in healthy subjects (n=5)
: 9.1 ?3.8 ppm
- remained similar during repeated aspirations
-> indicating continuous production
Estimated NO release into one maxillary sinus : 20 nmol /min
Fig. 1
Sinus NO concentrations ;
- not significantly changed by local instillation of control substrate D-NAME
- reduced by NOS inhibitor L-NAME by 78% compared with D-NAME
( * Nasal NO concentrations (1.2 ?0.2ppm) were only reduced by 22% following nasal inhalation of L-NAME )
Fig. 2
Age-dependency of NO values
- observed when sampling air continuously from one nostril in group of healthy subjects
- NO levels were increased with age
- low in newborns, and reaching levels similar to those in adults at the age of 10
Fig. 3
* higher sampling flow rate using continuous method (0.7 l/min)
- generally lower NO concentrations than in sampling of air into a syringe (0.08 l/min)

Tissue expression of NOS
Polyclonal & monoclonal antibodies against mouse macrophage NOS
- staining in the epithelial cells of paranasal sinuses in all biopsies
( strongest in the apical part of the epithelial cells )
- staining always disappeared if the antibodies were preincubated with mouse macrophage lysate
Patchy staining in nasal epithelium
Monoclonal antibodies against neuronal and endothelial NOS
- did not stain the sinus or nasal epithelium
With in situ mRNA hybridization
; both human hepatocyte NOS probes - labelled the epithelial cells in the sinus
nasal epithelium exhibited a low grain density ( higher than background level )
human neuronal & endothelial NOS probes did not label the epithelium
same probes labelled human brain tissue and vascular tissue

Discussion

large & continuous production of NO in human maxillary sinuses
> conc. of NO in the range of highest permissible atmospheric pollution levels (25ppm)
NO - originate from the epithelial cells lining the sinus
( since strong NOS immunoreactivity was found apically in those cells.)
- sinus NO values were reduced by local instillation of an NOS inhibitor into the sinus
in situ hybridization and immunohistochemical studies
: NOS sharing mRNA sequence homology with human hepatocyte inducible NOS
-abundant in normal sinus epithelial cells
bronchial epithelial cells ; express inducible NOS
- in inflammatory conditions
- in vitro after stimulation with certain cytokines
- not under basal conditions

* NO excretion per min. into one single maxillary sinus
- exceeded the total normal NO output from entire lower airways
Sinus NOS : differs from the NOS found in normal lower airway epith.
- shows mRNA sequence homology with human hepatocyte inducible NOS
- produce large amounts of NO
- properties associated with constitutive isoforms of NOS

@ process regulating the expression and activity of sinus NOS- remains to be studied.
In nasal epithelium ; only weak & patchy immunostaining for inducible NOS
minor decrease in nasal NO concentrations upon nasally inhaled L-NAME
-> much of NO found in nasal air ; produced in the paranasal sinuses
nasal NO concentrations will be reduced
- in conditions altering the communication bet. the sinuses & nasal cavity
- in situations where one or several sinuses are filled with fluid or poorly developed
age-dependent increase in nasal NO levels
: to follow the development and pneumatization of paranasal sinuses

# The concentration of NO in normal paranasal sinuses
; greatly exceed those that are bacteriostatic to S.aureus
- indicating a role for NO in sinus host defense
sterility of the sinuses (compared to nasal mucosa)
- explained by the differences in epithelial NO production
NO produced in the sinuses will continuously enter the nasal cavity
and have biological effects in more distal parts of the airways following inhalation
-> sinus-derived NO ; affect pulmonary blood flow or act in an 'aerocrine' fashion (participate in the first line of defence against airbone infectious agents)
Airway-derived NO ; have an alternative or complementary role in host difense
- increase ciliary beat frequency in bovine respiratory epithelium
- apical location of airway NOS (cilia are anchored to the same area of cell)
Excess production of NO - may be pathogenic;
* asthmatic airway epithelia express an inducible NOS
* asthmatics show elevated NO levels in exhaled air
- the production site within the host cell will determine whether the NO has a damaging effect on the host cell
; in the sinus, the enzyme seems to be located mainly apically in epithelial cells,
whereas inducible NOS staining in asthmatic epithelium is seen in the cytosol.
-> In sinus epithelial cells, most NO may be released extracellulary,
whereas in asthmatic bronchial epithelium, intracellular NO levels may be high

Conclusion
- There is a continuous and large production of NO in the paranasal sinuses in healthy subjects.
- An enzyme showing mRNA sequence homology with human hepatocyte inducible NOS, is constitutively expressed, and located apically in epithelial cells of the sinuses.
- NO may serve important host defense functions in these enigmatic cavities.

NITRIC OXIDE

Richard A. Robbins, Matthew B. Grigham
summarized by Jeong Hoon Oh, MD
Int J Biochem Cell Bio Vol. 29 No. 6 857-860 1997

Synthesis & Degradation
formed when one of the chemically equivalent guanido groups of the essential amino acid L-arginine is oxidized by five electrons
- catalyzed by a group of enzymes (nitric oxide synthase(NOS)) & several co-factors
( only L-arginine is cleaved ( not D-arginine ))
# NOS exist in several isoforms -> variation in NOS activity
* Constitutive NOS (cNOS, Type III NOS) : generally exist in endothelial cells
- constitutively expressed & Calcium-dependent
- cNOS accounts for the baseline production of the small amounts of NO from endothelial cells
* Brain NOS (bNOS, nNOS, Type I NOS) : predominantly in neural tissue
- constitutively expressed & Calcium-dependent
- produces picomolar amounts of NO
* Inducible form NOS (iNOS, Type II NOS) : detected in a variety of tissues & organs in addition to vascular endothelium
- not expressed in most tissues, but is induced by bacterial lipopolysaccharide(LPS) or cytokines (ex. TNF, IL-1, IFN)
- mech. of activation is different to constitutive NOS, but 50% homologous with cNOS or bNOS at the protein level
- induction-> formation of much larger amounts (>1000-fold) of NO for longer periods of time
# several co-factors necessary for NOS activity
; flavones(FAD, FMN), tetrahydrobiopterin, NADPH
# major regulator of iNOS activity
; transcriptional regulation of iNOS mRNA
( Nuclear factor -k?appears to play a major role in regulating iNOS transcription )
# Inhibitors of NOS ; synthetic & naturally occuring
- most inhibitors have substitutions made at the guanido group of arginine
( compete for the active site on NOS with arginine )
; ex. L-N-monomethyl-L-arginine (L-NMMA), L-nitroarginine methyl ester (L-NAME), aminoguanidine

Biological Function
# endothelial-dependent control of vascular tone & mediates vascular smooth muscle relaxation
- by increasing formation of cyclic guanyl monophosphate
# endothelium-independent vascular smooth muscle relaxation in cerebral & other arteries
# in the brain, act as a neural messenger mediating the action of glutamate acting at NMDA receptors
-> excess NO accounts for a major portion of the neural damage following strokes
# modulate both acute & chronic inflammatory reactions
- NO may suppress inflammation by reducing lymphocyte activation
# relaxes tracheal muscle and reduces metacholine-induced bronchoconstriction
- There is evidences of NO functions as the neurotransmitter of the inhibitory non-adrenergic & non-cholinergic bronchodilator response
@ NO is a potent vasodilator and may narrow airways by dilating the bronchial vessels
- airway blood vessel dilation and edema proposed to account for the airway obstruction in asthma
# participate in host defense by mediating antimicrobial activity and cytotoxicity for tumor cells
also increases ciliary beat frequency

Medical Applications
# Excessive production of NO -> hypotention seen in septic shock
( NOS inhibitors result in an increase in blood pressure in septic shock )
# Beneficial during respiratory distress syndrome
- inhalation of NO would improve ventilation-perfusion mismatching by selective vasodilation of well-ventilated alveoli.
# NO levels in exhaled breath are increased in some disorders such as asthma