The Importance Of Being NNOS? Unique Localization Of Neuronal Nitric Oxide Synthase To Cilia And Investigating A Nitric Oxide Synthase-1 Polymorphism In Primary Ciliary Dyskinesia

Abstract

Within the respiratory tract, motile cilia beat in a coordinated and directional manner to power mucociliary clearance of Background: inhaled particles and pathogens. Primary Ciliary Dyskinesia (PCD) is a multigenetic autosomal recessive disorder, in which defective cilia and impaired mucociliary clearance increases patient susceptibility to respiratory infection. Nitric Oxide (NO) production is important for normal columnar epithelial cell function and defense against infection but low nasal NO concentrations are consistently reported in PCD patients. It is suggested that mechanochemical dynein ATPases interact with NO Synthase (NOS) proteins and their molecular uncoupling may be responsible for low NO production. Genetic linkage studies have not identified the genes as PCD gene candidates, yet the NOS cause of low NO in PCD remains undetermined. NOS isoenzymes, neuronal (nNOS), inducible (iNOS) and endothelial (eNOS), encoded by the genes respectively, are responsible NOS1-3 for NO production. Whilst iNOS and eNOS have been identified in ciliated epithelium, nNOS immunoreactivity has not. Interestingly, various polymorphisms are associated with low NO concentrations in other ciliopathies, moreover, reduced NO in Cystic Fibrosis, NOS Acute Chest Syndrome and Asthma is associated particularly with more than 12 AAT allelic repeats within intron-13. NOS1 To localize all three NOS isoenzymes, in particular nNOS, within respiratory epithelium and investigate whether the intron-13 Aims: NOS1 AAT copy number correlates with low NO in PCD. NOS isoenzymes were immuno-localized in nasal polypectomy/turbinectomy samples (n=6) by a standard ABC method with Method: DAB chromogen detection. The AAT repeat region (Genbank AF085229) was amplified from gDNA by polymerase chain reaction NOS1 (PCR) from patients that we diagnosed as non-PCD (n=12) or PCD (n=13). PCR products were analysed by gel electrophoresis, gene sequencing and gene scanning methods. Allelic AAT copy numbers were compared with patient nasal NO concentrations (NiOx, Aerocrine) (as per ). Sullivan et al._2001_AJRCCM_vol.164_p.2186 nNOS, iNOS and eNOS immunoreactivity was present in ciliated epithelium. Most strikingly nNOS localized specifically to the Results: ciliated compartment, a novel finding confirmed by peptide absorbtion. PCD and non-PCD patients exhibited mean nasal NO concentrations of 33ppb (SEM±5) and 594ppb (SEM±113) respectively (p=0.0001). The allelic sum of AAT repeats did not correlate NOS1