P256 P2X4 and nNOS Expression in Human Ciliated Airway Epithelium
Background Calcium (Ca2+) and nitric oxide (NO) modulate ciliary beat frequency (CBF). NO synthase (NOS) isoenzymes, responsible for NO production, localise in human airway epithelium and constitutive NOS activity is dependant on Ca2+. Adenosine triphosphate gated purinergic ion channels, comprised of P2X1–7 subunits, govern Ca2+ influx and P2X4 reportedly localises to rabbit airway cilia. Also, P2X proteins co-localise with neuronal NOS (nNOS) in guinea pig cochlea outer hair cells and rat hypothalamus. We hypothesised that P2X4 and nNOS co-localise in human airway cilia and are involved in modulating CBF.
Objectives To determine P2X1,3–7 mRNA expression and P2X4 and nNOS localisation in human nasal epithelium, and assess their possible interaction in CBF modulation.
Methods and Results Relative to b-actin expression, consistent and moderate P2X4,6 mRNA and variable P2X3,5,7 mRNA were detected by RT-PCR in (n=4) human primary airway epithelium cultured at air-liquid interface (ALI). In ALI cultured primary epithelium P2X4 localised (n=3) to cell membranes and cytoplasm and nNOS localised to cilia (n=3) by immunofluorescence. P2X4 localised to the ciliary tips (n=2), whilst nNOS localised to the proximal portion of cilia (specificity confirmed by blocking peptide) in nasal polyp paraffin wax sections by immunohistochemistry (n=6). High speed video microscopy confirmed a 30 minute baseline CBF at 37oC (12.9 Hz SD±0.8, n=5) on nasal epithelium biopsies (n=2) and 30 minutes of NOS inhibition [with 1 mM: L-N-Ornithine, 1400W and S-Methyl-L-thiocitrulline] reduced baseline CBF by 20% to 9.6 Hz SD±0.9 (p<0.001) but the P2X4 inhibitor [10 µM brilliant blue G] had no effect.
Conclusion P2X4 and nNOS are expressed in human airway cilia but do not co-localise. NOS inhibition reduced CBF whilst P2X4 inhibition did not, suggesting that blocking P2X4 activity alone is not sufficient to modify NOS activity or CBF .