(412) Novel Approaches to Identifying Ion Transport Dysfunction During the Nasal Potential Difference Assay

Background: Ion transport across the airway epithelium is vital for respiratory health. Defective ion transport is directly responsible for the morbidity and mortality in cystic fibrosis (CF) and is present in a diverse array of pulmonary diseases, including eosinophilic asthma and chronic obstructive pulmonary disease. We hypothesize that certain defects in ion transport that are present during CF and other airway diseases are not detectable using the standard nasal potential difference (NPD) assay methodology, but these defects may be observed by altering the standard protocol. The goal of this project is to explore unique measurements of ion transport in vivo in order to detect novel defects in the functional properties of CFTR and other ion channels that are relevant to the health of the airways. We aim to detect these defects with high specificity and sensitivity.

Methods: The standard procedure for the measurement of CFTR function using NPD measurements consists of the sequential perfusions of Ringer’s solution, amiloride (inhibits sodium transport) in Ringer’s solution, amiloride in buffered solution with gluconate substitution of chloride (gluconate buffer, establishes chloride gradient), amiloride/isoproterenol (activates CFTR) in gluconate buffer, and amiloride/isoproterenol/ATP (activates alternative chloride channels) in gluconate buffer. To further explore the potential of the NPD assay, we vary the sequence and composition of the perfused solutions from this in order to obtain unique measurements of ion transport function. Solutions with diverse salt and mannitol content allow for alterations in tonicities/osmolarities for use during the assay in order to study the effects of these parameters on the activity of ion channels. Amiloride, isoproterenol, ATP, and lubiprostone (a synthetic prostaglandin that activates CFTR) are dissolved in these buffers in various combinations.

Results: During a single catheter placement in a nostril, it is possible to stimulate ion transport, remove the stimuli and allow the nasal epithelium to return to baseline, and then re-stimulate, resulting in similar responses as the initial stimulation, in a matter of minutes. This technique allows us to explore the impact of different sequences in quick succession. Varying the strength of the chloride gradient has dramatic impacts on the subsequent responses of CFTR and calcium-activated chloride channels to stimulations. Increasing the sodium concentration above that found in normal Ringer’s solution results in inhibition of ion transport, while increasing the osmolarity using mannitol does not. Lubiprostone is able to stimulate CFTR-mediated ion transport in vivo.

Conclusions: There is great potential for the use of novel NPD assays to diagnosis ion transport dysfunction in airway diseases. These new measures may also prove useful for the evaluation of therapeutics that modulate ion channel activities.

Acknowledgements: This work was supported by the Cystic Fibrosis Foundation (BRATCH16I0 to P.E.B. and ZEITLI2010 to P.L.Z.), National Institutes of Health (R01HL155325 to P.E.B), and the Department of Pediatrics at National Jewish Health.

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