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Emerging Concepts in Ion Channel Biophysics
Poster Abstracts
98
24-POS
Board 24
Hemichannel Closure Triggered by Extracellular ATP at Millimolar Concentration
Impairs Airways Ciliary Activity
Karla Droguett
, Mariana Rios, Llilian Arzola, Manuel Villalón, Nelson P. Barrera.
Pontificia Universidad Católica de Chile, Santiago, Chile.
Mucociliary clearance (MCC) in the airway epithelium protects the lungs against contaminants
and microorganisms present in the inspired air. However, in inflammatory chronic respiratory
diseases the MCC is impaired, which in turn contributes to airway inflammation and respiratory
disease progression. Extracellular ATP is a signalling molecule in the epithelium regulating both
basal ciliary beat frequency (CBF) and increased CBF, associated to [Ca
2+
]
i
, after mechanical
stimulation. In addition, the ATP effect on CBF is dependent on pannexin and connexin
hemichannels (Panx/Cx HCs) activity, through an autocrine purinergic mechanism involving
ATP release and Ca
2+
entry. Under pathophysiological conditions of the airways, such as asthma,
high levels of ATP had been measured in broncho alveolar fluid, however it is unclear how these
ATP concentrations affect ciliary activity and MCC. Our goal was to determine the cellular
effect of 1 mM ATP on primary cultures of mouse tracheal epithelium. A series of experimental
methods were carried out, hemichannel functionality through the uptake of ethidium bromide
(EtBr), CBF and ciliary beating forces using atomic force microscopy, and [Ca
2+
]
i
measurements
by fluorimetric assays. The ATP addition reduced the uptake rate of EtBr, effect that was also
observed in the presence of carbenoxolone (100 μM), a Panx/Cx HCs inhibitor. The
Ca
2+
ionophore ionomycin (10 μM) increases the uptake rate of EtBr, which was blocked by
ATP. Furthermore, ATP lowered CBF, ciliary forces and [Ca
2+
]
i
. These results show that high
levels of extracellular ATP, measured in chronic respiratory diseases, trigger epithelial
hemichannel closure that impairs airway ciliary activity and MCC. Funded by CONICYT-
FONDECYT 3150652 (KD), CONICYT 21160416 (LlA) and CONICYT-DPI20140080.