Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, C73
Alveolar epithelial CNGA1 channels mediate cGMP-stimulated, amiloride-insensitive lung liquid absorption
W. Wilkinson1, A. Benjamin2, I. De Proost1, M. Orogo-Wenn2, D. Riccardi1, D. Walters2, P. Kemp1
1. School of Biosciences, Cardiff University, Cardiff, United Kingdom. 2. Division of Clinical Sciences, St George's University of London, London, United Kingdom.
Impairment in lung liquid absorption can lead to severe respiratory symptoms such as those observed in pulmonary oedema (1). In the adult lung two principle pathways are involved in cation transport which drives liquid absorption; a well established amiloride-sensitive, Na+ channel (ENaC) (2,3), and more controversially, an age-dependent, amiloride-insenstive pathway which has been suggested to be mediated by cyclic-nucleotide gated (CNG) channels (4). We set out to elucidate the exact molecular identity of this channel and its physiological role in lung liquid absorption. Using immunohistochemistry we show that CNGA1, but not CNGA2 or CNGA3 channel subunits are expressed in the rat lung alveolar epithelium, predominantly in type I pneumocytes. We also developed an in situ assay of lung liquid absorption, briefly, male Wistar rats were given a terminal anaesthetic (i.p. injection of 1:1 ratio of hypnorm and hypnovel) and ventilated whilst the chest was opened and the pulmonary circulation was perfused. The lungs were then instilled with a liquid containing an impermeant tracer which the concentration of which was calculated in later samples to calculate changes in liquid volume, and thus rate of lung liquid absorption (Jv) during control and drug application periods. Application of 1mM 8Br-cGMP increased significantly Jv from -0.92±0.12 ml.h-1.g-1 to -1.35±0.09 ml.h-1.g-1 (n=10; P<0.01). When 8Br-cGMP was applied in the presence of 100μM l-cis-diltiazem or 100nM pseudechetoxin (PsTx), a specific toxin inhibitor of CNGA1 channels there was no significant difference between control and test periods; -0.86±0.03 ml.h-1.g-1 (control) vs. -1.00±0.09 ml.h-1.g-1 (1mM 8Br-cGMP + 100μM l-cis-diltiazem; n=4; P>0.1) and -1.21±0.16 ml.h-1.g-1 (control) vs. -1.13±0.10 ml.h-1.g-1 (1 mM8Br-cGMP + 100nM PsTx; n=4; P>0.5). Using heterologous recombination of CNGA1 and αβγ-ENaC channels in HEK293 cells we showed that 100nM PsTx had no effect on ENaC channels and but did inhibit CNGA1 currents. We also showed that Amiloride had no effect on CNGA1 channels suggesting there is no pharmacological overlap between these channels. In situ, importantly 8Br-cGMP was able to stimulate some lung liquid absorption even in the presence of a maximal inhibitory dose of amiloride (50μM). Furthermore the CNGA1 channel antagonists had little effect on terbutaline-stimulated lung liquid absorption, which could be completely ablated by amiloride. Taken together, these results confirm the importance of CNG channels in cGMP stimulated lung liquid absorption, show the molecular identity of this channels to be CNGA1, further highlight the likely importance of alveolar type I pneumocytes in lung liquid regulation and reinforce the idea that there are two non-overlapping pathways by which liquid absorption can be stimulated in the adult lung and thus may be targeted in the treatment of diseases characterised by lung liquid overload.
Where applicable, experiments conform with Society ethical requirements