Alveolar epithelial CNGA1 channels mediate cGMP-stimulated, amiloride-insensitive, lung liquid absorption

Abstract

Impairment of lung liquid absorption can lead to severe respiratory symptoms, such as those observed in pulmonary oedema. In the adult lung, liquid absorption is driven by cation transport through two pathways: a well-established amiloride-sensitive Na(+) channel (ENaC) and, more controversially, an amiloride-insensitive channel that may belong to the cyclic nucleotide-gated (CNG) channel family. Here, we show robust CNGA1 (but not CNGA2 or CNGA3) channel expression principally in rat alveolar type I cells; CNGA3 was expressed in ciliated airway epithelial cells. Using a rat in situ lung liquid clearance assay, CNG channel activation with 1 mM 8Br-cGMP resulted in an approximate 1.8-fold stimulation of lung liquid absorption. There was no stimulation by 8Br-cGMP when applied in the presence of either 100 μM L: -cis-diltiazem or 100 nM pseudechetoxin (PsTx), a specific inhibitor of CNGA1 channels. Channel specificity of PsTx and amiloride was confirmed by patch clamp experiments showing that CNGA1 channels in HEK 293 cells were not inhibited by 100 μM amiloride and that recombinant αβγ-ENaC were not inhibited by 100 nM PsTx. Importantly, 8Br-cGMP stimulated lung liquid absorption in situ, even in the presence of 50 μM amiloride. Furthermore, neither L: -cis-diltiazem nor PsTx affected the β(2)-adrenoceptor agonist-stimulated lung liquid absorption, but, as expected, amiloride completely ablated it. Thus, transport through alveolar CNGA1 channels, located in type I cells, underlies the amiloride-insensitive component of lung liquid reabsorption. Furthermore, our in situ data highlight the potential of CNGA1 as a novel therapeutic target for the treatment of diseases characterised by lung liquid overload.

Physiological effect of protein kinase C on ENaC-mediated lung liquid regulation in the adult rat lung

“…It is well documented that amiloride specifically blocks LL absorption (2,23), as shown in our model when a Ϫ102% change (decrease) in LL absorption was observed after addition of amiloride (34). When PMA and ionomycin were given in conjunction with amiloride, no significant change in J v compared with that observed with administration of amiloride alone was seen.…”

“…In our model, we have shown that terbutaline increased LL absorption by 157% (34). Administration of PMA and ionomycin to the lungs, together with ␤ 2 -mediated activation of ENaC, significantly reduced J v compared with administration of terbutaline (P ϭ 0.017; Fig.…”

“…Activation of ENaC with the ␤-adrenergic agonist terbutaline [see data previously published by our group (34)] in the presence of PMA and ionomycin resulted in an inhibition of the increased absorption rate normally produced by terbutaline (Fig. 5A), so that there was no statistical difference between the control rate and the test rates (P ϭ 0.12).…”

“…M amiloride (an ENaC blocker) caused the cessation of LL absorption in our preparation (34). If PKC is acting via a different channel, one would expect it to have an additive blocking effect (which would produce net secretion of LL) when activated in conjunction with ENaC inhibition.…”

Knockout Mice Reveal a Major Role for Alveolar Epithelial Type I Cells in Alveolar Fluid Clearance

“…Regulation of alveolar fluid homeostasis is based on active ion transport across the alveolar epithelium (1). Polarized localization and function of the basolateral Na-K-ATPase (Na pump) and apical sodium channels (2) are of crucial importance in transepithelial ion transport and the accompanying fluid clearance across the alveolar epithelial barrier (3,4).…”