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.

Background and PurposeHyperglycaemia increases glucose concentrations in airway surface liquid and increases the risk of pulmonary Pseudomonas aeruginosa infection. We determined whether reduction of blood and airway glucose concentrations by the anti‐diabetic drug dapagliflozin could reduce P. aeruginosa growth/survival in the lungs of diabetic mice.Experimental ApproachThe effect of dapagliflozin on blood and airway glucose concentration, the inflammatory response and infection were investigated in C57BL/6J (wild type, WT) or leptin receptor‐deficient (db/db) mice, treated orally with dapagliflozin prior to intranasal dosing with LPS or inoculation with P. aeruginosa. Pulmonary glucose transport and fluid absorption were investigated in Wistar rats using the perfused fluid‐filled lung technique.Key ResultsFasting blood, airway glucose and lactate concentrations were elevated in the db/db mouse lung. LPS challenge increased inflammatory cells in bronchoalveolar lavage fluid from WT and db/db mice with and without dapagliflozin treatment. P. aeruginosa colony‐forming units (CFU) were increased in db/db lungs. Pretreatment with dapagliflozin reduced blood and bronchoalveolar lavage glucose concentrations and P. aeruginosa CFU in db/db mice towards those seen in WT. Dapagliflozin had no adverse effects on the inflammatory response in the mouse or pulmonary glucose transport or fluid absorption in the rat lung.Conclusion and ImplicationsPharmacological lowering of blood glucose with dapagliflozin effectively reduced P. aeruginosa infection in the lungs of diabetic mice and had no adverse pulmonary effects in the rat. Dapagliflozin has potential to reduce the use, or augment the effect, of antimicrobials in the prevention or treatment of pulmonary infection.

-In pulmonary epithelia, ␤-adrenergic agonists regulate the membrane abundance of the epithelial sodium channel (ENaC) and, thereby, control the rate of transepithelial electrolyte absorption. This is a crucial regulatory mechanism for lung liquid clearance at birth and thereafter. This study investigated the influence of the gaseous signaling molecule hydrogen sulfide (H 2S) on ␤-adrenergic agonistregulated pulmonary sodium and liquid absorption. Application of the H2S-liberating molecule Na2S (50 M) to the alveolar compartment of rat lungs in situ decreased baseline liquid absorption and abrogated the stimulation of liquid absorption by the ␤-adrenergic agonist terbutaline. There was no additional effect of Na2S over that of the ENaC inhibitor amiloride. In electrophysiological Ussing chamber experiments with native lung epithelia (Xenopus laevis), Na 2S inhibited the stimulation of amiloride-sensitive current by terbutaline. ␤-adrenergic agonists generally increase ENaC abundance by cAMP formation and activation of PKA. Activation of this pathway by forskolin and 3-isobutyl-1-methylxanthine increased amiloride-sensitive currents in H441 pulmonary epithelial cells. This effect was inhibited by Na 2S in a dose-dependent manner (5-50 M). Na2S had no effect on cellular ATP concentration, cAMP formation, and activation of PKA. By contrast, Na 2S prevented the cAMP-induced increase in ENaC activity in the apical membrane of H441 cells. H441 cells expressed the H 2S-generating enzymes cystathionine-␤-synthase, cystathionine-␥-lyase, and 3-mercaptopyruvate sulfurtransferase, and they produced H 2S amounts within the employed concentration range. These data demonstrate that H 2S prevents the stimulation of ENaC by cAMP/PKA and, thereby, inhibits the proabsorptive effect of ␤-adrenergic agonists on lung liquid clearance.

The aim of this work was to determine whether PKC activation is involved in the physiological regulation of LL volume in a whole lung preparation. Rat lungs were perfused with a modified Ringer solution, and the lumen was filled with the same solution without glucose. LL volume was measured during a control period and after modulating drugs were administered, and net LL transepithelial movement (Jv) was calculated. When the PKC activator PMA (10 Ϫ5 M) and the Ca 2ϩ ionophore ionomycin (10 Ϫ6 M) were instilled into the lung together, Jv was significantly reduced (P ϭ 0.03). This reduction was blocked by the PKC inhibitor chelerythrine chloride (10 Ϫ6 M; P ϭ 0.56) and by a second PKC inhibitor GF109203X (10 Ϫ5 M; P ϭ 0.98). When PMA and ionomycin were added with the ␤-adrenergic agonist terbutaline, the terbutaline-induced increase in Jv was abolished. Addition of PMA and ionomycin with the epithelial Na ϩ channel (ENaC) blocker amiloride had no additional inhibitory effect. Together, these results suggest that PKC is likely to be involved in LL absorption, and the ability of PMA/ionomycin to block the terbutaline-induced increase in Jv suggests that the downstream target of PKC is ENaC.calcium-dependent protein kinase C; ionomycin; phorbol 12-myristate 13-acetate TIGHT CONTROL OF LUMINAL lung liquid (LL) volume is essential for normal pulmonary function. An excess or deficiency of LL in the airway and the alveoli leads to pathology, so mechanisms to control LL volume are critical. In the airway, there is a dynamic balance between secretion and absorption of liquid (30), and some of the cellular mechanisms involved have been shown to be present in alveolar cells (13). Cl Ϫ transport seems to underlie liquid secretion into the lumen, while Na ϩ transport has been shown to be essential for liquid absorption. The Na ϩ absorptive mechanisms have received more attention than the Cl Ϫ secretory mechanisms, and the airway epithelium has been better studied than the alveolar barrier, although the latter contributes by far more surface area (by a ratio of Ն500:1, if not 850:1) (18).The principal channel responsible for Na ϩ absorption is the epithelial Na ϩ channel (ENaC), which has been the focus of much research. In the perinatal period, activation of lung ENaC is very sensitive to epinephrine, which causes a dramatic reversal of net transepithelial liquid flow (J v ), thereby facilitating the change at birth from a prenatal secreting phenotype to an absorption phenotype (9, 17). Hummler et al. (15) demonstrated that ␣-ENaC-deficient (Ϫ/Ϫ) mice were unable to survive beyond 40 h after birth. These animals died from acute respiratory failure with liquid-filled lungs, providing conclusive evidence that ENaC is vital for the liquid absorption at birth. During postnatal life, there is a dynamic balance between the secretory and absorptive processes. Although liquid absorption predominates in the liquid-filled lung, a secretory mechanism persists and can be demonstrated to be present under certain circumstances (25). The me...

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