Active re-absorption of Na + across the alveolar epithelium is essential to maintain lung fluid balance. Na + entry at the luminal membrane is predominantly via the amiloride-sensitive Na + channel (ENaC) down its electrochemical gradient. This gradient is generated and maintained by basolateral Na + extrusion via Na + ,K + -ATPase an energy-dependent process. Several kinases and factors that activate them are known to regulate these processes; however, the role of AMP-activated protein kinase (AMPK) in the lung is unknown. AMPK is an ultra-sensitive cellular energy sensor that monitors energy consumption and down-regulates ATP-consuming processes when activated. The biguanide phenformin has been shown to independently decrease ion transport processes, influence cellular metabolism and activate AMPK. The AMP mimetic drug 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) also activates AMPK in intact cells. Western blotting revealed that both the α1 and α2 catalytic subunits of AMPK are present in Na + transporting H441 human lung epithelial cells. Phenformin and AICAR increased AMPK activity in H441 cells in a dose-dependent fashion, stimulating the kinase maximally at 5-10 mM (P = 0.001, n = 3) and 2 mM (P < 0.005, n = 3), respectively. Both agents significantly decreased basal ion transport (measured as short circuit current) across H441 monolayers by approximately 50% compared with that of controls (P < 0.05, n = 4). Neither treatment altered the resistance of the monolayers. Phenformin and AICAR significantly reduced amiloride-sensitive transepithelial Na + transport compared with controls (P < 0.05, n = 4). This was a result of both decreased Na + ,K + -ATPase activity and amiloride-sensitive apical Na + conductance. Transepithelial Na + transport decreased with increasing concentrations of phenformin (0.1-10 mM) and showed a significant correlation with AMPK activity. Taken together, these results show that phenformin and AICAR suppress amiloride-sensitive Na + transport across H441 cells via a pathway that includes activation of AMPK and inhibition of both apical Na + entry through ENaC and basolateral Na + extrusion via the Na + ,K + -ATPase. These are the first studies to provide a cellular signalling mechanism for the action of phenformin on ion transport processes, and also the first studies showing AMPK as a regulator of Na + absorption in the lung.

Background and purpose: AMP-activated protein kinase (AMPK) is activated by metformin, phenformin, and the AMP mimetic, 5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside (AICAR). We have completed an extensive study of the pharmacological effects of these drugs on AMPK activation, adenine nucleotide concentration, transepithelial amiloridesensitive (I amiloride ) and ouabain-sensitive basolateral (I ouabain ) short circuit current in H441 lung epithelial cells. Experimental approach: H441 cells were grown on permeable filters at air interface. I amiloride , I ouabain and transepithelial resistance were measured in Ussing chambers. AMPK activity was measured as the amount of radiolabelled phosphate transferred to the SAMS peptide. Adenine nucleotide concentration was analysed by reverse phase HPLC and NAD(P)H autofluorescence was measured using confocal microscopy. Key results: Phenformin, AICAR and metformin increased AMPK (a1) activity and decreased I amiloride . The AMPK inhibitor Compound C prevented the action of metformin and AICAR but not phenformin. Phenformin and AICAR decreased I ouabain across H441 monolayers and decreased monolayer resistance. The decrease in I amiloride was closely related to I ouabain with phenformin, but not in AICAR treated monolayers. Metformin and phenformin increased the cellular AMP:ATP ratio but only phenformin and AICAR decreased cellular ATP. Conclusions and implications: Activation of a1-AMPK is associated with inhibition of apical amiloride-sensitive Na þ channels (ENaC), which has important implications for the clinical use of metformin. Additional pharmacological effects evoked by AICAR and phenformin on I ouabain, with potential secondary effects on apical Na þ conductance, ENaC activity and monolayer resistance, have important consequences for their use as pharmacological activators of AMPK in cell systems where Na þ K þ ATPase is an important component.

Elevation of intracellular cAMP increases fluid re-absorption in the lung by raising amiloride-sensitive Na ؉ transport through the apically localized epithelial, amiloride-sensitive Na ؉ channel (ENaC). However, the signaling pathways mediating this response are still not fully understood. We show that inhibition of proteintyrosine kinase (PTK) with Genistein and protein kinase A (PKA) with KT5720, decreased forskolin-stimulated amiloride-sensitive short circuit current (I sc ) across H441 adult human lung epithelial cell monolayers. KT5720 also decreased basal I sc . Transport of Na ϩ through the amiloride-sensitive Na ϩ channel (ENaC), 2 found in the apical membrane of polarized lung epithelial cells, is considered the rate-limiting step for transepithelial Na ϩ movement and the regulation of lung fluid re-absorption via osmosis (1). ENaC comprises three subunits ␣, ␤, and ␥ (2). It has been shown that the ␣-subunit is capable of forming an Na ϩ -conducting pore and that ␤ and ␥ augment its conductance (2). Channels may be formed when ␣ENaC is expressed independently of the ␤ and ␥ subunits (3, 4). For this reason, expression of the ␣-subunit is considered of critical importance. This has been demonstrated in ␣ENaC knock-out mice where death of newborn mice lacking ␣ENaC was shown to be the result of an inability to clear their lungs of fluid (5). A more recent study has extended these findings and indicated that the low mRNA abundance level of ␣ENaC in nasal epithelium of premature infants is associated with respiratory failure (6).At birth, the lungs undergo a transition from a fluid-filled to that of an air-filled (post-natal) state, a requirement for normal breathing and efficient gas-exchange. In the fetal sheep and guinea pig lung, a surge in plasma adrenaline around and during the time of birth has been shown to correlate with increased amiloride-sensitive fluid re-absorption (7, 8). The ability of adrenaline to mediate lung fluid re-absorption has also been shown to be retained throughout adult life (9). Adrenaline is thought to act through the cAMP second messenger system, and its effects on fluid absorption can be mimicked by agents that increase intracellular cAMP such as forskolin (10 -12). In the rat fetal distal lung cell, the action of cAMP is thought to increase the recruitment of ENaC subunits to the apical membrane by increased trafficking via a Brefeldin A-sensitive pathway (13). This phenomenon is also upheld in adult human bronchiolar epithelial H441 cells (14) where the apical conductance of ENaC is also increased by cAMP (14 -16). However, which ENaC subunits are recruited to the apical membrane and the signaling pathways involved remains unclear in lung cells. Elevation of cAMP by ␤ 2 -adrenoreceptor agonists is classically associated with activation of protein kinase A (PKA). However, evidence indicates that the effect of ␤ 2 -adrenoreceptor activation on the trafficking of ENaC may not involve a direct effect of PKA and may utilize alternative pathways that involve protein-tyrosine k...