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1 Department of Basic Medical Sciences, Physiology, St Georges' Hospital Medical School, University of London, Cranmer Terrace, Tooting, London SW17 0RE, UK
2 Division of Molecular Physiology, Wellcome Trust Biocentre, University of Dundee, Dow Street, Dundee DD1 5EH, UK
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-ß-D-ribofuranoside (AICAR) also activates AMPK in intact cells. Western blotting revealed that both the 
1 and 2 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.
(Received 15 April 2005;
accepted after revision 25 May 2005;
first published online 26 May 2005)
Corresponding author D. Baines: Department Basic Medical Sciences, Physiology, St Georges' Hospital Medical School, University of London, London SW17 0RE, UK. Email: d.baines{at}sghms.ac.uk
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