title: Phenformin and 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) activation of AMP-activated protein kinase inhibits transepithelial Na+ transport across H441 lung cells.
abstract: 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-beta-D-ribofuranoside (AICAR) also activates AMPK in intact cells. Western blotting revealed that both the alpha1 and alpha2 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.
authors.affiliation: Department Basic Medical Sciences, Physiology, St Georges' Hospital Medical School, University of London, London SW17 0RE, UK.
authors.forename: Alison M
authors.initials: AM
authors.lastname: Woollhead
authors.forename: John W
authors.initials: JW
authors.lastname: Scott
authors.forename: D Grahame
authors.initials: DG
authors.lastname: Hardie
authors.forename: Deborah L
authors.initials: DL
authors.lastname: Baines
mesh_terms: D055372:AMP-Activated Protein Kinases; D000620:Aminoimidazole Carboxamide / Q000008:administration & dosage / Q000031:analogs & derivatives*; D001693:Biological Transport, Active / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D006801:Humans; D008168:Lung / Q000187:drug effects / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000187:drug effects* / Q000378:metabolism*; D010629:Phenformin / Q000008:administration & dosage*; D017346:Protein Serine-Threonine Kinases / Q000187:drug effects* / Q000378:metabolism*; D020545:Respiratory Mucosa / Q000378:metabolism; D012265:Ribonucleotides / Q000008:administration & dosage*; D012964:Sodium / Q000378:metabolism*
chemical_list: D009097:Multienzyme Complexes; D012265:Ribonucleotides; D000620:Aminoimidazole Carboxamide; D012964:Sodium; D010629:Phenformin; C526181:PRKAA2 protein, human; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases; C526180:PRKAA1 protein, human; C031143:AICA ribonucleotide