Phenformin and 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) activation of AMP-activated protein kinase inhibits transepithelial Na⁺ transport across H441 lung cells

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.