Adenosine monophosphate activated protein kinase (AMPK) is activated by a decrease in cellular energy and an increase in the cellular AMP:ATP ratio. The AMP mimetic AICAR (2mM, 1hour) and the mitochondrial complex 1 inhibitor metformin (2mM, 4 hours) activated adenosine monophosphate activated protein kinase (AMPK) and reduced transepithelial amiloride-sensitive Na⁺ transport (10mM amiloride) across H441 human airway epithelial cell monolayers to 55 10% and 75 4% of control respectively, p < 0.05, n = 4. This effect was reversed by the AMPK inhibitor Compound C (80mM). AICAR and metformin also inhibited amiloride-sensitive apical Na⁺ conductance to 38 8% and 68 11% of control, p < 0.05, n = 4 respectively. AICAR inhibited the activity of two amiloride-sensitive Na+ channels (ENaC) in the apical membrane of H441 cells, a non-selective 18pS channel and a highly Na+ selective, 5 pS ENaC-like channel. Activation of AMPK with AICAR was not associated with changes in the apical abundance of the a, b or g?ENaC subunits. Immunoprecipitation of phosphatidylinositol 4,5-bisphosphate (PIP2), present in the membrane of lung epithelial cells revealed that it was associated with a, b and?g?ENaC proteins in control monolayers. However, treatment with AICAR inhibited the interaction of PIP2 with b and g ENaC proteins. These data indicated that AICAR activation of AMPK inhibited ENaC channel activity (at least in part) by compromising its interaction with PIP2. Exposure to physiological factors that effect cellular energy status such as hypoxia (3% O₂, 5% CO₂) for 1 hour or glucose deprivation (< 0.4 mM) for 2 hours increased phospho/total acetyl CoA carboxylase (ACC) (a target for AMPK mediated phosphorylation) as analysed by western blotting from 0.06 0.04 to 1.02 0.11 and 0.93 0.3 to 3.5 0.8 densitometry units, P < 0.05, n = 4 and n = 3, respectively in H441 cells. In addition, phospho/total AMPK was correspondingly increased by both treatments. Exposure to hypoxia also inhibited amiloride-sensitive transepithelial Na⁺ transport to 70 3% of control, p < 0.05, n = 3. In the presence of STO-609 (53 mM), an inhibitor of CaMKK (an upstream kinase of AMPK), an apparent rise in phopho/total AMPK and ACC remained after hypoxia and glucose deprivation but the effect of did not reach significance n = 3. These data indicate that glucose deprivation and hypoxia result in activation of AMPK in H441 cells and that CaMKK may play a role in phosphorylation of AMPK in these cells.

Supported by the BBSRC and St George's University of London