Investigation of how activation of AMP activated protein kinase (AMPK) inhibits Na+ transport across H441 lung epithelial cells.

The airways of the lung are lined with a thin layer of fluid that is important in preventing infection and damage from inhaled agents. The volume of this fluid layer is controlled by the transport of sodium and chloride across the cells that line the surface of the airways through selective channels and pumps. Increased transport of sodium out of the airway is linked with dehydration of the fluid layer and is a contributary factor to the disease, Cystic Fibrosis. Conversely, a decrease in sodium transport is associated with fluid accumulation in the airways as in respiratory distress syndrome of the newborn and high altitude pulmonary oedema. We have recently discovered that activation of a molecule that senses low cellular energy levels (adenosine monophosphate-activated protein kinase (AMPK), decreases sodium transport across the lung epithelial cell. AMPK may therefore have a significant regulatory role in these diseases. How AMPK decreases sodium transport is unknown but it appears to reduce the amount of sodium entering the cell via the amiloride-sensitive sodium channel (ENaC) and the amount of sodium that is extruded from the cell via the Na+K+ATPase pump. It is the purpose of this project to investigate if activation of AMPK decreases sodium transport and the activity of ENaC and Na+K+ATPase by regulating the abundance of these proteins in the cell membrane. We will do this by using a combination of functional and molecular approaches in human H441 lung epithelial cells in vitro. Identifying the cellular mechanism by which this molecule regulates sodium transport is a critical first step in elucidating how it could regulate Na+ transport in vivo. This will help us develop more appropriate strategies for future research and the development of treatments for lung diseases which exhibit dissordered fluid volume.

The luminal surface of the airways is lined with a thin layer of fluid essential for pulmonary defence and its volume is regulated by transepithelial transport of Na+. This is generated by basolateral extrusion of Na+ by the energy-consuming Na+K+ATPase which reduces intracellular Na+ concentration and creates a diffusion gradient for Na+ entry at the luminal membrane primarily via the amiloride-sensitive epithelial Na+ channel (ENaC). The subsequent net transport of Na+ from the lumen to the interstitium creates an osmotic gradient that drives fluid absorption. We have recently discovered that activation of adenosine monophosphate-activated kinase (AMPK) with biguanides (metformin/phenformin) or the AMP mimetic drug (AICAR) decreased transepithelial Na+ transport across the lung epithelial cell. These findings are important because changes in transepithelial Na+ transport underlie diseases such as cystic fibrosis, respiratory distress syndrome and high altitude pulmonary oedema. AMPK may therefore have a significant regulatory role in these diseases. We found that activation of AMPK decreased apical Na+ entry via ENaC and extrusion via Na+K+ATPase. This has important therapeutic implications. However, before we can investigate the potential effects of AMPK in vivo, we need to better understand the cellular mechanisms by which AMPK elicits its effects on Na+ transport. This project will test the hypothesis that AMPK decreases Na+ transport by regulating the abundance and activity of Na+K+ATPase and ENaC channel proteins in the lung epithelial cell membrane. We will do this by using a combination of functional and molecular approaches in human H441 lung epithelial cells in vitro. We will investigate what AMPK subunits are activated, where they are localised and if they associate directly with ENaC, Na+K+ATPase or Nedd4-2 (an important regulator of ENaC activity) to change their abundance and activity in the lung membrane.