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

Lead Research Organisation: St George's University of London
Department Name: Basic Medical Sciences

Abstract

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.

Technical Summary

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.
 
Description We advanced our understanding of the cellular mechanisms by which AMP activated protein kinase (AMP) inhibited Na+ transport highlighting new potential therapeutic targets/strategies. We showed that 1. AMPK-?1 is the predominant isoform in airway epithelial cells and is found in cytosolic and membrane domains. 2. We showed that phospholipids in the cell membrane were important in the regulation of Na+ transport by AMPK. 3. We also showed that hypoxia regulates Na+ transport by AMPK-dependent and independent pathways.
Exploitation Route This project has lead to collaborations with other groups working on Na+ transport. We have provided expertise and resources to support further research into key signalling molecules (eg Nedd4-2) , the role of epithelial Na+ channels in other tissues (eg galvanotaxis) and signalling pathways in other epithelial tissues (eh ERK5). Our findings have been cited by others investigating the role of AMPK in other tissues. Our findings have informed our current project because metformin (an activator of AMPK) has had important beneficial effects on epithelial function and resistance to infection.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Findings from this project have contributed to our understanding of the role of AMPK and its regulation in airway epithelium. This has informed subsequent research projects and pharmacological activators of AMPK are being used to investigate their benefit in the treatment of respiratory disease. In addition, work from this project has received over 90 citations to date.
First Year Of Impact 2008
Sector Pharmaceuticals and Medical Biotechnology
Impact Types Societal

 
Title Fluorescent ENaC proteins 
Description Plasmids generated by our group encoding epithelial Na+ proteins linked to fluorescent molecules. 
Type Of Material Biological samples 
Year Produced 2011 
Provided To Others? Yes  
Impact Two high impact publications Further requests for tools 2019 
 
Description The gasotransmitter hydrogen sulphide decreases Na(+) transport across pulmonary epithelial cells. 
Organisation Justus Liebig University Giessen
Country Germany 
Sector Academic/University 
PI Contribution Providing functional assay
Collaborator Contribution Providing drugs, background and preliminary outputs for subsequent functional study
Impact Althaus M, Urness KD, Clauss WG, Baines DL, Fronius M (2012). The gasotransmitter hydrogen sulphide decreases Na(+) transport across pulmonary epithelial cells. Br J Pharmacol. 166(6):1946-1963
Start Year 2011