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.
Publications
Albert AP
(2008)
AICAR decreases the activity of two distinct amiloride-sensitive Na+-permeable channels in H441 human lung epithelial cell monolayers.
in American journal of physiology. Lung cellular and molecular physiology
Althaus M
(2012)
The gasotransmitter hydrogen sulphide decreases Na? transport across pulmonary epithelial cells.
in British journal of pharmacology
Badshah II
(2014)
Erk5 is a mediator to TGFß1-induced loss of phenotype and function in human podocytes.
in Frontiers in pharmacology
Baines D
(2013)
Kinases as targets for ENaC regulation.
in Current molecular pharmacology
Baines DL
(2010)
Lipopolysaccharide modifies amiloride-sensitive Na+ transport processes across human airway cells: role of mitogen-activated protein kinases ERK 1/2 and 5.
in Pflugers Archiv : European journal of physiology
Greenwood IA
(2009)
KCNQ-encoded channels regulate Na+ transport across H441 lung epithelial cells.
in Pflugers Archiv : European journal of physiology
Ismail NA
(2014)
The phosphorylation of endogenous Nedd4-2 In Na(+)-absorbing human airway epithelial cells.
in European journal of pharmacology
Kalsi KK
(2019)
Metformin attenuates the effect of Staphylococcus aureus on airway tight junctions by increasing PKC?-mediated phosphorylation of occludin.
in Journal of cellular and molecular medicine
Mace OJ
(2008)
AICAR activates AMPK and alters PIP2 association with the epithelial sodium channel ENaC to inhibit Na+ transport in H441 lung epithelial cells.
in The Journal of physiology
Nofziger C
(2009)
PPARgamma agonists inhibit vasopressin-mediated anion transport in the MDCK-C7 cell line.
in American journal of physiology. Renal physiology
Nofziger C
(2008)
Vasopressin regulates the phosphorylation state of AMP-activated protein kinase (AMPK) in MDCK-C7 cells.
in Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology
Patkee WR
(2016)
Metformin prevents the effects of Pseudomonas aeruginosa on airway epithelial tight junctions and restricts hyperglycaemia-induced bacterial growth.
in Journal of cellular and molecular medicine
Tan CD
(2011)
Cleavage of endogenous ?ENaC and elevated abundance of aENaC are associated with increased Na? transport in response to apical fluid volume expansion in human H441 airway epithelial cells.
in Pflugers Archiv : European journal of physiology
Tan CD
(2012)
AMP-activated protein kinase (AMPK)-dependent and -independent pathways regulate hypoxic inhibition of transepithelial Na+ transport across human airway epithelial cells.
in British journal of pharmacology
Woollhead AM
(2007)
Pharmacological activators of AMP-activated protein kinase have different effects on Na+ transport processes across human lung epithelial cells.
in British journal of pharmacology
Yang HY
(2013)
The epithelial sodium channel mediates the directionality of galvanotaxis in human keratinocytes.
in Journal of cell science
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-a1 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+ and glucose 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 continue to inform our current projects because metformin (an activator of AMPK) has had important beneficial effects on epithelial function and resistance to infection and glucose transport and metabolism is known to affect AMPK activity. |
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 and shared with UNC for CF research |
Description | Marsico lung Institute/Dept Cell Biology and Physiology |
Organisation | University of North Carolina at Chapel Hill |
Country | United States |
Sector | Academic/University |
PI Contribution | PhD student working on research question visited UNC. We provided a clear research question and data to support outcome. We provided expertise in CF gene therapy, codon optimised CFTR. We have prepared manuscript containing collaborative output and have submitted for publication. |
Collaborator Contribution | Providing technical expertise in ddPCR and analysis of mixed sex cell populations (non-CF and CF) by identification of sex determining AMELX or AMELY genes. |
Impact | Manuscript submitted for publication |
Start Year | 2018 |
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 |