title: Pharmacological activators of AMP-activated protein kinase have different effects on Na+ transport processes across human lung epithelial cells.
abstract: OBJECTIVE
  AMP-activated protein kinase (AMPK) is activated by metformin, phenformin, and the AMP mimetic, 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR). We have completed an extensive study of the pharmacological effects of these drugs on AMPK activation, adenine nucleotide concentration, transepithelial amiloride-sensitive (I(amiloride)) and ouabain-sensitive basolateral (I(ouabain)) short circuit current in H441 lung epithelial cells.
  
  
  METHODS
  H441 cells were grown on permeable filters at air interface. I(amiloride), I(ouabain) and transepithelial resistance were measured in Ussing chambers. AMPK activity was measured as the amount of radiolabelled phosphate transferred to the SAMS peptide. Adenine nucleotide concentration was analysed by reverse phase HPLC and NAD(P)H autofluorescence was measured using confocal microscopy.
  
  
  RESULTS
  Phenformin, AICAR and metformin increased AMPK (alpha1) activity and decreased I(amiloride). The AMPK inhibitor Compound C prevented the action of metformin and AICAR but not phenformin. Phenformin and AICAR decreased I(ouabain) across H441 monolayers and decreased monolayer resistance. The decrease in I(amiloride) was closely related to I(ouabain) with phenformin, but not in AICAR treated monolayers. Metformin and phenformin increased the cellular AMP:ATP ratio but only phenformin and AICAR decreased cellular ATP.
  
  
  CONCLUSIONS
  Activation of alpha1-AMPK is associated with inhibition of apical amiloride-sensitive Na(+) channels (ENaC), which has important implications for the clinical use of metformin. Additional pharmacological effects evoked by AICAR and phenformin on I(ouabain), with potential secondary effects on apical Na+ conductance, ENaC activity and monolayer resistance, have important consequences for their use as pharmacological activators of AMPK in cell systems where Na+K+ATPase is an important component.
authors.affiliation: Centre for Ion Channels and Cell Signalling, Division of Basic Medical Sciences, St George's, University of London, London, UK.
authors.forename: A M
authors.initials: AM
authors.lastname: Woollhead
authors.forename: J
authors.initials: J
authors.lastname: Sivagnanasundaram
authors.forename: K K
authors.initials: KK
authors.lastname: Kalsi
authors.forename: V
authors.initials: V
authors.lastname: Pucovsky
authors.forename: L J
authors.initials: LJ
authors.lastname: Pellatt
authors.forename: J W
authors.initials: JW
authors.lastname: Scott
authors.forename: K J
authors.initials: KJ
authors.lastname: Mustard
authors.forename: D G
authors.initials: DG
authors.lastname: Hardie
authors.forename: D L
authors.initials: DL
authors.lastname: Baines
mesh_terms: D055372:AMP-Activated Protein Kinases; D000227:Adenine Nucleotides / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000584:Amiloride; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives* / Q000494:pharmacology; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004847:Epithelial Cells; D053503:Epithelial Sodium Channels / Q000187:drug effects; D005453:Fluorescence; D006801:Humans; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D008168:Lung; D008687:Metformin / Q000494:pharmacology*; D018613:Microscopy, Confocal; D009097:Multienzyme Complexes / Q000187:drug effects* / Q000378:metabolism; D010042:Ouabain; D010629:Phenformin / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000187:drug effects* / Q000378:metabolism; D012265:Ribonucleotides / Q000494:pharmacology*; D012964:Sodium / Q000378:metabolism*
chemical_list: D000227:Adenine Nucleotides; D053503:Epithelial Sodium Channels; D007004:Hypoglycemic Agents; D009097:Multienzyme Complexes; D012265:Ribonucleotides; D000620:Aminoimidazole Carboxamide; D000249:Adenosine Monophosphate; D010042:Ouabain; D000584:Amiloride; D000255:Adenosine Triphosphate; D008687:Metformin; D012964:Sodium; D010629:Phenformin; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases; C526180:PRKAA1 protein, human; C031143:AICA ribonucleotide