title: AMP-activated protein kinase (AMPK) activator A-769662 increases intracellular calcium and ATP release from astrocytes in an AMPK-independent manner.
abstract: To test the hypothesis that, given the role of AMP-activated protein kinase (AMPK) in regulating intracellular ATP levels, AMPK may alter ATP release from astrocytes, the main sources of extracellular ATP (eATP) within the brain.
  
  
  
  Measurements of ATP release were made from human U373 astrocytoma cells, primary mouse hypothalamic (HTAS) and cortical astrocytes (CRTAS) and wild-type and AMPK α1/α2 null mouse embryonic fibroblasts (MEFs). Cells were treated with drugs known to modulate AMPK activity: A-769662, AICAR and metformin, for up to 3 hours. Intracellular calcium was measured using Fluo4 and Fura-2 calcium-sensitive fluorescent dyes.
  
  
  
  In U373 cells, A-769662 (100 μM) increased AMPK phosphorylation, whereas AICAR and metformin (1 mM) induced a modest increase or had no effect, respectively. Only A-769662 increased eATP levels, and this was partially blocked by AMPK inhibitor Compound C. A-769662-induced increases in eATP were preserved in AMPK α1/α2 null MEF cells. A-769662 increased intracellular calcium in U373, HTAS and CRTAS cells and chelation of intracellular calcium using BAPTA-AM reduced A-769662-induced eATP levels. A-769662 also increased ATP release from a number of other central and peripheral endocrine cell types.
  
  
  
  AMPK is required to maintain basal eATP levels but is not required for A-769662-induced increases in eATP. A-769662 (>50 μM) enhanced intracellular calcium levels leading to ATP release in an AMPK and purinergic receptor independent pathway.
authors.affiliation: Institute of Biomedical and Clinical Sciences, University of Exeter Medical School, Exeter, UK.|Division of Molecular and Clinical Medicine, Ninewells Hospital and Medical School, University of Dundee, Dundee, UK.
authors.forename: Julia M
authors.initials: JM
authors.lastname: Vlachaki Walker
authors.affiliation: Institute of Biomedical and Clinical Sciences, University of Exeter Medical School, Exeter, UK.
authors.forename: Josephine L
authors.initials: JL
authors.lastname: Robb
authors.affiliation: Institute of Biomedical and Clinical Sciences, University of Exeter Medical School, Exeter, UK.
authors.forename: Ana M
authors.initials: AM
authors.lastname: Cruz
authors.affiliation: Institute of Biomedical and Clinical Sciences, University of Exeter Medical School, Exeter, UK.
authors.forename: Amrinder
authors.initials: A
authors.lastname: Malhi
authors.affiliation: Institute of Biomedical and Clinical Sciences, University of Exeter Medical School, Exeter, UK.
authors.forename: Paul G
authors.initials: PG
authors.lastname: Weightman Potter
authors.affiliation: Division of Molecular and Clinical Medicine, Ninewells Hospital and Medical School, University of Dundee, Dundee, UK.
authors.forename: Michael L J
authors.initials: MLJ
authors.lastname: Ashford
authors.affiliation: Division of Molecular and Clinical Medicine, Ninewells Hospital and Medical School, University of Dundee, Dundee, UK.
authors.forename: Rory J
authors.initials: RJ
authors.lastname: McCrimmon
authors.affiliation: Institute of Biomedical and Clinical Sciences, University of Exeter Medical School, Exeter, UK.
authors.forename: Kate L J
authors.initials: KLJ
authors.lastname: Ellacott
authors.affiliation: Institute of Biomedical and Clinical Sciences, University of Exeter Medical School, Exeter, UK.|Division of Molecular and Clinical Medicine, Ninewells Hospital and Medical School, University of Dundee, Dundee, UK.
authors.forename: Craig
authors.identifier: 0000-0002-4263-0866
authors.initials: C
authors.lastname: Beall
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000103:Acetyl-CoA Carboxylase / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D001713:Biphenyl Compounds; D020013:Calcium Signaling / Q000187:drug effects*; D002460:Cell Line; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D004622:Embryo, Mammalian / Q000166:cytology; D004789:Enzyme Activation / Q000187:drug effects; D020536:Enzyme Activators / Q000494:pharmacology*; D006801:Humans; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D007031:Hypothalamus / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D009419:Nerve Tissue Proteins / Q000819:agonists / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011753:Pyrones / Q000494:pharmacology*; D013876:Thiophenes / Q000494:pharmacology*
chemical_list: D001713:Biphenyl Compounds; D020536:Enzyme Activators; D007004:Hypoglycemic Agents; D009419:Nerve Tissue Proteins; D011753:Pyrones; D013876:Thiophenes; D000255:Adenosine Triphosphate; D055372:AMP-Activated Protein Kinases; D000103:Acetyl-CoA Carboxylase; C512408:4-hydroxy-3-(4-(2-hydroxyphenyl)phenyl)-6-oxo-7H-thieno(2,3-b)pyridine-5-carbonitrile
keywords: A-769662; AMPK; ATP; BV-2; C2C12; GT1-7; H4IIE; INS-1; SH-SY5Y; U373; cortical astrocytes; hypothalamic astrocytes; intracellular calcium