title: Extracellular ATP through P2 receptors activates AMP-activated protein kinase and suppresses superoxide generation in cultured mouse podocytes.
abstract: Podocytes are an important constituent of the glomerular filtration barrier. The function of these glomerular cells is affected by extracellular nucleotides through P2 receptors. The activation of P2 receptors may lead to the activation of NAD(P)H oxidase, the key enzyme in oxidative stress, with the intracellular pathways leading to intracellular ATP depletion associated with an increase in the intracellular AMP:ATP ratio. This deregulation of the energy balance activates AMP-activated protein kinase (AMPK) to restore energy homeostasis. We investigated whether P2 receptor activation influences NAD(P)H oxidase-dependent rate of superoxide anion (O(2)(•-)) generation and AMPK activity in cultured mouse podocytes. The rate of O(2)(•-) generation was measured by chemiluminescence and changes in AMPK activity were determined by immunoblotting against AMPKα-Thr(172)-P. The addition of 100 μM ATP induced a rapid and transient decrease in rate of O(2)(•-) generation and increased AMPK phosphorylation with maximal effects in the first minute (2.44±0.09 versus 1.62±0.06 nmol/mg protein/min, P<0.05 and 0.64±0.04 versus 0.97±0.07, P<0.05, respectively). Both parameters returned to control levels at 10 min. Suramin (300 μM, P2 receptor antagonist) and compound C (100μM, AMPK inhibitor) completely, and STO-609 (25 μM, CaMKK-β inhibitor) partially, prevented ATP action in rate of O(2)(•-) generation and AMPK phosphorylation. Various ATP analogues (10 μM) mimicked the effects of ATP on rate of O(2)(•-) generation and AMPK phosphorylation. The data indicate that extracellular ATP, acting through P2 receptors upstream of CaMKK-β, modulates podocyte function through simultaneous effects on AMPK and NAD(P)H oxidase activities. This mechanism may play a role in restoring energy homeostasis after oxidative stress.
authors.affiliation: Mossakowski Medical Research Centre Polish Academy of Sciences, Laboratory of Molecular and Cellular Nephrology, Gdańsk, Poland. apiwkowska@cmdik.pan.pl
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Piwkowska
authors.forename: Dorota
authors.initials: D
authors.lastname: Rogacka
authors.forename: Maciej
authors.initials: M
authors.lastname: Jankowski
authors.forename: Stefan
authors.initials: S
authors.lastname: Angielski
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D005110:Extracellular Space / Q000378:metabolism*; D051379:Mice; D050199:Podocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism*
chemical_list: D018048:Receptors, Purinergic P2; D013481:Superoxides; D000255:Adenosine Triphosphate; D055372:AMP-Activated Protein Kinases