Extracellular ATP through P2 receptors activates AMP-activated protein kinase and suppresses superoxide generation in cultured mouse podocytes

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(center dot-)) generation and AMPK activity in cultured mouse podocytes. The rate of O-2(center dot-) generation was measured by chemiluminescence and changes in AMPK activity were determined by immunoblotting against AMPK alpha-Thr(172)-P. The addition of 100 mu M ATP induced a rapid and transient decrease in rate of O-2(center dot-) 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 mu M, P2 receptor antagonist) and compound C (100 mu M, AMPK inhibitor) completely, and STO-609 (25 mu M, CaMKK-beta inhibitor) partially, prevented ATP action in rate of O-2(center dot-) generation and AMPK phosphorylation. Various ATP analogues (10 mu M) mimicked the effects of ATP on rate of O-2(center dot-) generation and AMPK phosphorylation. The data indicate that extracellular ATP, acting through P2 receptors upstream of CaMKK-beta, 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. (C) 2011 Elsevier Inc. All rights reserved.