Andrea Huwiler

Extracellular nucleotides can activate a common purinoceptor mediating various cell responses. In this study we report that stimulation of rat mesangial cells with ATP and UTP leads to a rapid activation of the protein kinase B/Akt (PKB) pathway. Time‐course studies reveal a rapid and transient phosphorylation of both Ser⁴⁷³ and Thr³⁰⁸ of PKB with a maximal effect after 5 min of stimulation. The response is concentration‐dependent with a maximal effect at 30 μM of ATP and UTP. Western blot analysis of mesangial cells reveals the expression of the isoenzymes PKB‐α and PKB‐γ, but not the PKB‐β. ATP and UTP also activate the upstream located PI 3‐kinase‐dependent kinase. Furthermore, the ATP‐ and UTP‐induced PKB phosphorylation is abolished by two inhibitors of the PI 3‐kinase. In addition, suramin, a putative P2Y₂ receptor antagonist, and pertussis toxin, an inhibitor of G_i/G_o activation, markedly block ATP‐ and UTP‐induced PKB phosphorylation. A series of ATP and UTP analogues were tested for their ability to stimulate PKB phosphorylation. UTP, ATP and γ‐thio‐ATP are the only compounds capable of activating PKB. Stress‐induced apoptosis of mesangial cells is reduced by the stable ATP analogue, γ‐thio‐ATP, and this inhibitory effect is reversed in the presence of LY 294002. In summary, these results demonstrate that extracellular nucleotides are able to activate the PI 3‐kinase/PDK/PKB cascade via the P2Y₂‐receptor and a pertussis toxin‐sensitive G_i protein. Moreover, in mesangial cells this cascade may have an important role in the antiapoptotic response but not in the mitogenic or inflammatory response produced by extracellular nucleotides. British Journal of Pharmacology (2002) 136, 520–529; doi:10.1038/sj.bjp.0704748