¹Pharmazentrum Frankfurt, Klinikum der Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany

Correspondence: Andrea Huwiler, Pharmazentrum Frankfurt, Klinikum der Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany. E-mail: Huwiler@em.uni-frankfurt.de

Received 23 November 2001; Revised 13 February 2002; Accepted 5 April 2002.

Abstract

1. 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 M of ATP and UTP. Western blot analysis of mesangial cells reveals the expression of the isoenzymes PKB- and PKB-, but not the PKB-.
2. 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.
3. A series of ATP and UTP analogues were tested for their ability to stimulate PKB phosphorylation. UTP, ATP and -thio-ATP are the only compounds capable of activating PKB.
4. Stress-induced apoptosis of mesangial cells is reduced by the stable ATP analogue, -thio-ATP, and this inhibitory effect is reversed in the presence of LY 294002.
5. 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.