Extracellular ATP and UTP activate the protein kinase B/Akt cascade via the P2Y(2) purinoceptor in renal mesangial cells.
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 Ser(473) and Thr(308) of PKB with a maximal effect after 5 min of stimulation. The response is concentration-dependent with a maximal effect at 30 microM of ATP and UTP. Western blot analysis of mesangial cells reveals the expression of the isoenzymes PKB-alpha and PKB-gamma, but not the PKB-beta. 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 P2Y(2) 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 gamma-thio-ATP are the only compounds capable of activating PKB. Stress-induced apoptosis of mesangial cells is reduced by the stable ATP analogue, gamma-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 P2Y(2)-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.
Huwiler
Andrea
A
Pharmazentrum Frankfurt, Klinikum der Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany. Huwiler@em.uni-frankfurt.de
Rölz
Waltraud
W
Dorsch
Simone
S
Ren
Shuyu
S
Pfeilschifter
Josef
J
D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D000704:Analysis of Variance; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D016718:Arachidonic Acid / Q000378:metabolism; D015153:Blotting, Western; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004789:Enzyme Activation; D005110:Extracellular Space / Q000378:metabolism; D005920:Glomerular Mesangium / Q000166:cytology* / Q000187:drug effects; D007527:Isoenzymes / Q000378:metabolism; D037342:Pertussis Toxin / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000502:physiology*; D058473:Receptors, Purinergic P2Y2; D013498:Suramin / Q000494:pharmacology; D014544:Uridine Triphosphate / Q000378:metabolism* / Q000494:pharmacology
D000255:Adenosine Triphosphate; D016718:Arachidonic Acid; D007527:Isoenzymes; D037342:Pertussis Toxin; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D018048:Receptors, Purinergic P2; D058473:Receptors, Purinergic P2Y2; D013498:Suramin; D014544:Uridine Triphosphate; C546387:P2ry2 protein, rat; C494920:Akt1 protein, rat