Huwiler Andrea, Rölz Waltraud, Dorsch Simone, Ren Shuyu, Pfeilschifter Josef
Pharmazentrum Frankfurt, Klinikum der Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany.
Br J Pharmacol. 2002 Jun;136(4):520-9. doi: 10.1038/sj.bjp.0704748.
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.
细胞外核苷酸可激活一种介导多种细胞反应的共同嘌呤受体。在本研究中，我们报告用ATP和UTP刺激大鼠系膜细胞会导致蛋白激酶B/Akt（PKB）途径的快速激活。时间进程研究显示，PKB的Ser(473)和Thr(308)均迅速且短暂地磷酸化，刺激5分钟后达到最大效应。该反应呈浓度依赖性，ATP和UTP浓度为30 microM时效应最大。系膜细胞的蛋白质印迹分析显示存在同工酶PKB-α和PKB-γ，但不存在PKB-β。ATP和UTP还激活位于上游的PI 3激酶依赖性激酶。此外，PI 3激酶的两种抑制剂可消除ATP和UTP诱导的PKB磷酸化。另外，苏拉明（一种假定的P2Y(2)受体拮抗剂）和百日咳毒素（一种G(i)/G(o)激活抑制剂）可显著阻断ATP和UTP诱导的PKB磷酸化。测试了一系列ATP和UTP类似物刺激PKB磷酸化的能力。UTP、ATP和γ-硫代-ATP是仅有的能够激活PKB的化合物。稳定的ATP类似物γ-硫代-ATP可减少应激诱导的系膜细胞凋亡，且在LY 294002存在时这种抑制作用会逆转。总之，这些结果表明细胞外核苷酸能够通过P2Y(2)受体和百日咳毒素敏感的G(i)蛋白激活PI 3激酶/PDK/PKB级联反应。此外，在系膜细胞中，该级联反应可能在抗凋亡反应中起重要作用，但在细胞外核苷酸产生的促有丝分裂或炎症反应中不起作用。
