Karkoulias Georgios, Mastrogianni Orthodoxia, Lymperopoulos Anastasios, Paris Herve, Flordellis Christodoulos
Department of Pharmacology, School of Medicine, University of Patras, 26504 Rio Patras, Greece.
Cell Signal. 2006 May;18(5):729-39. doi: 10.1016/j.cellsig.2005.06.014. Epub 2005 Aug 10.
Previous study carried out on PC12 cells expressing each alpha(2)-adrenergic receptor subtype individually (PC12/alpha(2A), /alpha(2B) or /alpha(2C)) have shown that epinephrine causes activation of PI3K and phosphorylation of Erk 1/2. The signal transduction mechanisms whereby each alpha(2)-AR subtype triggers these actions were investigated in the present study. In all three clones, epinephrine-induced phosphorylation of MAPK or Akt was abolished by prior treatment with ketoconazole, but not with indomethacin or nordihydroguaiaretic acid. On the other hand, treatment of the clones with epinephrine caused a rapid increase of AA release, which was fully abolished by the PLC inhibitor U73122, but was unaffected by the PLA(2) inhibitor quinacrine. The effects of epinephrine on MAPK and Akt were mimicked by cell exposure to exogenous AA. Furthermore, whereas U73122 abolished the effects of epinephrine, quinacrine only prevented the effects of epinephrine, suggesting that AA release through PLC and its metabolites are responsible for MAPK and Akt activation by alpha(2)-ARs. Treatment with 1,10-phenanthroline, CRM197, or tyrphostin AG1478 suppressed MAPK and Akt phosphorylation by epinephrine or AA, in a subtype-specific manner. Furthermore, conditioned culture medium from epinephrine-treated PC12/alpha(2) induced MAPK and Akt phosphorylation in wild-type PC12. Inhibition of NGFR tyrosine phosphorylation had no effect but the src inhibitor PP1 abolished MAPK and Akt phosphorylation in all three clones. Our results provide evidence for a putative pathway by which alpha(2)-ARs activate MAPK and Akt in PC12 cells, involving stimulation of PLC, AA release, AA metabolism by cytochrome P450-dependent epoxygenase, stimulation of matrix metalloproteinases and subtype-specific transactivation of EGFR through src activation and heparin-binding EGF-like growth factor release.
先前对单独表达每种α₂-肾上腺素能受体亚型的PC12细胞（PC12/α₂A、/α₂B或/α₂C）进行的研究表明，肾上腺素会导致PI3K激活和Erk 1/2磷酸化。本研究对每种α₂-肾上腺素能受体亚型触发这些作用的信号转导机制进行了研究。在所有三个克隆中，酮康唑预处理可消除肾上腺素诱导的MAPK或Akt磷酸化，但吲哚美辛或去甲二氢愈创木酸预处理则无此作用。另一方面，用肾上腺素处理这些克隆会导致花生四烯酸（AA）释放迅速增加，PLC抑制剂U73122可完全消除这种增加，但PLA₂抑制剂喹吖因对此无影响。细胞暴露于外源性AA可模拟肾上腺素对MAPK和Akt的作用。此外，虽然U73122消除了肾上腺素的作用，但喹吖因仅能预防肾上腺素的作用，这表明通过PLC及其代谢产物释放的AA负责α₂-肾上腺素能受体激活MAPK和Akt。用1,10-菲咯啉、CRM197或酪氨酸磷酸化抑制剂AG1478处理可亚型特异性地抑制肾上腺素或AA诱导的MAPK和Akt磷酸化。此外，肾上腺素处理的PC12/α₂条件培养基可诱导野生型PC12中的MAPK和Akt磷酸化。抑制NGFR酪氨酸磷酸化无作用，但src抑制剂PP1可消除所有三个克隆中的MAPK和Akt磷酸化。我们的结果为α₂-肾上腺素能受体在PC12细胞中激活MAPK和Akt的假定途径提供了证据，该途径涉及PLC的刺激、AA释放、细胞色素P450依赖性环氧化酶对AA的代谢、基质金属蛋白酶的刺激以及通过src激活和肝素结合表皮生长因子样生长因子释放对EGFR的亚型特异性反式激活。
