Nabemoto Maiko, Mashimo Masato, Someya Akiyoshi, Nakamura Hiroyuki, Hirabayashi Tetsuya, Fujino Hiromichi, Kaneko Masayuki, Okuma Yasunobu, Saito Takeshi, Yamaguchi Naoto, Murayama Toshihiko
Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba 260-8675, Japan.
Eur J Pharmacol. 2008 Aug 20;590(1-3):1-11. doi: 10.1016/j.ejphar.2008.04.064. Epub 2008 May 10.
The phospholipase A(2) (PLA(2))-prostanoid cascade is involved in cannabinoid receptor-mediated neuronal functions. We investigated the signaling mechanism for the release of arachidonic acid by cannabinoids, 2-arachidonoyl glycerol (2-AG) and HU210, in rat PC12 cells and in primary cultured cells from the mouse cerebellum. The effect of selective inhibitors for signaling pathways and/or enzymes (alpha type cytosolic PLA(2) (cPLA(2)alpha), G protein, Src kinases, phospholipase C, protein kinase C) was assessed. Methods included translocation of the chimeric protein GFP-cPLA(2)alpha, the activities of Src family kinases, Ca(2+)-dependent fluorescence and cyclic AMP accumulation. Treatment with 2-AG and HU210 at greater concentrations than 3 muM caused the release of arachidonic acid, and the response was inhibited by AM251 (an antagonist of cannabinoid CB(1) receptor) and by pyrrophenone (a selective inhibitor of cPLA(2)alpha) in PC12 cells. The cannabinoid treatment caused the intracellular translocation of cPLA(2)alpha and an increase in the intracellular Ca(2+) level. Treatment with HU210 caused tyrosine phosphorylation of Src and Fyn, and increased their kinase activities. Pretreatment with inhibitors of tyrosine kinases or phospholipase C abolished the cannabinoids-induced release of arachidonic acid and Ca(2+) response, and protein kinase C inhibitor reduced the release of arachidonic acid. 2-AG caused the release of arachidonic acid from cultured cells of the mouse cerebellum via similar mechanisms. These data reveal that cannabinoids activated cPLA(2)alpha in a Src-phospholipase C-protein kinase C-dependent manner probably via cannabinoid CB(1) receptor and/or CB(1)-like receptor in neuronal cells.
磷脂酶A(2)（PLA(2)）-前列腺素级联反应参与大麻素受体介导的神经元功能。我们研究了大麻素、2-花生四烯酸甘油酯（2-AG）和HU210在大鼠PC12细胞和小鼠小脑原代培养细胞中释放花生四烯酸的信号传导机制。评估了信号通路和/或酶（α型胞质PLA(2)（cPLA(2)α）、G蛋白、Src激酶、磷脂酶C、蛋白激酶C）的选择性抑制剂的作用。方法包括嵌合蛋白GFP-cPLA(2)α的转位、Src家族激酶的活性、钙依赖性荧光和环磷酸腺苷积累。在PC12细胞中，用浓度高于3μM的2-AG和HU210处理会导致花生四烯酸释放，且该反应被AM251（大麻素CB(1)受体拮抗剂）和吡洛芬（cPLA(2)α的选择性抑制剂）抑制。大麻素处理导致cPLA(2)α的细胞内转位和细胞内钙水平升高。用HU210处理导致Src和Fyn的酪氨酸磷酸化，并增加它们的激酶活性。用酪氨酸激酶或磷脂酶C抑制剂预处理可消除大麻素诱导的花生四烯酸释放和钙反应，蛋白激酶C抑制剂可减少花生四烯酸的释放。2-AG通过类似机制导致小鼠小脑培养细胞中花生四烯酸的释放。这些数据表明，大麻素可能通过神经元细胞中的大麻素CB(1)受体和/或类CB(1)受体以Src-磷脂酶C-蛋白激酶C依赖性方式激活cPLA(2)α。
