| Summary: | 博士 === 高雄醫學大學 === 藥學研究所博士班 === 95 === Recently, the involvement of anandamide and PAF in endogenous cannabinoid system have been suggested. The present study was to investigate the inflammatory regulation of anandamide and its related molecular mechanisms. Efforts were made to examine whether anandamide and 2-O-methyl PAF altered Ca2+ levels and caused Ca2+-dependent cell death in Madin-Darby canine kidney (MDCK) cells. [Ca2+]i and cell death were measured using the fluorescent dyes fura-2 and WST-1 respectively. In MDCK cells, anandamide (>5 ?嵱) and 2-O-methyl PAF (> or =15 ?嵱) induced [Ca2+]i rises by causing Ca2+ release from endoplasmic reticulum and Ca2+ influx from extracellular space. Furthermore, anandamide (100 ?嵱 – 200 ?嵱) can cause Ca2+-dependent cytotoxicity in a concentration-dependent manner.
In the endotoxic model, the effects of anandamide on nitrite, tumor necrosis factor-alpha (TNF-α) and interleukins (IL)-6, and -10 formation in lipopolysaccharide (LPS) -treated RAW 264.7 macrophages and the possible anti-inflammatory mechanisms involved were also clarified. RAW 264.7 macrophages were simultaneously incubated with anandamide at final concentration of 100- 400 (?嵱) and LPS (1 microg/ml) for 24 h on a mechanical shaker.
These results suggest that anandamide and 2-O-methyl PAF may exert cell signaling regulation, the cAMP、protein kinase A、phospholipase A2 and Ca2+ signal were involved the regulation in MDCK cells. Anandamide and 2-O-methyl PAF induced effects apprar to be regulated by different second messengers. In LPS stimulated RAW 264.7 macrophages, anandamide regulates inflammatory responses by a mechanism that involves the attenuation of nitrite induction, free radical generation and modulation of inflammatory cytokines.
Keywords anandamide, 2-O-methyl PAF , MDCK cells , LPS , RAW macrophages, Cytokines
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