The regulation of the gene expression of the enzymes in arachidonate cascades by dietary factors

为了确定花生四烯酸级联的营养调节作用，我们通过补充n-6和n-3必需脂肪酸来修饰Madin-Darby犬肾（MDCK）细胞。对膜磷脂的酰基部分进行了分析，以了解必需脂肪酸的代谢。此外，花生四烯酸酯的激活机制在细胞信号转导中存在磷酸二酯和钙离子载体。n-6和n-3必需脂肪酸的细胞相互作用分析表明，n-3脂肪酸不能抑制花生四烯酸向膜磷脂的掺入。相反，n-6亚油酸显著阻断亚油酸向花生四烯酸的转化。进一步的研究表明，n-3必需脂肪酸对γ -亚麻酸的主要抑制位点是γ -亚麻酸链延伸到多同- γ -亚麻酸。虽然细胞反应中活化的前列腺素合成随膜磷脂中花生四烯酸水平的变化而变化，但它们之间的关系并不一定是直接的。用n-6必需脂肪酸修饰的培养细胞前列腺素（PG）内过氧化物合成酶活性明显低于用n-3必需脂肪酸修饰的细胞。这些发现暗示了n-6必需脂肪酸对前列腺素合成的反馈抑制。在50 nM PMA和0.1 muM A23187的刺激下，合成的PGE_2和前列腺素合成活性在刺激24h后达到最高水平。另一方面，Western blotting和免疫染色结果显示，24h时表达蛋白水平高于3h。用聚合酶链反应（PCR）扩增PG内过氧化物合酶亚型特异性cdna。有丝分裂原反应型II型对佛波酯的反应增加，并在3h左右达到最大水平，而不是24h。我们还利用MDCK细胞研究和表征了哺乳动物磷脂酶D参与细胞信号传导。少

To determine the nutritional regulation of arachidonate cascades, Madin-Darby canine kidney (MDCK) cells were modified by supplementation with n-6 and n-3 essential fatty acids. The acyl moiety of membrane phospholipids was analyzed for obtaining the insight into the metabolism of essential fatty acids. Moreover, the activation mechanism of arachidonate cascades in cell signaling in the presence of both phorbol diesters and calcium ionophore. The analysis of the cellular interaction of n-6 and n-3 essential fatty acids revealed that n-3 fatty acids failed to inhibit the incorporation of arachidonic acid into membrane phospholipids. In contrast, n-6 linoleic acid significantly blocked the conversion of linoleic acid to arachidonic acid. Additional study demonstrated that the predominant inhibitory site by n-3 essential fatty acid was the chain elongation of gamma-linolenic acid to dohomo-gamma-linolenic acid. Although prostanoid synthesis activated in cell response changed depending on … More the level of arachidonic acid in membrane phospholipids, the relationship between them was not necessarily straightforward. The cultured cells modified with n-6 essential fatty acids showed the more reduced prostaglandin (PG) endoperoxide synthase activity than those with n-3 essential fatty acids. These findings implicated the feedback inhibition of prostanoid synthesis caused by n-6 essential fatty acids.On stimulation with 50 nM PMA and 0.1 muM A23187, the synthesized PGE_2 and prostanoid synthetic activities reached to a maximal level after 24h of the stimulation. On the other hand, the expressed protein level was higher at 24h than at 3h as determined by Western blotting and immunostaining. cDNAs specific for PG endoperoxide synthase isoforms were amplified by polymerase chain reaction (PCR). In response to phorbol diesters, mitogen-responsive type II increased and reached to a maximum level around 3h rather than 24h. We also studied and characterize the mammalian phospholipase D involved in cell signaling using MDCK cells. Less