McKinney M, Richelson E
Biochem Pharmacol. 1986 Jul 15;35(14):2389-97. doi: 10.1016/0006-2952(86)90466-1.
Inhibitors of arachidonate metabolism and perturbants of the oxidation-reduction state of the cell were employed to develop a pharmacologic profile for muscarinic receptor-mediated cyclic GMP formation in murine neuroblastoma cells (clone N1E-115). Several lipoxygenase inhibitors [eicosatetraynoic acid (ETYA), nordihydroguaiaretic acid (NDGA), FPL 57231, FPL 55712, BW755c, propylgallate, and AA861] blocked the elevation of [3H]cyclic GMP induced by muscarinic receptor activation. The cyclooxygenase inhibitors indomethacin and ibuprofen were two orders of magnitude less potent in blocking the muscarinic receptor-mediated [3H]cyclic GMP response than in blocking cyclooxygenase in other systems. ETYA and NDGA did not affect the muscarinic inhibition of the prostaglandin E1-mediated increases in [3H]cyclic AMP levels in N1E-115 cells. ETYA did not have a reproducible effect on the muscarinic receptor-induced release of inositol phosphates. Thus, these lipoxygenase inhibitors appeared to be selective for the effector system coupled to the low-affinity muscarinic agonist-receptor conformation, i.e. that which induces cyclic GMP formation. Other effective inhibitors of the cyclic GMP response were methylene blue, catalase, bromphenacyl bromide, retinal, dithiothreitol, quinacrine, and oxidized glutathione. The antioxidant alpha-tocopherol in the concentration range of 100 microM to 1 mM potentiated the receptor response. Arachidonic acid itself was an inhibitor of the muscarinic receptor-mediated cyclic GMP response (IC50 = 45 microM). Linoleic acid and oleic acid were less potent (IC50 = 130 and 190 microM, respectively), and stearic acid was ineffective. When arachidonic acid was air-oxidized, its inhibitory potency was increased 10-fold. Most but not all of the spontaneously-produced oxidative metabolites, separable by reverse-phase high pressure liquid chromatography, were inhibitory to the receptor response. Enzymatically synthesized 12-hydroxyeicosatetraenoic acid and 15-hydroxyeicosatetraenoic acid inhibited the muscarinic receptor [3H]cyclic GMP response, with IC50 values of 17 and 8 microM respectively. Catalase was effective in blocking the muscarinic cyclic GMP response (IC50 = 5 microM) while having no effect on either the muscarinic receptor-induced inositol phosphate release or the reduction of cyclic AMP levels. Thus, the effector system for increasing cyclic GMP in these cells displays may of the expected characteristics for the involvement of a lipoxygenase or a related enzyme that oxidatively metabolizes arachidonate in order to activate the guanylate cyclase.(ABSTRACT TRUNCATED AT 400 WORDS)
采用花生四烯酸代谢抑制剂和细胞氧化还原状态扰动剂，来建立小鼠神经母细胞瘤细胞（克隆N1E - 115）中，毒蕈碱受体介导的环鸟苷酸形成的药理学特征。几种脂氧合酶抑制剂[二十碳四炔酸（ETYA）、去甲二氢愈创木酸（NDGA）、FPL 57231、FPL 55712、BW755c、没食子酸丙酯和AA861]可阻断毒蕈碱受体激活诱导的[3H]环鸟苷酸升高。环氧化酶抑制剂吲哚美辛和布洛芬在阻断毒蕈碱受体介导的[3H]环鸟苷酸反应方面，其效力比在其他系统中阻断环氧化酶的效力低两个数量级。ETYA和NDGA不影响毒蕈碱对N1E - 115细胞中前列腺素E1介导的[3H]环腺苷酸水平升高的抑制作用。ETYA对毒蕈碱受体诱导的肌醇磷酸释放没有可重复的影响。因此，这些脂氧合酶抑制剂似乎对与低亲和力毒蕈碱激动剂 - 受体构象偶联的效应系统具有选择性，即诱导环鸟苷酸形成的构象。其他有效的环鸟苷酸反应抑制剂有亚甲蓝、过氧化氢酶、溴苯甲酰溴、视黄醛、二硫苏糖醇、奎纳克林和氧化型谷胱甘肽。浓度范围为100微摩尔至1毫摩尔的抗氧化剂α - 生育酚可增强受体反应。花生四烯酸本身是毒蕈碱受体介导的环鸟苷酸反应的抑制剂（IC50 = 45微摩尔）。亚油酸和油酸的效力较低（IC50分别为130和190微摩尔），而硬脂酸无效。当花生四烯酸被空气氧化时，其抑制效力增加10倍。大多数但并非所有可通过反相高压液相色谱分离的自发产生的氧化代谢物，均对受体反应有抑制作用。酶促合成的12 - 羟基二十碳四烯酸和15 - 羟基二十碳四烯酸抑制毒蕈碱受体[3H]环鸟苷酸反应，IC50值分别为17和8微摩尔。过氧化氢酶可有效阻断毒蕈碱环鸟苷酸反应（IC50 = 5微摩尔），而对毒蕈碱受体诱导的肌醇磷酸释放或环腺苷酸水平降低均无影响。因此，这些细胞中增加环鸟苷酸的效应系统可能显示出脂氧合酶或相关酶参与氧化代谢花生四烯酸以激活鸟苷酸环化酶的预期特征。（摘要截于400字）
