Chloromethyl ketones block induction of nitric oxide synthase in murine macrophages by preventing activation of nuclear factor-kappa B.
Chloromethyl ketones block induction of nitric oxide synthase in murine macrophages by preventing activation of nuclear factor-kappa B.
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DOI：
10.4049/jimmunol.154.9.4741
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发表时间：
1995-05
影响因子：
4.4
通讯作者：
Hongkyun Kim;Hee Sung Lee;Kyung Taek Chang;Tae Hong Ko;K. Baek;N. Kwon
中科院分区：
文献类型：
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作者：
Hongkyun Kim;Hee Sung Lee;Kyung Taek Chang;Tae Hong Ko;K. Baek;N. Kwon
N-α-甲苯磺酰基-L-苯丙氨酸氯甲基酮（TPCK）和N-α-甲苯磺酰基-L-赖氨酸氯甲基酮（TLCK），丝氨酸蛋白酶抑制剂，阻断免疫细胞的许多细胞毒性功能，包括超氧阴离子产生、细胞因子释放、细胞介导的细胞溶解和一氧化氮（NO）相关的巨噬细胞功能。TPCK和TLCK以剂量依赖性方式抑制IFN-γ/LPS诱导的小鼠腹腔巨噬细胞NO生成（EC 50：TPCK约20 μ M，TLCK约30 μ M）。25 μ M TPCK和80 μ M TLCK的存活率超过91%。当TPCK处理延迟到激活1小时，抑制作用显着降低。激活2 h后，TPCK不再有效。添加TNF-α或IFN-γ/LPS激活的巨噬细胞培养物的条件培养基均不能阻止TPCK的抑制作用。TPCK和TLCK都没有减少巨噬细胞裂解物的NO酶促生成。从TPCK处理的细胞裂解物不产生NO，即使在补充必要的辅因子NO合酶。免疫印迹分析表明，同时处理的TPCK与IFN-γ/LPS废除NO合成酶的表达，而延迟添加的TPCK是部分有效或无效的。此外，TPCK处理降低了NO合酶mRNA的浓度，而不降低mRNA的稳定性。因此，丝氨酸蛋白酶抑制剂直接阻断了NO合酶表达的早期事件。电泳迁移率变动分析表明，TPCK阻断激活的核因子-κ B，一个转录因子所必需的NO合成酶的诱导。TPCK还阻断I κ B从胞质组分的消失，以及NF-κ B亚基p50和p65的核转位。延迟加入TPCK 10 min可部分抑制NF-κ B B活化过程，并可部分恢复NO的产生。因此，TPCK通过阻断NF-κ B活化来抑制NO合酶诱导。
N-alpha-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK), serine protease inhibitors, block many cytotoxic functions of immune cells including superoxide anion production, cytokine release, cell-mediated cytolysis, and nitric oxide (NO)-related macrophage functions. IFN-gamma/LPS-induced NO production from murine peritoneal macrophages was inhibited by TPCK and TLCK in a dose-dependent manner (EC50s: approximately 20 microM for TPCK and approximately 30 microM for TLCK). Viability exceeded 91% with 25 microM TPCK and with 80 microM TLCK. When TPCK treatment was delayed until 1 h of activation, the inhibitory effect was markedly reduced. After 2 h of the activation, TPCK was not effective anymore. Addition of either TNF-alpha or conditioned media from IFN-gamma/LPS-activated macrophage culture did not prevent the inhibitory effect of TPCK. Neither TPCK nor TLCK reduced enzymatic NO production from macrophage lysates. Lysates from TPCK-treated cells did not generate NO even after supplementing necessary cofactors for NO synthase. Immunoblotting analysis showed that simultaneous treatment of TPCK with IFN-gamma/LPS abolished the NO synthase expression, whereas delayed addition of TPCK was either partially effective or not effective at all. Furthermore, TPCK treatment reduced the concentration of mRNA for NO synthase without decreasing mRNA stability. Thus, the serine protease inhibitors directly blocked an early event in expression of NO synthase. Electrophoretic mobility shift assay indicated that TPCK blocked the activation of nuclear factor-kappa B, a transcription factor necessary for NO synthase induction. TPCK also blocked disappearance of I kappa B from cytosolic fraction, and nuclear translocation of NF-kappa B subunits p50 and p65. Delaying the addition of TPCK by 10 min partially prevented the inhibition of the NF-kappa B activation process and allowed partial resuming of NO production. Thus, TPCK inhibited NO synthase induction by blocking NF-kappa B activation.