Kim H, Lee H S, Chang K T, Ko T H, Baek K J, Kwon N S
Department of Biochemistry, College of Medicine, Chung-Ang University, Seoul, Korea.
J Immunol. 1995 May 1;154(9):4741-8.
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.
N-α-对甲苯磺酰-L-苯丙氨酸氯甲基酮（TPCK）和N-α-对甲苯磺酰-L-赖氨酸氯甲基酮（TLCK）作为丝氨酸蛋白酶抑制剂，可阻断免疫细胞的多种细胞毒性功能，包括超氧阴离子生成、细胞因子释放、细胞介导的细胞溶解以及与一氧化氮（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/脂多糖（LPS）诱导的小鼠腹腔巨噬细胞产生NO（半数有效浓度：TPCK约为20 μM，TLCK约为30 μM）。在25 μM TPCK和80 μM TLCK处理下，细胞活力超过91%。当TPCK处理延迟至激活后1小时，其抑制作用显著降低。激活2小时后，TPCK不再有效。添加肿瘤坏死因子-α（TNF-α）或IFN-γ/LPS激活的巨噬细胞培养的条件培养基均不能阻止TPCK的抑制作用。TPCK和TLCK均未降低巨噬细胞裂解物中酶促产生的NO。即使在补充一氧化氮合酶所需的辅助因子后，TPCK处理的细胞裂解物也不产生NO。免疫印迹分析表明，TPCK与IFN-γ/LPS同时处理可消除一氧化氮合酶的表达，而延迟添加TPCK则部分有效或完全无效。此外，TPCK处理降低了一氧化氮合酶的mRNA浓度，而不降低mRNA稳定性。因此，丝氨酸蛋白酶抑制剂直接阻断了一氧化氮合酶表达的早期事件。电泳迁移率变动分析表明，TPCK阻断了核因子-κB的激活，核因子-κB是诱导一氧化氮合酶所需的转录因子。TPCK还阻断了IκB从细胞质组分中的消失以及NF-κB亚基p50和p65的核转位。将TPCK的添加延迟10分钟可部分阻止对NF-κB激活过程的抑制，并使NO产生部分恢复。因此，TPCK通过阻断NF-κB激活来抑制一氧化氮合酶的诱导。
