Shiokoshi Takahiro, Ohsaki Yoshinobu, Kawabe Junichi, Fujino Takayuki, Kikuchi Kenjiro
First Department of Medicine, Asahikawa Medical College, Asahikawa, Japan.
J Hypertens. 2002 Mar;20(3):455-61. doi: 10.1097/00004872-200203000-00021.
Cytokines from inflammatory cells do not produce nitric oxide, but stimulate the production of nitric oxide in vascular smooth muscle cells (VSMC). Thromboxane A2 (TXA2) has been believed to have a key role in atherosclerogenesis and post-angioplasty restenosis.
To determine whether cytokine-induced nitric oxide production is regulated by the TXA2/prostaglandin H2 (PGH2) receptor.
We studied the interleukin-1beta (IL-1beta)-induced production of nitric oxide in rat VSMCs using the TXA2/PGH2 receptor antagonists, seratrodast and Bay-u3405, and an agonist, U-46619. Nitrite formation was measured colorimetrically. IL-1beta increased nitrite formation in a time-dependent manner. The nitrite concentration was 1.7 times greater in the presence of seratrodast than that without it. Nitrite accumulation was increased by Bay-u3405, but was decreased in the presence of U-46619, to 44% of that in its absence. Western and Northern blotting showed that seratrodast increased the levels of expression of inducible nitric oxide synthase (iNOS) protein and mRNA in a dose-dependent manner, whereas U-46619 decreased them. We speculated that VSMCs produced TXA2, thereby decreasing nitric oxide production; therefore we measured the accumulation of TXB2 using an enzyme immunoassay. Untreated VSMCs produced about 20 pg/mg protein of TXB2. This was increased by the addition of IL-1beta, to 152.1 +/- 43.0 pg/mg protein after a 24 h incubation; the expression of cyclooxygenase-2 (COX-2) protein was also increased, but there was no effect on the expression of COX-1 and TXA2 synthase. U-63557A, a TXA2 synthase inhibitor, increased the accumulation of nitrite to 1.3-fold that in its absence.
These data suggest that the expression of iNOS and the production of nitric oxide are regulated by the TXA2/PGH2 receptor in IL-1beta-stimulated VSMCs. The endogenous production of TXA2 by the induction of COX-2 from IL-1beta-stimulated VSMCs probably downregulated the production of nitric oxide in VSMCs. TXA2/PGH2 receptor inhibitors may contribute to the reduction in formation of atherosclerosis in lesions with vascular injury by enhancing the production of nitric oxide by VSMCs.
炎症细胞产生的细胞因子本身不产生一氧化氮，但可刺激血管平滑肌细胞（VSMC）产生一氧化氮。血栓素A2（TXA2）被认为在动脉粥样硬化形成和血管成形术后再狭窄中起关键作用。
确定细胞因子诱导的一氧化氮产生是否受TXA2/前列腺素H2（PGH2）受体调控。
我们使用TXA2/PGH2受体拮抗剂塞曲司特和Bay-u3405以及激动剂U-46619，研究白细胞介素-1β（IL-1β）诱导大鼠VSMC产生一氧化氮的情况。采用比色法测定亚硝酸盐形成。IL-1β以时间依赖性方式增加亚硝酸盐形成。在存在塞曲司特的情况下，亚硝酸盐浓度比不存在时高1.7倍。Bay-u3405增加亚硝酸盐积累，但在存在U-46619时亚硝酸盐积累减少，降至不存在时的44%。蛋白质印迹法和Northern印迹法显示，塞曲司特以剂量依赖性方式增加诱导型一氧化氮合酶（iNOS）蛋白和mRNA的表达水平，而U-46619则使其降低。我们推测VSMC产生TXA2，从而减少一氧化氮产生；因此我们使用酶免疫测定法测量TXB2的积累。未处理的VSMC产生约20 pg/mg蛋白的TXB2。添加IL-1β后其增加，孵育24小时后增至152.1±43.0 pg/mg蛋白；环氧合酶-2（COX-2）蛋白表达也增加，但对COX-1和TXA2合酶的表达无影响。TXA2合酶抑制剂U-63557A使亚硝酸盐积累增加至不存在时的1.3倍。
这些数据表明，在IL-1β刺激的VSMC中，iNOS的表达和一氧化氮的产生受TXA2/PGH2受体调控。IL-1β刺激的VSMC通过诱导COX-2内源性产生TXA2，可能下调VSMC中一氧化氮的产生。TXA2/PGH2受体抑制剂可能通过增强VSMC产生一氧化氮，有助于减少血管损伤病变中动脉粥样硬化的形成。
