de Frutos T, de Miguel L S, García-Durán M, González-Fernández F, Rodríguez-Feo J A, Montón M, Guerra J, Farré J, Casado S, López-Farré A
Hypertension and Cardiovascular Research Laboratory, Fundación Jiménez Díaz, 28040 Madrid, Spain.
Am J Physiol. 1999 Oct;277(4):H1317-25. doi: 10.1152/ajpheart.1999.277.4.H1317.
Despite the evidence that cytokines stimulate nitric oxide (NO) production by inducible nitric oxide synthase (iNOS), several reports recently demonstrated that the hypotensive response related to endothelial nitric oxide synthase (eNOS) activity could be inhibited by the same cytokines. The aim of the present work was to analyze whether NO generated by vascular smooth muscle cells (VSMC) could modify eNOS protein expression in endothelial cells. Bovine aortic endothelial cells (BAEC) and bovine VSMC (BVSMC) in coculture were used for the study. Interleukin-1beta (IL-1beta, 10 ng/ml)-treated BVSMC, which expressed iNOS protein, decreased eNOS protein expression in BAEC. The presence of NO antagonists N(omega)-nitro-L-arginine methyl ester (10(-3) mol/l) or N(G)-monomethyl-L-arginine (10(-3) mol/l) prevented the decrease in eNOS protein expression induced by IL-1beta-treated BVSMC. Surprisingly, two different NO donors, 3-morpholinosydnonimine (10(-4) mol/l) and S-nitroso-N-acetyl-D,L-penicillamine (10(-4) mol/l), failed to modify eNOS expression in BAEC, suggesting the existence of a diffusible mediator released from IL-1beta-treated BVSMC that acts on endothelial cells by reducing eNOS expression. The presence of NO antagonists reduced tumor necrosis factor-alpha (TNF-alpha) production by IL-1beta-stimulated BVSMC. This effect was also produced in the presence of a protein kinase G inhibitor, guanosine-5'-O-(2-thiodiphosphate) trilithium salt. A polyclonal antibody against TNF-alpha prevented eNOS expression in the BAEC-BVSMC coculture. In conclusion, NO by itself failed to modify eNOS protein expression in endothelial cells but increased TNF-alpha generation by IL-1beta-stimulated BVSMC and, in this way, reduced eNOS expression in the endothelium.
尽管有证据表明细胞因子可通过诱导型一氧化氮合酶（iNOS）刺激一氧化氮（NO）生成，但最近有几份报告表明，与内皮型一氧化氮合酶（eNOS）活性相关的降压反应可被相同的细胞因子抑制。本研究的目的是分析血管平滑肌细胞（VSMC）产生的NO是否会改变内皮细胞中eNOS蛋白的表达。共培养的牛主动脉内皮细胞（BAEC）和牛VSMC（BVSMC）用于该研究。表达iNOS蛋白的经白细胞介素-1β（IL-1β，10 ng/ml）处理的BVSMC降低了BAEC中eNOS蛋白的表达。NO拮抗剂N（ω）-硝基-L-精氨酸甲酯（10⁻³ mol/l）或N（G）-单甲基-L-精氨酸（10⁻³ mol/l）的存在可防止经IL-1β处理的BVSMC诱导的eNOS蛋白表达降低。令人惊讶的是，两种不同的NO供体，3-吗啉代-sydnonimine（10⁻⁴ mol/l）和S-亚硝基-N-乙酰-D，L-青霉胺（10⁻⁴ mol/l），未能改变BAEC中eNOS的表达，这表明存在一种从经IL-1β处理的BVSMC释放的可扩散介质，其通过降低eNOS表达作用于内皮细胞。NO拮抗剂的存在降低了IL-1β刺激的BVSMC产生的肿瘤坏死因子-α（TNF-α）。在蛋白激酶G抑制剂鸟苷-5'-O-（2-硫代二磷酸）三锂盐存在的情况下也产生了这种效果。抗TNF-α的多克隆抗体可防止BAEC-BVSMC共培养中eNOS的表达。总之，NO本身未能改变内皮细胞中eNOS蛋白的表达，但增加了IL-1β刺激的BVSMC产生的TNF-α，并以此方式降低了内皮中eNOS的表达。
