School of Medicine of Ribeirão Preto, University of São Paulo - USP, SP, Brazil.
Nitric Oxide. 2012 Jun 30;27(1):59-66. doi: 10.1016/j.niox.2012.04.005. Epub 2012 Apr 24.
Nitric oxide (NO) has been pointed out as being the main mediator involved in the hypotension and tissue injury taking place during sepsis. This study aimed to investigate the cellular mechanisms implicated in the acetylcholine (ACh)-induced relaxation detected in aortic rings isolated from rats submitted to cecal ligation and perforation (CLP group), 6h post-CLP. The mean arterial pressure was recorded, and the concentration-effect curves for ACh were constructed for endothelium-intact aortic rings in the absence (control) or after incubation with one of the following NO synthase inhibitors: L-NAME (non-selective), L-NNA (more selective for eNOS), 7-nitroindazole (more selective for nNOS), or 1400W (selective for iNOS). The NO concentration was determined by using confocal microscopy. The protein expression of the NOS isoforms was quantified by Western blot analysis. The prostacyclin concentration was indirectly analyzed on the basis of 6-keto-prostaglandin F(1α) (6-keto-PGF(1α)) levels measured by enzyme immunoassay. There were no differences between Sham- and CLP-operated rats in terms of the relaxation induced by acetylcholine. However, the NOS inhibitors reduced this relaxation in both groups, but this effect remained more pronounced in the CLP group as compared to the Sham group. The acetylcholine-induced NO production was higher in the rat aortic endothelial cells of the CLP group than in those of the Sham group. eNOS protein expression was larger in the CLP group, but the iNOS protein was not verified in any of the groups. The basal 6-keto-PGF(1α) levels were higher in the CLP group, but the acetylcholine-stimulated levels did not increase in CLP as much as they did in the Sham group. Taken together, our results show that the augmented NO production in sepsis syndrome elicited by cecal ligation and perforation is due to eNOS up-regulation and not to iNOS.
一氧化氮（NO）已被指出是参与脓毒症期间发生的低血压和组织损伤的主要介质。本研究旨在探讨在盲肠结扎和穿孔（CLP 组）后 6 小时分离的大鼠主动脉环中，乙酰胆碱（ACh）诱导松弛所涉及的细胞机制。记录平均动脉压，并构建内皮完整的主动脉环在不存在（对照）或孵育以下一种 NO 合酶抑制剂后的 ACh 浓度-效应曲线：L-NAME（非选择性）、L-NNA（更选择性的 eNOS）、7-硝基吲唑（更选择性的 nNOS）或 1400W（选择性的 iNOS）。通过共聚焦显微镜测定 NO 浓度。通过 Western blot 分析定量测定 NOS 同工型的蛋白表达。根据酶免疫测定法测量的 6-酮-前列腺素 F1α（6-keto-PGF1α）水平间接分析前列环素浓度。在 Sham 和 CLP 操作的大鼠之间，乙酰胆碱诱导的松弛没有差异。然而，NOS 抑制剂在两组中均降低了这种松弛，但与 Sham 组相比，CLP 组的这种作用更为明显。CLP 组大鼠主动脉内皮细胞中乙酰胆碱诱导的 NO 产生高于 Sham 组。CLP 组的 eNOS 蛋白表达较大，但在任何一组中均未检测到 iNOS 蛋白。CLP 组的基础 6-酮-PGF1α 水平较高，但 CLP 组中乙酰胆碱刺激的水平没有像 Sham 组那样增加。总之，我们的结果表明，盲肠结扎和穿孔引起的脓毒症综合征中 NO 产生的增加是由于 eNOS 的上调，而不是 iNOS。
