Thomae K R, Joshi P C, Davies P, Pitt B R, Billiar T R, Simmons R L, Nakayama D K
Department of Surgery, University of North Carolina at Chapel Hill, USA.
Surgery. 1996 Jan;119(1):61-6. doi: 10.1016/s0039-6060(96)80215-7.
We recently demonstrated that rat pulmonary artery smooth muscle (RPASM) generates maximal nitric oxide (NO) when exposed to inflammatory cytokines, such as tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma. Our hypothesis is that NO produced by cytokine-stimulated RPASM has local cytotoxic effects on endothelium. Accordingly, we designed a pulmonary smooth muscle and endothelial coculture experiment in which the effects of NO on endothelium can be distinguished from the direct effects of cytokines.
RPASM cells were incubated with a mixture of TNF-alpha (500 units/ml) and IFN-gamma (100 units/ml) for 24 hours. This cytokine mixture was then removed and the NO-producing smooth muscle cells were incubated in a coculture transwell system with rat pulmonary artery endothelial (RPAE) cells. Subsequent NO production (as measured by nitrite concentration in cell supernatants), and the number of viable attached endothelial cells were then measured at 48 hours.
RPASM continued to produce large amounts of NO, in the absence of further cytokine stimulation, after a 24-hour exposure to TNF-alpha and IFN-gamma. This RPASM-generated NO decreased the number of viable attached endothelial cells after 24 hour RPASM-RPAE coculture by 57%. The competitive stereospecific inhibitor of inducible NO synthase (iNOS), NG-monomethyl-L-arginine (NMA), returned the inducible NO production to basal levels and reversed the cytotoxic effects on endothelial cells. The number of viable attached endothelial cells returned to control levels.
The NO produced by cytokine-activated RPASM has local cytotoxic effects on RPAE in coculture. Such NO produced in the vasculature may be a factor in the origin of acute lung injury under conditions of trauma and sepsis.
我们最近证实，大鼠肺动脉平滑肌（RPASM）在暴露于炎性细胞因子，如肿瘤坏死因子（TNF）-α和干扰素（IFN）-γ时会产生最大量的一氧化氮（NO）。我们的假设是，细胞因子刺激的RPASM产生的NO对内皮具有局部细胞毒性作用。因此，我们设计了一项肺平滑肌和内皮共培养实验，其中NO对内皮的作用可以与细胞因子的直接作用区分开来。
将RPASM细胞与TNF-α（500单位/毫升）和IFN-γ（100单位/毫升）的混合物孵育24小时。然后去除该细胞因子混合物，将产生NO的平滑肌细胞在共培养转孔系统中与大鼠肺动脉内皮（RPAE）细胞一起孵育。随后在48小时时测量后续的NO产生量（通过细胞上清液中的亚硝酸盐浓度测量）以及存活附着的内皮细胞数量。
在暴露于TNF-α和IFN-γ 24小时后，在没有进一步细胞因子刺激的情况下，RPASM继续产生大量NO。在RPASM-RPAE共培养24小时后，这种由RPASM产生的NO使存活附着的内皮细胞数量减少了57%。诱导型NO合酶（iNOS）的竞争性立体特异性抑制剂NG-单甲基-L-精氨酸（NMA）使诱导型NO产生恢复到基础水平，并逆转了对内皮细胞的细胞毒性作用。存活附着的内皮细胞数量恢复到对照水平。
细胞因子激活的RPASM产生的NO在共培养中对RPAE具有局部细胞毒性作用。在脉管系统中产生的这种NO可能是创伤和脓毒症条件下急性肺损伤发生的一个因素。
