Vara E, Arias-Díaz J, García C, Hernández J, Balibrea J L
Department of Biochemistry (Facultad de Medicina), Universidad Complutense, Madrid, Spain.
Arch Surg. 1995 Dec;130(12):1279-85; discussion 1286. doi: 10.1001/archsurg.1995.01430120033005.
Tumor necrosis factor alpha (TNF-alpha)-induced inhibition of surfactant synthesis seems to participate in the pathogenesis of the adult respiratory distress syndrome.
To examine the ability of human type II pneumocytes to produce nitric oxide (NO) in the presence of TNF-alpha and, in addition, to explore the role of this radical in the transduction of the cytokine signal.
Multiple organ donors were the source of lung tissue specimens. Type II pneumocytes were isolated by enzymatic digestion, adherence separation of macrophages, and gradient purification. After 24-hour preculture, cells were cultured for 24 hours in the presence or absence of TNF-alpha (100 ng/mL), sodium nitroprusside (100 mumol/L), N omega-nitro-L-arginine methyl ester (NAME) (1 mmol/L), methylene blue (10 mumol/L),8-bromo-3',5'-cyclic guanosine monophosphate (8-Br-cGMP) (1 mmol/L), prostaglandin E2 (PGE2) (0.1 mumol/L), indomethacin (30 mumol/L), and combinations. The NO release to the medium and cGMP and PGE2 contents of the cells were measured.
The incorporation of 14C-labeled glucose (D-[U-14C]glucose) into phosphatidylcholine and phosphatidylglycerol was selectively inhibited either by 8-Br-cGMP or in the presence of TNF-alpha, PGE2, or nitroprusside, all of which caused an increase in the intracellular levels of cGMP. The inhibitory effect of TNF-alpha was partially reverted by indomethacin, NAME, N-monomethyl arginine, or methylene blue. The inhibitory effect of PGE2 was partially reverted by NAME, while that of nitroprusside was reverted by methylene blue, but not by indomethacin. Tumor necrosis factor alpha induced an increase in PGE2 (4.31 +/- 0.27 vs 1.65 +/- 0.17-pg/microgram protein, n = 10, P < .01) and cGMP (0.238 +/- 0.012 vs 0.109 +/- 0.014-pmol/microgram protein, n = 10, P < .01) cell content and in the NO release to the medium (3.10 +/- 0.14 vs 1.19 +/- 0.11-nmol/microgram protein, n = 10, P < .01). The basal NO release to the medium was also increased in the presence of PGE2. The NAME, which blocked NO generation and cGMP increase, did not affect PGE2 production in response to TNF-alpha. However, indomethacin, which blocked PGE2 production, also blunted NO generation and cGMP increase.
The NO generation, secondary to PGE2 production, seems responsible for the TNF-alpha-induced inhibition of phosphatidylcholine synthesis by human type II pneumocytes. Nitric oxide seems to exert this effect through activation of guanylyl cyclase.
肿瘤坏死因子α（TNF-α）诱导的表面活性剂合成抑制似乎参与了成人呼吸窘迫综合征的发病机制。
研究人Ⅱ型肺泡上皮细胞在TNF-α存在时产生一氧化氮（NO）的能力，并探讨该自由基在细胞因子信号转导中的作用。
多器官供体为肺组织标本来源。通过酶消化、巨噬细胞贴壁分离和梯度纯化分离出Ⅱ型肺泡上皮细胞。在24小时预培养后，细胞在有或无TNF-α（100 ng/mL）、硝普钠（100 μmol/L）、Nω-硝基-L-精氨酸甲酯（NAME）（1 mmol/L）、亚甲蓝（10 μmol/L）、8-溴-3'，5'-环鸟苷单磷酸（8-Br-cGMP）（1 mmol/L）、前列腺素E2（PGE2）（0.1 μmol/L）、吲哚美辛（30 μmol/L）及其组合的情况下培养24小时。检测细胞向培养基中释放的NO以及细胞内cGMP和PGE2的含量。
8-Br-cGMP或在TNF-α、PGE2或硝普钠存在时，14C标记的葡萄糖（D-[U-14C]葡萄糖）掺入磷脂酰胆碱和磷脂酰甘油的过程被选择性抑制，所有这些情况均导致细胞内cGMP水平升高。TNF-α的抑制作用可被吲哚美辛、NAME、N-单甲基精氨酸或亚甲蓝部分逆转。PGE2的抑制作用可被NAME部分逆转，而硝普钠的抑制作用可被亚甲蓝逆转，但不能被吲哚美辛逆转。肿瘤坏死因子α导致细胞内PGE2（4.31±0.27对1.65±0.17 - pg/μg蛋白，n = 10，P <.01）和cGMP（0.238±0.012对0.109±0.014 - pmol/μg蛋白，n = 10，P <.01）含量增加，以及细胞向培养基中释放的NO增加（3.10±0.14对1.19±0.11 - nmol/μg蛋白，n = 10，P <.01）。在PGE2存在时，细胞向培养基中释放的基础NO也增加。阻断NO生成和cGMP增加的NAME不影响TNF-α刺激下的PGE2产生。然而，阻断PGE2产生的吲哚美辛也减弱了NO生成和cGMP增加。
继发于PGE2产生的NO生成似乎是人Ⅱ型肺泡上皮细胞中TNF-α诱导的磷脂酰胆碱合成抑制的原因。一氧化氮似乎通过激活鸟苷酸环化酶发挥这种作用。
