Hesthammer Ronja, Eide Torunn, Thorsen Einar, Svardal Asboørn M, Djurhuus Rune
Norwegian Centre for Maritime and Diving Medicine, Haukeland University Hospital, Bergen, Norway.
Department of Clinical Science, University of Bergen, Norway.
Undersea Hyperb Med. 2019;46(4):509-519.
Nitric oxide (NO) may protect against gas bubble formation and risk of decompression sickness. We have previously shown that the crucial co-factor tetrahydrobiopterin (BH4) is oxidized in a dose-dependent manner when exposed to hyperoxia similar to diving conditions but with minor effects on the NO production by nitric oxide synthase. By manipulating the intracellular redox state, we further investigated the relationship between BH4 levels and production of NO in human endothelial cells (HUVECs). HUVECs were cultured with and without ascorbic acid (AA) and the glutathione (GSH) synthesis inhibitor buthionine sulfoximine, prior to hyperoxic exposure. The levels of biopterins and GSH were determined in cell lysates while the production of NO was determined in intact cells. Omitting AA resulted in a 91% decrease in BH4 levels (0.49 ± 0.08 to 0.04 ± 0.01 pmol/10⁶ cells, p⟨0.001) at 20 kPa oxygen (O2), and 88% decrease (0.24 ± 0.03 to 0.03 ± 0.01 pmol/10⁶ cells, p=0.01) after exposure to 60 kPa O2. The NO generation was decreased by 23% (74.5 ± 2.2 to 57.3 ± 5.6 pmol/min/mg protein, p⟨0.001) at 20 kPa O2, but no significant change was observed at 60 kPa O2. GSH depletion had no effects on the NO generation. No correlation was found between NO generation and the corresponding intracellular BH4 concentration (p=0.675, r=-0.055) or the BH4 to BH2 ratio (p=0.983, r=0.003), determined across 18 in vitro experiments. Decreased BH4 in HUVECs, due to hyperoxia or lack of ascorbic acid, does not imply corresponding decreases in NO generation.
一氧化氮（NO）可能预防气泡形成及减压病风险。我们之前已经表明，关键辅助因子四氢生物蝶呤（BH4）在暴露于类似于潜水条件的高氧环境时会以剂量依赖方式被氧化，但对一氧化氮合酶产生NO的影响较小。通过操纵细胞内氧化还原状态，我们进一步研究了人内皮细胞（HUVECs）中BH4水平与NO产生之间的关系。在高氧暴露之前，将HUVECs分别在有和没有抗坏血酸（AA）以及谷胱甘肽（GSH）合成抑制剂丁硫氨酸亚砜胺的情况下进行培养。在细胞裂解物中测定生物蝶呤和GSH的水平，而在完整细胞中测定NO的产生。在20 kPa氧气（O2）条件下，省略AA导致BH4水平下降91%（从0.49±0.08降至0.04±0.01 pmol/10⁶细胞，p<0.001），在暴露于60 kPa O2后下降88%（从0.24±0.03降至0.03±0.01 pmol/10⁶细胞，p = 0.01）。在20 kPa O2时，NO生成减少了23%（从74.5±2.2降至57.3±5.6 pmol/分钟/毫克蛋白质，p<0.001），但在60 kPa O2时未观察到显著变化。GSH耗竭对NO生成没有影响。在18个体外实验中，未发现NO生成与相应的细胞内BH4浓度（p = 0.675，r = -0.055）或BH4与BH2的比率（p = 0.983，r = 0.003）之间存在相关性。由于高氧或缺乏抗坏血酸导致HUVECs中BH4减少，并不意味着NO生成会相应减少。
