Hampl V, Cornfield D N, Cowan N J, Archer S L
Dept of Medicine, VA Medical Center, Minneapolis, MN 55417, USA.
Eur Respir J. 1995 Apr;8(4):515-22.
There is indirect, contradictory evidence both for increased and reduced synthesis of the endothelium-derived vasodilator, nitric oxide, in the pulmonary circulation during acute hypoxia. Therefore, we decided to directly measure the effect of acute hypoxia on nitric oxide production by cultured pulmonary endothelium. Because increases in the intracellular free calcium concentration are known to initiate nitric oxide synthesis, we also studied cytosolic calcium levels. We measured the accumulation of the stable nitric oxide metabolite, nitrite, in the fluid used to superfuse the cultured bovine pulmonary artery endothelial cells at an oxygen tension (PO2) of either 20.3 (normoxia) or 4.9 kPa (hypoxia) (152 or 37 mmHg). Intracellular calcium levels were measured with dual-excitation microfluorimetry after loading the cells with the fluorescent calcium indicator, fura 2. Basal NO synthesis, measured as nitrite accumulation over 10 min, was significantly higher under hypoxic than normoxic conditions (8.3 +/- 2.2 versus 4.6 +/- 0.8 nM). Hypoxia transiently increased cytosolic calcium concentration (from 113 +/- 10 to 231 +/- 45 nM). Ryanodine and thapsigargin (which deplete intracellular calcium stores), but not the removal of extracellular calcium, inhibited the hypoxic increase in cytosolic calcium, indicating that it resulted primarily from release of intracellular calcium. Bradykinin-elicited NO synthesis was potentiated by hypoxia. Bradykinin-induced increase in cytosolic calcium was not inhibited by hypoxia. We conclude that hypoxia acutely increases cytosolic calcium levels and basal and bradykinin-stimulated nitric oxide synthesis in pulmonary artery endothelium.
关于急性低氧期间肺循环中内皮源性血管舒张剂一氧化氮的合成增加还是减少，存在间接的、相互矛盾的证据。因此，我们决定直接测量急性低氧对培养的肺内皮细胞一氧化氮产生的影响。由于已知细胞内游离钙浓度的增加会启动一氧化氮的合成，我们还研究了胞质钙水平。我们测量了在氧分压（PO2）为20.3（常氧）或4.9 kPa（低氧）（152或37 mmHg）的条件下，用于灌注培养的牛肺动脉内皮细胞的液体中稳定的一氧化氮代谢产物亚硝酸盐的积累。在用荧光钙指示剂fura 2加载细胞后，用双激发微荧光法测量细胞内钙水平。以10分钟内亚硝酸盐积累量衡量的基础一氧化氮合成，在低氧条件下显著高于常氧条件（8.3±2.2对4.6±0.8 nM）。低氧使胞质钙浓度短暂升高（从113±10到231±45 nM）。ryanodine和毒胡萝卜素（耗尽细胞内钙储备），但不是去除细胞外钙，抑制了低氧引起的胞质钙增加，表明其主要源于细胞内钙的释放。低氧增强了缓激肽诱导的一氧化氮合成。低氧并未抑制缓激肽诱导的胞质钙增加。我们得出结论，低氧急性增加肺动脉内皮细胞的胞质钙水平以及基础和缓激肽刺激的一氧化氮合成。
