Effects of hypoxia-reoxygenation on superoxide production and change of intra-cellular Ca^<++> of neutrophils

用细胞色素C还原方法研究了低氧复氧对中性粒细胞产生超氧阴离子(O2^-)的影响。从健康成人外周静脉血中分离出中性粒细胞。对照组给予氧气雾化(220 Mm Hg)。缺氧组中性粒细胞用氮气处理30min。复氧组中性粒细胞在氮气预处理30min后吸氧15min。用细胞色素C还原法测定佛波醇醋酸酯刺激前后超氧阴离子的产生。低氧使O_2~(2+)产生显著减少。复氧使未受刺激的中性粒细胞产生O_2~(2+)显著增加。实验二研究了缺氧复氧对中性粒细胞NADPH氧化酶活性和细胞内钙离子的影响。对照组中性粒细胞的细胞膜用空气(PO_2 220 Mm Hg)处理。缺氧组用氮气处理细胞膜30min(P_2O_2 28 Mm Hg)。复氧组在氮气预处理30分钟(PO_2 480 Mm Hg)后，再用氧气处理细胞膜15分钟。氧疗组：氧疗15min(P0_2 480 Mm Hg)。细胞色素C还原法测定NADPH氧化酶活性。低氧导致NADPH氧化酶活性显著降低。复氧引起NADPH氧化酶活性显著升高。这些结果表明，NADPH氧化酶在缺氧-复氧过程中被激活，细胞内钙离子随着复氧的增加而增加。提示细胞内钙离子的增加激活了中性粒细胞中的NADPH氧化酶。

A study was made on the effects of hypoxia-reoxygenation on superoxide anion (O_2^-) production in neutrophils with the cytochrome C reduction method. Neutrophils were separated from the peripheral venous blood of healthy adults. The control group was treated with air PO_2 220mmHg). In the hypoxia group, neutrophils were treated with N_2 for 30 min. In the reoxygenation group, neutrophils were treated with oxygen for 15 min after pretreatment with N_2 for 30 min. Superoxide anion production was measured with the cytochrome C reduction method, after stimulation of phorbol myristate acetate or without such stimulation. Hypoxia caused a significant decrease of O_2^- production. Reoxygenation caused a significant increase of O_2^- production from unstimulated neutrophils. These results suggest that neutrophils were activated by hypoxia-reoxygenation.As the second study, the effects of hypoxia-reoxygenation on NADPH oxidase activity and the change of intracellular Ca ion neutrophils were investigated. In the control group cell membranes of neutrophils were treated with air(PO_2 220mmHg). In the hypoxia group, cell membranes was treated with N_2 for 30 min (PO_2 28mmHg). In the reoxygenation group, cell membranes were treated with oxygen for 15 min after pretreatment with N_2 for 30 min (PO_2 480mmHg. In the oxygenation group, that was treated with oxygen for 15 min (PO_2 480mmHg). NADPH oxidase activity was measured with the cytochrome C reduction method. Hypoxia caused a significant decrease of NADPH oxidase activity. Reoxygenation caused significant increase of NADPH oxidase activity. These results suggest that NADPH oxidase was activated by hypoxia-reoxygenation.Intracellular Ca ion was increased with reoxygenation. This finding suggested the increased intracellular Ca ion activates NADPH oxidase in the neutrophils.