Marshall C, Mamary A J, Verhoeven A J, Marshall B E
Department of Anesthesia, University of Pennsylvania Medical School, Philadelphia 19143, USA.
Am J Respir Cell Mol Biol. 1996 Nov;15(5):633-44. doi: 10.1165/ajrcmb.15.5.8918370.
An NADPH-oxidase complex containing at least two protein components (gp91-phox and p22-phox) and a unique low redox potential (-245 mV) cytochrome b-245 is the source of superoxide generated for bacterial killing in neutrophils and has been suggested as the oxygen sensor in the carotid body. In pure cultures of smooth muscle cells from calf small pulmonary arteries (300 microns diameter) the presence of the 91 kD protein specific to this cytochrome was demonstrated by Western blot analysis with monoclonal antibody 48. Low-temperature-difference spectrophotometry of homogenates of these cells demonstrated the characteristic cytochrome b-245 spectrum when titrated between redox potentials of -152 and -345 mV, consistent with the low redox potential form. When these same cells were exposed to hypoxia (approximately 40 mmHg), superoxide production increased significantly from 1.4 +/- 0.2 to 73 +/- 12 nmoles.min-1 mg-1 protein. Hypoxic generation of superoxide was inhibited by the NADPH-oxidase inhibitor diphenyleneiodonium (DPI: 10 microM) but not by the mitochondrial inhibitor myxathiazole (10 microM). The hypoxic superoxide increase was significantly greater than that observed from smooth muscle cells from large pulmonary arteries or from large or small systemic arteries. Fluorescence immunocytochemistry revealed the presence of the NADPH-oxidase protein in the walls of pulmonary arteries in rat lung slices, and confocal microscopy showed the complex to be widely distributed in the vicinity of the arterial smooth muscle walls. In hypoxia or norepinephrine (NP)-induced vasoconstriction of pulmonary artery rings from cats, the sensitivity to inhibition by DPI was observed to be significantly greater for hypoxia (ED50 = 0.8 microM) than for NP-induced (ED50 = 13.4 microM) constriction. Together these observations demonstrate that the unique cytochrome b-245 containing NADPH-oxidase is present in pulmonary artery smooth muscle and that an NADPH-oxidase or NADH-oxidoreductase complex is activated to release superoxide by hypoxic conditions. It is concluded that a trans-membrane NADPH-oxidase is the most likely and that activation of this system may be involved in the initiation of hypoxic pulmonary vasoconstriction.
一种含有至少两种蛋白质成分（gp91 - phox和p22 - phox）以及独特的低氧化还原电位（-245 mV）细胞色素b - 245的NADPH氧化酶复合物，是中性粒细胞中产生用于杀灭细菌的超氧化物的来源，并且有人提出它是颈动脉体中的氧传感器。在用单克隆抗体48进行的蛋白质印迹分析中，证实了来自小牛小肺动脉（直径300微米）的平滑肌细胞纯培养物中存在这种细胞色素特有的91 kD蛋白质。当在-152至-345 mV的氧化还原电位之间滴定这些细胞的匀浆时，低温差分光光度法显示出特征性的细胞色素b - 245光谱，这与低氧化还原电位形式一致。当这些相同的细胞暴露于低氧（约40 mmHg）时，超氧化物产生量从1.4±0.2显著增加至73±12纳摩尔·分钟-1·毫克-1蛋白质。NADPH氧化酶抑制剂二苯碘鎓（DPI：10 microM）可抑制低氧诱导的超氧化物产生，但线粒体抑制剂粘噻唑（10 microM）则不能。低氧诱导的超氧化物增加显著大于从大肺动脉或大、小体循环动脉的平滑肌细胞中观察到的增加。荧光免疫细胞化学显示大鼠肺切片中肺动脉壁存在NADPH氧化酶蛋白，共聚焦显微镜显示该复合物广泛分布于动脉平滑肌壁附近。在猫肺动脉环的低氧或去甲肾上腺素（NP）诱导的血管收缩中，观察到DPI对低氧诱导收缩的抑制敏感性（ED50 = 0.8 microM）显著高于对NP诱导收缩的抑制敏感性（ED50 = 13.4 microM）。这些观察结果共同表明，肺动脉平滑肌中存在含有独特细胞色素b - 245的NADPH氧化酶，并且低氧条件可激活NADPH氧化酶或NADH氧化还原酶复合物以释放超氧化物。结论是跨膜NADPH氧化酶最有可能参与其中，并且该系统的激活可能与低氧性肺血管收缩的起始有关。
