He L, Dinger B, Sanders K, Hoidal J, Obeso A, Stensaas L, Fidone S, Gonzalez C
Dept. of Physiology, University of Utah School of Medicine, 410 Chipeta Way, Salt Lake City, 84108 UT, USA.
Am J Physiol Lung Cell Mol Physiol. 2005 Dec;289(6):L916-24. doi: 10.1152/ajplung.00015.2005.
Membrane potential in oxygen-sensitive type I cells in carotid body is controlled by diverse sets of voltage-dependent and -independent K(+) channels. Coupling of Po(2) to the open-closed state of channels may involve production of reactive oxygen species (ROS) by NADPH oxidase. One hypothesis suggests that ROS are produced in proportion to the prevailing Po(2) and a subset of K(+) channels closes as ROS levels decrease. We evaluated ROS levels in normal and p47(phox) gene-deleted [NADPH oxidase knockout (KO)] type I cells using the ROS-sensitive dye dihydroethidium (DHE). In normal cells, hypoxia elicited an increase in ROS, which was blocked by the specific NADPH oxidase inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF, 3 mM). KO type I cells did not respond to hypoxia, but the mitochondrial uncoupler azide (5 microM) elicited increased fluorescence in both normal and KO cells. Hypoxia had no effect on ROS production in sensory and sympathetic neurons. Methodological control experiments showed that stimulation of neutrophils with a cocktail containing the chemotactic peptide N-formyl-Met-Leu-Phe (1 microM), arachidonic acid (10 microM), and cytochalasin B (5 microg/ml) elicited a rapid increase in DHE fluorescence. This response was blocked by the NADPH oxidase inhibitor diphenyleneiodonium (10 microM). KO neutrophils did not respond; however, azide (5 microM) elicited a rapid increase in fluorescence. Physiological studies in type I cells demonstrated that hypoxia evoked an enhanced depression of K+ current and increased intracellular Ca2+ levels in KO vs. normal cells. Moreover, AEBSF potentiated hypoxia-induced increases in intracellular Ca2+ and enhanced the depression of K+ current in low O(2). Our findings suggest that local compartmental increases in oxidase activity and ROS production inhibit the activity of type I cells by facilitating K+ channel activity in hypoxia.
颈动脉体中对氧敏感的I型细胞的膜电位受多种电压依赖性和非依赖性钾通道调控。氧分压（Po₂）与通道开闭状态的耦合可能涉及NADPH氧化酶产生活性氧（ROS）。一种假说认为，ROS的产生与当前的Po₂成比例，并且随着ROS水平降低，一部分钾通道会关闭。我们使用对ROS敏感的染料二氢乙锭（DHE）评估正常和p47（phox）基因缺失[NADPH氧化酶敲除（KO）]的I型细胞中的ROS水平。在正常细胞中，缺氧会引起ROS增加，这被特异性NADPH氧化酶抑制剂4-（2-氨基乙基）苯磺酰氟（AEBSF，3 mM）阻断。KO I型细胞对缺氧无反应，但线粒体解偶联剂叠氮化物（5 μM）在正常和KO细胞中均引起荧光增加。缺氧对感觉神经元和交感神经元的ROS产生没有影响。方法学对照实验表明，用含有趋化肽N-甲酰甲硫氨酰-亮氨酰-苯丙氨酸（1 μM）、花生四烯酸（10 μM）和细胞松弛素B（5 μg/ml）的混合物刺激中性粒细胞会引起DHE荧光迅速增加。这种反应被NADPH氧化酶抑制剂二苯基碘鎓（10 μM）阻断。KO中性粒细胞无反应；然而，叠氮化物（5 μM）引起荧光迅速增加。对I型细胞的生理学研究表明，与正常细胞相比，缺氧在KO细胞中引起钾电流增强抑制和细胞内钙离子水平升高。此外，AEBSF增强了缺氧诱导的细胞内钙离子增加，并增强了低氧条件下钾电流的抑制。我们的研究结果表明，氧化酶活性和ROS产生的局部区域增加通过促进缺氧时钾通道活性来抑制I型细胞的活性。
