MITOCHONDRIAL-FUNCTION IN TYPE-I CELLS ISOLATED FROM RABBIT ARTERIAL CHEMORECEPTORS
MITOCHONDRIAL-FUNCTION IN TYPE-I CELLS ISOLATED FROM RABBIT ARTERIAL CHEMORECEPTORS
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DOI：
10.1113/jphysiol.1992.sp019114
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发表时间：
1992-05-01
影响因子：
5.5
通讯作者：
BISCOE, TJ
中科院分区：
文献类型：
--
作者：
DUCHEN, MR;BISCOE, TJ
1. 在这篇论文和随附的论文（Duchen和Biscoe，1992）中，我们检验了线粒体电子传递的氧敏感性形成颈动脉体（主要外周动脉氧传感器）中转导的基础的假设。 我们在这里描述了分离的I型细胞的线粒体NAD（P）H的自发荧光的变化，伴随着P（o2）的变化。 NAD（P）H自发荧光（激发，340-360 nm;发射峰，450 nm）随着缺氧而增加，反映了NAD（P）H/NAD（P）比率的升高。 自发荧光的逐渐增加被视为对P（o2）的逐渐降低的响应，表明线粒体功能在低于约60 mmHg的P（o2）时逐渐改变。 缺氧和氰化物对NAD（P）H自发荧光的相互排斥反应表明NAD（P）H自发荧光可能来源于线粒体. 氧化的黄素蛋白在450 nm激发时发出荧光，发射峰在550 nm。 在这些条件下获得的小信号随着解偶联剂的增加而增加，并且随着P（o2）的下降而显示出梯度下降，反映了FADH/FAD比率的上升。 缺氧使[Ca ~（2+）]i升高。 缺氧引起的线粒体功能的变化并不继发于这种上升。 短暂的K+诱导的去极化导致[Ca 2 +]i的瞬时增加。 同时，NAD（P）H自发荧光迅速降低，随后增加，其持续时间远远超过[Ca 2 +]i的升高。 这种自发荧光的延迟增加小于缺氧的增加，即使K+诱导的去极化提高[Ca 2 +]i比缺氧。 在无Ca 2+溶液中，去极化引起的变化被消除，而与缺氧相关的变化被消除. K+诱导的去极化自发荧光的变化似乎反映了（i）线粒体Ca 2+摄取后呼吸刺激引起的NAD（P）H氧化和（ii）线粒体Ca 2+依赖性激活的NAD（P）还原。 这种激活可以持续几分钟后，只有100毫秒的去极化，而伴随缺氧的变化密切跟随P（o2）的变化的时间过程。 在相同条件下，在类似分离的大鼠或小鼠嗜铬细胞和小鼠背根神经节神经元中，未观察到自发荧光或[Ca 2 +]i的可测量变化，直到P（o2）降至约5 mmHg以下。 羰基氰化物对三氟甲氧基苯腙（FCCP）增加O2消耗，氧化线粒体NADH，从而降低自发荧光（Δ-F（FCCP））。 缺氧或CN-阻断电子传递减少O2消耗，增加线粒体NADH/NAD和自发荧光（Δ F（CN））。 因此，具有FCCP的自体荧光的分数变化Δ-F（FCCP）/（Δ-F（FCCP + F（CN））是静息O2消耗的量度。 在I型细胞中，平均值为0.27（标准差+/- 0.08，n = 18）。 这与类似解离的小鼠背根神经节（DRG）神经元形成对比，其平均比率为0.53（S.D. +/- 0.14，n = 14），培养DRG细胞（平均值0.64，S.D. +/- 0.16，n = 16）或新鲜分离的嗜铬细胞（平均值0.45 +/- 0.05，n = 10）。 这表明I型细胞具有异常高的静息O2消耗，接近其最大容量。 这些数据表明：（i）线粒体功能在P（o2）值的生理相关范围内发生变化;（ii）组织的高耗氧反映了I型细胞的高耗氧。 它们与专门的线粒体电子传递赋予这些细胞不寻常的氧敏感性的假设一致。
1. In this, and the accompanying paper (Duchen & Biscoe, 1992), we test the hypothesis that the oxygen sensitivity of mitochondrial electron transport forms a basis for transduction in the carotid body, the primary peripheral arterial oxygen sensor. We here describe for isolated type I cells the changes in autofluorescence of mitochondrial NAD(P)H that accompany changes in P(o2).2. NAD(P)H autofluorescence (excitation, 340-360 nm; emission peak, 450 nm) increased with anoxia, reflecting a rise in the NAD(P)H/NAD(P) ratio. Graded increases in autofluorescence were seen in response to graded decreases in P(o2), suggesting that mitochondrial function is progressively altered below a P(o2) of about 60 mmHg.3. A mitochondrial origin for the NAD(P)H autofluorescence was suggested by the mutual exclusion of the responses to anoxia and cyanide.4. Oxidized flavoproteins fluoresce when excited at 450 nm with an emission peak at 550 nm. The small signals obtained under these conditions increased with uncoupler and showed a graded decrease with falling P(o2) reflecting a rise in the FADH/FAD ratio.5. Hypoxia raises [Ca2+]i. The hypoxia-induced changes in mitochondrial function were not secondary to this rise. A brief K+-induced depolarization leads to a transient increase in [Ca2+]i. At the same time there is a rapid decrease in NAD(P)H autofluorescence followed by an increase that far outlasts the rise in [Ca2+]i. This delayed increase in autofluorescence was smaller than was the increase with anoxia, even though K+-induced depolarization raised [Ca2+]i more than does anoxia. In Ca2+-free solutions the depolarization-induced changes were abolished, while those associated with hypoxia were maintained.6. The changes of autofluorescence with K+-induced depolarization appear to reflect (i) oxidation of NAD(P)H by stimulation of respiration following mitochondrial Ca2+ uptake and (ii) reduction of NAD(P) by the Ca2+-dependent activation of mitochondrial dehydrogenases. This activation could last several minutes following only 100 ms depolarization, while the changes accompanying hypoxia closely followed the time course of the change in P(o2).7. In similarly isolated rat or mouse chromaffin cells and mouse dorsal root ganglion neurons under identical conditions, no measurable change in autofluorescence or in [Ca2+]i was seen until the P(o2) fell below about 5 mmHg.8. Carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP) increases O2 consumption, oxidizing mitochondrial NADH and hence decreasing autofluorescence, (DELTA-F(FCCP)). Blockade of electron transport by anoxia or CN- decreases O2 consumption, increasing mitochondrial NADH/NAD and autofluorescence (DELTA-F(CN)). The fractional change in autofluorescence with FCCP, DELTA-F(FCCP)/(DELTA-F(FCCP + F(CN)), is thus a measure of resting O2 consumption. In type I cells, this gave a mean of 0.27 (S.D. +/- 0.08, n = 18). This contrasts with similarly dissociated mouse dorsal root ganglion (DRG) neurons, for which the mean ratio was 0.53 (S.D. +/- 0.14, n = 14), with DRG cells in culture (mean of 0.64, S.D. +/- 0.16, n = 16) or with freshly dissociated chromaffin cells (mean 0.45 +/- 0.05, n = 10). This suggests that the type I cells have an unusually high resting O2 consumption, near their maximal capacity.9. These data suggest that (i) mitochondrial function alters over the physiologically relevant range of P(o2) values and (ii) that the high oxygen consumption of the tissue reflects that of the type I cell. They are consistent with the hypothesis that specialized mitochondrial electron transport confers on these cells their unusual oxygen sensitivity.