Kimura C, Oike M, Ito Y
Department of Pharmacology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
Am J Physiol Heart Circ Physiol. 2000 Nov;279(5):H2310-8. doi: 10.1152/ajpheart.2000.279.5.H2310.
To investigate the possible cellular mechanisms of the ischemia-induced impairments of cerebral microcirculation, we investigated the effects of hypoxia/reoxygenation on the intracellular Ca(2+) concentration (Ca(2+)) in bovine brain microvascular endothelial cells (BBEC). In the cells kept in normal air, ATP elicited Ca(2+) oscillations in a concentration-dependent manner. When the cells were exposed to hypoxia for 6 h and subsequent reoxygenation for 45 min, the basal level of Ca(2+) was increased from 32.4 to 63.3 nM, and ATP did not induce Ca(2+) oscillations. Hypoxia/reoxygenation also inhibited capacitative Ca(2+) entry (CCE), which was evoked by thapsigargin (DeltaCa(2+): control, 62.3 +/- 3.1 nM; hypoxia/reoxygenation, 17.0 +/- 1.8 nM). The impairments of Ca(2+) oscillations and CCE, but not basal Ca(2+), were restored by superoxide dismutase and the inhibitors of mitochondrial electron transport, rotenone and thenoyltrifluoroacetone (TTFA). By using a superoxide anion (O(2)(-))-sensitive luciferin derivative MCLA, we confirmed that the production of O(2)(-) was induced by hypoxia/reoxygenation and was prevented by rotenone and TTFA. These results indicate that hypoxia/reoxygenation generates O(2)(-) at mitochondria and impairs some Ca(2+) mobilizing properties in BBEC.
为了研究缺血诱导的脑微循环损伤可能的细胞机制，我们研究了缺氧/复氧对牛脑微血管内皮细胞（BBEC）细胞内钙离子浓度（[Ca²⁺]i）的影响。在正常空气中培养的细胞中，ATP以浓度依赖的方式引发钙离子振荡。当细胞暴露于缺氧6小时并随后复氧45分钟时，[Ca²⁺]i的基础水平从32.4 nM增加到63.3 nM，并且ATP不再诱导钙离子振荡。缺氧/复氧还抑制了由毒胡萝卜素引发的容量性钙离子内流（CCE）（Δ[Ca²⁺]i-CCE：对照组，62.3±3.1 nM；缺氧/复氧组，17.0±1.8 nM）。超氧化物歧化酶以及线粒体电子传递抑制剂鱼藤酮和噻吩甲酰三氟丙酮（TTFA）可恢复钙离子振荡和CCE的损伤，但不能恢复基础[Ca²⁺]i。通过使用对超氧阴离子（O₂⁻）敏感的荧光素衍生物MCLA，我们证实缺氧/复氧诱导了O₂⁻的产生，而鱼藤酮和TTFA可防止这种产生。这些结果表明，缺氧/复氧在线粒体处产生O₂⁻并损害BBEC中的一些钙离子动员特性。
