Mitochondrial membrane potential changes in osteoblasts treated with parathyroid hormone and estradiol.
Mitochondrial membrane potential changes in osteoblasts treated with parathyroid hormone and estradiol.
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DOI：
10.1006/excr.1997.3570
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发表时间：
1997-06
影响因子：
3.7
通讯作者：
M. Troyan;V. R. Gilman;C. Gay
中科院分区：
文献类型：
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作者：
M. Troyan;V. R. Gilman;C. Gay
这项研究评估了用已知调节成骨细胞的激素处理培养的成骨细胞后线粒体膜电位的变化。一种荧光碳菁染料5，5‘，6，6’-tetrachloro-1，1‘，3，3’-tetraethylbenzimidazolocarbocyanine+++碘，也被称为JC-1，被用作探针。当线粒体膜电位为高负值时，JC-1在585 nm(橙红色)处发射光子，当膜电位降低时，JC-1在527 nm(绿色)处发射光子。成骨细胞在无血清介质中冲洗5min，然后负载1×10(-6)M JC-1 10min。用激光扫描共聚焦显微镜系统检测JC-1荧光的分布和强度。激素治疗包括甲状旁腺激素(PTH；10(-8)M)、17β-雌二醇(10(-8)M)和甲状腺素(T4；10(-8)M)。钾离子载体呋喃霉素(10(-6)M)被用作对照，因为已知它可以在不干扰pH梯度的情况下干扰线粒体的电化学梯度。瓦林霉素引起绿色/红色比率的显著、迅速的增加(比未经处理的值高22.5%)，这表明在所有被评估的样本中，线粒体膜电位都降低了。甲状旁腺素使所有受检细胞的膜电位降低，但不太明显，但显著(7-14%)。已知甲状旁腺素通过多种方式影响成骨细胞，并抑制线粒体呼吸；结果证实了这一效果。对于雌二醇，一半的细胞有显著的反应，记录到膜电位下降了6%到13%；另一半细胞没有反应。甲状腺激素不改变线粒体膜电位。在甲状旁腺激素和雌二醇治疗后，在20 S内可检测到对呋喃霉素的应答，但发生的速度较慢，超过200至300 S。对甲状旁腺素和雌二醇的反应可能是由于线粒体摄取细胞内钙离子所致。
This study assessed mitochondrial membrane potential changes in cultured osteoblasts treated with hormones known to regulate osteoblasts. A fluorescent carbocyanine dye, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine++ + iodide, also called JC-1, was used as a probe. JC-1 emits photons at 585 nm (orange-red) when the membrane potential in mitochondria is highly negative, but when the potential becomes reduced emission occurs at 527 nm (green). Osteoblasts were rinsed in serum-free medium for 5 min, then loaded with 1 x 10(-6) M JC-1 for 10 min. The distribution and intensity of JC-1 fluorescence were evaluated with a laser-scanning confocal microscope system. Hormone treatments included parathyroid hormone (PTH; 10(-8) M), 17beta-estradiol (10(-8) M), and thyroxine (T4; 10(-8) M). The potassium ionophore valinomycin (10(-6) M) was used as a control since it is known to disrupt the electrochemical gradient of mitochondria without interfering with the pH gradient. Valinomycin caused a profound, rapid increase (22.5% above untreated values) in the green/red ratio, which indicated a lowering of the mitochondrial membrane potential in all samples evaluated. PTH caused a less pronounced, but significant (7-14%), reduction in membrane potential in all cells examined. PTH is known to affect osteoblasts in a number of ways and is inhibitory to mitochondrial respiration; the results confirm this effect. For estradiol, half of the cells responded at a significant level, with a membrane potential reduction of 6 to 13% being recorded; the other half did not respond. Thyroxine did not alter mitochondrial membrane potential. Responses were detectable within 20 s for valinomycin, but occurred at a slower rate, over 200 to 300 s, following PTH and estradiol treatment. Responses to PTH and estradiol could be due to mitochondrial uptake of cytosolic Ca2+.