Monteith G R, Blaustein M P
Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Am J Physiol. 1999 May;276(5):C1193-204. doi: 10.1152/ajpcell.1999.276.5.C1193.
The role of mitochondria in Ca2+ homeostasis is controversial. We employed the Ca2+-sensitive dye rhod 2 with novel, high temporal and spatial resolution imaging to evaluate changes in the matrix free Ca2+ concentration of individual mitochondria ([Ca2+]m) in agonist-stimulated, primary cultured aortic myocytes. Stimulation with 10 microM serotonin (5-HT) evoked modest cytosolic Ca2+ transients [cytosolic free Ca2+ concentration ([Ca2+]cyt) <500 nM; measured with fura 2] and triggered contractions in short-term cultured myocytes. However, 5-HT triggered a large mitochondrial rhod 2 signal (indicating pronounced elevation of [Ca2+]m) in only 4% of cells. This revealed heterogeneity in the responses of individual mitochondria, all of which stained with MitoTracker Green FM. In contrast, stimulation with 100 microM ATP evoked large cytosolic Ca2+ transients (>1,000 nM) and induced pronounced, reversible elevation of [Ca2+]m (measured as rhod 2 fluorescence) in 60% of cells. This mitochondrial Ca2+ uptake usually lagged behind the cytosolic Ca2+ transient peak by 3-5 s, and [Ca2+]m declined more slowly than did bulk [Ca2+]cyt. The uptake delay may prevent mitochondria from interfering with rapid signaling events while enhancing the mitochondrial response to large, long-duration elevations of [Ca2+]cyt. The responses of arterial myocytes to modest physiological stimulation do not, however, depend on such marked changes in [Ca2+]m.
线粒体在钙离子稳态中的作用存在争议。我们使用对钙离子敏感的染料罗丹明2，并结合新颖的、具有高时空分辨率的成像技术，来评估激动剂刺激的原代培养主动脉肌细胞中单个线粒体的游离钙离子浓度（[Ca2+]m）的变化。用10微摩尔血清素（5-HT）刺激可诱发适度的胞质钙离子瞬变（胞质游离钙离子浓度（[Ca2+]cyt）<500纳摩尔；用fura 2测量），并在短期培养的肌细胞中引发收缩。然而，5-HT仅在4%的细胞中触发了强烈的线粒体罗丹明2信号（表明[Ca2+]m显著升高）。这揭示了单个线粒体反应的异质性，所有线粒体均用MitoTracker Green FM染色。相比之下，用100微摩尔ATP刺激可诱发较大的胞质钙离子瞬变（>1000纳摩尔），并在60%的细胞中诱导[Ca2+]m显著且可逆的升高（以罗丹明2荧光测量）。这种线粒体钙离子摄取通常比胞质钙离子瞬变峰值滞后3 - 5秒，且[Ca2+]m下降比整体[Ca2+]cyt更慢。摄取延迟可能会阻止线粒体干扰快速信号事件，同时增强线粒体对[Ca2+]cyt大幅、长时间升高的反应。然而，动脉肌细胞对适度生理刺激的反应并不依赖于[Ca2+]m的如此显著变化。
