Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota 55905, USA.
Am J Physiol Cell Physiol. 2012 Aug 1;303(3):C244-56. doi: 10.1152/ajpcell.00414.2011. Epub 2012 Jun 6.
Regulation of cytosolic Ca(2+) concentration (Ca(2+)) in airway smooth muscle (ASM) is a key aspect of airway contractility and can be modulated by inflammation. Mitochondria have tremendous potential for buffering Ca(2+), helping prevent Ca(2+) overload, and modulating other intracellular events. Here, compartmentalization of mitochondria to different cellular regions may subserve different roles. In the present study, we examined the role of Ca(2+) buffering by mitochondria and mitochondrial Ca(2+) transport mechanisms in the regulation of Ca(2+) in enzymatically dissociated human ASM cells upon exposure to the proinflammatory cytokines TNF-α and IL-13. Cells were loaded simultaneously with fluo-3 AM and rhod-2 AM, and Ca(2+) and mitochondrial Ca(2+) concentration (Ca(2+)) were measured, respectively, using real-time two-color fluorescence microscopy in both the perinuclear and distal, perimembranous regions of cells. Histamine induced a rapid increase in both Ca(2+) and Ca(2+), with a significant delay in the mitochondrial response. Inhibition of the mitochondrial Na(+)/Ca(2+) exchanger (1 μM CGP-37157) increased Ca(2+) responses in perinuclear mitochondria but not distal mitochondria. Inhibition of the mitochondrial uniporter (1 μM Ru360) decreased Ca(2+) responses in perinuclear and distal mitochondria. CGP-37157 and Ru360 significantly enhanced histamine-induced Ca(2+). TNF-α and IL-13 both increased Ca(2+), which was associated with decreased Ca(2+) in the case of TNF-α but not IL-13. The effects of TNF-α on both Ca(2+) and Ca(2+) were affected by CGP-37157 but not by Ru360. Overall, these data demonstrate that in human ASM cells, mitochondria buffer Ca(2+) after agonist stimulation and its enhancement by inflammation. The differential regulation of Ca(2+) in different parts of ASM cells may serve to locally regulate Ca(2+) fluxes from intracellular sources versus the plasma membrane as well as respond to differential energy demands at these sites. We propose that such differential mitochondrial regulation, and its disruption, may play a role in airway hyperreactivity in diseases such as asthma, where Ca(2+) is increased.
细胞浆钙离子浓度(Ca(2+))的调节是气道平滑肌（ASM）收缩性的一个关键方面，可被炎症所调节。线粒体具有巨大的缓冲Ca(2+)的潜力，有助于防止钙超载，并调节其他细胞内事件。在这里，线粒体在不同细胞区域的区室化可能发挥不同的作用。在本研究中，我们研究了线粒体钙缓冲作用以及线粒体钙转运机制在酶解人 ASM 细胞暴露于促炎细胞因子 TNF-α和 IL-13 时对Ca(2+)调节中的作用。同时用 fluo-3 AM 和 rhod-2 AM 装载细胞，并分别使用实时双色荧光显微镜测量细胞核周和远端、膜周区域的Ca(2+)和线粒体钙浓度(Ca(2+))。组胺诱导Ca(2+)和Ca(2+)快速增加，线粒体反应有明显延迟。线粒体 Na(+)/Ca(2+)交换体抑制剂（1 μM CGP-37157）增加核周线粒体的Ca(2+)反应，但不增加远端线粒体的Ca(2+)反应。线粒体单转运体抑制剂（1 μM Ru360）降低核周和远端线粒体的Ca(2+)反应。CGP-37157 和 Ru360 显著增强组胺诱导的Ca(2+)。TNF-α和 IL-13 均增加Ca(2+)，而 TNF-α引起的Ca(2+)降低，但 IL-13 则不然。CGP-37157 而非 Ru360 影响 TNF-α对Ca(2+)和Ca(2+)的作用。总之，这些数据表明，在人 ASM 细胞中，线粒体在激动剂刺激后缓冲Ca(2+)，其增强与炎症有关。ASM 细胞不同部位Ca(2+)的差异调节可能有助于局部调节细胞内来源与质膜的 Ca(2+)流，并对这些部位的差异能量需求做出反应。我们提出，这种差异线粒体调节及其破坏可能在哮喘等疾病中气道高反应性中发挥作用，其中Ca(2+)增加。
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