Glucose-stimulated calcium dynamics in islets of Langerhans in acute mouse pancreas tissue slices.
Glucose-stimulated calcium dynamics in islets of Langerhans in acute mouse pancreas tissue slices.
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DOI：
10.1371/journal.pone.0054638
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Rupnik MS
中科院分区：
文献类型：
--
作者：
Stožer A;Dolenšek J;Rupnik MS
在胰岛内的内分泌细胞中，钙离子将细胞刺激与激素分泌偶联。自从现代荧光测定法出现以来，许多采用主要分离的小鼠胰岛的体外研究已经研究了各种促分泌素对细胞质钙的影响，主要是在胰岛素分泌β细胞中。由于技术限制，这些研究的见解本质上局限于最外层细胞的一个相当小的亚群。结果似乎还取决于各种因素，如培养条件和持续时间，并且并不总是容易与体内发现相一致。主要的争议在于钙振荡的类型、钙波的存在以及同步活动的水平。在这里，我们开始结合联合收割机的原位急性小鼠胰腺组织切片制备与非侵入性荧光钙标记和随后的共聚焦激光扫描显微镜，利用一种创新的方法，使许多细胞的反应表征从所有层的胰岛现有的争议，以揭示新的光。我们的实验可重复地表明，稳定的快速钙振荡持续高原，而不是缓慢的振荡作为主要类型的反应，在急性组织切片，钙波是同步振荡的机械基板。我们还发现了间接证据，即使是大幅度的钙信号是不够的，代谢活化是必要的，以确保细胞同步后，刺激葡萄糖。我们的新方法有助于解决现有的争议，并显示出帮助回答重要生理问题的潜力，使其成为可预见未来的首选方法之一。
In endocrine cells within islets of Langerhans calcium ions couple cell stimulation to hormone secretion. Since the advent of modern fluorimetry, numerous in vitro studies employing primarily isolated mouse islets have investigated the effects of various secretagogues on cytoplasmic calcium, predominantly in insulin-secreting beta cells. Due to technical limitations, insights of these studies are inherently limited to a rather small subpopulation of outermost cells. The results also seem to depend on various factors, like culture conditions and duration, and are not always easily reconcilable with findings in vivo. The main controversies regard the types of calcium oscillations, presence of calcium waves, and the level of synchronized activity. Here, we set out to combine the in situ acute mouse pancreas tissue slice preparation with noninvasive fluorescent calcium labeling and subsequent confocal laser scanning microscopy to shed new light on the existing controversies utilizing an innovative approach enabling the characterization of responses in many cells from all layers of islets. Our experiments reproducibly showed stable fast calcium oscillations on a sustained plateau rather than slow oscillations as the predominant type of response in acute tissue slices, and that calcium waves are the mechanistic substrate for synchronization of oscillations. We also found indirect evidence that even a large amplitude calcium signal was not sufficient and that metabolic activation was necessary to ensure cell synchronization upon stimulation with glucose. Our novel method helped resolve existing controversies and showed the potential to help answer important physiological questions, making it one of the methods of choice for the foreseeable future.
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