Varadi Aniko, Rutter Guy A
Henry Wellcome Laboratories for Integrated Cell Signalling, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom.
Endocrinology. 2004 Oct;145(10):4540-9. doi: 10.1210/en.2004-0241. Epub 2004 Jun 24.
Elevated glucose concentrations cause Ca2+ influx and the exocytotic release of insulin from pancreatic islet beta-cells. Whether increases in cytosolic free Ca2+ concentration also mobilize Ca2+ from intracellular stores (Ca2+-induced Ca2+ release) is unresolved. Endoplasmic reticulum-targeted cameleons have previously been used to explore the involvement of endoplasmic reticulum (ER) Ca2+ release in these cells, albeit with differing conclusions. Cameleons comprise two spectrally shifted green fluorescent proteins, enhanced cyan and yellow fluorescent protein, whose orientation is affected by Ca2+, changing intramolecular fluorescence resonance energy transfer. By measuring pH in the cytosol and ER lumen, we demonstrate that high K+ concentrations (>20 mm) acidify both compartments in clonal MIN6 beta-cells when external bicarbonate concentrations are low (<5 mm), interfering with measurements using Ycam-2 and Ycam-4ER. However, when intracellular pH is strongly buffered (24 mm HCO3-), glucose or cell depolarization increases ER [Ca2+] monitored with Ycam-4ER. KCl-induced increases in ER [Ca2+] were diminished when intracellular stores were sensitized with 1 mm caffeine and inhibited by pretreatment with ryanodine. Furthermore, preincubation with ryanodine tended to slow the falling phase of the ER Ca2+ transient after cell depolarization with KCl and reduced the peak cytosolic [Ca2+]. By contrast, stimulation with glucose increased ER [Ca2+] both in the absence and presence of caffeine or ryanodine. These observations suggest that Ca2+-induced ER Ca2+ release can occur in beta-cells under some conditions but may not be essential for glucose-stimulated insulin secretion.
血糖浓度升高会导致钙离子内流，并促使胰岛β细胞通过胞吐作用释放胰岛素。胞质游离钙离子浓度的升高是否也会从细胞内储存库动员钙离子（钙离子诱导的钙离子释放）尚不清楚。此前，内质网靶向的钙指示剂已被用于探究内质网（ER）钙离子释放在这些细胞中的作用，尽管得出了不同的结论。钙指示剂由两种光谱发生偏移的绿色荧光蛋白组成，即增强型青色荧光蛋白和黄色荧光蛋白，其方向受钙离子影响，会改变分子内荧光共振能量转移。通过测量细胞质和内质网腔中的pH值，我们发现，当外部碳酸氢盐浓度较低（<5 mM）时，高钾浓度（>20 mM）会使克隆的MIN6β细胞中的这两个区室酸化，从而干扰使用Ycam-2和Ycam-4ER进行的测量。然而，当细胞内pH值被强烈缓冲（24 mM HCO3-）时，葡萄糖或细胞去极化会增加用Ycam-4ER监测到的内质网[Ca2+]。当用1 mM咖啡因使细胞内储存库致敏时，氯化钾诱导的内质网[Ca2+]增加会减弱，并被ryanodine预处理所抑制。此外，用ryanodine预孵育往往会减缓用氯化钾使细胞去极化后内质网钙离子瞬变的下降阶段，并降低细胞质[Ca2+]峰值。相比之下，无论是否存在咖啡因或ryanodine，葡萄糖刺激都会增加内质网[Ca2+]。这些观察结果表明，在某些条件下，钙离子诱导的内质网钙离子释放可能会在β细胞中发生，但对于葡萄糖刺激的胰岛素分泌可能并非必不可少。
