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Ca²⁺-Induced Ca²⁺ Release in Pancreatic Islet ß-Cells: Critical Evaluation of the Use of Endoplasmic Reticulum-Targeted "Cameleons"

Henry Wellcome Laboratories for Integrated Cell Signalling and Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom

Address all correspondence and requests for reprints to: Dr. G. A. Rutter, Henry Wellcome Laboratories for Integrated Cell Signalling and Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom. E-mail: g.a.rutter{at}bris.ac.uk.

Elevated glucose concentrations cause Ca²⁺ influx and the exocytotic release of insulin from pancreatic islet ß-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 ß-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 ß-cells under some conditions but may not be essential for glucose-stimulated insulin secretion.