Ca2+-induced Ca2+ release in pancreatic islet beta-cells: critical evaluation of the use of endoplasmic reticulum-targeted "cameleons".
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.
Varadi
Aniko
A
Henry Wellcome Laboratories for Integrated Cell Signalling, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom.
Rutter
Guy A
GA
D000818:Animals; D002021:Buffers; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D045604:Extracellular Fluid / Q000378:metabolism; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D005947:Glucose / Q000494:pharmacology; D007202:Indicators and Reagents; D007425:Intracellular Membranes / Q000378:metabolism*; D007515:Islets of Langerhans / Q000378:metabolism*; D007552:Isotonic Solutions / Q000494:pharmacology; D008164:Luminescent Proteins; D009994:Osmolar Concentration; D011189:Potassium Chloride / Q000494:pharmacology; D011993:Recombinant Fusion Proteins
D002021:Buffers; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D007552:Isotonic Solutions; C492605:Krebs-Ringer HEPES bicarbonate solution; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C492603:Ycam-2 fluorescent protein; C492604:Ycam-4ER; D011189:Potassium Chloride; D005947:Glucose; D002118:Calcium