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RESEARCH ARTICLE |
1 Department of Medical Cell Biology, Uppsala University, Biomedicum, Box 571, SE-751 23 Uppsala, Sweden
2 Department of Biophysics, National T. Shevchenko University of Kiev, Kiev, Ukraine
*Author for correspondence (e-mail: erik.gylfe{at}medcellbiol.uu.se)
Accepted March 5, 2001
The store-operated pathway for Ca2+ entry was studied in individual mouse pancreatic ß-cells by measuring the cytoplasmic concentrations of Ca2+ ([Ca2+]i) and Mn2+ ([Mn2+]i) with the fluorescent indicator fura-2. Influx through the store-operated pathway was initially shut off by pre-exposure to 20 mM glucose, which maximally stimulates intracellular Ca2+ sequestration. To avoid interference with voltage-dependent Ca2+ entry the cells were hyperpolarized with diazoxide and the channel blocker methoxyverapamil was present. Activation of the store-operated pathway in response to Ca2+ depletion of the endoplasmic reticulum was estimated from the sustained elevation of [Ca2+]i or from the rate of increase in [Mn2+]i due to influx of these extracellular ions. Increasing concentrations of the inositol 1,4,5-trisphosphate-generating agonist carbachol or the sarco(endo)plasmatic reticulum Ca2+-ATPase inhibitor cyclopiazonic acid (CPA) cause gradual activation of the store-operated pathway. In addition, the carbachol- and CPA-induced influx of Mn2+ depended on store filling in a graded manner. The store-operated influx of Ca2+/Mn2+ was inhibited by Gd3+ and 2-aminoethoxydiphenyl borate but neither of these agents discriminated between store-operated and voltage-dependent entry. The finely tuned regulation of the store-operated mechanisms in the ß-cell has direct implications for the control of membrane potential and insulin secretion.
Key words: Pancreatic ß-cell, Store-operated, Calcium channels, Insulin secretion
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