Regulation of organelle Ca²+ in individual pancreatic β-cells

Sammanfattning: The Ca2+ion plays a fundamental role in the control of a variety of cellular functions, including insulinsecretion from pancreatic β-cells. Whereas the concentration of free Ca2+ in the cytoplasm ([Ca2+]i) ismaintained at low levels (5O-100 nM), some organelles contain high concentrations of the ion. ThisCa2+ is important both for organelle function and for the regulation of [Ca2+]i. In the present study the concentration of Ca2+ in the cytoplasm and organelles of individual mouse pancreatic β-cells was estimated with dual-wavelength microfluorometry and the Ca2+-sensitive indicators fura-2 and furaptra. Measuring the increase of [Ca2+]i resulting from intracellular mobilisation of the ion, mostorganelle Ca2+ (>90%) was found in acidic compartments released when combining the Ca2+ionophore Br-A23187 with a protonophore. Only 3-4% of the Ca2+ was recovered from a poolsensitive to inhibition of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA). Organelle Ca2+ was also measured in situ after the establishment of a new technique based on the incorporation of furaptra into β-cells followed by its removal from the cytoplasm by controlled plasma membranepermeabilisation with digitonin or α-toxin. The Ca2+ detected with furaptra was located essientially in the endoplasmic reticulum (ER), being sensitive to SERCA inhibition. Glucose was found to promote the accumulation of Ca2+ in the ER of intact β-cells by a high-affinity mechanism not requiring but accelerated by elevation of [Ca2+]i. In permeabilised β-cells exposed to 50-200 nM Ca2+ ATP caused rapid Ca2+ filling of the ER, reaching 200-500 μM. Half-maximal and maximal Ca2+ filling occurred at3.5-45 μM and 1 mM ATP, respectively. Whereas inositol 1,4,5-trisphosphate (IP3) was found to potently stimulate the mobilisation of Ca2+ from the ER, ryanodine receptor agonists and nicotinic acid adenine dinucleotide phosphate lacked such an effect. Preexposure to a high glucose concentrationresulted in increased maximal ATP-induced Ca2+ filling of the ER and in enhanced mobilisation ofCa2+ in response to IP3.