Mobilization of Ca2+ stores in individual pancreatic beta-cells permeabilized or not with digitonin or alpha-toxin.
The concentration of free Ca2+ in the cytoplasm and organelles of individual mouse pancreatic beta-cells was estimated with dual wavelength microfluorometry and the indicators Fura-2 and furaptra. Measuring the increase of cytoplasmic Ca2+ resulting from intracellular mobilization of the ion in ob/ob mouse beta-cells, most organelle calcium (92%) was found in acidic compartments released when combining the Ca2+ ionophore Br-A23187 with a protonophore. Only 3-4% of organelle calcium was recovered from a pool sensitive to the Ca(2+)-ATPase inhibitor thapsigargin. Organelle Ca2+ was also measured directly in furaptra-loaded beta-cells after controlled plasma membrane permeabilization. The permeabilizing agent alpha-toxin was superior to digitonin in preserving the integrity of intracellular membranes, but digitonin provided more reproducible access to intracellular sites. After permeabilization, the thapsigargin-sensitive fraction of Ca2+ detected by furaptra was as high as 90%, suggesting that the indicator essentially measures Ca2+ in endoplasmic reticulum (ER). Both alpha-toxin- and digitonin-permeabilized cells exhibited ATP-dependent uptake of Ca2+ into thapsigargin-sensitive stores with half-maximal and maximal filling at 6-11 microM and 1 mM ATP respectively. Most of the thapsigargin-sensitive Ca2+ was mobilized by inositol 1,4,5-trisphosphate (IP3), whereas caffeine, ryanodine, cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate lacked effects both in beta-cells from ob/ob mice and normal NMRI mice. Mobilization of organelle Ca2+ by 4-chloro-3-methylphenol was attributed to interference with the integrity of the ER rather than to activation of ryanodine receptors. The observations emphasize the importance of IP3 for Ca2+ mobilization in pancreatic beta-cells, but question a role for ryanodine receptor agonists.
Tengholm
A
A
Department of Medical Cell Biology, Uppsala University, Sweden.
Hellman
B
B
Gylfe
E
E
D000246:Adenosine Diphosphate Ribose / Q000031:analogs & derivatives / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001427:Bacterial Toxins / Q000494:pharmacology*; D002110:Caffeine / Q000494:pharmacology; D000001:Calcimycin / Q000031:analogs & derivatives / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002451:Cell Compartmentation; D002463:Cell Membrane Permeability / Q000187:drug effects; D036563:Cyclic ADP-Ribose; D003593:Cytoplasm / Q000378:metabolism; D004072:Digitonin / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006460:Hemolysin Proteins / Q000494:pharmacology*; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007476:Ionophores / Q000494:pharmacology; D007515:Islets of Langerhans / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008820:Mice, Obese; D009249:NADP / Q000031:analogs & derivatives / Q000378:metabolism; D015388:Organelles / Q000378:metabolism; D012433:Ryanodine / Q000378:metabolism / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology
D001427:Bacterial Toxins; D004791:Enzyme Inhibitors; D006460:Hemolysin Proteins; D007476:Ionophores; D019837:Ryanodine Receptor Calcium Release Channel; C021091:staphylococcal alpha-toxin; D036563:Cyclic ADP-Ribose; D012433:Ryanodine; D000246:Adenosine Diphosphate Ribose; D000001:Calcimycin; D002110:Caffeine; D009249:NADP; C024376:NAADP; D019284:Thapsigargin; C033170:4-bromo-A-23187; D015544:Inositol 1,4,5-Trisphosphate; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D004072:Digitonin; D002118:Calcium