Mobilization of Ca2+ stores in individual pancreatic beta-cells permeabilized or not with digitonin or alpha-toxin

Cell Calcium. 2000 Jan;27(1):43-51. doi: 10.1054/ceca.1999.0087.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Diphosphate Ribose / analogs & derivatives
  • Adenosine Diphosphate Ribose / pharmacology
  • Adenosine Triphosphate / metabolism
  • Adenosine Triphosphate / pharmacology
  • Animals
  • Bacterial Toxins / pharmacology*
  • Caffeine / pharmacology
  • Calcimycin / analogs & derivatives
  • Calcimycin / pharmacology
  • Calcium / metabolism*
  • Calcium-Transporting ATPases / antagonists & inhibitors
  • Cell Compartmentation
  • Cell Membrane Permeability / drug effects
  • Cyclic ADP-Ribose
  • Cytoplasm / metabolism
  • Digitonin / pharmacology*
  • Enzyme Inhibitors / pharmacology
  • Hemolysin Proteins / pharmacology*
  • Inositol 1,4,5-Trisphosphate / metabolism
  • Ionophores / pharmacology
  • Islets of Langerhans / cytology
  • Islets of Langerhans / drug effects
  • Islets of Langerhans / metabolism*
  • Mice
  • Mice, Obese
  • NADP / analogs & derivatives
  • NADP / metabolism
  • Organelles / metabolism
  • Ryanodine / metabolism
  • Ryanodine / pharmacology
  • Ryanodine Receptor Calcium Release Channel / metabolism
  • Thapsigargin / pharmacology

Substances

  • Bacterial Toxins
  • Enzyme Inhibitors
  • Hemolysin Proteins
  • Ionophores
  • Ryanodine Receptor Calcium Release Channel
  • staphylococcal alpha-toxin
  • Cyclic ADP-Ribose
  • Ryanodine
  • Adenosine Diphosphate Ribose
  • Calcimycin
  • Caffeine
  • NADP
  • NAADP
  • Thapsigargin
  • 4-bromo-A-23187
  • Inositol 1,4,5-Trisphosphate
  • Adenosine Triphosphate
  • Calcium-Transporting ATPases
  • Digitonin
  • Calcium