The endoplasmic reticulum is a glucose-modulated high-affinity sink for Ca2+ in mouse pancreatic beta-cells.
The regulation of organelle free Ca2+ was analysed in individual mouse pancreatic beta-cells loaded with the fluorescent low-affinity indicator furaptra. Removal of the cytoplasmic indicator by controlled digitonin permeabilization of the plasma membrane resulted in a sudden increase of the 340 nm/380 nm fluorescence excitation ratio followed by a gradual decay, reflecting the emptying of Ca2+ from organelle pools. Subsequent introduction of 3 mM ATP caused rapid refilling of a Ca2+ pool, which represented the endoplasmic reticulum (ER) in being mobilized with inositol 1,4,5-trisphosphate (IP3) and the sarco(endo)plasmic reticulum Ca2+-ATPase inhibitor thapsigargin. The concentration of Ca2+ in the ER observed immediately after permeabilization depended on the glucose concentration in a hyperbolic fashion with half-maximal filling at about 6 mM of the sugar. Glucose promotion of Ca2+ sequestration in the ER involved a high-affinity mechanism not requiring but accelerated by a rise of the cytoplasmic Ca2+ concentration. Glucose also exerted a long-term action on the ER storage of Ca2+, maintaining the set-point for its maximal concentration and preserving the response to IP3. The results indicate that the ER has an important role in the glucose-stimulated beta-cell by serving as a high-affinity sink for Ca2+, irrespective of the prevailing concentration of cytoplasmic Ca2+.
Tengholm
A
A
Department of Medical Cell Biology, Uppsala University, Biomedical Centre, Box 571, SE-751 23 Uppsala, Sweden.
Hellman
B
B
Gylfe
E
E
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000502:physiology*; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives*; D005947:Glucose / Q000494:pharmacology*; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D007515:Islets of Langerhans / Q000502:physiology*; D007700:Kinetics; D051379:Mice; D008820:Mice, Obese; D015388:Organelles / Q000187:drug effects / Q000502:physiology; D019284:Thapsigargin / Q000494:pharmacology
D005456:Fluorescent Dyes; C058776:2-(2-(5-carboxy)oxazole)-5-hydroxy-6-aminobenzofuran-N,N,O-triacetic acid; D019284:Thapsigargin; D015544:Inositol 1,4,5-Trisphosphate; D000255:Adenosine Triphosphate; D005947:Glucose; D002118:Calcium; D016257:Fura-2