Measurements of membrane potential, transmembrane 45Ca fluxes, cytoplasmic free Ca2+ concentration and insulin release by transplantable rat insulinoma cells maintained in tissue culture.
Regulation of insulin release, membrane potential, transmembrane 45Ca fluxes and cytoplasmic free Ca2+ concentration, [Ca2+]i, was examined using suspensions of transplantable NEDH rat insulinoma cells previously cultured for 2-3 days to eliminate necrotic tumour cells and counter prior hypoglycaemia. Insulinoma cells displayed a resting [Ca2+]i of 94 +/- 8 nM (n = 17) and released 104 +/- 15 ng insulin 10(-6) cells (n = 7) during 60 min incubations with uptake of 2.7 +/- 0.2 nmol 45Ca 10(-6) cells (n = 7). High concentrations of glucose did not affect membrane potential, transmembrane 45Ca fluxes, [Ca2+]i or insulin release by insulinoma cells. K+ at 25 mM depolarised the plasma membrane, induced a small increase in 45Ca efflux and increased [Ca2+]i by 65%. This modest action was not associated with demonstrable effects on 45Ca uptake and insulin release. The effect of 25 mMK+ on [Ca2+]i was counteracted by D-600, but this blocker of voltage-activated Ca2+ channels and verapamil lacked effects on transmembrane 45Ca fluxes and insulin release. The Ca2+-calmodulin antagonist, trifluoroperazine, was also without effect on 45Ca fluxes and insulin release. Ca2+ ionophore ionomycin increased [Ca2+]i, whereas A23187 and X537A did not affect transmembrane 45Ca fluxes. Moreover, insulin release was independent of extracellular Ca2+ over the range 0-20.4 mM despite marked affects on transmembrane 45Ca fluxes and a greater than 4-fold change of [Ca2+]i. Dibutyryl cyclic AMP increased insulin release by 55% without affecting transmembrane 45Ca fluxes or [Ca2+]i. The phosphodiesterase inhibitor, theophylline, also enhanced insulin release by 10-36% with no change of 45Ca uptake. The effectiveness of theophylline was independent of extracellular Ca2+ over the range 0-10.2 mM. These results indicate that inappropriate Ca2+ regulation is a key pathogenic feature underlying the inappropriate insulin secretion of rat insulinoma cells.
Flatt
P R
PR
Department of Biochemistry, University of Surrey, Guildford, UK.
Abrahamsson
H
H
Arkhammar
P
P
Berggren
P O
PO
Rorsman
P
P
Swanston-Flatt
S K
SK
D007516:Adenoma, Islet Cell / Q000378:metabolism*; D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism*; D007340:Insulinoma / Q000378:metabolism*; D007476:Ionophores / Q000494:pharmacology; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D010190:Pancreatic Neoplasms / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology; D051381:Rats; D013806:Theophylline / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000378:metabolism
D002121:Calcium Channel Blockers; D007328:Insulin; D007476:Ionophores; D003994:Bucladesine; D013806:Theophylline; D005947:Glucose; D011188:Potassium; D002118:Calcium