Effect of secretagogues on cytosolic free Ca2+ and insulin release at different extracellular Ca2+ concentrations in the hamster clonal beta-cell line HIT-T15.
We have examined the relationship between extracellular Ca2+, cytosolic free Ca2+ and insulin release in the clonal beta-cell line HIT-T15. Glucose-stimulated insulin release was dependent on the extracellular Ca2+ concentration in a dose-related manner; the threshold medium Ca2+ concentration for glucose-stimulated insulin release was 0.5 mM. Both forskolin and 12-O-tetradecanoylphorbol 13-acetate (TPA) increased insulin release in the presence of glucose at all extracellular Ca2+ concentration tested (0.1-2.5 mM) but not in the absence of Ca2+. Thus, the threshold medium Ca2+ concentration for glucose-stimulated insulin release was reduced to 0.1 mM by forskolin or TPA. Step-wise increases in the medium Ca2+ concentration in the presence of an initiator of insulin release resulted in a dose-related increase in cytosolic free Ca2+. In the presence of 10 mM glucose, cytosolic free Ca2+ in HIT cells was increased from 60 +/- 5 nM in Ca2+-free medium to 290 +/- 46 nM in medium containing 2.5 mM Ca2+. The effects of increasing extracellular Ca2+ in the presence of 40 mM K+ were similar but considerably more pronounced. Inclusion of either TPA or forskolin in the incubation medium had no significant effect on the steady-state cytosolic free Ca2+ levels in the absence of glucose but in the presence of 10 mM glucose forskolin caused modest (11-18%) increases in steady-state cytosolic free Ca2+ levels at extracellular Ca2+ concentrations of 0.25 mM or above. In contrast, in the presence of glucose TPA significantly reduced the steady-state levels of cytosolic free Ca2+ by 17-21% at extracellular Ca2+ concentrations of 0.25 mM or above. These data provide further evidence that insulin release mediated by activation of beta-cell protein kinases involves primarily an increase in sensitivity of the secretory system to intracellular Ca2+.
Hughes
S J
SJ
Nuffield Department of Clinical Biochemistry, John Radcliffe Hospital, Headington, Oxford, U.K.
Chalk
J G
JG
Ashcroft
S J
SJ
D000634:Aminoquinolines; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D005456:Fluorescent Dyes; D005947:Glucose / Q000502:physiology; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism*; D011188:Potassium / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000634:Aminoquinolines; D005456:Fluorescent Dyes; D007328:Insulin; D005576:Colforsin; D005947:Glucose; D013755:Tetradecanoylphorbol Acetate; C035739:Quin2; D011188:Potassium; D002118:Calcium