Effect of secretagogues on cytosolic free Ca2+ and insulin release in the hamster clonal beta-cell line HIT-T15.
We have examined the effect of secretagogues on cytosolic free Ca2+ (Cai) in the hamster clonal beta-cell line HIT-T15 using the Ca2+-binding fluorescent indicator Quin 2. Stimulation of HIT cells by glucose increased Cai in a dose-dependent manner; raising the medium glucose concentration from zero to 2 mM increased Cai by 36%, from 89 +/- 4 to 121 +/- 6 nM (mean +/- S.E.M., n = 23). Further raising the medium glucose concentration to 10 mM increased Cai to 139 +/- 6 nM. Cai was maximum and plateaued at 4 min after each addition of glucose. Addition of 40 mM K+ to the medium rapidly depolarized the HIT cells and increased Cai to 407 +/- 48 nM. The increases in Cai in response to glucose of K+ were blocked by the simultaneous presence of verapamil (50 microM). Stimulation by glucose or K+ also increased insulin release in parallel incubations of Quin 2-loaded HIT cells. Carbamylcholine chloride, forskolin or the phorbol ester 12-O-tetradecanoylphorbol acetate had no significant effect on Cai in glucose-stimulated HIT cells monitored 5 min after the addition of each test agent, despite increasing insulin release by 241, 239 and 216% respectively. These data support the hypothesis that potentiators of insulin release which activate cAMP-dependent protein kinase or protein kinase C do not increase Cai but sensitize the secretory mechanism to Ca2+.
Hughes
S J
SJ
Nuffield Department of Clinical Biochemistry, John Radcliffe Hospital, Headington, Oxford.
Ashcroft
S J
SJ
D000634:Aminoquinolines / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D002478:Cells, Cultured; D002999:Clone Cells; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology*; D007328:Insulin / Q000378:metabolism*; D007515:Islets of Langerhans / Q000166:cytology / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014700:Verapamil / Q000494:pharmacology
D000634:Aminoquinolines; D005456:Fluorescent Dyes; D007328:Insulin; D005576:Colforsin; D002217:Carbachol; D014700:Verapamil; D005947:Glucose; D013755:Tetradecanoylphorbol Acetate; C035739:Quin2; D011188:Potassium; D002118:Calcium