Dioctanoylglycerol regulation of cytosolic Ca2+ by protein kinase C-independent mechanism in HIT T-15 islet cells.
The effect of activators of protein kinase C (PKC) on cytosolic concentration of free Ca2+ [( Ca2+]i) was assessed in insulin-secreting islet cell line HIT T-15. Dioctanoylglycerol (DiC8) and 12-O-tetradecanoylphorbol-13-acetate (TPA) evoked activation of PKC. Basal [Ca2+]i was 65-160 nM. DiC8 induced triphasic increases in [Ca2+]i; phase 2 was the most prominent and consistent one. With 25-150 microM DiC8, [Ca2+]i increased in a dose-dependent manner during phase 2; half-maximal stimulatory dose was 53 microM. TPA did not evoke any increase in [Ca2+]i. Staurosporine, sphingosine, and H7, which are inhibitors of PKC, did not block DiC8-induced rise in [Ca2+]i. DiC8-induced rise in [Ca2+]i was also seen in cells that had been depleted of PKC by prior exposure to TPA. DiC8-induced rise in [Ca2+]i still occurred in the presence of the Ca(2+)-channel blocker verapamil or when the extracellular Ca2+ had been reduced from 2.5 mM to 30 nM by EGTA. Three immediate metabolites of DiC8, monooctanoylglycerol, octanoate, and glycerol, did not evoke any change in [Ca2+]i. Monooleoylglycerol and R59022, which induce increases in endogenous diacylglycerol (DAG) by inhibiting DAG kinase, evoked increases in [Ca2+]i. DiC8 did not cause any change in inositol 1,4,5-trisphosphate levels. DiC8 evoked biphasic increases in insulin release; the second-phase increase in [Ca2+]i preceded the late phase of insulin secretion. Exogenous DAGs should be used with caution in assessing PKC function. Changes in the generation in DAGs must be included among the mechanisms by which Ca2+ homeostasis is regulated in islet cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Thomas
T P
TP
Department of Internal Medicine, University of Michigan, Ann Arbor.
Martin
D B
DB
Pek
S B
SB
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D006224:Cricetinae; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004075:Diglycerides / Q000494:pharmacology*; D000431:Ethanol / Q000494:pharmacology; D007515:Islets of Langerhans; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D008647:Mesocricetus; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013110:Sphingosine / Q000494:pharmacology; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014700:Verapamil / Q000494:pharmacology
D000470:Alkaloids; D004075:Diglycerides; D007546:Isoquinolines; D010879:Piperazines; C044295:1,2-dioctanoylglycerol; D000431:Ethanol; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D014700:Verapamil; D011493:Protein Kinase C; D019311:Staurosporine; D013110:Sphingosine; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium