Year-end Sale % OFF 
Abstract
In studying the regulation of insulin secretion by phorbol esters, we examined their effects on the cytosolic free Ca2+ concentration ([Ca2+]i), using the Ca2+ indicator fura-2 in the rat insulin-secreting beta-cell line RINm5F. [Ca2+]i was measured in parallel with the rate of insulin release. 50 nM 12-O-tetradecanoylphorbol-13-acetate (TPA), which may act via protein kinase C, stimulated insulin release and caused an increase in [Ca2+]i. Ca2+-free conditions eliminated the increase in [Ca2+]i and resulted in a reduced stimulation of insulin release by TPA. The Ca2+ channel blocker nitrendipine (300 nM) inhibited both the increase in [Ca2+]i and the increased rate of insulin secretion. Another phorbol ester, 4 beta-phorbol 12,13-didecanoate, which activates protein kinase C, also induced an increase in [Ca2+]i and in the rate of insulin release, while 4 alpha-phorbol 12,13-didecanoate, which fails to stimulate protein kinase C, was without effect. Further studies with bis-oxonol as an indicator of membrane potential showed that TPA depolarized the beta-cell plasma membrane. From these results, it is concluded that TPA depolarizes the plasma membrane, induces the opening of Ca2+ channels in the RINm5F beta-cell plasma membrane, increases [Ca2+]i, and results in insulin secretion. The action of TPA was next compared with that of a depolarizing concentration of KC1 (25 mM), which stimulates insulin secretion simply by opening Ca2+ channels. TPA consistently elicited less depolarization, a smaller rise of [Ca2+]i, but a greater release of insulin than KC1. Therefore an additional action of TPA is suggested, which potentiates the action of the elevated [Ca2+]i on insulin secretion.
Highlights
Phorbol Ester-stimulated Insulin Secretiobny RINm5F Insulinoma Cells Is Linked with MembraneDepolarization and an Increase in Cytosolic Free Ca2’ Concentration*
Phorbol esters, we examined their effects on the cyto- tetradecanoylphorbol-13-acetate(TPA), were shown to actisolic free Caa+concentration ([Ca2+]i),using the Ca2+ vate protein kinase C directly by substituting for diacylglycindicator fura-2 in the rat insulin-secreting @-celline erol [4, 7, 8]
RINmSF.[Ca2+]i was measured in parallel with the rate of insulin release. 50 nM 12-0-tetradecanoylphorbol-13-acetate (TPA), which may act via protein kinase C, stimulated insulin release and caused an increase in [Ca2+]i.Ca2+-freeconditions eliminated the increase in [Ca2+]iand resultedin a reduced stimulation of insulin release by TPA
Summary
Cellsof the insulin-secreting p-cell line RINm5F [30]were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 pg/ml streptomycin, and 100 units/ml penicillin at 37°C in a 95% air plus 5% COzatmosphere. The cell number/well was obtained by counting after the incubation, and the rate of insulin release was expressed as picograms of insulin/1O3cells/30 min. The single cells were suspended at 2 X 10' cells/ml in RPMI 1640 medium with 5% fetal bovine serum and 10 mM HEPES and maintained at room temperature prior to loading. For fura-2 loading the cell suspension was centrifuged, resuspended in KRB with 1p~ fura-2 acetoxymethyl ester at 6 x lo cells/ml, and incubated with continuous shaking at 37 "C for 30 min. The fura-2-loaded cells obtained were washed and resuspended in KRB at X lo6cells/ml, and 3 ml of the suspension was placed in each quartz cuvette. The dispersed single cells were suspended in RPMI 1640 mediumwith 5% fetal bovine serum and 10.
Sign-in/Register to view highlights & summary 
Published Version
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
