Ca2+ and electrolyte mobilization following agonist application to the pancreatic β cell line HIT

Abstract

We have investigated intracellular Ca2+ mobilization in oscillations of cytoplasmic Ca2+ in response to glucagon-like peptide 1 (GLP-1) and glucose in clonal HIT insulinoma cells with a confocal laser-scanning microscope (CLSM). We also used electron probe X-ray microanalysis to determine the GLP-1- and glucose-induced changes in electrolyte levels in the cytoplasm and insulin granules of the cells. GLP-1 produced 10- to 35-s duration oscillations in cytoplasmic Ca2+ concentration ([Ca2+]i), both with and without Ca2+ in the extracellular solution, suggesting that Ca2+ is mobilized from intracellular Ca2+ stores, namely secretory granules. Glucose caused 1- to 3-min duration oscillatory increases in [Ca2+]i when the extracellular solution contained Ca2+. When the cells were cultured without Ca2+ (no Ca2+ added, 1 mM EGTA), an oscillatory [Ca2+]i increase of amplitude and short duration (12-35 s) was produced by 11 mM glucose, and the oscillation was inhibited by ruthenium red. X-ray microanalysis showed that stimulation with glucose increased the total Ca concentration in the cytoplasm and decreased it in the insulin granules with and without Ca2+ in the extracellular solution. The application of glucose significantly decreased K, and increased Na and C1 in the cytoplasm when the extracellular solution contained Ca2+. Our result also suggests that the [Ca2+]i oscillation induced by glucose is involved in the release of Ca2+ from intracellular Ca2+ stores through the ryanodine receptor, which is blocked by ruthenium red, and/or through the inositol trisphosphate receptor that may be present in the membrane of insulin granules.

The Multiple Actions of GLP-1 on the Process of Glucose-Stimulated Insulin Secretion

“…3C) (62). Our studies suggest that GLP-1 application results in oscillations in [Ca 2ϩ ] i in HIT-T15 cells and that these oscillations are not abolished (although they are diminished in amplitude) by removal of extracellular Ca 2ϩ or by VDCC blockade (Fig.…”

Epac Activates the Small G Proteins Rap1 and Rab3A to Achieve Exocytosis

“…Interplay between calcium and cAMP in human sperm appears to be different because cAMP achieves exocytosis in the presence of high concentrations of EGTA, which chelates extracellular calcium, preventing an influx from the outside, but not of low concentrations of 1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid-acetoxymethyl ester, which chelates intracellular calcium, preventing mobilization from stores (8). Interestingly, cAMP-elevating agents such as GLP-1 stimulate the release of calcium stored in insulin-containing secretory granules (85). In this study we show that cAMP/Epac achieve intracellular calcium mobilization in human sperm via a pathway involving Rap1 and a PLC.…”

Involvement of the ryanodine-sensitive Ca 2+ store in GLP-1-induced Ca 2+ oscillations in insulin-secreting HIT cells

“…RyR is a large homotetrameric molecule that contains the calcium release channel of the sarcoplasmic reticulum, and Ca 2+ release is stimulated by ATP, cyclic ADP-ribose and caffeine, and is inhibited by Mg 2+ , H + , ruthenium red and calmodulin [27]. It was revealed that the existence of RY2 in the pancreatic B cells is involved in intracellular Ca 2+ mobilization [18,34], and we have reported that oscillatory [Ca 2+ ] i elevation induced by glucose in Ca 2+ -free medium is blocked by ruthenium red [33]. However, limited information is available for spatial and temporal resolution of repetitive [Ca 2+ ] i elevations evoked by GLP-1 in single cells of pancreatic B cells.…”

“…For the Ca 2+ -free experiments, we used a physiological salt solution where Ca 2+ was omitted and 2 mM EGTA was added. Glucose (11 mM) was added to the perfusate for the experiments to measure [Ca 2+ ] i changes [33]. Then, the cells in the dish were transferred to the stage of a confocal laser-scanning microscope (Leica Fluovert, Heidelberg, Germany and Zeiss LSM 510-16, Germany).…”