Ca2+-induced Ca2+ release from the endoplasmic reticulum amplifies the Ca2+ signal mediated by activation of voltage-gated L-type Ca2+ channels in pancreatic β-cells

Stimulus-secretion coupling in pancreatic beta -cells involves membrane depolarization and Ca2+ entry through voltage-gated L-type Ca2+ channels, which is one determinant of increases in the cytoplasmic free Ca2+ concentration ([Ca2+](i)). We investigated how the endoplasmic reticulum (ER)-associated Ca2+ apparatus further modifies this Ca2+ signal. When fura-2-loaded mouse beta -cells were depolarized by KCI in the presence of 3 mM glucose, [Ca2+](i) increased to a peak in two phases. The second phase of the [Ca2+](i) increase was abolished when ER Ca2+ stores were depleted by thapsigargin, The steady-state [Ca2+]i measured at 300 s of depolarization was higher in control cells compared with cells in which the ER Ca2+ pools were depleted. The amount of Ca2+ presented to the cytoplasm during depolarization as estimated from the integral of the increment in [Ca2+](i) over time (integral Delta [Ca2+](i)dt) was similar to 30% higher compared with that in the Ca pool-depleted cells. neothapsigargin, an inactive analog, did not affect [Ca2+](i) response. Using Sr2+ in the extracellular medium and exploiting the differences in the fluorescence properties of Ca2+- and Sr2+-bound fluo-3, we found that the incoming Sr2+ triggered Ca2+ release from the ER, Depolarization-induced [Ca2+](i) response was not altered by U73122, an inhibitor of phosphatidylinositol-specific phospholipase C, suggesting that stimulation of the enzyme by Ca2+ is not essential for amplification of Ca2+ signaling, [Ca2+](i) response was enhanced when cells were depolarized in the presence of 3 mM glucose, forskolin, and caffeine, suggesting involvement of ryanodine receptors in the amplification process. Pretreatment with ryanodine (100 muM) diminished the second phase of the depolarization-induced increase in [Ca2+](i). We conclude that Ca2+ entry through L-type voltage-gated Ca2+ channels triggers Ca2+ release from the ER and that such a process amplifies depolarization-induced Ca2+ signaling in beta -cells.