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

Stimulus-secretion coupling in pancreatic beta -cells involves membrane dep olarization and Ca2+ entry through voltage-gated L-type Ca2+ channels, whic h is one determinant of increases in the cytoplasmic free Ca2+ concentratio n ([Ca2+](i)). We investigated how the endoplasmic reticulum (ER)-associate d Ca2+ apparatus further modifies this Ca2+ signal. When fura-2-loaded mous e 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, T he steady-state [Ca2+]i measured at 300 s of depolarization was higher in c ontrol cells compared with cells in which the ER Ca2+ pools were depleted. The amount of Ca2+ presented to the cytoplasm during depolarization as esti mated from the integral of the increment in [Ca2+](i) over time (integral D elta [Ca2+](i)dt) was similar to 30% higher compared with that in the Ca po ol-depleted cells. neothapsigargin, an inactive analog, did not affect [Ca2 +](i) response. Using Sr2+ in the extracellular medium and exploiting the d ifferences in the fluorescence properties of Ca2+- and Sr2+-bound fluo-3, w e found that the incoming Sr2+ triggered Ca2+ release from the ER, Depolari zation-induced [Ca2+](i) response was not altered by U73122, an inhibitor o f phosphatidylinositol-specific phospholipase C, suggesting that stimulatio n of the enzyme by Ca2+ is not essential for amplification of Ca2+ signalin g, [Ca2+](i) response was enhanced when cells were depolarized in the prese nce of 3 mM glucose, forskolin, and caffeine, suggesting involvement of rya nodine receptors in the amplification process. Pretreatment with ryanodine (100 muM) diminished the second phase of the depolarization-induced increas e in [Ca2+](i). We conclude that Ca2+ entry through L-type voltage-gated Ca 2+ channels triggers Ca2+ release from the ER and that such a process ampli fies depolarization-induced Ca2+ signaling in beta -cells.