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 β-cells involves membrane depolarization and Ca²⁺ entry through voltage-gated L-type Ca ²⁺ 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 β-cells were depolarized by KCl 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 [Ca ²⁺l_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 [Ca ²⁺]_i over time (∫Δ[Ca²⁺] _i·dt) was ∼30% higher compared with that in the Ca ²⁺ 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 μM) diminished the second phase of the depolarization-induced increase in [Ca ²⁺]_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 Ca ²⁺ signaling in β-cells.