Ca²⁺ influx does more than provide releasable Ca²⁺ to maintain repetitive spiking in human umbilical vein endothelial cells

We investigated why oscillations of intracellular Ca²⁺ concentrations ([Ca²⁺]_i) in endothelial cells challenged by submaximal histamine run down in Ca²⁺-free medium despite stores retaining most of their Ca²⁺. One explanation is that only a small subpopulation of the Ca²⁺ stores oscillate and are completely emptied of Ca²⁺. To investigate if influx refills an empty store subpopulation, we differentiated between cations entering the cell and those released from internal stores by using extracellular Sr²⁺ as a Ca²⁺ surrogate; we distinguished between [Sr²⁺]_i and [Ca²⁺]_i by using the larger effect of Sr²⁺ on fura 2 fluorescence at 360 nm (F₃₆₀). Ca²⁺ was still available for release when oscillations had run down since oscillations promptly reappeared on addition of Sr²⁺_o and these were predominantly of Ca²⁺ (indicated by F₃₆₀ changes). Also, totally depleting Ca²⁺ stores inhibited Sr²⁺-induced oscillations, suggesting that Sr²⁺ entry leads to Ca²⁺ release. In contrast, Ba²⁺_o was unable to stimulate oscillations. Finally, oscillations generated by photolytic release of inositol trisphosphate (IP₃) analogues were similarly sensitive to extracellular Ca²⁺ and Sr²⁺. We conclude that stores (or a subpopulation) are not completely depleted of Ca²⁺ when oscillations run down in Ca²⁺-free medium. Bivalent cation entry therefore maintains sensitivity to IP₃, possibly by maintaining luminal bivalent cation levels.