Regulation of Ca²⁺ movements by cyclovirobuxine D in ECV304 endothelial cells

In Fura-2/loaded ECV304 endothelial cells cyclovirobuxine D promoted a transient increase in cytosolic free Ca²⁺ originating from both an intracellular pool sensitive to the endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin and the extracellular space. The intracellular pool was apparently different from that mobilized by other agents acting through IP ₃ generation. The integrity of the plasma membrane was an absolute requirement. In cells treated with digitonin, cyclovirobuxine D did not promote any Ca²⁺ release from the intracellular stores even at high concentrations and in the absence or presence of thapsigargin or sodium azide, the inhibitors of the endoplasmic reticular or mithocondrial Ca²⁺ uptake. Furthermore, cyclovirobuxine D was effective in halting the persistent increase in cytosolic Ca²⁺ caused by thapsigargin, inhibiting the operation of the "capacitative" Ca²⁺ membrane channels as demonstrated by the decrease in the extent of both Ca²⁺-overshoot and Mn²⁺ influx. Additional effects of cyclovirobuxine D included a depolarization of plasma membrane apparently related to an enhanced influx of Na⁺ from the extracellular space. The results obtained indicate that cyclovirobuxine D markedly affects intracellular Ca²⁺ homeostasis in ECV304 endothelial cells by both promoting a discharge of intracellular pools and by interfering with the operation of store-dependent channels via plasma membrane depolarization.