Depletion of intracellular Ca²⁺ stores sensitizes the flow-induced Ca²⁺ influx in rat endothelial cells

Hemodynamic shear stress elicits a rise in endothelial [Ca²⁺]_i, which may serve as a key second messenger to regulate many flow-associated physiological and biochemical processes. In the present study, we used Mn²⁺ quenching of fluorescent dye Fluo3 as an assay to investigate the Ca²⁺ influx of rat aortic endothelial cells in response to flow. We found that the Ca²⁺ signaling in response to flow could be greatly influenced by the status of intracellular Ca²⁺ stores. Depletion of intracellular Ca²⁺ stores by thapsigargin (4 μmol/L) or cyclopiazonic acid (10 μmol/L) drastically sensitized the Ca²⁺ influx in response to flow. Ca²⁺-mobilizing agonist bradykinin (100 nmol/L) or ATP (100 μmol/L) had similar sensitizing effect. The effect of bradykinin or ATP was blocked by Xestospongin C and U73122, suggesting that the sensitization was related to the IP₃-mediated store depletion. On the other hand, the Mn²⁺ quenching in response to flow was greatly reduced by ochratoxin A (100 nmol/L), an agent that could increase the filling state of intracellular Ca²⁺ stores. In addition, we found that depletion-sensitized Ca²⁺ influx in response to flow was mediated by a PKG-inhibitable cation channel and that the influx was affected by membrane potential and K⁺ channel activity. In conclusion, the present study argues for a critical role of intracellular Ca²⁺ status in determining the Ca²⁺ signaling in response to flow and it provides a general mechanistic explanation for the stimulatory role of blood-borne agonists on flow-induced Ca²⁺ influx.