Flow-induced calcium transients and release of endothelium-derived relaxing factor in cultured vascular endothelial...

Cytoplasmic Ca2+ response to flow and its relation to the release of endothelium-derived relaxing factor (EDRF) by endothelial cells were studied in vitro. Intracellular Ca2+ concentration ([Ca2+]i) and EDRF were measured using Fura-2 and photometric fluorescence microscopy, and a bioassay method using a rabbit aortic ring, respectively. Cultured endothelial cells were exposed to flow in Hanks' balanced salt solution containing various concentrations of ATP in a specially designed apparatus. At ATP levels over 100 nM, the application of flow to cells led to an apparent increase in [Ca2+]i and when flow stopped [Ca2+]i returned to the resting level. When the flow rate was increased stepwise, [Ca2+]i increased flow-rate dependently at around 500 nM ATP. At a constant flow rate, endothelial cells were then exposed to the perfusate, the ATP concentration of which was increased stepwise, and the simultaneous changes in [Ca2+]i and release of EDRF were measured. Both [Ca2+]i and EDRF increased as ATP concentrations increased, and there was a proportional relationship between the two. The range of the ATP-induced increase in [Ca2+]i was similar to that of the flow-rate dependent changes observed at 500 nM ATP. These results suggest that vascular endothelial cells have a mechanism by which changes in flow rate or shear stress are recognized at an appropriate concentration of extracellular ATP and the information is converted into changes in [Ca2+]i. Moreover, the flow-induced changes in [Ca2+]i may be involved in the regulation of EDRF release by endothelial cells.