EFFECT OF EXTRACELLULAR ATP LEVEL ON FLOW-INDUCED CA++ RESPONSE IN CULTURED VASCULAR ENDOTHELIAL-CELLS

Cultured vascular endothelial cells loaded with the highly fluorescent Ca++-sensitive dye Fura-2 were exposed to the flow of a fluid containing various concentrations of ATP (0, 0.5, 1,5 μM) in an apparatus designed on the basis of fluid dynamics, and simultaneous changes in intracellular free Ca++ concentration were monitored by photometric fluorescence microscopy. The flow rate of the perfusate was altered from 0 to 6.3 to 22.8 to 39.0 cm/sec, inducing shear stress on the cell surface of 0, 2.9, 10.4, and 17.9 dynes/cm2, respectively. Although no significant change in intracellular Ca++ level was observed at ATP levels below 100 nM, at an ATP level of 500 nM, the intracellular Ca++ level increased together with an increase in the flow rate of the perfusate. At this level of ATP, the intracellular Ca++ levels at flow rates of 0, 6.3, 22.8, and 39.0 cm/sec were 44.8 ± 7.3, 60.3 ± 10.7, 74.0 ± 5.8 and 89.4 ± 6.4 nM (mean ± SD; n=8), respectively. At ATP levels over 1 μM, the flow-rate dependency of Ca++ response became less clear than that observed at the ATP level of 500 nM. These Ca++ responses to changes in flow rate disappeared when extracellular Ca++ was chelated by adding 2 mM of EGTA to the perfusate. These results suggest that the vascular endothelial cell has a mechanism that elevates the intracellular Ca++ level in accord with the flow rate at appropriate ATP concentrations, and that changes in intracellular Ca++ level under this mechanism seem to be chiefly caused by the influx of extracellular Ca++ into cells. © 1991.