Flow-induced changes in Ca2+ signaling of vascular endothelial cells: effect of shear stress and ATP

The effect of hemodynamic flow on apparent cytosolic free Ca2+ concentration ([Ca2+]i) of cultured bovine aortic endothelial cells was examined in the absence and presence of adenine nucleotides using microfluorimetric analysis of fura-2 fluorescence. In the absence of adenine nucleotides, flow-induced shear stress produced little change (less than 10 nM) in [Ca2+]i. Similar results were obtained using calf pulmonary artery and human umbilical vein endothelial cells. However, addition of the adenine nucleotides ATP, ADP, or AMP under flow conditions produced a transient peak increase in [Ca2+]i that was followed by a sustained elevation. The rank order of potency for the peak response was ADP greater than or equal to ATP much much greater than AMP. Adenosine was without effect on [Ca2+]i. Washout of ATP resulted in the immediate return of [Ca2+]i to basal values, indicating that the effect of ATP was rapidly reversible. Decreasing the flow rate to zero during the sustained phase also resulted in an immediate decrease of [Ca2+]i. Similar results were obtained with ADP and AMP but not with the nonhydrolyzable adenine nucleotide analogues alpha,beta-methyleneadenosine-5'-diphosphate, beta,gamma-imidoadenosine-5'-triphosphate, or beta,gamma-methyleneadenosine-5'-triphosphate. Furthermore, the rate of [Ca2+]i decrease upon cessation of flow during the sustained phase of the response to ATP was inversely proportional to the ATP concentration. These results suggest that hydrolysis of ATP to adenosine by the ectonucleotidase is responsible for the termination of the ATP response under zero-flow conditions. Evaluation of the dose- and flow-dependent response of the cells to ATP indicates that convective-diffusive transport of ATP may play an important role in regulation of endothelial cell [Ca2+]i in presence of ectonucleotidase activity and could have important consequences for the regulation of blood flow in the vasculature.