Role of transport dependent calcium signaling in nitric oxide production and endothelial shear stress responses

In this study, I compared the calcium response to shear stress between the ECs from large vessels (Bovine Aortic Endothelial Cells: BAECs)and microvessels (Rat AdrenoMedullary Endothelial Cells: RAMECs); characterized the interplay between [Ca²⁺]i and endothelial nitric oxide synthase (eNOS) activity in BAECs; and developed a 2-D model of transport-dependent intracellular calcium signaling in endothelial cells to evaluate the effects of spatial colocalization of eNOS and capacitive calcium entry (CCE) channels in caveolae on eNOS activiation in response to shear stress and ATP. In RAMECs, the calcium response to the onset of shear stress was heterogeneous in time and space. Shear stress induced calcium waves that originated from one or several cells and propagated to neighboring cells. The initiation and the propagation of calcium waves in RAMECs were significantly suppressed under conditions in which either purinergic receptors were blocked by suramin or extracellular ATP was degraded by apyrase. Exogenously applied ATP produced similarly heterogeneous responses. In BAECs, the onset of shear stress elicited a transient increase in intracellular calcium concentration that was spatially uniform, synchronous, and dose dependent. When BAECs were perfused in PBS containing Ca²⁺, a step increase in shear stress elilicted a transient increase in [Ca²⁺]i, followed by a sustained plateau which decayed slowly to near baseline. Elimination of extracellular Ca²⁺ with EGTA did not affect the initial calcium peak, while the [Ca²⁺]i plateau was reduced by 20%. Despite the similarity in the calcium responses, nitric oxide (NO) production in the presence of extracellular calcium is more than twice that in the absence of extracellular calcium. Similar results were observed in BAECs in response to stimulation with ATP. To achieve a quantitative understanding the Ca²⁺ and NO signaling mechanism, we developed a mathematical model that incorporates the cell morphology as well as endothelial calcium signaling processes. The model predicts that spatial segregation of calcium channels in endothelial cells can create microdomains where calcium concentration differs significantly from the spatial average calcium concentration. This transport-dependent calcium signaling specificity effect is enhanced in ECs elongated by flow by increasing the spatial segregation of the caveolar signaling domains. Our simulation significantly advances the understanding of how Ca²⁺, despite its many potential actions, can mediate selective activation of signaling pathways. We show that diffusion limited calcium transport allows functional compartmentalization of signaling pathways based on the spatial arrangements of Ca²⁺ sources and targets.