Ph.D. Thesis Defense - Role of Transport Dependent Calcium Signaling in Nitric Oxide Production and Endothelial Shear Stress Responses
Date: August 10, 2007
Time: 10:30 AM
Location: Bossone Research Enterprise Center, Room: 702
Speaker(s):
Dihui Hong, M.S.
Advisors: Kenneth Barbee, Ph.D., Donald Buerk, Ph.D., and Dov Jaron, Ph.D.
Details:
Endothelial cells (ECs) are subject to the shear stress generated by blood flowing past their apical surfaces. Changes in fluid shear stress could be sensed directly by cells and elicit a cascade of responses which include the elevation of intracellular Ca2+ concentration ([Ca2+]i) and production of nitric oxide (NO). This study 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 number and size of the responding groups of cells did not depend on the magnitude of shear stress, nor did the magnitude of the calcium change in the responding 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. The number of responding cells was dependent on ATP concentration, but the magnitude of the calcium change was not. Our data suggest that shear stress stimulates RAMECs to release ATP, causing the increase in intracellular calcium concentration via purinergic receptors in cells that are heterogeneously sensitive to ATP. The propagation of the calcium signal is also mediated by ATP, and the spatial pattern suggests a locally elevated ATP concentration in the vicinity of the initially responding cells.
In BAECs, the onset of shear stress elicited a transient increase in intracellular calcium concentration that was spatially uniform, synchronous, and dose dependent. The amplitude of calcium response in BAECs was significantly suppressed under conditions in which either purinergic receptors were blocked by suramin or extracellular ATP was degraded by apyrase. When BAECs were perfused in PBS containing Ca2+, a step increase in shear stress from 0 to 20 dyn/cm2 elilicted a transient increase in [Ca2+]i attaining peak amplitude in 30 s, followed by a sustained plateau which decayed slowly to near baseline after 5 min. 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 (reflected by the change in relative DAF fluorescence with time (d(F/F0)/dt)) 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 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.
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