Hypercholesterolemia is considered a major risk factor for the development of atherosclerosis. Early stage atherosclerosis is characterized by endothelial dysfunction, which is described by impaired or reduced availability of nitric oxide (NO). However, the mechanism by which cholesterol leads to decreased NO production remains unclear. NO is produced by the endothelium in response to agonist stimulation and shear stress, although the mechanisms of NO production have yet to be fully determined due to experimental difficulties in measuring NO, especially under flow conditions. In order to understand the effect of cholesterol enrichment on shear stress-induced NO production, we first designed a device to measure shear stress-induced NO directly and in real-time from endothelial cells in vitro. With this device we were able to establish a mechanism for shear stress-induced NO production through ATP autocrine signaling and activation of store operated calcium channels (SOCs) which are responsible for the influx of extracellular calcium following store depletion termed capacitative calcium entry (CCE). To further understand the mechanism of ATP stimulation and subsequent activation of CCE in NO production, we investigated ATP-induced eNOS phosphorylation in cells under static conditions. We found that the mechanism of ATP-induced eNOS phosphorylation is through a complex interplay between CCE and protein kinase C (PKC). In addition, our results suggest an important co-localization effect of SOCs and eNOS that is responsible for maximal activation of eNOS.

Hypercholesterolemia is considered a major risk factor for the development of atherosclerosis. Early stage atherosclerosis is characterized by endothelial dysfunction, which is described by impaired or reduced availability of nitric oxide (NO). However, the mechanism by which cholesterol leads to decreased NO production remains unclear. NO is produced by the endothelium in response to agonist stimulation and shear stress, although the mechanisms of NO production have yet to be fully determined due to experimental difficulties in measuring NO, especially under flow conditions. In order to understand the effect of cholesterol enrichment on shear stress-induced NO production, we first designed a device to measure shear stress-induced NO directly and in real-time from endothelial cells in vitro. With this device we were able to establish a mechanism for shear stress-induced NO production through ATP autocrine signaling and activation of store operated calcium channels (SOCs) which are responsible for the influx of extracellular calcium following store depletion termed capacitative calcium entry (CCE). To further understand the mechanism of ATP stimulation and subsequent activation of CCE in NO production, we investigated ATP-induced eNOS phosphorylation in cells under static conditions. We found that the mechanism of ATP-induced eNOS phosphorylation is through a complex interplay between CCE and protein kinase C (PKC). In addition, our results suggest an important co-localization effect of SOCs and eNOS that is responsible for maximal activation of eNOS.