Characterisation of store-operated calcium entry in a vascular endothelial cell line and impact on the production of nitric oxide

Store-operated calcium entry (SOCE) is a principal mechanism for extracellular calcium entry in non-excitable cell types, and is primarily facilitated by the calcium- release activated calcium (CRAC) channel; itself comprised of the pore-forming Orai-1 and calcium-sensing Stromal interaction molecule (STIM)-1 proteins. Depletion of endoplasmic reticulum (ER) calcium stores initiates STIM-1 translocation to defined ER-plasma membrane puncta, and subsequent Orai-STIM interaction and opening of Orai. The importance of this mechanism in calcium signalling in diverse tissue types is becoming increasingly clear. The vascular endothelium is a dynamic tissue, involved in the maintenance of vascular homeostasis and haemostasis. Many endothelium-derived bioactive agents, such as endothelin-1, prostaglandins, and the potent vasodilator nitric oxide (NO), are known to be produced via calcium- dependent mechanisms. However, the role of the CRAC channel in the vascular endothelium is poorly defined with little known about downstream targets of calcium influx through CRAC channels. The dysregulation of NO production by endothelial nitric oxide synthase (eNOS) is a major contributory factor in many vascular disease states, yet the calcium channel responsible for eNOS activation has yet to be identified. Within this thesis, I establish the endothelial cell line sEnd.1 as a new model system for studying CRAC channel signalling in the vascular endothelium, defining sEnd.1 SOCE as being CRAC channel-dependent. Inhibition of CRAC channels with an array of inhibitors, and knock-down of STIM-1, both reduced ATP- and TG-induced SOCE. The sEnd.1 model system was subsequently used to identify calcium entry through the CRAC channel as the elusive activation mechanism for eNOS. Through real-time imaging with the fluorescent NO dye DAF-2-DA, we established that NO production is non-linear, with a slow initial increase preceding a faster NO production phase. These kinetics, with a characteristic delay before fast production have, to our knowledge, not previously been reported. The time taken to reach the fast phase of NO production could be manipulated through changes in both local and bulk calcium rises, which indicated roles for both elements of calcium signalling in eNOS activation. eNOS regulation by calcium is complex, occurring not only through direct binding of calcium-calmodulin, but additionally through changing post-translational modifications, which in turn regulate the calcium-dependency of eNOS, such as phosphorylation of Ser1177. We propose that the delay in fast production of NO is due to the time taken to alter eNOS post-translational modifications, which thus remove inhibition on eNOS. Activation of CRAC channels increased phosphorylation of residue Ser1177 via calcium-calmodulin kinase II (CaMKII), with a similar time course to that required to reach the fast phase of NO production. Inhibition of CaMKII increased the time taken to reach fast activation. In conclusion this thesis presents a new model system for investigation of CRAC channel signalling in the endothelium. Furthermore, we clearly identify a critical endothelial pathway as being regulated by CRAC channels, by demonstrating the production of NO in response to both ATP and TG, which stimulate calcium entry through CRAC channels.