Sustained endothelial nitric-oxide synthase activation requires capacitative Ca²⁺ entry

Endothelial nitric-oxide synthase (eNOS), a Ca²⁺/calmodulin-dependent enzyme, is critical for vascular homeostasis. While eNOS is membrane- associated through its N-myristoylation, the significance of membrane association in locating eNOS near sources of Ca²⁺ entry is uncertain. To assess the Ca²⁺ source required for eNOS activation, chimera containing the full-length eNOS cDNA and HA-tagged aequorin sequence (EHA), and MHA (myristoylation-deficient EHA) were generated and transfected into COS-7 cells. The EHA chimera was primarily targeted to the plasma membrane while MHA was located intracellularly. Both constructs retained enzymatic eNOS activity and aequorin-mediated Ca²⁺ sensitivity. The plasma membrane- associated EHA and intracellular MHA were compared in their ability to sense changes in local Ca²⁺ concentration, demonstrating preferential sensitivity to Ca²⁺ originating from intracellular pools (MHA) or from capacitative Ca²⁺ entry (EHA). Measurements of eNOS activation in intact cells revealed that the eNOS enzymatic activity of EHA was more sensitive to Ca²⁺ influx via capacitative Ca²⁺ entry than intracellular release, whereas MHA eNOS activity was more responsive to intracellular Ca²⁺ release. When eNOS activation by CCE was compared with that generated by an equal rise in [Ca²⁺](i) due to the Ca²⁺ ionophore ionomycin, a 10-fold greater increase in NO production was found in the former condition. These results demonstrate that EHA and MHA chimera are properly targeted and retain full functions of eNOS and aequorin, and that capacitative Ca²⁺ influx is the principle stimulus for sustained activation of eNOS on the plasma membrane in intact cells.