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

Endothelial nitric-oxide synthase (eNOS), a Ca2+/calmodulin-dependent enzym e, is critical for vascular homeostasis. While eNOS is membrane-associated through its N-myristoylation, the significance of membrane association in l ocating eNOS near sources of Ca2+ entry is uncertain. To assess the Ca2+ so urce required for eNOS activation, chimera containing the full-length eNOS cDNA and HA-tagged aequorin sequence (EHA), and MHA (myristoylation deficie nt EHA) were generated and transfected into COS-7 cells. The EHA chimera wa s primarily targeted to the plasma membrane while MHA was located intracell ularly. Both constructs retained enzymatic eNOS activity and aequorin-media ted Ca2+ sensitivity. The plasma membrane-associated EHA and intracellular MHA were compared in their ability to sense changes in local Ca2+ concentra tion, demonstrating preferential sensitivity to Ca2+ originating from intra cellular 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 i ntracellular release, whereas MHA eNOS activity was more responsive to intr acellular 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 condi tion. These results demonstrate that EHA and MHA chimera are properly targe ted and retain full functions of eNOS and aequorin, and that capacitative C a2+ influx is the principle stimulus for sustained activation of eNOS on th e plasma membrane in intact cells.