Sustained Endothelial Nitric-oxide Synthase Activation Requires Capacitative Ca2+ Entry

作者：Sui Lin, Kent A. Fagan, Kai-Xun Li, Philip W. Shaul, Dermot M.F. Cooper, David M. Rodman · 发表于：Journal of Biological Chemistry · 年份：2000 · DOI：10.1074/jbc.275.24.17979 · 被引用次数：173 · 研究领域：Nitric Oxide and Endothelin Effects、Receptor Mechanisms and Signaling、Renin-Angiotensin System Studies

Endothelial nitric-oxide synthase (eNOS), a Ca(2+)/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 Ca(2+) entry is uncertain. To assess the Ca(2+) 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 Ca(2+) sensitivity. The plasma membrane-associated EHA and intracellular MHA were compared in their ability to sense changes in local Ca(2+) concentration, demonstrating preferential sensitivity to Ca(2+) originating from intracellular pools (MHA) or from capacitative Ca(2+) entry (EHA). Measurements of eNOS activation in intact cells revealed that the eNOS enzymatic activity of EHA was more sensitive to Ca(2+) influx via capacitative Ca(2+) entry than intracellular release, whereas MHA eNOS activity was more responsive to intracellular Ca(2+) release. When eNOS activation by CCE was compared with that generated by an equal rise in [Ca(2+)](i) due to the Ca(2+) ionophore ionomycin, a 10-fold greater increase in NO production was found in the former condition. These results demonstrate that EHA and MHA ch...