Sustained endothelial nitric-oxide synthase activation requires capacitative Ca2+ entry.
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 chimera are properly targeted and retain full functions of eNOS and aequorin, and that capacitative Ca(2+) influx is the principle stimulus for sustained activation of eNOS on the plasma membrane in intact cells.
Lin
S
S
Cardiovascular Pulmonary Research Laboratory, Division of Pulmonary Sciences and Critical Care Medicine, Department of Pharmacology, Dallas, Texas, USA.
Fagan
K A
KA
Li
K X
KX
Shaul
P W
PW
Cooper
D M
DM
Rodman
D M
DM
D000331:Aequorin; D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000201:enzymology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006632:Histamine / Q000494:pharmacology; D015759:Ionomycin; D008163:Luminescent Measurements; D019814:Myristic Acid / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D011994:Recombinant Proteins / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology; D014162:Transfection
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D019814:Myristic Acid; D009569:Nitric Oxide; D000331:Aequorin; D015759:Ionomycin; D019284:Thapsigargin; D006632:Histamine; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D002118:Calcium