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Sustained endothelial nitric-oxide synthase activation requires capacitative Ca2+ entry.
1Cardiovascular Pulmonary Research Laboratory, Division of Pulmonary Sciences and Critical Care Medicine, Department of Pharmacology, Dallas, Texas, USA.
The Journal of Biological Chemistry
|June 13, 2000
Summary
Capacitative calcium entry, not intracellular release, primarily activates membrane-bound endothelial nitric-oxide synthase (eNOS). This finding clarifies how eNOS senses calcium signals for sustained NO production in vascular homeostasis.
Area of Science:
- Molecular Biology
- Cellular Physiology
- Biochemistry
Background:
- Endothelial nitric-oxide synthase (eNOS) is crucial for vascular homeostasis and is regulated by Ca(2+)/calmodulin.
- eNOS membrane association via N-myristoylation is known, but its role in proximity to Ca(2+) sources is unclear.
- Understanding eNOS localization and calcium sensing is vital for elucidating its activation mechanisms.
Purpose of the Study:
- To investigate the role of membrane association in eNOS activation by different calcium sources.
- To determine whether plasma membrane localization influences eNOS sensitivity to capacitative calcium entry versus intracellular calcium release.
- To identify the primary stimulus for sustained eNOS activation in intact cells.
Main Methods:
- Generated EHA (full-length eNOS-aequorin chimera) and MHA (myristoylation-deficient mutant) constructs.
- Transfected constructs into COS-7 cells to assess targeting and function.
- Measured eNOS activity and calcium sensitivity using aequorin luminescence and NO production assays.
Main Results:
- EHA localized to the plasma membrane, while MHA remained intracellular.
- Both EHA and MHA retained eNOS enzymatic activity and calcium sensitivity.
- Plasma membrane-localized EHA showed preferential sensitivity to capacitative calcium entry, whereas intracellular MHA responded more to intracellular calcium release. Sustained eNOS activation was significantly higher with capacitative calcium entry compared to ionomycin-induced calcium rise.
Conclusions:
- EHA and MHA chimeras are correctly targeted and retain eNOS and aequorin functions.
- Capacitative calcium influx is the principal stimulus for sustained activation of plasma membrane-associated eNOS.
- Membrane association directs eNOS to specific calcium signaling microdomains, influencing its activation.