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Functional Relevance of Golgi and Plasma Membrane Localized Endothelial Nitric Oxide Synthase (eNOS) in Reconstituted Endothelial cells
Author(s) -
Zhang Qian,
Church Jarrod E,
Jagnandan Davin,
Catravas John D,
Sessa William C,
Fulton David
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a721-a
Subject(s) - enos , golgi apparatus , nitric oxide synthase type iii , nitric oxide , chemistry , microbiology and biotechnology , nitric oxide synthase , medicine , biochemistry , endocrinology , biology , endoplasmic reticulum
Endothelial nitric oxide synthase (eNOS) is regulated by post‐translational modifications which target eNOS to the plasma membrane (PM) and the perinuclear/Golgi region. It has been shown in COS‐7 cells that targeting of eNOS to the Golgi or PM regulates the mechanism and degree of eNOS activation. However, little is known about the functional significance of eNOS targeting in endothelial cells (EC). Our goal was to isolate these two pools of enzyme in EC and determine their functional significance in response to agonist stimulation and manipulation of membrane cholesterol levels. Using an RNAi strategy, we generated stable populations of EC that had greater than 90% inhibition of eNOS expression and lacked the ability to produce NO. Reconstitution of these eNOS “knockdown” EC with Golgi and PM targeted eNOS restored the ability of EC to produce NO. Calcium‐dependent agonists were the more efficient stimulus for the PM‐restricted eNOS in EC. In contrast, Golgi eNOS was less responsive to both calcium and Akt‐dependent agonists. eNOS restricted to the PM was more sensitive to manipulation of membrane cholesterol levels and was significantly attenuated by modified LDL. Methyl‐β‐cyclodextrinβ(CD) significantly reduced and CD‐cholesterol complexes significantly increased NO release from PM eNOS, but not Golgi eNOS. Similarly, modified LDL substantially reduced the activity of PM eNOS but not Golgi eNOS (HL74279).