Functional relevance of Golgi- and plasma membrane-localized endothelial NO synthase in reconstituted endothelial cells.
OBJECTIVE
We have previously shown in COS-7 cells that targeting of endothelial NO synthase (eNOS) to the Golgi or plasma membrane (PM) regulates the mechanism and degree of eNOS activation. However, little is known about the functional significance of eNOS targeting in endothelial cells (ECs). The goal of the current study was to isolate these 2 pools of enzyme in ECs and determine their functional significance in response to agonist stimulation and manipulation of membrane cholesterol levels.


RESULTS
Using an RNA interference strategy, we generated stable populations of ECs that had >90% inhibition of eNOS expression and lacked the ability to produce NO. Reconstitution of these eNOS "knockdown" ECs with Golgi- and PM-targeted eNOS restored the ability of ECs to produce NO. Calcium-dependent agonists were the more efficient stimulus for the PM-restricted eNOS in ECs. 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 low-density lipoprotein.


CONCLUSIONS
Within ECs, the PM is the most efficient location to produce NO but is more vulnerable to cholesterol levels and modified low-density lipoprotein.
Zhang
Qian
Q
Vascular Biology Center, Department of Pharmacology, Medical College of Georgia, Augusta, GA 30912, USA.
Church
Jarrod E
JE
Jagnandan
Davin
D
Catravas
John D
JD
Sessa
William C
WC
Fulton
David
D
D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology*; D002522:Chlorocebus aethiops; D002784:Cholesterol / Q000494:pharmacology; D042783:Endothelial Cells / Q000201:enzymology* / Q000648:ultrastructure; D004789:Enzyme Activation; D006056:Golgi Apparatus / Q000201:enzymology*; D008077:Lipoproteins, LDL / Q000494:pharmacology; D009569:Nitric Oxide / Q000096:biosynthesis; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D034622:RNA Interference
D008077:Lipoproteins, LDL; C099252:oxidized low density lipoprotein; D009569:Nitric Oxide; D002784:Cholesterol; D052250:Nitric Oxide Synthase Type III; D002118:Calcium