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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:1015.)
© 2006 American Heart Association, Inc.

Vascular Biology

Functional Relevance of Golgi- and Plasma Membrane-Localized Endothelial NO Synthase in Reconstituted Endothelial Cells

Qian Zhang; Jarrod E. Church; Davin Jagnandan; John D. Catravas; William C. Sessa; David Fulton

From the Vascular Biology Center and Department of Pharmacology (Q.Z., J.E.C., D.J., J.D.C., D.F.), Medical College of Georgia, Augusta; and Department of Pharmacology and Molecular Cardiobiology Division (W.C.S.), Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Conn.

Correspondence to David Fulton, PhD, Vascular Biology Center, Medical College of Georgia, 1459 Laney Walker Blvd, Augusta, GA 30912. E-mail dfulton{at}mcg.edu

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.

Methods and 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.

To determine the functional significance of subcellular targeting in endothelial cells, endogenous eNOS was depleted using virally delivered RNAi and reconstituted with Golgi- and PM-restricted eNOS. We found that the PM is the most efficient location to produce NO but is more vulnerable to cholesterol levels and modified LDL.

Key Words: eNOS • cholesterol • Golgi • plasma membrane • RNAi

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