Regulation of Endothelial Nitric Oxide Synthase by Subcellular Localization and

Abstract

Endothelial nitric oxide synthase (eNOS) is regulated by post-translational modifications that 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 (ECs). Our first goal was to isolate these two pools of enzyme in ECs 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. This approach can be broadly applied to endothelial cells from a number of different species and from different vascular beds and should have broad utility. Using these cells we found that the PM is the optimal location within the cell to produce NO, but it is also the most vulnerable to changes in cholesterol and oxidized LDL. 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. The functional significance of the increased NO produced by the PM eNOS is reflected in the greater ability to elicit endothelium-dependent relaxation, greater suppression of vWF secretion, a key regulator of platelet aggregation, and inhibition of endothelial cell proliferation. Mechanistically, PM eNOS induces more nitrosylation of proteins such as NSF, but this is related to the amount of NO being produced, rather than its intracellular location. Increased superoxide formation in endothelial cells (ECs) has been identified as a causative factor in endothelial dysfunction by reducing nitric oxide (NO) bioavailability, uncoupling eNOS. A major source of intracellular superoxide is the NADPH oxidase (Nox) family of enzymes. In experiments to address the effect of superoxide on local eNOS activity, we found that Nox5 increased eNOS activity paradoxically in both cotransfected COS-7 cells and transduced bovine aortic ECs determined by chemiluminescence to measure the NO metabolite. Nox5 also activated eNOS in human aortic ECs as detected by a cGMP reporter assay that measured the release of biologically functional NO from cells in the presence of superoxide dismutase (SOD). To establish the functional significance of this observation in blood vessels, the endothelium of mouse aorta was tranduced with Nox5 or control adenoviruses. Nox5 potently inhibited Achinduced relaxation, potentiated contractile responses to phenylephrine. In precontracted blood vessels, acute exposure to SOD induced significant vascular relaxation in vessels exposed to Nox5 versus control and unmasked the ability of Nox5 to activate eNOS in blood vessel endothelium. These results are in contrast to a number of described mechanisms for eNOS inhibition and provide valuable clues that in complex diseases such as diabetes and hypertension that ROS production is not the sole cause of endothelial cell dysfunction.