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Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:959-961
doi: 10.1161/01.ATV.0000217612.29550.8b
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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:959.)
© 2006 American Heart Association, Inc.

Editorials

Where Is Endothelial Nitric Oxide Synthase More Critical: Plasma Membrane or Golgi?

Zheng-Gen Jin

From the Cardiovascular Research Institute and Department of Medicine, University of Rochester School of Medicine, Rochester, NY.

Correspondence to Zheng-Gen Jin, Cardiovascular Research Institute and Department of Medicine, University of Rochester School of Medicine, 601 Elmwood Avenue, Box 679, Rochester, NY 14642. E-mail zheng-gen_jin{at}urmc.rochester.edu


Key Words: endothelial nitric oxide synthase • nitric oxide • endothelial cells • subcellular targeting • plasma membrane • Golgi • cholesterol

Vascular endothelium–derived nitric oxide (NO), originally identified as endothelium-derived relaxing factor,1–3 plays a pivotal role in regulation of vascular homeostasis.4 NO is a major regulator of vascular tone and blood pressure, and has multiple antiatherogenic roles including antiinflammatory, antithrombotic, antiproliferative, and antioxidant effects.4–6 Loss of the bioavailability of endothelium-derived NO is the hallmark of endothelial dysfunction and is implicated in the pathogenesis of cardiovascular disease such as hypertension and atherosclerosis.7,8 Therefore, it is of great interest to understand the molecular mechanisms regulating NO production by endothelium, which is likely to provide new insight into endothelial function in health and disease.

See page 1015

Endothelial NO synthase (eNOS) is a highly regulated, calcium (Ca2+)/calmodulin (CaM)-dependent enzyme responsible for the physiological production of NO in the vasculature.9,10 In endothelial cells (ECs), NO is formed from its precursor L-arginine via the enzymatic activation of eNOS with cofactors such as tetrahydrobiopterin (BH4). eNOS activation and subsequent NO production is stimulated by a variety of physical stimuli such as fluid shear stress generated by blood flow and by many humoral factors including acetylcholine, vascular endothelial growth factor (VEGF), bradykinin, estrogen, insulin, and angiopoietin.10–12 Increasing evidence suggest that eNOS is regulated by subcellular localization,9,13,14 posttranslational modifications such as phosphorylation at serine 1179 (S1179) by Akt,15–17 and interactions with several regulatory proteins such as heat shock protein 90 (HSP90) and caveolin-1.18,19 In particular, subcellular localization of eNOS is critical for optimal coupling of extracellular stimulation to NO production.10 In ECs, eNOS appears to localize at peripheral aspects of the Golgi complex and cholesterol-rich microdomains of the plasma membrane (PM) such as caveolae. Cotranslational N-myristoylation and posttranslational cysteine palmitoylation of eNOS determine its membrane targeting.9,13 It has been proposed that eNOS membrane localization may bring eNOS in close proximity to factors that are required for its proper function, such as L-arginine, BH4, CaM, and Akt.20 However, the functional significance of eNOS subcellular targeting in ECs at the physiological and pathological settings is still not clear.21

In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology, Zhang et al has demonstrated that the PM is the most efficient location for eNOS to produce NO in ECs.22 The conclusion is supported by the compelling data from a series of well-designed and executed experiments. First, the authors generated new eNOS "knockdown" ECs by using stably retrovirus-infected small interference RNA (siRNA) to decrease endogenous eNOS expression. Then the authors reconstituted these cells with wild-type (WT) eNOS or Golgi- and PM-targeted eNOS mutants using a silent eNOS construct, which is impervious to the siRNA while retaining the identical amino acid sequence of the protein. With these new ECs, the authors found that the PM-eNOS was much more sensitive to Ca2+ mobilizing agents such as thapsigargin and Akt-dependent agonists such as angiopoietin to produce NO compared with WT-eNOS and Golgi-eNOS. These results clearly indicate that within ECs the PM is the most efficient location for eNOS to produce NO in response to various agonists.

Several mechanisms may possibly account for the functional differences between the PM-eNOS and the Golgi-eNOS, such as phosphorylation state, interactions with regulatory proteins, and the availability of its substrate and cofactors (Figure). In this study Zhang et al found that the PM-eNOS was highly phosphorylated on S1179, S617, and S116 compared with the Golgi-eNOS in the basal condition. This finding is consistent with the previous report showing that eNOS mutants deficient in palmitoylation and myristoylation affect its subcellular localization, phosphorylation, and activity.23 Interestingly, the authors also observed that there was significantly more HSP90 (but not caveolin-1) bound to the PM-eNOS versus the Golgi-eNOS. One of the possible mechanisms to link the increase of HSP90 binding and hyperphosphorylation of eNOS is that HSP90 may increase Akt- or other protein kinase–mediated eNOS phosphorylation and facilitate CaM association with eNOS.24 Indeed, it has been shown that statin-induced phosphorylation of eNOS at S1179 is directly dependent on the ability of HSP90 to recruit Akt in the eNOS complex.25 Alternatively, HSP90 may protect eNOS from dephosphorylation by reducing the accessibility to protein phosphatases.26 Although binding of HSP90 to eNOS in response to VEGF and fluid shear stress increases eNOS activity by facilitating CaM-induced displacement of caveolin-1 from endogenous eNOS,27 it remains unclear whether there is any change in caveolin-1 interaction with the PM eNOS versus the Golgi-PM in response to agonists.


Figure 1
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  		Putative mechanisms for the functional difference of the PM- and the Golgi-eNOS. In endothelial cells, eNOS is mainly localized in caveolae, a cholesterol-enriched PM microdomains, and the Golgi complex, in which eNOS is associated with caveolin-1. Ca2+ mobilizing agents such as thapsigargin increase intracellular Ca2+ level and result in Ca2+/CaM binding to eNOS that stimulates eNOS activation and subsequently NO production. Other stimuli such as angiopoietin mediate Akt and other protein kinase activation leading to eNOS phosphorylation that increases eNOS activation even in low level of intracellular Ca2+. The PM-localized eNOS is more efficient to produce NO in response to various agonists compared with that by Golgi-eNOS, which may be attributable to higher phosphorylation and CaM affinity of the PM-eNOS relative to the Golgi-eNOS. HSP90 binding to eNOS may facilitate Akt-induced eNOS phosphorylation at S1179. However, the PM-eNOS is much sensitive to the modification of cholesterol levels in cells such as by the treatment of mLDL. In addition, subcellular localization of eNOS is dynamic, and eNOS can traffic between the PM and the Golgi complex. Of note, the availability of eNOS substrate L-arginine and its cofactors such as BH4 in particular subcellular localization (not shown) may affect the ability of eNOS to produce NO as well.

Another important finding in this article is that the PM-eNOS is more sensitive to cholesterol change and modified low-density lipoprotein (mLDL) challenge than the Golgi-eNOS. Cholesterol is one of the main components in caveolae, a specialized microdomain of the PM.14 ECs incubated with cholesterol not only have increased caveolae numbers but also release more NO in response to Ca2+ ionophore,28 whereas agents that lower the cholesterol content of caveolae such as cyclodextrin and oxidized LDL cause a decease of eNOS activity to fluids shear stress29 and acetylcholine.30 Extending these previous reports, Zhang et al in this study showed that manipulation of cholesterol levels by cyclodextrin and mLDL had a great impact on activation of the PM-eNOS but not the Golgi-eNOS. This finding is of clinical relevance because a key process in the early pathogenesis of hypercholesterolemia-induced vascular disease and atherosclerosis is diminished NO production by EC.14 In addition to hypercholesterolemia, several other proatherogenic factors such as angiotensin II31 and oscillatory shear stress32 have recently been shown to cause eNOS mislocalization and eNOS dysfunction in intact vessels. Because the change of cholesterol levels in ECs selectively influences the PM-eNOS activity, the authors also raised an intriguing question of whether a Golgi-targeted eNOS would offer more vascular protection than a PM-eNOS in the model of endothelial dysfunction in vascular disease. However, it remains to be clarified whether the Golgi eNOS–derived NO contributes to maintain vascular homeostasis and whether the Golgi-eNOS would improve endothelial function in vitro and in vivo.

The underlying mechanisms for the different sensitivity of subcellular eNOS to cholesterol modification are largely unknown. The authors found no change in basal or ionomycin-stimulated intracellular Ca2+ levels, phosphorylation states, and protein–protein interaction (eNOS association with HSP90 and caveolin-1) and the PM eNOS localization in cholesterol-enriched membrane microdomains. There is an obvious discrepancy between these results and the data reported by Blair et al,30 which showed that oxidized LDL displaced eNOS from caveolae through depletion of caveolae cholesterol and resulted in impaired eNOS activation. It will be important to determine whether there is any change in caveolae cholesterol content after exposure of mLDL. On another note, in Zhang’s report, the PM-eNOS appears targeting to two fractions, the light membrane fraction (presumably the caveolae fraction) and the heavy membrane fraction (non-caveolae fraction of the PM). This observation is also contradictory to many reports showing that endogenous eNOS in the PM fraction is mainly localized in caveolae.14 It is unclear whether the difference is attributable to overexpression of the PM-eNOS in cells or the fractionation procedures. Because Ca2+-independent inducible NOS (iNOS) and Ca2+/CaM-insensitive eNOS mutants have no significant difference in NO production with or without cholesterol manipulation, the authors hypothesized that the changes in the association of CaM with eNOS might be the potential mechanism whereby membrane cholesterol levels affect eNOS activation. The hypothesis was supported indirectly by a very recent report from the same group, that showed that the activity of Ca2+/CaM-independent eNOS mutant was unaffected by intracellular targeting.33

In summary, the studies by Zhang et al provide an important novel concept that the PM-eNOS are more efficient to produce NO in response to agonists but more vulnerable to cholesterol levels and mLDL than the Golgi-eNOS. The data clearly advance our understanding of the functional roles for eNOS subcellular targeting and of the mechanisms underlying eNOS inactivation and endothelial dysfunction in disease, but numerous questions remain to be answered. For example, it will be important to determine whether high phosphorylation state of the PM-eNOS in basal condition does indeed contribute to the sensitivity to calcium mobilizing agents, using eNOS phosphorylation-site mutants. And what are the molecular mechanisms underlying more HSP90 binding to the PM-eNOS? Why is the PM-eNOS more vulnerable to mLDL? It will be interesting to know whether physiological stimuli such as fluid shear stress stimulate the Golgi-eNOS activation to produce bioavailable NO from endothelium. In addition, it remains to be determined whether the mechanisms described are operative in intact vessels, especially in pathological conditions. By addressing these issues, the eventual hope is that we might design better therapeutic strategies to prevent endothelial dysfunction and atherosclerotic vascular disease.


*    Acknowledgments
 
This work is supported by grant RO1 HL080611 from the National Heart, Lung, and Blood Institute, National Institute of Health, and the Thomas R. Lee Career Development Award from the American Diabetes Association (1-06-CD-13).


*    References
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Related Article:

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, and David Fulton
Arterioscler. Thromb. Vasc. Biol. 2006 26: 1015-1021. [Abstract] [Full Text] [PDF]



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