Functional reconstitution of endothelial nitric oxide synthase reveals the importance of serine 1179 in endothelium-dependent vasomotion.

Department of Pharmacology, Vascular Cell Signaling and Therapeutics Program, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Conn 06536, USA.
Circulation Research (Impact Factor: 11.02). 06/2002; 90(8):904-10.
Source: PubMed


Phosphorylation of endothelial nitric oxide synthase (eNOS) at serine 1179 can activate the enzyme, leading to NO release. Because eNOS is important in regulating vascular tone, we investigated whether phosphorylation of this residue is involved in vasomotion. Adenoviral transduction of endothelial cells (ECs) with the phosphomimetic S1179DeNOS markedly increased basal and vascular endothelial cell growth factor (VEGF)-stimulated NO release compared with cells transduced with wild-type virus. Conversely, adenoviral transduction of ECs with the non-phosphorylatable S1179AeNOS suppressed basal and stimulated NO release. Using a novel method for luminal delivery of adenovirus, transduction of the endothelium of carotid arteries from eNOS knockout mice with S1179DeNOS completely restored NO-mediated dilatation to acetylcholine (ACh), whereas vasomotor responses in arteries transduced with S1179AeNOS were significantly attenuated. Basal NO release was also significantly reduced in arteries transduced with S1179AeNOS, compared with S1179DeNOS. Thus, our data directly demonstrate that phosphorylation of eNOS at serine 1179 is an important regulator of basal and stimulated NO release in ECs and in intact blood vessels.

Download full-text


Available from: Jun Yu, Dec 30, 2013
  • Source
    • "These results clearly demonstrate that among NO-synthases eNOS plays by far the most important role in BP regulation. However, in contrast to large arteries of eNOS-KO where no compensation for the lack of eNOS is observed [1] [6] [10], other endothelium-dependent vasodilators such as prostaglandins, nNOS or endothelium-derived hyperpolarizing factor compensate for the lack of eNOS in coronary, femoral, mesenteric, cerebral and skeletal resistance arteries [11e16]. These data indicate that different vascular-bed specific mechanisms regulate arterial tone in the absence of vascular eNOS and raise the question to what extent hypertension in eNOS-KO is caused by the lack of endothelial eNOS expression. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The aim of the study was to evaluate the possible contribution of non-endothelial eNOS to the regulation of blood pressure (BP). To accomplish this, a double transgenic strain expressing eNOS exclusively in the vascular endothelium (eNOS-Tg/KO) has been generated by endothelial-specific targeting of bovine eNOS in eNOS-deficient mice (eNOS-KO). Expression of eNOS was evaluated in aorta, myocardium, kidney, brain stem and skeletal muscle. Organ bath studies revealed a complete normalization of aortic reactivity to acetylcholine, phenylephrine and the NO-donors in eNOS-Tg/KO. Function of eNOS in resistance arteries was demonstrated by acute i.v. infusion of acetylcholine and the NOS-inhibitor L-NAME. Acetylcholine decreased mean arterial pressure in all strains but eNOS-KO responded significantly less sensitive as compared eNOS-Tg/KO and C57BL/6. Likewise, acute i.v. L-NAME application elevated mean arterial pressure in C57BL/6 and eNOS-Tg/KO, but not in eNOS-KO. In striking contrast to these findings, mean, systolic and diastolic BP in eNOS-Tg/KO remained significantly elevated and was similar to values of eNOS-KO. Chronic oral treatment with L-NAME increased BP to the level of eNOS-KO only in C57BL/6, but had no effect on hypertension in eNOS-KO and eNOS-Tg/KO. Taken together, functional reconstitution of eNOS in the vasculature of eNOS-KO not even partially lowered BP. These data suggest that the activity of eNOS expressed in non-vascular tissue might play a role in physiologic BP regulation.
    Full-text · Article · Feb 2015 · Biochemical and Biophysical Research Communications
  • Source
    • "Previous studies have demonstrated that Ser1179 phosphorylation (human eNOS Ser1177) is critical to eNOS activity [30]. Specifically, phosphorylation of Ser1179 or the mutation of Ser1179 to aspartic acid results in a 2–3 fold increase in eNOS activity [31]. However, it is still unclear whether eNOS phosphorylation alters its structural and functional properties, including eNOS dimer structure. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Endothelial nitric oxide synthase (eNOS) is a multifunctional enzyme with roles in diverse cellular processes including angiogenesis, tissue remodeling, and the maintenance of vascular tone. Monomeric and dimeric forms of eNOS exist in various tissues. The dimeric form of eNOS is considered the active form and the monomeric form is considered inactive. The activity of eNOS is also regulated by many other mechanisms, including amino acid phosphorylation and interactions with other proteins. However, the precise mechanisms regulating eNOS dimerization, phosphorylation, and activity remain incompletely characterized. We utilized purified eNOS and bovine aorta endothelial cells (BAECs) to investigate the mechanisms regulating eNOS degradation. Both eNOS monomer and dimer existed in purified bovine eNOS. Incubation of purified bovine eNOS with protein phosphatase 2A (PP2A) resulted in dephosphorylation at Serine 1179 (Ser1179) in both dimer and monomer and decrease in eNOS activity. However, the eNOS dimer∶monomer ratio was unchanged. Similarly, protein phosphatase 1 (PP1) induced dephosphorylation of eNOS at Threonine 497 (Thr497), without altering the eNOS dimer∶monomer ratio. Different from purified eNOS, in cultured BAECs eNOS existed predominantly as dimers. However, eNOS monomers accumulated following treatment with the proteasome inhibitor lactacystin. Additionally, treatment of BAECs with vascular endothelial growth factor (VEGF) resulted in phosphorylation of Ser1179 in eNOS dimers without altering the phosphorylation status of Thr497 in either form. Inhibition of heat shock protein 90 (Hsp90) or Hsp90 silencing destabilized eNOS dimers and was accompanied by dephosphorylation both of Ser1179 and Thr497. In conclusion, our study demonstrates that eNOS monomers, but not eNOS dimers, are degraded by ubiquitination. Additionally, the dimeric eNOS structure is the predominant condition for eNOS amino acid modification and activity regulation. Finally, destabilization of eNOS dimers not only results in eNOS degradation, but also causes changes in eNOS amino acid modifications that further affect eNOS activity.
    Full-text · Article · Aug 2014 · PLoS ONE
  • Source
    • "As seen in Fig. 2B, WT ECs responded to VEGF with the formation of stress fibers, and this effect was reduced in eNOS 2/2 ECs. Moreover, the necessity of eNOS for VEGF-induced changes in cytoskeletal reorganization was shown by the rescuing the eNOS knockout phenotype by adenoviral reexpression of eNOS (Ad-eNOS; Gratton et al., 2003b; Scotland et al., 2002) but not a control adenovirus encoding bgalactosidase (Ad-b-Gal; Fig. 2C). Finally, to rule out potential artifacts due to culturing ECs or interactions between ECs and the extracellular matrix, the formation of stress fibers was examined by 'en face' imaging of F-actin in endothelial layers of intact blood vessels isolated from WT or eNOS 2/2 mice. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Transient disruption of endothelial adherens junctions and cytoskeletal remodeling are responsible for increases in vascular permeability induced by inflammatory stimuli and vascular endothelial growth factor (VEGF). Nitric oxide (NO) produced by endothelial NO synthase (eNOS) is critical for VEGF induced changes in permeability in vivo, however, the molecular mechanisms by which endogenous NO modulates endothelial permeability is not clear. Here we show that the lack of eNOS reduces VEGF induced permeability, an effect mediated by enhanced Rac-GTPase activation and stabilization of cortical actin. The loss of NO, increased the recruitment of the Rac-GEF, Tiam-1, to adherens junctions and VE-cadherin and reduced Rho-activation and stress fiber formation. In addition, NO deficiency reduced VEGF-induced VE-cadherin phosphorylation, and impaired the localization, but not the activation, of c-src to cell junctions. The physiological role of eNOS activation is clear since VEGF, histamine and inflammation induced vascular permeability is reduced in mice with bearing the knockin mutation of the key phosphorylation site S1176. Thus, NO is critical for Rho GTPase dependent regulation of cytoskeletal architecture leading to reversible changes in vascular permeability.
    Full-text · Article · May 2014 · Journal of Cell Science
Show more