Immunohistochemical localization of endothelial nitric oxide synthase in human villous and extravillous trophoblast populations and expression during syncytiotrophoblast formation in vitro

eNOS (endothelial nitric oxide synthase) catalyses the conversion of L-arginine into L-citrulline and NO. Evidence has been presented previously that eNOS is associated with the CAT (cationic amino acid transporter)-1 arginine transporter in endothelial caveolae, and it has been proposed that eNOS–CAT-1 association facilitates the delivery of extracellular L-arginine to eNOS. Definitive proof of a protein–protein interaction between eNOS and CAT-1 is lacking, however, and it is also unknown whether the two proteins interact directly or via an adaptor protein. In the present study, we raised a polyclonal antibody against CAT-1, and show using reciprocal co-immunoprecipitation protocols that eNOS and CAT-1 do indeed form a complex in BAECs (bovine aortic endothelial cells). In vitro binding assays with GST (glutathione S-transferase)–CAT-1 fusion proteins and eNOS show that the two proteins interact directly and that no single CAT-1 intracellular domain is sufficient to mediate the interaction. Overexpression of CAT-1 in BAECs by adenoviral-mediated gene transfer results in significant increases in both L-arginine uptake and NO production by the cells. However, whereas increased L-arginine transport is reversed completely by the CAT-1 inhibitor, L-lysine, increased NO release is unaltered, suggesting that NO production in this in vitro model is independent of CAT-1-mediated transport. Furthermore, eNOS enzymic activity is increased in lysates of CAT-1-overexpressing cells accompanied by increased phosphorylation of eNOS at Ser-1179 and Ser-635, and decreased association of eNOS with caveolin-1. Taken together, these data suggest that direct interaction of eNOS with CAT-1 enhances NO release by a mechanism not involving arginine transport.

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Can endothelial hemoglobin-α regulate nitric oxide vasodilatory signaling?

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00315.2016 ◽

2017 ◽

Vol 312 (4) ◽

pp. H854-H866 ◽

Cited By ~ 2

Author(s):

Jaimit Parikh ◽

Adam Kapela ◽

Nikolaos M. Tsoukias

Keyword(s):

Mathematical Modeling ◽

Nitric Oxide ◽

Nitric Oxide Synthase ◽

Endothelial Nitric Oxide Synthase ◽

Cellular Models ◽

No Signaling ◽

Oxide Synthase ◽

Endothelial Nitric Oxide

We used mathematical modeling to investigate nitric oxide (NO)-dependent vasodilatory signaling in the arteriolar wall. Detailed continuum cellular models of calcium (Ca2+) dynamics and membrane electrophysiology in smooth muscle and endothelial cells (EC) were coupled with models of NO signaling and biotransport in an arteriole. We used this theoretical approach to examine the role of endothelial hemoglobin-α (Hbα) as a modulator of NO-mediated myoendothelial feedback, as previously suggested in Straub et al. ( Nature 491: 473–477, 2012). The model considers enriched expression of inositol 1,4,5-triphosphate receptors (IP3Rs), endothelial nitric oxide synthase (eNOS) enzyme, Ca2+-activated potassium (KCa) channels and Hbα in myoendothelial projections (MPs) between the two cell layers. The model suggests that NO-mediated myoendothelial feedback is plausible if a significant percentage of eNOS is localized within or near the myoendothelial projection. Model results show that the ability of Hbα to regulate the myoendothelial feedback is conditional to its colocalization with eNOS near MPs at concentrations in the high nanomolar range (>0.2 μM or 24,000 molecules). Simulations also show that the effect of Hbα observed in in vitro experimental studies may overestimate its contribution in vivo, in the presence of blood perfusion. Thus, additional experimentation is required to quantify the presence and spatial distribution of Hbα in the EC, as well as to test that the strong effect of Hbα on NO signaling seen in vitro, translates also into a physiologically relevant response in vivo. NEW & NOTEWORTHY Mathematical modeling shows that although regulation of nitric oxide signaling by hemoglobin-α (Hbα) is plausible, it is conditional to its presence in significant concentrations colocalized with endothelial nitric oxide synthase in myoendothelial projections. Additional experimentation is required to test that the strong effect of Hbα seen in vitro translates into a physiologically relevant response in vivo

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Interactive effects of in vitro binge-like alcohol and ATP on umbilical endothelial nitric oxide synthase post-translational modifications and redox modulation

Reproductive Toxicology ◽

10.1016/j.reprotox.2013.11.006 ◽

2014 ◽

Vol 43 ◽

pp. 94-101 ◽

Cited By ~ 4

Author(s):

Kaviarasan Subramanian ◽

Vishal D. Naik ◽

Kunju Sathishkumar ◽

Onkar B. Sawant ◽

Shannon E. Washburn ◽

...

Keyword(s):

Nitric Oxide ◽

Nitric Oxide Synthase ◽

Endothelial Nitric Oxide Synthase ◽

Interactive Effects ◽

Redox Modulation ◽

Post Translational Modifications ◽

Oxide Synthase ◽

Endothelial Nitric Oxide

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Endothelial Nitric Oxide Synthase Expression Is Progressively Increased in Primary Cerebral Microvascular Endothelial Cells During Hyperbaric Oxygen Exposure

Oxidative Medicine and Cellular Longevity ◽

10.4161/oxim.2.1.7697 ◽

2009 ◽

Vol 2 (1) ◽

pp. 7-13 ◽

Cited By ~ 12

Author(s):

Xiongfei Xu ◽

Zhongzhuang Wang ◽

Quan Li ◽

Xiang Xiao ◽

Qinglin Lian ◽

...

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Nitric Oxide Synthase ◽

Hyperbaric Oxygen ◽

Endothelial Nitric Oxide Synthase ◽

Microvascular Endothelial Cells ◽

Oxide Synthase ◽

Endothelial Nitric Oxide ◽

Enos Protein

Exposure to hyperbaric oxygen (HBO) can lead to seizures. Many studies have demonstrated that there exist a very close relationship between the alteration of cerebral blood flow (CBF) and the onset of seizures. Nitric oxide (NO) may play a key role in the change of CBF during exposure, and modulation of endothelial nitric oxide synthase (eNOS)-derived NO by HBO is responsible for early vasoconstriction, whereas late HBO-induced vasodilation depends upon a large amount of NO from both eNOS and neuronal nitric oxide synthase (nNOS). To investigate the effect of HBO on the activity and expression of eNOS in cerebral microvascular endothelial cells (CMEC) in vitro, primarily cultured CMEC from neonatal rats were exposed to oxygen at 500 kPa [5 atmosphere absolute (ATA)] for 10, 20, 30, 60 and 120 minutes (min), then eNOS activity, protein and mRNA contents in cells were detected. Our results showed that immediately after exposure, 30, 60 and 120 min HBO exposures did not alter NOS activity. When detected no matter immediately or six hours (h) after exposure, these exposures also did not alter eNOS protein and mRNA levels. However, when detected 24 h after exposure, 30, 60 and 120 min exposures upregulated eNOS protein content by 39%, 60% and 40% respectively. 10 and 20 min exposures upregulated eNOS mRNA content by about 15%, while 30, 60 and 120 min exposures upregulated it by about 20–30%. The increased eNOS protein and mRNA contents at 24 h after exposure may reflect new protein synthesis for eNOS. Our studies showed that with the exposing protocols we used, HBO did induce eNOS expression increase in CMEC. However, compared with the decrease of CBF in vivo, which occurred in a relative short time after rat was exposed to HBO above 4 ATA, the responses of eNOS in CMEC in vitro were a little slow. Thus we considered that for the vasodilation in the late period of HBO exposure before seizure, the effect of NO produced by eNOS was limited.

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