Exp. Biol. Med. 2009;234:132-139
doi:10.3181/0807-RM-233
© 2009 Society for Experimental Biology and Medicine
Role of Protease-Activated Receptor-1 in Endothelial Nitric Oxide Synthase-Thr495 Phosphorylation
Vabren L. Watts and
Evangeline D. Motley1
Department of Cardiovascular Biology, Meharry Medical College, Nashville, Tennessee 37208
1 Department of Cardiovascular Biology, Meharry Medical College, 1005 D. B. Todd Blvd., Nashville, TN 37208. E-mail: emotley{at}mmc.edu
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Abstract
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Protease activated receptors (PARs) are G protein-coupled receptors
that are known to regulate endothelial nitric oxide synthase
(eNOS) activity in part by phosphorylating the enzyme at various
sites. Ser1177 is a positive regulatory site, which leads to
the enhanced production of nitric oxide (NO), a vasodilator
of arteries. Thr495 is a negative regulatory site, which inhibits
NO production. We have shown that thrombin, a PAR agonist, mediates
eNOS-Ser1177 phosphorylation through G
q and a calcium and protein
kinase C (PKC)-
sensitive, but phosphatidylinositol 3-kinase
(PI3K)/Akt-independent pathway. However, the mechanism for eNOS-Thr495
phosphorylation by PAR agonists is unknown. We used a specific
synthetic PAR-1 activating peptide, TFLLR, and thrombin to assess
the role of PAR-1 involvement in the phosphorylation of eNOS-Thr495
in human umbilical vein endothelial cells (HUVECs). Using Western
blot analysis and the Griess Reagent assay, we found that both
agonists phosphorylated Thr495 in a time- and dose-dependent
manner and significantly decreased nitrite production, respectively.
Pretreatment of cells with the PAR-1 inhibitor, SCH-79797, resulted
in a significant decrease in thrombin- and TFLLR-induced phosphorylation
of eNOS-Thr495 and an increase in nitrite production. We further
demonstrated that inhibition of Rho with C3 exoenzyme or dominant
negative (dn) RhoA, and inhibition of Rho-Kinase (ROCK) with
Y-27632 caused a significant decrease in thrombin and TFLLR-induced
Thr495 phosphorylation. Blockade of the Rho/ROCK pathway also
caused an increase in nitrite production. This suggests that
PAR-1 regulates eNOS activity via phosphorylation of eNOS-Thr495,
which is dependent upon activation of the Rho/ROCK pathway.
These findings will be beneficial in further understanding the
signaling pathways that regulate eNOS-induced NO production,
which plays an important role in endothelial dysfunction associated
with cardiovascular disease.
Keywords: signaling, thrombin, protease-activated receptor, nitric oxide, endothelial NO synthase, Rho, ROCK
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Introduction
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Hypertension is characterized by the inability of blood vessels
to properly vasodilate resulting in a narrowed vessel lumen
under high pressure. One of the underlying causes of hypertension
is endothelial dysfunction, which is characterized by the endothelium’s
failure to produce and/or release nitric oxide (NO), a potent
vasodilator. Thrombin and certain proteases play very important
roles in endothelial physiology and pathophysiology through
activation of a family of G-protein coupled receptors (GPCRs)
termed PARs (
1). PARs are coupled to three G protein families—G
q,
G
12/13, and G
i/o—and are capable of activating various
signaling molecules such as PKC, Akt, and Rho (
2). There are
four members of the PAR family, and thrombin activates PAR-1,
–3, and –4; trypsin activates PAR-2, which can be
transactivated by PAR-1 (
3). Only PAR-1 and –2 have been
recognized as having functional significance in human endothelial
cells (
3). Endothelial thrombin/PAR functions include regulations
of endothelium-dependent contraction/relaxation, endothelial
permeability/barrier function, leukocyte adhesion, angiogenesis,
endothelial cell migration and proliferation (
4). However, the
detailed molecular mechanisms by which PARs regulate these functions
remain largely unclear.
Endothelial nitric oxide synthase (eNOS) is a constitutively expressed enzyme that is responsible for oxidizing L-arginine to yield NO. Various phosphorylation sites located on eNOS can regulate its enzymatic activity. Phosphorylation at site Ser1177/1179 (human/bovine) causes an increase in eNOS activity, while phosphorylation at site Thr495/497 (human/bovine) causes a decrease in eNOS activity (5). A recent in vivo study showed that mice injected with rapamycin, a drug that is known to induce hypertension, resulted in a significant increase in phosphorylation at eNOS site Thr495 and a decrease in NO production in mice aortic endothelial cells (6). Therefore, this study suggests a role for the phosphorylation of eNOS-Thr495 in endothelial dysfunction and hypertension.
Recently, we have shown that thrombin via PAR activation regulates eNOS activity through phosphorylation of site Ser1179 in bovine aortic endothelial cells (BAECs). We found that PAR activation mediated this phosphorylation via a Ca2+, and protein kinase C (PKC)- dependent pathway (7). We also demonstrated in BAECs that activation of PAR by thrombin leads to phosphorylation of eNOS at site Thr497. However, the mechanism by which PAR activation mediates phosphorylation of this negative regulatory site is unknown.
We used human umbilical vein endothelial cells (HUVECs) to study the mechanism involved in PAR-1-induced eNOS-Thr495 phosphorylation. In our studies, we demonstrated that PAR-1 agonists phosphorylate eNOS-Thr495, not Ser1177, via a Rho/Rho-kinase (ROCK)-dependent pathway. We also demonstrated that this pathway plays a pivotal role in inhibiting nitrite production, a stable metabolite of NO. These findings will be beneficial in further understanding the signaling pathways that regulate eNOS-induced NO production, which plays an important role in endothelial dysfunction associated with cardiovascular disease.
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Materials and Methods
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Reagents.
Thrombin was purchased from Sigma Chemicals. TFLLR and SCH-79797
were purchased from Tocris. Dominant negative RhoA was kindly
donated by Dr. George E. Davis of University of Missouri School
of Medicine. C3-transferase was purchased from Cytoskeleton
Inc. Y-27635 was purchased from Calbiochem. M199 containing
15% FBS, penicillin-streptomycin, and amphotericin B were purchased
from Invitrogen. Endothelial cell growth supplement and antibodies
against eNOS-Thr495 and -Ser1177, total eNOS, and RhoA were
purchased from Cell Signaling Technology.
Cell Culture.
Primary cultured HUVECs harvested from umbilical cords were obtained from the Cardiovascular Medicine Division at Vanderbilt University Medical Center. The cells were grown in M199 containing 15% FBS, penicillin-streptomycin, amphotericin B, and endothelial cell growth supplement. Experiments were performed in 6-, 12-, and 96-well plates. Cells from passage 2–6 were grown to about 90% confluency. Media containing 5% FBS was added for 16–24 hrs before each experiment to ensure a quiescent cell state.
Immunoblotting.
Cell lysates were subjected to SDS-PAGE and phosphorylated proteins were electrophoretically transferred to a nitrocellulose membrane as previously described (7). The membranes were then exposed to primary antibodies overnight at 4°C. After incubation with the peroxidase-linked secondary antibody for 1 hr at room temperature, immunoreactive proteins were visualized by an Amersham Biosciences ECL Direct Detection System (chemiluminescence reaction kit) purchased from General Electric Healthcare (7).
Adenovirus Infection.
The generation and characterization of adenovirus encoding GFP and dominant-negative mutants of RhoA-GFP are described in detail elsewhere (8). HUVECs were infected with the adenovirus for 2 days as previously described before treatment with thrombin.
Nitrite Colorimic Assay.
The Griess Reagent Assay was used to measure nitrite production from the medium of stimulated HUVECs. In a 96-well plate, samples were mixed with an equal volume of Griess reagent (0.1% naphthalethylenediamine dihydrochloride and 1% sulphanilamide in a 1:1 volume). The optical density was read at 540 nm using a Spectramax 250 microplate reader. Nitrite was compared to total protein extracted from whole cell lysates using 1X SDS lysis buffer. Total protein quantification was performed at an optical density of 280 nm using the Protein A-280 application of the NanoDrop ND-1000 spectrophotometer (NanoDrop, Wilmington, DE, http://www.nanodrop.com).
Statistical Analysis of Data.
The data from each study are representative of three or more independent experiments. Intensity of the bands from Western blots was measured by UN-SCAN-IT Gel Automated Digitizing System Version 5.1. Next the data was analyzed with Prism 5.0 statistical program software using two-tailed Student’s t test analysis. Data from Western blots and Griess assays are presented as mean ± SD with a significance level of P <0.05, represented by an asterisk (*), pound (#), or diamond (
).
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Results
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Effects of Thrombin on eNOS Phosphorylation and Nitrite Production.
We first wanted to examine whether or not thrombin phosphorylates
eNOS at site Thr495, a catalytically negative regulatory site.
In Figure 1A
, we demonstrated in HUVECs that thrombin stimulates
eNOS-Thr495 phosphorylation in a concentration-dependent manner,
which is significant in the range of 0.5–10 U/ml. Thrombin
also induced eNOS-Thr495 phosphorylation in a time-dependent
manner (Fig. 1B
). Significant phosphorylation of site Thr495
was observed between 2–30 mins (data not shown) with maximal
phosphorylation occurring at 10 mins. Our lab has shown that
thrombin induces significant phosphorylation of site Ser1177
(Fig. 1B
) between 1–2 mins (
7). In addition to measuring
eNOS phosphorylation, we also measured nitrite production from
the media of cells that were stimulated with thrombin. We observed
a significant decrease in nitrite production with thrombin.
As a positive control we used the potent vasodilator, bradykinin,
since it is known to release a sufficient amount of NO from
endothelial cells (
9) (Fig. 1C
). We observed a significant increase
in nitrite production from the media of HUVECs stimulated with
bradykinin (Fig. 1C
).
Effects of PAR-1 Activating Peptide on eNOS-Thr495 Phosphorylation and Nitrite Production.
Since thrombin can interact with more than one receptor, we used TFLLR, a more specific activating PAR-1 peptide, to determine its role in the phosphorylation of eNOS-Thr495. TFLLR increased phosphorylation of eNOS-Thr495 in a concentration-dependent manner with significant phosphorylation occurring between 0.5 µM–10 µM (Fig. 2A). Even though we observed significant phosphorylation of eNOS-Thr495 between 5–30 mins (data not shown) with TFLLR, maximal phosphorylation was seen at 5 mins (Fig. 2B). We also observed that TFLLR did not cause phosphorylation of site Ser1177, suggesting that this site is activated by a different PAR (Fig. 2B). Nitrite levels were significantly decreased in cells that were stimulated with TFLLR (Fig. 2C). This data suggest that activation of PAR-1 plays an important role in phosphorylation of eNOS at site Thr495, not Ser1177, and causes inhibition of NO production.
Effects of PAR-2 Activating Peptide on eNOS-Thr495 Phosphorylation and Nitrite Production.
We also used SLIGRL, a PAR-2 activating peptide, to determine if phosphorylation of eNOS-Thr495 is mediated through PAR-2. Cells stimulated with SLIGRL showed no significant phosphorylation of eNOS-Thr495; however, maximal phosphorylation of eNOS-Ser1177 was observed at a concentration of 50 µM with significant phosphorylation occurring between 0.5 and 10 mins (Fig. 3A and B). SLIGRL also caused a significant increase in nitrite production (Fig. 3C). This data suggest that PAR-2 induces eNOS activation and NO production via phosphorylation of site Ser1177, not Thr495. This data further demonstrates that phosphorylation of eNOS-Thr495 is independent of PAR-2 activation.
Effects of PAR-1 Inhibitor, SCH-79797, on Thrombin and TFLLR Mediated eNOS-Thr495 Phosphorylation and Nitrite Production.
To verify PAR-1 involvement in thrombin-induced Thr495 phosphorylation of eNOS, we inhibited activation of PAR-1 by using the PAR-1 selective antagonist, SCH-79797 (SC). Cells were pretreated with SC (10 µM) one hour prior to stimulation with the agonist. Since bradykinin has been shown to induce eNOS-Thr495 phosphorylation via the bradykinin receptor, B1, we used bradykinin as a positive control (10, 11). Our results show that bradykinin-induced eNOS-Thr495 phosphorylation was not inhibited by SC (Fig. 4A). Phosphorylation of eNOS-Thr495 in cells stimulated with thrombin or TFLLR in the presence of SC was attenuated. However, nitrite levels were increased in cells stimulated with both thrombin and TFLLR in the presence of the PAR-1 inhibitor (Fig. 4B). SC did not inhibit nitrite production induced by bradykinin, which lead us to believe that PAR-1 is responsible for inhibiting eNOS activity and NO production through the phosphorylation of eNOS at site Thr495.
Effects of Rho Inhibitor, C3 Transferase, and Dominant Negative RhoA on Thrombin and TFLLR Mediated eNOS-Thr495 Phosphorylation and Nitrite Production.
Activation of the RhoA/ROCK cascade has been implicated in endothelial dysfunction (12–14). We used cell permeable C3 transferase, which inhibits Rho through ADP ribosylation, and dnRho to determine if Rho was involved in the PAR-1 agonist-induced phosphorylation of eNOS-Thr495. Pretreatment with 10 µg/ml of C3 transferase caused complete inhibition of thrombin- and TFLLR-induced phosphorylation of eNOS at site Thr495 (Fig. 5A). Bradykinin was used as a positive control and did not induce any significant change in Thr495 phosphorylation in the presence of C3. In another set of experiments, we overexpressed dnRhoA-GFP or GFP alone to further determine the role of RhoA in PAR-1-induced phosphorylation of eNOS-Thr495 in HUVECs. DnRhoA caused almost a two-fold inhibition of thrombin- and TFLLR-induced phosphorylation of eNOS at site Thr495 (Fig. 5B). Both C3 and overexpression of dnRhoA caused an increase in nitrite production from cells stimulated with PAR agonists (Fig. 5C and 5D). In addition, we observed a significant decrease in nitrite production with bradykinin. This study further demonstrates that thrombin and a PAR-1 specific agonist induce eNOS-Thr495 phosphorylation and inhibit NO production via a Rho-dependent signaling pathway.
Effects of ROCK Inhibitor, Y-27632, on eNOS-Thr495 Phosphorylation and Nitrite Production Induced by Thrombin and TFLLR.
Because ROCK is activated downstream of Rho (12), we used Y-27632, a ROCK inhibitor, to determine ROCK involvement in PAR-1 mediated eNOS-Thr495 phosphorylation. Inhibition of ROCK significantly decreased thrombin- and TFLLR-induced eNOS-Thr495 phosphorylation; however, there was no effect on bradykinin-induced phosphorylation (Fig. 6A). An increase in nitrite production was observed with thrombin and TFLLR in presence of the Y-27632 (Fig. 6B), suggesting that PAR-1 mediates eNOS-Thr495 phosphorylation through ROCK activation. As observed with C3, we also saw a significant decrease in bradykinin-induced nitrite production in the presence of the ROCK inhibitor.
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Discussion
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The major novel findings in this study are that thrombin induces
phosphorylation of eNOS at residue Thr495 via a PAR-1 and Rho/ROCK-dependent
pathway, which leads to a decrease in NO production. A scheme
of the proposed signaling pathway is shown in Figure 7
.
Only recently has information been provided on eNOS-Thr495 regulation involving thrombin (7, 14). Although previous studies have shown that thrombin phosphorylates eNOS at various sites (7, 14), little is known about which PAR(s) are activated by thrombin to phosphorylate these sites. Here, we have explored the importance of PAR-1 in the phosphorylation of thrombin-induced eNOS-Thr495 in HUVECs. In addition, we also observed the importance of PAR-2 in eNOS-Ser177 phosphorylation. Only PAR-1 and PAR-2 have been shown to have functional significance in endothelial cells. Infusing rodents with TFLLR leads to a biphasic blood pressure response; a rapid/transient hypotensive response (mediated by NO) is followed by a more sustained hypertensive response (15). Infusing rodents with SLIGRL, PAR2-activating peptide, solely leads to an hypotensive response (15). In previous studies, we have shown that BAECs pretreated with thrombin, a PAR-1 agonist, caused a rapid/transient phosphorylation of the positive eNOS regulatory site (Ser 1177 human/1179 bovine) as well as an increase in cGMP production, another bio-marker for NO (7). In our studies with HUVECs, we found that thrombin was capable of transient eNOS-Ser1177 phosphorylation, but our nitrite results suggested that this phosphorylation was not strong enough to induce an increase in NO production. This phenomenon may vary among cell lines, which leaves room for investigation. Here we demonstrate that a PAR-1, not PAR-2, agonist induced phosphorylation of eNOS at its inhibitory site (Thr495), which lead to a significant decrease in nitrite production (6). Our results correlate with the aforementioned in vivo studies.
PAR-1 is coupled to three different G-protein families, which gives rise to various signaling pathways, including that of Rho and Rho-kinase/ROCK (12), which plays a major role in cardiovascular pathologies such as hypertension and atherosclerosis. Inhibition of this pathway leads to rapid phosphorylation of eNOS-Ser1177 and eNOS activation (13, 16). Constitutively active RhoA and ROCK increase phosphorylation of thrombin-induced eNOS-Thr495 phosphorylation (14). In our studies, we showed that inhibition of RhoA and ROCK, with dnRho-GFP and Y-27632, negatively effects both thrombin- and TFLLR-induced eNOS-Thr495 phosphorylation and increased nitrite production. We did not see these inhibitory effects with bradykinin because it deactivates eNOS through an ERK-dependent pathway (17), not Rho/ROCK. This directly supports the notion that thrombin mediates phosphorylation of the eNOS inhibitory site via Rho/ROCK, which is activated downstream of PAR-1. The involvement of Rho/ROCK in PAR-1 induced Thr495 phosphorylation also strongly suggests that G12/13 mediates this response. Phosphorylation of eNOS-Thr495 indicates a decrease in eNOS enzymatic activity, thereby preventing the catalyzation of L-arginine to yield NO.
In conclusion, thrombin induces phosphorylation of eNOS-Thr495 and inhibition of NO production via PAR-1 and a Rho/ROCK-dependent pathway. These findings will be beneficial in further understanding the signaling pathways that regulate eNOS-induced NO production, which plays an important role in endothelial dysfunction associated with cardiovascular disease.
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Figure 1. Effects of thrombin on eNOS phosphorylation and nitrite production. A) HUVECs were stimulated with the indicated concentrations of thrombin for 10 mins. B) HUVECs were stimulated with 10 U/ml of thrombin at the indicated time points and phosphorylation of eNOS-Thr495 and -Ser1177 was determined using Western blot analysis with phosphospecific antibodies. The data shown are presented as mean ± SD derived from three separate experiments, normalized to total eNOS. In panel A, * P < 0.05 vs. control; # P < 0.05 vs. 0.1–0.5 U/ml thrombin. In panel B, * P < 0.05 vs. respective control; # P < 0.05 vs. 0.5 and 1 min for site Thr495; P < 0.05 vs. 5 and 10 mins for site Ser1177. C) Nitrite production was measured from the media of HUVECs treated with thrombin (10 U/ml) for 10 mins and bradykinin (20 µM) for 20 mins using the Griess Reagent assay. Means are derived from ten separate experiments, normalized to total protein from whole cell lysate. Basal: 6.493 ± 0.128; bradykinin: 9.50 ± 0.171; thrombin: 5.056 ± 0.233. *** P < 0.0001 vs. basal.
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Figure 3. Effects of PAR-2 activating peptide on phosphorylation on eNOS-Thr495 and nitrite production. A) HUVECs were stimulated with the indicated concentrations of SLIGRL for 1 min. B) HUVECs were stimulated with 50 µM of SLIGRL at indicated time points and phosphorylation of eNOS was determined using Western blot analysis with phosphospecific antibodies. The data are presented as mean ± SD for three separate experiments. In panel A, * P < 0.05 vs. control; # P < 0.05 vs. 1 µM SLIGRL; P < 0.05 vs. 10–20 µM SLIGRL. In panel B, * P < 0.05 vs. respective control. C) Nitrite production was measured from the media of HUVECs treated with SLIGRL (50 µM) for 1 min using the Griess Reagent assay. Means are derived from three separate experiments, normalized to total protein from whole cell lysate. Basal: 5.736 ± 0.646; SLIGRL: 12.03 ± 0.002. *** P < 0.0001 vs. basal.
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Figure 4. Effects of PAR-1 inhibitor, SCH-79797, on thrombin and TFLLR mediated eNOS-Thr495 phosphorylation and nitrite production. A) Cells were pretreated in the absence or presence of SCH-79797 (10 µM) one hour prior to stimulation with 20 µM of bradykinin for 20 mins, 10 U/ml of thrombin for 10 mins, or 10 µM of TFLLR for 5 mins. Phosphorylation of eNOS-Thr495 was determined using Western blot analysis with phosphospecific antibodies, normalized to total eNOS. The data are presented as mean ± SD for three separate experiments. In panel A, * P < 0.05 vs. unstimulated control; # P < 0.05 vs. respective stimulated control. B) Nitrite production was measured from the treatment groups in panel A using the Griess Reagent assay. Means are derived from three separate experiments, normalized to total protein from whole cell lysate. * P < 0.05 vs. unstimulated control; # P < 0.05 vs. respective stimulated control.
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Figure 5. Effects of Rho inhibitor, C3 transferase, and dominant negative RhoA on thrombin and TFLLR mediated eNOS-Thr495 phosphorylation and nitrite production. A) HUVECs were stimulated with 20 µM of bradykinin for 20 mins, 10 U/ml of thrombin for 10 mins, or 10 µM of TFLLR for 5 mins either in the absence or presence of C3 transferase (10 U/ml). B) HUVECs were infected with adenovirus encoding dominant negative (dn) RhoA (20 moi) for 48 hrs and then stimulated with 10 U/ml of thrombin for 10 mins or 10 µM of TFLLR for 5 mins. GFP adenovirus was used to define basal levels of transformation. Phosphorylation of eNOS-Thr495 and expression of RhoA was determined using Western blot analysis with antibodies against respective proteins. eNOS-Thr495 phosphorylation was normalized to total eNOS. The data are presented as mean ± SD for three separate experiments. In panels A and B, * P < 0.05 vs. unstimulated control; # P < 0.05 vs. respective stimulated control. C and D) Nitrite production was measured from treatment groups in panels A and B using the Griess Reagent assay. Means are derived from three separate experiments, normalized to total protein from whole cell lysate. * P < 0.05 vs. unstimulated control; # P < 0.05 vs. respective stimulated control.
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Figure 6. Effects of ROCK inhibitor, Y-27632, on eNOS-Thr495 phosphorylation and nitrite production induced by thrombin and TFLLR. A) HUVECs were stimulated with 20 µM of bradykinin for 20 mins, 10 U/ml of thrombin for 10 mins, or 10 µM of TFLLR for 5 mins either in the absence or presence of Y-27632 (50 µM). Phosphorylation of eNOS-Thr495 was determined using Western blot analysis with phosphospecific antibodies, normalized to total eNOS. The data are presented as mean ± SD for three separate experiments. * P < 0.05 vs. unstimulated control; # P < 0.05 vs. respective stimulated control. B) Nitrite production was measured from the treatment groups in panel A using the Griess Reagent assay. Means are derived from three separate experiments, normalized to total protein from whole cell lysate. * P < 0.05 vs. unstimulated control; # P < 0.05 vs. respective stimulated control.
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Figure 7. Proposed signal transduction cascade by which PAR-1 activation leads to eNOS-Thr495 phosphorylation.
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Acknowledgments
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We would like to thank Dr. Douglass Vaughn at Vanderbilt University
Medical Center Division of Cardiology for providing us with
HUVECs, and Dr. Joey Barnett at Vanderbilt University Medical
Center for providing us with dnRho, which was a generous gift
from George E. Davis at the University of Missouri School of
Medicine. We would also like to thank Dr. Shyamali Mukherjee
of Meharry Medical College for assisting us with the Nitrite
Colorimic Assays.
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Footnotes
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We would like to acknowledge that Dr. Motley was supported by
the NIH-SCORE 3SO6GM08037–32.
Received for publication July 30, 2008.
Accepted for publication November 20, 2008.
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References
|
|---|
- Hirano K. The roles of proteinase-activated receptors in the vascular physiology and pathophysiology. Arterioscler Thromb Vasc Biol 27:27–36, 2007.[Abstract/Free Full Text]
- Macfarlane SR, Seatter MJ, Kanke T, Hunter GD, Plevin R. Proteinase-activated receptors. Pharmacol Rev 53:245–282, 2001.[Abstract/Free Full Text]
- O’Brien PJ, Prevost N, Molino M, Holinger MK, Woolkalis MJ, Woulfe DS, Brass LF. Thrombin responses in human endothelial cells. Contributions from receptors other than PAR1 include the transactivation of PAR2 by thrombin-cleaved PAR1. J Biol Chem 275:13502–13509, 2000.[Abstract/Free Full Text]
- Minami T, Sugiyama A, Wu SQ, Abid R, Kodama T, Aird WC. Thrombin and phenotypic modulation of the endothelium. Arterioscler Thromb Vasc Biol 24:41–53, 2004.[Abstract/Free Full Text]
- Fulton D, Gratton JP, Sessa WC. Post-translational control of endothelial nitric oxide synthase: why isn’t calcium/calmodulin enough? J Pharmacol Exp Ther 299:818–824, 2001.[Abstract/Free Full Text]
- Long C, Cook LG, Hamilton SL, Wu G, Mitchell BM. FK506 binding protein 12/12.6 depletion increases endothelial nitric oxide synthase threonine 495 phosphorylation and blood pressure. Hypertension 49:569–576, 2007.[Abstract/Free Full Text]
- Motley ED, Eguchi K, Patterson M, Palmer P, Suzuki H, Eguchi S. Mechanism of endothelial nitric oxide synthase phosphorylation and activation by Thrombin. Hypertension 49:429–431, 2007.[Free Full Text]
- Bayless KJ, Davis GE. The Cdc42 and Rac1 GTPases are required for capillary lumen formation in three-dimensional extracellular matrices. J Cell Sci 115:1123–1136, 2002.[Abstract/Free Full Text]
- Palmer RM, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327:524–526, 1987.[Medline]
- Fleming I, Fisslthaler B, Dimmeler S, Kemp BE, Busse R. Phosphorylation of Thr495 regulates Ca2+/Calmodulin-dependent endothelial nitric oxide synthase activity. Circ Res 88:E68–E75, 2001.[Medline]
- Korkmaz Y, Bloch W, Steinritz D, Baumann MA, Addicks K, Schneider K, Raab WH. Bradykinin mediates phosphorylation of eNOS in odontoblasts. J Dent Res 85:536–541, 2006.[Abstract/Free Full Text]
- Eto M, Barandiér C, Rathgeb L, Kozai T, Joch H, Yang Z, Lüscher TF. Thrombin suppresses endothelial nitric oxide synthase and upregulates endothelin-converting enzyme-1 expression by distinct pathways: role of Rho/ROCK and mitogen-activated protein kinase. Circ Res 89:583–590, 2001.[Abstract/Free Full Text]
- Ming XF, Viswambharan H, Barandier C, Ruffieux J, Kaibuchi K, Rusconi S, Yang Z. Rho GTPase/Rho kinase negatively regulates endothelial nitric oxide synthase phosphorylation through the inhibition of protein kinase B/Akt in human endothelial cells. Mol Cell Biol 22:8467–8477, 2002.[Abstract/Free Full Text]
- Sugimoto M, Nakayama M, Goto TM, Amano M, Komori K, Kaibuchi K. Rho-kinase phosphorylates eNOS at threonine 495 in endothelial cells. Biochem Biophys Res Commun 361:462–467, 2007.[Medline]
- Steinberg SF. The cardiovascular actions of protease-activated receptors. Mol Pharmacol 67:2–11, 2004.[Medline]
- Trebicka J, Hennenberg M, Laleman W, Shelest N, Biecker E, Schepke M, Nevens F, Sauerbruch T, Heller J. Atorvastatin lowers portal pressure in cirrhotic rats by inhibition of RhoA/Rho-kinase and activation of endothelial nitric oxide synthase. Hepatology 46:242–253, 2007.[Medline]
- Bernier SG, Haldar S, Michel T. Bradykinin-regulated interactions of the mitogen-activated protein kinase pathway with the endothelial nitric-oxide synthase. J Biol Chem 275:30707–30715, 2000.[Abstract/Free Full Text]
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Abstract
Introduction
