JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M405547200 on August 27, 2004

J. Biol. Chem., Vol. 279, Issue 45, 46637-46643, November 5, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/45/46637    most recent
M405547200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Anter, E.
Right arrow Articles by Keaney, J. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Anter, E.
Right arrow Articles by Keaney, J. F., Jr.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Activation of Endothelial Nitric-oxide Synthase by the p38 MAPK in Response to Black Tea Polyphenols*

Elad Anter{ddagger}, Shane R. Thomas{ddagger}§, Eberhard Schulz{ddagger}, Oz M. Shapira¶, Joseph A. Vita{ddagger}, and John F. Keaney, Jr.{ddagger}||

From the {ddagger}Evans Memorial Department of Medicine, the Whitaker Cardiovascular Institute and the Department of Cardiothoracic Surgery, Boston University School of Medicine, Boston, Massachussetts 02118

Received for publication, May 18, 2004 , and in revised form, August 17, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Black tea improves endothelial function in patients with coronary artery disease. We sought to determine the responsible components of black tea and elucidate the underlying cell signaling mechanisms. We exposed porcine aortic endothelial cells to components of black tea and found that the polyphenol fraction acutely enhanced nitric oxide bioactivity. This effect involved endothelial nitric-oxide synthase (eNOS) phosphorylation at Ser-1177 and dephosphorylation at Thr-495, consistent with increased eNOS activity. These effects were calcium-dependent, as removal of extracellular calcium prevented eNOS phosphorylation at Ser-1177, whereas inhibition of intracellular calcium mobilization with TMB-8 blunted Thr-495 dephosphorylation. Black tea polyphenol-induced eNOS activation appeared dependent upon the phosphatidylinositol 3-kinase-Akt pathway, as it was significantly inhibited by LY294002 and a dominant negative Akt, respectively. Pharmacological inhibition of p38 mitogen-activated protein kinase (p38 MAPK) with either SB202190 or SB203580 as well as overexpression of a dominant negative p38 MAPK{alpha} attenuated both eNOS activation and phosphorylation changes in response to black tea polyphenols. Inhibition of p38 MAPK{alpha} also blunted Akt activation in response to black tea polyphenols, suggesting that p38{alpha} MAPK is upstream of Akt in this pathway. Finally, a constitutively active mutant of MKK6bE, an upstream kinase for p38 MAPK, enhanced both the basal and stimulated activity of Akt, leading to increased eNOS activity. Taken together, these data identify the p38 MAPK as an upstream component of Akt-mediated eNOS activation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The general appreciation that oxidative stress is involved in the pathogenesis of cardiovascular diseases has prompted interest in natural classes of antioxidants. One such class of natural antioxidants is the flavonoid family, a group of polyphenolic substances found mainly in fruit, vegetables, wine, and tea (1). Being the second most consumed beverage world-wide aside from water, tea has become a subject of many epidemiological studies, suggesting an inverse relationship between tea consumption and cardiovascular diseases (for review, see Ref 2). One proposed mechanism for the benefit of such dietary flavonoids is their antioxidant properties that include the scavenging of reactive oxygen species (3) and the inhibition of lipid peroxidation (4). This latter property of flavonoids suggests they may inhibit low density lipoprotein oxidation, an event implicated in atherosclerosis (5). However, clinical studies in healthy human subjects indicate that tea flavonoids have a limited impact on this process ex vivo (6).

There is compelling evidence that another target for flavonoids is the endothelium (reviewed in Refs. 1 and 7). Normally, the endothelium regulates vasomotor tone, platelet activity, leukocyte adhesion, and vascular smooth muscle proliferation via the release of several paracrine factors, including nitric oxide (NO)1 (8). These functions of the endothelium are impaired in the setting of atherosclerosis and its associated vascular diseases (8). Moreover, individuals with the greatest impairment of endothelial function are at the greatest risk of cardiovascular events (9), suggesting that endothelial dysfunction is, in part, responsible for the clinical activity of atherosclerosis. In this regard, we have found that endothelial NO bioactivity is enhanced in patients with atherosclerosis by either the acute or chronic administration of black tea (10). The purpose of the current investigation was to determine the components of black tea responsible for this effect and to elucidate the underlying cell signaling events involved in the effect of black tea.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—M-199 medium, L-glutamine, penicillin, streptomycin, and fetal bovine serum (FBS) were obtained from Invitrogen. We obtained LY294002, SB202190, SB203580, p38 inhibitor III, W7 hydrochloride, and TMB-8 hydrochloride from Calbiochem. Polyclonal antibodies directed against Akt, phospho-Akt (Ser-473), phospho-GSK-3{alpha}/{beta} (Ser-21/9), p38 mitogen-activated protein kinase (MAPK), phospho-p38 MAPK (Thr-180 and Tyr-182), phospho-MAPK-activated protein kinase-2 (phospho-MAPKAPK-2) (Thr-222), extracellular signal-regulated kinase (ERK), phospho-ERK (Thr-202 and Tyr-204), and anti-hemagglutinin were obtained from Cell Signaling Technology (Beverly, MA). Polyclonal antibodies directed against phospho-eNOS (Thr-495) and phospho-eNOS (Ser-1179) were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-eNOS monoclonal antibody was obtained from Transduction Laboratories (Lexington, KY). L-[3H]arginine (1 mCi; 53mCi/mmol) was obtained from PerkinElmer Life Sciences, and cGMP assay kits were from Cayman (Ann Arbor, MI). Black tea polyphenols and black tea fractions were provided by Unilever, Inc. All other reagents were obtained from Sigma.

Endothelial Cell Culture—Porcine aortic endothelial cells (PAECs) were harvested and cultured on fibronectin-coated tissue culture flasks in M-199 medium supplemented with 20% FBS, 50 µg/ml heparin sulfate, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. For experiments, confluent PAECs were used between passages 4 and 10. Prior to all experiments, PAECs were placed in serum-free and phenol-free media (Opti-MEM, Invitrogen) for 6 h and then washed twice in HEPES-buffered physiologic salt solution (PSS) containing 22 mM HEPES (pH 7.4), 124 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.5 mM CaCl2, 0.16 mM Na2PO4, 0.4 mM NaH2PO4, 5 mM NaHCO3, and 5.6 mM D-glucose. Cells were then incubated in HEPES-buffered PSS 30 min prior to the addition of the black tea compounds as indicated.

L-[3H]Arginine to L-[3H]Citrulline Conversion Assay—Determination of eNOS catalytic activity in intact PAECs was performed by measurement of the conversion of L-[3H]arginine to L-[3H]citrulline, which was sensitive to L-nitro-arginine methyl ester (L-NAME). Confluent PAECs in 100-mm culture dishes were washed and incubated in PSS for 30 min followed by the addition of 10 µM L-arginine plus 3.3µCi of L-[3H]arginine. PAECs were then treated with the agonist of interest or buffer for 15 min and then washed with PSS and lysed with 200 µl of 100% ethanol, after which 2 ml of stop buffer (20 mM sodium acetate anhydrous, pH 5.5, 1 mM L-citrulline, 2 mM EDTA, and 2 mM EGTA) was added. The lysates was then subjected to anion exchange chromatography using 2-ml Dowex AG50W-X8 columns (Bio-Rad) equilibrated with stop buffer. L-Citrulline was eluted three times with 2 ml of stop buffer, and the eluent was collected for the determination of L-[3H]citrulline by liquid scintillation counting. Data are reported as the extent of conversion of L-[3H]arginine to L-[3H]citrulline that is sensitive to pretreatment of the PAECs for 30 min with L-NAME (500 µM) and is expressed as dpm/106 PAECs.

Estimation of Endothelium-derived NO—Confluent PAECs in six-well plates were equilibrated for 30 min in PSS supplemented with 250 µM 3-isobutyl-1-methylxanthine (IBMX) and 250 µM L-arginine in the absence or presence of L-NAME (500 µM) or the inhibitor of interest. Equilibrated cells were then exposed to the black tea compound or vehicle alone for 30 min before the cells were lysed by the addition of 6% ice-cold trichloroacetic acid. Cell lysates were then subjected to centrifugation at 12,000 x g for 10 min, and determination of cGMP in supernatants was performed as described previously (11). The cell pellet protein content was determined by the BCA protein assay (Pierce) after solubilization with 1 N KOH.

Immunoprecipitation and Western Blotting—Endothelial cells were washed with PSS and incubated in lysis buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM {beta}-glycerophosphate, 1 mM Na3VO4, 1 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride for 30 min on ice followed by brief sonication. Cell lysates were centrifuged (12,000 x g for 15 min at 4 °C), and the supernatant was either resuspended in loading buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 200 mM dithiothreitol, 20% glycerol, and 0.2% bromphenol blue) or used for immunoprecipitation. For eNOS immunoprecipitation, lysates were incubated with eNOS monoclonal antibody (5 µg/ml) rotating for 16 h at 4 °C followed by a 1-h incubation with protein A/G-agarose. Following centrifugation, the immunoprecipitates were washed three times with lysis buffer, resuspended in loading buffer, boiled for 5 min, and subjected to SDS-polyacrylamide gel electrophoresis. Resolved proteins were transferred at 4 °C to a nitrocellulose membrane (Hybond; Amersham Biosciences), and the membrane was blocked with 4% nonfat dry milk in TBS-Tween buffer (20 mM Tris-HCl, pH 7.4, 135 mM NaCl, and 0.1% Tween) for 30 min. Blocked membranes were incubated with primary antibody (1:1000 dilution) in 4% nonfat dry milk in TBS-Tween buffer at 4 °C for 16 h. Membranes were washed with TBS-Tween and incubated at room temperature for 1 h with a horseradish peroxidase-conjugated secondary antibody (1:7500 dilution) in TBS-Tween containing 4% nonfat dry milk. Membranes were washed in TBS-Tween, and proteins were detected using an enhanced chemiluminescence detection kit (New England Biolabs, Beverly, MA).

Recombinant Adenoviral Transfection—The recombinant adenoviral vectors expressing dominant negative Akt (T308A and S473A), which cannot be activated by phosphorylation (12), and the constitutively active Akt (Myr-Akt) was kindly provided by Ken Walsh, Boston University (13). Recombinant adenovirus-expressing activated MKK6 (MKK6bE) was a kind gift from Dr. Jiahuia Han, Scripps Institute (14). The adenoviral vector expressing dominant negative p38 MAP kinase {alpha}-isoform was a kind gift of Dr. Yinbin Wang, UCLA (14). The transfection efficiency of the dominant-negative Akt and Myr-Akt was confirmed by immunoblotting for total protein, and transfection of the MKK6bE adenovirus was determined by immunoblotting for the hemagglutinin tag. Transfection of the FLAG-tagged dominant negative isoform of p38 MAPK was assessed by immunoblotting for the FLAG protein. PAECs were infected in medium without FBS for 2 h, washed, and incubated in fresh medium with 10% FBS for 24–36 h prior to experimentation.

Statistical Analysis—All numerical data are presented as means ± S.E. Western blots shown are representative of three or more independent experiments. For parametric data, comparisons among treatment groups were performed with one-way analysis of variance and an appropriate post hoc comparison. Instances involving only two comparisons were evaluated with a Student's t test. Instances involving more then two comparisons were evaluated using analysis of variance. Statistical significance was accepted if the null hypothesis was rejected with p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The Polyphenolic Fraction of Black Tea Activates eNOS—We first assessed dose- and time-dependent effects of black tea on endothelial NO production by the increase in cellular cGMP content (Fig. 1, A and B). Black tea-induced cGMP accumulation appeared to result from eNOS activation, as it was also characterized by the enhanced conversion of L-[3H]arginine to L-[3H]citrulline and was sensitive to the NOS inhibitor L-NAME (Fig. 1C). Treatment with inactive enantiomer, D-NAME, had no effect on the black tea-induced eNOS activation (data not shown). The effect of black tea was entirely reproduced by the polyphenolic fraction (Fig. 1, C and D) over a physiologic concentration range (~50–200 ng/ml) (10, 15). Black tea enhanced eNOS activity by 4–5-fold (80 ± 12% of the maximal eNOS stimulation afforded by 1 µM A23187 [GenBank] ; p < 0.05). Because polyphenols can produce low concentrations of H2O2 (16) that can lead to eNOS activation (17), we tested the effect of black tea polyphenols in the presence of polyethylene glycol catalase (100 units/ml) and found no effect on eNOS activity (data not shown), excluding H2O2 as the mechanism for our observations.



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 1.
Black tea promotes eNOS activity in PAECs. A, PAECs were incubated for 30 min in PSS containing 250 µM IBMX and 200 µM L-arginine. Cells were then exposed to the indicated concentrations of black tea for 5 min, and cGMP accumulation was determined as described under "Experimental Procedures." B, PAECs were incubated with black tea (100 ng/ml) for the indicated time intervals, and cGMP accumulation was determined. C and D, PAECs were exposed to buffer alone as control (CTL), black tea (TEA; 100 ng/ml), black tea polyphenols (BTP; 100 ng/ml), or A23187 [GenBank] (1 µM) in the presence or absence of 500 µM L-NAME, and L-[3H]citrulline production (C) or cGMP accumulation (D) was determined as described under "Experimental Procedures." Data represent the mean ± S.E. of 10–12 independent experiments; *, p < 0.05 versus with L-NAME.

 
Black Tea Polyphenols Change the Phosphorylation Status of eNOS—The phosphorylation status of eNOS at residues Ser-1177 (18, 19) and Thr-495 (17, 20) has important implications for its enzymatic activity. Therefore, PAECs were exposed to black tea polyphenols, and eNOS phosphorylation was examined. Under resting conditions, PAECs exhibit little or no phosphorylation of eNOS at Ser-1177 but significant phosphorylation at Thr-495 (Fig. 2A). Treatment with black tea polyphenols resulted in a time-dependent increase in eNOS phosphorylation at Ser-1177 and dephosphorylation at Thr-495 (Fig. 2A), which is consistent with eNOS activation. We next investigated the upstream kinases involved in eNOS phosphorylation in response to black tea polyphenols.



View larger version (52K):
[in this window]
[in a new window]
  		FIG. 2.
Black tea polyphenols induce eNOS and Akt phosphorylation. A, PAECs were treated with black tea polyphenols (100 ng/ml) for the indicated times, cells were lysed, eNOS was immunoprecipitated, and phosphorylation status at Ser-1177 and Thr-495 was assessed by immunoblotting with phosphorylation-specific antibodies. An anti-eNOS monoclonal antibody was used to determine total eNOS in the immunoprecipitates. B, PAECs were treated as described for panel A, and the total lysates were analyzed for the phosphorylation status of Akt (P-Akt; Ser-473), GSK-3{alpha}/{beta} (P-GSK-3{alpha}; Ser-21/9), and total Akt. Blots are representative of 4–5 independent experiments

 
Black Tea Polyphenols Activate eNOS through a Mechanism Involving the Phosphoinositide 3-Kinase (PI 3-K)/Akt Signaling Pathway—Phosphorylation of eNOS on Ser-1177 is characteristic of the serine kinase Akt (18, 19, 21, 22). To probe the involvement of Akt, PAECs were treated with black tea polyphenols, and the lysates were assayed for Akt activation using an antibody specific for activated Akt (i.e. phosphorylated at Ser-473). We observed time-dependent activation of Akt by black tea polyphenols, which closely approximated the time course of eNOS phosphorylation at Ser-1177 (Fig. 2B). To specifically address the role of Akt in black tea polyphenol-induced eNOS stimulation, we overexpressed a dominant negative mutant Akt isoform via adenoviral vector transfection. Dominant negative Akt significantly attenuated black tea polyphenol-induced changes in eNOS Ser-1177 phosphorylation (Fig. 3A), and its activation was determined both by the increase in cellular cGMP (Fig 3B) and the conversion of L-[3H] arginine to L-[3H] citrulline (Fig. 3C).



View larger version (34K):
[in this window]
[in a new window]
  		FIG. 3.
Polyphenol-induced eNOS activation involves Akt. A, PAECs were transfected (multiplicity of infection of 50) with an adenovirus containing either dominant negative Akt (dN Akt) LacZ (–) for 24–36 h. PAECs were then equilibrated in PSS for 30 min followed by treatment with 100 ng/ml black tea polyphenols or vehicle for 5 min. The cells were then lysed, eNOS was immunoprecipitated, phosphorylation status was assessed, and total eNOS was detected as described for Fig. 2A. Supernatants were analyzed for phospho-GSK-3{alpha}/{beta} (P-GSK3{alpha}; Ser-21/9), and total Akt was assessed by immunoblotting. B, PAECs treated as described for panel A were incubated for 30 min in PSS containing 250 µM IBMX and 200 µM L-arginine prior to a 5-min treatment with buffer alone or black tea polyphenols (BTP; 100 ng/ml), and NO bioactivity was determined by cGMP accumulation. C, PAECs were treated as described for panel B, and eNOS catalytic activity was determined as L-[3H]citrulline formation as described under "Experimental Procedures." Data represent the mean ± S.E. of 4–6 independent experiments and are expressed as the percentage of the values obtained for black tea polyphenols alone. *, p < 0.05 versus no polyphenols and LacZ adenovirus.

 
Because Akt is a downstream target of PI 3-K (23), we examined the role of PI 3-K in black tea polyphenol-induced eNOS activation. Treatment of PAECs with the PI 3-K inhibitor LY294002 significantly attenuated the black tea polyphenol-induced changes in eNOS phosphorylation and Akt activation (Fig. 4A). This effect had important implications for eNOS activity, because both cellular cGMP accumulation and L-[3H]arginine conversion to L-[3H]citrulline in response to black tea polyphenols were abrogated (Fig. 4, B and C). These data implicate PI 3-K as an upstream signal for both Ser-1177 phosphorylation and Thr-495 dephosphorylation in the response to black tea polyphenols.



View larger version (35K):
[in this window]
[in a new window]
  		FIG. 4.
Black tea polyphenol-induced eNOS activation is PI 3-kinase-dependent. A, PAECs in PSS were incubated in the absence or presence of LY294002 (20 µM) for 30 min prior to incubation with buffer alone or black tea polyphenols (BTP; 100 ng/ml for 5 min). PAECs were then lysed, eNOS was immunoprecipitated, and total eNOS and its phosphorylation status were assessed as described for Fig. 2A. Supernatants were analyzed for phosphorylated and total Akt by immunoblotting. B, PAECs were incubated in PSS containing 250 µM IBMX and 200 µM L-arginine with (+) or without (–) LY294002 (LY;20 µM) for 30 min prior to buffer or black tea polyphenol (BTP) treatment (100 ng/ml; 5 min), and NO bioactivity was determined by cGMP accumulation as described for Fig. 1. C, PAECs in PSS were incubated as described for panel A before treatment with buffer alone or black tea polyphenols (BTP) (100 ng/ml; 15 min), and eNOS catalytic activity was determined by L-[3H]citrulline formation as described under "Experimental Procedures." Data represent the mean ± S.E. of 6–8 independent experiments and are expressed as the percentage of the values obtained for black tea polyphenols alone. *, p < 0.05 versus vehicle alone

 
Black Tea Polyphenol-induced eNOS Activation Is Dependent on Both Calcium and Calmodulin—Because eNOS activation can be either calcium-dependent or -independent (24), we investigated the role of calcium on eNOS activation by black tea polyphenols. Stimulation of PAECs with black tea polyphenols in a calcium-free PSS solution selectively attenuated eNOS Ser-1177 phosphorylation with no effect on Thr-495 dephosphorylation (Fig. 5A). This condition induced only a 2-fold increase in cGMP synthesis (p < 0.05 versus without polyphenols) compared with a 5-fold increase in the presence of extracellular calcium (Fig. 5B; p < 0.05 versus with extracellular calcium). In contrast, inhibition of intracellular calcium release with TMB8 had no impact on Ser-1177 phosphorylation in response to black tea polyphenols and completely abrogated Thr-495 dephosphorylation (Fig. 5A). This condition was associated with a 2.5-fold increase in cGMP (p < 0.05 versus without polyphenols) that was significantly less than that without TMB8 (p < 0.05 versus polyphenols alone; Fig. 5B). The combined inhibition of both intracellular and extracellular calcium transients completely inhibited all responses to black tea polyphenols (Fig. 5B). Thus, both intracellular and extracellular calcium are required for full changes in eNOS phosphorylation and activation in response to black tea polyphenols.



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 5.
The effect of calcium and calmodulin on eNOS activation by black tea polyphenols. A and B, PAECs were incubated for 30 min in PSS with (+) or without (–) CaCl2 (EC Ca2+) or TMB8 as indicated. PAECs were then treated with or without or black tea polyphenols (BTP; 100 ng/ml) for 5 min prior to the assessment of eNOS phosphorylation status (A) or cGMP accumulation (B) as described in the Fig. 4 legend. C and D, PAECs were incubated in the presence (+) or absence (–) of W7 (10 µM) prior to incubation with 100 ng/ml black tea polyphenol (BTP) (5 min) followed by assessment of eNOS phosphorylation and cGMP accumulation as described in the Fig. 4 legend. Data represent the mean ± S.E. of 3–6 independent experiments and are expressed as the percentage of the values with black tea polyphenols in calcium-containing buffer. *, p < 0.05 versus vehicle control with extracellular calcium; {dagger}, p < 0.05 versus with BTP and extracellular calcium alone.

 
We next examined the role of calmodulin in the black tea polyphenol-mediated eNOS stimulation. Inhibition of calmodulin with W7 had no impact on changes in eNOS phosphorylation induced by black tea polyphenols (Fig. 5C), whereas eNOS activation was completely inhibited as judged by cGMP accumulation (Fig. 5D). Thus, although calmodulin is required for eNOS activation, it is not required for polyphenol-induced changes in eNOS phosphorylation.

The Role of p38MAP Kinase in the Response to Black Tea Polyphenols—Previous data implicate MAP kinases in eNOS activation by high density lipoprotein (25) and H2O2 (26). Therefore, we probed lysates with antibodies specific for the activated forms ERK, p38 MAPK, and c-Jun N-terminal kinase. Lysates from black tea polyphenol-treated PAECs demonstrated rapid phosphorylation of p38 MAPK and its down-stream target, MAPKAPK-2 (Fig. 6A), which was temporally reminiscent of eNOS phosphorylation at Ser-1177 (Fig. 2A). Consistent with this notion, pharmacological inhibition of p38 MAPK with either SB202190 or SB203580 attenuated polyphenol-induced eNOS phosphorylation on Ser-1177 (Fig. 6B), and eNOS activation was measured by cGMP accumulation (Fig. 6C) and the conversion of L-[3H]arginine to L-[3H]citrulline (Fig. 6D). In contrast, we did not observe any reproducible activation of ERK or c-Jun N-terminal kinase in response to black tea polyphenols (data not shown).



View larger version (37K):
[in this window]
[in a new window]
  		FIG. 6.
Role of p38 MAPK in eNOS activation by black tea polyphenols. A, PAECs were treated with 100 ng/ml black tea polyphenols for the indicated times followed by cell lysis and the assessment of phosphorylated forms of p38 MAPK (Thr-180 and Tyr-182) and MAPKAPK-2 (Thr-222) as well as total p38 MAPK by immunoblotting. B, PAECs in PSS were incubated (30 min) in the presence (+) or absence (–) of SB202190 (5 µM) or SB203580 (7.5 µM) as indicated prior to treatment with buffer alone or black tea polyphenols (BTP; 100 ng/ml for 5 min). Cells were then lysed, eNOS was immunoprecipitated, and eNOS phosphorylation status and total eNOS were determined by immunoblotting as described for Fig. 2A. Activation of p38 MAPK was assessed by immunoblotting supernatants for phospho-MAPKAPK-2 (P-MAPKAPK-2). C and D, PAECs in PSS containing 250 µM IBMX and 200 µM L-arginine were incubated with vehicle (CTL), SB202190 (5 µM), or SB203580 (7.5 µM) as indicated prior to treatment with buffer alone or black tea polyphenols (BTP; 100 ng/ml). After 5 min, cells lysates were analyzed for cGMP (C) or L-[3H]citrulline formation (D) as described in the Fig. 5 legend. Data represent the mean ± S.E. of 8–12 independent experiments and are expressed as the percentage of the values obtained for black tea polyphenols alone. *, p < 0.05 versus control without black tea polyphenols; {dagger}, p < 0.05 versus control with black tea polyphenols.

 
To confirm the role of p38MAPK, we overexpressed a dominant negative isoform of p38{alpha} MAPK and found an inhibition of Akt and eNOS Ser-1177 phosphorylation in response to black tea polyphenols (Fig. 7A) that was associated with a significant inhibition of eNOS activation (Fig. 7B). Using the converse approach, the activation of p38MAPK with a constitutively active mutant of MKK6 (MKK6bE) enhanced both basal and black tea polyphenol-induced Akt activation and eNOS Ser-1177 phosphorylation (Fig. 7C), which is consistent with an observed increased eNOS activity (Fig. 7D).



View larger version (30K):
[in this window]
[in a new window]
  		FIG. 7.
The role of p38 MAPK in black tea polyphenol-induced eNOS activation. A, PAECs were transfected (multiplicity of infection of 50) with dominant negative p38 MAPK{alpha} (dN p38{alpha}) or LacZ for 24–36 h. PAECs were then equilibrated in PSS (30 min) followed by treatment with 100 ng/ml black tea polyphenols (BTP) or vehicle for 5 min. The cells were then lysed, and the lysates were probed for the phosphorylation of eNOS (Ser-1177 and Thr-495), Akt (P-Akt), p38 MAPK, and MAPKAPK-2 (P-MAPKAPK) using immunoblotting with phosphorylation-specific antibodies. Total eNOS, Akt, and FLAG (dN P38{alpha}) were also assessed by immunoblotting. B, PAECs transfected as for panel A were treated with buffer alone or with black tea polyphenols (BTP; 100 ng/ml) for 30 min prior to the assessment of eNOS catalytic activity by L-[3H]citrulline formation as described under "Experimental Procedures." C, PAECs were transfected (multiplicity of infection of 50) with LacZ or a constitutively active mutant of MKK6 (MKK6bE) for 24–36 h, equilibrated in PSS (30 min), and treated with 100 ng/ml black tea polyphenols or vehicle for 5 min. Cells were then lysed, and the lysates were probed for total and phosphorylated eNOS, Akt (P-Akt), and p38 MAPK (P-p38). Expression of MKK6bE was assessed by immunoblotting for the hemagglutinin tag (HA-Tag). D, PAECs, as described for panel C, were treated with buffer alone or with black tea polyphenols (BTP; 100 ng/ml) for 30 min prior to the assessment of eNOS catalytic activity as described for panel B. Data represent the mean ± S.E. of 6–8 independent experiments and are expressed as the percentage of the values obtained for BTP with the LacZ adenovirus. *, p < 0.05 versus LacZ alone; {dagger}, p < 0.05 versus black tea polyphenols with LacZ alone.

 
Because there is evidence that Akt can regulate p38 MAPK (27), we examined the relation between Akt and p38 in our system. Infection of PAECs with MKK6bE lead to activation of p38 MAPK, Akt, and eNOS (Fig. 8, A and B). The inhibition of PI 3-K with LY294002 had no impact on MKK6bE-induced p38 MAPK activation (Fig. 8A) but significantly attenuated Akt and eNOS activation (Fig. 8, A and B), suggesting that PI 3-K is a downstream target of p38 MAPK in this system. Consistent with this finding, neither the dominant negative nor the constitutively active Akt isoforms had any impact on black tea polyphenol-induced p38 MAPK activation (Fig. 9, A and B). However, the overexpression of dominant negative Akt increased basal p38 MAPK phosphorylation, whereas constitutively active Akt had the opposite effect (Fig. 9, A and B). Consistent with prior reports (27), these data suggest that p38 MAPK and Akt may share a reciprocal regulatory relationship that may have important implications for eNOS activation.



View larger version (41K):
[in this window]
[in a new window]
  		FIG. 8.
The p38MAPK activates Akt via PI 3-kinase. A, PAECs were transfected with constitutively active MKK6 (MKK6bE) or LacZ as described in the Fig. 7 legend for 24–36 h. The cells were then incubated for 30 min with vehicle or LY294002 (LY; 20 µM) and lysed, and the lysates were probed for phosphorylation of eNOS (Ser-1177 and Thr-495), Akt, and p38 MAPK using immunoblotting with phosphorylation-specific antibodies. Total eNOS, Akt, and hemagglutinin (HA; MKK6bE tag) were also assessed by immunoblotting. Blots are representative of three independent experiments. B, PAECs, treated as described for panel A, were assessed for eNOS catalytic activity as described in "Experimental Procedures." Data are mean ± S.E. of three experiments. *, p < 0.05 versus vehicle and LacZ control. BTP, black tea polyphenol.

 



View larger version (49K):
[in this window]
[in a new window]
  		FIG. 9.
Akt down-regulates basal P38 MAPK activity. PAECs were transfected (multiplicity of infection of 50) with an adenovirus containing LacZ or dominant negative (dN Akt) (A) or constitutively active (Myr Akt)(B) forms of Akt. PAECs were then equilibrated in PSS (30 min) followed by treatment with 100 ng/ml black tea polyphenols (BTP) or vehicle for 5 min. Cells were then lysed, and the lysates were probed for phosphorylation of p38 MAPK (P-p38), its downstream target MAPKAPK-2 (P-MAPKAPK-2), and the Akt target GSK3{alpha} (P-GSK3{alpha}). Data is representative of three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we examined the short term effects of black tea on NO bioactivity in cultured endothelial cells. We found that black tea promoted both eNOS catalytic activity and NO bio-activity in cultured endothelial cells. This activity of black tea could be entirely reproduced by the polyphenol fraction and appeared to involve coordinated eNOS phosphorylation at Ser-1177 and dephosphorylation at Thr-495. Upstream mediators for these events included PI 3-kinase and Akt, as well as a prominent role for p38 MAPK. In support of the latter, we found that both pharmacological inhibition by SB 202190 or SB203580 and the overexpression of dominant negative p38 MAPK significantly inhibited the response to black tea polyphenols. Moreover, we could qualitatively reproduce the effect of black tea with a constitutively active mutant of MKK6 that produced p38 MAPK activation independent of polyphenol exposure. The activation of p38 MAPK led to an Akt-dependent increase in eNOS activity. If we consider that the range of black tea polyphenol levels utilized in this study corresponds to that achieved in vivo with the consumption of 1–7 cups of black tea (10, 15), these data suggest that the findings presented here are applicable to clinical studies with black tea.

In this regard, there is data showing that the polyphenols found in black tea have physiological actions on the endothelium in vivo. Patients with coronary artery disease exhibit impaired endothelial function in both the coronary (28) and brachial (29) arteries. We have demonstrated previously that both acute and chronic tea consumption improves flow-mediated, endothelium-dependent, arterial relaxation in the brachial artery (10). In light of observations that brachial artery flow-mediated dilation is dependent on endothelium-derived NO (30), the data presented here provide some insight into these clinical studies. Indeed, flow-mediated activation of eNOS involves Akt-dependent phosphorylation (22), and our data indicate that black tea polyphenols also induce Akt activation (Fig. 2B). Thus, one might speculate that black tea and shear could produce synergistic Akt activation, thereby explaining the effect of black tea on endothelial function. Thus, our data provide evidence for direct effects of black tea polyphenols on endothelial cells that could, in part, explain the clinical indicating observations that tea improves endothelial function in patients with vascular disease.

Our findings that the polyphenolic fraction of black tea promotes eNOS activity are in agreement with published data on other sources of polyphenols. For example, resveretrol is a polyphenol found in red wine that increases both the expression and activity of eNOS in endothelial cells (31). A recent study with the green tea constituent epigallocatechin-3-gallate demonstrated activation of eNOS through a mechanism that involved both Akt and protein kinase A (32). With regard to the former, we found that the polyphenolic fraction of black tea activated Akt in a PI-3K-dependent manner. The major contribution of this study is the identification of p38 MAPK as an upstream mediator of Akt activation in response to polyphenols. Indeed, these findings prompt speculation that p38 MAPK may be involved in the regulation of eNOS activity in response to other stimuli as well.

Although it is generally thought that MAP kinases do not play a crucial role in eNOS regulation, our studies are reminiscent of reports on estrogen demonstrating that it enhances endothelial NO release in a MAP kinase-dependent manner (33). Although that data implicated ERK in estrogen-mediated eNOS activation, we only observed p38 MAPK activation in response to black tea polyphenols. Consistent with this observation, the inhibition of p38 MAPK attenuated eNOS activation in response to black tea polyphenols, whereas ERK inhibition had no impact (data not shown). The p38 MAPK has been generally shown to be activated in response to inflammatory cytokines, endotoxins, and osmotic stress, but its role in endothelial cells is incompletely defined. Our data suggest a novel role for p38 MAPK as an upstream mediator of Akt-dependent eNOS activation. It is important to note, however, that we could only approximate the extent of polyphenol-induced eNOS activation with constitutively active MKK6. The precise mechanism(s) for these observations is not clear, but may indicate that p38 MAPK and Akt activate eNOS in a cooperative manner rather than a strictly linear kinase cascade. A full accounting of the p38 MAPK role in eNOS regulation will require further study.

We observed that the effect of black tea polyphenols involved coordinated phosphorylation of eNOS on Ser-1177 and dephosphorylation at Thr-495, two events that have been reported previously to correlate directly with eNOS activity status (17, 20, 34). However, the relative importance of Ser-1177 phosphorylation versus Thr-495 dephosphorylation with respect to the stimulation of eNOS activity remains controversial. Our data sheds some light on this issue, as transfection with dominant negative Akt abolished only eNOS Ser-1177 phosphorylation, yet eNOS catalytic activation was inhibited by ~50% (Fig. 3, B and C). Considering that dominant negative Akt did not alter polyphenol-induced Thr-495 dephosphorylation, these data suggest that dephosphorylation provides an independent contribution to eNOS stimulation. In support of this hypothesis, the inhibition of intracellular calcium release with TMB8 abrogated Thr-495 dephosphorylation without altering events at Ser-1177, and this manipulation also produced ~50% inhibition of the response to black tea polyphenols. These data are in general agreement with bradykinin-induced eNOS stimulation that results, in part, through dephosphorylation at Thr-495, which appears to sensitize the enzyme to calcium/calmodulin (20, 34). Furthermore, there is also recent data that Thr-495 phosphorylation dictates, in part, the relative production of NO or superoxide from the enzyme (35). Thus, our data indicate that Thr-495 dephosphorylation is dependent upon intracellular calcium release and that this event contributes importantly to eNOS activation in response to black tea polyphenols.

The data presented here also add to our understanding of eNOS regulation with regard to calcium. Considerable data indicate that eNOS is activated in association with changes in cytosolic calcium concentration in response to agonists such as bradykinin (34), estradiol (36), and vascular endothelial growth factor (37). Although it is known that both intracellular and extracellular calcium play a role in eNOS activation (for review, see Ref. 38), their relative contributions to phosphorylation events are not yet clear. In this study, black tea polyphenol-induced eNOS stimulation was calcium-calmodulindependent, although calmodulin did not play a role in modulating eNOS phosphorylation. Rather, we found that eNOS Ser-1177 phosphorylation required extracellular calcium, but Thr-495 dephosphorylation was dependent upon the availability of intracellular calcium. With regard to the latter, previous studies indicate that eNOS dephosphorylation at Thr-495 may be mediated by protein phosphatase 1 (34) or calcineurin (20) in a calcium-dependent manner. In preliminary studies, we did not find any effect of cyclosporin A on Thr-495 dephosphorylation by black tea polyphenols, suggesting that calcineurin is not involved in black tea polyphenol-induced Thr-495 dephosphorylation. Experiments with calyculin A were confounded by the toxicity of the compound to endothelial cells. Thus, the nature of the calcium-dependent phosphatase induced by black tea polyphenols remains to be uncovered.

In summary, the data presented here indicate that black tea stimulates eNOS catalytic activity largely as a consequence of its polyphenolic fraction. The mechanisms of black tea polyphenol-induced eNOS stimulation involve p38 MAPK-mediated activation of the PI 3-K/Akt pathway that leads to the concerted phosphorylation of eNOS at Ser-1177 and dephosphorylation at Thr-495. This action of black tea polyphenols affords calmodulin-dependent eNOS activation and enhanced NO bioactivity. These data suggest that one cardiovascular benefit of black tea is that the activation of eNOS that may, in part, explain clinical trials supporting reduced cardiovascular disease as a function of increased black tea consumption.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants DK55656, HL60886, HL67206, and HL68758, the Juvenile Diabetes Research Foundation, and Unilever. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Supported by C. J. Martin Postdoctoral Research Fellowship Grant 007158 from the Australian National Health and Medical Research Council. Back

|| This work was performed while this author was an Established Investigator of the American Heart Association. To whom correspondence should be addressed: Whitaker Cardiovascular Inst., Boston University School of Medicine, 715 Albany St., Rm. W507, Boston, MA 02118. Tel.: 617-638-4885; Fax: 617-638-5437; E-mail: jkeaney{at}bu.edu.

1 The abbreviations used are: NO, nitric oxide; eNOS, endothelial nitric-oxide synthase; PAEC, porcine aortic endothelial cells; MAP, mitogen-activated protein; MAPK, MAP kinase; MAPKAPK-2, MAPK-activated protein kinase 2; MKK6, MAPK kinase 6; MKK6bE, recombinant adenovirus-expressing activated MKK6; ERK, extracellular signal-regulated kinase; GSK3, glycogen synthase kinase 3; FBS, fetal bovine serum; PSS, physiologic salt solution; L-NAME, L-nitro-arginine methyl ester; IBMX, 3-isobutyl-1-methylxanthine; PI 3-K, phosphoinositide 3-kinase; TBS, Tris-buffered saline. Back


   ACKNOWLEDGMENTS
 
We thank Nikhiel Rau for technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Duffy, S. J., and Vita, J. A. (2003) Curr. Opin. Lipidol. 14, 21–27[CrossRef][Medline] [Order article via Infotrieve]
  2. Higdon, J. V., and Frei, B. (2003) Crit. Rev. Food Sci. Nutr. 43, 89–143[CrossRef][Medline] [Order article via Infotrieve]
  3. Robak, J., and Gryglewski, R. J. (1988) Biochem. Pharmacol. 37, 837–841[CrossRef][Medline] [Order article via Infotrieve]
  4. van Acker, F. A., Schouten, O., Haenen, G. R., van der Vijgh, W. J., and Bast, A. (2000) FEBS Lett. 473, 145–148[CrossRef][Medline] [Order article via Infotrieve]
  5. Boullier, A., Bird, D. A., Chang, M. K., Dennis, E. A., Friedman, P., Gillotre-Taylor, K., Horkko, S., Palinski, W., Quehenberger, O., Shaw, P., Steinberg, D., Terpstra, V., and Witztum, J. L. (2001) Ann. N. Y. Acad. Sci. 947, 214–222[Abstract/Free Full Text]
  6. Princen, H. M., Van Duyvenvoorde, W., Buytenhek, R., Blonk, C., Tijburg, L. B., Langius, J. A., Meinders, A. E., and Pijl, H. (1998) Arterioscler. Thromb. Vasc. Biol. 18, 833–841[Abstract/Free Full Text]
  7. Vita, J. A. (2003) J. Nutr. 133, (suppl.) 3293S–3297S[Abstract/Free Full Text]
  8. Widlansky, M. E., Gokce, N., Keaney, J. F., Jr., and Vita, J. A. (2003) J. Am. Coll. Cardiol. 42, 1149–1160[Abstract/Free Full Text]
  9. Gokce, N., Keaney, J. F., Jr., Hunter, L. M., Watkins, M. T., Nedeljkovic, Z. S., Menzoian, J. O., and Vita, J. A. (2003) J. Am. Coll. Cardiol. 41, 1769–1775[Abstract/Free Full Text]
  10. Duffy, S. J., Keaney, J. F., Jr., Holbrook, M., Gokce, N., Swerdloff, P. L., Frei, B., and Vita, J. A. (2001) Circulation 104, 151–156[Abstract/Free Full Text]
  11. Huang, A., Vita, J. A., Venema, R. C., and Keaney, J. F., Jr. (2000) J. Biol. Chem. 275, 17399–17406[Abstract/Free Full Text]
  12. Alessi, D. R., Andjelkovic, M., Caudwell, B., Cron, P., Morrice, N., Cohen, P., and Hemmings, B. A. (1996) EMBO J. 15, 6541–6551[Medline] [Order article via Infotrieve]
  13. Fujio, Y., and Walsh, K. (1999) J. Biol. Chem. 274, 16349–16354[Abstract/Free Full Text]
  14. Wang, Y., Huang, S., Sah, V. P., Ross, J., Jr., Brown, J. H., Han, J., and Chien, K. R. (1998) J. Biol. Chem. 273, 2161–2168[Abstract/Free Full Text]
  15. Lee, M. J., Wang, Z. Y., Li, H., Chen, L., Sun, Y., Gobbo, S., Balentine, D. A., and Yang, C. S. (1995) Cancer Epidemiol. Biomarkers Prev. 4, 393–399[Abstract]
  16. Halliwell, B., Clement, M. V., Ramalingam, J., and Long, L. H. (2000) IUBMB Life 50, 251–257[CrossRef][Medline] [Order article via Infotrieve]
  17. Thomas, S. R., Chen, K., and Keaney, J. F., Jr. (2002) J. Biol. Chem. 277, 6017–6024[Abstract/Free Full Text]
  18. Dimmeler, S., Fleming, I., Fisslthaler, B., Hermann, C., Busse, R., and Zelher, A. (1999) Nature 399, 601–605[CrossRef][Medline] [Order article via Infotrieve]
  19. Fulton, D., Gratton, J., McCabe, T., Fontana, J., Fujio, Y., Walsh, K., Franke, T., Papapetropoulos, A., and Sessa, W. C. (1999) Nature 399, 597–601[CrossRef][Medline] [Order article via Infotrieve]
  20. Harris, M. B., Ju, H., Venema, V. J., Liang, H., Zou, R., Michell, B. J., Chen, Z. P., Kemp, B. E., and Venema, R. C. (2001) J. Biol. Chem. 276, 16587–16591[Abstract/Free Full Text]
  21. Michell, B. J., Griffiths, J. E., Mitchelhill, K. I., Rodriguez-Crespo, I., Tiganis, T., Bozinovski, S., de Montellano, P. R., Kemp, B. E., and Pearson, R. B. (1999) Curr. Biol. 9, 845–848[CrossRef][Medline] [Order article via Infotrieve]
  22. Gallis, B., Corthals, G. L., Goodlett, D. R., Ueba, H., Kim, F., Presnell, S. R., Figeys, D., Harrison, D. G., Berk, B. C., Aebersold, R., and Corson, M. A. (1999) J. Biol. Chem. 274, 30101–30108[Abstract/Free Full Text]
  23. Fruman, D. A., Meyers, R. E., and Cantley, L. C. (1998) Annu. Rev. Biochem. 67, 481–507[CrossRef][Medline] [Order article via Infotrieve]
  24. Ayajiki, K., Kindermann, M., Hecker, M., Fleming, I., and Busse, R. (1996) Clin. Res. 78, 750–758
  25. Mineo, C., Yuhanna, I. S., Quon, M. J., and Shaul, P. W. (2003) J. Biol. Chem. 278, 9142–9149[Abstract/Free Full Text]
  26. Cai, H., Li, Z., Davis, M. E., Kanner, W., Harrison, D. G., and Dudley, S. C., Jr. (2003) Mol. Pharmacol. 63, 325–331[Abstract/Free Full Text]
  27. Gratton, J. P., Morales-Ruiz, M., Kureishi, Y., Fulton, D., Walsh, K., and Sessa, W. C. (2001) J. Biol. Chem. 276, 30359–30365[Abstract/Free Full Text]
  28. Ludmer, P. L., Selwyn, A. P., Shook, T. L., Wayne, R. R., Mudge, G. H., Alexander, R. W., and Ganz, P. (1986) N. Engl. J. Med. 315, 1046–1051[Abstract]
  29. Anderson, T. J., Uehata, A., Gerhard, M. D., Meredith, I. T., Knab, S., Delagrange, D., Lieberman, E. H., Gamz, P., Creager, M. A., Yeung, A. C., and Selwyn, A. P. (1995) J. Am. Coll. Cardiol. 26, 1235–1241[Abstract]
  30. Lieberman, E. H., Gerhard, M. D., Uehata, A., Selwyn, A. P., Ganz, P., Yeung, A. C., and Creager, M. A. (1996) Am. J. Cardiol. 78, 1210–1214[CrossRef][Medline] [Order article via Infotrieve]
  31. Wallerath, T., Deckert, G., Ternes, T., Anderson, H., Li, H., Witte, K., and Forstermann, U. (2002) Circulation 106, 1652–1658[Abstract/Free Full Text]
  32. Lorenz, M., Wessler, S., Follmann, E., Michaelis, W., Dusterhoft, T., Baumann, G., Stangl, K., and Stangl, V. (2004) J. Biol. Chem. 279, 6190–6195[Abstract/Free Full Text]
  33. Chen, Z., Yuhanna, I. S., Galcheva-Gargova Z, Karas, R. H., Mendelsohn, M. E., and Shaul, P. W. (1999) J. Clin. Investig. 103, 401–406[Medline] [Order article via Infotrieve]
  34. Fleming, I., Fisslthaler, B., Dimmeler, S., Kemp, B. E., and Busse, R. (2001) Circ. Res. 88, E68–E75[CrossRef][Medline] [Order article via Infotrieve]
  35. Lin, M. I., Fulton, D., Babbitt, R., Fleming, I., Busse, R., Pritchard, K. A., Jr., and Sessa, W. C. (2003) J. Biol. Chem. 278, 44719–44726[Abstract/Free Full Text]
  36. Goetz, R. M., Thatte, H. S., Prabhakar, P., Cho, M. R., Michel, T., and Golan, D. E. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 2788–2793[Abstract/Free Full Text]
  37. Papapetropoulos, A., Garcia-Cardena, G., Madri, J. A., and Sessa, W. C. (1997) J. Clin. Investig. 100, 3131–3139[Medline] [Order article via Infotrieve]
  38. Fleming, I., and Busse, R. (1999) Cardiovasc. Res 43, 532–541[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Nutr.Home page
P. Gresele, P. Pignatelli, G. Guglielmini, R. Carnevale, A. M. Mezzasoma, A. Ghiselli, S. Momi, and F. Violi
Resveratrol, at Concentrations Attainable with Moderate Wine Consumption, Stimulates Human Platelet Nitric Oxide Production
J. Nutr., September 1, 2008; 138(9): 1602 - 1608.
[Abstract] [Full Text] [PDF]

Home page
 J. Nutr.Home page
D. Grassi, A. Aggio, L. Onori, G. Croce, S. Tiberti, C. Ferri, L. Ferri, and G. Desideri
Tea, Flavonoids, and Nitric Oxide-Mediated Vascular Reactivity
J. Nutr., August 1, 2008; 138(8): 1554S - 1560S.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
W.-H. Kan, J.-T. Hsu, Z.-F. Ba, M. G. Schwacha, J. Chen, M. A. Choudhry, K. I. Bland, and I. H. Chaudry
p38 MAPK-dependent eNOS upregulation is critical for 17{beta}-estradiol-mediated cardioprotection following trauma-hemorrhage
Am J Physiol Heart Circ Physiol, June 1, 2008; 294(6): H2627 - H2636.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
G. E. Mann, D. J. Rowlands, F. Y.L. Li, P. de Winter, and R. C.M. Siow
Activation of endothelial nitric oxide synthase by dietary isoflavones: Role of NO in Nrf2-mediated antioxidant gene expression
Cardiovasc Res, July 15, 2007; 75(2): 261 - 274.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J.-a Kim, G. Formoso, Y. Li, M. A. Potenza, F. L. Marasciulo, M. Montagnani, and M. J. Quon
Epigallocatechin Gallate, a Green Tea Polyphenol, Mediates NO-dependent Vasodilation Using Signaling Pathways in Vascular Endothelium Requiring Reactive Oxygen Species and Fyn
J. Biol. Chem., May 4, 2007; 282(18): 13736 - 13745.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
M. A. Potenza, F. L. Marasciulo, M. Tarquinio, E. Tiravanti, G. Colantuono, A. Federici, J.-a Kim, M. J. Quon, and M. Montagnani
EGCG, a green tea polyphenol, improves endothelial function and insulin sensitivity, reduces blood pressure, and protects against myocardial I/R injury in SHR
Am J Physiol Endocrinol Metab, May 1, 2007; 292(5): E1378 - E1387.
[Abstract] [Full Text] [PDF]

Home page