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L-Theanine promotes nitric oxide production in endothelial cells through eNOS phosphorylation

Authors:
L-Theanine promotes nitric oxide production in endothelial cells
through eNOS phosphorylation
Jamila H. Siamwala
a,1
, Paul M. Dias
b,1
, Syamantak Majumder
a
, Manoj K. Joshi
b
, Vilas P. Sinkar
b
,
Gautam Banerjee
b
, Suvro Chatterjee
a,
a
Vascular Biology Lab, AU-KBC Research Centre, Anna University, MIT Campus, Chennai, India
b
Unilever R&D, Bangalore, India
Received 31 August 2011; received in revised form 23 February 2012; accepted 28 February 2012
Abstract
Consumption of tea (Camellia sinensis) improves vascular function and is linked to lowering the risk of cardiovascular disease. Endothelial nitric oxide is the
key regulator of vascular functions in endothelium. In this study, we establish that L-theanine, a non-protein amino-acid found in tea, promotes nitric oxide (NO)
production in endothelial cells. L-theanine potentiated NO production in endothelial cells was evaluated using Griess reaction, NO sensitive electrode and a NO
specific fluorescent probe (4-amino-5-methylamino-2',7'-difluororescein diacetate). L-Theanine induced NO production was partially attenuated in presence of
L-NAME or L-NIO and completely abolished using eNOS siRNA. eNOS activation was Ca
2+
and Akt independent, as assessed by fluo-4AM and immunoblotting
experiments, respectively and was associated with phosphorylation of eNOS Ser 1177. eNOS phosphorylation was inhibited in the presence of ERK1/2 inhibitor,
PD-98059 and partially inhibited by PI3K inhibitor, LY-294002 and Wortmanin suggesting PI3K-ERK1/2 dependent pathway. Increased NO production was
associated with vasodilation in ex ovo (chorioallantoic membrane) model. These results demonstrated that L-theanine administration in vitro activated ERK/
eNOS resulting in enhanced NO production and thereby vasodilation in the artery. The results of our experiments are suggestive of L-theanine mediated vascular
health benefits of tea.
© 2013 Elsevier Inc. All rights reserved.
Keywords: Nitric oxide; L-theanine; eNOS; Endothelial cells; Vasodilation
1. Introduction
Tea (Camellia sinensis) has been shown to have vasculoprotective,
anti-oxidative, anti-thrombogenic, anti-inammatory and lipid low-
ering properties [1]. Epidemiological studies [2,3] and meta analysis
[4,5] illustrate a strong positive link between tea consumption and
improvement of cardiovascular health as assessed by endothelium
mediated vasodilation [6]. Both black and green teas are known to
improve endothelial functions by enhancing endothelial NO produc-
tion [7]. Inhibition of endothelial function and, particularly, pertur-
bation of eNOS-derived NO production, has been implicated in a
number of cardiovascular diseases [8,9]. NO production by eNOS is a
tightly regulated event, inuenced by a series of post-translational
modications which include modulation of phosphorylation state by
kinases and phosphatases, protein-protein interactions, and avail-
ability of requisite substrates and cofactors for optimum eNOS activity
[10]. Tea, as a beverage, is known to improve cardiovascular health by
inuencing eNOS activity [1,8]. However, the bioactive ingredients in
tea inuencing this cardioprotective effect remains to be conrmed.
L-theanine (γ-glutamylethylamide), a non-protein amino acid,
comprises 0.52 % of the dry weight of both green and black tea
(Camellia sinensis)[11], is claimed to induce mental relaxation [12]
and neuro-protection [13].L-theanine is readily bioavailable on
consumption [14] and even crosses the bloodbrain barrier [15].In
the present study, we found that L-theanine promotes NO
production in endothelial cells. Such effects of L-theanine are
brought about by its effect on NO signaling cascade. We show that
these benets are mediated by phosphorylation of eNOS-Ser 1177
and involve PI3K/ERK pathway.
2. Materials and methods
2.1. Materials
L-theanine (99.9%) was procured from Sunphenone (MN, USA). Bradykinin,
Propidium iodide (PI), eNOS siRNA, DAR 4M, L-NAME (N
G
-nitro-L-arginine methyl
ester) and Calcium Ionophore A23187 were purchased from Sigma Chemical (MO,
USA), DAF FM-DA (4-amino-5-me thylamino-2',7'-diuororescein diacetate) was
purchased from Invitrogen (OR, USA), Annexin V-FITC apoptosis detection kit &
Available online at www.sciencedirect.com
Journal of Nutritional Biochemistry 24 (2013) 595 605
Conict of interest: None declared.
Corresponding author. AU-KBC Research Centre, M.I.T Campus of Anna
University, Chromepet, Chennai-600044, India. Tel.: + 91 44 2223 4885x48,
+91 44 2223 2711x48; fax: +91 44 2223 1034.
E-mail address: soovro@yahoo.ca (S. Chatterjee).
1
Authors have contributed equally.
0955-2863/$ - see front matter © 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jnutbio.2012.02.016
Fig. 1
596 J.H. Siamwala et al. / Journal of Nutritional Biochemistry 24 (2013) 595605
antibodies were purchased from Calbiochem, EMD Chemicals (Darmstadt, Germany).
BAPTA-AM and uo-4AM were obtained from Sigma and Invitrogen respectively.
Polyclonal antibodies specic for eNOS, ERK1/2, Akt and its phosphorylated forms were
from Cell Signaling Technology (MA, USA). Fertilized white leghorn chicken eggs were
obtained from local Poultry Research Centre, Nandanam, Chennai, India. All other
chemicals were of the analytical grade and were obtained commercially.
2.2. Cell lines
eNOS GFP transfected ECV304 cell line was a kind gift from Dr. Vijay Shah (Mayo
Clinic, Canada). The endothelial cell line EA.hy926 was procured from American Type
Culture Collection (ATCC; VA, USA).
2.3. Harvesting primary endothelial cells
Human umbilical vein endothelial cells (HUVECs) were isolated from freshly
acquired human umbilical cord as described elsewhere [16]. The procedure was
approved from Institution's Research Ethics Committee and informed consents were
obtained from the parents. Bovine aortic endothelial cells (BAEC) were isolated from
the pulmonary arteries of freshly slaughtered bovine from government-authorized
abattoirs. BAECs were harvested as described elsewhere [17]
.
HUVEC were cultured in
M199 supplemented with endothelial growth factor, 10% fetal bovine serum (FBS)
(v/v) and 1% penicillin (w/v) and streptomycin (w/v). The BAEC, eNOS GFP and
EA.hy926 cells were cultured in DMEM supplemented with 10% FBS (v/v) and 1%
penicillin (w/v) and streptomycin (w/v).
2.4. Cell viability assay
L-theanine was dissolved in phosphate-buffered saline (PBS) (pH 7.4) for all the
experiments performed. The 1 × PBS was subsequently used as vehicle control for all
the experiments. EA.hy926 cells were treated with varying concentration of L-theanine
(0.01 μM, 0.1 μM, 1.0 μM, 10 μM) for 30 min and after 24 h of incubation cell viability
was determined using CMFDA as described elsewhere [18]
.
2.5. Cell apoptosis assay
EA.hy926 cells were split on coverslips to 60% conuency and were subsequently
treated with different concentrations of L-theanine (0.01 μM, 0.1 μM, 1.0 μM, 10 μM).
After 24 h, the cell apoptosis was measured using Annexin V and propidium iodide
following the manufacturer's instructions (Abcam). Briey the cells were treated with
the binding buffer and subsequently, with Annexin V and propidium iodide for 5 min in
dark. The positive control H
2
O
2(
150 M) was added 30 min before measuring apoptosis.
The images were acquired using the Olympus IX71 inverted uorescence microscope.
The Annexin V positive cells were counted manually from 10 different elds of images
taken from four sets of experiments.
2.5. Measurement of total NO production by Greiss assay
The assay was done using Griess reagent. About 1×10
6
conuent ECV-eNOS-GFP
cells or BAEC were treated with different concentrations of L-theanine (0.01 μM, 0.1 μM,
1.0 μM, 10 μM) for 30 min. The culture supernatent was collected and assayed for
nitrites using Greiss reagent according to Nims et al. [19].
Fig. 1. L-Theanine activates NO production. A signicant increase in NO production in endothelial cells was observed with different concentrations (0.0110 μM) of L-theanine using
different methodologies (A) Flow cytometry based measurement of NO using DAF FM uorescence in the presence or absence of L-NAME. Bradykinins (BK), VEGF were used as positive
control. (B) Amperometric measurement of NO using NO specic electrode in the presence or absence of L-NIO. B (A) NO was measured amperometrically directly from primary BAEC
in the presence or absence of L-Arginine (L-Arg) or L-NAME. (B) The culture supernatant were analysed for nitrates/ nitrites using Griess assay in HUVEC. C. Real-time detection of NO
production in HUVEC cells using ow-cytometry. A gate made on DAF FM positive cells (black box) for enumeration for different incubation periods (1,2,5,10,15 and 30 min) with
L-theanine (0.1 μM). *Pb.01 vs. vehicle control or #Pb.01 vs. L-NAME. The graphs are representative of the mean of 4 independent experiments. *Pb.01 vs. vehicle control analysed
using one way ANOVA .
597J.H. Siamwala et al. / Journal of Nutritional Biochemistry 24 (2013) 595605
2.6. Amperometric measurement of nitric oxide using a nitric oxide sensitive electrode
2.6.1. NO production from cell monolayer
The levels of NO released by endothelial cells grown on coated cover slips was
measured using amperometric probe (Apollo 4000 analyzer) as described previously
[18]. Briey, conuent cells were pre-treated with L-arginine (100 μM) for 30 min.
Electrode was allowed to equilibrate in PBS for at least 30 min at this stage. The
electrode was placed in the well so that the tip of the electrode was 1 mm above the
surface of the cells. Once equilibrated, different concentrations of L-theanine (0.01 μM,
0.1 μM, 1.0 μM, 10 μM) was added slowly without disturbance, followed by a real-time
acquisition of NO production through a single-board computer that displays the
experimental data.
2.6.2. NO production from the blood vessels
The experiment was performed as described elsewhere from the vitelline arteries
in 4th day embryo [20]
.
Blood vessel measurement from chorioallantoic membrane
(CAM) was performed with NO sensitive electrode (200 M size, 0.1 nm sensitive range
electrode) attached to Apollo 4000 Free radical analyzer. A window was made on the
broad side (air sac) of the egg. NO probe was placed close to vitelline artery and
L-theanine (1 μM) or L-NIO (1 μM) treated discs were placed on the blood vessel in
close proximity of the probe. NO pattern was monitored for 30 min at 37°C, under
enclosed condition. Electrode was calibrated with SNAP solution prior to experiments.
2.7. Measurement of intracellular NO using DAF FM fluorescence by flow-cytometry
DAF FM-DA based measurement of intracellular NO involving uorescence assisted
cell segregation of responding endothelial population has been described elsewhere [21].
Briey, EA.hy926 cells (2×10
4
/well) were seeded in 24 well microtiter plates. After
adherence, cells were starved for 12 hrs in serum free low glucose media (5 mM). Cells
were loaded with DAF FM-DA (1 μM) in PBS for 30 min and washed twice with serum-
free medium. Subsequently, cells were stimulated with L-theanine (0.00110 μM),
bradykinin (25 nM), vascular endothelial growth factor (VEGF) (25 nM) or glutamic acid
(1 μM) in presence or absence of L-NAME (10 μM; 30 min pre-incubation). Comparable
vehicle control (DMSO in PBS) was used to nullify the effect on morphological changes.
The cells were washed (3×) with PBS, followed by trypsinization and xation with 2%
paraformaldehyde for 15 min. A population of 10,000 cells were gated and segregated
based on theirrelative uorescence intensities using FACS Calibur (Becton Dickenson, San
Diego, CA, USA). The mean yield of 2 distinct populations was measured and compared
with the respective population in untreated cells.
2.8. Calcium measurement using fluo-4AM
EA.hy926 cells were seeded on glass coverslips and cultured overnight. The media
was removed and the cells were washed trice with PBS. The cells were pretreated with
or without BAPTA-AM (3.5 μM for 5 min). After wash in PBS, the cells were loaded with
uo-4AM (5 M) for 15 min followed by wash with PBS to remove any free dye and
incubated for 15 min in PBS to allow complete de-esterication of AM esters. Fluo-4-
pretreated cells were then treated with Calcium Ionophore (A23187; 50 μM ) followed
by stimulation with calcium chloride (CaCl
2
; 0.0011μM in PBS for 15 min. For the
theanine experiments, cells were treated or not treated with BAPTA-AM and then
loaded with uo-4AM. The dye-loaded cells were treated with theanine (1 M) or
calcium ionophore (1 M) or CaCl
2
(1 M) alone or in combination, followed by spectro-
uorometric measurements at 488/530 nm ex/em using TECAN-Innite M1000.
Multichannel dispenser was used to avoid well to well variations.
2.9. RNA interference and DAR 4M imaging
ECV-eNOS-GFP cells were transfected with siRNAs specically designed to target
human eNOS using Li pofectamine 2000 (Invitrogen) reagent accordi ng to the
manufacture's protocols. 44 hours after transfection, the cells were loaded with
200μl of 0.1 μM diaminorhodamine 2 diacetate 4M (Molecular Probe, USA) and
incubated for 30 min, followed by treatment with L-theanine (1 μM). The images were
acquired using the Olympus IX71 inverted uorescence microscope.
2.10. Vasodilation assay
Fertilised white leghorn chicken eggs incubated for four days (37°C at constant
swirling) were collected from the Poultry Research Centre. The egg shell was cut open
to expose the vitelline arteries for video recording. After 1 min 10 μlofL-theanine
(1 μM) or L-NIO (10 μM) dissolved in 1× PBS was added directly on the blood vessel
and recorded for 1 min. The 0, 15, 30, 45, 60 second images were used to calculate the
area of a xed portion of blood vessel. The change in area of the blood vessel after
addition of L-theanine was calculated with respect to 0 sec.
2.11. Western blot analysis
EA.hy926 (1x10
6
cells/well) were treated with 1 μML-theanine or PD or
Wortmanin or LY compound and incubated for different time points (030 min). PD
or Wortmanin or LY compound were dissolved in DMSO as indicated. Cells were
harvested in cold lysis buffer and protein levels normalised using bicinchoninic acid
method. 50 μg protein equivalent was loaded in each of the wells and separated by
polyacrylamide gel electrophoresis, electrotransferred and immunodetected for the
proteins of interest.
2.12. Statistical analyses
Data are presented as mean ±S.E.M. Data were analyzed using one-way analysis of
variance (ANOVA) test, Student's ttest and Tukey post hoc tests as appropriate using
the SIGMA STAT software package. All the experiments were performed in triplicate
(n=3) unless otherwise specied.
Fig. 2. Detection of Apoptosis: (A) and (B) 1×10
6
M cells were split on glass coverslips followed by treatment with different concentrations of L-theanine (0.01 μM, 0.1 μM, 1 μM, 10 μM)
for 30 min. After 24 h of incubation cell apoptosis was measured using Annexin V and Propidium iodide (1 mg/ml). The cells were treated with positive control, hydrogen peroxide
(H
2
O
2
) (150 M) for 30 min before the apoptosis measurement. (C) Cell viability assay was carried out using a cell tracker CMFDA (5 μM). The results are presented as mean +SD of
three wells from a representative experiment. The levels of signicance of treated vs. vehicle control determined by one way ANOVA (*Pb.05).
598 J.H. Siamwala et al. / Journal of Nutritional Biochemistry 24 (2013) 595605
3. Results
3.1. Effects of different concentrations of L-theanine on NO production
Treatment of human endothelial cell-line (EA.hy926) with
L-theanine increased intracellular NO production (Fig. 1AA). This
increase was partially abolished in presence of L-NAME (10 μM).
Similarly, exposure of L-theanine to chick embryo vessels showed
enhanced NO release, which was partially abolished in presence of
L-NIO (10 μM), an eNOS specic inhibitor (Fig. 1AB). Variable
response to agonist stimulation is been reported in different
endothelial cell-types [13]. Thus, we evaluated NO production in
different cell-type under L-theanine treatment. The trend of NO
produced were similar in different endothelial cell types as assessed
by FACS analysis (Fig. 1AA) in EA.hy926, electrode based detection
in BAEC (Fig. 1BA) and Griess-based assay in HUVEC (Fig. 1BB).
L-glutamic acid (GA) (1 μM), a L-theanine analog, showed NO levels
similar to untreated control, ruling out non-specic effects of
amino-acid (negative control) (Fig. 1AA). The kinetics of NO
response was maximum at 2 min of L-theanine treatment as
accessed by ow cytometry (Fig. 1C). However, such response was
compromised in presence of higher concentration of L-theanine
(10 μM) (Fig. 1B). As the levels of free-thiols in the cells remained
unchanged (Fig. 2A), any possibility of these effects arising due to
loss of cell viability and proliferation was ruled out. Annexin V
positive cells were also lesser or equal to the vehicle control cells
for all concentrations of L-theanine and signicantly lower than the
hydrogen peroxide treated cells, thereby indicating L-theanine to be
non-apoptotic (Fig. 2B).
3.2. Pulse response to L-theanine
In order to determine if L-theanine pulsation would result in
signal desensitization, which would result in progressive loss of
response to any subsequent addition of L-theanine, endothelial
cells were pulsated with 0.1 μML-theanine at regular 30 min
intervals. It was observed that pulsation with L-theanine resulted
in progressive increase in NO production, as accessed by ow
cytometry (Fig. 3). When 0.1 μML-theanine was added to EC, NO
levels increased to 2.8 and dropped to 1.56 after 30 min, whereas
L-theanine pulse for the second time induced further NO
production followed by a drop (3.1). The third response to L-
theanine was even higher (3.18) than the rst peak and second
peak (Fig. 3). The drop (2.5) was also higher than the rst and
second drops (Fig. 3).
Fig. 4. eNOS silencing reduces L-theanine induced NO production. Cells were transfected with small-interfering (si) RNA for siRNA (eNOS siRNA) or with vehicle control and treated
with L-Theanine (1 μM) for the 30 min. Cells were incubated with DAR 4M probe for 15 min and images taken with Olympus XL71 inverted uorescence microscope at
20× magnication.
Fig. 3. Pulse response to L-theanine: EA.hy926 (2×10
5
) cells were serum starved and
loaded with DAF FM-DA (1 μM). In presence of the dye, cells were pulsated trice
with L-theanine (1 μM) and monitored for NO production at regular interval (1, 2, 5,
10, 15 and 30 min) by ow cytometry. Arrows indicate the time of addition of
L-theanine.
599J.H. Siamwala et al. / Journal of Nutritional Biochemistry 24 (2013) 595605
3.3. Source of NO production
L-Theanine treatment increased intracellular NO production
compared to that of untreated control. However, this response was
completely abrogated in endothelial cells treated with eNOS siRNA in
presence or absence of L-theanine treatment (Fig. 4), suggesting eNOS
dependent NO production.
3.4. Vasodilation in response to L-theanine
One of the prime functions of nitric oxide in the blood vessels is
vasodilation. In CAM based vasodilation model, L-theanine treated
blood vessel showed an increase in the marked area compared to
control. A specic inhibitor of eNOS, L-NIO abolished L-theanine
induced blood vessel dilation (Fig. 5).
3.5. L-Theanine mediated eNOS phosphorylation is PI3K/ERK
pathway dependant
Calcium experiment with a calcium specic dye showed no change
in the intracellular calcium levels at the given L-theanine concentra-
tions suggesting calcium independent activation of eNOS (Fig. 6).
Calcium ionophore along with calcium chloride (1 M) increased the
uorescence signicantly in the presence or absence of cell permeable
Fig. 5. L-Theanine mediated NO production induces vasodilation. A. (A-D) Represents the setup of the vasodilation assay and the videography. B. 4th day White Leghorn eggs were
broken and L-theanine or L-NIO applied locally followed by videography using a Sony Handycam attached to the stereomicroscope with an adapter for 1 min. Snapshots were taken
from the video every 15 sec for 1 min. Using image J software, 4 different widths on the selected blood were measured before and after addition of compounds. The difference in area
due to vasodilation was calculated with respect to 0. Four independent experiments on different eggs (n =4) showed similar results. The levels of signicance of treated vs. vehicle
control was determined by one way ANOVA (*Pb.05).
600 J.H. Siamwala et al. / Journal of Nutritional Biochemistry 24 (2013) 595605
calcium chelator, BAPTA-AM. The calcium levels of cell treated with
theanine and calcium chloride were similar to cells treated with
BAPTA-AM along with theanine and calcium chloride, thereby
excluding theanine's role as a calcium ionophore (Fig. 6). Treatment
of EA.hy926 cells with L-theanine dose-dependently increased eNOS
Ser1177 phosporylation (Fig. 7AA). This increase occurred at 25 min
of L-theanine treatment (Fig. 7AC) and coincided with NO production
(Fig. 1AA). Pre-treatment of EA.hy926 cells with Wortmanin (an
inhibitor of PI3K), and PD-98059 (an inhibitor of MAPK) inhibited
VEGF and L-theanine stimulated NO production and eNOS Ser1177
phosporylation, suggesting involvement of PI3K and MAPK depen-
dent pathway (Fig. 7BA). However, L-theanine mediated eNOS
Ser1177 phosporylation was independent of Akt (Ser473) phospor-
ylation (Fig. 7BC), whereas VEGF mediated eNOS phosporylation was
Akt (Ser473) dependent (Fig. 7BC), suggesting involvement of
different kinase(s). L-theanine treatments for 115 min induced
eNOS Ser1177 phosporylation (Fig. 7C) and ERK phosphorylation
(Fig. 8A) at 1 and 2 min, followed by a drop in phosporylation levels at
5 and 15 min respectively. Such phosporylation was inhibited in the
presence of PI3K inhibitor, LY-294002 (Fig. 8C) and ERK1/2 inhibitor,
PD-98059 (Fig. 8E).
3.6. L-Theanine mediated NO production is PI3K/ERK
pathway dependent
To ascertain the effects of L-theanine treatment on NO production
in presence of ERK1/2 inhibitor, PD-98059 and PI3K inhibitor,
LY-294002 on NO production, the cells were pre-incubated with
inhibitors alone or in combination followed by L-theanine treatment.
NO production of the pre-treated cells was analyzed for DAF FM
uorescence using ow-cytometry. As demonstrated in Fig. 9, the
L-theanine induced NO production was signicantly reduced in
presence of ERK1/2 and PI3K inhibitors alone and in combination as
observed by reduced uorescence. Further the 2 and 5 min increase in
NO production was completely abolished in presence of PD-98059
and LY-294002 (Fig.9).
4. Discussions
Tea has been shown to improve cardiovascular health [3]. This
improvement in cardiovascular health has been attributed to
enhanced endothelial function. Homeostatic regulation of endothelial
function involves NO which in turn is governed by either NO
production or improved NO bioavailability [1,7]. Both green and
black tea have been shown to promote NO production via activation
of eNOS, involving mechanism such as protein phosporylation and
de-phosporylation, translocation and calcium mobilization; or en-
hanced NO bioavailability as a result of protein inhibition, protein-
protein interaction or activation of antioxidant enzymes. However,
there is no evidence of any amino acid or its derivative in tea
activating eNOS as an agonist. In present study, we establish the
efcacy of L-theanine in promoting NO production in endothelial cells
and elucidate its mechanism of eNOS activation.
L-theanine has been implicated in reduction of blood pressure in
hypertensive rats [22]. Also, in a randomized, double-blind, and
placebo-controlled study, Rogers et al. showed that L-theanine
antagonizes caffeine induced blood pressure [23]. However, the
molecular mechanism underlying such benet remains ill-dened.
The present study demonstrates that L-theanine increase NO
production in endothelial cells (Figs. 1AC). L-theanine stimulated
intracellular NO production was determined using ow cytometry
based DAF FM-DA uorescence (Fig. 1AA). NO being a gas diffuses or
interacts with other biomolecules and the NO released was measured
using NO-specic electrode (Fig. 1BA). A part of NO released get
converted to nitrites and nitrates and accumulates in culture super-
natents. This was measured using Greiss assay (Fig. 1BB). Such
subsequencial measurement is essential, as there would be cross
specicities in all the currently used bio-assays to measure NO
production [24].
Variable responses to agonist stimulation amongst different cell
types and cell-lines are reported previously [25]. The study also
highlights that primary endothelial cells, HUVEC, is more responsive
to L-theanine in producing NO than the immortalized cell line,
EA.hy926 (Fig. 1C). L-theanine efcacy was compared with well
known agonist for eNOS activation such as Bradykinin and VEGF (Fig.
1Aa). L-theanine promoted NO production at micromolar concentra-
tions, while VEGF and bradykinin exerted similar effects at nanomolar
range [26,27]. Similar correlation between NO produced and NO
released was observed in our experiments when estimated for the
nitrite/nitrate content in the culture supernatant (Fig. 1BB). To
ascertain, such effect in in vivo condition, we monitored ex ovo NO
production using NO specic electrode (Fig. 1AB). Enhanced NO
production was observed in L-theanine treated conditions, whose
effect was abolished in presence of L-NIO, a specic inhibitor for eNOS.
Another report by Nagasawa et al. demonstrated that L-theanine
confers neuroprotective effects by increasing the expression levels of
PLC-beta1 and gamma1, and thereby minimized apoptosis in the
neural cells [28]. In line with those results, the present study supports
the anti-apoptotic property of L-theanine. Although cell viability
study with CMFDA (Fig. 2) under L-theanine did not show any signs of
cell death, the results of the Annexin V experiments indicated that L-
theanine reduces even the basal level of apoptosis in the cells.
eNOS is an important regulator of cardiovascular homeostasis and
plays a crucial role in the state of blood vessel vasodilation and hence
blood pressure regulation [29]. Numerous in vitro/ex vivo studies
demonstrate defect in the NO signalling pathway in isolated
atherosclerotic blood vessels in rabbits [30], pigs [31], rats [32],
primates [33] and humans [34], where basal as well as stimulated NO
release appeared to be affected, which in-turn affect the vasodilatory
property, suggesting compromised NO production in such patholo-
gies. Improved vasodilation results in vessel expansion allowing
optimal blood ow. To validate our in vitro observation, we
performed functional bioassay for artery dilation ex vivo using CAM
model. In CAM vasodilation model, we show that L-theanine was able
to induce vasodilation in the major arteries of the 4th day old chick
embryo (Fig. 5).
To elucidate the source of L-theanine mediated NO production;
eNOS expression was suppressed using eNOS-siRNA. eNOS GFP cell
line was used in these experiments, since it allows measurement of
Fig. 6. NO production is calcium independent. EA.hy926 cells were treated with or
without BAPTA-AM (3.5 M) and then loaded with Fluo-4AM (5 mM). The dye loaded
cells were treated with Theanine (1 M) or calcium ionophore (1 M) or CaCl
2
(1 M)
alone or in combination, followed by spectro-uorometric measurements at 488/530
nm ex/em after 5 min using TECAN-Innite M1000. The results are expressed as
relative uorescence unit (RFU).The levels of signicance of treated vs. vehicle control
determined by one way ANOVA (*Pb.05).
601J.H. Siamwala et al. / Journal of Nutritional Biochemistry 24 (2013) 595605
NO production and sub-cellular localization of eNOS simultaneously.
In our experiment silencing eNOS using a siRNA eNOS, abolished
eNOS-GFP uorescence, suggesting knock down of eNOS expression.
Treatment of L-theanine in such eNOS knockdown cells abolished NO
production as determined by DAR uorescence (Fig. 4), supporting
eNOS dependant NO production.
We also explored the molecular mechanism of L-theanine
mediated vaso-promotion. L-theanine induced NO production in 2
5 min. The increase saturated at 10 min after which it started to
decline (Fig. 1C). This resembled VEGF and bradykinin actions on
eNOS [27,28]. Malinski et al. using a highly selective NO [35]
electrode, reported that bradykinin treatment of BAEC activates
eNOS within seconds, and enzyme activity returns to baseline within
5 min. Additionally, phosphorylation of NO synthase in endothelial
cells in response to bradykinin is maximal around 5 min of agonist
exposure and persists for at least 20 min, long after the initial phase of
enzyme activation has decayed. It has been demonstrated that the
initial phase of NO production (sec scale) under bradykinin is calcium
dependent while later phase (min scale) is associated with the
phosphorylation of eNOS [36]. We did not observe such bi-phasic
response of Ca
2+
under L-theanine treatment. L-theanine did not
inuence intra-cellular Ca
2+
levels, further fortifying only phosphor-
ylation dependent eNOS activation as a primary mechanism. eNOS
modulation is the most important post-translational modication and
Fig. 7. L-Theanine increases eNOS (Ser 1177) phosphorylation. A. (A) and (B) eNOS phosphorylation (Ser1177, Ser113 and Thr495) was measured in EA.hy926 cells treated with
different doses of L-theanine (0.0011μM) for 5 min. (C) and (D) EA.hy926 cells treated with L-theanine (0.1 μM) for 1, 2, 5, 15 and 30 min. B. HUVECs treated with L-theanine (Th) (0.1
μM) or VEGF (V) (50 ng/ml) without/with PI3K inhibitors (wortmannin (WT) , 200 nM; 30 min pretreatment) or ERK inhibitor (PD-98059, 1 μM; 2h pre-incubation) and eNOS and Akt
phosphorylation was determined using anti-phospho eNOS antibody or anti-phospho Akt antibodies. The graphs were obtained by dividing the phospho with the total protein and are
representative of 3 independent experiments. The levels of signicance of treated vs. vehicle control determined by one way ANOVA (*Pb.05).
602 J.H. Siamwala et al. / Journal of Nutritional Biochemistry 24 (2013) 595605
Fig. 8. ERK1/2 / PI3K is upstream of L-theanine induced eNOS phosphorylation. A. EA.hy926 cells treated with/without the PI3K inhibitor (LY-294002 , 50 μM; 1 hr pre-incubation) or
ERK inhibitor (PD-98059, 1 μM; 2h pre-incubation) followed by L-theanine (1 μM) for 1, 2, 5, 15 min. The cells were lysed and analysed using immune-blotting. The blots were probed
with anti-phospho ERK1/2 and anti ERK1/2 and normalised with non-phosporylated protein. The Western blots are presented along with the corresponding densitometry data. The
levels of signicance of treated vs. control determined by one way ANOVA *Pb.05 vs. vehicle control.
Fig. 9. NO production is dependent on PI3K/ERK1/2 pathway. EA.hy926 cells treated with/without the ERK inhibitor (PD-98059, 1 μM; 2h pre-incubation) or PI3K inhibitor (LY-294002,
50 μM; 1h pre-incubation). Real-time detection of NO production in EA.hy926 cells was monitored by ow-cytometry. A gate made on DAF FM positive cells for enumeration for
different incubation periods (0,1,2,5 and 15 min) with L-theanine (0.1 μM). The levels of signicance of treated vs. control determined by one way ANOVA *Pb.05 vs. vehicle control.
Level of signicance for inhibition expressed as # Pb.05 vs. stimulated response.
603J.H. Siamwala et al. / Journal of Nutritional Biochemistry 24 (2013) 595605
the primary mechanism of modulating NO activity [37].The
phosphorylating sites of eNOS are spread across the calmodulin
domain, reductase domain and the oxygenase domain, the most
signicant being Ser1177, Ser113 and Thr495. Phosphorylation of
Thr495 is associated with calcium dependence of eNOS resulting in
enhanced NO production [37,38]. Treatment of EA.hy926 cells with L-
theanine resulted in increased eNOS Ser1177 phosporylation;
however, the phosporylation state of eNOS Ser113 or Thr497
remained unchanged (Fig. 7A) further conrming calcium indepen-
dent mechanism [39], as eNOS Thr495 phosporylation is associated
with Ca/Calmodulin interaction. Such change in L-theanine depen-
dent eNOS Ser1177 phosporylation correlated well with increase in
NO production (Fig. 1AA).
Both AMPK and Akt regulate activity of eNOS by phosporylation of
eNOS at Ser1177, resulting in increased production of NO in
endothelial cells [38,39]. At least in terms of its kinetics, the L-
theanine mediated increase in NO production and eNOS phosphor-
ylation was analogous to that mediated by VEGF. We therefore
compared the effect of L-theanine and VEGF induced signaling
cascade. Pre-treatment of EA.hy926 cells with Wortmanin (an
inhibitor of PI3K) partially suppressed eNOS Ser1177 phosporylation,
suggesting PI3K dependent pathway (Fig. 7B) [40]. This prompted us
to explore PI3K linked Akt dependent pathway. L-theanine mediated
eNOS Ser1177 phosporylation was independent of Akt (Ser473)
phosporylation, whereas VEGF mediated eNOS phosporylation was
Akt (Ser473) dependent (Fig. 7B). These intriguing ndings led us to
new line of investigation which did not involve the traditional PI3K-
Akt-eNOS pathway. Further, L-theanine treatment for 115 min
resulted in eNOS Ser1177 (Fig. 7A) and ERK phosphorylation
(Fig. 8BA), showing similar kinetics at 1 and 2 min. This was followed
by a drop in the p-ERK1/2 levels at 5 and 15 min respectively. Such
phosporylation of eNOS was inhibited in the presence of ERK1/2
inhibitor, PD-98059 (Fig. 8E) and partially inhibited by PI3K inhibitor,
LY-294002 (Fig. 8C). Further treatment with specic inhibitors for
ERK1/2 and PI3K pathway (PD-98059 and LY-294002) completely
blocked L-theanine induced NO production. All our results suggest
that L-theanine promotes NO production in endothelial cell via PI3K-
Akt independent and PI3K-ERK1/2 dependent pathway.
The PI3K/Akt and MEK/ERK signal transduction cascades play
important role in transmitting signals from membrane receptors to
downstream targets that regulate critical cellular responses. PI3K and
MEK pathway have been reported to cross talk. The cross-talks are
known to be down-stream of Tyrosine Kinase Receptor (RTK) [41].
From the results of our experiments, we hypothesis, L-theanine would
trigger activation of RTK pathway, subsequently leading to eNOS
phosporylation. Since agonist such as Bradykinin, VEGF, insulin,
sphingosine-1-phosphate activate eNOS phosphorylation on serine,
threonine and tyrosine residues through kinase activities [37],we
investigated L-theanine effects on a range of kinases. We observed
that L-Theanine cross-talks with both PI3K and MAPK signaling,
leading to activation of eNOS.
Flavonoids in tea, particularly catechins (ECG and EGCG) have
evidence to give sustained eNOS phosporylation (5 to 30 min), while
L-theanine induced eNOS phosporylation remains transient (25
min). Extrapolating in vitro evidence to in vivo efcacy, we have
shown that L-theanine would induce vasodilation in ex ovo (CAM)
model, further clinical efcacy in human's remains to be investigated.
To the best of our knowledge, this is the rst report on the efcacy
of L-theanine in promoting NO production in endothelial cells. Both
green and black tea has been shown to improve vascular function by
improving NO dependent endothelial function. The avonoids have
been implicated in vascular health benets of tea [8,9]. Our results
show that L-theanine may partially contribute to this effect. Future
work should be directed towards understanding L-theanine mediated
effects on vascular and neuronal health.
Acknowledgments
We acknowledge Vimal V., Ph.D. student for helping us with the
photography of the vasodilation model. This study was nancially
supported by Unilever R&D, Bangalore, India to S .Chatterjee.
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