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An Extract From Brown Rice Inhibits Signal
Transduction of Angiotensin II in Vascular SmoothMuscle Cells
Background
Health benefits of brown rice over white rice have been described
previously. However, whether the outer bran of rice contains an
ingredient useful to prevent cardiovascular diseases remains
unknown. The subaleurone layer of rice, which is usually lostby milling brown rice for whitening, is rich in varied nutrients,suggesting that some ingredient contained within this layer may bebeneficial for the cardiovascular system.
Methods
To assess potential benefits of the subaleurone layer toward
pathological vascular remodeling, we examined the effects of thelayer extracts from Japanese rice (Oryza sativa var. japonica) on
angiotensin II (Ang II)-induced signal transduction and hypertrophy
in cultured rat vascular smooth muscle cells (VSMCs).

White rice has long been consumed as a staple food in many
countries. However, it has been reported that white riceconsumption may increase the risk for type II diabetes.1 In
contrast, brown rice intake seems to decrease risks of cardiovascular disease and diabetes,2 suggesting that the potential
health benefit of rice is lost during rice milling. The subaleurone layer of rice located between the white center and outerbran is usually lost in the milling process. This layer contains various important nutrients, and recent developments
in pressure-equalizing rice polishing technology have enabled producers to retain this layer on certain types of white
rice (http://www.toyo-rice.jp/english/index.html). However,despite proposed health benefits, knowledge about whether
1Department of Internal Medicine, School of Medicine, Wakayama Medical

University, Wakayama, Japan; 2Cardiovascular Research Center and Departmentof Physiology, Temple University School of Medicine, Philadelphia, Pennsylvania,
USA; 3Department of Materials Engineering, Nagaoka National College
of Technology, Nagaoka, Japan; 4Department of Materials Science andEngineering, Wakayama National College of Technology, Wakayama, Japan.
Correspondence: Satoru Eguchi (seguchi@temple.edu)
Received 27 October 2010; first decision 10 November 2010;
accepted 29 December 2010.
© 2011 American Journal of Hypertension, Ltd.
530Results
Pretreatment of the ethyl acetate extract (100 μg/ml), but not otherextracts, inhibited Ang II (100 nmol/l)-induced immediate signaltransduction events. Also, the extract diminished c-Fos expression
and hypertrophic protein accumulation induced by Ang II in the cells.
Conclusion
These data suggest that an ingredient in the ethyl acetate extractfrom the subaleurone layer of rice has a protective effect towardcardiovascular diseases by interfering with signal transduction
induced by Ang II.
Keywords: angiotensin II; blood pressure; hypertension; rice;signal transduction; vascular smooth muscle cell
American Journal of Hypertension, advance online publication 17 February 2011;
doi:10.1038/ajh.2011.10

this rice layer contains an ingredient that prevents cardiovascular diseases remains unknown.
Angiotensin II (Ang II), the major bioactive peptide of
the renin–angiotensin system, is strongly implicated in various cardiovascular diseases such as hypertension, atherosclerosis, and heart failure.3 In addition to inducing arterial
contraction, Ang II is an important growth factor causing
hypertrophy and or hyperplasia of vascular smooth muscle cells (VSMCs). Most of the known actions of Ang II are
mediated through the angiotensin type 1 receptor (AT1),
which is coupled to multiple heterotrimeric G proteins.4
We and others have previously reported that Ang II induces
hypertrophy and migration of VSMCs through the epidermal
growth factor receptor (EGFR) and Rho-kinase (ROCK),
which are mainly activated through Gq and G12/13, respectively.5–7 The activation of the EGFR and ROCK by Ang II
can be monitored by phosphorylation of the receptor andthe substrate myosin phosphatase target subunit-1 (MYPT1)
in VSMCs, respectively.6 To assess a cardiovascular protective effect of the rice subaleurone layer, we have investigatedthe effects of its extracts on Ang II–induced signal transduction events and subsequent hypertrophy and migration in
cultured VSMCs.
maY 2011 | VOLUME 24 NUMBER 5 | 530-533 | AMERICAN JOURNAL OF HYPERTENSION

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Hirotoshi Utsunomiya1, Akira Takaguri2, Allison M. Bourne2, Katherine J. Elliott2, Shin-ichi Akazawa3,
Yoshiharu Okuno4, Ryohei Kono1 and Satoru Eguchi2

brief communications

Rice Extract Inhibits Angiotensin II

Methods

Extraction of the rice layer. The subaleurone layer of Japaneserice (1.2 kg) was refluxed with methanol at 60 °C to produce amethanol extract. The extract was filtered and evaporated using
a rotary evaporator under reduced pressure to yield a viscous
mass (140 g). This extract was suspended in water (1 liter) andtransfered to a 2–l funnel for separation. The suspended solution was extracted three times with 300 ml of hexane, dichloromethane, and ethyl acetate, successively. Each soluble fraction was concentrated under reduced pressure at 40 °C to givehexane (60.0 g), dichloromethane (1.11 g), and ethyl acetate(2.19 g) extracts. Each extract was reconstituted in 100% dimethyl sulfoxide at 100 mg/ml. Extracts prepared at three distinct
times were utilized to confirm the findings.
Cell culture. VSMCs were prepared from thoracic aorta ofSprague-Dawley rats by the explant method as described previously.8 All animal procedures conformed to the Guide for
the Care and Use of Laboratory Animals published by the
US National Institutes of Health (NIH Publication No. 85-23,
revised 1996) and Temple University. VSMCs were subcultured in Dulbecco’s modified Eagle’s medium containing 10%
fetal bovine serum, penicillin, and streptomycin. VSMCs frompassages 3–12 used in the experiments typically showed >99%
positive immunostaining of smooth muscle α-actin antibody.
Cells at 80–90% confluence in culture wells were made quiescent by incubation with serum-free medium for 2–3 days. To
avoid any potential phenotypic selection and genetic alteration,
VSMCs were renewed every 2–3 months and VSMCs from frozen stock were never used. The results were confirmed in atleast two distinct cell lines. For experiments, the serum-free
media was exchanged and VSMCs were incubated with fresh
serum-free media for 30 min. Cells were then pretreated withthe rice layer extract (final concentration 10, 30, or 100 μg/
ml in Dulbecco’s modified Eagle’s medium with 0.1% dimethyl
sulfoxide) or the vehicle (0.1% dimethyl sulfoxide) as a control
for 1h before Ang II stimulation.
Immunoblotting. Immunoblotting was performed as previously described.8 Quiescent VSMCs grown on 6-well plates(~5 × 105 cells/well) were stimulated with Ang II for specified
durations. The reaction was terminated by the replacement of
AMERICAN JOURNAL OF HYPERTENSION | VOLUME 24 NUMBER 5 | may 2011

medium with 100 μl of 1 × SDS sample buffer. Each 40 μl of thecell lysates was subjected to SDS-PAGE gel electrophoresis andelectrophoretically transferred to a nitrocellulose membrane.
The membranes were then exposed to primary antibodies
overnight at 4 °C. After incubation with the peroxidase-linked
secondary antibody for 1 h at room temperature, immunoreactive proteins were visualized by a chemiluminescence reaction
kit. The results were quantified by densitometry in the linear
range of film exposure using CanoScan N670U (Canon, Lake
Success, NY) and Un-Scan-It Gel 5.3 software (Silk Scientific,
Orem, UT), and the ratios EGFR-p/EGFR and MYPT1-p/
MYPT1 were calculated. An example of data supporting the linearity has been demonstrated.7 Unless stated otherwise, results
were expressed as % increase in which the response to Ang II
is defined as 100% because the basal signals are more varied
depending on film exposure than the stimulated signals.
Protein assay. To assess Ang II–induced VSMC hypertrophy
directly, we have been utilizing an assay measuring cell protein accumulation but not a radiolabeled leucine incorporation
assay to avoid unnecessary use of a radioactive compound.6,9Consistent with a highly cited past report,10 incubationof VSMCs with 100 nmol/l Ang II for 3 days in serum-freeDulbecco’s modified Eagle’s medium resulted in increases incell protein and volume without any significant change in cellproliferation/viability.6,9 Thus, the protein assay was performedin the present study to estimate protein synthesis as previously
described.6,9 VSMC on 12-well culture plates were incubatedwith serum-free Dulbecco’s modified Eagle’s medium for 2days and were further incubated with or without 100 nmol/l
Ang II for 3 days. After aspiration of the medium, cells were
washed twice in ice-cold Hank’s balanced salt solution, and thetotal amount of cellular protein was measured using a BCA
protein assay kit (Pierce, Rockford, IL).
Measurement of intracellular Ca2+. Intracellular Ca2+ wasmeasured as described previously by using fura 2 as an indicator.6 VSMCs (~105 cells/well) subcultured on coverslips
were loaded with 3 μmol/l fura 2-AM at room temperature for
45 min. The fura 2 fluorescence was acquired at a frequency of
1 Hz and the intracellular Ca2+ values were then obtained as
described.6
Statistical analysis. Data are presented as means ± s.e.m.
Significance between groups was evaluated using one-way
analysis of variance followed by Student’s t-test with Stat View
software (SAS Institute, Cary, NC). P < 0.05 was considered to
be statistically significant.
Results

We investigated which extracts (each 100 μg/ml for 1 h treatment) from the subaleurone layer affect Ang II–induced EGFR
transactivation as assessed by its Tyr1068 phosphorylation in
VSMCs. The ethyl acetate extract, but not other extracts (hexane extract, dichloromethane extract, or methanol extract), has
an inhibitory effect on the phosphorylation (data not shown).
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Reagents. The subaleurone layer of Japanese rice (Oryza sativa
var. japonica) was separated with pressure-equalizing ricepolishing technology developed by Toyo Rice, Tokyo, Japan(http://www.toyo-rice.jp/english/index.html). Ang II was purchased from Sigma-Aldrich (St Louis, MO). Antibodies forTyr1068-phosphorylayed EGFR and Thr853-phosphorylated
MYPT1 were purchased from Invitrogen (4478G: Carlsbad,
CA) and Upstate (36-003; Millipore, Billerica, MA), respectively. Antibodies for EGFR and c-Fos were purchased fromSanta Cruz Biotechnology (sc-03 and sc-52; Santa Cruz, CA).
Antibody for MYPT1 was purchased from Covance (PRB457C; Princeton, NJ). Antibody for GAPDH was purchasedfrom Chemicon, Billerica, MA (MRB374; Millipore).

brief communications
b

**

100

†

†

EGFR-p
MYPT-p

†

50

0

−

+

−

+

−

+

Ang llCa2+ (340 nm/380 nm)

Phosphorylation (%)

a

Rice Extract Inhibits Angiotensin II

Discussion
4
DMSO
2
Rice-exAng ll0

EGFR-p

0

100

(s)

200

EGFR

MYPT
0

−

+

−

+

100

Ang ll
c-Fos
GAPDH

DMSO

Rice-ex(µg/ml)

400

*

†

200

0

Basal Ang ll Basal Ang ll
DMSO

Rice-ex

Figure 1 | An ethyl acetate extract of the subaleurone layer of rice inhibitedangiotensin II (Ang II)-induced signal transduction in vascular smoothmuscle cells. The cells pretreated with the extract (Rice-ex) (a–d) 100 μg/­mlor (a) 30 μg/ml for 1 h were stimulated with 100 nmol/l Ang II for (a) 2 min,(c) 1 h, (d) 3 days or (b) indicated durations. Stimulations of epidermal
growth factor receptor (EGFR) or myosin phosphatase target subunit-1(MYPT1) (a) phosphorylation, (b) intracellular Ca2+ mobilization, (c) c-Fos
induction, and (d) protein synthesis were evaluated as described in the
Methods section. (a). Data are mean ± s.e.m. of three experiments. *P < 0.05
compared to the basal control. †P < 0.05 compared with the stimulated
control. (b) Representative data from each n = 6. (c) Representative blot fromn = 3. (d) Data are mean ± s.e. (n = 3). *P < 0.05 compared to the basal control.
†P < 0.05 compared with the stimulated control. DMSO, dimethyl sulfoxide.

Pretreatment of cultured VSMCs with the ethyl acetate extract
for 1 h at 30 μg/ml partially and 100 μg/ml almost completely
inhibited Ang II–induced phosphorylation of EGFR, whereas
only 100 μg/ml partially inhibited Ang II–induced phosphorylation of MYPT1. The extract had no effects on expressions of
EGFR and MYPT1 (Figure 1a). No significant inhibition wasobserved with 10 μg/ml of the extract. Preliminary experimentswith the ethyl acetate extract (100 μg/ml) for 30-min treatment
showed weaker inhibitory effect on Ang II–induced EGFR
phosphorylation.
We have shown that intracellular Ca2+ elevation throughthe Gq-coupled AT1 receptor is essential for Ang II–induced
EGFR transactivation.6,8 As shown in Figure 1b, the acetate
extracts almost completely inhibited intracellular Ca2+ elevation induced by Ang II. Because c-Fos induction is implicatedin the Ang II–induced vascular remodeling,11 we further
examined the effects of the acetate extract on the c-Fos induction. As shown in Figure 1c, pretreatment of the acetate
extract partially inhibited Ang II–induced c-Fos expression.
Finally, we investigated whether the acetate extract inhibits
Ang II–induced cell hypertrophy by measuring protein accumulation in response to Ang II. Again, the extract significantly
inhibited Ang II–induced protein accumulation (Figure 1d).
No morphological difference or reduction of total protein wasobserved in VSMCs treated with or without the extract for
72 h, thus excluding a toxicity of the extract.
532

Acknowledgments: This work was supported by National Institute of Healthgrant, HL076770 (S.E.), by an American Heart Association Established
Investigator Award, 0740042N (S.E.), and by a Grant-in-Aid for Science, Sports,
and Culture, Japan (H.U.).

Disclosure: The authors declared no conflict of interest.
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2.
3.
4.
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6.

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C

30

d
Cell Protein (µg/well)

MYPT-p

VSMC hypertrophy induced by Ang II is believed to be pivotal for progression of hypertension.3,5,12 Thus, inhibition of
hypertrophic signal transduction events represents a potentially
important therapeutic strategy for prevention of hypertensive
multiorgan damage. Although our data were collected in cultured vascular cells with limited concentrations and time points,
we have shown that the ethyl acetate extract from subaleurone
layer of Japanese rice significantly inhibited key signaling events
that have been linked to vascular remodeling in vivo.5
A number of past publications have reported that certain
extracts or purified chemicals from plants have significant
inhibitory effects on signal transduction events and subsequent downstream functions induced by Ang II in VSMCs.13–
15 Those plant-derived biochemicals act on either Ang II
binding to the receptor16 or the downstream signal transduction.13–15 However, to the best of our knowledge, no past publication is available reporting if any extract from a staple grain
has an inhibitory effect toward Ang II or reporting the chemical property of the rice subaleurone layer.
Our past research has pointed out the critical role of Gqmediated intracellular Ca2+ elevation for downstream signaltransduction of Ang II, including EGFR transactivation.6,8
However, G12/13 activation mainly mediates MYPT1 phosphorylation by Ang II through ROCK, and Gq makes only a partial
contribution to the phosphorylation in VSMCs.7 Therefore, it
is likely that the ethyl acetate extract interferes with the signaling events between the receptor binding and the Ca2+ elevation such as Gq or phospholipase C. This also explains thepartial inhibition of c-Fos induction by the extract because
both the EGFR cascade and the ROCK cascade are implicated in the induction.11,17 Accordingly, further studies arenecessary to confirm that the rice subaleurone layer contains
a specific ingredient useful toward preventing cardiovasculardiseases and to determine its detailed mechanism in signal
interference.

Rice Extract Inhibits Angiotensin II

7.

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12. Touyz RM, Schiffrin EL. Signal transduction mechanisms mediating thephysiological and pathophysiological actions of angiotensin II in vascular smoothmuscle cells. Pharmacol Rev 2000; 52:639–672.
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acid inhibits vascular smooth muscle cell proliferation induced by angiotensin II
in stroke-prone spontaneously hypertensive rats. Hypertens Res 2005; 28:369–377.
14. Xiao JH, Zhang YL, Feng XL, Wang JL, Qian JQ. Effects of isoliensinine on
angiotensin II-induced proliferation of porcine coronary arterial smooth muscle
cells. J Asian Nat Prod Res 2006; 8:209–216.
15. Zhou CH, Wu XH, Wu YQ. Nobiletin, a dietary phytochemical, inhibits vascularsmooth muscle cells proliferation via calcium-mediated c-Jun N-terminal kinases
pathway. Eur J Pharmacol 2009; 615:55–60.
16. Caballero-George C, Vanderheyden PM, Apers S, Van den Heuvel H, Solis PN,
Gupta MP, Claeys M, Pieters L, Vauquelin G, Vlietinck AJ. Inhibitory activity on
binding of specific ligands to the human angiotensin II AT(1) and endothelin 1
ET(A) receptors: bioactive benzo[c]phenanthridine alkaloids from the root of
Bocconia frutescens. Planta Med 2002; 68:770–775.
17. Yamakawa T, Tanaka S, Numaguchi K, Yamakawa Y, Motley ED, Ichihara S,
Inagami T. Involvement of Rho-kinase in angiotensin II-induced hypertrophy of
rat vascular smooth muscle cells. Hypertension 2000; 35:313–318.

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Suzuki H, Kimura K, Shirai H, Eguchi K, Higuchi S, Hinoki A, Ishimaru K, Brailoiu E,
Dhanasekaran DN, Stemmle LN, Fields TA, Frank GD, Autieri MV, Eguchi S.
Endothelial nitric oxide synthase inhibits G12/13 and rho-kinase activated by
the angiotensin II type-1 receptor: implication in vascular migration. Arterioscler
Thromb Vasc Biol 2009; 29:217–224.
8. Eguchi S, Numaguchi K, Iwasaki H, Matsumoto T, Yamakawa T, Utsunomiya H,
Motley ED, Kawakatsu H, Owada KM, Hirata Y, Marumo F, Inagami T. Calciumdependent epidermal growth factor receptor transactivation mediates theangiotensin II-induced mitogen-activated protein kinase activation in vascularsmooth muscle cells. J Biol Chem 1998; 273:8890–8896.
9. Nakashima H, Frank GD, Shirai H, Hinoki A, Higuchi S, Ohtsu H, Eguchi K, Sanjay A,
Reyland ME, Dempsey PJ, Inagami T, Eguchi S. Novel role of protein kinase
C-delta Tyr 311 phosphorylation in vascular smooth muscle cell hypertrophy byangiotensin II. Hypertension 2008; 51:232–238.
10. Geisterfer AA, Peach MJ, Owens GK. Angiotensin II induces hypertrophy, nothyperplasia, of cultured rat aortic smooth muscle cells. Circ Res 1988;
62:749–756.
11. Eguchi S, Iwasaki H, Hirata Y, Frank GD, Motley ED, Yamakawa T, Numaguchi K,
Inagami T. Epidermal growth factor receptor is indispensable for c-Fos expression
and protein synthesis by angiotensin II. Eur J Pharmacol 1999; 376:203–206.

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