Tài liệu Báo cáo Y học: Enhancement by a-tocopheryl hemisuccinate of nitric oxide production induced by lypopolysaccharide and interferon-c through the upregulation of protein kinase C in rat vascular smooth muscle cells docx

Enhancement by a-tocopheryl hemisuccinate of nitric oxide
production induced by lypopolysaccharide and interferon-c
through the upregulation of protein kinase C in rat vascular
smooth muscle cells
Kentaro Kogure, Motoki Morita, Susumu Hama, Sawa Nakashima, Akira Tokumura and Kenji Fukuzawa
1
Faculty of Pharmaceutical Sciences, University of Tokushima, Japan
The effect of a-tocopheryl hemisuccinate (TS) on lipo-
polysaccharide (LPS)/interferon-c (IFN)-induced nitric
oxide production in rat vascular smooth muscle cells
(VSMC) was examined. The LPS/IFN-induced NO pro-
duction was enhanced by TS but not by the other
a-tocopherol (a-T) derivatives a-tocopheryl acetate (TA)
and a-tocopheryl nicotinate (TN), or a-T itself. a-T, TA
and TN inhibited the enhancement by TS of LPS/IFN-
induced NO production. The enhancing effect of TS was
observed in the presence of LPS, but not IFN, suggesting
that TS participates in the LPS-stimulated signal pathway
leading to NO production. Protein kinase C (PKC)
inhibitors, but not protein kinase A inhibitors, inhibited the
enhancing effect of TS on LPS/IFN-induced NO produc-
tion. Furthermore, TS enhanced the amount of PKCa in
VSMC. From these results, we concluded that the enhan-
cing effect of LPS/IFN-induced NO production was
caused by upregulation of PKC in VSMC.
Keywords: a-tocopheryl hemisuccunate; a-tocopherol; nitric
oxide; vascular smooth muscle cells; protein kinase C.
The nonantioxidant function of a-tocopherol (a-T) has been
proposed [1–6]. It has been reported that a-T nonanti-
oxidatively prevented the proliferation of smooth muscle
cells through inhibition of protein kinase C (PKC), and the

transcription of some genes such as CD36 and collagenase.
We are interested in a-tocopheryl hemisuccinate (TS) as a
key compound for clarification of the nonantioxidant
function of a-T. TS is a naturally occurring amphiphilic
compound initially isolated from a green barley extract that
stimulates release of prolactin and growth hormone from
pituitary cells in vitro [7]. Since then, various biological
activities of TS such as inhibition of acetylcholine esterase
activity [8] and induction of apoptosis in various cancer cells
have been reported [9–16]. Furthermore, it has been reported
that TS increased activities of c-Jun NH
2
-terminal kinases
(JNK), extracellular signal-regulated kinases (ERK) and
mitogen-activated protein kinase kinase (MEK1/2) [14,15].
In vascular smooth muscle cells (VSMC), NO is known
to play a critical role in vasodilatory function and athero-
sclerotic processes [17], and inducible NO production in the
VSMC system has been well studied [18]. Recently, Kim
et al. reported that TS itself induced nitric oxide (NO)
production and inducible NO synthase (iNOS) expression
in U937 human monoblasts through activation of nuclear
factor-kappa B (NF-jB) [19]. However, because TS has
been reported to inhibit NF-jB activation in various cell
lines, such as human Jurkat T cells and human umbilical
vein endothelial cells [20–23], the mechanism of the TS effect
still remains unclear. Therefore, we investigated in detail the
effect of TS on NO production in the VSMC system. The
investigation would further give useful information about its
nonantioxidant function and therapeutic possibilities for

vascular diseases.
In atherosclerotic plaques, cytokines such as tumor
necrosis factor-a (TNF-a) and interleukin-1 secreted from
macrophages and foam cells have been implicated in the
pathogenetic events [24]. On the other hand, because these
cytokines are responsible for iNOS expression through
NF-jB factor activation, they are supposed to elicit the
NO-dependent vasodilation to improve the decreased blood
flow in the vascular lesions. To investigate the enhancing
effect of TS on NO production under the atherosclerosis-
like conditions, we used the system containing lipopolysac-
charide (LPS) known as a stimulant of iNOS expression
through a similar signaling cascade to these cytokines
[24,25]. In this study, interferon-c (IFN), which was also
reported to be secreted from T-cells in the lesions of
Correspondence to K. Fukuzawa, Faculty of Pharmaceutical
Sciences, University of Tokushima, Shomachi-1,
Tokushima 770-8505, Japan.
Fax: + 81 88 633 9572,
E-mail:
Abbreviations: AsA, ascorbic acid; BHA, butylhydroxyl anisol; ERK,
extracellular signal-regulated kinase; IFN, interferon-c;iNOS,indu-
cible nitric oxide synthase; JNK, c-Jun NH
2
-terminal kinase; LPS,
lipopolysaccharide; MEK, mitogen-activated protein kinase kinase;
MyD88, myeloid differentiation protein; NF-jB, nuclear factor-kappa
B; NO, nitric oxide; PKA, protein kinase A; PKC, protein kinase C;
PP2A, protein phosphatase-2A; a-T, a-tocopherol; TA, a-tocopheryl
acetate; TN, a-tocopheryl nicotinate; TRAF, tumor necrosis factor

receptor; TS, a-tocopheryl hemisuccinate; VSMC, vascular smooth
muscle cells.
Enzymes: nitric oxide synthase (EC 1.14.13.39); protein kinase
(EC 2.7.1.37).
(Received 11 December 2001, revised 18 February 2002,
accepted 21 March 2002)
Eur. J. Biochem. 269, 2367–2372 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02894.x
atherosclerosis, was further used together with LPS to
strengthen LPS function.
MATERIALS AND METHODS
Materials
RRR-a-Tocopheryl hemisuccinate (TS), RRR-a-tocopheryl
acetate (TA) and (+/–)-a-tocopheryl nicotinate (TN) were
purchased from Sigma Chemical Co. (St Louis, MO, USA)
(Fig. 1). LPS was obtained from DIFCO Laboratories
(Detroit, MI). Recombinant rat IFN was purchased from
PEPRO TECH EC (London, UK). RRR-a-Tocopherol
(a-T) waskindlyprovidedby Eisai Co.(Tokyo, Japan).Other
reagents were of the highest grade commercially available.
Treatment of VSMC with TS
VSMC were isolated from rat thoracic aorta using the
proteases elastase and collagenase as described previously
[26]. The VSMC (1 · 10
6
cells) were seeded into 35-mm
dishes, and were cultured for 24 h in Dulbecco’s modified
Eagle’s medium with 10% fetal bovine serum in a CO
2
-
incubator at 37 °CwithCO

2
in humidified air. Then, the
medium containing serum was removed, and the cells were
washed with phosphate buffered saline. Next, 2 mL of the
medium containing TS without serum was added to the
dishes. After treatment for 24 h with TS, LPS and IFN at
final concentrations of 10 lgÆmL
)1
and 100 UÆmL
)1
were
also added to the dishes for induction of NO production.
Then, 48 h after the addition of LPS/IFN, the cells were
subjected to various assays.
Nitrite analysis
The amount of NO was determined as production of nitrite,
because NO generated by various stimuli was quickly
oxidized to nitrite. Nitrite in the culture medium was
measured colorimetrically using Griess reagent (1% sulfa-
nilamide, 0.1% N-1-naphthyl-ethylenediamine dihydro-
chloride) [27]. Nitrite diazotiates the aryl amine, and then
the diazotiated product forms an azochromophore by
coupling with naphthyl-ethylenediamine. Absorbance was
measured at 550 nm in a Shimadzu UV-1600 spectropho-
tometer, and nitrite concentration was determined using
sodium nitrite as a standard.
Western blotting of inducible NO synthase
After removal of the culture medium for analysis of nitrite,
cells were collected from the dish into a sample tube using a
cell scraper. Buffer (2% SDS, 20% glycerol, 50 m

M
Tris/
HCl, pH 6.8) to dissolve cells was added to the cells, and
then the cell suspension was sonicated in a bath-type
sonicator for 5min. The amount of protein of the solubilized
cells was determined with a bicinchoninic acid protein assay
kit (PIERCE, Rockford, IL, USA) using bovine serum
albumin as a standard. The solubilized cells were subjected
to 10% SDS/polyacrylamide gel electrophoresis, and pro-
teins were transferred electrophoretically to a poly(vinylidene
difluoride) membrane. The membrane was treated with a
rabbit anti-iNOS polyclonal antibody or rabbit anti-PKC
polyclonal antibody at 1 : 1000 dilution and anti-(rabbit
IgG) Ig horseradish peroxidase conjugated antibody at
1 : 5000 dilution as a primary antibody and a secondary
antibody, respectively. The blots were detected with an
enhanced chemiluminescence kit (Amersham International
Plc, UK) and exposed to photographic films. The results of
Western blot analysis were representative pictures of at least
three independent experiments.
Statistical analysis
Data were expressed as the mean ± standard deviation of at
least three independent experiments. Statistical significance
was assessed by multiple-comparison test (Fisher’s protec-
ted least significant difference method). A P value of < 0.01
was considered to be statistically significant.
RESULTS
Effect of TS on LPS/IFN-induced NO production
in VSMC
The addition of LPS induced small, but significant NO

production, but additions of both LPS and IFN induced
detectably high levels of NO in VSMC. As shown in
Fig. 2A, approximately 20 l
M
of NO was produced by
10 lgÆmL
)1
of LPS and 100 UÆmL
)1
of IFN. Under these
conditions, detectable induction of iNOS protein was
observed by Western blotting (Fig. 2B). Thus, in subsequent
experiments, we used LPS and IFN 10 lgÆmL
)1
and
100 UÆmL
)1
, respectively, to induce the NO production in
VSMC. These concentrations are relatively high in com-
parison with those used for induction of NO in macroph-
ages but are similar to the amounts used in cells with low
sensitivity to LPS and IFN [28].
NO production was not induced by 10 l
M
TS alone in the
absence of LPS/IFN. However, treatment with TS 24 h
before the addition of LPS/IFN enhanced LPS/IFN-induced
NO production about twofold after a 48-h incubation
Fig. 1. Structures of a-tocopherol (a-T) and T derivatives a-tocopheryl
hemisuccinate (TS), a-tocopheryl acetate (TA) and a-tocopheryl nicoti-

nate (TN).
2368 K. Kogure et al. (Eur. J. Biochem. 269) Ó FEBS 2002
(Fig. 2A). The enhancing effects of TS at concentrations
higher than 10 l
M
were almost the same as that with 10 l
M
TS. Because TS has been reported to induce apoptosis in
various cell lines [8–15,29], the inhibitory effect of TS on the
cell growth at higher concentrations might prevent the
increase in the enhancing effect of TS on LPS/IFN-induced
NO production in VSMC. Therefore, we used a concentra-
tion of 10 l
M
TS in this study, except in some cases.
Furthermore, TS enhanced the amount of iNOS protein
induced by LPS/IFN (Fig. 2B), as well as NO production.
Accordingly, we concluded that the enhancing effect of TS
on LPS/IFN-induced NO production is caused by enhance-
ment of iNOS protein induction.
Effects of a-T and its derivatives on the enhancement
by TS of LPS/IFN-induced NO production
We compared the effects of a-T and its derivatives TA and
TN with that of TS on LPS/IFN-induced NO production.
The additions of a-T, TA and TN did not induce NO
production in the absence of LPS/IFN, like TS (data not
shown). Furthermore, a-T, TA and TN, even at 50 l
M
,did
not enhance the LPS/IFN-induced NO production in

VSMC in contrast to TS (Fig. 3).
We further examined the effects of a-T, TA and TN on
the enhancement by 10 l
M
TS of LPS/IFN-induced NO
production. As shown in Fig. 3, 50 l
M
a-T significantly
lowered the TS-enhanced NO production. In addition, a-T
reduced the amount of TS-enhanced iNOS protein induced
by LPS/IFN (data not shown). It is noteworthy that TA and
TN also decreased the enhancing effect of TS on LPS/IFN-
induced NO production.
Because the antioxidant a-T prevented the enhancing
effect of TS on LPS/IFN-induced NO production (Fig. 3),
we examined the effect of antioxidants such as butylhyd-
roxyl anisol (BHA) and ascorbic acid (AsA) on the
enhancing effect of TS on LPS/IFN-induced NO produc-
tion. Unexpectedly, neither BHA nor AsA affected the
TS-enhanced NO production (data not shown).
Effects of TS on NO productions induced by single
additions of various concentrations of LPS and IFN
NO production is reported to be stimulated by LPS and
IFN through independent signal pathways; LPS and IFN
stimulate cells by activating NF-jBandtheinterferon
regulatory factor-1, respectively [25,29–31]. To determine
which signal pathway of LPS or IFN was stimulated
with TS, we examined the effects of TS on the NO
production with LPS alone or IFN alone. As shown in
Fig. 4, addition of LPS alone (10 mgÆmL

)1
)orIFN
alone (100 UÆmL
)1
) induced the NO productions but
were very feeble as 1.1-fold and 1.4-fold of control,
respectively. These results indicate that the detectable
high NO production needs the presence of both LPS and
IFN in our experimental conditions. TS even at a high
concentration of 50 l
M
did not enhance IFN-induced
NO production (Fig. 4A). On the other hand, LPS-
dependent NO production was enhanced at 50 l
M
of TS
(Fig. 4B). Addition of 10 l
M
TS also stimulated, but not
significantly, the LPS-dependent NO production (data
not shown). The TS-stimulated NO production increased
with an increase in the concentration of LPS. From these
results, TS was suggested to participate in the pathway
stimulated with LPS, and IFN is necessary for expansion
of the TS effect. In addition, the TS (50 l
M
)-induced
acceleration of NO production in the LPS alone system
was inhibited by a-T, TA and TN (Fig. 4B, insert) as in
the LPS/IFN combination system (Fig. 3). However,

because the acceleration effect of TS on LPS-dependent
NO production system was very small, the inhibiting
Fig. 3. Effects of a-T and its derivatives (TA and TN) on LPS/IFN-
induced NO production in VSMC treated without or with 10 l
M
TS.
The concentrations of a-T, TA and TN were 50 l
M
.Otherexperi-
mental conditions were as described in Fig. 2. Values are means ± SD
(n ¼ 3). *P £ 0.01.
Fig. 2. Enhancements by TS of LPS/IFN-induced NO production (A)
and iNOS induction (B) in VSMC. The concentrations of LPS and IFN
were 10 lgÆmL
)1
and 100 UÆmL
)1
, respectively. (A) NO was measured
using the Griess reagent as nitrite. The amount of TS added alone was
10 l
M
. The doses of TS coadded with LPS/IFN are shown under the
columns. Values are means ± SD (n ¼ 3). *P £ 0.01. (B) Induced
iNOS in VSMC was detected by Western blotting using a rabbit anti-
iNOS Ig. The concentration of TS was 10 l
M
.
Ó FEBS 2002 Tocopheryl succinate-enhanced NO production (Eur. J. Biochem. 269) 2369
effects of a-T and its derivatives were not as clear as the
effects observed in the system with LPS/IFN.

Effects of protein kinase inhibitors
on the enhancement by TS of LPS/IFN-induced
NO production
Protein kinases such as PKA and PKC, and various factors
such as tumor necrosis factor receptor 6 (TRAF6) and
myeloid differentiation protein (MyD88) are reported to
participate in LPS-induced signal transduction [25,29–31].
BecauseTSwassuggestedtoaffectmainlythesignal
pathway stimulated with LPS, we examined the effects of
inhibitors of PKA and PKC on TS-enhanced NO produc-
tion. As shown in Fig. 5A, the PKA inhibitors KT-5720
and H8 and the PKC inhibitors Ro31-2880 and
GF109203X did not affect apparently LPS/IFN-induced
NO production. In the combination system of 10 l
M
TS
with LPS/IFN, the PKA inhibitors KT-5720 and H8 had no
effect, but the PKC inhibitors Ro31-2880 and GF109203X
significantly inhibited NO production. The degrees of
inhibition of NO production with Ro31-2880 and
GF109203X were approximately 70 and 30%, respectively.
Effects of TS on the amount of PKCa
We further examined the effects of TS on the amounts of
PKCa and PKCb by Western blotting. As shown in
Fig. 5B, PKCa was increased slightly by treatment with
LPS/IFN. Addition of 10 l
M
TS to the LPS/IFN-system
enhanced the amount of PKCa. However, we could not
detect PKCb in control cells, and no change of PKCb

involved with and without TS was observed in the LPS/IFN
treated cells (data not shown). The amounts of other
proteins related with the LPS-stimulated signal pathway,
such as TRAF6 and MyD88 were not affected by the
addition of TS in this study (data not shown). These results
suggested that TS induced upregulation of PKCa.
DISCUSSION
In this study, to obtain information about the mode of
actions of TS, we examined the effect of TS on LPS/IFN-
induced NO production in VSMC. We found that TS
activated LPS/IFN-induced NO production in VSMC
through enhancement of iNOS protein synthesis, although
TS itself did not induce NO production in the absence of
LPS/IFN (Fig. 2). Previously, we found that TS was taken
up immediately into the VSMC, but TS was not hydrolyzed
to a-T and succinic acid [32]. Accordingly, the enhancement
of LPS/IFN-induced NO production is attained by TS itself
rather than a derivative.
TS enhanced LPS-dependent but not IFN-dependent NO
production, indicating that TS activated a LPS-stimulated
signal pathway (Fig. 4). PKC is a key kinase in the LPS-
stimulated signal pathway [30,31]. The findings that PKC
inhibitors, not PKA inhibitors, inhibited TS-enhanced NO
production, and the level of NO production inhibited with
PKC inhibitor Ro31-2880 was almost the same as that of
LPS/IFN-induced NO production without TS (Fig. 5A),
suggesting that the enhancement of NO production with TS
was strongly dependent on PKC activity. Furthermore, an
increase in the amount of PKCa by TS treatment (Fig. 5B)
suggested that TS enhanced LPS-dependent NO production

through upregulation of PKCa.
Fig. 5. Effects of PKA inhibitors (KT-5720 and H8) and PKC inhibitors
(Ro31-8220 and GF109203X) on the enhancement by TS of LPS/IFN-
induced NO production in VSMC (A), and Western blot analysis of
PKCa in control VSMC, VSMC treated with LPS/IFN and VSMC
treated with TS and LPS/IFN (B). KT: KT-5720, Ro: Ro31-8220, GF:
GF109203X. The concentrations of KT, H8, Ro, GF and TS were 1,
20,5,10and10l
M
, respectively. Values are means ± SD (n ¼ 3).
*P £ 0.01. Other experimental conditions were as described in Fig. 2.
Fig. 4. Effects of TS on NO production
induced with various amount of IFN (A) or LPS
(B) in VSMC. VSMC was treated without
(open column) or with 50 l
M
TS (closed
column). The insert graph of (B) shows the
inhibition effects of a-T, TA and TN on the
enhancement by 50 l
M
TS of LPS-induced
NO production. Values are means ± SD
(n ¼ 3). *P £ 0.01.
2370 K. Kogure et al. (Eur. J. Biochem. 269) Ó FEBS 2002
TS enhanced the LPS/IFN-induced NO production but
TA and TN did not (Fig. 3), although their antioxidative
OH-moiety is masked. We hypothesized that an am-
phiphilic characteristic of TS is important for its enhancing
effect, because among the a-T derivatives examined only TS

has an amphiphilic structure of the polar carboxyl moiety
and the hydrophobic isoprene moiety. The enhancing effect
of TS on LPS/IFN-induced NO production was inhibited
by the coexistence of a-T, TA and TN (Figs 3 and 4), but
not of the antioxidants BHA and AsA. These results
suggested that active oxygens and free radicals did not
participate in the TS effect, and that the inhibitory effect of
a-T was mediated by a nonantioxidative reaction. a-T was
reported to decrease PKCa activity due to activation of
protein phosphatase 2A (PP2A) in smooth muscle cells [2–
6]. These studies suggest that the inhibitory effect of a-T
was due to reduction of accelerated PKC activity with TS in
VSMC. TA and TN also showed the inhibition effect like a-
T on TS-activated NO production, suggesting that the
action target of both TA and TN is the same as that of a-T.
It is very interesting that a-T and TS showed opposite
effects on PKC in this study, although the structures of
both are very similar. Perhaps, the negatively charged
carboxyl moiety of TS is important for upregulation of
PKC. Recently, Neuzil et al. reported the opposite findings
to ours. They proposed that TS-induced apoptosis in
hepatopoietic and cancer cell lines is caused by the
prevention of PKC activity due to activation of PP2A,
similar to a-T activation of PP2A [13]. The reason for the
inconsistency between our results and those of Neuzil et al.
is unclear; it may be caused by differences in the response,
delivery and distribution of TS in each cell line.
Recently, we found that TS-induced apoptosis of VSMC
was caused by the stimulation of superoxide production due
to the activation of NADPH oxidase [32]. As the activation

of NADPH oxidase is reported to be associated with the
activation of PKC [33,34], the PKC-dependent mechanism
of TS-induced enhancement of NO production observed in
this study are consistent with the mechanism of TS-induced
apoptosis. Yu et al. reported recently that activation of
ERK is required for TS-induced apoptosis of human breast
cancer cells [15]. Kim et al. reported that the addition of TS
alone induced NO production in human U937 monoblasts
possibly by the activation of NF-jB [19]. Further study is
necessary to clarify the relationship between the activation
of these factors participating in cell survival signaling
pathwaysandTSeffects.
In this study, we found for the first time that TS
enhanced LPS/IFN-induced NO production in VSMC,
and that the TS effect would be induced through upreg-
ulation of PKCa. In addition, we found that the a-T
derivatives TA and TN inhibited TS-enhanced NO
production in a manner similar to a-T. From these results,
we assumed that the nonantioxidative function of TS is
based on its unique structure.
ACKNOWLEDGEMENTS
This work was supported by Grant No. 12771436 from the Japanese
Society for the Promotion of Science, and in part by a research grant
from the Faculty of Pharmaceutical Sciences, The University of
Tokushima.
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