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

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

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

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-

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

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.

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

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

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