Enhancement by a-tocopheryl hemisuccinate of nitric oxideproduction induced by lypopolysaccharide and interferon-c through the upregulation of protein kinase C in rat vascular smooth mus
Trang 1Enhancement 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 Fukuzawa1
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 NH2-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: fukuzawa@ph.tokushima-u.ac.jp
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)
Trang 2atherosclerosis, was further used together with LPS to
strengthen LPS function
M A T E R I A L S A N D M E T H O D S
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) was kindly provided by 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· 106 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 CO2
-incubator at 37C with CO2in 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)1and 100 UÆmL)1were
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 mM 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
R E S U L T S
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 lM of NO was produced by
10 lgÆmL)1of LPS and 100 UÆmL)1of 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 lMTS 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).
Trang 3(Fig 2A) The enhancing effects of TS at concentrations
higher than 10 lMwere almost the same as that with 10 lM
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 lMTS 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 lM, 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 lM TS of LPS/IFN-induced NO
production As shown in Fig 3, 50 lM 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-jB and the interferon 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) or IFN 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 lM did not enhance IFN-induced
NO production (Fig 4A) On the other hand, LPS-dependent NO production was enhanced at 50 lM of TS (Fig 4B) Addition of 10 lMTS 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 lM)-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 (TAand 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 Other experi-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)1and 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
Trang 4effects 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]
Because TS was suggested to affect mainly the signal
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 lMTS 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 lMTS 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
D I S C U S S I O N
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 PKAinhibitors (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, 10 and 10 l 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.
Trang 5TS 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 a-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
pathways and TS effects
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
A C K N O W L E D G E M E N T S
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.
R E F E R E N C E S
1 Traber, M.G & Packer, L (1995) Vitamin E: beyond antioxidant function Am J Clin Nutr 62, 1501S–1509S.
2 Azzi, A., Boschoboinik, D., Cle´ment, S., O¨zer, N.K., Ricciarelli, R., Stocker, A., Tashinato, A & ¸Sirikc¸i, O¨ (1997) Signalling functions of a-tocopherol in smooth muscle cells Intern J Vit Nutr Res 67, 343–349.
3 O¨zer, N.K & Azzi, A (2000) Effect of vitamin E on the devel-opment of atherosclerosis Toxicology 148, 179–185.
4 Azzi, A & Stocker, A (2000) Vitamin E: non-antioxidant roles Prog Lipid Res 39, 231–255.
5 Azzi, A., Breyer, I., Feher, M., Pastori, M., Ricciarelli, R., Spy-cher, S., Staffieri, M., Stocker, A., Zimmer, S & Zingg, J.-M (2000) Specific cellular responses to a-tocopherol J Nutr 130, 1649–1652.
6 Azzi, A., Breyer, I., Feher, M., Ricciarelli, R., Stocker, A., Zimmer, S & Zingg, J.-M (2001) Nonantioxidant functions
of a-tocopherol in smooth muscle cells J Nutr 131, 378S– 381S.
7 Badamchian, M., Spangelo, B.L., Bao, Y., Hagiwara, Y., Hagiwara, H., Ueyama, H & Goldstein, A.L (1994) Isolation of a vitamin E analog from a green barley leaf extract that stimulates release of prolactin and growth hormone from rat anterior pitui-tary cells in vitro J Nutr Biochem 5, 145–150.
8 Chelliah, J., Smith, J.D & Fariss, M.W (1994) Inhibition of cholinesterase activity by tetrahydroaminoacridine and the hemi-succinate esters of tocopherol and cholesterol Biochim Biophys Acta 1206, 17–26.
9 Turley, J.M., Fu, T., Ruscetti, F.W., Mikovits, J.A., Bertolette, D.C III & Birchenall-Roberts, M.C (1997) Vitamin E succinate induces Fas-mediated apoptosis in estrogen receptor-negative human breast cancer cells Cancer Res 57, 881–890.
10 Yu, W., Israel, K., Liao, Q.Y., Aldaz, C.M., Sanders, B.G & Kline, K (1999) Vitamin E succinate (VES) induces Fas sensitivity
in human breast cancer cells: role for M r 43,000 Fas in VES-triggered apoptosis Cancer Res 59, 953–961.
11 Neuzil, J., Svensson, I., Weber, T., Weber, C & Brunk, U.T (1999) a-Tocopheryl succinate-induced apoptosis in Jurkat T cells involves caspase-3 activation, and both lysosomal and mitoch-ondrial destabilization FEBS Lett 445, 295–300.
12 Yamamoto, S., Tamai, H., Ishisaka, R., Kanno, T., Arita, K., Kobuchi, H & Utsumi, K (2000) Mechanism of a-tocopheryl succinate-induced apoptosis of promyelocytic leukemia cells Free Rad Res 33, 407–418.
13 Neuzil, J., Weber, T., Schro¨der, A., Lu, M., Ostermann, G., Gellert, N., Mayne, G.C., Olejnicka, B., Ne`gre-Salvayre, A., Stı´-cha, M., Coffey, R.J & Weber, C (2001) Induction of cancer cell apoptosis by a-tocopheryl succinate: molecular pathways and structural requirements FASEB J 15, 403–415.
14 You, H., Yu, W., Sanders, B.G & Kline, K (2001) RRR-a-Tocopheryl succinate induces MDA-MB-435 and MCF-7 human breast cancer cells to undergo differentiation Cell Growth Differ.
12, 471–480.
15 Yu, W., Liao, Q.-Y., Hantash, F.M., Sanders, B.G & Kline, K (2001) Activation of extracellular signal-regulated kinase and c-Jun NH 2 -terminal kinase but not p38 mitogen-activated protein kinases is required for RRR-a-tocopheryl succinate-induced apoptosis of human breast cancer cells Cancer Res 61, 6569– 6576.
16 Neuzil, J., Weber, T., Terman, A., Weber, C & Brunk, U.T (2001) Vitamin E analogues as inducers of apoptosis: implications for their potential antineoplastic role Redox Report 6, 143–151.
17 Jeremy, J.Y., Rowe, D., Emsley, A.M & Newby, A.C (1999) Nitric oxide and the proliferation of vascular smooth muscle cells Cardiovasc Res 43, 580–594.
Trang 618 Hecker, M., Cattaruzza, M & Wagner, A.H (1999) Regulation of
inducible nitric oxide synthase gene expression in vascular smooth
muscle cells Gen Pharmacol 32, 9–16.
19 Kim, S.-J., Bang, O.-S., Lee, Y.-S & Kang, S.-S (1998)
Produc-tion of inducible nitric oxide is required for monocytic
differ-entiation of U937 cells induced by vitamin E-succinate J Cell Sci.
111, 435–441.
20 Suzuki, Y.J & Packer, L (1993) Inhibition of NF-jB activation
by vitamin E derivatives Biochem Biophys Res Commun 193,
277–283.
21 Suzuki, Y.J & Packer, L (1993) Inhibition of NF-jB DNA
binding activity by a-tocopheryl succinate Biochem Mol Biol.
Int 31, 693–700.
22 Erl, W., Weber, C., Wardemann, C & Weber, P.C (1997)
a-Tocopheryl succinate inhibits monocytic cell adhesion to
endothelial cells by suppressing NF-jB mobilization Am.
J Physiol 273, H634–H640.
23 Nakamura, T., Goto, M., Matsumoto, A & Tanaka, I (1998)
Inhibition of NF-jB transcriptional activity by a-tocopheryl
suc-cinate Biofactors 7, 21–30.
24 Ross, R (1999) Mechanism of disease-atherosclerosis-an
inflam-matory disease New Engl J Med 340, 115–126.
25 Zhang, F.X., Kirschning, C.J., Mancinelli, R., Xu, X.-P., Jin, Y.,
Faure, E., Mantovani, A., Rothe, M., Muzio, M & Arditi, M.
(1999) Bacterial lipopolysaccharide activates nuclear factor-jB
through interleukin-1 signaling mediators in cultured human
dermal endothelial cells and mononuclear phagocytes J Biol.
Chem 274, 7611–7614.
26 Tokumura, A., Iimori, M., Nishioka, Y., Kitahara, M., Sakashita,
M & Tanaka, S (1994) Lysophosphatidic acids induce
pro-liferation of cultured vascular smooth muscle cells from rat aorta.
Am J Physiol 267, C204–C210.
27 Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L.,
Wishnok, J.S & Tannenbaum, S.R (1982) Analysis of nitrate,
nitrite, and [ 15 N]nitrate in biological fluids Anal Biochem 126,
131–138.
28 Hong, Y., Suzuki, S., Yatoh, S., Mizutani, M., Nakajima, T., Bannai, S., Sato, H., Soma, H., Okuda, Y & Yamada, N (2000) Effect of hypoxia on nitric oxide production and its synthase gene expression in rat smooth muscle cells Biochem Biophys Res Commun 268, 329–332.
29 Saura, M., Zaragoza, C., Bao, C., McMillan, A & Lowenstein, C.J (1999) Interaction of interferon regulatory factor-1 and nuclear factor jB during activation of inducible nitric oxide synthase transcription J Mol Biol 289, 459–471.
30 Chen, C.-C., Wang, J.-K & Lin, S.-B (1998) Antisense oligo-nucleotides targeting protein kinase C-a-, -bI, or -d but not -g inhibit lipopolysaccharide-induced nitric oxide synthase expres-sion in RAW 264.7 macrophages: Involvement of a nuclear factor kB-dependent mechanism J Immunol 161, 6206– 6214.
31 Chen, C.-C., Chiu, K.-T., Sun, Y.-T & Chen, W.-C (1999) Role
of the cyclic AMP-protein kinase a pathway in lipopolysaccharide-induced nitric oxide synthase expression in RAW 264.7 macro-phages J Biol Chem 274, 31559–31564.
32 Kogure, K., Morita, M., Nakashima, S., Hama, S., Tokumura, A.
& Fukuzawa, K (2001) Superoxide is responsible for apoptosis in rat vascular smooth muscle cells induced by a-tocopheryl hemi-succinate Biochim Biophys Acta 1528, 25–30.
33 Kramer, I.M., Verhoeven, A.J., van der Bend, R.L., Weening, R.S & Roos, D (1988) Purified protein kinase C phosphorylates
a 47-kDa protein in control neutrophil cytoplasts but not
in neutrophil cytoplasts from patients with the autosomal form
of chronic granulomatous disease J Biol Chem 263, 2352– 2357.
34 Wang, J.-P., Tsao, L.-T., Raung, S.-L., Lin, P.-L & Lin, C.-N (1999) Stimulation of respiratory burst by cyclocommunin in rat neutrophils is associated with the increase in cellular
Ca 2+ and protein kinase C activity Free Rad Biol Med 26, 580–588.