From the Institutes of Pharmacology and
¶ Medical Technology, College of Medicine, National Taiwan
University, No.1, Jen-Ai Road, 1st Section, Taipei 10018, Taiwan
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ABSTRACT |
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The signaling pathway involved in protein kinase
C (PKC) activation and role of PKC isoforms in lipopolysaccharide
(LPS)-induced nitric oxide (NO) release were studied in primary
cerebellar astrocytes. LPS caused a dose- and
time-dependent increase in NO release and inducible NO
synthase (iNOS) expression. The tyrosine kinase inhibitor, genestein,
the phosphatidylcholine-phospholipase C inhibitor, D609, and the
phosphatidate phosphodrolase inhibitor, propranolol, attenuated the LPS
effects, whereas the PI-PLC inhibitor, U73122, had no effect. The PKC
inhibitors (staurosporine, Ro 31-8220, Go 6976, and calphostin C) also
inhibited LPS-induced NO release and iNOS expression. However, long
term (24 h) pretreatment of cells with 12-O-tetradecanoyl
phorbol-13-acetate (TPA) did not affect the LPS response. Previous
results have shown that TPA-induced translocation, but not
down-regulation, of PKC occurs in astrocytes (Chen, C. C., and
Chen, W. C. (1996) Glia 17, 63-71), suggesting possible involvement of PKC
in LPS-mediated effects. Treatment with
antisense oligonucleotides for PKC
or
, another isoform abundantly expressed in astrocytes, demonstrated the involvement of
PKC
, but not
, in LPS-mediated effects. Stimulation of cells for
1 h with LPS caused activation of nuclear factor (NF)-kB in the
nuclei as detected by the formation of a NF-kB-specific DNA-protein complex; this effect was inhibited by genestein, D609, propranolol, or
Ro 31-8220 or by PKC
antisense oligonucleotides, but not by long
term TPA treatment. These data suggest that in astrocytes, LPS might
activate phosphatidylcholine-phospholipase C and
phosphatidylcholine-phospholipase D through an upstream protein
tyrosine kinase to induce PKC activation. Of the PKC isoforms present
in these cells, only activation of PKC
by LPS resulted in the
stimulation of NF-kB-specific DNA-protein binding and then initiated
the iNOS expression and NO release. This is further evidence
demonstrating that different members of the PKC family within a single
cell are involved in specific physiological responses.
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INTRODUCTION |
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Nitric oxide (NO),1 a bioactive free radical, is involved in various physiological and pathological processes in many systems (1). Low concentrations of NO play a role in neurotransmission and vasodilation. However, when secreted at higher concentrations, NO is implicated in the pathogenesis of stroke and other degenerative diseases, such as demyelinating conditions and ischemic and traumatic injury (2). NO is formed enzymatically from L-arginine by nitric-oxide synthase (NOS). NOS enzymes are classified into two groups. One type (cNOS) is constitutively present in several cell types (e.g. neurons and endothelial cells) and is regulated predominantly at the post-transcriptional level by calmodulin in a Ca2+-dependent manner (2). In contrast, the inducible form (iNOS), expressed in various cell types, including vascular smooth muscle cells, macrophages, hepatocytes, and astrocytes, is induced in response to proinflammatory cytokines and bacterial lipopolysaccharide (LPS) (3-6). Cellular NO release following iNOS induction in astrocytes and microglia has been implicated in oligodendrocyte degeneration in demyelinating diseases and in neuronal death during trauma (7-9).
The mechanism of the signal transduction cascade involved in the
induction of iNOS in response to LPS and cytokines is an active area of
investigation. Although LPS-produced iNOS induction in primary
astrocytes has been reported (6, 10), the molecular events involved are
not understood. Previous reports have shown a potential role for
tyrosine kinase in LPS-produced iNOS induction (11, 12). The murine
iNOS promotor contains 24 transcriptional factor binding sites,
including those for NF-kB and activator protein-1 (13, 14). Proteins of
the NF-kB family appear to be essential for the enhanced iNOS gene
expression seen in macrophages exposed to LPS (15), and the p65 NF-kB
also seems to be responsible for iNOS induction in astrocytes (16). In
the present study, the intracellular signaling pathway by which LPS
induces iNOS expression in primary astrocytes was studied. The results
show that LPS might activate phosphatidylcholine-phospholipases C and D
(PC-PLC and PC-PLD) via tyrosine phosphorylation to produce PKC and
NF-kB activation, iNOS expression, and, finally, NO release. Of the PKC
isoforms ,
,
,
, and
expressed in astrocytes (17, 18),
only PKC
is involved in the regulation of LPS-induced NF-kB
activation, iNOS expression, and NO release.
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EXPERIMENTAL PROCEDURES |
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Materials--
Affinity-purified rabbit polyclonal antibody to
iNOS was obtained from Transduction Laboratories (Lexington, KY). Basal
modified Eagle's medium, fetal calf serum, glutamine, gentamycin,
penicillin, and streptomycin were purchased from Life Technologies,
Inc. Rabbit polyclonal antibodies to PKC and
and the NF-kB probe
were purchased from Santa Cruz Biotechnology. TPA was from L. C.
Services Corp. (Woburn, MA). LPS (from Escherichia coli
serotype 0127:B8), staurosporine, pyrolidine dithiocarbamate,
sulfanilamide, and N-(1-naphthyl)-ethylenediamine were from
Sigma. Genestein, calphostin C, Go 6976, and Ro 31-8220 were from
Calbiochem (San Diego, CA). D609, U73122, and U73343 were from RBI
(Natick, MA). T4 polynucleotide kinase was from New England Biolabs
(Beverly, MA). Poly(dI·dC) was from Amersham Pharmacia Biotech.
Reagents for SDS-polyacrylamide gel electrophoresis were from Bio-Rad.
[
-32P]ATP (3000 Ci/mmol) was from NEN Life Science
Products. The horseradish peroxidase-labeled donkey anti-rabbit second
antibody and ECL detecting reagent were purchased from Amersham
Pharmacia Biotech.
Primary Cultures of Astrocytes--
Glial cell cultures were
prepared from the cerebellum of 8-day Wistar rats as described
previously (17). Briefly, the cerebella were dissected and dissociated
by mechanical chopping and trypsinization to obtain a cell suspension.
Cells were plated at a density of 105 cells/well in
poly-L-lysine-precoated 12-well plates for the nitrite
assay and at a density of 107 cells/10-cm dish for iNOS and
PKC and
isoform expression tests and the NF-kB gel shift assay.
Cultures were maintained in basal modified Eagle's medium supplemented
with 10% fetal calf serum, 2 mM glutamine, and 50 µg/ml
gentamycin, which was changed twice each week. Cells grown in an
atmosphere of 5% CO2/95% humidified air at 37 °C were
used after 10-12 days in culture, at which time they consisted of
confluent glial cells, which stained positively for glial fibrillary
acidic protein (17).
Determination of NO Concentration-- NO production in culture supernatant was evaluated by measuring nitrite, its stable degradation product, using the Griess reagent. The basal modified Eagle's medium was changed to phenol red-free medium before the cells were stimulated with LPS (1 µg/ml) for 24 h, and then the isolated supernatant was centrifuged and mixed with an equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthylethylene diamine dihydrochloride, 2% phosphoric acid) and incubated at room temperature for 10 min before the absorbance was measured at 550 nm in a microplate reader. Sodium nitrite (NaNO2) was used as a standard. In pretreatment experiments, cells were incubated with genestein (a tyrosine kinase inhibitor), U73122 (a PI-PLC inhibitor), D609 (a PC-PLC inhibitor), propranolol (a phosphatidate phosphohyrolase inhibitor), staurosporine, calphostin C, Go 6976, or Ro 31-8220 (PKC inhibitors) for 30 min or with TPA for 24 h before the addition of LPS.
Preparation of Cell Extracts and Western Blot Analysis of iNOS
and PKC and
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Following treatment with LPS, or pretreatment
with inhibitors, TPA or antisense oligonucleotides (see below) followed
by LPS, the cells were harvested and collected. For studies of iNOS
expression or PKC
and
expression (antisense oligonucleotides
treatment), cell homogenates were prepared and subjected to
SDS-polyacrylamide gel electrophoresis using 7.5% (iNOS) or 10% (PKC
isoform) running gels, and then the proteins were transferred to
nitrocellulose paper, and immunoblot analyses were performed as
described previously (17). Briefly, the membrane was incubated
successively at room temperature with 0.1% milk in Tris-buffered
saline/Tween 20 (TTBS) for 1 h, with rabbit antibodies specific
for iNOS or PKC
or
for 1 h and with horseradish
peroxidase-labeled anti-rabbit antibody for 30 min. After each
incubation, the membrane was washed extensively with TTBS. The
immunoreactive band was detected with ECL detecting reagents and
developed with Hyperfilm-ECL.
Synthesis of PKC and
Antisense Oligonucleotides and
Treatment of Cells with Oligonucleotides--
Phosphorothioate
oligodeoxynucleotides were synthesized in trityl-on mode using an
Applied Biosystems model 391 DNA synthesizer, as described previously
(19), using A, G, C, and T phosphoramidites, controlled pore glass
supports, and sulfuring reagent purchased from Glen Research Corp.
(Sterling, VA). The oligodeoxynucleotides were deblocked and cleaved
from the solid support using concentrated ammonia water by a standard
procedure. After evaporation of the ammonia, the deprotected
oligodeoxynucleotides were purified on Sep-Pak C18 cartridges
(Millipore, Milford, MA), as reported previously (20). Control
sequences (scrambled versions of the antisense oligonucleotides) with
the same base composition as the PKC
antisense oligonucleotides were
also synthesized. The sequences of the PKC
and
antisense
oligonucleotides and scrambled PKC
controls are listed in Table
I.
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Preparation of Nuclear Extracts and the Electrophoretic Mobility
Shift Assay (EMSA)--
Control cells or cells pretreated with
genestein, D609, propranolol or Ro 31-8220 for 30 min, TPA for 24 h, or antisense oligonucleotides for 9 days were treated with 1 µg/ml
LPS for 1 h. Nuclear extracts were then isolated as described
previously (21). Briefly, cells were washed with ice-cold
phosphate-buffered saline (PBS) and pelleted. The cell pellet was
resuspended in hypotonic buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM NaF, and 1 mM NaVO4) and
incubated for 15 min on ice, and then the cells were lysed by the
addition of 0.5% Nonidet P-40, followed by vigorous vortexing for
10 s. The nuclei were pelleted and resuspended in extraction
buffer (20 mM HEPES, pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 1 mM NaF, and
1 mM Na3VO4), and the tube
vigorously shaken at 4 °C for 15 min on a shaking platform. The
nuclear extracts were then centrifuged, and the supernatants were
aliquoted and stored at 80 °C.
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RESULTS |
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Signaling Pathways for LPS-induced NO Production and 130-kDa iNOS Expression-- Exposure of cells to LPS resulted in concentration- and time-dependent nitrite production and expression of the 130-kDa iNOS (Fig. 1). Using a 24-h exposure period, maximum nitrite release (65 ± 9 nmol/105 cells/24 h; n = 3) was obtained at 10 µg/ml LPS, and the basal nitrite release was 2 ± 1 nmol/105 cells/24 h (n = 3) (Fig. 1A). When cells were treated with 1 µg/ml LPS for various times, nitrite release was significant at 12 h (8 ± 5 nmol/105 cells/24 h; n = 3) and maximal at 48 h (Fig. 1B). In the following NO release experiments, the cells were treated with 1 µg/ml LPS for 24 h.
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Inhibitory Effect of PKC Inhibitors and Lack of Effect of Long Term
TPA Treatment on LPS-Induced NO production and iNOS
Expression--
LPS-induced nitrite production and iNOS expression
were both inhibited by D609 and propranolol, indicating the involvement of the PC-PLC and PC-PLD pathways. Both pathways can increase diacylglycerol levels and then activate PKC. To determine whether activation of PKC by LPS was involved in the regulation of LPS-induced NO production, PKC inhibitors were used. Pretreatment of cells for 30 min with 100 nM staurosporine, 0.5 µM Ro
31-8220, 3 µM Go 6976, or 100 nM calphostin
C inhibited LPS-induced nitrite production by 48, 73, 47, or 26%,
respectively; iNOS protein expression was also inhibited by Ro 31-8220
(Fig. 3A). When cells were
treated with 1 µM TPA for 24 h, NO release (10 ± 1 nmol/105 cells/24 h; n = 4) was seen;
under these conditions, the LPS-induced nitrite production was 66 ± 5 nmol/105 cells/24 h (n = 7), which was
greater than with LPS alone (48 ± 5 nmol/105 cells/24
h; n = 3), although not statistically significant by the t test (Fig. 3B). Similar results were
obtained for iNOS expression (Fig. 3B). Previous studies
have shown that in macrophages, MDCK cells and astrocytes, PKC is
translocated but not down-regulated by TPA treatment (21). The
inhibition of the LPS-induced effects by PKC inhibitors, but not by
long term TPA treatment, suggested the possible involvement of PKC
in LPS-induced NO production.
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Inhibitory Effect of PKC Antisense Oligonucleotides but Not
PKC
Antisense Oligonucleotides on LPS-Induced NO Production and iNOS
Expression--
To further study the involvement of PKC
and the
lack of involvement of other isoforms in the LPS-induced NO release and
iNOS expression, PKC
antisense and scrambled control
oligonucleotides and antisense oligonucleotides for PKC
, another
isoform abundantly expressed in astrocytes (17), were used. Following
treatment of primary astrocyte cultures with PKC
or PKC
antisense
oligonucleotides for 9 days, the expression levels of PKC
or
were determined by Western blotting. As shown in Fig.
5A, 10 µM of
PKC
or
antisense oligonucleotides caused a specific reduction in
the level of the corresponding immunoreactive isoform protein,
e.g. PKC
antisense oligonucleotides specifically
inhibited the expression of PKC
protein but had no effect on the
expression of PKC
. Because cerebellar astrocytes grow confluently
after 10-12 days in culture, PKC
or
antisense oligonucleotides
were added for entire culture periods, and the reduction in the level
of PKC
and PKC
was similar to those shown in Fig.
5A.
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Induction of NF-kB in the Nuclei of LPS-stimulated Astrocytes and
the Inhibitory Effect of PKC Antisense Oligonucleotides--
In
resting cells, the NF-kB heterodimer is held in the cytosol by binding
to IkB (22); after stimulation of the cells with various agents, the
cytosolic NF-kB/IkB complex dissociates and free NF-kB translocates to
the nuclei. pyrolidine dithiocarbamate, an antioxidant that acts as a
specific inhibitor of NF-kB activation (23), blocks the ability of
astrocytes to produce nitrite production and the nuclear binding
activity for NF-kB normally seen in response to
LPS.2 Thus, activation of
NF-kB is critical in the induction of iNOS by LPS in astrocytes. We
performed an EMSA using oligonucleotides containing NF-kB recognition
site-like sequences present in the iNOS gene (13) and nuclear extracts
prepared from LPS-stimulated cells. In nuclear extracts of unstimulated
astrocytes, one faint NF-kB-specific DNA-protein complex was
identified, the intensity of which markedly increased following
exposure of the cells to 1 µg/ml LPS for 10 min and was even greater
after 1 h of treatment (Fig.
6A). For the EMSA, cells were
treated with LPS for 1 h.
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DISCUSSION |
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In both cultured astrocytes and C6 glioma cells, LPS induces NO production (6, 24). In macrophages, activation by LPS requires the presence of a 53-kDa glycoprotein, mCD14 (25), which is attached to the cell surface by a glycosylphosphatidylinositol moiety (26). A second form of CD14, soluble CD14, is found at high concentrations in the serum (27), and can confer LPS responsiveness upon cells that lack mCD14, including astrocytoma cells (28). Complexes formed between LPS and either mCD14 or soluble CD14 are thought to lead to transfer of bound LPS to a distinct signaling molecule, which may be either transmembrane or intracellular (29). The formation of LPS/CD14 complexes is accelerated by LPS-binding protein (30), which is also present at high concentrations in normal serum and thus can contribute to the serum effects (31). Recent studies have demonstrated the presence of CD14 mRNA and protein in rat astrocytes and that LPS-produced iNOS induction requires membrane and soluble forms of CD14 (32). Thus, in astrocytes, formation of a LPS/LPS-binding protein complex might allow binding to, and activation of, CD14, and then trigger signal transduction to initiate the expression of iNOS and NO release.
Although PKC has been shown to be involved in LPS-induced iNOS expression and NO production in macrophages (33, 34), it has been reported to be unnecessary for iNOS induction by LPS in astrocytes (11). However, in the present study, four PKC inhibitors, calphostin C, Go 6976, Ro 31-8220, and staurosporine, inhibited LPS-stimulated NO production and iNOS expression, indicating that PKC activation is an obligatory event in the LPS-mediated regulation of NO release and iNOS expression in astrocytes. PKC is activated by the physiological activator diacylglycerol, which can be generated either directly by the action of PLC or indirectly by a pathway involving the production of phosphatidic acid by PLD, followed by a dephosphorylation reaction catalyzed by phosphatidate phosphohydrolase. Normally, the PLC involved in the production of diacylglycerol is PI-PLC, but PC-PLC may also be involved (36, 37). The PC-PLC inhibitor, D609, and the phosphatidate phosphohydrolase inhibitor, propranolol, both inhibited LPS-induced iNOS expression and NO production, whereas the PI-PLC inhibitor, U73122, had no effect. Thus, LPS may act through the PC-PLC and PC-PLD pathways, but not the PI-PLC pathway, to induce PKC activation in astrocytes; this contrasts with the situation in RAW 264.7 macrophages in which the PI-PLC and PC-PLC pathways, but not the PC-PLD pathway, are involved (38). The mechanism involved in the activation of PC-PLC and PC-PLD is still unknown but may involve tyrosine phosphorylation (37, 39). Genestein also inhibited LPS-induced iNOS induction and NO production in astrocytes. The tyrosine kinase involved might be p53/p56lyn, because, in monocytes, LPS activates this kinase, which is associated with CD14 (40, 41). Thus, in astrocytes, the LPS/LPS-binding protein complex binds to CD14 and then might activate PC-PLC and PC-PLD via an upstream protein tyrosine phosphorylation to elicit PKC activation and, finally, iNOS expression and NO production.
Although PKC inhibitors attenuated LPS-induced iNOS expression and NO
production, long term TPA pretreatment, which down-regulates PKC,
, and
, but not PKC
, in astrocytes (17, 18), had no effect,
indicating the possible involvement of PKC
in LPS-mediated effects.
To confirm the involvement of PKC
, PKC
antisense oligonucleotides and the scrambled controls or antisense oligonucleotides for PKC
, which is abundantly expressed in astrocytes and down-regulated by TPA,
were used. The specificity of the PKC
and PKC
antisense oligonucleotides was demonstrated (Fig. 5A), and the results
showed inhibition of LPS-stimulated iNOS expression and NO production by PKC
antisense oligonucleotides but not by PKC
antisense or control oligonucleotides. Thus, a crucial role for PKC
in the LPS-induced stimulation of NO production and iNOS expression has been
demonstrated. The PKC family, which consists of
phospholipid-dependent serine/threonine kinases, is
believed to play a major role in cellular functions. Molecular cloning
has shown that it consists of at least 12 isoforms with different
tissue expressions (36), which have been shown to be related to
specialized cell functions (36). Primary cerebellar astrocytes express
the new types of PKC
,
, and
, which are not expressed in
neuronal granule cells (17,
18).3 PKC
and
, but not
PKC
, have been shown to be involved in the regulation of
receptor-mediated PI hydrolysis (17, 18). However, in this study,
PKC
, but not other isoforms, was shown to be involved in the
LPS-induced iNOS expression and NO production. This is further evidence
that different members of the PKC family within a single cell elicit
specific physiological responses. However, PKC
, which is abundantly
expressed in RAW 264.7 macrophages (21), was not involved in the
LPS-induced iNOS expression and NO production (38).
The transcriptional factor, NF-kB, is critical in the induction of iNOS
by LPS in macrophages (13, 14). In astrocytes, the NF-kB blocker,
pyrolidine dithiocarbamate, inhibits LPS-induced NO production and iNOS
expression,2 indicating that NF-kB is also critical in the
induction of iNOS by LPS in these cells. LPS increased the levels of
the NF-kB-specific DNA-protein complex in nuclear extracts (Fig.
6A); this activation was inhibited by genestein, D609,
propranolol, or Ro 31-8220 but not by long term TPA treatment.
Furthermore, PKC antisense oligonucleotides, but not the scrambled
controls, attenuated this activation, indicating the involvement of
PKC
in the LPS-stimulated up-regulation of iNOS in astrocytes.
Direct activation of PKC by TPA does induce NF-kB activation, NO
production, and iNOS expression in astrocytes (Figs. 4 and
7A). Similar findings have been reported in peritoneal macrophages, hepatocytes, and human umbilical vein endothelial cells
(42-44) but not in RAW 264.7 macrophages, in which TPA alone did not
induce NF-kB activation and NO production (38, 45). Although the role
of PKC
has been clearly demonstrated, other LPS-activated components
are also involved in the co-stimulation of NF-kB and iNOS expression,
because direct activation of PKC by TPA induced less NF-kB activation
and NO production than LPS stimulation (Figs. 1B, 4, and
7A).
Glial cells, including astrocytes, microglia, and oligodendrocytes, are involved in lesion and plaque formation in multiple sclerosis and experimental allergic encephalomyelitis, a model for multiple sclerosis. Multiple sclerosis is a central nervous system disorder with immune-mediated destruction of myelin and the myelin-producing cells, oligodendrocytes. The presence of iNOS in tissues of patients with multiple sclerosis and in animals with experimental allergic encephalomyelitis suggests that NO may play a role in the central nervous system autoimmune diseases (35, 46, 47). Rodent astrocytes and microglia express a high level of iNOS and release significant amounts of NO within hours of LPS stimulation (Refs. 6, 7, and 10 and the present study). Astrocytes are the major cell population in the central nervous system; therefore, induction of iNOS in astrocytes may be an important source of NO in central nervous system inflammatory disorders associated with neuronal and oligodendrocytes death (9).
In summary, the signaling pathway involved in the LPS-induced
activation of PKC in primary astrocytes was explored, and the PKC
isoform , but not other isoforms, was found to be involved in the
regulation of LPS-induced NF-kB activation, iNOS expression, and NO
release. This is the first study showing the involvement of these two
mechanisms in LPS-stimulated NO release in such cells.
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FOOTNOTES |
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* This work was supported by a research grant from the National Science Council of Taiwan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel: 886-2-23970800, ext. 8321; Fax: 886-2-23947833; E-mail: ccchen{at}ha.mc.ntu.edu.tw.
1 The abbreviations used are: NO, nitric oxide, iNOS, inducible nitric- oxide synthase; LPS, lipopolysaccharide; PC-PLC, phosphatidylcholine-phospholipase C; PC-PLD, phosphatidylcholine-phospholipase D; PI-PLC, phosphoinositide-phospholipase C; NF-kB, nuclear factor kB; EMSA, electrophoretic mobility shift assay; PKC, protein kinase C; TPA, 12-O-tetradecanoyl phorbol-13-acetate.
2 C.-C. Chen, J.-K. Wang, D.-Y. Lin, and W.-C. Chen, unpublished data.
3 W.-C. Chen, and C.-C. Chen, unpublished data.
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REFERENCES |
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