(Received for publication, October 18, 1996, and in revised form, January 6, 1997)
From the Department of Pediatrics, Medical University of South Carolina, Charleston, South Carolina 29425
Nitric oxide produced by inducible nitric-oxide
synthase (iNOS) in different brain cells in response to various
cytokines plays an important role in the pathophysiology of stroke and
other neurodegenerative diseases. This study underlines the importance of cAMP in inhibiting the induction of NO production by
lipopolysaccharide (LPS) and cytokines in rat primary astrocytes.
Compounds (forskolin, 8-bromo-cAMP, and
(Sp)-cAMP) that increase cAMP and activate
protein kinase A (PKA) were found to inhibit LPS- and cytokine-mediated production of NO as well as the expression of iNOS, whereas compounds (H-89 and (Rp)-cAMP) that decrease cAMP and PKA
activity stimulated the production of NO and the expression of iNOS in
rat primary astrocytes. Forskolin, but not the inactive analogue
1,9-dideoxyforskolin, inhibited NO production and iNOS expression in a
dose-dependent manner in astrocytes. The inhibition of LPS-
and/or cytokine-induced NO production in rat C6 glial cells
by forskolin suggest that similar to astrocytes, iNOS expression in
C6 cells is also regulated by similar mechanisms. In
contrast, in rat peritoneal macrophages the cAMP analogues stimulated
the LPS- and cytokine-induced production of NO. In vitro,
the PKA had no effect on iNOS activity in LPS-treated astrocytes or
macrophages, suggesting that PKA modulates the intracellular signaling
events associated with the induction of iNOS biogenesis rather than the
post-translational modification of iNOS. The compounds which activate
PKA activity, blocked the activation of NF- in astrocytes but
stimulated the activation of NF-
in macrophages. This
differential regulation of NF-
activation in two different cell
types (astrocytes and macrophages) by the same second messenger (cAMP)
indicates that intracellular events or pathways in the activation of
NF-
may be different. Moreover, this inhibition of iNOS
expression in LPS- and cytokine-treated astrocytes by cAMP may be of
therapeutic potential in NO-mediated cytotoxicity in neurodegenerative
diseases.
Nitric oxide (NO),1 a bioactive free
radical, is involved in various physiological and pathological
processes in many organ systems (1). At low concentration NO has been
shown to play a role in neurotransmission and vasodilation and NO
secreted at higher concentrations is implicated in having a role in the
pathogenesis of stroke and other neurodegenerative diseases (2). This
is of particular importance in conditions associated with infiltrating macrophages and production of proinflammatory cytokines such as demyelinating conditions (e.g. multiple sclerosis,
experimental allergic encephalopathy, and X-adrenoleukodystrophy) and
in ischemic and traumatic injuries (3-6). NO is enzymatically formed
from L-arginine by nitric-oxide synthase (NOS). Basically,
the NOS are classified into two groups. One type, constitutively
expressed (cNOS) in several cell types (e.g. neurons,
endothelial cells) is regulated predominantly at the
post-transcriptional level by calmodulin in a
calcium-dependent manner (2). In contrast, the inducible
form (iNOS), expressed in various cell types including smooth muscle
cells, macrophages, keratinocytes, hepatocytes, and brain cells is
induced in response to a series of proinflammatory cytokines including
interleukin-1 (IL-1
), tumor necrosis factor-
(TNF-
),
interferon-
(IFN-
), and bacterial lipopolysaccharide (LPS)
(7-10). The cytokine induced production of iNOS and NO in astrocytes
and microglia has been implicated in oligodendrocyte degeneration in
demyelinating diseases (4, 6) and neuronal death during trauma
(11).
Efforts at understanding the mechanism of signal transduction cascade
for induction of iNOS in response to LPS and cytokines are an active
area of investigation. Despite a large number of observations
describing the induction of NO by cytokines, the molecular events
leading to the induction of iNOS in cytokines induced astrocytes are
not understood. The differential effect of Rolipram, an inhibitor of
type IV phosphodiesterase on the production of TNF- and NO in a
murine macrophage cell line suggests a role for cAMP in LPS-induced
immune response (12). An increase in the intracellular cAMP
concentration by Rolipram increased the production of NO and decreased
the production of TNF-
in a LPS-stimulated murine macrophage cell
line (12). The cAMP-dependent protein kinase (protein
kinase A, PKA) is an integral constituent of the protein kinase cascade
that links a number of extracellular signals to a variety of cellular
functions (13). Some of the effects of PKA are known to antagonize the
effects stimulated by growth factors whose receptors are protein
tyrosine kinases (e.g. the effect of cAMP on
insulin-stimulated glycogen and triacylglyceride formation, and the
antagonism of mitogen-activated protein kinase pathway by PKA)
(14-16). Inhibition of LPS- and cytokine-mediated NO production by
tyrosine kinase inhibitors suggests the involvement of putative protein
tyrosine kinase(s) in the signaling events transducing iNOS expression
(17, 18). The inhibition of iNOS induction by an inhibitor of NF-
(pyrrolidine dithiocarbamate) but not the expression stimulated by
8-bromo-cAMP suggested the participation of more than one pathway in
the induction of iNOS (19).
To understand the mechanism of LPS- and cytokine-mediated induction of
iNOS and its possible role in neurodegenerative diseases, we examined
the intracellular events for the induction of iNOS in astrocytes. We
report here that cAMP-dependent protein kinase (PKA)
significantly inhibits LPS- and cytokine-mediated activation of
NF- and the expression of iNOS in rat primary astrocytes. However, cAMP analogues stimulate the expression of iNOS and activation of NF-
in rat peritoneal macrophages. To our knowledge, this is
the first example where cAMP regulates the activation of NF-
and
induction of iNOS differentially in two cell types (astrocytes and
macrophages) of the same animal species (rat).
Recombinant rat IFN-, DMEM/F-12 medium, fetal
bovine serum, and NF-
DNA-binding protein detection kit were from
Life Technologies, Inc. Human IL-1
was from Genzyme. Mouse
recombinant TNF-
was obtained from Boehringer Mannheim, Germany. LPS
(Escherichia coli), NADPH, FAD, tetrahydrobiopterin,
Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide), and Dowex 50W were from Sigma.
NG-Methyl-L-arginine,
forskolin, 1,9-dideoxyforskolin, 8-Br-cAMP, (Sp)-cAMP, isoproterenol,
3-isobutyl-1-methylxanthine, PKItide, and antibodies against mouse
macrophage iNOS were obtained from Calbiochem.
L-[2,3,4,5,-H3]Arginine was purchased from
Amersham. [
-32P]ATP (3000 Ci/mmol) was from DuPont
NEN.
Astrocytes were prepared from rat cerebral tissue as described by McCarthy and DeVellis (20). Cells were maintained in DMEM/F-12 medium containing 10% fetal bovine serum. After 10 days of culture astrocytes were separated from microglia and oligodendrocytes by shaking for 24 h in an orbital shaker at 240 rpm. The shaking was repeated two more times after a gap of 1 or 2 weeks time before subculturing to ensure the complete removal of all the oligodendrocytes and microglia. Cells were trypsinized, subcultured, and used in various experiments as described. Cells were stimulated with LPS or different cytokines in serum-free DMEM/F-12 medium. C6 glial cells obtained from ATCC was also maintained and induced with different stimuli as described.
Assay for NO SynthesisSynthesis of NO was determined by assay of culture supernatants for nitrite, a stable reaction product of NO and molecular oxygen. Briefly, 400 µl of culture supernatant was allowed to react with 200 µl of Griess reagent (9, 10) and incubated at room temperature for 15 min. The optical density of the assay samples was measured spectrophotometrically at 570 nm. Fresh culture media served as the blank in all experiments. Nitrite concentrations were calculated from a standard curve derived from the reaction of NaNO2 in the assay.
Assay of NOS ActivityNOS activity was measured directly by production of L-[2,3,4,5-3H]citrulline from L-[2,3,4,5-3H]arginine (10). In these experiments, 50 µl of astrocyte homogenate was incubated at 37 °C in the presence of 50 mM Tris-HCl (pH 7.8), 0.5 mM NADPH, 5 µM FAD, 5 µM tetrahydrobiopterin, and 12 µM L-[2,3,4,5-3H]arginine (118 mCi/mmol) in a total volume of 200 µl. The reactions were stopped by the addition of 800 µl of ice-cold 20 mM HEPES (pH 5.5) followed by the addition of 2 ml of Dowex 50W equilibrated in the same buffer. The samples were then centrifuged and the concentration of L-[3H]citrulline was determined in the supernatant by liquid scintillation counting. Protein was measured by the procedure of Bradford (21).
Protein Kinase A AssayCell extracts were assayed for PKA activity as described (22) by measuring the phosphorylation of Kemptide (0.17 mM) in the presence or absence of PKI peptide (15 µM). PKA activity was calculated as the amount of Kemptide phosphorylated in the absence of PKI peptide minus that phosphorylated in the presence of PKI peptide.
Immunoblot Analysis for iNOSFollowing 24 h incubation in the presence or absence of different stimuli, astrocytes were scraped off, washed with Hank's buffer, and homogenized in 50 mM Tris-HCl (pH 7.4) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 5 µg/ml pepstatin A, and 5 µg/ml leupeptin). After electrophoresis the proteins were transferred onto a nitrocellulose membrane, and the iNOS band was visualized by immunoblotting with antibodies against mouse macrophage iNOS and 125I-labeled protein A.
RNA Isolation and Northern Blot AnalysisStimulated primary
astrocytes were taken out from culture dishes directly by adding
Ultraspec-II RNA reagent (Biotecx Laboratories Inc.) and total RNA was
isolated according to the manufacturer's protocol. For Northern blot
analyses, 20 µg of total RNA was electrophoresed on 1.2% denaturing
formaldehyde-agarose gels, electrotransferred to Hybond-Nylon Membrane
(Amersham), and hybridized at 68 °C with 32P-labeled
cDNA probe using Express Hyb hybridization solution (Clontech) as
described by the manufacturer. The cDNA probe was made by
polymerase chain reaction amplification using two primers (forward
primer: 5-CTC CTT CAA AGA GGC AAA AAT A-3
; reverse primer: 5
-CAC TTC
CTC CAG GAT GTT GT-3
) (23).2 After
hybridization filters were washed two or three times in solution I
(2 × SSC, 0.05% SDS) for 1 h at room temperature followed by solution II (0.1 × SSC, 0.1% SDS) at 50 °C for another
hour. The membranes were then dried and exposed with x-ray films
(Kodak). The same filters were stripped and rehybridized with probes
for glyceraldehyde-3-phosphate dehydrogenase. The relative mRNA
content for iNOS was measured after scanning the bands with a Bio-Rad (Model GS-670) imaging densitometer.
Nuclear extracts from stimulated or
unstimulated astrocytes (1 × 107 cells) were prepared
using the method of Dignam et al. (25) with slight
modification. Cells were harvested, washed twice with ice-cold
phosphate-buffered saline, and lysed in 400 µl of buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 2 mM
MgCl2, 0.5 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 5 µg/ml pepstatin
A, and 5 µg/ml leupeptin) containing 0.1% Nonidet P-40 for 15 min on
ice, vortexed vigorously for 15 s, and centrifuged at 14,000 rpm
for 30 s. The pelleted nuclei were resuspended in buffer B (20 mM HEPES, pH 7.9, 25% (v/v) glycerol, 0.42 M
NaCl, 1.5 mM MgCl2, 0.2 mM EDTA,
0.5 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 5 µg/ml pepstatin
A, and 5 µg/ml leupeptin). After 30 min on ice, lysates were
centrifuged at 14,000 rpm for 10 min. Supernatants containing the
nuclear proteins were diluted with 20 µl of modified buffer C (20 mM HEPES, pH 7.9, 20% (v/v) glycerol, 0.05 M
KCl, 0.2 mM EDTA, 0.5 mM dithiothreitol, and
0.5 mM phenylmethylsulfonyl fluoride) and stored at
70 °C until use. Nuclear extracts were used for the
electrophoretic mobility shift assay using the NF-
DNA-binding
protein detection system kit (Life Technologies, Inc.) according to the
manufacturer's protocol.
Primary astrocytes in serum-free DMEM/F-12 were treated
with different activators and inhibitors of PKA 15 min before the addition of 1 µg/ml LPS. Fig. 1 shows that the
compounds (forskolin, 8-bromo-cAMP, and
(Sp)-cAMP) known to increase intracellular cAMP inhibited the LPS-stimulated NO production as nitrite (Fig.
1A), iNOS activity as conversion of arginine to citrulline
(Fig. 1B), expression of iNOS protein (Fig. 1C)
and mRNA (Fig. 1D), and activated the PKA activity (Fig.
1E). The inactive forskolin analogue, 1,9-dideoxyforskolin (10 µM), neither inhibited the LPS-induced iNOS activity
nor stimulated the PKA activity (Table I). Other PKA
activators like -adrenergic receptor agonist, isoproterenol (10 µM), and cAMP phosphodiesterase inhibitor,
3-isobutyl-1-methylxanthine (1 mM), also inhibited LPS-stimulated NO production and iNOS activity (Table I). On the other
hand, LPS-stimulated NO production, iNOS activity, and expression of
iNOS protein and mRNA were increased by PKA inhibitors (H-89 and
(Rp)-cAMP) (Fig. 1). However, in the absence of
LPS neither PKA activators nor PKA inhibitors had any effect on the production of NO (data not shown). This inhibition of NO production by
cAMP was not only confined to astrocytes, but forskolin was also found
to inhibit LPS- and cytokine-induced NO production in rat
C6 glial cells (Fig. 2). The decrease in
LPS-induced iNOS expression with the increase in cAMP level and the
increase in LPS-induced iNOS expression with the decrease in cAMP level
clearly delineate cAMP and cAMP-dependent protein kinase as
important regulators of iNOS biosynthesis in glial cells.
|
Dose Dependence of Forskolin Inhibition of the LPS Stimulation of iNOS
Astrocytes were incubated with different
concentrations of forskolin 15 min before the addition of 1 µg/ml
LPS, and after 24 h the iNOS activity was measured as nitrite
concentrations in the supernatant and conversion of arginine to
citrulline in the cellular homogenates (Fig. 3). The
level of nitrite and iNOS activity were inhibited to a similar degree
at all the concentrations of forskolin tested. The lowest dose of
forskolin found to inhibit iNOS activity and NO production
significantly (by 30%) was 0.1 µM. At 10 µM forskolin, NO production and iNOS activity were
inhibited by about 90%. Higher doses of forskolin (50-100
µM) did not result in further significant inhibition of
iNOS (data not shown). This may be due to the fact that PKA was already
completely activated in extracts of cells incubated with 10 µM forskolin. The PKA activity increased with the
increase in forskolin concentration. The reciprocal relationship of
production of NO and iNOS activity with PKA activity supports the
conclusion that PKA may play a pivotal role in the regulation of iNOS
expression in astrocytes.
Modulation of LPS- and/or Cytokine-mediated iNOS Expression by Compounds Modulating Intracellular Levels of cAMP in Rat Primary Astrocytes
Primary astrocytes were stimulated with TNF-,
IL-1
, and IFN-
alone or in different combinations for 24 h
and iNOS was measured. TNF-
, IL-1
, and IFN-
individually were
able to induce iNOS activity, protein, and mRNA, however, when
tested in combinations between them or with LPS, the magnitude of
induction was significantly higher (Figs. 4 and
5). Forskolin, the activator of PKA, completely inhibited the cytokine-induced expression of iNOS, whereas H-89, a
specific inhibitor of PKA, stimulated the cytokine-induced expression of iNOS (Fig. 4). Similarly, the induction of iNOS by several combinations of cytokines and LPS were also inhibited by forskolin (Fig. 5) suggesting that augmentation of the cellular levels of cAMP
and the activation of cAMP-dependent protein kinase may
represent a general counter-regulatory mechanism for down-regulation of iNOS expression in astrocytes.
Augmentation of LPS-induced NO Production by cAMP Derivatives in Rat Peritoneal Macrophages
Since cAMP derivatives inhibited the LPS- and cytokine-induced NO production in rat primary astrocytes, we examined the effect of these derivatives on NO production in rat resident macrophages. In contrast to the inhibition of NO production observed in astrocytes, both forskolin and 8-bromo-cAMP stimulated the LPS-induced NO production in macrophages (data not shown). Previously other investigators have also shown the stimulation of LPS-induced production of NO by forskolin or 8-Br-cAMP in rat macrophages (12, 26), mesangial cells (27), and murine 3T3 fibroblasts (28). On the other hand, H-89, the specific inhibitor of PKA, inhibited LPS-induced NO production in macrophages, suggesting also the involvement of the PKA pathway in LPS-induced NO production in macrophages. These results suggest that cAMP may regulate the LPS-induced expression of iNOS in astrocytes and macrophages by different mechanisms.
Effect of Phosphorylation by a Catalytic Subunit of PKA on iNOS Activity in Rat Primary Astrocytes and MacrophagesTo investigate the mechanism of activation of iNOS by PKA possibly by post-translational phosphorylation, the homogenates of astrocytes or macrophages stimulated with 1.0 µg/ml LPS for 24 h in serum-free DMEM/F-12 were used as substrates for phosphorylation by a catalytic subunit of PKA. Phosphorylation of homogenates of either astrocytes or macrophages by different amounts of a catalytic subunit of PKA for different time intervals did not result in any change in iNOS activity as measured by the formation of L-[3H]citrulline from L-[3H]arginine (data not shown). These observations suggest that PKA does not directly activate or inhibit iNOS from either astrocytes or macrophages by post-translational phosphorylation.
Differential Regulation of LPS-induced NF-Increase in the levels
of intracellular cAMP inhibits the induction of NO production in
astrocytes but stimulates the production of NO in macrophages. Since
the activation of NF- is necessary for the induction of iNOS (29,
30), we examined the effect of cAMP on LPS-induced activation of
NF-
in astrocytes and macrophages to understand the basis of this
differential regulation of NO production by cAMP. Both astrocytes and
macrophages were treated with forskolin or H-89, alone or with LPS, and
the nuclear proteins were extracted. NF-
activation was evaluated
by the formation of a distinct and specific complex in a gel-shift
DNA-binding assay. Treatment of astrocytes or macrophages with 1.0 µg/ml LPS resulted in the activation of NF-
(Fig.
6, A and B). This gel shift assay
detected a specific band in response to LPS that was competed off by an
unlabeled probe. Forskolin or H-89 alone at different concentrations
failed to induce NF-
in astrocytes. However, in astrocytes,
forskolin markedly inhibited the LPS-induced activation of NF-
,
whereas H-89 stimulated this activation (Fig. 6A). On the
other hand, in macrophages, consistent with previous observations (31,
32), forskolin alone induced the DNA binding activity of NF-
and
stimulated LPS-induced activation of NF-
, and H-89, the
specific inhibitor of PKA, inhibited LPS-induced activation of
NF-
. These results demonstrate differential regulation of
NF-
activation by cAMP in astrocytes and macrophages.
Recent studies have provided evidence that cAMP induces the expression of iNOS in LPS- and cytokine-stimulated glomerular mesangial cells (33), smooth muscle cells (34), cardiac myocytes (35), murine 3T3 fibroblasts (28), and peritoneal macrophages (26). The increase in cAMP as a result of inhibition of phosphodiesterase (an enzyme that degrades cAMP), or an exogenous supply of cAMP derivatives or compounds that enhance intracellular levels of cAMP cause induction of iNOS. Contrary to these results we have observed that intracellular levels of cAMP negatively regulate the expression of iNOS in LPS or cytokine-stimulated primary astrocytes from rat brain. This conclusion is based on the following observations. 1) LPS induced the expression of iNOS mRNA, protein, and activity as compared with no effect on the PKA activity in astrocytes. Treatment of LPS-stimulated astrocyte cultures with forskolin or 8-Br-cAMP or (Sp)-cAMP results in an increase in PKA activity and the down-regulation of the expression of iNOS mRNA, protein, and activity. 2) Inhibition of the PKA activity with H-89 or with (Rp)-cAMP results in an increase in the expression of iNOS mRNA, protein, and activity in LPS-stimulated astrocytes. The reciprocal relationship between activation of PKA by forskolin and inhibition of NO production and iNOS activity also supports the conclusion that cAMP levels negatively regulate the expression of iNOS in astrocytes. Similar results were observed with rat C6-glial cells. In contrast, in agreement with previous observations (12, 26) the treatment of LPS-stimulated macrophages with forskolin, 8-Br-cAMP, and (Sp)-cAMP augmented the NO production. Moreover, addition of PKA inhibitors (H-89 or (Rp)-cAMP) to LPS-stimulated macrophages resulted in inhibition of NO production.
A comparative study of induction of iNOS by different pathways in 3T3
fibroblast cells reported that stimulation of PKC by tetradecanoylphorbol-13-acetate or activation of PKA by cAMP elevating agents or cytokine/receptor tyrosine kinase (e.g.
transforming growth factor-1) converge in the activation of
NF-
(28). The presence of the consensus sequence for the binding
of NF-
(36, 37) and interferon regulatory factor (29, 38) in the
iNOS gene have been shown to be functionally important for the
induction of iNOS. Although, the signal transduction pathways effective
in iNOS induction converge at NF-
activation, they differ
significantly in different cell types. For example, in murine RAW 264.7 cells, neither the PKA nor the PKC pathways are able to activate
NF-
(39). In cardiac myocytes, iNOS is activated by the PKA
pathway (35), whereas, in human Jurkat T-cells the PKC pathway but not
the PKA pathway activates NF-
(40). Studies reported in this
article clearly establish that the induction of iNOS in both
macrophages and astrocytes converge at the activation of NF-
.
However, interestingly, the intracellular events for the activation of
NF-
in astrocytes and macrophages are quite different. In
astrocytes, activators of PKA inhibit LPS-induced NF-
activation
as well as induction of iNOS and inhibitors of PKA enhance LPS-induced
activation of NF-
and induction of iNOS. This is contrary to the
observations with macrophages (Fig. 6) (31, 32). In macrophages, cAMP
enhances the activation of NF-
and the induction of iNOS.
The signaling events transduced by LPS and cytokines for the induction
of iNOS are not completely established so far. LPS is shown to bind
cell-surface receptor CD14 (41) and induce iNOS probably via activation
of NF- (29, 30). The inhibition of LPS-mediated NF-
activation by cAMP in astrocytes and stimulation of NF-
activation by cAMP in macrophages clearly suggest that the differential
effect of cAMP on NO production in astrocytes and macrophages is due to
the differential effect on NF-
activation. The basis for this
differential regulation of NF-
by cAMP in astrocytes and
macrophages is not understood at the present time. One potential
intracellular target of LPS signaling in cells is the activation of the
MAP kinase pathway. It has been shown by several groups that
augmentation of intracellular cAMP blocks the signaling pathway from
Ras to MAP kinase in cells such as fibroblasts and fat cells by
phosphorylation of Raf (an upstream member of MAP kinase pathway) (15,
16, 22). Phosphorylation of Raf decreases its affinity for and prevents
its interaction with Ras, another upstream proximal member of the MAP
kinase pathway (15, 16). This lack of interaction effectively prevents
translocation of Raf-1 to the membrane by Ras and subsequent activation
of Raf by other as yet unidentified molecules (15, 16). If a similar mechanism also exists in astrocytes, the inhibition of LPS-induced activation of NF-
and expression of iNOS by augmentation of intracellular cAMP in rat primary astrocytes may involve the inhibition of a LPS-mediated signal transduction event, i.e. activation
of Raf. In addition, it has been reported that Ik
can be
phosphorylated by Raf, resulting in release of a p50/p65 heterodimer
(42). Thus inhibition of Raf may lead to a decrease in the release of p50/p65 heterodimer and its translocation into the nucleus. However, in
macrophages, cAMP alone activates NF-
and also stimulates LPS-mediated activation of NF-
suggesting that the regulation of
NF-
by cAMP in macrophages is entirely different from that in
astrocytes. Increase in intracellular cAMP may inhibit the activation
of Raf in macrophages, however, the activation of NF-
may not
involve Raf signaling. In addition to inhibiting the MAP kinase
signaling pathway, cAMP may also modulate the activation of NF-
as well as the transcription of iNOS via a cAMP-dependent protein kinase-mediated phosphorylation of the cAMP-response
element-binding protein (13). Therefore, it will be interesting to
study the role of cAMP in the signaling pathways from Ras to MAP kinase in astrocytes or macrophages to explain the observed differential regulation of NF-
and iNOS and to explore the role of
cAMP-response element-binding protein, if any, in LPS- and
cytokine-mediated activation of NF-
and expression of iNOS.
NO is a diffusible free radical that plays many roles in diverse
pathological conditions. Once iNOS is induced in vitro, NO release can continue for hours or days (43-45) and its presence can be
detrimental to neurons and oligodendrocytes (4, 46). NO and
peroxynitrite (reaction product of NO and
O2) are potentially toxic molecules
that may mediate toxicity through the formation of iron-NO complexes of
iron containing enzyme systems (47), oxidation of protein sulfhydryl
groups (48), nitration of proteins and nitrosylation of nucleic acid,
and DNA strand break (49). Although monocytes/macrophages are the
primary source of iNOS in inflammation, LPS and other cytokines also
induce a similar response in astrocytes (50, 51). 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 oligodendrocyte death (4, 24). The studies reported in this article
indicate that use of cAMP analogues or cAMP phosphodiesterase
inhibitors may represent a possible avenue of research for therapeutics
directed against the nitric oxide-mediated brain disorders,
particularly in demyelinating conditions.
We thank Jan Ashcraft for technical help and Dr. Avtar K. Singh for helpful suggestions and reviewing the manuscript.