cAMP and purinergic P2y
receptors upregulate and enhance inducible NO synthase mRNA and
protein in vivo
Stan S.
Greenberg,
Xinfang
Zhao,
Ji-Fang
Wang,
Li
Hua, and
Jie
Ouyang
Departments of Medicine and Physiology, The Alcohol Research Center,
Louisiana State University Medical Center, New Orleans, Louisiana 70112
 |
ABSTRACT |
Adenosine 3',5'-cyclic monophosphate
(cAMP) and purinergic P2y receptor
agonists upregulate inducible nitric oxide (NO) synthase (iNOS) but
inhibit Escherichia coli endotoxin
lipopolysaccharide (LPS)- and cytokine-mediated upregulation of iNOS in
cultured cells. We examined the effects of cAMP and
P2y receptor agonists on the iNOS
system in vivo. Intratracheal administration of dibutyryl-cAMP (DBcAMP,
0.1 and 1 mg/kg), a P2y receptor
agonist [2-methylthioadenosine 5'-triphosphate (MeS-ATP), 5 mg/kg], or LPS (0.6 mg/kg) to rats 2 h before bronchoalveolar
lavage (BAL) increased iNOS mRNA (competitor-equalized reverse
transcription-polymerase chain reaction) and iNOS protein (Western
blot) in rat alveolar macrophages compared with the effects of sterile
phosphate-buffered saline (0.5 ml it). At equal levels of upregulation
of iNOS mRNA, 1) LPS, but not DBcAMP
or MeS-ATP, upregulated nuclear transcription factor-
B (NF-
B) and
2) iNOS protein and formation of NO
were greater in alveolar macrophages from LPS- and MeS-ATP-treated rats
than from DBcAMP-treated rats. Administration of DBcAMP or MeS-ATP 15 min before LPS did not inhibit LPS-induced alveolar macrophage-derived
iNOS mRNA, iNOS protein, and NO. Diethyldithiocarbamate (DETC, 5 mg/kg
it) inhibited LPS-induced iNOS mRNA but did not affect upregulation of
iNOS mRNA produced by the other agonists. We conclude that an
LPS-dependent and -independent pathway of iNOS mRNA induction exists in
vivo. The former is activated by LPS and most cytokines, is associated with upregulation of NF-
B and inhibited by DETC, and elicits an
inflammatory response. The latter, activated by DBcAMP and MeS-ATP, is
not associated with upregulation of NF-
B, inhibition by DETC, or
activation of inflammation. The two systems are additive in vivo rather
than antagonistic. Speculatively, if the LPS-independent iNOS pathway
exists in humans, the iNOS in tissues from patients taking drugs
affecting cAMP or P2y receptors
may be iatrogenic rather than pathogenetic in origin.
transcription factors; tumor necrosis factor-
; diethyldithiocarbamate; adenosine 3',5'-cyclic
monophosphate; nitric oxide; lipopolysaccharide
 |
INTRODUCTION |
ALVEOLAR MACROPHAGES (AM) are the first line of defense
against airborne-derived pathogens. Exposure of AM to pathogenic
organisms or their lipopolysaccharide (LPS) membrane coat activates
this phagocytic cell for production of cellular mediators that aid in
the killing and/or phagocytosis of the foreign organism. Among these mediators are cytokines, arachidonic acid metabolites, and oxygen- and nitrogen-derived free radicals, including nitric oxide (NO)
and peroxynitrite (20, 28, 31, 32, 44, 45, 48, 49). NO, a free radical,
is the smallest known bioactive molecule produced by almost every cell
in almost all species, including humans (17, 28). High concentrations
of this free radical, such as the concentrations produced in AM by the
inducible isozyme of NO synthase (iNOS), can destroy bacteria,
parasites, and specific types of tumor cells (17, 20, 28, 31, 46). The
binding of NO to iron molecules can inhibit the enzymatic activity of these pathogens as well as their host cells (17, 28). Moreover, by
inhibiting the iron-responsive elements on iNOS mRNA or iNOS protein
and by posttranslational mechanisms involving the ADP ribosylation of
proteins, NO can prevent the formation of new enzymes or modify mRNA
and cellular proteins and, thereby, their functions (7, 10, 17, 28). In
view of the diverse nature and multiple sites of action of NO within
the cell, it is important to define the factors that modulate the
regulation of iNOS in vivo.
iNOS (EC 1.14.13.39) is an isoform of the NOS family of enzymes. It is
usually absent in resting cells and can be induced by cytokines and
bacterial cell wall products such as the interleukins, tumor necrosis
factor-
(TNF-
), and Escherichia
coli endotoxin LPS (4, 17, 28). However, recent studies
suggest that a rapid increase and decrease in iNOS mRNA may occur
through the action of a cycloheximide-inhibitable protein (16), which
acts by prolonging the lifetime of iNOS mRNA (23). These proteins may
be promoters or enhancers of iNOS transcription. Nevertheless, the
question of whether some cells can constitutively express iNOS remains
unanswered. However, the induction of iNOS appears to be primarily
regulated at the level of gene transcription by cytokines and LPS as a
result of their ability to activate protein and tyrosine kinases (7,
13, 16, 17, 19, 24, 25, 28) and promoters and nuclear transcription
factors such as nuclear factor-
B (NF-
B) and inducible regulatory
factor type 1 (IRF-1) (3, 27, 29, 47, 48). Glucocorticoids, which inhibit LPS-induced gene expression for iNOS, appear to inhibit gene
expression by interfering with an adenosine 3',5'-cyclic monophosphate (cAMP)-responsive enhancer (CREB) of transcription (1).
Recent studies, using cultured cells and cell lines, also demonstrated
that many of the cell-signaling pathways involved in the expression of
iNOS appear to be modulated by purinergic P2y receptors and by cAMP (2, 8,
12, 14, 17, 24, 26, 30, 34, 37-39, 44).
Compounds that stimulate P2y
receptors or upregulate the cAMP system act synergistically with LPS to
upregulate iNOS protein in the RAW 264.7 murine macrophage cell line
(43), in cultured rat vascular smooth muscle (14, 24) and mesangial
cells (38), and in cardiac myocytes in cell culture (39) by enhancement of translation or by inhibition of the degradation of iNOS mRNA and/or protein. Moreover, prolonged incubation of murine
fibroblasts with cAMP in cell culture induces iNOS mRNA and iNOS
protein (6) while augmenting cytokine-stimulated NO synthesis in
cultured cardiac myocytes (26) and vascular smooth muscle cells (30). However, cAMP and a P2y receptor
agonist [2-methylthioadenosine 5'-triphosphate
(MeS-ATP)] did not directly affect iNOS but suppressed LPS-mediated upregulation of iNOS mRNA and the production of NO in
isolated astrocytes (37) and in cultured murine macrophages (8, 12).
These data suggest that a cytokine-independent pathway exists for cAMP
and P2y receptor agonists to
upregulate or downregulate the iNOS system by inhibition or
facilitation of the degradation or stability of iNOS mRNA in vitro (12,
29, 39). Nevertheless, the role of these autacoids as modulators of
iNOS in vivo remains undefined. Thus we examined the effects of
upregulation of the cAMP system and purinergic
P2y receptor stimulation on iNOS
mRNA, iNOS protein, and NO production of rat AM in vivo and the
interaction of these agonists with LPS on the NOS system. We also
tested the ability of these agonists to upregulate NF-
B.
 |
MATERIALS AND METHODS |
Direct effects of autacoids.
Conventional male Sprague-Dawley rats (Hilltop Farms, Scottsdale, PA),
weighing 225-250 g, were housed at the vivarium of the Louisiana
State University Medical Center at New Orleans under a 12:12-h
dark-light cycle and were allowed food and water ad libitum. On the
morning of the experiment, the rats were anesthetized with ether, the
trachea was isolated, and the animals were given 0.5 ml of sterile
phosphate-buffered saline (PBS) or test compounds dissolved in 0.5 ml
of PBS by intratracheal (it) gavage. The test compounds used were
E. coli endotoxin (LPS serotype
026:B6, 0.6 mg/kg; Difco, Detroit, MI), dibutyryl-cAMP (DBcAMP, 0.1 or
1 mg/kg; Research Biochemicals, Cleveland, OH), MeS-ATP (5 mg/kg;
Research Biochemicals), albuterol (0.5 mg/kg; University Hospital, New Orleans, LA), or isoproterenol (0.2 µg/kg; Sigma Chemical, St. Louis,
MO). The neck wounds were closed, and the animals were allowed to
recover. Two hours after intratracheal administration of PBS, LPS, or
the autacoids, the rats were anesthetized with ether. A thoracotomy was
performed, and blood was obtained by cardiac puncture for analyses of
TNF-
and
and
[reactive nitrogen
intermediates (RNI)]. The heart and lungs were removed, and the
lung was subjected to bronchoalveolar lavage (BAL) with 30 ml of PBS.
The BAL fluid was analyzed for TNF-
and RNI. The AM and recruited
neutrophils [polymorphonuclear leukocytes (PMN)] were
isolated from the BAL fluid and used for determination of the total
cell count and the differential percentage of AM and PMN iNOS and
TNF-
mRNA and protein and ex vivo RNI production by the freshly
isolated AM (20, 31, 46).
The intratracheal administration of drugs into the lung and evaluation
of the AM at 2 h postinjection were chosen because TNF-
, the initial
cytokine elicited by intratracheal administration of LPS, reaches its
peak concentration and is compartmentalized within the lung, so it does
not activate the systemic cytokine cascade (20, 31, 46). Thus BAL fluid
TNF-
concentrations can be used as an index of the degree of
activation of the local inflammatory response within the lung.
Moreover, intratracheal administration limits the interaction of the
autacoids to the resident cells within the alveolar space, thereby
eliminating the complexity of data interpretation resulting from the
activation of circulating monocytes and macrophages and other
cytokines.
Indirect effect of autacoids on LPS-induced iNOS.
The experiments described above were repeated in rats
(n = 9-12/group) pretreated with
DBcAMP (0.1 or 1 mg/kg it) or MeS-ATP (5 mg/kg it) 15 min before
administration of LPS (0.6 mg/kg it).
Effect of diethyldithiocarbamate on effects of autacoids and LPS on
iNOS.
Rats (n = 5-6/group) were given
diethyldithiocarbamate (DETC, 5 mg/kg it; Alexis Biochemical, San
Diego, CA) to inhibit NF-
B (15, 36) 30 min before intratracheal
administration of the autacoids or LPS (direct effects) or 30 min
before intratracheal administration of LPS and 15 min before
intratracheal administration of the autacoids (indirect effects), and
the experiments described above on the cell counts and the iNOS and
TNF-
systems were repeated 2 h after the LPS injection.
Cell counts and differential.
Cell counts were performed on washed cells with a hemocytometer using a
Motorola video system (Cole-Palmer, Chicago, IL). Differentials were
performed on Cyto-Spin preparations stained with Diff-Quik (Baxter,
McGraw Park, IL). Viability was always >95%, as determined by trypan
blue exclusion (20, 31, 46).
Cell separation.
The AM were isolated from the BAL fluid of individual lavage samples
using Polymorph-Prep (Nycomed, GIBCO, Grand Island, NY) and
Ficoll-Hypaque (specific gravity 1.077; Sigma Chemical). The 2-h
samples were >99% pure AM. Cell viability, as determined by trypan
blue exclusion, was >95%. Isolation and purification of these cells
have been described in detail (20, 31, 46). Freshly isolated AM
(106 cells /0.5 ml) from each
of the experimental groups were immediately frozen in liquid nitrogen
and assayed for mRNA for iNOS, homogenized and assayed for iNOS
protein, or incubated in
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered salt solution (pH 7.4) containing (in mM) 128 NaCl, 4.9 KCl, 1.2 MgCl2, 1.6 CaCl2, 10 dextrose, 18.7 NaHEPES-HEPES buffer, 1.18 NaH2PO4,
and 0.13 L-arginine for 1 h. The cells were immediately removed by centrifugation at 5,000 g for 15 min at 4°C, and the
incubate was assayed for RNI with ozone chemiluminescence or TNF-
with the WEHI assay or with an enzyme-linked immunosorbent assay (31,
46).
Measurement of RNI.
Plasma, BAL fluid, or AM incubates (10-50 µl) were added to 200 ml of a reducing solution (2.3% vanadium chloride in 2 N HCl at
98°C) under a stream of ultrapure nitrogen gas. The nitrate was
converted to nitrite, which was subsequently converted to free NO.
Determination of the NO formed from RNI was made by measuring the
specific chemiluminescence resulting from the reaction of NO with
machine-generated ozone using an NO-NOX analyzer (model 821, Dasibi Environmental, Glendale, CA). Conversion of standard solutions of nitrite to NO was >99%, whereas conversion of nitrate to NO was 94-96% compared with calibrated standards of NO gas (20, 31, 46). Conversion of
S-nitroso-N-acetylpenicillamine to NO was also >99%.
Assay of mRNA for iNOS and TNF-
.
Transcripts for iNOS and TNF-
were measured by cDNA-equalized
reverse transcription-polymerase chain reaction (cERT-PCR) in lavaged
cells, as previously described (20, 31, 45). Briefly, the total RNA of
the AM was isolated using TRIzol reagent (GIBCO, Gaithersburg, MD).
Total cDNA was obtained by reverse transcription of total RNA and
labeled with
[32P]dCTP. Total
cDNA (10 ng) was amplified together with competitor (1 pg) by using the same specific iNOS or TNF-
primers and
[32P]dCTP. Primer
sequences for iNOS and TNF-
were as follows:
5'-AATGGCAACATCAGGTCGGCCATCACT-3' (iNOS-A) and
5'-GCTGTGTGTCACAGAAGTCTCGAACTC-3' (iNOS-B);
5'-AAGTTCCCAAATGGCCTCCCTCTCATC-3' (TNF-
-A) and
5'-GGAGGTTGACTTTCTCCTGGTATGAGA-3' (TNF-
-B). Amounts of iNOS and TNF-
cDNA were determined by phosphorimager scan and
quantitation of the smear and signal bands normalized to the competitor. The total cDNA formed (in ng) equaled the gray value of the
smear / total gray value of the standard × 330 × 4 × [dCTP], where 330 is the average molecular weight
of dNTP (dATP, dGTP, dCTP, dTTP) and [dCTP] is dCTP
concentration. The amount of iNOS or TNF-
cDNA (in pg/ng cDNA)
equaled (iNOS or TNF-
gray value
background) /
(competitor gray value
background) × (1 pg competitor/10 ng total cDNA). The results were expressed as picograms of iNOS or
TNF-
mRNA per nanogram of cDNA and were compared with standard curves plotted by addition of known amounts of iNOS or TNF-
mRNA standards and their competitors to cell samples
(n = 8) devoid of any measurable iNOS
or TNF-
mRNA.
Measurement of iNOS enzyme by Western blot.
BAL fluids (0.5 ml) containing ~3-5 × 106 cells were centrifuged at
1,500 g at 4°C for 1 min. The
pellets were then homogenized with 50 ml of homogenization buffer
[20 mM tris(hydroxymethyl)aminomethane (Tris) · HCl, pH 7.5, 0.25 M sucrose, 2 mM ethylene
glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic
acid, 2 mM EDTA, 0.02% leupeptin, 1 mM phenylmethylsulfonyl fluoride,
and 0.1% Triton X-100]. The homogenates were incubated for 1 h
at 4°C and centrifuged at 3,000 revolutions /min for 30 min at
4°C in a tabletop refrigerated centrifuge (model TJ-9, Beckman
Instruments, Fullerton, CA). The supernatants were stored at
20°C. The concentration of protein in the homogenates was
assayed with the bicinchoninic acid method (18). Protein samples (50 µg) were separated on 10% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis gels. Proteins were electrophoretically transferred to
nitrocellulose using a semidry transfer cell (Bio-Rad, Hercules, CA).
The transfer buffer was 48 mM Tris · HCl and 39 mM
glycine buffer (pH 9.2) containing 0.037% sodium dodecyl sulfate and
20% methanol. Nonspecific sites were blocked with blocking solution
containing 5% (wt /vol) nonfat milk and 0.05% Tween 20 in 80 mM
Na2HPO4,
20 mM
NaH2PO4,
and 100 mM NaCl (pH 7.5) for 1 h at room temperature. The
nitrocellulose membranes were then incubated with polyclonal anti-rat
iNOS antibody (Transduction Lab, Louisville, KY) at a 1:5,000 dilution
in PBS containing 1% nonfat milk and 0.05% Tween 20 overnight at
4°C. After the membranes were washed three times for 10 min each,
they were incubated for 1 h at room temperature with the horseradish peroxidase-linked secondary antibody (1:5,000 dilution in 1% nonfat milk and 0.05% Tween 20 in PBS) and three times for 10 min each in
0.05% Tween 20 in PBS. The bound antibody on the membrane was detected
by the enhanced chemiluminescence method according to the
manufacturer's instructions (Amersham, Arlington Heights, IL).
Exposure times of immunoblots to Hyperfilm were 1 min. The immunoblots
were exposed to Hyperfilm for 1-60 min. The gel image was scanned
by using Deskscan II. The band of specific iNOS protein was quantitated
by NIH Image. The amount of iNOS protein was evaluated as the mean gray
value of the bands, which is calculated as gel units. After the gray
value of the background film was calibrated and adjusted to zero, the
iNOS signal band was selected and measured. The amount of iNOS protein
was evaluated as the mean gray value of the selected area corrected for
the percent change in S18 or
-actin protein that was measured in the
same lane.
Assay for NF-
B.
Nuclear extracts of AM from PBS-, LPS-, DBcAMP (1 mg/kg)-, and
MeS-ATP-treated rats were prepared by standard methods (41), and the
activation of NF-
B was assayed with the electrophoretic mobility
shift assay (EMSA) (41). For NF-
B the oligonucleotide sequence was
5'-AGT TGA GGG GAC TTT CCC AGG C-3' and 3'-TCA ACT CCC CTG AAA GGG TCC G-5'. The single strand of duplex was end labeled with
[
-32P]ATP using T4
polynucleotide kinase, and strands were annealed with each other as the
DNA probe of NF-
B. Nuclear extracts (5-20 µg) were
preincubated in 10 µl of reaction mixture containing 10 mM
Tris · HCl (pH 7.5), 50 mM NaCl, 1 mM EDTA, 0.2 mM
dithiothreitol, 5-10% glycerol, and 1-2 µg of poly(dI-dC).
After 5 min at room temperature, 1 µl of
[
-32P]ATP-labeled
oligonucleotide duplex probe was added, and the incubation was
continued for another 20 min. In competition experiments the unlabeled
competitor DNA (100-fold molar excess) was included during the
preincubation period. HeLa nuclear extract was used as a
positive control and compared with antibodies to authentic NF-
B
(Santa Cruz Biotechnologies, Santa Cruz, CA). After electrophoresis on
a 4% nondenaturing acrylamide gel (in 0.5× TBE
running buffer at 100 V for 3 h), dried gels were read on a
phosphorimager.
TNF-
assays.
The TNF-
bioactive and immunoreactive proteins contained in the
first 5-ml recovery sample of BAL fluid and AM incubates were measured
using the WEHI assay and an enzyme-linked immunosorbent assay kit
(Genzyme, Cambridge, MA), respectively, as described previously in
detail (13), as indexes of the inflammatory response. The bioreactive
TNF-
(expressed in units of activity) reflects the biologic activity
of the total TNF-
protein that is capable of killing the L929 cells.
The immununoreactive TNF-
is the amount of specific immunoreactive
TNF-
protein in the BAL fluid and AM incubates (31).
Statistical analyses of data.
Each experiment was replicated with 6-22 rats /group. Data were
analyzed with analysis of variance for a randomized complete block or
completely random sample design. Differences between and among means
were analyzed with Dunnett's and Duncan's tests. Biochemical data
were analyzed with multivariate analysis of variance, and means were
compared with Newman-Keuls test.
P
0.05 was accepted for
statistical significance of mean differences.
 |
RESULTS |
BAL fluid cell counts and differential.
The control rats given PBS had a total BAL fluid cell content of
15.2 ± 1.3 (n = 22) to
18.9 ± 0.8 × 106 (SE)
cells (n = 11), 99-99.8% of
which were also AM (Tables 1 and
2). This did not differ from the cell
type and distribution obtained from the BAL fluid of untreated rats
given anesthetic before lavage (Table 1). Treatment of rats with LPS
(0.6 mg/kg it) increased the total number of cells by increasing the
influx of PMN into the lung (Table 1). In contrast, pretreatment of rats with DBcAMP, MeS-ATP, isoproterenol, and albuterol did not significantly affect the number of AM or PMN recovered in the BAL fluid
(Table 1). Pretreatment of rats with DETC inhibited LPS-stimulated AM
as well as PMN recruitment into the alveolar space, thereby decreasing
the total cell number without affecting the relative distribution of AM
and PMN (Table 2). Although DETC slightly enhanced the accumulation of
neutrophils within the lung, the effect was minor and did not override
the lack of effect of DBcAMP, MeS-ATP, or isoproterenol on the AM or
PMN (Table 2). Administration of DBcAMP or MeS-ATP with LPS slightly
decreased the total number of AM in the BAL fluid, thus increasing the
percentage but not the number of LPS-stimulated PMN in the alveolar
space (Table 1). Conversely, pretreatment of rats given the combination of autacoid mimetic and LPS with DETC decreased the total number of
cells in the BAL fluid, with a greater effect on PMN. Thus the relative
distribution of AM and PMN returned to that seen with DETC and LPS
alone (Table 2).
Direct effects of LPS and autacoids on the iNOS system of rat AM.
Treatment of rats with LPS, DBcAMP, and MeS-ATP upregulated iNOS
mRNA in AM within 2 h after intratracheal administration. The content
of iNOS mRNA generated in AM from rats treated with DBcAMP (1.0 mg/kg)
or MeS-ATP was equal to or greater than that produced by LPS (Fig.
1). LPS, DBcAMP, and MeS-ATP increased the levels of iNOS protein within AM 2 h after intratracheal administration to rats. The levels of iNOS protein produced by the low and high dose
of DBcAMP for equivalent levels of iNOS mRNA produced in the AM were
lower than those produced in the AM from LPS-treated rats (Fig. 1).
PBS, isoproterenol, and albuterol did not induce iNOS mRNA or iNOS
protein (Fig. 1). Means ± SE from 5-22 individual experiments
are summarized in Fig. 2.

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Fig. 1.
Representative gels of inducible NO synthase (iNOS) mRNA and iNOS
protein in alveolar macrophages (AM) from rats treated with
phosphate-buffered saline (PBS), autacoid mimetics, or
2-adrenoceptor agonists. AM
were obtained from bronchoalveolar lavage (BAL) fluid, and total mRNA
was isolated and subjected to cDNA-equalized reverse
transcription-polymerase chain reaction (cERT-PCR) or iNOS protein was
isolated using Western blot analyses and a polyclonal antibody to iNOS.
Gel density of iNOS signal was determined with computerized gel
densitometry. Top: cERT-PCR of iNOS
mRNA in AM from BAL fluid 2 h after intratracheal treatment in vivo
with PBS (0.5 ml; lane 1),
lipopolysaccharide (0.6 mg/kg; lane
2), dibutyryl-cAMP (0.1 and 1 mg/kg;
lanes 3 and
4), 2-methylthioadenosine
5'-triphosphate (5 mg/kg; lane
5), isoproterenol (0.2 µg/kg; lane
6), or albuterol (0.5 mg/kg; lane
7). Lane 0, positive
control for iNOS mRNA (top) or iNOS
protein (bottom);
lane M, kilobase marker for RNA or
molecular mass marker for iNOS protein. CP, competitor DNA (cDNA).
Ratio of mRNA to cDNA corrected for background represents differences
in iNOS mRNA. The less dense the cDNA, the more iNOS mRNA is present.
Bottom: the less dense the band, the
less iNOS protein is present. For calculations see
MATERIALS AND METHODS.
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Fig. 2.
Effect of PBS, autacoid mimetics, and lipopolysaccharide (LPS) on rat
AM iNOS mRNA (top) and iNOS protein
(bottom).
Top: ratio of iNOS mRNA to competitor
DNA corrected for background (ordinate) represents iNOS mRNA expressed
as pg mRNA/ng cDNA. Bottom: Western
blot gel density of iNOS protein in 50 mg of protein for AM from BAL
fluid 2 h after intratracheal treatment of rats in vivo with PBS, LPS,
dibutyryl-cAMP (DBcAMP), 2-methylthioadenosine 5'-triphosphate
(MeS-ATP), isoproterenol (Iso), or albuterol (Alb), as described in
Fig. 1 legend. Values are means ± SE for 5-22 rats /group.
* Significantly different (P < 0.05) from PBS.
Significantly different
(P < 0.05) from LPS.
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RNI production.
A basal level of RNI existed in the BAL fluid of rats treated with PBS
(Fig. 3,
top). The RNI generated by AM from
PBS-treated rats incubated ex vivo for 1 h did not differ from that of
the buffer in the absence of cells (Fig. 3,
bottom). Pretreatment of rats with
LPS, DBcAMP, or MeS-ATP increased RNI levels in BAL fluid and the ex
vivo incubates of AM (Fig. 3,
bottom). However, the increase in
RNI induced by the autacoid mimetics was less than that in the BAL
fluid and incubates of AM from LPS-treated rats. The two
-adrenoceptor agonists that did not induce mRNA transcription or
iNOS protein did not affect RNI levels in the BAL fluid or in the ex
vivo incubates of AM (Fig. 3).

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Fig. 3.
Effect of intratracheal administration of PBS (0.5 ml), LPS (0.6 mg/kg), DBcAMP (0.1 and 1.0 mg/kg), MeS-ATP (5 mg/kg), Iso (0.2 µg/kg), or Alb (0.5 mg/kg) on reactive nitrogen intermediate (RNI)
levels in BAL fluid (top) and 1-h ex
vivo incubates of rat AM (bottom).
Values are means ± SE for 5-22 rats/group.
* Significantly different (P < 0.05) from PBS.
Significantly different
(P < 0.05) from LPS.
|
|
Effect of LPS and autacoids on TNF-
.
Low background levels of TNF-
mRNA were present in freshly isolated
AM from rats treated with PBS, DBcAMP, MeS-ATP, albuterol, or
isoproterenol and did not differ among the treatment groups (P > 0.67). TNF-
mRNA and
immunoreactive and biologically active TNF-
protein were only
increased when measured in AM from LPS-treated rats (Fig.
4).

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Fig. 4.
Effect of intratracheal administration of PBS (0.5 ml), LPS (0.6 mg/kg), DBcAMP (0.1 and 1.0 mg/kg), MeS-ATP (5 mg/kg), Iso (0.2 µg/kg), or Alb (0.5 mg/kg) on biologically active and immunoreactive
tumor necrosis factor- (TNF- ) in BAL fluid
(top) and 1-h ex vivo incubates of
rat AM (bottom). Values are means ± SE for 5-22 rats/group. * Significantly different
(P < 0.05) from PBS.
Significantly different
(P < 0.05) from LPS.
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Effect of DBcAMP and MeS-ATP on LPS-stimulated iNOS mRNA, iNOS
protein, and RNI.
The contents of LPS-induced iNOS mRNA and iNOS protein in AM from rats
pretreated with the autacoid mimetics were greater than those produced
by administration of the autacoid mimetic or LPS individually to rats
(Fig. 5). The magnitude of DBcAMP (1 mg/kg)- or MeS-ATP-stimulated enhancement of LPS-mediated upregulation of iNOS mRNA and iNOS protein in rat AM was additive rather than synergistic with that of LPS (Figs. 5 and
6). However, DBcAMP (0.1 mg/kg)-induced
enhancement of LPS-mediated upregulation of iNOS mRNA and iNOS protein
was synergistic with LPS in AM from rats pretreated with this low dose
of DBcAMP. LPS-induced increases of RNI in BAL fluid and ex vivo
incubates of AM were also enhanced in an additive and synergistic
manner in rats pretreated with DBcAMP or MeS-ATP (Fig.
7). Pretreatment of rats with PBS,
isoproterenol, or albuterol did not affect LPS-mediated upregulation of
iNOS mRNA, iNOS protein, or RNI (Figs. 5-7).

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Fig. 5.
Representative gels of iNOS mRNA
(top) and iNOS protein
(bottom) of autacoid-induced
enhancement of LPS-mediated upregulation of iNOS mRNA and iNOS protein
obtained from rat AM 2 h after intratracheal administration of LPS.
Lane 1, PBS (0.5 ml/kg);
lane 2, PBS + LPS (0.6 mg/kg);
lanes 3 and
4, DBcAMP (0.1 and 1 mg/kg) + LPS;
lane 5, MeS-ATP (5 mg/kg) + LPS;
lane 6, Iso (0.2 µg/kg) + LPS;
lane 0, positive control for iNOS mRNA
(top) or iNOS protein
(bottom); lane
M, kilobase marker for iNOS mRNA or molecular mass
markers for identification of iNOS protein. PBS or autacoids were
administered intratracheally 15 min before intratracheal administration
of LPS. For details see Fig. 1 legend. For calculations see
MATERIALS AND METHODS.
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Fig. 6.
Autacoid-induced enhancement of LPS-mediated upregulation of iNOS mRNA
and iNOS protein from rat AM 2 h after intratracheal administration of
LPS. Top: ratio of iNOS mRNA to
competitor DNA corrected for background (ordinate).
Bottom: Western blot gel density of
iNOS protein in AM obtained from BAL fluid 2 h after treatment of
rats in vivo with intratracheal PBS. Rats were pretreated with PBS,
DBcAMP, MeS-ATP, or Iso as described in Fig. 5 legend. Values are means ± SE for 5-17 rats /group. * Significantly different
(P < 0.05) from LPS alone.
Significantly different
(P < 0.05) from LPS + autacoid
mimetic (P < 0.05).
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Fig. 7.
Autacoid-induced enhancement of LPS-mediated upregulation of RNI in BAL
fluid (top) and ex vivo incubates of
AM (bottom). Values are means ± SE for 5-17 rats/group. * Significantly different
(P < 0.05) from LPS alone.
Significantly different
(P < 0.05) from LPS + autacoid
mimetic (P < 0.05).
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|
Effect of DBcAMP and MeS-ATP on LPS-stimulated TNF-
.
Pretreatment of rats with DBcAMP or MeS-ATP did not affect
LPS-induced increases in AM TNF-
mRNA (Fig.
8, top).
However, each of these autacoids inhibited the release of biologically active and immunoreactive TNF-
in the BAL fluid and ex vivo
incubates of AM (Fig. 8, top and
bottom). Pretreatment of rats with
PBS, isoproterenol, or albuterol did not affect LPS-mediated
upregulation of TNF-
mRNA in AM or TNF-
protein levels in the BAL
fluid and ex vivo incubates of AM (Fig. 8).

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Fig. 8.
Autacoid-induced inhibition of LPS-mediated upregulation of
biologically active and immunoreactive TNF- in BAL fluid
(top) and 1-h ex vivo incubates of
rat AM (bottom). Values are means ± SE for 5-17 rats/group. * Significantly different
(P < 0.05) from LPS alone.
Significantly different
(P < 0.05) from LPS + autacoid
mimetic. For details see Fig. 5 legend.
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|
Effect of LPS, DBcAMP, and MeS-ATP on NF-
B.
The pathway utilized by LPS and the autacoid mimetics was explored on a
limited basis. Nuclear extracts of AM from rats treated with PBS, LPS,
DBcAMP (1 mg/kg), and MeS-ATP were studied at 2 h, since preliminary
studies showed that the peak in vivo change in NF-
B occurred at this
time in AM (unpublished observations). LPS increased NF-
B, as
determined by EMSA (Fig. 8) and verified by the supershift assay (data
not shown). In contrast, concentrations of DBcAMP and MeS-ATP that
increased iNOS mRNA did not increase NF-
B compared with PBS-treated
rats (Fig. 9), which also did not express
iNOS mRNA (Fig. 1).

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Fig. 9.
Effects of intratracheal administration of PBS (0.5 ml), LPS (0.6 mg/kg), DBcAMP (1 mg/kg), and MeS-ATP (5 mg/kg) on nuclear factor- B
(NF- B) DNA binding activity in nuclear extracts of AM. Rats were
killed 2 h after drug administration. AM were isolated from BAL fluid,
and nuclear extracts were isolated. Nuclear extracts (5 µg/well) were
analyzed by electrophoretic mobility shift assay for NF- B DNA
binding. Gel is representative of 4 separate experiments.
|
|
Effects of DETC on LPS, DBcAMP, and MeS-ATP.
Pretreatment of rats with DETC inhibited LPS-mediated upregulation of
iNOS mRNA, iNOS protein, and RNI levels in the BAL fluid and ex vivo
incubates of AM but did not affect DBcAMP- and MeS-ATP-mediated increases in these parameters (Figs. 10
and 11). Moreover, administration of DETC
to rats pretreated with DBcAMP or MeS-ATP before intratracheal administration of LPS abolished the component of the enhanced iNOS mRNA
and iNOS protein attributable to LPS (Figs. 10 and
12). The residual response to the
coadministration of these autacoid mimetics and LPS remaining in
DETC-pretreated rats equaled that produced by DBcAMP or MeS-ATP alone
(Figs. 10 and 12). Pretreatment of rats with DETC did not affect the
ability of LPS to upregulate TNF-
mRNA, immunoreactive or
biologically active (data not shown) TNF-
protein, or DBcAMP or
MeS-ATP to suppress LPS-mediated upregulation of TNF-
biologically
active (data not shown) and immunoreactive (Fig.
13) TNF-
protein in BAL fluid and ex
vivo incubates from AM.

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Fig. 10.
A: representative cERT-PCR and Western
blot gels (50 µg of applied protein) showing effects of PBS (0.5 ml
it; lanes 1, 3, 5, 6, and
9) and diethyldithiocarbamate (DETC,
5 mg/kg it; lanes 2, 4, 7, 8, and
10) on rat AM iNOS mRNA and iNOS
protein induced by intratracheal administration of PBS
(lanes 1 and
2), LPS (0.6 mg/kg;
lanes 3 and
4), DBcAMP (0.1 mg/kg;
lanes 5 and
7), DBcAMP (1 mg/kg;
lanes 6 and
8), or MeS-ATP (5 mg/kg;
lanes 9 and
10). DETC was given 30 min before
autacoids or LPS. Lane 0, iNOS mRNA or
iNOS protein standards; lane M,
kilobase marker for iNOS mRNA. B:
representative cERT-PCR and Western blot gels (50 µg of applied
protein) showing effect of DETC (5 mg/kg it; lanes 2, 4, 6, and 8) on
autacoid-induced enhancement of LPS-mediated iNOS mRNA and protein of
AM from rats pretreated in vivo with DBcAMP (0.1 mg/kg it) or MeS-ATP
(5 µg/kg it) 15 min before administration of LPS (0.6 mg/kg it).
Lane 0, iNOS mRNA and cDNA or iNOS
protein standards; lane 1, LPS in
PBS-pretreated rats; lane 2, LPS in
DETC-pretreated rats; lanes 3 and
4, DBcAMP in PBS- and DETC-pretreated
rats; lane 5, DBcAMP facilitation of
LPS in PBS-pretreated rats; lane 6,
impairment in DBcAMP facilitation of LPS in DETC-pretreated rats;
lane 7, MeS-ATP facilitation of LPS in
PBS-pretreated rats; lane 8,
impairment in MeS-ATP facilitation of LPS in DETC-pretreated rats;
lane M, kilobase marker for iNOS mRNA
and cDNA. For details see Fig. 1 legend.
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Fig. 11.
Left: effect of DETC on direct effects
of LPS and autacoid mimetics on iNOS mRNA
(top) and iNOS protein
(bottom). DETC was given 30 min
before PBS (0.5 ml/kg it), LPS (0.6 mg/kg it), DBcAMP (0.1 and 1 mg/kg
it), or MeS-ATP (5 mg/kg it). Values are means ± SE from
6-14 experiments. Right: effect
of intratracheal administration of DETC on PBS-, LPS-, DBcAMP-, and
MeS-ATP-mediated changes in RNI levels in BAL fluid
(top) and 1-h ex vivo incubates of
rat AM (bottom). Values are means ± SE from 5-14 rats/group. * Significantly different
(P < 0.05) from autacoid or LPS in
absence of DETC.
Significantly different
(P < 0.05) from response to LPS in
presence of DETC.
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Fig. 12.
Left: effect of DETC on enhancement by
autacoid mimetics of LPS-mediated upregulation of iNOS mRNA
(top) and iNOS protein
(bottom). DETC was given 15 min
before PBS (0.5 ml/kg it), DBcAMP (0.1 mg/kg it), or MeS-ATP (5 mg/kg)
and, thus, 30 min before intratracheal administration of LPS. Values
are means ± SE for 6-14 experiments.
Right: effect of intratracheal
administration of DETC on enhancement by autacoid mimetics of
LPS-mediated upregulation of RNI levels in BAL fluid
(top) and 1-h ex vivo incubates of
rat AM (bottom). Values are means ± SE for 5-14 rats /group. * Significantly different
(P < 0.05) from control.
Significantly different
(P < 0.05) from LPS.
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Fig. 13.
Modification by DETC (crosshatched bars) on LPS (0.6 mg/kg)-mediated
upregulation of TNF- mRNA alone or in presence of DBcAMP (0.1 and
1.0 mg/kg) or MeS-ATP (5 mg/kg;
top), on immunoreactive (hatched
bars) TNF- in BAL fluid (middle),
or on ex vivo incubates of rat AM
(bottom). DETC was administered 15 min before intratracheal administration of DBcAMP or MeS-ATP (5 mg/kg),
which was administered 15 min before intratracheal LPS. Values are
means ± SE for 6-14 rats/group. * Significantly
different (P < 0.05) from control.
Significantly different
(P < 0.05) from LPS.
|
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 |
DISCUSSION |
New findings of the study.
This is the first study to demonstrate that in vivo administration of
low doses of DBcAMP and the P2y
receptor partial agonist MeS-ATP directly stimulated transcription of
iNOS mRNA and translation of the mRNA to active iNOS protein, which in
turn produced RNI in the BAL fluid and in the 1-h ex vivo incubates of
rat AM. Furthermore, intratracheal administration of small doses of
these compounds before administration of LPS enhanced the ability of
LPS to upregulate iNOS mRNA and iNOS protein in the freshly isolated AM
and the resultant RNI in the BAL fluid and 1-h ex vivo incubates of the recovered AM. In contrast to LPS, the autacoid mimetics upregulated the
iNOS system without eliciting activation of the inflammatory process,
since TNF-
was not increased and recruitment of PMN into the
alveolar space did not occur. These data suggest that local regulation
of iNOS transcription in AM in the rat may result from the activity of
endogenous cAMP and P2y receptor
stimulation of the AM in the mouse and rat (12, 34, 44) and potentially different tissues in several species (2, 14, 17, 24, 26, 28-30,
37-39). It may be argued that if this postulate were correct, the
-adrenoceptor agonists should have increased iNOS mRNA and iNOS
protein, whereas they were devoid of effects on the iNOS system in rat
AM in vivo. However, isoproterenol and albuterol were used as negative
controls to test for nonspecific irritation of the lung and thereby
activation of the AM because rat AM are essentially devoid of
-adrenoceptors. Furthermore, LPS selectively increased NF-
B and
DETC, an inhibitor of NF-
B, and free radical-mediated activation of
NF-
B (15, 29, 36, 47) selectively attenuated LPS-mediated
upregulation of transcription of iNOS mRNA. Thus the data also support
the postulate that the cell-signaling pathway utilized by the autacoid
mimetics differed from that utilized by LPS. This finding is in
agreement with that which demonstrated that dexamethasone
differentially inhibited cAMP- but not interleukin-1
-induced
upregulation of iNOS in renal mesangial cells (33). Moreover,
posttranscriptional and translation mechanisms may be differentially
modulated by the autacoid and LPS pathways. This conclusion is based on
the finding that disparities existed between the amount of iNOS protein
and generated NO produced in the LPS-, MeS-ATP-, and DBcAMP-stimulated
AM that contained equal levels of iNOS mRNA.
For equivalent amounts of iNOS mRNA produced by the high dose of
DBcAMP, MeS-ATP, and LPS, the amounts of iNOS protein and RNI generated
by AM from the rats treated with DBcAMP were slightly but significantly
lower than the amounts produced by LPS. Moreover, the high dose of
DBcAMP interacted with LPS in an additive manner, whereas the low dose
of DBcAMP interacted with LPS in a synergistic fashion. These findings,
in agreement with others using cultured cells (12, 29, 30, 33), further
support the conclusion that the autacoid mimetics and LPS upregulate
the iNOS system via distinct cell-signaling pathways (12, 29).
Effects of DETC.
LPS-induced upregulation of the iNOS system is dependent on activation
of a tyrosine kinase (13, 17, 28) or an isozyme of protein kinase C
(17, 25, 28) and subsequent activation of NF-
B (this study; 17, 28, 29, 47) that is inhibited by DETC and pyrrolidine dithiocarbamate (15,
36, 43). We also demonstrated that in vivo administration of the
protein kinase C inhibitors staurosporine and chelerythrine to rats did
not affect DBcAMP- and MeS-ATP-induced upregulation of iNOS mRNA (21).
This study demonstrated that pretreatment of rats with LPS, but not
with DBcAMP or MeS-ATP, selectively upregulated NF-
B- and
DETC-inhibited LPS-mediated transcription of iNOS mRNA and iNOS protein
in AM in vivo. The cell-signaling pathways utilized by the autacoids and LPS must differ, since a dose of DETC that inhibited LPS-mediated upregulation of iNOS mRNA and iNOS protein failed to affect the iNOS
mRNA or the iNOS protein induced by the autacoids, which did not
upregulate NF-
B. Moreover, in experiments in which LPS was
administered with the autacoid mimetics, the iNOS mRNA, iNOS protein,
and RNI remaining after the pretreatment of rats with DETC equaled that
produced by the autacoid mimetics alone. These findings support the
conclusion that the pathway utilized by DBcAMP and MeS-ATP to
upregulate transcription of iNOS in rat AM in vivo differed from that
utilized by LPS and appears to be independent of NF-
B.
Mechanism of action of DBcAMP and MeS-ATP.
It is possible that these autacoids increased endogenous constitutive
iNOS mRNA by preventing its degradation by messenger ribonucleases, as
it does in cardiac myocytes in cell culture (39). iNOS mRNA was
undetectable in AM from PBS-treated rats. Although this may have
resulted from the limits of sensitivity of the assay (10-5 pg/ng
cDNA), this is likely, since we used 2-4 × 106 AM/ml to measure iNOS mRNA and
protein, 10 times the number of cells in which iNOS mRNA and iNOS
protein can be detected. Induction of gene transcription is a
prerequisite for the control of iNOS enzyme activity (17, 28), and iNOS
mRNA is not expressed in the absence of the autacoids or LPS.
Endogenous cAMP and ATP also exist in AM and should be capable of
activating the machinery to prevent the degradation or stimulate
upregulation of any constitutive iNOS mRNA. Despite the absence of iNOS
mRNA and iNOS protein in AM from PBS-treated rats, iNOS mRNA and iNOS
protein were increased in the AM 2 h after intratracheal administration
of DBcAMP or MeS-ATP. The increase in iNOS mRNA in the AM from DBcAMP-
and MeS-ATP-treated rats did not differ from that produced by LPS, which activates the transcription of iNOS mRNA and has only minor inhibitory effects on the degradation of iNOS mRNA (17, 28). Thus it is
unlikely that the autacoids increase the levels of iNOS mRNA in rat AM
in vivo by preventing its degradation.
Several P2y receptor agonists act
synergistically with LPS to stimulate macrophage membrane guanosine
5'-triphosphatase (GTPase) activity (12). MeS-ATP is a partial
agonist at the P2y receptor but is
devoid of stimulatory effects on GTPase activity (12). Thus it is also
unlikely that stimulation of membrane GTPase can explain the ability of
these autacoid mimetics to upregulate the iNOS system and enhance the
effects of LPS on the iNOS system of the rat AM in vivo. Finally,
inhibition of protein synthesis with cycloheximide inhibits LPS- and
cytokine-mediated upregulation of iNOS transcription (16, 23). Because
cAMP, and possibly P2y receptor
stimulation, can activate protein synthesis, it is possible that they
upregulate a transcription factor or a protein promoter of
transcription, which in turn activates the formation of iNOS mRNA (3,
17, 27, 28, 48) and, once formed, may subsequently prevent its
degradation (39). Alternatively, each of the autacoid mimetics can
upregulate protein kinase A (PKA), which may result in the activation
of PKA-dependent transcription factors or inhibition of a repressor of
transcription (3). This also may upregulate iNOS transcription (7).
Further studies are required to test these postulates in vivo.
Differences between effects of autacoid mimetics and LPS on iNOS
protein.
Translation of iNOS protein was reduced in AM from the rats treated in
vivo with DBcAMP and MeS-ATP compared with LPS-treated rats.
Dimerization of the iNOS subunits is essential for the
posttranslational activation of iNOS activity (9). Among the factors
promoting dimerization are tetrahydrobiopterin
(BH4), arginine, and heme (9,
22). LPS increases arginine transport and upregulates BH4 in AM (4, 17, 28). DBcAMP and
MeS-ATP are devoid of these actions (17, 21, 28). Moreover, NO limits
the dimerization of iNOS by diminishing the availability of heme iron
to the enzyme (10, 17, 28). cAMP increases translation of iNOS mRNA to protein (17, 28). The low and high doses of DBcAMP were synergistic and
additive, respectively, with LPS-mediated upregulation of iNOS protein,
yet were additive with LPS-induced increases in the AM content of iNOS
mRNA. Because LPS can upregulate
BH4 and arginine transport to AM,
we speculate that these factors may have contributed to the synergistic
effect of DBcAMP and Mes-ATP on LPS-mediated upregulation of iNOS
protein. Thus we speculate that the lower amounts of iNOS protein and
subsequent generation of RNI measured in AM from rats treated with the
autacoid mimetics may result, in part, from limited posttranslational
dimerization of iNOS. The inability of MeS-ATP to synergize with LPS
cannot be explained, unless the maximal rate of iNOS formation was
reached at the endogenous concentrations of arginine in the AM (17, 28). Alternatively, it is possible that the autacoids and LPS induced
two distinct isozymes of iNOS protein similar to those found in the rat
kidney (35). The synergism between the low dose of DBcAMP- and
LPS-mediated upregulation of iNOS protein in AM may have resulted from
a sensitivity of the LPS-induced isozyme to DBcAMP but not to MeS-ATP.
The mechanism of action of the autacoids on the iNOS system remains to
be elucidated.
Similarities and differences between studies.
Our findings that in vivo administration of DBcAMP and MeS-ATP
upregulated transcription of iNOS mRNA and increased iNOS protein and
RNI are in agreement with some in vitro studies using peripheral macrophages, fibroblasts, vascular smooth muscle cells, and cardiac myocytes in cell culture (6, 14, 24, 26, 29, 38, 39, 44). However, our
results differ from those studies that only demonstrated inhibitory
effects of these and related compounds when they are used in vitro in
high concentrations with a combination of LPS and cytokines (8,
12, 17, 28, 37, 38). Moreover, our findings that DBcAMP- and
MeS-ATP-induced upregulation of the iNOS system is independent of
upregulation of NF-
B and refractory to inhibition by DETC differ
from most data obtained in cell culture (6, 8, 12, 14, 17, 24, 26, 28,
29, 37-39, 44). Several factors can explain these differences,
including the dose or concentration of the autacoid mimetics used and
the cell type under study. However, the most important factor is
probably the difference between the use of an in vivo and a cell
culture model.
Dose and concentration effects.
MeS-ATP is a partial agonist of
P2y receptors, so low doses may
act selectively as an agonist, whereas higher doses may act as an
antagonist. Moreover, in vitro studies that demonstrated cAMP- and
MeS-ATP-induced inhibition of LPS-mediated upregulation of iNOS mRNA
and iNOS protein utilized high micromolar and millimolar concentrations
of cAMP or DBcAMP or 0.1 mM MeS-ATP (8, 12, 37, 38). In contrast, low
concentrations of prostaglandin E2
and DBcAMP increased iNOS mRNA in cultured RAW 264.7 macrophages and
enhanced interferon-
- (34) and TNF-
-mediated upregulation of iNOS
mRNA and iNOS protein (44). Thus low and physiological levels of cAMP
and P2y receptor stimulation may
upregulate the iNOS system, whereas high concentrations of these
autacoids may inhibit the iNOS system.
Cell culture vs. in vivo studies and cell types.
The use of cell lines and primary cells in culture may also modify the
ex vivo cell-signaling pathways used to upregulate transcription of
iNOS mRNA and its translation to iNOS protein. Peptide growth factors
and the cell culture process itself cause many enzymes and proteins to
revert to their fetal phenotype (5). Many cell lines and primary cells
in culture exhibit phenotypic and genotypic transformations that may
hinder the extrapolation of the cell-signaling pathways found to their
tissues of origin in vivo (5, 43). For example, TNF-
is a potent
inhibitor of endothelial cell growth in vitro but is angiogenic in vivo (42). Also, differences exist in the induction mechanisms for iNOS mRNA
between rat aortic smooth muscle cells in culture and isolated aortic
strips (43). Finally, LPS, DBcAMP, and MeS-ATP upregulate iNOS
transcription and RNI production within 15 min after their
administration in vivo (20, 21, 31, 45). Yet, continuous exposure for
at least 10-48 h to a fixed concentration of these compounds is
required to stimulate the iNOS system or to modify the effect of
cytokines on the iNOS system (6, 8, 12, 14, 17, 24, 26, 28-30, 38,
39, 43). Thus the effects of these autacoids on the iNOS system may not
be expressed in vitro, or their cell-signaling pathways utilized to
induce iNOS mRNA and iNOS protein may differ when tested in vivo and in
cell culture.
Conclusions.
We conclude that at least two distinct cell-signaling pathways exist
for in vivo induction of iNOS transcription in rat AM. The first is a
cytokine- and LPS-stimulated pathway that involves stimulation of the
transcription factor NF-
B, is inhibitable by DETC (15, 36), and is
associated with activation of the inflammatory response. The second
pathway can be activated by DBcAMP and the
P2y receptor partial agonist
MeS-ATP. It is not associated with upregulation of NF-
B, it is
refractory to inhibition by DETC, and it does not elicit an
inflammatory response in the lung. Speculatively, this second pathway
may involve activation of PKA (21). DBcAMP and MeS-ATP administered
with LPS enhance LPS-mediated upregulation of iNOS mRNA, iNOS protein,
and NO in an additive and synergistic fashion. These data support the
conclusion that in vivo induction of iNOS in rat lung AM may be
selectively regulated by drugs and endogenous autacoids that increase
cAMP or activate P2y receptors.
Also, significant qualitative and quantitative differences appear to
exist between rates and mechanisms of upregulation of the transcription
of iNOS mRNA in vivo and in macrophages subjected to cell culture. This
suggests that the process of cell culture may modify the cell-signaling
pathway involved in the upregulation of iNOS, in agreement with the
findings in isolated aortic strips and aortic smooth muscle cells in
culture (43). The mechanism(s) by which cAMP and MeS-ATP increase iNOS
mRNA and iNOS protein remains to be defined.
Perspectives
Although cellular regulation of rat and human iNOS mRNA and iNOS
protein has many similarities, differences also exist (17, 28). Thus
the findings reported here may be significant for veterinary and
clinical medicine as well as for basic research. First, if cAMP and
P2y receptor stimulation
upregulate the transcription of iNOS in humans and higher mammals, then
medications that upregulate these systems may also upregulate iNOS.
This may be beneficial in asthma and hypertension, where drugs that
elicit increases in cAMP or ATP will also stimulate low levels of iNOS,
contributing to maintained dilation of airway or vascular smooth muscle
without further stimulation of inflammation. However, it may also be
deleterious in other heart and lung diseases in which oxygen free
radicals are elevated, since this could result in high levels of the
more toxic peroxynitrite (28, 32). Also, this finding makes it more
difficult to determine the role of iNOS in diseased tissue in animals
and humans. For example, NO in the exhaled air of asthmatic patients is
derived primarily from iNOS, in contrast to the exhaled NO obtained
from normal control subjects, in whom the NO is derived primarily from
constitutive NOS (49). iNOS is also increased in platelets from
patients with coronary atherosclerosis (11) as well as transplanted
hearts from patients with some forms of heart failure (17, 28). These
patients usually are treated with medications that can modulate the
cAMP and purinergic receptor pathways. Thus the existence of iNOS in
these patients may not be related to the pathogenesis of disease but
rather may result from the effects of medications on the iNOS system.
Thus it is important that the effect of the drugs taken by the patients
or tissue donors be considered in the design, execution, and evaluation of experiments when the iNOS system is evaluated. Finally, the cell-signaling pathways involved in iNOS regulation differ in aortic
smooth muscle cells in culture and isolated aortic strips in a muscle
chamber (43). Cell culture, in vitro, and in vivo models each have
their strengths and weaknesses. However, when cell-signaling pathways
are found in culture and/or in vitro, they may not represent
the cell-signaling pathway that is active in vivo without rigorous
testing and confirmation. This caveat applies not only to the iNOS
system but to any system in which the cells undergo changes in
phenotype or genotype in culture. Thus an understanding of the role of
NF-
B and other transcription factors in vivo is needed to understand
the physiological cell-signaling pathways involved in the regulation of
iNOS in animals and humans and the sites of their dysregulation in
disease states.
 |
ACKNOWLEDGEMENTS |
This research was supported in part by National Institute on
Alcohol Abuse and Alcoholism Grants AA-09816 (S. S. Greenberg) and
PO5-AA-09803 (J. J. Spitzer).
 |
FOOTNOTES |
This work was presented in part at the UCLA Meeting on the Biochemistry
and Molecular Biology of Nitric Oxide, Sunset Village, Los Angeles, CA,
13-17 July 1996, and as an oral communication at the November 1996 Sessions of the American Heart Association. Abstracts of this work have
been published (Circ. Suppl. 94, II56, 1996 and Proc. Conf. Biochem. Mol. Biol. Nitric Oxide,
1996).
Address for reprint requests: S. S. Greenberg, Dept. of Medicine, Sect.
of Cardiology, LSU Medical School, 1542 Tulane Ave., Rm. 334, New
Orleans, LA 70112.
Received 1 October 1996; accepted in final form 6 June 1997.
 |
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