1 Biomedical Sciences Graduate Program, 2 Departments of Pharmacology and Medicine, University of California at San Diego, La Jolla, California 92093
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ABSTRACT |
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Treatment of cultured adult rat cardiac
fibroblasts with interleukin-1 (IL-1
) induces the inducible
nitric oxide synthase (iNOS) expression, increases nitric oxide (NO)
and cGMP production, and attenuates cAMP accumulation in response to
isoproterenol by ~50%. Reduced cAMP accumulation is due to NO
production: the effect is mimicked by NO donors and prevented by
NG-monomethyl-L-arginine, an NOS
inhibitor. Effects of NO are not restricted to the
-adrenergic
response; the response to forskolin is similarly diminished. NO donors
only slightly (12%) decrease forskolin-stimulated adenylyl cyclase
(AC) activity in cardiac fibroblast plasma membranes, suggesting that
the main effect of NO is not a direct one on AC. An inhibitor of
soluble guanylyl cyclase inhibits the effects of IL-1
and NO donors;
inhibition of cGMP-dependent protein kinase is without effect.
3-Isobutyl-1-methylxanthine, a nonspecific phosphodiesterase (PDE)
inhibitor, and erythro-9-(2-hydroxy-3-nonyl)adenine, a specific
inhibitor of the cGMP-stimulated PDE (PDE2), completely restore cAMP
accumulation in sodium nitroprusside-treated fibroblasts and largely
reverse the attenuated response in IL-1
-treated fibroblasts. Although NO reportedly acts by reducing AC activity in some cells, in
cardiac fibroblasts NO production decreases cAMP accumulation largely
by the cGMP-mediated activation of PDE2.
nitric oxide; interleukin-1; phosphodiesterase 2; guanosine
3',5'-cyclic monophosphate; soluble guanylyl cyclase
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INTRODUCTION |
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NITRIC OXIDE (NO) regulates diverse physiological processes such as vascular tone, immune defense, and neurotransmission (6, 27). NO is synthesized from L-arginine by nitric oxide synthases (NOS). Of the three isoforms of NOS that have been identified, two are constitutively expressed in cells, whereas a third isoform, the inducible NOS (iNOS), is expressed only after stimulation of cells with inflammatory cytokines or lipopolysaccharides (1). The induction of iNOS by inflammatory cytokines has a protective effect by causing the production of large quantities of NO that can kill microorganisms such as bacteria (1, 11). NO is an important mediator within the cardiovascular system, where it can modulate the function of the heart and vessels (1, 18). In some pathological settings such as myocardial infarction, allograft rejection, or sepsis, the induction of iNOS and the subsequent increase in NO production may be deleterious and may be associated with depressed contractile responses and the death of cardiac myocytes (19, 23).
Most studies of NO modulation in the heart have focused on cardiac
myocytes and endothelial cells, whereas very little attention has been
given to the cardiac fibroblasts. Cardiac fibroblasts are the major
nonmyocyte constituent of cardiac tissue, comprising two-thirds of the
cell number and one-fifth of the cell mass in the heart
(14). Our laboratory has observed that the signaling systems in cardiac fibroblasts are distinct from those of cardiac myocytes. We have previously reported that cardiac fibroblasts express
2-adrenergic receptors that couple to
Gs-adenylyl cyclase (AC) but lack
1
receptors, the subtype that predominates on cardiac myocytes (15,
22). We have also reported that interleukin (IL)-1
induces
iNOS expression in cardiac fibroblasts and that the
2-adrenergic response (cAMP elevation, cAMP-dependent
protein kinase activation) enhances the expression by stabilizing iNOS mRNA (15). Under such conditions, cardiac fibroblasts can
be major producers of NO. In a variety of cell types, NO reportedly alters the activities of AC and phosphodiesterase (PDE), thus functionally linking the cAMP and NO pathways. Such interactions could
be important in cardiac fibroblasts, in which elevated cAMP can
modulate proliferation and collagen production (3, 4, 10).
Accordingly, we have continued our studies of cAMP metabolism in
cardiac fibroblasts by considering whether NO derived from iNOS or from
pharmacologic donors of NO affects the
-adrenergic response. We
report here that NO accumulation in cardiac fibroblasts decreases cAMP
accumulation in response to
-adrenergic stimulation, in large part
by causing the activation of a cGMP-stimulated phosphodiesterase (PDE2).
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MATERIALS AND METHODS |
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Materials.
Rat recombinant IL-1 was purchased from Sigma-Aldrich (St. Louis,
MO). Collagenase I and trypsin were from Worthington (Freehold, NJ).
S-nitroso-N-acetylpenicillamine (SNAP) was
purchased from Alexis Biochemicals (San Diego, CA). A monoclonal
antibody to iNOS was obtained from Transduction Laboratories
(Lexington, KY). [
-32P]ATP and [3H]cAMP
were from Perkin Elmer Lifesciences (Boston, MA). All other reagents
and chemicals were of reagent grade from Sigma-Aldrich or
Calbiochem-Novabiochem (La Jolla, CA).
Isolation of adult ventricular fibroblasts. Rat cardiac fibroblasts were isolated from adult male Sprague-Dawley rats (250-275 g) as recently described (15). Briefly, the hearts were minced and placed in a trypsin/collagenase digestion solution. The digestions were pooled, centrifuged, and resuspended in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and penicillin-streptomycin (100 U/ml). The cells were plated for 30 min, after which unattached cells were rinsed off. All cells used in experiments were from passages 2 through 4. The purity of these cultures was >95% as determined by immunostaining (15).
Western blot analysis.
Fibroblasts (on 60-mm culture dishes) were lysed at 4°C in a buffer
containing 50 mM -glycerolphosphate (pH 7.5 at 4°C), 1 mM EGTA, 10 mM MgCl2, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 10 µg/ml leupeptin. Equal amounts of total protein per lane were loaded and separated on a 7.5%
SDS-polyacrylamide gel and then transferred to an Immobilon-P membrane
(Millipore, Bedford, MA). After being blocked in 5% nonfat milk, the
membrane was incubated with a monoclonal iNOS antibody overnight at
4°C, followed by a series of washes and incubation with a secondary antibody coupled to horseradish peroxidase for 1 h at 20°C. iNOS was detected by using enhanced chemiluminescence (Amersham Life Science).
Measurement of nitrite levels. NO production was estimated by measuring nitrite accumulation in the culture medium. Fibroblasts were plated on 60-mm culture dishes and grown to 80-90% confluency. For experiments, cells were incubated in phenol red-free DMEM supplemented with 1.5 mM L-arginine, 0.1 mg/ml BSA, 10 µg/ml leupeptin, and 100 U/ml penicillin-streptomycin with vehicle or drug added for 24 h. Nitrite in the medium was measured by the method of Griess et al (13).
Assay of cGMP accumulation.
Cells were treated with IL-1 for 24 h; 1 mM
3-isobutyl-1-methylxanthine (IBMX) was added to each plate 45 min
before the assay was terminated. In experiments with a NO donor, the
cells were pretreated for 15 min with 1 mM IBMX and then stimulated with 1 mM sodium nitroprusside (SNP) for 45 min. All experiments were
terminated by aspiration of the medium and addition of ice-cold 5%
trichloroacetic acid (TCA). The TCA extracts were extracted four times
with water-saturated ether, and the cGMP content was determined by RIA
(16). Data are expressed as picomoles of cGMP per
milligram of protein.
Assay of cAMP accumulation.
Cardiac fibroblasts were incubated with DMEM without serum for 2 h
and then treated as described in RESULTS, after which the medium was aspirated and ice-cold 5% TCA was added. The extracts were
purified over Dowex-50, and the cAMP content was determined by the
method of Gilman (12). Data in the figures are expressed as percentages of maximal responses in the particular experimental protocol. Actual mean cyclic contents were as follows: basal, 4 ± 1; isoproterenol (1 µM, 5 min), 48 ± 6; and forskolin (30 µM,
5 min), 193 ± 8 pmol/mg protein. IBMX (1 mM), a nonspecific PDE
inhibitor, increased the effect of isoproterenol 26-fold; the
PDE2-specific inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA; 0.2 mM), doubled the -adrenergic response.
AC assay.
Membranes were prepared by Dounce homogenization of the cells in
buffer A (50 mM HEPES, pH 7.6, 5 mM MgCl2, 1 mM
EGTA, 0.3 mM PMSF, and 10 µg/ml leupeptin). The membranes were
collected by centrifugation at 3000 g for 5 min and
resuspended in buffer A. Membrane protein (40 µg) was
incubated with 1 mM SNP or vehicle on ice for 30 min, and AC activity
was assayed by monitoring the conversion of [-32P]ATP
to [
-32P]cAMP at 30°C for 10 min (24).
Protein determination. Protein content was estimated by the method of Bradford (5) by using BSA as a standard.
Data analysis. Analysis and graphing of data were performed with Prism 2.0 (GraphPad Software, San Diego, CA). Data are expressed as means ± SE. Statistical analysis was performed by ANOVA for multiple group comparison and by Student's t-test for direct comparison of two groups. P values <0.05 were considered significant.
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RESULTS |
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Induction of functional iNOS by IL-1; effect on the
-adrenergic and forskolin responses.
We investigated whether IL-1
treatment results in increased
expression of iNOS by immunoblotting with an antibody against iNOS.
Expression of iNOS was not detected in unstimulated cells; however,
treatment with IL-1
(10 ng/ml) caused an increase in the iNOS
protein expression in a time-dependent manner (Fig.
1, A and B). The
iNOS protein band, molecular mass ~130 kDa, was first detected after
exposure to IL-1
for 6 h, reaching a maximum after 24 h of
treatment. Moreover, treatment for 24 h with IL-1
caused a
concentration-dependent increase in NO production with an
EC50 of 3 ng/ml (~0.17 nM) (Fig. 1C).
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Rationale for subsequent studies.
Having established that exposure to IL-1 causes iNOS induction and
depresses responses to
-adrenergic agonists and forskolin, we
designed experiments to elucidate the mechanism of this effect by
answering the following questions. 1) Is the effect on cAMP accumulation due to iNOS induction and NO production, or is it due to
other effects of IL-1
? 2) Is the effect on an
identifiable component of hormone-sensitive AC? 3)
Are the expected cellular consequences of NO production required for
the depression of cAMP accumulation? If cGMP production is involved,
does it mediate the effect via cGMP-dependent protein kinase (PKG), by
modulating cAMP hydrolysis or by other means?
NO dependence of the IL-1 effect, mimicry by NO donors, and
reversibility.
Exposure to IL-1
(10 ng/ml, 24 h) reduced cAMP accumulation in
response to isoproterenol and forskolin (Fig. 3, A and
B). Inclusion of L-NMMA fully prevented the
effect of IL-1
to reduce these responses (Fig. 3, A and
B). These data indicate that these effects of IL-1
are
dependent on enzymatic NO production and, thus, on the induction of iNOS.
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Effect of IL-1/NO does not localize to hormone-sensitive AC.
IL-1
reduced responses to both isoproterenol and forskolin (Fig. 3,
A and B). Because forskolin directly
activates AC and also activates the
s-AC
complex, we conclude that this NO-dependent inhibition of cAMP
accumulation is not occurring at the level of the
-adrenergic
receptor, on a mechanism regulating receptor refractoriness, or at the
receptor-Gs protein interface.
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Roles of soluble guanylyl cyclase, cGMP, and PKG.
A major target of NO is the soluble guanylyl cyclase (sGC); NO
activates sGC and thereby enhances production of cGMP (9). Treatment of cardiac fibroblasts with IL-1 stimulated NO production (Fig. 2); treatment with either a NO donor or IL-1
enhanced
accumulation of cGMP (Fig. 6, A
and B). The competitive inhibitor of NO synthase, L-NMMA (1 mM) inhibited cGMP accumulation in response to
IL-1
(10 ng/ml, 24 h): control, 39 ± 32;
L-NMMA, 32 ± 25; IL-1
, 641 ± 81; and IL-1
plus L-NMMA, 46 ± 24 pmol cGMP/mg (means ± SE, n = 3; Fig. 6, A and B). These
data support the idea that cGMP produced by IL-1
-treated cells
results from the effect of NO to activate sGC.
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Role of cyclic nucleotide PDEs.
Because the NO/cGMP effect does not appear to require PKG activation,
we turned our attention to the other major mediator of cGMP's effects,
the cyclic nucleotide PDE activities that are modulated by cGMP. To
investigate whether enhanced PDE activity mediates the inhibition of
isoproterenol-stimulated cAMP production, we determined the effects of
PDE inhibitors in fibroblasts treated with either SNP or IL-1.
Treatment of cells with 1 mM SNP for 45 min, in the absence of a PDE
inhibitor, resulted in the expected 50% decrease in cAMP accumulation
in response to isoproterenol (Fig.
8A). Pretreatment of cells
with 1 mM IBMX, a nonspecific PDE inhibitor (Fig. 8B), or
0.2 mM EHNA, a PDE2 specific inhibitor (Fig. 8C), completely
restored cAMP accumulation. We also investigated whether specific
inhibitors of other PDE isoforms (types 1, 3, and 4) could restore the
attenuated cAMP accumulation in response to isoproterenol. However,
neither 25 µM vinpocetine (PDE1) nor 10 µM milrinone (PDE3) or 10 µM rolipram (PDE4) restored cAMP accumulation in SNP-treated cells,
verifying that the NO/cGMP response reflects the altered activity of an
EHNA-sensitive isoform, PDE2, specifically.
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DISCUSSION |
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We have examined the effect of NO production on the -adrenergic
response in cardiac fibroblasts. Our data show that induction of iNOS
by IL-1
treatment attenuates the increase in intracellular cAMP in
response to isoproterenol or forskolin. This is similar to some
previous reports. For instance, Chung et al. (7) reported that isoproterenol-stimulated cAMP accumulation in rat ventricular myocytes was inhibited by conditioned medium containing cytokines from
activated immune cells. However, these workers also reported that the
effect was not apparent when the cells were stimulated with forskolin,
suggesting that the effect of NO occurred at the level of the
-adrenergic receptor or G protein coupling in the myocytes
(7). Balligand et al. (2) reported that
incubation of adult rat cardiac myocytes with macrophage-conditioned
medium was accompanied by induction of iNOS, an increase in NO
production, and a depression of the contractile response to
-adrenergic stimulation. Joe et al. (19) reported a
decrease in the
response but not in the forskolin response (cAMP
accumulation) in rat cardiac myocytes expressing iNOS, suggesting that
some of the effect of NO was occurring proximal to AC.
Our data in cardiac fibroblasts are similar to the extent that we
observed that IL-1 induces iNOS and causes NO production, resulting
in a decrease in isoproterenol-stimulated cAMP accumulation. In
contrast to the case in myocytes (7, 19), we found that forskolin-stimulated cAMP accumulation is also attenuated in
IL-1
-treated fibroblasts, suggesting that the
-adrenergic
receptor and Gs are not the targets of NO in these cells.
If data on myocytes prove to be correct, then this is another way in
which cardiac myocytes and fibroblasts differ (22). Our
data also indicate that activation of the Gi pathway
(defined ± PTx) is not the mechanism by which NO exerts its
effect on cAMP accumulation.
There is no mechanism proposed that accounts for the effect of NO on
-adrenergic-Gs coupling reported in myocytes (7, 19). There is, however, a developing literature on effects of NO
on AC. Tao et al. (26) demonstrated that NO can inhibit
both hormone- and forskolin-stimulated AC activity in N18TG2-cultured neuroblastoma cells. Specifically, NO seems to selectively inhibit AC5
and AC6 with no effect on isoforms 1 or 2 (17). We found that pharmacologically produced NO caused a small (12%) but
significant reduction in forskolin-stimulated AC activity in membranes
isolated from cardiac fibroblasts. This inhibition is insufficient to
fully account for the 50% decrease in responses to forskolin or
isoproterenol that we observe in intact fibroblasts. In addition, we
did not detect this inhibition of AC activity by NO in intact cardiac fibroblasts in the presence of a PDE inhibitor. This difference can be
explained by several factors. The release of NO from NO donors can be
influenced by a number of variables, including the incubation medium,
light, temperature, and pH. For example, the differences in medium or
the temperature at which experiments were performed could be factors in
the different effects of NO on AC activity in whole cells vs.
membranes. Preliminary RT-PCR and Western analysis indicate expression
of AC2, 3, 4, 5, 6, and 7 in the fibroblasts (unpublished observation).
Even though the NO-sensitive isoforms (5 and 6) are present in the
fibroblasts, our data (little effect of NO on hormone-sensitive AC
activity) suggest that these two forms are a small component of the
total AC activity. Thus the major functional isoforms of AC present in
cardiac fibroblasts are likely to be NO insensitive.
One target of cGMP is the cGMP-stimulated PDE (PDE2), to which binding
of cGMP causes activation, resulting in a decrease in cAMP content in
cells (20). Not all workers find that this pathway is
involved in cytokine action. Chung et al. (7) reported that cytokine-induced inhibition of cAMP accumulation in cardiac myocytes was not altered by IBMX. Joe et al. (19) reported
that IBMX only partly reversed the attenuation of the -adrenergic response in cardiac myocytes expressing iNOS. Our data are very clear:
the NO-induced decrease in cAMP accumulation in response to
isoproterenol or forskolin reflects enhanced degradation of cAMP in
cardiac fibroblasts. The mechanism that we propose is shown in Fig.
10: IL-1
induces iNOS; iNOS makes
NO, which activates the sGC; and the resultant cGMP activates PDE2,
thereby reducing cAMP accumulation.
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In nitroprusside-treated cells, the same scheme would apply, with
the NO activating sGC with the same sequelae. Again, the evidence is
similar: preincubation with IBMX or EHNA completely restored
isoproterenol-stimulated cAMP accumulation to control levels,
suggesting that the effect of NO is attributable to PDE2 activation by
cGMP. However, in cells treated with IL-1 to induce iNOS, IBMX or
EHNA only partially restored cAMP accumulation to control levels: PDE
inhibitors reduced the NO effect from ~55 to 15% inhibition of
maximal isoproterenol-stimulated cAMP accumulation. These results are
also in agreement with the data obtained from experiments where
guanylyl cyclase was inhibited by ODQ: inhibiting cGMP production
completely restored isoproterenol-stimulated cAMP accumulation in
SNP-treated cells and largely, but not completely, restored
isoproterenol-stimulated cAMP accumulation in IL-1
treated cells.
These results suggest that some of the effect of iNOS induction is not
dependent on acute cGMP production and PDE2 activation. The main
difference between the two experimental protocols is duration of NO
exposure (acute vs. iNOS induction over 24 h). In experiments with
SNP, the cells were exposed to the NO donor for 45 min before
isoproterenol stimulation; in IL-1
stimulated cells, the cells were
exposed to NO production for an extended period of time. We detected
significant iNOS protein after 6 h of IL-1
treatment; thus when
we stimulated the cells with isoproterenol after 24 h of IL-1
treatment, the cells had been exposed to increasing levels of NO
production by iNOS for at least 18 h. It is clear that the effects
on cAMP accumulation by IL-1
treatment are due to an increase in NO
production and not to some other effect of IL-1
, because the cAMP
accumulation can be completely restored by the inclusion of an NOS
inhibitor. Nevertheless, the prolonged exposure to NO may cause
transcriptional changes in the cells, possibly due to the activation of
PKG (9). Activation of PKG may induce proteins that
influence second messenger generation. It is also possible that chronic
NO and cGMP production could result in downregulation of portions of
the response pathway, such as the
-adrenergic receptor,
Gs, or AC. Thus the acute effects of NO production on cAMP
accumulation are through the activation of PDE2, whereas chronic NO and
cGMP production also increase PDE2 activity but may also bring other
factors into play that also reduce the
-adrenergic response to a
lesser extent.
In conclusion, we have demonstrated that cardiac fibroblasts can produce large quantities of NO in response to immune activation and the consequent induction of iNOS. The resulting NO depresses cAMP accumulation within the cardiac fibroblast. The mechanism of this effect is not via an inhibition of AC by NO or via stimulation of PKG by the resultant cGMP. Rather, the effect is via stimulation of sGC by NO and stimulation of PDE2 by the elevated cGMP.
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ACKNOWLEDGEMENTS |
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This work was supported by grants from the University of California San Diego Academic Senate and the California Tobacco-Related Diseases Research Program and by a predoctoral fellowship to Å. B. Gustaffson from the American Heart Association, Western States Affiliates.
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FOOTNOTES |
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Address for reprint requests and other correspondence: L. L. Brunton, Dept. of Pharmacology, 0636, Univ. of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093 (E-mail: lbrunton{at}ucsd.edu).
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.
10.1152/ajpcell.00299.2001
Received 2 July 2001; accepted in final form 22 January 2002.
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