Cardiovascular Research Center and Arthritis Unit, Massachusetts General Hospital, and Departments of Medicine and Anesthesia, Harvard Medical School, Charlestown, Massachusetts 02129; and Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, and Cardiac Unit, University Hospital Gasthuisberg, University of Leuven, B-3000 Leuven, Belgium
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ABSTRACT |
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Exposure of rat
pulmonary artery smooth muscle cells (rPASMC) to cytokines leads to
nitric oxide (NO) production by NO synthase 2 (NOS2). NO stimulates
cGMP synthesis by soluble guanylate cyclase (sGC), a heterodimer
composed of 1- and
1-subunits. Prolonged exposure of rPASMC to NO decreases sGC subunit mRNA and protein levels.
The objective of this study was to determine whether levels of NO
produced endogenously by NOS2 are sufficient to decrease sGC expression
in rPASMC. Interleukin-1
(IL-1
) and tumor necrosis factor-
(TNF-
) increased NOS2 mRNA levels and decreased sGC subunit mRNA
levels. Exposure of rPASMC to IL-1
and TNF-
for 24 h
decreased sGC subunit protein levels and NO-stimulated sGC enzyme
activity.
L-N6-(1-iminoethyl)lysine
(NOS2 inhibitor) or
1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (sGC inhibitor) partially prevented the cytokine-mediated decrease in
sGC subunit mRNA levels. However, cytokines also decreased sGC subunit
mRNA levels in PASMC derived from NOS2-deficient mice. These results
demonstrate that levels of NO and cGMP produced in cytokine-exposed
PASMC are sufficient to decrease sGC subunit mRNA levels. In addition,
cytokines can decrease sGC subunit mRNA levels via NO-independent mechanisms.
interleukin-1; tumor necrosis factor-
; nitric oxide; guanosine 3',5'-cyclic monophosphate
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INTRODUCTION |
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NITRIC OXIDE (NO) regulates many vascular smooth muscle cell functions, including vascular tone, as well as cellular proliferation, apoptosis, migration, and synthesis of extracellular matrix. NO is synthesized from oxygen and L-arginine by a family of NO synthases (NOSs) (12). Two NOS isoforms initially cloned from brain (NOS1) and endothelium (NOS3) are expressed in a variety of cell types and are activated by increases in intracellular calcium. A third NOS isoform, NOS2 or inducible NOS, is typically found in cells exposed to cytokines or lipopolysaccharide and produces high levels of NO in a calcium-independent manner. The NOS2 gene is highly expressed in inflammatory states and contributes to the hemodynamic sequelae of sepsis (20).
NO acts in part by stimulating soluble guanylate cyclase (sGC) to
produce the intracellular second messenger cGMP. In vascular smooth
muscle cells, sGC is a heterodimer composed of 1- and
1-subunits (11). cGMP in turn interacts
with a variety of intracellular targets, including cGMP-dependent
protein kinase, leading to vasorelaxation and modulation of other
vascular cell functions. The action of cGMP is limited by conversion to
GMP by phosphodiesterases.
Although the mechanisms regulating production of NO have been
extensively characterized, the mechanisms controlling responsiveness to
NO are less completely understood. In prior studies, we and others
observed that prolonged incubation of cells with NO donor compounds
decreased sGC expression (3, 14, 18, 21). The objective of
this study was to test the hypothesis that levels of NO produced
endogenously by NOS2 are sufficient to impair sGC function in vascular
smooth muscle cells. To induce NOS2 and increase endogenous NO levels,
vascular smooth muscle cells were exposed to the proinflammatory
cytokines interleukin-1 (IL-1
) and tumor necrosis factor-
(TNF-
). We report here that cytokines decreased sGC subunit mRNA and
protein levels as well as NO-stimulated sGC enzyme activity. Inhibitors
of NOS and sGC partially blocked the effect of cytokines on sGC subunit
gene expression, suggesting a role for endogenously produced NO and
cGMP in the regulation of sGC function. However, the incomplete effect
of NOS and sGC inhibitors on the cytokine-induced decrease in sGC
subunit mRNA levels, as well as observations in pulmonary artery smooth
muscle cells (PASMC) from NOS2-deficient mice, suggests that a
NO-independent mechanism also participates in the modulation of sGC
subunit gene expression by cytokines.
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METHODS |
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This study was approved by the Committee for Research Animal Studies at Massachusetts General Hospital.
Reagents.
IL-1 and TNF-
were purchased from Genzyme (Cambridge, MA) and
from R&D Systems (Minneapolis, MN).
L-N6-(1-iminoethyl)lysine
(L-NIL),
1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), and actinomycin D were obtained from Calbiochem-Novabiochem (La
Jolla, CA). Sodium nitroprusside (SNP) and
NG-nitro-L-arginine methyl ester
(L-NAME) were purchased from Sigma Chemical (St. Louis, MO).
Cell culture experiments. Cultures of rat PASMC (rPASMC) were prepared from explants of endothelium- and adventitia-stripped pulmonary arteries of adult Sprague-Dawley rats, as described by Yu et al. (22). Cells were maintained in RPMI 1640 medium with 10% NuSerum (Collaborative Biomedical Products, Bedford, MA), penicillin, and streptomycin. The cells were used between passages 3 and 10.
With use of similar methods, cultures of murine PASMC (mPASMC) were prepared from NOS2-deficient mice. These mice were generously provided by Dr. Carl Nathan (Cornell University).RNA blot hybridization.
RNA was extracted from cultured rPASMC using the guanidine
isothiocyanate-cesium chloride method (16). In some
studies, RNA was extracted by the acid guanidinium-phenol-chloroform
technique using TRIzol Reagent (Life Technologies, Gaithersburg, MD)
(2). Ten micrograms of total cellular RNA were
fractionated in 1.3% agarose-formaldehyde gels containing ethidium
bromide, transferred to nylon membranes (MAGNA CHARGE; Micron
Separations, Westborough, MA), and cross-linked under ultraviolet
light. The membranes containing RNA from rPASMC were hybridized with
32P-radiolabeled rat sGC 1- and
1-subunit cDNA probes, washed, and exposed to X-ray
films as previously described (1, 3, 8). cDNAs encoding
sGC
1- and
1-subunits were kindly
provided by Dr. M. Nakane (Abbott Laboratories, Abbott Park, IL). For
some experiments, the membranes were also hybridized with a
radiolabeled 0.3-kb XbaI-PstI restriction
fragment containing exon 24 of the rat NOS2 gene (Genbank accession no.
AJ230484) (6).
Immunoblotting.
rPASMC were homogenized in buffer containing 50 mM Tris · HCl
(pH 7.6), 1 mM EDTA, 1 mM dithiothreitol, and 2 mM phenylmethylsulfonyl fluoride. Cell supernatants containing 30 µg of protein were
fractionated by 8% SDS-PAGE, transferred to nitrocellulose filters
(Micron Separations), and incubated with a subunit-specific polyclonal antibody directed either against the rat sGC 1-subunit
(generated as described below) or against the rat sGC
1-subunit (3). Bound antibodies were
detected using horseradish peroxidase-protein A (Boehringer Mannheim)
and enhanced chemiluminescence (Amersham Life Sciences).
sGC enzyme activity. sGC enzyme activity was measured using methods adapted from Mittal (10), as previously described (3, 8). Briefly, cell extracts containing 30 µg of protein were incubated in a reaction mixture including 1 mM GTP with and without 1 mM SNP. The reaction was terminated by addition of HCl and boiling, and newly synthesized cGMP in the reaction mixture was measured using a commercial radioimmunoassay kit (Biomedical Technologies). The enzyme activity is expressed as picomoles of cGMP produced per minute per milligram protein in the cell extracts.
Nitrite measurements. rPASMC were plated in 24-well dishes (105 cells/well). On the following day, cells were washed twice in DMEM without phenol red with 10% NuSerum and then incubated in the same medium with and without cytokines in the presence and absence of L-NIL. Samples of culture medium were harvested after 8 h, and nitrite concentrations were measured using the Griess reaction and a standard curve using sodium nitrite as previously described (5). Nitrite production is expressed as nanomoles nitrite produced per hour per 105 cells.
Statistics. sGC enzyme activities and nitrite production rates were compared by a factorial model of analysis of variance. When significant differences were detected, Fisher's analysis was used post hoc to compare groups. The differences were considered significant if P < 0.05.
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RESULTS |
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Cytokines decreased sGC subunit mRNA
levels in rPASMC.
To investigate the effects of cytokines on sGC subunit gene expression,
rPASMC were incubated with IL-1 and TNF-
or with IL-1
alone
for up to 24 h, and the levels of sGC subunit mRNAs were
determined by RNA blot hybridization. Exposure of rPASMC to the
combination of IL-1
(20 ng/ml) and TNF-
(100 ng/ml) decreased sGC
1- and
1-subunit mRNA levels in a
time-dependent fashion (Fig. 1). The
combination of cytokines decreased sGC subunit mRNA levels beginning at
4 h, with minimum mRNA levels achieved at 8 h. Levels of sGC
subunit mRNAs remained depressed for at least 24 h after exposure.
Similar time-dependent decreases in sGC subunit mRNAs were
observed when rPASMC were incubated with IL-1
(20 ng/ml) alone (Fig.
1). Incubation of rPASMC with TNF-
(100 ng/ml) alone decreased sGC
1- and
1-subunit mRNA levels in a
time-dependent manner, with minimum mRNA levels achieved at 16 h
(data not shown). sGC subunit mRNA levels were consistently less in
cells incubated with the combination of IL-1
and TNF-
for 8 h than in cells incubated with either cytokine alone for 8 h.
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Cytokines decreased sGC subunit protein levels and
enzyme activity in rPASMC.
To investigate whether the cytokine-mediated decrease in sGC subunit
mRNA levels was associated with decreases in subunit protein
expression, rPASMC were incubated with the combination of IL-1 and
TNF-
for 24 h, and sGC subunit protein levels were measured
using immunoblot techniques. Exposure of rPASMC to the cytokine
combination decreased both
1- (82 kDa) and
1- (70 kDa) subunit protein levels (Fig.
2).
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Cytokines decreased stability of sGC subunit
mRNAs in rPASMC via a
transcription-dependent mechanism.
To investigate whether changes in gene transcription or mRNA stability
contribute to the cytokine-induced decrease in sGC subunit mRNA levels,
rPASMC were incubated in the presence and absence of cytokines with and
without the transcription inhibitor actinomycin D. Incubation of rPASMC
with actinomycin D (1 µM) blocked gene transcription as indicated by
the rapid decrease in c-jun mRNA levels (data not shown).
Incubation of rPASMC with actinomycin D for 8 h decreased sGC
1- and
1-subunit mRNA levels (Fig.
4). Levels of sGC subunit mRNAs were
greater in rPASMC incubated with actinomycin D alone than in rPASMC
incubated for 8 h with the combination of IL-1
and TNF-
.
Because sGC subunit mRNA levels declined more rapidly in
cytokine-treated cells than in cells in which transcription was
inhibited, it is likely that cytokines act, at least in part, by
decreasing sGC subunit mRNA stability. However, sGC subunit mRNA levels
did not differ in rPASMC exposed to actinomycin D alone compared with
rPASMC exposed to cytokines in the presence of actinomycin D. These
findings suggest that the cytokine-induced destabilization of sGC
subunit mRNAs is dependent on ongoing gene transcription.
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Cytokines decreased sGC subunit mRNA
levels in rPASMC via a NO-dependent mechanism.
Exposure of rPASMC to the combination of IL-1 and TNF-
increased
NOS2 mRNA levels in a time-dependent fashion (see Fig. 1). The time of
onset and the duration of the induction of NOS2 gene expression were
similar to the time of onset and the duration of the decrease in sGC
subunit mRNA levels. Because prolonged incubation of rPASMC with NO
donor compounds has been observed to decrease sGC subunit mRNA levels
(3, 18), we investigated whether the cytokine-induced
decrease in sGC subunit mRNAs requires increased NO production by NOS2.
rPASMC were preincubated for 30 min with and without L-NIL
(0.5 or 1 mM), a selective NOS2 inhibitor, and then exposed to IL-1
(20 ng/ml) and TNF-
(100 ng/ml) for 8 h. Exposure of rPASMC to
the cytokines led to accumulation of nitrite in the culture medium, an
effect that was blocked by incubation with L-NIL (Fig.
5). Preincubation of the cells with L-NIL attenuated the decrease in sGC subunit mRNAs produced
by the cytokines (Fig. 6). Similarly,
L-NAME (10 mM), a nonselective NOS inhibitor, completely
inhibited nitrite release and partially prevented the cytokine-induced
decreases in sGC subunit mRNA levels (data not shown). These results
demonstrate that IL-1
and TNF-
decrease sGC subunit mRNA levels
in part via a NO-dependent mechanism.
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Cytokines decreased sGC subunit mRNA
levels in PASMC from NOS2-deficient mice.
The observations that NOS and sGC inhibitors only partially blocked the
ability of cytokines to decrease sGC subunit mRNA levels suggested that
the cytokine-induced decrease in sGC subunit mRNA levels was also in
part mediated via a NOS2-independent mechanism. To further investigate
this possibility, smooth muscle cells were cultured from the pulmonary
arteries of mice deficient in NOS2 (9). mPASMC were
incubated with IL-1 (20 ng/ml) and TNF-
(100 ng/ml) for 8 h,
and changes in sGC
1-subunit mRNA levels were
quantitated by RNA blot hybridization (Fig.
8). Incubation of mPASMC with IL-1
and
TNF-
decreased sGC
1-subunit mRNA levels. Preincubation with ODQ did not prevent the cytokine-induced decrease in
sGC subunit mRNA levels. These results support the hypothesis that
cytokines can decrease sGC subunit mRNA levels via NO- and cGMP-independent mechanisms.
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DISCUSSION |
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The activity of sGC determines the quantity of cGMP synthesized in
response to NO and influences the magnitude of the response to this
vasodilator. Understanding the mechanisms responsible for regulation of
sGC function is likely to provide important insights into how
responsiveness to NO is determined. In this report, we observed that
exposure of rPASMC to cytokines induced NOS2 gene expression and
decreased sGC 1- and
1-subunit mRNA levels. Decreased sGC subunit mRNA levels were accompanied by decreased
1- and
1-subunit protein concentrations
and decreased NO-stimulated sGC enzyme activity. It is of interest that
in extracts prepared from rPASMC exposed to cytokines for 24 h,
the ability of NO to induce cGMP synthesis was completely inhibited,
whereas sGC subunit protein levels remained detectable, although at
diminished levels. These observations suggest that cytokines may impair
sGC enzyme-specific activity as well as decrease sGC subunit protein levels.
A variety of stimuli have been reported to modulate sGC function in
vascular smooth muscle cells as well as in other cell types. Agents
that increase intracellular cAMP concentrations decrease sGC subunit
mRNA levels and enzyme function in rat fetal lung fibroblasts
(19), aortic smooth muscle cells (15), and pheochromocytoma (PC12) cells (8). Similarly, nerve growth factor (NGF) decreases sGC 1- and
1-subunit mRNA and protein levels as well as enzyme
activity in PC12 cells via a Ras-dependent mechanism (8).
In prior studies, we (3) and others (14, 18)
observed that incubation of vascular smooth muscle cells with agents
that elevate intracellular cGMP, most notably NO donor compounds,
decreased sGC subunit mRNA and protein levels as well as enzyme
activity. However, it was uncertain whether endogenous production of NO
and cGMP was sufficient to decrease sGC subunit mRNA levels in cultured
vascular smooth muscle cells. In the current study, we observed that
agents that inhibit NOS2 (L-NAME and L-NIL) or
that inhibit sGC (ODQ) were able to attenuate the decrease in sGC
subunit mRNA levels in cytokine-treated rPASMC. These results demonstrate that levels of NO and cGMP produced endogenously by vascular smooth muscle cells are sufficient to modulate sGC subunit gene expression.
However, we also observed that concentrations of L-NAME and
L-NIL that blocked NO synthesis (measured as nitrite
release into the culture medium) were insufficient to completely
prevent the decrease in sGC subunit mRNA levels. Similarly,
concentrations of ODQ sufficient to block cGMP release into the
perfusate (in response to NO production by NOS2; data not shown) did
not completely prevent cytokine-induced decrease in sGC subunit mRNA
levels. To confirm that modulation of sGC subunit gene expression by
cytokines was in part independent of the cytokine-induced NOS2
expression, PASMC were prepared from NOS2-deficient mice. Incubation of
NOS2-deficient mPASMC with cytokines decreased sGC
1-subunit mRNA levels. These results suggest that
decreases in sGC subunit gene expression in cytokine-treated vascular
smooth muscle cells can occur in the absence of NOS2 induction. The NO-
and cGMP-independent mechanisms by which cytokines decrease sGC subunit
mRNA levels in vascular smooth muscle cells remain to be elucidated.
In prior studies, we reported that exposure of rPASMC to NO donor
compounds destabilized mRNAs encoding the sGC 1- and
1-subunits via a mechanism that is transcription
dependent (3). sGC subunit mRNAs were similarly
destabilized in NGF-treated PC12 cells (8). In this
report, we noted that sGC subunit mRNA levels decreased more rapidly in
rPASMC exposed to cytokines than in rPASMC in which transcription was
terminated with actinomycin D. These findings suggest that cytokines
decreased sGC subunit mRNA stability. Moreover, pretreatment with
actinomycin D prevented the cytokine-induced decrease in sGC subunit
mRNA levels, suggesting that cytokine-induced sGC subunit mRNA
destabilization was transcription dependent. We considered the
possibility that actinomycin D blocked sGC subunit mRNA destabilization
by inhibiting induction of NOS2 and NO production. However, we observed
that actinomycin D blocked sGC
1-subunit mRNA
destabilization in cytokine-treated NOS2-deficient mPASMC (data not
shown). The observation that three different types of signals (NO, NGF,
and cytokines) modulate sGC subunit mRNA levels at the level of mRNA
stability suggests that shared mechanisms may be responsible for
regulating sGC subunit mRNA levels in response to a variety of stimuli.
Other investigators have reported that incubation of vascular smooth
muscle cells with inflammatory stimuli decreased sGC enzyme activity
(13, 17). Papapetropoulos et al. (13)
observed that incubation of rat aortic smooth muscle cells with IL-1
(10 U/ml) or lipopolysaccharide (1 µg/ml) decreased sGC
1-subunit mRNA levels without altering
1-subunit protein levels. Similarly, Scott and Nakayama
(17) reported that exposure of rPASMC to lipopolysaccharide (1-200 µg/ml) for 24 h decreased sGC
1-subunit mRNA levels and that this decrease in sGC
subunit mRNA levels was blocked by pretreatment with a NOS inhibitor.
In the latter report, lipopolysaccharide decreased
1-subunit protein levels but not
1-subunit protein levels. These reports differ from our findings that both
1- and
1-subunits are
decreased in PASMC exposed to IL-1
and TNF-
. Differences in the
observations reported previously and our findings may be attributable
to variations in the type and dose of inflammatory mediators used.
Several groups have reported recently that alterations in sGC levels modulate the ability of NO to dilate preconstricted vascular rings. Fullerton and colleagues (4) proposed that sGC function was impaired in pulmonary arteries from rats exposed to lipopolysaccharide, with decreased vasodilation in response to an NO donor compound but preserved vasodilation in response to 8-bromo-cGMP. Kloss et al. (7) observed that SNP-induced relaxation was less in aortic rings from 16-mo-old spontaneously hypertensive rats compared with rings from normotensive Wistar-Kyoto rats and attributed this impairment to decreased vascular sGC subunit mRNA and protein levels.
Extrapolation of our findings in cultured vascular smooth muscle cells to the intact animal suggests that the vasculature when exposed to inflammatory mediators can adapt to elevated NO levels by decreasing one of the NO receptors sGC. Cytokine-mediated changes in the function of other components of the NO-cGMP signal transduction system, such as phosphodiesterases and cGMP-dependent protein kinase, may also have important roles in regulating vascular responsiveness to NO. Cytokine-induced desensitization of vascular responsiveness to NO may represent a homeostatic mechanism preventing excessive signaling via cGMP and may serve to limit the vasodilation associated with sepsis.
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ACKNOWLEDGEMENTS |
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We thank Dr. R. Ullrich for assistance in the preparation of mouse PASMC and Dr. W. M. Zapol for advice and support.
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FOOTNOTES |
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* M. Takata and G. Filippov contributed equally to this work.
This work was supported by National Heart, Lung, and Blood Institute Grants HL-42397 (M. Takata and F. Ichinose), T32-HL-07208 (G. Filippov), and HL-55377 (K. D. Bloch). H. Liu is a recipient of a Physician Scientist Development Award from the American Heart Association. S. Janssens is a Clinical Investigator of the National Science Foundation, Belgium, and holder of a chair financed by Zeneca. K. D. Bloch is an Established Investigator of the American Heart Association.
Address for reprint requests and other correspondence: K. D. Bloch, Cardiovascular Research Center, Massachusetts General Hospital-East, 149 13th St., Charlestown, MA 02129 (E-mail: blochk{at}helix.mgh.harvard.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.
Received 20 May 2000; accepted in final form 1 September 2000.
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