Cytokines decrease sGC in pulmonary artery smooth muscle cells via NO-dependent and NO-independent mechanisms

Masao Takata*, Galina Filippov*, Heling Liu, Fumito Ichinose, Stefan Janssens, Donald B. Bloch, and Kenneth D. Bloch

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


    ABSTRACT
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INTRODUCTION
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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 alpha 1- and beta 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-1beta (IL-1beta ) and tumor necrosis factor-alpha (TNF-alpha ) increased NOS2 mRNA levels and decreased sGC subunit mRNA levels. Exposure of rPASMC to IL-1beta and TNF-alpha 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-1beta ; tumor necrosis factor-alpha ; nitric oxide; guanosine 3',5'-cyclic monophosphate


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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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 alpha 1- and beta 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-1beta (IL-1beta ) and tumor necrosis factor-alpha (TNF-alpha ). 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.


    METHODS
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This study was approved by the Committee for Research Animal Studies at Massachusetts General Hospital.

Reagents. IL-1beta and TNF-alpha 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 alpha 1- and beta 1-subunit cDNA probes, washed, and exposed to X-ray films as previously described (1, 3, 8). cDNAs encoding sGC alpha 1- and beta 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).

Blots containing RNA extracted from mPASMC were hybridized with a 0.8-kb EcoRI-HindIII restriction fragment of an expressed sequence tag (EST) clone encoding the mouse sGC alpha 1-subunit cDNA (accession no. AI592293). This cDNA was identified by screening the nonredundant database of GenBank + European Molecular Biology Laboratory (EMBL) + DNA Data Bank of Japan (DDBJ) EST divisions of the National Center for Biotechnology Information using the sequence of the rat sGC alpha 1-subunit cDNA (accession no. U60835). cDNA sequences in accession no. AI592293 were 87% homologous to sequences in the 3'-untranslated region of the sGC alpha 1-subunit cDNA. No significant homology was detected between this EST clone and other sequences in the database of nonredundant GenBank + EMBL + DDBJ + Protein Data Base (PDB) sequences. The EST clone was obtained from the IMAGE Consortium (Genome Systems, St. Louis, MO), and its nucleotide sequence was confirmed.

Equal loading of RNA on gels was confirmed by staining 28S and 18S ribosomal RNA with ethidium bromide. All RNA blots shown are representative of at least three experiments.

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 alpha 1-subunit (generated as described below) or against the rat sGC beta 1-subunit (3). Bound antibodies were detected using horseradish peroxidase-protein A (Boehringer Mannheim) and enhanced chemiluminescence (Amersham Life Sciences).

To prepare an antiserum directed against the sGC alpha 1-subunit, a XbaI-NsiI restriction fragment of the rat alpha 1-subunit cDNA was ligated into the prokaryotic expression plasmid pMAL-c2 (New England Biolabs, Beverly, MA), and the plasmid was used to transform Escherichia coli. A fusion protein containing maltose-binding protein and amino acids 1-632 of the rat sGC alpha 1-subunit was purified by affinity chromatography. Rabbit polyclonal antiserum directed against the fusion protein was generated as described previously (3).

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.


    RESULTS
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INTRODUCTION
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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-1beta and TNF-alpha or with IL-1beta 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-1beta (20 ng/ml) and TNF-alpha (100 ng/ml) decreased sGC alpha 1- and beta 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-1beta (20 ng/ml) alone (Fig. 1). Incubation of rPASMC with TNF-alpha (100 ng/ml) alone decreased sGC alpha 1- and beta 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-1beta and TNF-alpha for 8 h than in cells incubated with either cytokine alone for 8 h.


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Fig. 1.   Modulation of nitric oxide synthase 2 (NOS2) and soluble guanylate cyclase (sGC) subunit mRNA levels in rat pulmonary artery smooth muscle cells (rPASMC) incubated with cytokines. RNA was extracted from untreated rPASMC (C) and from rPASMC incubated with interleukin-1beta (IL-1beta ) or the combination of IL-1beta and tumor necrosis factor-alpha (TNF-alpha ) for 4, 8, 16, and 24 h. RNA was fractionated in agarose gels containing ethidium, transferred to membranes, and hybridized with radiolabeled sGC subunit cDNA probes as well as a radiolabeled genomic fragment of the rat NOS2 gene. A photograph of 28S ribosomal RNA in the ethidium-containing agarose gel is shown to confirm equal loading of RNA samples. Incubation of rPASMC with IL-1beta or the combination of IL-1beta and TNF-alpha decreased sGC subunit mRNA levels and increased NOS2 mRNA levels.

NOS2 mRNA was not detected in rPASMC that were not exposed to cytokines. Exposure of rPASMC to the combination of IL-1beta and TNF-alpha increased NOS2 mRNA levels within 4 h, with maximum levels evident at 8 h. In rPASMC exposed to IL-1beta alone, NOS2 mRNA appeared later and maximum levels were less than in cells exposed to the combination of cytokines (Fig. 1).

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-1beta and TNF-alpha for 24 h, and sGC subunit protein levels were measured using immunoblot techniques. Exposure of rPASMC to the cytokine combination decreased both alpha 1- (82 kDa) and beta 1- (70 kDa) subunit protein levels (Fig. 2).


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Fig. 2.   sGC subunit protein levels in rPASMC exposed to cytokines. Protein was extracted from untreated rPASMC and rPASMC exposed to IL-1beta and TNF-alpha for 24 h (in triplicate), fractionated using SDS-PAGE, and transferred to nitrocellulose membranes. Immunoreactive alpha 1-subunit (82-kDa) and beta 1-subunit (70-kDa) proteins were detected using sGC subunit-selective polyclonal antisera. Incubation of rPASMC with cytokines decreased sGC alpha 1- and beta 1-subunit protein levels. (In the bottom panel, the band above the beta 1-subunit represents residual antibody bound to the alpha 1-subunit.)

To determine whether cytokine-induced changes in sGC subunit protein levels correlated with changes in enzyme function, basal and NO-stimulated sGC enzyme activity were measured in cell extracts from rPASMC exposed to the combination of IL-1beta and TNF-alpha for 24 h (Fig. 3). In untreated rPASMC, basal sGC enzyme activity was low (15 ± 7 pmol cGMP · min-1 · mg protein-1) and increased 15-fold in the presence of SNP. In rPASMC incubated with the cytokines for 24 h, basal sGC enzyme activity did not differ from that in untreated rPASMC (19 ± 10 pmol cGMP · min-1 · mg protein-1), but incubation with SNP failed to augment sGC enzyme activity. These results demonstrate that exposure of rPASMC to IL-1beta and TNF-alpha for 24 h decreases sGC subunit protein levels as well as the ability of the holoenzyme to augment cGMP synthesis in response to NO.


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Fig. 3.   sGC enzyme activity in rPASMC exposed to cytokines. Extracts prepared from untreated rPASMC (control) and rPASMC exposed to IL-1beta and TNF-alpha for 24 h were assayed (in triplicate) for sGC enzyme activity in the absence (basal sGC enzyme activity) and presence of sodium nitroprusside (SNP, 1 mM) [nitric oxide (NO)-stimulated sGC enzyme activity]. sGC enzyme activity is expressed as picomoles of cGMP produced per minute per milligram protein in the soluble cell extract (means ± SE). *P < 0.05 vs. NO-stimulated activity in extracts from cytokine-treated cells and vs. basal enzyme activity in extracts from control or cytokine-exposed cells. NO-stimulated sGC enzyme activity was decreased in rPASMC exposed to cytokines for 24 h.

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 alpha 1- and beta 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-1beta and TNF-alpha . 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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Fig. 4.   Cytokines decreased sGC subunit mRNA stability. rPASMC pretreated with or without actinomycin D (AD) for 30 min were incubated in the absence (-) and presence (+) of the combination of IL-1beta and TNF-alpha for 8 h. RNA was extracted, fractionated on agarose gels containing ethidium, transferred to nylon membranes, and hybridized with radiolabeled sGC subunit cDNA probes. Ethidium bromide staining of 28S rRNA in the agarose gel confirmed equal loading of RNA samples. sGC subunit mRNA levels decreased more rapidly in cytokine-treated rPASMC than in rPASMC in which transcription was terminated with actinomycin D, suggesting that cytokines decrease sGC subunit mRNA stability. The cytokine-induced decrease in sGC subunit mRNA levels was prevented by pretreatment with actinomycin D.

Cytokines decreased sGC subunit mRNA levels in rPASMC via a NO-dependent mechanism. Exposure of rPASMC to the combination of IL-1beta and TNF-alpha 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-1beta (20 ng/ml) and TNF-alpha (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-1beta and TNF-alpha decrease sGC subunit mRNA levels in part via a NO-dependent mechanism.


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Fig. 5.   L-N6-(1-iminoethyl)lysine (L-NIL) blocked nitrite release by rPASMC incubated with cytokines. rPASMC (105 cells/well of a 24-well plate) were incubated for 8 h in 1 ml of phenol-free medium without (control) and with IL-1beta and TNF-alpha in the presence and absence of L-NIL (1 mM). Nitrite release was measured using the Greiss reaction and is expressed as nanomoles of nitrite released per hour per 105 cells (means ± SE). *P < 0.001 vs. untreated rPASMC, L-NIL-treated rPASMC, and rPASMC exposed to cytokines in the presence of L-NIL. L-NIL blocked nitrite release by rPASMC exposed to cytokines.



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Fig. 6.   Attenuation of the cytokine-induced decrease in sGC subunit mRNA levels by NOS2 inhibition. rPASMC pretreated with and without L-NIL (0.5 or 1 mM) for 30 min were incubated in the absence and presence of the combination of IL-1beta and TNF-alpha for 8 h. RNA was extracted, fractionated on agarose gels containing ethidium, transferred to nylon membranes, and hybridized with radiolabeled sGC subunit cDNA probes. Ethidium bromide staining of 28S rRNA in the agarose gel confirmed equal loading of RNA samples. Inhibition of NOS2 partially prevented the cytokine-induced decrease in sGC subunit mRNA levels.

To determine whether the cytokine-induced decrease in sGC subunit mRNA levels was dependent on increased cGMP concentrations (associated with NO production by NOS2), rPASMC were preincubated with ODQ (5 µM), a sGC inhibitor, for 30 min and then were exposed to IL-1beta and TNF-alpha or to 0.5 mM SNP for 8 h. Pretreatment with ODQ nearly completely inhibited the ability of SNP to decrease sGC subunit mRNA levels (data not shown and Ref. 3) and partially prevented the cytokine-induced decrease in sGC subunit mRNA levels (Fig. 7). Similarly, the decrease in sGC subunit mRNA levels observed in rPASMC incubated with either IL-1beta or TNF-alpha alone was attenuated in the presence of ODQ (data not shown). These results demonstrate that both IL-1beta and TNF-alpha decrease sGC subunit mRNA levels in part via a cGMP-dependent mechanism.


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Fig. 7.   Attenuation of the cytokine-induced decrease in sGC subunit mRNA levels by inhibition of sGC. rPASMC pretreated with and without 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ, 5 µM) for 30 min were incubated in the absence and presence of the combination of IL-1beta and TNF-alpha for 8 h. RNA was extracted, fractionated on agarose gels containing ethidium, transferred to nylon membranes, and hybridized with radiolabeled sGC subunit cDNA probes. Ethidium bromide staining of 28S rRNA in the agarose gel confirmed equal loading of RNA samples. Inhibition of sGC enzyme activity partially prevented the cytokine-induced decrease in sGC subunit mRNA levels.

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-1beta (20 ng/ml) and TNF-alpha (100 ng/ml) for 8 h, and changes in sGC alpha 1-subunit mRNA levels were quantitated by RNA blot hybridization (Fig. 8). Incubation of mPASMC with IL-1beta and TNF-alpha decreased sGC alpha 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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Fig. 8.   Cytokines decreased sGC alpha 1-subunit mRNA levels in PASMC from NOS2-deficient mice. Murine PASMC (mPASMC) pretreated with and without ODQ (5 µM) for 30 min were incubated in the absence and presence of the combination of IL-1beta and TNF-alpha for 8 h. In addition, mPASMC were incubated with SNP (0.5 mM) for 8 h. RNA was extracted, fractionated on agarose gels containing ethidium, transferred to nylon membranes, and hybridized with radiolabeled mouse sGC alpha 1-subunit cDNA probe. Ethidium bromide staining of 28S rRNA in the agarose gel confirmed equal loading of RNA samples. (RNA from untreated mPASMC is shown in the lane marked C.) Exposure of mPASMC to cytokines decreased sGC alpha 1-subunit mRNA levels in an ODQ-insensitive manner.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
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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 alpha 1- and beta 1-subunit mRNA levels. Decreased sGC subunit mRNA levels were accompanied by decreased alpha 1- and beta 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 alpha 1- and beta 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 alpha 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 alpha 1- and beta 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 alpha 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-1beta (10 U/ml) or lipopolysaccharide (1 µg/ml) decreased sGC alpha 1-subunit mRNA levels without altering alpha 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 alpha 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 alpha 1-subunit protein levels but not beta 1-subunit protein levels. These reports differ from our findings that both alpha 1- and beta 1-subunits are decreased in PASMC exposed to IL-1beta and TNF-alpha . 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.


    ACKNOWLEDGEMENTS

We thank Dr. R. Ullrich for assistance in the preparation of mouse PASMC and Dr. W. M. Zapol for advice and support.


    FOOTNOTES

* 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.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bloch, KD, Filippov G, Sanchez LS, Nakane M, and de la Monte SM. Pulmonary soluble guanylate cyclase, a nitric oxide receptor, is increased during the perinatal period. Am J Physiol Lung Cell Mol Physiol 272: L400-L406, 1997[Abstract/Free Full Text].

2.   Chomczynski, P, and Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156-159, 1987[ISI][Medline].

3.   Filippov, G, Bloch DB, and Bloch KD. Nitric oxide decreases stability of mRNAs encoding soluble guanylate cyclase subunits in rat pulmonary artery smooth muscle cells. J Clin Invest 100: 942-948, 1997[Abstract/Free Full Text].

4.   Fullerton, DA, McIntyre RC, Jr, Hahn AR, Agrafojo J, Koike K, Meng X, Banerjee A, and Harken AH. Dysfunction of cGMP-mediated pulmonary vasorelaxation in endotoxin-induced acute lung injury. Am J Physiol Lung Cell Mol Physiol 268: L1029-L1035, 1995[Abstract/Free Full Text].

5.   Hevel, JM, and Marletta MA. Nitric-oxide synthase assays. Methods Enzymol 233: 250-258, 1994[ISI][Medline].

6.   Keinanen, R, Vartiainen N, and Koistinaho J. Molecular cloning and characterization of the rat inducible nitric oxide synthase (iNOS) gene. Gene 234: 297-305, 1999[ISI][Medline].

7.   Kloss, S, Bouloumie A, and Mulsch A. Aging and chronic hypertension decrease expression of rat aortic soluble guanylyl cyclase. Hypertension 35: 43-47, 2000[Abstract/Free Full Text].

8.   Liu, H, Force T, and Bloch KD. Nerve growth factor decreases soluble guanylate cyclase in rat pheochromocytoma PC12 cells. J Biol Chem 272: 6038-6043, 1997[Abstract/Free Full Text].

9.   Macmicking, JD, Nathan C, Hom G, Chartrain N, Fletcher DS, Trumbauer M, Stevens K, Xie QW, Sokol K, and Hutchinson N. Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase. Cell 81: 641-650, 1995[ISI][Medline].

10.   Mittal, CK. Determination of adenylate cyclase and guanylate cyclase activities in cells of the immune system. Methods Enzymol 132: 422-428, 1986[Medline].

11.   Nakane, M, Arai K, Saheki S, Kuno T, Buechler W, and Murad F. Molecular cloning and expression of cDNAs coding for soluble guanylate cyclase from rat lung. J Biol Chem 265: 16841-16845, 1990[Abstract/Free Full Text].

12.   Nathan, C, and Xie QW. Nitric oxide synthases: roles, tolls, and controls. Cell 78: 915-918, 1994[ISI][Medline].

13.   Papapetropoulos, A, Abou-Mohamed G, Marczin N, Murad F, Caldwell RW, and Catravas JD. Downregulation of nitrovasodilator-induced cyclic GMP accumulation in cells exposed to endotoxin or interleukin-1 beta. Br J Pharmacol 118: 1359-1366, 1996[Abstract].

14.   Papapetropoulos, A, Go CY, Murad F, and Catravas JD. Mechanisms of tolerance to sodium nitroprusside in rat cultured aortic smooth muscle cells. Br J Pharmacol 117: 147-155, 1996[Abstract].

15.   Papapetropoulos, A, Marczin N, Mora G, Milici A, Murad F, and Catravas JD. Regulation of vascular smooth muscle soluble guanylate cyclase activity, mRNA, and protein levels by cAMP-elevating agents. Hypertension 26: 696-704, 1995[Abstract/Free Full Text].

16.   Sambrook, J, Fritsh EF, and Maniatis T. Molecular Cloning: A Laboratory Manual (2nd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1989.

17.   Scott, WS, and Nakayama DK. Escherichia coli lipopolysaccharide downregulates soluble guanylate cyclase in pulmonary artery smooth muscle. J Surg Res 80: 309-314, 1998[ISI][Medline].

18.   Scott, WS, and Nakayama DK. Sustained nitric oxide exposure decreases soluble guanylate cyclase mRNA and enzyme activity in pulmonary artery smooth muscle. J Surg Res 79: 66-70, 1998[ISI][Medline].

19.   Shimouchi, A, Janssens SP, Bloch DB, Zapol WM, and Bloch KD. cAMP regulates soluble guanylate cyclase beta 1-subunit gene expression in RFL-6 rat fetal lung fibroblasts. Am J Physiol Lung Cell Mol Physiol 265: L456-L461, 1993[Abstract/Free Full Text].

20.   Thiemermann, C. Nitric oxide and septic shock. Gen Pharmacol 29: 159-166, 1997[Medline].

21.   Ujiie, K, Hogarth L, Danziger R, Drewett JG, Yuen PS, Pang IH, and Star RA. Homologous and heterologous desensitization of a guanylyl cyclase-linked nitric oxide receptor in cultured rat medullary interstitial cells. J Pharmacol Exp Ther 270: 761-767, 1994[Abstract].

22.   Yu, FS, Lee SL, Lanzillo JJ, and Fanburg BL. Endothelial cell inhibition of hypoxia-induced stimulation of serotonin uptake by vascular smooth muscle cells. Am Rev Respir Dis 139: 1144-1148, 1989[ISI][Medline].


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