Attenuation of cAMP accumulation in adult rat cardiac fibroblasts by IL-1beta and NO: role of cGMP-stimulated PDE2

Åsa B. Gustafsson1 and Laurence L. Brunton2

1 Biomedical Sciences Graduate Program, 2 Departments of Pharmacology and Medicine, University of California at San Diego, La Jolla, California 92093


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Treatment of cultured adult rat cardiac fibroblasts with interleukin-1beta (IL-1beta ) 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 beta -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-1beta 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-1beta -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-1beta ; phosphodiesterase 2; guanosine 3',5'-cyclic monophosphate; soluble guanylyl cyclase


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta 2-adrenergic receptors that couple to Gs-adenylyl cyclase (AC) but lack beta 1 receptors, the subtype that predominates on cardiac myocytes (15, 22). We have also reported that interleukin (IL)-1beta induces iNOS expression in cardiac fibroblasts and that the beta 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 beta -adrenergic response. We report here that NO accumulation in cardiac fibroblasts decreases cAMP accumulation in response to beta -adrenergic stimulation, in large part by causing the activation of a cGMP-stimulated phosphodiesterase (PDE2).


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Rat recombinant IL-1beta 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). [alpha -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 beta -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-1beta 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 beta -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 [alpha -32P]ATP to [alpha -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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Induction of functional iNOS by IL-1beta ; effect on the beta -adrenergic and forskolin responses. We investigated whether IL-1beta 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-1beta (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-1beta for 6 h, reaching a maximum after 24 h of treatment. Moreover, treatment for 24 h with IL-1beta caused a concentration-dependent increase in NO production with an EC50 of 3 ng/ml (~0.17 nM) (Fig. 1C).


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Fig. 1.   Time-dependent increase in inducible nitric oxide synthase (iNOS) expression and concentration-dependent stimulation of nitric oxide (NO) production in response to interleukin (IL)-1beta . A: cardiac fibroblasts were treated with 10 ng/ml IL-1beta , and periodically samples were collected and subjected to SDS-PAGE followed by immunoblot analysis using a monoclonal antibody against iNOS. A 130-kDa band corresponding to iNOS was detected in IL-1beta -treated cells. B: iNOS protein bands (as in A) were quantified with NIH Image software and plotted as a percentage of maximal iNOS induction. Data are means ± SE, n = 3. C: cells were incubated with IL-1beta (10 ng/ml, 24 h), at which time nitrite accumulation in the medium was measured. Data are means ± SE, n = 3.

The induction of iNOS (24-h treatment with IL-1beta , 10 ng/ml) caused a 10-fold increase in NO production compared with untreated cells (Fig. 2). Addition of 1 mM NG-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of NO synthase, inhibited NO production by IL-1beta -treated cells. These data indicate that the NO is enzymatically produced and that its production can be readily inhibited pharmacologically.


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Fig. 2.   IL-1beta stimulates NO production. Cells were incubated with or without 10 ng/ml IL-1beta  ± 1 mM NG-monomethyl-L-arginine (L-NMMA) for 24 h. IL-1beta caused a significant increase in NO production, measured as nitrite released into the medium, over basal (* P < 0.0001, n = 4). L-NMMA completely inhibited IL-1beta -induced nitrite accumulation. Con, control.

Having defined the time course and magnitude of iNOS induction and NO production in IL-1beta -treated cells, we proceeded to determine whether IL-1beta treatment would affect the beta -adrenergic response. Treatment of cardiac fibroblasts with 10 ng/ml IL-1beta for 24 h resulted in a 55% decrease in isoproterenol-stimulated cAMP accumulation (Fig. 3A). Forskolin-stimulated cAMP accumulation was similarly attenuated after IL-1beta treatment (Fig. 3B).


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Fig. 3.   IL-1beta treatment inhibits cAMP accumulation. Cardiac fibroblasts were treated with diluent or 10 ng/ml IL-1beta for 24 h and then stimulated for 5 min with 1 µM isoproterenol (Iso) (A) or 30 µM forskolin (B). The cAMP content was determined as described in MATERIALS AND METHODS. IL-1beta treatment significantly decreased cAMP accumulation in response to Iso (* vs. **, P < 0.01, n = 4) and forskolin (* vs. **, P < 0.001). L-NMMA (1 mM) completely restored both Iso- and forskolin-stimulated cAMP accumulation. Data are means ± SE, n = 3. Forsk, forskolin.

Rationale for subsequent studies. Having established that exposure to IL-1beta causes iNOS induction and depresses responses to beta -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-1beta ? 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-1beta effect, mimicry by NO donors, and reversibility. Exposure to IL-1beta (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-1beta to reduce these responses (Fig. 3, A and B). These data indicate that these effects of IL-1beta are dependent on enzymatic NO production and, thus, on the induction of iNOS.

By way of substantiating this conclusion, we found that the effect of IL-1beta could be reproduced by exposing fibroblasts to NO donors. We examined the effect of two structurally different NO donors, SNP and SNAP, on isoproterenol-stimulated cAMP accumulation. Pretreatment of cells with SNP or SNAP for 45 min caused decreases in the beta -adrenergic response (Fig. 4A) comparable to that due to the iNOS induction occurring with a 24-h treatment with IL-1beta (Fig. 3A) Thus we could readily simulate the effect of iNOS induction by providing NO pharmacologically.


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Fig. 4.   NO donors reversibly attenuate Iso-stimulated cAMP accumulation. A: cardiac fibroblasts were incubated with diluent, 1 mM sodium nitroprusside (SNP), or 1 mM S-nitroso-N-acetylpenicillamine (SNAP) for 45 min. Cells were then treated with 1 µM Iso for 5 min and the cAMP levels were determined. Both SNP and SNAP significantly inhibited Iso-stimulated cAMP accumulation (* and **, P < 0.001). Data are means ± SE, n = 4. B: cells were preincubated with or without 1 mM SNP, and after 45 min the cells were washed once with fresh medium and then incubated in medium without SNP. The cells were stimulated with 1 µM Iso for 5 min at the indicated times after removal of SNP. Data are means ± SE, n = 4.

The issue of the reversibility of the effect of NO offers a clue as to mechanism because the interaction between heme and NO is rapidly reversible, whereas other NO-mediated modifications, such as tyrosine nitrosation, are not readily reversible in cells (25). To determine whether the inhibition of cAMP accumulation by NO was reversible, we investigated whether cardiac fibroblasts that had been treated with an NO donor could recover the ability to produce cAMP in response to isoproterenol. Cells were preincubated with 1 mM SNP for 45 min, washed once with medium, and incubated in fresh medium without SNP. At the indicated times after the removal of SNP, the cells were stimulated with isoproterenol for 5 min and the cAMP levels were determined. With no washout (i.e., at 0 min in Fig. 4B), isoproterenol-stimulated cAMP accumulation was inhibited by 55% by the SNP treatment, as expected. However, isoproterenol-stimulated cAMP accumulation was progressively less inhibited as time after removal of SNP increased. At 5, 10, and 20 min after SNP removal, the cardiac fibroblasts displayed 20, 12, and 8% inhibition of isoproterenol-stimulated cAMP accumulation, respectively, thus recovering with a half-time of ~5 min. At 30 min, the fibroblasts appeared to have completely recovered from the effects of SNP; the beta -adrenergic response was comparable to that of untreated cells. Thus the effect of exogenous NO seems fully reversible by the simple expedient of washing the cells, suggesting that long-lived covalent changes are not involved.

Effect of IL-1beta /NO does not localize to hormone-sensitive AC. IL-1beta reduced responses to both isoproterenol and forskolin (Fig. 3, A and B). Because forskolin directly activates AC and also activates the alpha s-AC complex, we conclude that this NO-dependent inhibition of cAMP accumulation is not occurring at the level of the beta -adrenergic receptor, on a mechanism regulating receptor refractoriness, or at the receptor-Gs protein interface.

It seemed possible that the reduced response to isoproterenol and forskolin could result from an activation of the Gi pathway by NO. We have demonstrated the existence of an active Gi pathway in these cells by measuring pertussis toxin (PTx)-sensitive inhibition of extracellular signal-regulated kinase (ERK) by lysophosphatidic acid (unpublished observation). We found that treatment of cells overnight with PTx, sufficient to inhibit this activation of Gi, did not alter the effect of SNP to decrease isoproterenol-stimulated cAMP accumulation (Fig. 5A). In fact, SNP treatment decreased cAMP accumulation to the same extent in cells that had been treated with PTx as in untreated cells, indicating that the inhibitory action of NO is not mediated through activation of the Gi pathway.


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Fig. 5.   Effects of NO are not mediated by Gi activation or adenylyl cyclase (AC) inhibition. A: pertussis toxin (PTx) treatment does not alter the effect of SNP. Cells were incubated with or without 0.1 µg/ml PTx for 18 h, after which cells were treated with 1 mM SNP for 45 min, and cAMP accumulation was assessed after a 5-min exposure to 1 µM Iso. PTx did not significantly alter the effect of SNP to decrease Iso-stimulated cAMP accumulation (* vs. **, P > 0.5; mean ± SE; n = 3). B: SNP has a small effect on AC activity. Membranes were incubated on ice for 30 min with or without 1 mM SNP. Forskolin-stimulated AC activity was assessed by using 20 µg of membrane protein per tube. The effect of SNP was slight but significant (* vs. **, P < 0.05). Data are means ± SE, n = 3; 100% = 568 pmol · mg-1 · min-1.

Recent studies suggest that NO can inhibit AC activity directly in N18TG2 neuroblastoma cells and that the inhibition is specific to AC isoforms 5 and 6 (17, 21). Because types 5 and 6 are known to be expressed in the heart (8), we investigated whether NO would have an effect on AC activity in the membranes of rat cardiac fibroblasts. Using the same protocol employed by McVey et al. (21), we found that preincubation of membranes with 1 mM SNP for 30 min had a small but significant effect on AC activity in the fibroblasts: SNP treatment reduced activity to about 88% of control enzyme activity (Fig. 5B). This 12% diminution of AC activity by NO in cardiac fibroblasts is much smaller than the 50-75% reduction reported in membranes from N18TG2 cells (21). This small effect seems unlikely to account for the 50% decrease in isoproterenol-stimulated cAMP accumulation in response to NO that we observed in the intact fibroblast. To the extent that the NO effect described by McVey et al. (21) is diagnostic of the presence of AC5 and AC6, we also conclude that these isoforms are not major contributors to cAMP production by rat ventricular fibroblasts.

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-1beta stimulated NO production (Fig. 2); treatment with either a NO donor or IL-1beta 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-1beta (10 ng/ml, 24 h): control, 39 ± 32; L-NMMA, 32 ± 25; IL-1beta , 641 ± 81; and IL-1beta 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-1beta -treated cells results from the effect of NO to activate sGC.


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Fig. 6.   Effects of IL-1beta and NO are mediated via soluble guanylyl cyclase (sGC). A: cardiac fibroblasts were incubated with or without 10 µM 1H[1,2,4]oxadiazolo- [4,3-a]quinoxalin-1-one (ODQ), an inhibitor of sGC, for 1 h and then stimulated with 1 mM SNP for 45 min. cGMP content was then determined. SNP treatment significantly stimulated cGMP accumulation over control (* P < 0.001); ODQ inhibited SNP-stimulated cGMP accumulation. Data are means ± SE, n = 3. B: IL-1beta (10 ng/ml, 24 h) stimulated cGMP accumulation in the fibroblasts over control (* P < 0.001), which was completely inhibited in the presence of 10 µM ODQ. Data are means ± SE, n = 3. C: after treatments with ODQ (10 µM) and SNP (1 mM), as above, cells were stimulated with 1 µM Iso for 5 min and the cAMP content was determined. ODQ completely restored the beta -adrenergic response in SNP-treated fibroblasts (* vs. **, P < 0.001). Data are means ± SE, n = 3. D: cardiac fibroblasts were treated with IL-1beta (10 ng/ml, 24 h) and then incubated with 10 µM ODQ for 1 h before a 5-min stimulation with 1 µM Iso. ODQ partially restored the beta -adrenergic response (* vs. **, P < 0.001); a significant reduction in cAMP accumulation persisted compared with Iso-stimulated cells (** P < 0.01). Data are means ± SE, n = 3.

Indeed, 1H[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a specific inhibitor of sGC, inhibited cGMP production in response to SNP and IL-1beta (see Fig. 6, A and B). ODQ also fully restored isoproterenol-stimulated cAMP accumulation in SNP-stimulated cardiac fibroblasts (Fig. 6C), suggesting that sGC-cGMP mediates the effects of NO donors on cAMP production. Similarly, ODQ substantially restored isoproterenol-stimulated cAMP accumulation in IL-1beta -treated cells (Fig. 6D). We interpret this data to mean that NO-stimulated cGMP production mediates most of the effects of IL-1beta on cAMP accumulation.

Many effects of cGMP are mediated via activation of PKG. The effect of NO/cGMP to reduce cAMP is not one of those effects: treatment of cells with 1 µM KT-5823, an inhibitor of PKG, did not alter the effect of SNP to decrease isoproterenol-stimulated cAMP accumulation (Fig. 7). These data indicate that the inhibitory action of NO on cAMP accumulation is not mediated through activation of PKG.


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Fig. 7.   Effects of NO are not mediated via cGMP-dependent protein kinase (PKG) Cardiac fibroblasts were incubated with diluent or 1 µM KT-5823, an inhibitor of PKG, for 15 min, after which cells were treated with 1 mM SNP for 45 min. cAMP accumulation was then assessed after a 5-min stimulation with 1 µM Iso. KT-5823 treatment did not alter the effect of SNP (* vs. **, P > 0.05). Data are means ± SE, n = 3.

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-1beta . 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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Fig. 8.   Phosphodiesterase (PDE) inhibitors restore Iso-stimulated cAMP accumulation in SNP-treated cardiac fibroblasts. Cardiac fibroblasts were incubated with diluent (A), 1 mM 3-isobutyl-1-methylxanthine (IBMX), a nonspecific PDE inhibitor (B), or 0.2 mM erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), a PDE2-specific inhibitor (C), for 15 min before SNP treatment (1 mM, 45 min), after which cells were stimulated with 1 µM Iso for 5 min and the cAMP content was determined. A: SNP significantly reduced Iso-stimulated cAMP accumulation (* vs. **, P < 0.001). In the presence of IBMX (B) or EHNA (C), Iso-stimulated cAMP accumulation was completely restored (* vs. **, P > 0.05.). Data are means ± SE, n = 3.

The findings were similar, but not identical, in IL-1beta -treated cells. Neither IBMX nor EHNA completely reversed the attenuated cAMP response in IL-1beta -treated cells. Both inhibitors largely (to 85% of maximum) reversed the attenuation in cAMP accumulation in IL-1beta -treated fibroblasts (Fig. 9); thus most of the IL-1beta effect is attributable to activation of PDE2. There is, however, a small component of the IL-1beta effect on cAMP accumulation that is not restored by inhibitors of PDE2, which is in accord with the results obtained with the sGC inhibitor ODQ (Fig. 6D). As previously demonstrated (Fig. 3), an NO synthase inhibitor will fully restore cAMP accumulation, so the effect of IL-1beta is NO dependent. We hypothesize that this EHNA/ODQ-resistant component reflects an effect of long-term IL-1beta exposure on expression of other proteins involved in cAMP production and metabolism.


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Fig. 9.   PDE inhibitors largely restore Iso-stimulated cAMP accumulation in IL-1beta -treated cardiac fibroblasts. Cells were treated with IL-1beta (10 ng/ml, 24 h) and then incubated with diluent (A), 1 mM IBMX (B), or 0.2 mM EHNA (C) for 15 min before a 5-min stimulation with Iso (1 µM). IL-1beta treatment reduced the beta -adrenergic response to 50% of maximum (* vs. **, P < 0.001). PDE inhibitors restored the beta -adrenergic response to 85% of maximum, still a significant reduction (* vs. **, P < 0.05). Data are means ± SE, n = 3.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have examined the effect of NO production on the beta -adrenergic response in cardiac fibroblasts. Our data show that induction of iNOS by IL-1beta 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 beta -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 beta -adrenergic stimulation. Joe et al. (19) reported a decrease in the beta  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-1beta 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-1beta -treated fibroblasts, suggesting that the beta -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 beta -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 beta -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-1beta induces iNOS; iNOS makes NO, which activates the sGC; and the resultant cGMP activates PDE2, thereby reducing cAMP accumulation.


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Fig. 10.   Testable mechanisms by which IL-1beta , iNOS, and NO could affect cAMP accumulation. Solid arrows indicate the proposed pathway. Dashed lines indicate sites of action of pharmacologic inhibitors used diagnostically in our experiments. Shaded lines indicate pathways found not to play a role in the effect of NO on cAMP accumulation in cardiac fibroblasts.

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-1beta 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-1beta 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-1beta 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-1beta treatment; thus when we stimulated the cells with isoproterenol after 24 h of IL-1beta 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-1beta treatment are due to an increase in NO production and not to some other effect of IL-1beta , 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 beta -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 beta -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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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