Nitric Oxide Increases Tumor Necrosis Factor Production in Differentiated U937 Cells by Decreasing Cyclic AMP*

(Received for publication, May 30, 1996, and in revised form, November 1, 1996)

Shuibang Wang , Liang Yan , Robert A. Wesley and Robert L. Danner Dagger

From the Critical Care Medicine Department, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

Nitric oxide (NO) increases tumor necrosis factor (TNF) synthesis in human peripheral blood mononuclear cells by a cGMP-independent mechanism. NO has been shown to inhibit adenylate cyclase in cell membranes. Since cAMP down-regulates TNF transcription, we examined the possibility that NO enhances TNF synthesis by decreasing cAMP. U937 cells were induced to differentiate using phorbol myristate acetate (100 nM for 48 h) and then were incubated for 24 h with sodium nitroprusside (SNP) or S-nitroso-N-acetylpenicillamine (SNAP). These NO donors increased TNF production (7.0- and 15.6-fold, respectively, at 500 µM) in a dose-dependent manner (p = 0.002). However, SNP and SNAP did not elevate cGMP levels in U937 cell cultures, and the cGMP analog, 8-bromo-cGMP, had no effect on TNF production. In contrast, SNP (p = 0.001) and SNAP (p = 0.009) decreased intracellular cAMP levels by up to 51.5% over 24 h and, in the presence of a phosphodiesterase inhibitor, blunted isoproterenol-stimulated increases in cAMP by 21.8% (p = 0.004) and 27.6% (p = 0.008), respectively. H89, an inhibitor of cAMP-dependent protein kinase, dose dependently increased TNF production in phorbol myristate acetate-differentiated U937 cells in the absence (6.5-fold at 30 µM; p = 0.035), but not in the presence (p = 0.77) of SNAP. Conversely, the cAMP analog dibutyryl cAMP (Bt2cAMP) blocked SNAP-induced TNF production (p = 0.001). SNP and SNAP (500 µM) increased relative TNF mRNA levels by 57.5% (p = 0.045) and 66.2% (p = 0.001), respectively. This effect was prevented by Bt2cAMP. These results indicate that NO up-regulates TNF production by decreasing intracellular cAMP.


INTRODUCTION

Nitric oxide (NO)1 is a free-radical gas produced by many cell types (1-4). NO has a diverse repertoire of important functions (5-9) including neurotransmission (5, 10), vasodilatation (11), antiplatelet activity, and immune modulation (12-14). Most of these effects are mediated through a unique cGMP signaling pathway. NO covalently attacks the heme moiety of soluble guanylate cyclase, activating the enzyme, and thereby elevating intracellular cGMP concentrations (15-17). This increase in cGMP subsequently activates certain protein kinases, which phosphorylate target proteins involved in regulation of cell function (17-19). Although the role of cGMP as a NO second messenger is undisputed, some findings have led to speculation about the existence of cGMP-independent signal transduction pathways for NO.

First, NO is a free radical with the ability to react with a variety of enzymes besides soluble guanylate cyclase. NO has been shown to catalyze the covalent binding of NAD to glyceraldehyde-3-phosphate dehydrogenase (20), oxidize iron-containing proteins such as aconitase or ribonucleotide reductase (21-23), and nitrosylate tyrosine and cysteine residues in a variety of proteins (24-26). Second, some effects of NO cannot be reproduced with cell permeable cGMP analogs. For example, the synthesis of tumor necrosis factor alpha  (TNFalpha ), a proinflammatory cytokine implicated in tissue injury and shock (27), is increased in human peripheral blood mononuclear cells (28) and lipopolysaccharide-stimulated neutrophil preparations (29) by exogenous NO. Although NO increases cGMP concentrations in these cells, cGMP analogs have no effect on TNFalpha production (28, 29). Collectively, these investigations suggest that NO might use cGMP-independent signaling pathways for some of its cellular functions.

Recently, adenylate cyclase has been added to the list of enzymes that can be modified by NO (30). Treatment of cell membranes with NO decreases cAMP production by inhibiting calmodulin activation of type I adenylate cyclase, presumably through thiol nitrosylation at the calmodulin-binding site (30, 31). Notably, increases in cAMP in leukocytes activate cAMP-dependent protein kinase (PKA). This kinase phosphorylates transcription factors that bind to the cAMP-response element on the TNFalpha promoter, thereby inhibiting TNFalpha mRNA transcription (32-35). The effect of NO on type I adenylate cyclase suggests that NO might up-regulate TNFalpha synthesis in human monocytes by decreasing cAMP concentrations.

We investigated this question using U937 cells, a human monocytic cell line that differentiates into monocyte-macrophage-like cells and produces TNFalpha when exposed to phorbol myristate acetate (PMA) (36-38). The specific objectives were as follows: 1) to demonstrate that NO up-regulates TNFalpha production in PMA-differentiated U937 cells and test the cGMP-dependence of this effect; 2) to determine whether NO alters resting or stimulated cAMP concentrations in intact cells; 3) to investigate the effect of inhibitors or activators of PKA on NO-stimulated TNFalpha production in this system; and 4) to determine if NO-induced changes in TNFalpha mRNA levels were consistent with a cAMP mechanism.


EXPERIMENTAL PROCEDURES

Reagents and Cells

PMA, S-nitroso-N-acetylpenicillamine (SNAP), 8-bromo-cGMP, dibutyryl cGMP (Bt2cGMP), dibutyryl cAMP (Bt2cAMP), H89, and isoproterenol were all purchased from Calbiochem (San Diego, CA). Sodium nitroprusside (SNP) was from Elkins-Sinn (Cherry Hill, NJ). Ficoll-Hypaque, prostaglandin E2 (PGE2), and 3-isobutyl-1-methyl-xanthine (IBMX) were all obtained from Sigma. Dextran was from Amersham Life Science Inc. TRI REAGENT-LS used for RNA isolation was from Molecular Research Center, Inc. (Cincinnati, OH).

Human neutrophils were isolated from the venous blood of normal volunteers by dextran sedimentation and Ficoll-Hypaque density centrifugation as described previously (39). U937 cells obtained from ATCC (Rockville, MD) were cultured in RPMI 1640 supplemented with 25 mM HEPES, 10% heat-inactivated fetal calf serum, penicillin (100 units/ml), streptomycin (100 µg/ml), and 2% (w/v) L-glutamine (all from Biofluids, Inc., Rockville, MD). The cells were grown at 37 °C in a humidified atmosphere containing 5% CO2. Differentiation was induced by incubating U937 cells with PMA (100 nM) for 48 h. The cells were then washed three times with Hank's balanced salt solution without Ca2+ and Mg2+ (HBSS-) to remove residual PMA.

Measurement of TNFalpha Production

Differentiated U937 cells (5 × 105 cells/ml) were incubated with each of the following reagents for 24 h: increasing concentrations of SNP or SNAP (0-500 µM); 8-bromo-cGMP or Bt2cGMP (0-1000 µM); or Bt2cAMP (0-100 µM) in the presence of varying doses of SNAP (0-500 µM). In another experiment, differentiated U937 cells were pretreated for 6 h with varying concentrations of H89 (0-30 µM), a cell-permeable PKA inhibitor. The cells (5 × 105 cells/ml) were then washed three times with HBSS- and incubated for an additional 24 h in the absence or presence of SNAP (500 µM). TNFalpha release into the medium was measured using an enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN). All reagents were dissolved into RPMI 1640, and tested negative for endotoxin using a limulus amoebocyte lysate assay (BioWhittaker, Inc., Walkersville, MD).

Determination of Total cGMP

At a concentration of 4 × 106 cells/ml, differentiated U937 cells or freshly isolated human neutrophils were preincubated for 15 min in a shaking water bath at 37 °C with Hank's balanced salt solution with Ca2+ and Mg2+ (HBSS+) containing 1 mM IBMX. After addition of SNP or SNAP (0-1000 µM), these cells were incubated for 0-120 min followed by the addition of iced ethanol (65% (v/v) final concentration). The amount of cGMP present in the samples was quantitated by an enzyme immunoassay according to the manufacturer's protocol (Amersham).

Determination of cAMP

Differentiated U937 cells (5 × 105 cells/ml) were incubated in RPMI 1640 containing increasing doses of SNP or SNAP (0-500 µM) for 24 h. The cells were then scraped and spun for collection of cell pellets. Intracellular cAMP was extracted by lysing the cell pellets in 65% iced ethanol and centrifuging at 2000 × g for 15 min. The resultant supernatants were dried and cAMP was quantitated using an enzyme immunoassay according to the manufacturer's protocol (Amersham).

To investigate the effect of NO on agonist-stimulated cAMP responses, differentiated U937 cells (5 × 105 cells/ml) were pretreated for 15 min with HBSS- containing 500 µM SNP or SNAP in the presence of IBMX (1 mM). Adenylate cyclase agonists, isoproterenol (1 µM), or PGE2 (10 µM) were then added and total cAMP was extracted at several time points (0-15 min) and quantitated as described above.

Ribonuclease Protection Assay (RPA)

Differentiated U937 cells (5 × 105 cells/ml) were incubated under the following conditions for 3.5 h: RPMI 1640 alone or with 8-bromo-cGMP (100 µM), Bt2cAMP (100 µM), SNP (500 µM), SNAP (500 µM), or Bt2cAMP (100 µM) and SNAP (500 µM). RNA was isolated using TRI REAGENT-LS. The entire open reading frame of human TNFalpha cDNA, in the PAW711 plasmid, was kindly provided by Dr. Alice M. Wang (40). A 253-base pair TNFalpha gene fragment, obtained by digestion with the enzymes AvaI and HincII (Stratagene, La Jolla, CA), was subcloned into the pGEM3Z vector (Promega, Madison, WI). The recombined pGEM3Z plasmid was subsequently sequenced to confirm the presence of the TNFalpha fragment insert. Single-strand antisense mRNA probe labeled with 35S was prepared by in vitro transcription using SP6 polymerase. RPA was performed with a RPA II kit (Ambion Inc., Austin, TX) using 50 µg of total RNA according to the manufacturer's instructions. Results obtained were expressed as percentages of the concurrently run beta -actin controls.

Statistics

All data are expressed as mean ± S.E. All p values are two-sided. To analyze dose-response curves in each experiment, the data was first summarized using the nonparametric Sen-Theil estimate of regression slope (41). From these dose-response slope estimates, one sample t tests (against the hypothesis of H0: slope = 0) were done to determine whether the observed dose-response was significant. To determine if the TNFalpha response to doses of SNAP was monotonically decreasing as the Bt2cAMP dose increased, in addition to using the same technique as described above, we also used Page's nonparametric test (42) for ordered alternatives in a 2-way ANOVA layout (the first factor was the increasing doses of Bt2cAMP, the second factor was experiment). To evaluate the effects of NO on the isoproterenol-induced cAMP response in PMA-differentiated U937 cells, the area under the curve was determined for each condition/experiment. Then, nominal p values were computed for each of the two comparisons of interest: isoproterenol versus isoproterenol/SNAP, and isoproterenol versus isoproterenol/SNP. Last, these nominal p values were adjusted by multiplying each by 2, to take into account the multiple comparisons (Bonferroni adjustment) (43). When comparing relative TNFalpha mRNA levels, we used paired t tests.


RESULTS

Effect of NO on TNFalpha Production by PMA-differentiated U937 Cells

First, we confirmed previous reports (37, 38) that PMA-differentiated U937 cells produce TNFalpha (Fig. 1). Next, we demonstrated that exogenous NO donors, SNP or SNAP, increased TNFalpha release from PMA-differentiated U937 cells over a 24-h incubation period in a dose-dependent manner (p = 0.002 for both). At the highest concentrations examined (500 µM), SNP and SNAP increased TNFalpha production 7.0- and 15.6-fold, respectively (Fig. 1).


Fig. 1. Effect of NO on TNFalpha production. PMA-differentiated U937 cells were incubated with the indicated concentrations of SNP and SNAP for 24 h. TNFalpha release into the medium is presented as the mean ± S.E. of four independent experiments, each run in duplicate or triplicate.
[View Larger Version of this Image (11K GIF file)]


Effect of NO on Total cGMP in U937 Cell Cultures

Production of cGMP by PMA-differentiated U937 cells in response to exogenous NO donors was assessed using human neutrophils as a positive control (Fig. 2). After 2 h incubation in the presence of IBMX (1 mM), neither SNP nor SNAP elevated total cGMP in U937 cells (p > 0.1). In contrast, and as expected (28), either SNP or SNAP increased total cGMP production by human neutrophils in a dose-dependent manner (p = 0.012 and p = 0.004, respectively).


Fig. 2. Effect of NO on cGMP production. Human neutrophils and PMA-differentiated U937 cells were preincubated with IBMX (1 mM) for 15 min at 37 °C. Subsequently, the indicated concentrations of SNP or SNAP were added, and the cells were incubated for 2 h. Inset, the effect of SNAP (1 mM) on cGMP at earlier time points. Total cGMP is presented as the mean ± S.E. of three independent experiments, each run in duplicate.
[View Larger Version of this Image (27K GIF file)]


To exclude the possibility that cGMP was quickly degraded despite the presence of a phosphodiesterase (PDE) inhibitor, thereby masking an increase in cGMP production, total cGMP was also measured at earlier time points (0, 1, 3, 5, 10, and 15 min) in the presence of IBMX (1 mM) after exposure to SNAP (1 mM). SNAP increased total cGMP in neutrophils which reached a maximum at 15 min. However, this NO donor had no effect on cGMP production in PMA-differentiated U937 cells (Fig. 2, inset). Furthermore, in separate experiments, SNP or SNAP had no effect on total cGMP in naive U937 cells, or in U937 cells differentiated with retinoic acid with or without 1alpha ,25-dihydroxyvitamin D3 (data not shown).

Effect of cGMP Analogs on TNFalpha Production by PMA-differentiated U937 Cells

Cell-permeable analogs of cGMP, 8-bromo-cGMP, or Bt2cGMP were incubated with PMA-differentiated U937 cells for 24 h. The release of TNFalpha into the medium was not altered by 0-1 mM of either 8-bromo-cGMP (Fig. 3A, p = 0.33) or Bt2cGMP (Fig. 3B, p = 0.66).


Fig. 3. Effect of cGMP analogs on TNFalpha production. PMA-differentiated U937 cells were incubated with the indicated concentrations of 8-bromo-cGMP (A) or Bt2cGMP (B) for 24 h in separate experiments. TNFalpha released into the medium is presented as the mean ± S.E. of three independent experiments, each run in duplicate.
[View Larger Version of this Image (55K GIF file)]


Effect of NO on cAMP Levels in PMA-differentiated U937 Cells

To determine if NO altered cAMP levels, intact PMA-differentiated U937 cells were incubated in the presence of increasing concentrations of SNP or SNAP for 24 h. Either SNP or SNAP decreased intracellular cAMP levels (Fig. 4) in PMA-differentiated U937 cells in a dose-dependent manner (p = 0.001 and p = 0.009, respectively). Compared with culture medium alone, SNP or SNAP at the highest concentrations examined (500 µM) decreased intracellular cAMP by 37.8 and 51.5%, respectively.


Fig. 4. Effect of NO on intracellular cAMP concentrations. PMA-differentiated U937 cells were incubated with the indicated concentrations of SNP or SNAP for 24 h. Intracellular cAMP is presented as the mean ± S.E. of seven independent experiments, each run in duplicate.
[View Larger Version of this Image (12K GIF file)]


To confirm that NO decreases cAMP levels in PMA-differentiated U937 cells, the effect of NO on agonist-stimulated cAMP responses was examined in the presence of IBMX (1 mM). As shown in Fig. 5A, SNP or SNAP (500 µM) blunted isoproterenol-stimulated increases in total cAMP by 21.8% (p = 0.004) and 27.6% (p = 0.008), respectively. Furthermore, the inhibitory effects of SNP (p = 0.045) or SNAP (p = 0.011) were also demonstrated using PGE2 (10 µM) instead of isoproterenol to activate adenylate cyclase (Fig. 5B). This experiment was performed in HBSS- to avoid activation of Ca2+/calmodulin-dependent PDE I (44, 45).


Fig. 5. Effect of NO on agonist-stimulated cAMP responses. PMA-differentiated U937 cells were pretreated for 15 min with 500 µM SNP or SNAP in HBSS- with 1 mM IBMX. Isoproterenol (A) (1 µM) or PGE2 (B) (10 µM) were then added and total cAMP was extracted at the indicated time points and quantitated. Total cAMP is presented as the mean ± S.E. of three independent experiments, each run in duplicate.
[View Larger Version of this Image (22K GIF file)]


Effect of Inhibitors or Activators of PKA on TNFalpha Production by PMA-differentiated U937 Cells

Finding that NO but not cGMP increased TNFalpha production, and that NO decreased intracellular cAMP levels, we next examined the effect of cell-permeable agents that either inhibit (H89) or activate (Bt2cAMP) PKA on TNFalpha production. Preincubation of PMA-differentiated U937 cells with H89 increased TNFalpha production (6.5-fold at 30 µM) in a dose-dependent manner (Fig. 6, p = 0.035). The addition of SNAP elevated TNFalpha production and eliminated the dose effect of H89 (p = 0.77). Conversely, Bt2cAMP blocked SNAP-induced TNFalpha production (Fig. 7). With increasing concentrations of Bt2cAMP (0-100 µM), the dose-dependent effect of SNAP on TNFalpha production was abolished (p = 0.001). Cell viability by trypan blue exclusion was not decreased at the concentrations of Bt2cAMP employed (0-100 µM, data not shown).


Fig. 6. Effect of H89, a PKA inhibitor, on TNFalpha production. PMA-differentiated U937 cells were pretreated for 6 h with H89, washed with HBSS- and then incubated for an additional 24 h with or without SNAP (500 µM). TNFalpha released into the medium is presented as the mean ± S.E. of three independent experiments, each run in duplicate.
[View Larger Version of this Image (11K GIF file)]



Fig. 7. Effect of Bt2cAMP on NO-induced TNFalpha production. PMA-differentiated U937 cells were incubated with 0-100 µM Bt2cAMP in the presence of the indicated concentrations of SNAP for 24 h. TNFalpha released into the medium is presented as the mean ± S.E. of three independent experiments, each run in duplicate.
[View Larger Version of this Image (18K GIF file)]


Effect of NO and Cyclic Nucleotide Analogs on TNFalpha mRNA Levels in PMA-differentiated U937 Cells

Relative TNFalpha mRNA levels were measured using a RPA to investigate the effects of NO, cGMP, and cAMP on TNFalpha mRNA transcription (Fig. 8). SNP or SNAP increased TNFalpha mRNA levels by 57.3% (p = 0.045) and 66.2% (p = 0.001), respectively. An analog of cAMP, Bt2cAMP, decreased TNFalpha mRNA levels (p = 0.002), and prevented the effect of SNAP (Bt2cAMP versus Bt2cAMP and SNAP: p = 0.93). In contrast to the NO donors, 8-bromo-cGMP had no effect on TNFalpha mRNA levels (p = 0.52).


Fig. 8. Effect of NO on TNFalpha mRNA expression. PMA-differentiated U937 cells were incubated under the following conditions for 3.5 h: Bt2cAMP (100 µM); Bt2cAMP (100 µM) and SNAP (500 µM); RPMI 1640 alone; 8-bromo-cGMP (100 µM); SNP (500 µM); or SNAP (500 µM). Inset: lane 1, Bt2cAMP; lane 2, Bt2cAMP and SNAP; lane 3, RPMI 1640 alone; lane 4, 8-bromo-cGMP; lane 5, SNP; lane 6, SNAP. TNFalpha mRNA was quantitated using a RPA. Results of four independent experiments are expressed as a percentage of concurrently run beta -actin controls (mean ± S.E.).
[View Larger Version of this Image (59K GIF file)]



DISCUSSION

We demonstrated that NO increased TNFalpha production in PMA-differentiated U937 cells by decreasing intracellular cAMP levels, indicating that NO uses cAMP, rather than cGMP as a second messenger for some of its cellular effects. This conclusion is based on these findings: 1) two structurally dissimilar NO donors increased TNFalpha production in a dose-dependent manner; 2) both SNP and SNAP increased cGMP concentrations in human neutrophil cultures, but had no effect on cGMP concentrations in PMA-differentiated U937 cell cultures; 3) cell-permeable analogs of cGMP, 8-bromo-cGMP and Bt2cGMP, did not alter TNFalpha production by PMA-differentiated U937 cells; 4) SNP or SNAP not only decreased intracellular cAMP in a dose-dependent manner, but also blunted isoproterenol- and PGE2-stimulated cAMP responses in PMA-differentiated U937 cells; 5) an inhibitor of PKA, H89, increased TNFalpha release in the absence but not in the presence of SNAP; 6) conversely, an activator of PKA, Bt2cAMP, abolished the effect of SNAP on TNFalpha production; and 7) finally, NO donors and Bt2cAMP but not 8-bromo-cGMP caused changes in relative TNFalpha mRNA levels that were consistent with a cAMP mechanism for the observed effects of NO. Collectively, these experiments demonstrate that NO-induced up-regulation of TNFalpha production in this human cell line uses cAMP, not cGMP, as its second messenger.

Many of the known effects of NO have been attributed to its ability to generate cGMP through its action on soluble guanylate cyclase (15-18). However, we were unable to demonstrate that NO donors increase cGMP in either naive or differentiated U937 cells. It seems unlikely that our inability to detect NO-stimulated increases in cGMP was due to degradation of cGMP. U937 cells have extremely low cGMP hydrolytic activity and do not contain the cGMP-specific PDE isoenzyme (PDE V) (45, 46). Furthermore, our experiments were conducted in the presence of a potent, nonselective PDE inhibitor. These data indicate that U937 cells lack NO-sensitive soluble guanylate cyclase. Moreover, membrane permeable cGMP analogs, 8-bromo-cGMP and Bt2cGMP, were unable to mimic the effect of NO on TNFalpha production, a finding that has also been reported in human peripheral blood mononuclear cells and neutrophil preparations (28, 29). These results further suggest that NO regulates TNFalpha production in PMA-differentiated U937 cells by a cGMP-independent mechanism.

The ability of NO to decrease intracellular cAMP levels and blunt isoproterenol- and PGE2-stimulated cAMP responses provide direct evidence for our speculation that NO increases TNFalpha production in PMA-differentiated U937 cells by decreasing cAMP levels. Decreases in intracellular cAMP can result either from its reduced synthesis by adenylate cyclase or from increased catabolism due to increased PDE activity (47). The result that NO decreased cAMP concentrations in the presence of IBMX, a nonspecific PDE inhibitor, support the hypothesis that changes in cAMP levels were due to decreased synthesis, rather than increased catabolism by PDE. Interestingly, cGMP can either increase cAMP hydrolysis by activating PDE II (45, 46) or decrease cAMP hydrolysis by inhibiting PDE III (48, 49). Increased or decreased cAMP hydrolysis mediated by cGMP is unlikely in our experiments since U937 cells lack PDE II activity (45, 46), a PDE inhibitor was used, and as shown here, U937 cells do not produce cGMP in response to a NO signal. However, in other cell types that contain NO-sensitive soluble guanylate cyclase, the ability of NO to decrease cAMP production may be masked by decreased cAMP hydrolysis via cGMP-mediated inhibition of PDE III activity (48-51).

As already noted, NO has been shown to inhibit calmodulin-dependent adenylate cyclase activity in isolated cell membranes by oxidizing cysteine residues at the calmodulin-binding site (30). Other studies have shown that calcium ionophores potentiate cAMP responses in human peripheral blood mononuclear cells and neutrophils stimulated with isoproterenol and PGE2, and this potentiation was inhibited by calmodulin inhibitors (52-54). These observations indicate that the calmodulindependent adenylate cyclase subtype that is inhibited by NO is present in human leukocytes. Although this suggests a possible mechanism for NO modulation of intracellular cAMP levels, other possibilities exist. Substitution of cysteine residues for other amino acids in the beta 2-adrenergic receptor markedly shifts the dose-response curve to the right for isoproterenol-stimulated increases in intracellular cAMP concentrations (55). This suggests that NO could reduce agonist-stimulated cAMP responses by decreasing receptor affinity through the nitrosylation of key cysteine-containing domains.

Inhibitors and activators of PKA were used to further explore the possibility that NO was using a cAMP-dependent signaling pathway. H89, a specific cell-permeable inhibitor of PKA (56), dose-dependently increased TNFalpha release. This finding demonstrates in PMA-differentiated U937 cells that basal cAMP levels and the resulting degree of PKA activation are inhibitory of TNFalpha synthesis. Interestingly, H89, at the doses tested, did not further up-regulate SNAP-induced TNFalpha production, suggesting that PKA was maximally inactivated by the decrease in cAMP levels caused by NO. In contrast, Bt2cAMP only slightly suppressed basal TNFalpha release, but completely abolished SNAP-induced increases in TNFalpha production. This cAMP analog can permeate cell membranes and is resistant to hydrolysis by PDE (57), enabling it to persist in cell cultures and mimic prolonged elevations of intracellular cAMP. Toxicity caused by the butyrate moiety of the Bt2cAMP molecule was unlikely to be responsible for this effect, since the concentrations of Bt2cAMP used were relatively low and Bt2cGMP (up to 1 mM), which also contains a butyrate moiety did not alter TNFalpha production. Furthermore, cell viability by trypan blue exclusion was not decreased by Bt2cAMP. Together, these results reinforced our conclusion that NO increases TNFalpha production in PMA-differentiated U937 cells by decreasing cAMP levels.

Similarly, NO donors were also found to increase relative TNFalpha mRNA levels and Bt2cAMP completely prevented this effect. An analog of cGMP, 8-bromo-cGMP, had no effect. These results are consistent with a cAMP mechanism acting at the level of transcription for the observed effects of NO on TNFalpha production in PMA-differentiated U937 cells. Evidence is also available that cAMP can down-regulate TNFalpha expression at a post-transcriptional level in monocytes and macrophages (58-60).

Previously, NO was demonstrated to increase TNFalpha mRNA levels in HL-60 cells (61), but we found no change in TNFalpha mRNA levels in human neutrophil preparations (28). These inconsistent findings may be ascribed to the different methods employed to measure mRNA levels. In our present study, we measured TNFalpha mRNA levels using a RPA, which may be quantitatively more reliable than the reverse transcription polymerase chain reaction assay used in our previous experiments with neutrophils (28). Furthermore, differentiated U937 cells may contain more copies of TNFalpha mRNA than neutrophils. Besides the cAMP mechanism, our data do not exclude the possibility of additional mechanisms for the up-regulation of TNFalpha production by NO. For example, NO could activate or induce other transcription factors, such as NF-kappa B (29).

In conclusion, the present study indicates that NO increase TNFalpha production in PMA-differentiated U937 cells by decreasing intracellular cAMP. To our knowledge, this is the first demonstration in intact cells that NO signal transduction can use cAMP rather than cGMP to regulate cell function.


FOOTNOTES

*   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.
Dagger    To whom reprint requests or correspondence should be addressed: Critical Care Medicine Department, Bldg. 10, Rm. 7D43, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, MD 20892. Tel.: 301-496-9320; Fax: 301-402-1213.
1    The abbreviations used are: NO, nitric oxide; TNFalpha , tumor necrosis factor alpha ; PKA, cAMP-dependent protein kinase; PMA, phorbol myristate acetate; SNAP, S-nitroso-N-acetylpenicillamine; Bt2cGMP, dibutyryl cGMP; Bt2cAMP, dibutyryl cAMP; SNP, sodium nitroprusside; PGE2, prostaglandin E2; IBMX: 3-isobutyl-1-methylxanthine; HBSS-, Hank's balanced salt solution without Ca2+ and Mg2+; HBSS+, Hank's balanced salt solution with Ca2+ and Mg2+; PDE, phosphodiesterase; RPA, ribonuclease protection assay.

REFERENCES

  1. Pollock, J. S., Förstermann, U., Mitchell, J. A., Warner, T. D., Schmidt, H. H. H. W., Nakane, M., and Murad, F. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 10480-10484 [Abstract]
  2. Sessa, W. C., Harrison, J. K., Barber, C. M., Zeng, D., Durieux, M. E., D'Angelo, D. D., Lynch, K. R., and Peach, M. J. (1992) J. Biol. Chem. 267, 15274-15276 [Abstract/Free Full Text]
  3. Busse, R., and Mülsch, A. (1990) FEBS Lett. 275, 87-90 [CrossRef][Medline] [Order article via Infotrieve]
  4. Förstermann, U., Kleinnert, H., Gath, I., Schwarz, P., Closs, E. J., and Dunn, N. J. (1995) Adv. Pharmacol. 34, 171-186 [Medline] [Order article via Infotrieve]
  5. Bredt, D. S., Hwang, P. M., and Snyder, S. H. (1990) Nature 347, 768-770 [CrossRef][Medline] [Order article via Infotrieve]
  6. Shibuki, K., and Okada, D. (1991) Nature 349, 326-328 [CrossRef][Medline] [Order article via Infotrieve]
  7. Nathan, C. (1992) FASEB J. 6, 3051-3064 [Abstract/Free Full Text]
  8. Moncada, S., Palmer, R. M. J., and Higgs, E. A. (1991) Pharmacol. Rev. 43, 109-142 [Medline] [Order article via Infotrieve]
  9. Lowenstein, C. J., and Snyder, S. S. (1992) Cell 70, 705-707 [Medline] [Order article via Infotrieve]
  10. Garthwaite, J., Charles, S. L., and Chess-Williams, R. (1988) Nature 336, 385-388 [CrossRef][Medline] [Order article via Infotrieve]
  11. Palmer, R. M. J., Ashton, D. S., and Moncada, S. (1988) Nature 333, 664-666 [CrossRef][Medline] [Order article via Infotrieve]
  12. Mellion, B. T., Ignarro, L. J., Ohlstein, E. H., Pontecorvo, E. G., Hyman, A. L., and Kadowitz, P. J. (1981) Blood 57, 946-955 [Medline] [Order article via Infotrieve]
  13. Evans, C. H. (1995) Agents Actions Suppl. 47, 107-116 [Medline] [Order article via Infotrieve]
  14. Moilanen, E., and Vapaatalo, H. (1995) Ann. Med. 27, 359-367 [Medline] [Order article via Infotrieve]
  15. Ignarro, L. J., Degna, J. N., Baricos, W. H., Kadowitz, P. J., and Wolin, M. S. (1982) Biochem. Biophys. Acta 718, 49-59 [Medline] [Order article via Infotrieve]
  16. Craven, P. A., and DeRubertis, F. R. (1978) J. Biol. Chem. 253, 8433-8443 [Abstract]
  17. Ignarro, L. J. (1990) Pharmacol. Toxicol. 67, 1-7 [Medline] [Order article via Infotrieve]
  18. Stewart, A. G., Phan, L. H., and Grigoriadis, G. (1994) Microsurgery 15, 693-702 [Medline] [Order article via Infotrieve]
  19. Kuo, P. C., and Schroeder, R. A. (1995) Ann. Surg. 221, 220-235 [Medline] [Order article via Infotrieve]
  20. McDonald, L. J., and Moss, J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6238-6241 [Abstract]
  21. Drapier, J. C., and Hibbs, J. B., Jr. (1986) J. Clin. Invest. 78, 790-797 [Medline] [Order article via Infotrieve]
  22. Lepoivre, M., Chenais, B., Yapo, A., Lemaire, G., Thelander, L., and Tenu, J-P. (1990) J. Biol. Chem. 265, 14143-14149 [Abstract/Free Full Text]
  23. Stamler, J. S. (1994) Cell 78, 931-936 [Medline] [Order article via Infotrieve]
  24. Stamler, J. S., Simon, D. I., Osborne, J. A., Mullins, M. E., Jarak, O., Michel, T., Singel, D. J., and Lascalzo, J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 444-448 [Abstract]
  25. Arnell, D. K., and Stamler, J. S. (1995) Arch. Biochem. Biophys. 318, 279-285 [CrossRef][Medline] [Order article via Infotrieve]
  26. Lander, H. M., Ogiste, J. S., Pearce, S. F. A., Levi, R., and Novogrodsky, A. (1995) J. Biol. Chem. 270, 7017-7020 [Abstract/Free Full Text]
  27. Tracey, K. J., Beutler, B., Lowry, S. F., Merryweather, J., Wolpe, S., Milsark, I. W., Hariri, R. J., Fahey, T. J., III, Zentella, A., Albert, J. D., Shires, G. T., and Cerami, A. (1986) Science 234, 470-474 [Medline] [Order article via Infotrieve]
  28. Van Dervort, A. L., Yan, L., Madara, P. J., Cobb, P. J., Wesley, R. A., Corriveau, C. C., Tropea, M. M., and Danner, R. L. (1994) J. Immunol. 152, 4102-4109 [Abstract/Free Full Text]
  29. Lander, H. M., Sehajpal, P., Levine, D. M., and Novogrodsky, A. (1993) J. Immunol. 150, 1509-1516 [Abstract/Free Full Text]
  30. Duhe, R. J., Nielsen, M. D., Dittman, A. H., Villacres, E. C., Choi, E-J., and Storm, D. R. (1994) J. Biol. Chem. 269, 7290-7296 [Abstract/Free Full Text]
  31. Vorherr, T., Knöpfel, L., Hofmann, F., Mollner, S., Pfeuffer, T., and Carafoli, E. (1993) Biochemistry 32, 6081-6088 [Medline] [Order article via Infotrieve]
  32. Economou, J. S., Rhoades, K., Essner, R., Mcbride, W. H., Gasson, J. C., and Morton, D. L. (1989) J. Exp. Med. 170, 321-326 [Abstract]
  33. Newell, C. L., Deisseroth, A. B., and Lopez-Berestein, G. (1994) J. Leukocyte Biol. 56, 27-35 [Abstract]
  34. Righi, M. (1993) Funct. Neurol. 8, 359-363 [Medline] [Order article via Infotrieve]
  35. Zhong, W. W., Burke, P. A., Drotar, M. E., Chavali, S. R., and Frose, R. A. (1995) Immunology 84, 446-452 [Medline] [Order article via Infotrieve]
  36. Sundström, C., and Nilsson, K. (1976) Int. J. Cancer 17, 565-577 [Medline] [Order article via Infotrieve]
  37. Hass, R., Lonnemann, G., Männel, D., Topley, N., Hartmann, A., Köhler, L., Resch, K., and Goppelt-Strübe, M. (1991) Leukemia Res. 15, 327-339 [Medline] [Order article via Infotrieve]
  38. Taimi, M., Defacque, H., Commes, T., and Favero, J. (1993) Leukemia Res. 79, 229-235
  39. Danner, R. L., Joiner, K. A., and Parrilo, J. E. (1987) J. Clin. Invest. 80, 605-612 [Medline] [Order article via Infotrieve]
  40. Wang, A. M., Creasey, A. A., Ladner, M. B., Lin, L. S., Strickler, J., Arsdell, J. N., Yamamoto, R., and Mark, D. F. (1985) Science 228, 149-154 [Medline] [Order article via Infotrieve]
  41. Hollander, M., and Wolfe, D (1973) Nonparametric Statistical Methods, pp. 205-206, John Wiley & Sons, Inc., New York
  42. Hollander, M., and Wolfe, D. (1973) Nonparametric Statistical Methods, pp. 147-150, John Wiley & Sons, Inc., New York
  43. Miller, R. (1986) Beyond ANOVA, Basics of Applied Statistics, pp. 74-76, John Wiley & Sons, Inc., New York
  44. Barnes, P. J. (1995) Eur. Respir. J. 8, 457-462 [Abstract/Free Full Text]
  45. Torphy, T. J., Zhou, H-L., and Cieslinski, L. B. (1992) J. Pharmacol. Exp. Ther. 263, 1195-1205 [Abstract]
  46. Barnette, M. S., Grous, M., Burman, M., Cieslinski, L. B., Huang, L., and Torphy, T. J. (1992) Am. Rev. Resp. Dis. 145, A282-A283
  47. Gill, G. N., and Gim, W. (1979) Adv. Cyclic. Nucleotide Res. 10, 93-106 [Medline] [Order article via Infotrieve]
  48. Sonnebury, W. K., and Beavo, J. A. (1994) Adv. Pharmacol. 26, 87-114 [Medline] [Order article via Infotrieve]
  49. Beavo, J. A. (1995) Physiol. Rev. 75, 725-748 [Abstract/Free Full Text]
  50. Turner, N. C., Lamb, J., Worby, A., and Murray, K. J. (1994) Br. J. Pharmacol. 111, 1047-1052 [Abstract]
  51. Bowen, R., and Haslam, R. J. (1991) J. Cardiovasc. Pharmacol. 17, 424-433 [Medline] [Order article via Infotrieve]
  52. Iannone, M. A., Wolberg, G., and Zimmerman, T. P. (1991) Biochem. Pharmacol. 42, (Suppl.) S105-S111
  53. Ishitoya, J., and Takenawa, T. (1987) J. Immunol. 138, 1201-1207 [Abstract/Free Full Text]
  54. Dooper, M. W. S. M., Hoekstra, Y., Timmermans, A. D. E., Monchy, J. G. R., and Kauffman, H. F. (1994) Biochem. Pharmacol. 47, 289-294 [Medline] [Order article via Infotrieve]
  55. Savarese, T. M., and Fraser, C. M. (1992) Biochem. J. 283, 1-19 [Medline] [Order article via Infotrieve]
  56. Chijiwa, T., Mishima, A., Hagiwara, M., Sano, M., Hayashi, K., Inoue, T., Naito, K., Toshioka, T., and Hidaka, H. (1990) J. Biol. Chem. 265, 5267-5272 [Abstract/Free Full Text]
  57. Postenak, T., and Weiman, G. (1974) Methods Enzymol. 38, 399-417 [Medline] [Order article via Infotrieve]
  58. Kunkel, S. L., Spengler, M., May, M. A., Spengler, R., Larrick, J., and Remick, D. (1988) J. Biol. Chem. 263, 5380-5384 [Abstract/Free Full Text]
  59. Prabhakar, U., Lipshutz, D., Bartus, J. O., Slivjak, M. J., Smith, E. F., III, Lee, J., and Esser, K. M. (1994) Int. J. Immunopharmacol. 16, 805-816 [CrossRef][Medline] [Order article via Infotrieve]
  60. Seldon, P. M., Barnes, P. J., Meja, K., and Giembycz, M. A. (1995) Mol. Pharmacol. 48, 747-757 [Abstract]
  61. Magrinat, G., Nickmason, S., Shami, P. J., and Weinberg, J. B. (1992) Blood 180, 1880-1884

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.