Nitric Oxide Provokes Tumor Necrosis Factor-α Expression in Adult Feline Myocardium Through a cGMP-Dependent Pathway
Background—The mechanism(s) responsible for the persistent coexpression of tumor necrosis factor-α (TNF-α) and nitric oxide (NO) in the failing heart is unknown.
Methods and Results—To determine whether NO was sufficient to provoke TNF-α biosynthesis, we examined the effects of an NO donor, S-nitroso-N-acetyl penicillamine (SNAP), in buffer-perfused Langendorff hearts. SNAP (1 μmol/L) treatment resulted in a time- and dose-dependent increase in myocardial TNF-α mRNA and protein biosynthesis in adult cat hearts. The effects of SNAP were completely abrogated by a NO quenching agent, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (C-PTIO), and mimicked by sodium nitroprusside. Electrophoretic mobility shift assays demonstrated that SNAP treatment led to the rapid induction of nuclear factor kappa-beta (NF-κB) but not AP-1. The importance of the cGMP pathway in terms of mediating NO-induced TNF-α biosynthesis was shown by studies that demonstrated that 8-bromo-cGMP mimicked the effects of SNAP and that the effects of SNAP could be completely abrogated using a cGMP antagonist, 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), or protein kinase G antagonist (Rp-8-Br-cGMPS). SNAP and 8-Br-cGMP were both sufficient to lead to the site-specific phosphorylation (serine 32) and degradation of IκBα in isolated cardiac myocytes. Finally, protein kinase G was sufficient to directly phosphorylate IκBα on serine 32, a critical step in the activation of NF-κB.
Conclusions—These studies show that NO provokes TNF-α biosynthesis through a cGMP-dependent pathway, which suggests that the coincident expression of TNF-α and NO may foster self-sustaining positive autocrine/paracrine feedback inflammatory circuits within the failing heart.
Previous studies have shown that tumor necrosis factor-α (TNF-α) is expressed de novo within the myocardium in response to tissue injury.1 In most instances, the expression of TNF-α mRNA and protein within the myocardium is self-limited1 ; that is, after the removal of the inciting injury, both the TNF-α gene and protein expression are no longer evident within the myocardium.1 Nonetheless, in certain settings, such as chronic heart failure, TNF-α mRNA and protein are persistently expressed in the myocardium.2 However, the mechanisms that are responsible for persistent TNF-α expression within the myocardium are not known. On the basis of the observation that TNF-α and the inducible form of nitric oxide synthase (iNOS) are coexpressed in failing human hearts3 and of experimental studies showing that TNF-α upregulates iNOS in cardiac myocytes4 and that nitric oxide (NO) donors are sufficient to increase TNF-α production in various nonmyocyte cell types,5 we considered the possibility that TNF-α and NO might participate in self-sustaining positive autocrine/paracrine feedback circuits within the myocardium. Accordingly, to begin to address this question, in the present study, we systematically examined the effects of NO on TNF-α mRNA and protein biosynthesis in the adult heart. The results of this simple experimental study constitute an initial demonstration that NO is a sufficient stimulus to provoke TNF-α expression within the adult mammalian myocardium. Moreover, the results of this study show that NO provokes TNF-α biosynthesis through a novel cGMP-dependent pathway.
Myocardial TNF-α Biosynthesis Ex Vivo
Myocardial TNF-α biosynthesis was assessed ex vivo, under endotoxin-free conditions, using a modified Langendorff perfusion apparatus, as detailed previously.1 The heart was perfused with a recirculating Krebs-Henseleit bicarbonate buffer containing S-nitroso-N-acetyl penicillamine (SNAP) or diluent (control hearts). In preliminary control experiments using 10−4 to 10−9 mol/L SNAP, we determined that 10−6 mol/L SNAP produced stable nitrite concentrations of 2.8 to 3.1 μmol/L for at least 180 minutes. Insofar as this range of nitrite concentrations corresponded to the nitrite levels that have been observed after the breakdown of pathophysiologically relevant concentrations of NO6 and that they did not produce detectable quantities of peroxynitrite in isolated cardiac myocytes, we used 1 μmol/L SNAP for the majority of the experiments described herein.
Myocardial TNF mRNA and Protein Biosynthesis
Starting after the addition of SNAP (time 0), a 500 mg sample of myocardium was excised from the suspended heart (carefully sparing the large epicardial vessels) every 30 minutes for a total of 180 minutes. This sample was frozen in liquid nitrogen and stored at −70°C. Total RNA was extracted using the guanidinium thiocyanate/phenol method. For the detection of TNF-α mRNA, we developed a feline cDNA probe that was subsequently used for ribonuclease protection assays (see Data Supplement, which can be found at www.circulationaha.org). To detect TNF-α protein levels, 0.5 mL of recirculating buffer was collected every 30 minutes for a total of 180 minutes. TNF-α protein levels were determined by ELISA using a commercially available kit that recognizes feline TNF-α (Human Ultrasensitive Cytoscreen, Biosource).
Cellular Source for Myocardial TNF-α Biosynthesis
To determine whether NO was sufficient to induce TNF-α in cultured myocytes, we treated cultured cardiac myocytes with SNAP or diluent. The methods for isolating adult feline cardiac myocytes and the characteristics and purity of the cell culture system have been detailed previously.7 Total RNA was extracted 0, 1, and 6 hours after the addition of 1 μmol/L SNAP or diluent, and RNase protection assays were performed. Cytosolic TNF-α protein was determined at time 0 and at 1 and 6 hours after the addition of 1 μmol/L SNAP or diluent. Myocyte cultures were lysed with 200 μL of 0.05% Triton X-100 and harvested. A 100-μL aliquot of the cell lysate was used to determine total protein (Bicinchoninic Acid assay, Pierce), and the remaining cell lysate was used to measure TNF-α protein levels (ELISA).
Transcription Factors for TNF-α
To determine whether SNAP-induced myocardial TNF-α biosynthesis was mediated, at least in part, by the activation of NF-κB or AP-1, we performed electrophoretic mobility shift assays. Freshly isolated cat hearts were treated with 1 μmol/L SNAP or diluent for a total of 180 minutes. Starting at time 0 and every 30 minutes thereafter, we obtained myocardial biopsies (≈500 mg) from the SNAP-treated hearts. All myocardial samples were frozen and stored at −70°C. Electrophoretic mobility shift assays (EMSAs) were performed by incubating 10 μg of nuclear extracts from the SNAP-treated hearts with 8 fmol/L (20 000 cpm) of the double-stranded consensus sequence for either nuclear factor kappa-beta (NF-κB) (5′-AGTTGA-GGGGACTTTCCCAGGC-3′) or AP-1 (5′-CGCTTGATGACTC-AGCCGGAA-3′) (Data Supplement available at www.circulationaha.org). The specificity of binding was determined in “cold” competition experiments using 25× and 50× molar excess of the respective unlabeled oligonucleotides. To determine the components of the DNA-protein binding complexes, we performed supershift assays by incubating the nuclear extracts with 2 μg of anti-human polyclonal antibodies directed against the various components of NF-κB, including p50, p52, p65 (Rel A), Rel B, and cRel, for 15 minutes at room temperature before the addition of the labeled oligonucleotide consensus sequences.
Role of the cGMP Pathway in TNF-α Biosynthesis and NF-κB Activation
Myocardial cGMP Levels and Protein Kinase G Activity
In preliminary control experiments, we determined that SNAP (1 μmol/L) stimulation of Krebs-Henseleit bicarbonate–perfused hearts led to a 14-fold increase in cGMP levels within 2 minutes and a 2-fold increase in protein kinase G (PKG) levels, whereas no increase occurred in either cGMP or PKG in diluent-treated hearts (Data Supplement available at www.circulationaha.org).
Inhibition and Activation of cGMP
To determine whether the cGMP pathway was important in terms of SNAP-induced TNF-α biosynthesis, we examined the effects of the activation and inhibition of the cGMP/PKG pathway. Buffer-perfused hearts were pretreated for 60 minutes with either 10−5 mol/L 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), a specific inhibitor of guanylyl cyclase, or with Rp-8-Br-cGMPS (25 μmol/L), a specific inhibitor of PKG, before stimulating the hearts with SNAP. Next, we stimulated freshly isolated hearts for 180 minutes with 10−5 mol/L 8-Br-cGMP, a biologically active cGMP analog. As an additional control experiment, we also stimulated a separate group of hearts for 180 minutes with 10−7 mol/L atrial natriuretic factor, a peptide that activates cGMP through an NO-independent pathway. We measured TNF-α mRNA by ribonuclease protection assay, protein biosynthesis by ELISA, and NF-κB activation by EMSA.
Mechanism for cGMP-Induced NF-κB Activation
To determine whether SNAP stimulation and cGMP stimulation phosphorylated IκBα on serine 32 (Ser32), we stimulated 35S-methionine/cysteine-labeled myocyte cultures with 1 μmol/L SNAP or 10−5 mol/L 8-Br-cGMP and incubated the cell lysates with either a C-21 antibody that recognizes the terminal 21 amino acid residues of the carboxyl terminus of IκBα or a phosphospecific antibody that recognizes IκBα only when it is phosphorylated on Ser32 (Data Supplement available at www.circulationaha.org). The IκBα protein-antibody complex was immunoprecipitated using protein A-agarose beads, and the gels were then fixed, soaked in a fluorographic solution, dried, and exposed.
To determine whether PKG was sufficient to directly phosphorylate IκBα on Ser32, we used a simple cell-free in vitro assay system (Data Supplement). For these studies, an IκBα-glutathione-S-transferase (GST) protein was incubated with 1000 U of PKG for 10 minutes, and the samples were incubated overnight with the phosphospecific Ser32 antibody. Immune complexes were precipitated and analyzed by SDS-PAGE, and the gels were vacuum-dried and exposed to x-ray film at −70°C.
Data are expressed as mean±SEM. One-way ANOVA was used to test for differences between group means. When appropriate, post hoc multiple comparisons were performed to test for differences between control and experimental groups (Dunnett’s test) or between experimental groups (Newman-Keuls test). Two-way ANOVA was used to evaluate overall differences in the means between different groups as a function of time. Significant differences were said to exist at P<0.05.
Myocardial TNF-α Biosynthesis Ex Vivo
Myocardial TNF-α mRNA Biosynthesis
The salient finding shown by Figure 1A⇓ is that treatment with SNAP (1 μmol/L) resulted in a time-dependent increase in TNF-α mRNA biosynthesis. As shown, TNF-α mRNA expression was detectable within 30 minutes, and it increased ≈7-fold by 180 minutes. Similar qualitative findings were observed in 2 additional SNAP-treated hearts. In contrast, TNF-α mRNA was not detectable in the hearts (n=3) that were treated with diluent alone, as we reported previously.1
Myocardial TNF-α Protein Biosynthesis
Figure 1B⇑ shows that TNF-α protein synthesis was detectable as early as 90 minutes after SNAP (1 μmol/L) treatment, and it increased ≈19-fold by 180 minutes. In contrast, TNF-α protein levels were not detectable in the diluent-treated hearts. The inset of Figure 1B⇑ shows that the SNAP-induced (10−9 to 10−4 mol/L) increase in TNF-α protein biosynthesis was concentration-dependent (P<0.001 by ANOVA). Post hoc multiple comparison testing showed that there were significant differences (P<0.05) between these and control values for SNAP concentrations ≥10−9 mol/L.
Two additional control studies were performed to be certain that the SNAP-induced TNF-α biosynthesis was not a nonspecific effect of the NO donor chosen. First, we pretreated hearts (n=3) for 60 minutes with the NO-scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (C-PTIO) (10−4 mol/L) and then treated them with 1 μmol/L SNAP for 180 minutes. Second, we stimulated another group of hearts (n=3) for 180 minutes with 5 μmol/L sodium nitroprusside, a structurally dissimilar NO donor. As shown in Figure 2A⇓, pretreatment with C-PTIO completely abrogated TNF-α mRNA expression, whereas sodium nitroprusside treatment induced TNF-α mRNA synthesis. Figure⇑ 2B shows that C-PTIO prevented SNAP-induced TNF-α protein synthesis, whereas sodium nitroprusside provoked TNF-α protein synthesis.
Cellular Source for Myocardial TNF-α Biosynthesis
To determine whether SNAP stimulation was sufficient to provoke TNF-α biosynthesis in adult cardiac myocytes, we examined TNF-α mRNA and protein biosynthesis in diluent and SNAP-treated adult cardiac myocytes cultures. Figure 3A⇓ shows that TNF-α mRNA levels were barely detectable in the diluent-treated cultures, whereas TNF-α mRNA levels were increased in a time-dependent manner in the SNAP-treated (1 μmol/L) myocyte cultures. Similar qualitative results were observed in 2 additional primary myocyte isolations. Figure 3B⇓ shows that TNF-α protein levels were negligible in diluent-stimulated myocyte cultures (n=6 cultures per time point), whereas an 11- and 16-fold increase occurred at 1 and 6 hours, respectively, in TNF-α protein synthesis in the SNAP-treated (1 μmol/L) myocyte cultures (n=6 cultures per time point). Two-way ANOVA indicated that a statistically significant difference existed in the effect of SNAP on myocyte TNF-α synthesis when compared with diluent treatment (P<0.001).
Transcription Factors for TNF-α
To determine whether SNAP-induced TNF-α biosynthesis was related to the activation of NF-κB and/or AP-1, we performed EMSAs in SNAP-treated hearts. Figure 4A⇓ shows the temporal increase in NF-κB–DNA complexes in SNAP-treated (1 μmol/L) hearts. As shown, the NF-κB binding activity was increased at as early as 30 minutes, which is consistent with the rapid onset of TNF-α mRNA biosynthesis in the SNAP-treated hearts. After 180 minutes of SNAP stimulation, 3 separate binding complexes were detectable by EMSA; they were denoted C1 (slowest moving), C2, and C3 (fastest moving). Similar findings were obtained in 3 additional experiments. The specificity of the binding to the radiolabeled oligonucleotides was demonstrated by cold chase experiments, in which the C1 through C3 complexes decreased in intensity and eventually disappeared when a 25- and 50-fold molar excess of unlabeled oligonucleotide was added, respectively, to the nuclear extracts. To determine the specific components that comprised the NF-κB/Rel complexes, we also performed supershift assays using antibodies to the various known components of NF-κB and Rel (p50, p52, p65, RelB, and cRel). These studies showed that NF-κB protein complexes 2 and 3 were comprised of p50 and p65 proteins. In contrast to the findings observed with NF-κB, we did not observe any evidence of AP-1-DNA binding activity in nuclear extracts from the SNAP-treated hearts. As shown (Figure 4C⇓), angiotensin II (positive control) was sufficient to activate AP-1.
Role of the cGMP Pathway in TNF-α Biosynthesis and NF-κB Activation
Inhibition and Activation of cGMP
We next examined the effects of inhibiting and activating the cGMP/PKG pathway in our model system. For the series of studies that follow, TNF-α mRNA and protein levels are presented in Figures 5A⇓ and 5B⇓, respectively, and the corresponding NF-κB gel shift assays are presented in Figure⇑ 5C. When hearts were pretreated for 60 minutes with either ODQ (10−5 mol/L), an inhibitor of guanylyl cyclase, or Rp-8-Br-cGMPS (2.5×10−5 mol/L), an inhibitor of PKG, before stimulating them with 1 μmol/L SNAP for 180 minutes, TNF-α mRNA/protein biosynthesis and NF-κB activation were completely abrogated. Treating hearts with 10−5 mol/L 8-Br-cGMP or 10−7 mol/L atrial natriuretic factor resulted in TNF-α mRNA/protein biosynthesis and NF-κB activation. Similar qualitative results were obtained for at least 3 hearts in each experimental group. Finally, we showed that stimulating the cells with 10−6 to 10−2 mol/L 8-Br-cGMP led to a dose-dependent increase in TNF-α biosynthesis in isolated myocytes, whereas stimulation with 10−6 to 10−2 mol/L 8-Br-cAMP had no discernible effect on TNF-α biosynthesis (Data Supplement).
Mechanism for cGMP-Induced NF-κB Activation
Insofar as our studies suggested that the cGMP/PKG pathway was sufficient to activate NF-κB, we next sought to determine whether PKG was sufficient to activate NF-κB through an IκBα-dependent mechanism. As shown in Figure 6A⇓, both SNAP (1 μmol/L) and 8-Br-cGMP (10−5 mol/L) were sufficient to lead to the rapid (<30 minute) degradation (≈60% decrease) in the level of IκBα protein in cultured adult cardiac myocytes; they also led to the phosphorylation of IκBα on Ser32, which is consistent with the rapid degradation of the phosphorylated IκBα protein reported in nonmyocyte cell types.8 To confirm these observations in cultured cells, we next asked whether PKG, a serine/threonine kinase,9 was sufficient to phosphorylate IκBα on Ser32 in a simple cell-free assay system. The important finding shown by lane 2 in Figure 6B⇓ is that PKG was sufficient to phosphorylate IκBα at the critical Ser32 residue, whereas phosphorylation of IκBα was not detectable in the absence of either PKG (lane 1) or the IκBα-GST fusion protein (lane 3). Similar results were obtained in 3 additional experiments.
The major conclusion to be drawn from this study is that pathophysiologically relevant concentrations of NO are sufficient to provoke TNF-α biosynthesis in the adult mammalian heart. Three distinct but mutually complementary lines of evidence support this statement. First, treatment of isolated buffer-perfused adult cat hearts with concentrations of SNAP that are known to generate pathophysiologically relevant concentrations of NO6 resulted in a robust increase in TNF-α mRNA (Figure 1A⇑) and protein biosynthesis (Figure⇑ 1B). Consistent with previous observations from this laboratory, neither TNF-α mRNA nor protein biosynthesis were observed in diluent-treated hearts.1 The fact that we could completely inhibit the effects of SNAP-induced TNF-α mRNA (Figure⇑ 2A) and protein biosynthesis (Figure 2B⇑) with an NO scavenger (C-PTIO) and mimic the effects of SNAP with a structurally dissimilar NO donor (sodium nitroprusside) suggests that the effects of SNAP were related to the generation of NO and not to a nonspecific effect of the NO donor. Second, these findings were evident at the level of the intact ventricle and in the isolated cardiac myocyte itself. That is, stimulation of relatively pure (>98%) cultures of isolated adult cardiac myocytes with SNAP resulted in an 11- and 16-fold increase in TNF-α biosynthesis at 1 and 6 hours, respectively (Figure 3⇑). Third, SNAP stimulation resulted in the rapid (30 minutes) activation of NF-κB, an important transcription factor for TNF-α (Figure 4⇑). Importantly, the time course for activation of NF-κB was entirely consistent with the rapid de novo expression of TNF-α mRNA that we observed after SNAP stimulation. Moreover, our findings suggest that the SNAP-induced NF-κB/Rel complexes were comprised of p50/p65 heterodimers.
A second important finding of this study was that NO provoked TNF-α biosynthesis through a cGMP-dependent pathway. In preliminary control experiments (Data Supplement) we confirmed that SNAP stimulation of isolated hearts resulted in a rapid 14-fold increase in myocardial cGMP and a 2-fold increase in myocardial PKG activity. Importantly, we were able to completely abrogate TNF-α mRNA (Figure 5A⇑) and protein biosynthesis (Figure 5B⇑), as well as NF-κB activation (Figure 5C⇑), when we used specific inhibitors to block NO-induced guanylyl cyclase or PKG activity. To provide more direct evidence for the role of the cGMP pathway, 3 additional studies were performed. First, we could mimic the effects of SNAP on TNF-α mRNA and protein biosynthesis and NF-κB activation (Figure 5⇑) using 8-Br-cGMP, a biologically active cGMP analog. Second, when we stimulated hearts with atrial natriuretic factor, a peptide that stimulates cGMP through an NO-independent pathway, we could provoke TNF-α mRNA and protein biosynthesis and NF-κB activation (Figure 5⇑). Third, in additional control experiments (Data Supplement), we determined that the SNAP-induced TNF-α biosynthesis was not secondary to cross-talk and/or activation of cAMP, as has been described in rat aortic smooth muscle cells.10 To determine the mechanism for the observed cGMP/PKG-mediated activation of NF-κB, we demonstrated that the stimulation of isolated cardiac myocytes with SNAP or 8-Br-cGMP led to the degradation of IκBα and to the phosphorylation of IκBα at the critical Ser32 residue (Figure 6A⇑). Insofar as the above studies suggested that PKG might be acting as an IκBα kinase, we further demonstrated that PKG was sufficient to phosphorylate IκBα on its Ser32 residue in a simple, cell-free, in vitro kinase assay. Thus, in summary, the above results suggest that NO provokes TNF-α biosynthesis through a pathway that involves the activation of cGMP, PKG, and NF-κB.
Although the findings in the present study are consistent with earlier reports that have implicated NO and/or cGMP in TNF-α biosynthesis,5 11 they seem to disagree with previous reports which suggest that NO decreases NF-κB-DNA binding12 and suppresses TNF-α biosynthesis.13 14 There are several possible explanations for the observed differences. For example, the cell types and the nature and doses of the NO-donors used in the aforementioned studies were different from those used in the present study. Furthermore, apart from these more obvious methodological differences, there is, perhaps, a more intriguing explanation. That is, in all of the studies in which NO was shown to decrease NF-κB activation and/or TNF-α expression, the cells had been previously stimulated with agents that activate NF-κB before being treated with NO. Thus, in settings in which NF-κB is already activated, the primary effect of NO might be inhibitory, either by increasing the NF-κB–induced transcription of inhibitory IκB proteins or, alternatively, by stabilizing IκB.13 However, in the absence of prior NF-κB activation, the primary effect of NO might be stimulatory, as was observed in the present study. Indeed, this point of view is consonant with the recent observation in endothelial cells that low concentrations of NO are sufficient to activate NF-κB, whereas higher concentrations of NO inhibit NF-κB activation.15
The present study constitutes the initial demonstration that NO provokes TNF-α biosynthesis through the cGMP pathway. Although the exact biological significance of these findings is not known, the results of this study may provide new insight into one mechanism for the progressive cardiac decompensation that occurs after sustained myocardial injury. That is, given that NO provokes myocardial TNF-α biosynthesis and that TNF-α is sufficient to induce iNOS expression, the coincident expression of TNF-α and NO in the heart might foster self-sustaining positive autocrine/paracrine feedback inflammatory circuits within the myocardium that lead to the inappropriate overexpression of these 2 inflammatory mediators. Additionally, these studies suggest the intriguing possibility that cross-talk may exist between 2 signal transduction pathways that have, heretofore, been considered functionally distinct in the heart (ie, the adrenergic system and the cytokine system). Indeed, although it has long been recognized that TNF-α can induce the expression of iNOS in certain cell types, what is less well appreciated is that catecholamines stabilize iNOS mRNA in cardiac myocytes.16 Thus, both cytokines and adrenergic mediators may converge on and amplify the expression of NO in certain disease states. Whether either of these mechanisms provides a satisfactory explanation for the sustained expression of TNF-α and iNOS in the failing human heart will require further study.
The authors gratefully acknowledge the secretarial assistance of Jana Grana, as well as the spirited technical assistance of Dorellyn Lee-Jackson and Stacey Walker. The authors also thank Dr Andrew I. Schafer for his past and present support and guidance. This research was supported by research funds from the Department of Veterans Affairs and the National Institues of Health (grants P50 HL-O6H, RO1 HL58081-01, and RO1 HL61543-01).
Guest Editor for this article was Wilson S. Colucci, MD, Boston Medical Center, Boston, Mass.
Supplementary material for this article can be found Online at www.circulationaha.org
- Received February 18, 2000.
- Revision received April 7, 2000.
- Accepted April 10, 2000.
- Copyright © 2000 by American Heart Association
Kapadia S, Oral H, Lee J, et al. Hemodynamic regulation of tumor necrosis factor-α gene and protein expression in adult feline myocardium. Circ Res. 1997;81:187–195.
Torre-Amione G, Kapadia S, Lee J, et al. Tumor necrosis factor-α and tumor necrosis factor receptors in the failing human heart. Circulation. 1996;93:704–711.
Haywood GA, Tsao PS, von der Leyer HE, et al. Expression of inducible nitric oxide synthase in human heart failure. Circulation. 1996;93:1087–1094.
Balligand JL, Ungureanu D, Kelly RA, et al. Abnormal contractile function due to induction of nitric oxide synthesis in rat cardiac myocytes follows exposure to activated macrophage-conditioned medium. J Clin Invest. 1993;91:2314–2319.
Lander HM, Sehajpal P, Levine DM, et al. Activation of human peripheral blood mononuclear cells by nitric oxide-generating compounds. J Immunol. 1993;150:1509–1516.
Majid DS, Omoro SA, Chin SY, et al. Intrarenal nitric oxide activity and pressure natriuresis in anesthetized dogs. Hypertension. 1998;32:266–272.
Yokoyama T, Nakano M, Bednarczyk JL, et al. Tumor necrosis factor-α provokes a hypertrophic growth response in adult cardiac myocytes. Circulation. 1997;95:1247–1252.
Komalavilas P, Lincoln TM. Phosphorylation of the inositol 1,4,5-trisphosphate receptor by cyclic GMP-dependent protein kinase. J Biol Chem. 1994;269:8701–8707.
Cornwell TL, Arnold E, Boerth NJ, et al. Inhibition of smooth muscle cell growth by nitric oxide and activation of cAMP-dependent protein kinase by cGMP. Am J Physiol. 1994;267:C1405–C1413.
Peng HB, Spiecker M, Liao JK. Inducible nitric oxide: an autoregulatory feedback inhibitor of vascular inflammation. J Immunol. 1998;161:1970–1976.
Peng HB, Libby P, Liao JK. Induction and stabilization of IKBa by nitric oxide mediates inhibition of NF-kB. J Biol Chem. 1995;14214–14219.
Eigler A, Moeller J, Endres S. Exogenous and endogenous nitric oxide attenuates tumor necrosis factor synthesis in the murine macrophage cell line RAW 264.7. J Immunol. 1995;154:4048–4054.