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(Circulation. 1997;96:1937-1943.)
© 1997 American Heart Association, Inc.

Articles

Chronic Hypoxia Modulates the Interleukin-1ß–Stimulated Inducible Nitric Oxide Synthase Pathway in Cardiac Myocytes

Rachid Kacimi, PhD; Carlin S. Long, MD; ; Joel S. Karliner, MD

From the Cardiology Section of the Veterans Affairs Medical Center, the Cardiovascular Research Institute, and the Department of Medicine, University of California, San Francisco.

Correspondence to Dr Joel S. Karliner, Chief, Cardiology Section, VA Medical Center, 4150 Clement St, San Francisco, CA 94121. E-mail karliner.joel-s{at}sanfrancisco.va.gov

	Abstract

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Background We wished to determine whether the cytokine-inducible nitric oxide synthase (iNOS) pathway is modulated by chronic hypoxia in vitro.

Methods and Results We investigated the effects of the proinflammatory cytokine interleukin (IL)-1ß on expression of iNOS mRNA, iNOS protein, and NO production in cultured neonatal rat cardiomyocytes subjected to 1% O₂ for 48 hours. Among several cytokines tested, IL-1ß was the most effective in stimulating NO production, which was maximum at 48 hours. In parallel, IL-1ß induced expression of both iNOS mRNA and protein. Hypoxia alone had no effect on NO production, iNOS gene expression, or protein induction. However, chronic hypoxia decreased IL-1ß–stimulated NO production, mRNA expression, and protein level in cardiac myocytes. Radioligand binding and electrophoretic mobility shift assays showed that during chronic hypoxia, IL-1 receptor density and activity of the transcription factor NF-B induced by IL-1ß were decreased, which may account at least in part for the decrease in iNOS expression.

Conclusions These data indicate that IL-1ß induces iNOS gene expression, de novo synthesis of iNOS protein, and NO generation in neonatal rat cardiomyocytes and that chronic hypoxia appears to be a potent negative regulator of iNOS expression in these cells.

Key Words: interleukins • nitric oxide synthase • signal transduction • hypoxia

	Introduction

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Nitric oxide is a short-lived gaseous radical and the smallest biologically active metabolite yet identified. Recent work has implicated NO as the chemical mediator of myocardial depression during bacterial sepsis.^{1 2 3} Myocardial NO also increases in response to cytokines such as IL-1ß in both neonatal and adult cardiac myocytes. It has been suggested that the increase in cytokines that accompanies inflammatory processes such as viral myocarditis and cardiac transplant rejection^{3 4 5 6} is involved in the transient contractile dysfunction seen under these circumstances. Because similar contractile dysfunction often occurs after myocardial ischemia/reperfusion, it is likely that cytokines (and by extension NO) are also involved in the process of postischemic stunning.^{7 8 9 10} Although NO production has been reported to be induced in some cells in response to hypoxia, this phenomenon has not been documented in myocardial cells. Furthermore, the role of NO in ischemia/reperfusion has been reported to be both positive (related to decreased platelet adhesion and vasodilatation) and negative (attributed to the induction of free radicals, inactivation of mitochondrial enzymes, and decreased myocardial contractility).^{11 12 13 14 15 16 17 18} A more rational approach to myocardial salvage after occlusion requires a better understanding of potential interactions among variables known to be present during ischemia/reperfusion in the heart, namely, hypoxia, cytokines, and NO.

Chronic hypoxia and/or ischemia has been shown to modulate NO responses in different cell models,^{19 20 21 22 23 24} but the relationship between hypoxia and NOS regulation is not well understood. McQuillan et al²⁵ found a decrease in NO production and constitutive NOS mRNA expression in endothelial cells exposed to chronic hypoxia. In contrast, Arsher et al²⁶ recently showed that induction of iNOS is resistant to graded hypoxia (21% to 2.5% O₂) in mesangial cells. To the best of our knowledge, however, the effect of prolonged hypoxia on iNOS regulation has not previously been investigated in cardiac cells. We undertook the present study to determine whether induction of nitric oxide synthase by the proinflammatory cytokine IL-1ß is modulated by hypoxia in myocardial cells. Furthermore, it is believed that expression of the iNOS gene is controlled at the transcriptional level and is mediated at least in part by the transcription factor NF-B.^{27 28} To address the potential role of this factor in cardiac myocytes, we also examined the effect of IL-1ß on NF-B activation.

	Methods

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Induction of Hypoxia
Primary myocyte cell cultures were prepared by enzymatic dissociation of ventricular tissue from 1-day-old Sprague-Dawley rat pups as previously described in our laboratory.²⁹ Experiments at low oxygen tension were performed in an airtight Plexiglas humidified chamber (Anaerobic Environment, Sheldon), maintained at 37°C and continuously gassed with a mixture of 98%N₂/1% CO₂/1%O₂.²⁹ Cells were placed into the hypoxia chamber on culture day 4 and remained for 48 hours. Maintenance of the desired O₂ concentration was routinely monitored during incubation with an O₂ electrode (Controls Katharobic System). In concurrent experiments, normoxic conditions were created by placing cultured cells in a Forma Scientific incubator (gassed with 99% air/1% CO₂ at 37°C). We have previously shown that incubation of cells in 1% O₂ for up to 72 hours does not cause lactate dehydrogenase release or changes in pH, ATP levels, or cell counts.²⁹

Treatment of Cells and Determination of Nitrite Accumulation
After overnight incubation, serum-free medium was removed and fresh medium added. Cytokines, drugs, or vehicle was then added, and cells were returned to the incubator or placed in the hypoxia chamber. All reagents that could potentially contain endotoxin were analyzed for endotoxin content by the manufacturer. In each instance, the concentration used in our experiments was <0.1 ng/mL by the limulus amebocyte lysate assay. After incubation for 24 or 48 hours, aliquots of the incubation media were removed for determination of nitrite content by the Griess reaction.³⁰

RNA Extraction and RT-PCR Analysis
After treatment, cells were washed twice with cold PBS, and total RNA was prepared from cultured myocytes by acid guanidinium thiocyanate–phenol-chloroform extraction.³¹ RNA was quantified spectrophotometrically and electrophoresed on ethidium bromide–stained agarose gels to check its integrity.

First-strand cDNA synthesis was performed by reverse transcription of total myocyte RNA from cultures treated with IL-1ß or vehicle under normoxic or hypoxic conditions. Total RNA was reverse transcribed for 1 hour at 42°C in 20 µL of a mixture containing 20 pmol/L (final concentration) of oligo (dT) primers, 1xPCR buffer, 3 mmol/L MgCl₂, 0.5 mmol/L dNTPs, 20 U of RNase inhibitor, and 200 U of Moloney murine leukemia virus reverse transcriptase followed by incubation at 94°C for 5 minutes. The resulting cDNA was amplified by use of a set of sense (5'-CCCTTCCGAAGTTTCTGGCAGCAGC-3') and antisense (5'-GGCTGTCAGAGCCTCGTGGCTTTGG-3') iNOS primers (from Clontech Laboratories). The first-strand cDNA was also subjected to PCR with the housekeeping gene ß-actin–specific sense (5'-TTGTAACTGGGACGATATGG-3') and antisense (5'-GATCTTGATCTTCATGGTGCTAGG-3') primers (Clontech) according to the following thermocycling parameters: each cycle consisted of incubations at 94°C for 45 seconds, 65°C for 45 seconds, and 72°C for 2 minutes for a total of 35 cycles followed by a 7-minute extension at 72°C. Products were analyzed by agarose gel electrophoresis (2%) and visualized by ethidium bromide staining under ultraviolet light. Single PCR-amplified products of the expected size were obtained for iNOS (496 bp) and ß-actin (764 bp). X174 DNA/HaeIII fragments (DNA ladder) were used as DNA size markers (Promega Inc). The fidelity of the PCR product was confirmed by DNA sequencing analysis (Biomolecular Resource Center at the University of California at San Francisco).

Western Blot Analysis and EMSA
Cell lysates were subjected to SDS-PAGE by the method of Laemmli.³² Western blotting³³ was performed with a mouse monoclonal antibody against the synthetic COOH-terminal fragment of rat iNOS (Transduction Laboratories). Immune complexes were visualized with the ECL detection system (Amersham).

Nuclear extracts were prepared by the method described by Dignam et al.³⁴ An oligonucleotide probe for the NF-B consensus sequence (Promega) was end-labeled with [-³²P]ATP by incubation with T4 polynucleotide kinase at 37°C for 10 minutes. The labeled probe was separated from unincorporated nucleotide in a spin column (BioRad). EMSA experiments were performed by incubation of 10 µg of nuclear extracts in 20 µL of binding buffer (50 mmol/L Tris [pH 7.5], 250 mmol/L NaCl, 2.5 mmol/L DTT, 5 mmol/L MgCl₂, 2.5 mmol/L EDTA, 0.25 mg/mL poly[dI-dC], and 10% glycerol) for 10 minutes at room temperature. For competition experiments, an excess of unlabeled NF-B and nuclear protein were preincubated in the binding buffer for 10 minutes. ³²P-labeled oligonucleotide probe (20 000 to 50 000 cpm) was then added, and the reaction mixture was incubated for 20 minutes at room temperature. The reaction was stopped by addition of 2 µL of 10x loading buffer (250 mmol/L Tris [pH 7.5], 0.2% bromphenol blue, 0.2% xylene cyanol). Samples were electrophoresed in native 4% polyacrylamide gels in running buffer (0.5x Tris-borate-EDTA) at 100 V for 3 hours. The gels were then dried and exposed to autoradiographic x-ray film (X-Omat AR-5, Eastman Kodak Co) with an intensifying screen for 6 to 12 hours at -80°C.

Measurement of IL-1RA
Cardiac myocyte culture supernatant was harvested at 48 hours after treatment with human recombinant IL-1ß, hypoxia, or their combination, and IL-1RA (R&D Systems, Inc) was measured by sandwich ELISA according to the manufacturer's instructions. The sensitivity limit of the ELISA is 14 pg/mL.

IL-1R Binding Assay
IL-1R density and agonist affinity were determined by radioligand binding techniques. After 48 hours of treatment, cells were harvested and washed twice with ice-cold PBS buffer, and membranes were prepared as previously described in our laboratory.²⁹ Membranes were then incubated in buffer containing 25 pmol/L human recombinant [¹²⁵I]IL-1ß (2410 Ci/mmol) (Amersham Inc) with or without inclusion of 12 concentrations of unlabeled human recombinant IL-1ß (R&D Systems Inc), ranging from 1 pmol/L to 100 nmol/L. The buffer contained 50 mmol/L Tris, 10 mmol/L MgCl₂, 1 mmol/L EGTA, and 0.2% BSA at pH 7.4 in a total assay volume of 250 µL. Assays were performed in duplicate for 2 hours at 22°C; these conditions allowed complete equilibration of the receptors with the radioligand. The reaction was terminated by rapid vacuum filtration through Whatman GF/C filters, which were immediately washed three times with 6 mL of ice-cold incubation buffer each time. Filters were then counted in a gamma counter. Maximum receptor density and the dissociation constant were determined from competition binding experiments with an iterative nonlinear curve-fitting program.

Data Analysis
Values are expressed as mean±SEM. Differences in the means among the groups were tested by ANOVA where appropriate. If the F test showed an overall significance, post hoc comparison among multiple groups was performed with the Student-Newman-Keuls test. Values of P<.05 were considered significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
NO Production
There is considerable evidence that the proinflammatory cytokine IL-1ß stimulates NO production in neonatal rat cardiac myocytes.^{35 36} In initial experiments, we confirmed that a time-dependent increase in NO production is induced by IL-1ß (10 ng/mL) with significant stimulation at 48 hours (P<.0001, n=12) (Fig 1). The concentration of IL-1ß we used was based on the concentration-response data reported by Tsujino et al,³⁵ who observed a response plateau of NO production at 10 ng/mL in a similar neonatal rat ventricular myocyte preparation. In contrast, there is conflicting evidence regarding the effects of tumor necrosis factor- on NO induction in myocytes.^{36 37} We found that tumor necrosis factor- (10 ng/mL) had no effect on NO production in cardiac myocytes at either 24 or 48 hours (n=7); in addition, neither IL-6 (10 ng/mL) nor interferon- (10 ng/mL) stimulated NO production (data not shown).

View larger version (23K): [in this window] [in a new window]   Figure 1. Effect of chronic hypoxia on IL-1ß–stimulated NO production. Neonatal rat ventricular myocytes were incubated with vehicle (PBS/BSA, control) or IL-1ß (10 ng/mL) and subjected to 1% O2 (hypoxia) or normoxia for 24 or 48 hours. Thereafter, medium was removed and used for nitrite determination. Background values present in medium without cells were subtracted. Addition of cells caused no changes. Results are mean±SEM of seven experiments. *P<.02 vs normoxia; **P<.0001 vs normoxia; ¶P<.006 vs normoxia at 24 hours.

Chronic hypoxia (1% O₂, 48 hours) by itself did not influence NO release at any time studied (24 to 48 hours). Rather, we found that chronic hypoxia tended to decrease IL-1ß–induced NO production after 24 hours, with a marked reduction after 48 hours (P<.0001, n=7) (Fig 1).

To examine the possible influence of hypoxia and IL-1ß treatment on myocyte viability, we measured protein content, lactate dehydrogenase release (LDH kit from Sigma Inc), and cell death (Eukolight viability/cytotoxicity kit from Molecular Probes Inc). We found that hypoxia alone, IL-1ß alone, or their combination had no significant effects on these measures (data not shown).

To confirm the specificity of IL-1ß on iNOS induction, we used the NOS inhibitor L-NMMA. Fig 2 shows that coincubation of IL-1ß with 1 mmol/L L-NMMA completely blocked NO production induced by IL-1ß alone, confirming that NO accumulation induced by IL-1ß is due to an increase of iNOS activity in cardiac myocytes.

View larger version (33K): [in this window] [in a new window]   Figure 2. Inhibition of IL-1ß–stimulated NO release. Effects of 1 mmol/L L-NMMA (n=5), 1 µmol/L dexamethasone (DEX, n=3), 10 µg/mL cycloheximide (CYC, n=4), 100 nmol/L calphostin C (CAL, n=4), and 10 µmol/L genistein (GEN, n=4) on IL-1ß–stimulated nitrite production over 48 hours. Background values were subtracted as described above. n indicates number of separate experiments. One-way ANOVA was significant (F=13.48, P<.0001). **P<.05 vs IL-1ß stimulation by post hoc testing vs control (Student-Newman-Keuls).

There have been conflicting reports on the second messenger pathways involved in iNOS stimulation by cytokines. Both PKC and tyrosine kinase pathways have been implicated in several different cell types.^{38 39 40 41 42} The tyrosine kinase inhibitor genistein (10 µmol/L) inhibited IL-1ß–induced NO production, whereas the PKC inhibitor calphostin C did not (Fig 2). Furthermore, agonists known to augment PKC activity, angiotensin II (100 nmol/L), phorbol myristate acetate (100 nmol/L), and norepinephrine (2 µmol/L), did not stimulate NO production (data not shown). In additional experiments, the protein synthesis inhibitor cycloheximide (10 µg/mL) and the glucocorticoid dexamethasone (1 µmol/L) also inhibited IL-1ß–induced NO production (Fig 2), as reported by others.^{35 36}

iNOS Protein
To further clarify whether the induction of NO elicited by IL-1ß is due to activation of preformed NOS or an increase in iNOS protein, the amount of iNOS protein was measured by immunoblot analysis. A positive control (cell lysate from a stimulated macrophage cell line) showed the expected band of 130 kD, which corresponds to iNOS (Fig 3A). Although no iNOS protein was detectable in control myocytes, there was a clear induction of iNOS protein after a 48-hour treatment with IL-1ß (Fig 3A).

View larger version (34K): [in this window] [in a new window]   Figure 3. IL-1ß-induced iNOS protein expression and effect of chronic hypoxia. Neonatal ventricular myocytes were incubated with or without IL-1ß (10 ng/mL) for 48 hours under normoxic or hypoxic conditions. Cell lysates were subjected to SDS-PAGE followed by immunoblotting as described in "Methods." Mouse macrophage cell lysates (Transduction Laboratories) were used as positive control. Stimulation was for 12 hours with 10 ng/mL interferon- and 1 µg/mL lipopolysaccharide.39 A, IL-1ß induces iNOS protein expression. Positive control is shown on left, indicating position of 130-kD iNOS protein. iNOS protein was undetectable in controls. Data are representative of 10 separate experiments. B, Chronic hypoxia decreases IL-1ß–induced iNOS protein expression. Figure is representative Western blot analysis of three separate experiments.

Chronic hypoxia did not produce any detectable increase in iNOS protein (Fig 3B). Similar to the decrease in NO production, chronic hypoxia decreased iNOS protein induced by IL-1ß in cardiac myocytes (Fig 3B).

iNOS mRNA
Because the increase in iNOS protein could be due to an increase in gene transcription, we analyzed iNOS mRNA using specific PCR primers chosen on the basis of the published sequence of murine macrophage iNOS cDNA.⁴³ iNOS mRNA could not be detected in cultured neonatal myocytes under baseline conditions. However, stimulation of myocytes with IL-1ß (10 ng/mL) for 48 hours resulted in the appearance of the expected 496-bp amplified product (Fig 4). We used the housekeeping gene ß-actin as an internal control, which demonstrates successful first-strand cDNA synthesis for the control RNA (Fig 4). Fig 5 shows the effect of chronic hypoxia on iNOS mRNA expression performed with RT-PCR by coamplification of iNOS with ß-actin. No signal was detected after 48 hours of exposure to hypoxia in myocytes (Fig 5). However, as had been seen with both iNOS protein and NO accumulation, iNOS mRNA levels induced by IL-1ß were markedly reduced in the presence of chronic hypoxia (Fig 5).

View larger version (22K): [in this window] [in a new window]   Figure 4. RT-PCR analysis of iNOS mRNA induction. Myocytes were treated with IL-1ß (10 ng/mL) or vehicle (PBS/BSA, control) for 48 hours, and RT-PCR using primers for iNOS (496 bp) and ß-actin (764 bp) was performed as described in "Methods." Top, iNOS mRNA expression induced by IL-1ß; bottom, simultaneous amplification of housekeeping gene ß-actin as internal reference. Negative control reaction for RT-PCR using primers for iNOS or ß-actin was performed without addition of reverse transcriptase (lane labeled RT-). Mr indicates size marker lane for X174 RF DNA digested with HaeIII.

View larger version (51K): [in this window] [in a new window]   Figure 5. Effect of chronic hypoxia on iNOS mRNA expression. Myocytes were incubated with vehicle (PBS/BSA, control) or IL-1ß (10 ng/mL) and subjected to normoxia or hypoxia (1% O2) for 48 hours. RT-PCR analysis was performed by coamplification of primers for iNOS and ß-actin as described in "Methods." Positions of ß-actin (764 bp), which serves as internal amplification standard, and iNOS (496 bp) mRNAs are indicated by upper and lower arrows, respectively. Negative control reaction for RT-PCR using primers for iNOS and ß-actin was performed without addition of reverse transcriptase in the reaction (lane labeled RT-). Mr indicates DNA size marker lane as in Fig 4. Combination of chronic hypoxia with IL-1ß blunted induction of iNOS mRNA compared with IL-1ß alone by 50% as measured by densitometry analysis. Data are representative of five separate experiments.

NF-B Activity
The experiments described above indicate conclusively that IL-1ß increases NO production by increasing iNOS mRNA transcription and protein synthesis and that this effect is blunted by chronic hypoxia. An EMSA performed on nuclear extracts from myocytes treated with 10 ng/mL of IL-1ß showed an increase in the binding activity of NF-B at 6 hours. This effect was partially blocked by the tyrosine kinase inhibitor genistein (Fig 6A). We also showed that the increase of NF-B activity after 48 hours is blunted by chronic hypoxia (Fig 6C). The gel shift bands were specific NF-B–DNA–protein complexes, because the addition of excess unlabeled NF-B oligonucleotide to the nuclear extract specifically abolished the NF-B signal (Fig 6B).

View larger version (26K): [in this window] [in a new window]   Figure 6. A, EMSA of NF-B binding. Myocytes were treated with IL-1ß, IL-1ß+genistein, or vehicle (PBS/BSA/DMSO 0.005%) control for 6 hours. Nuclear extracts from cells were incubated with a 32P-labeled consensus NF-B binding site followed by electrophoresis through a 4% polyacrylamide gel in 0.5xTris-borate-EDTA. Lane 1, control; lane 2, IL-1ß; lane 3, IL-1ß+genestein. The tyrosine kinase inhibitor genistein inhibited NF-B binding stimulated by IL-1ß. Data are representative of three separate experiments. B, NF-B competition binding assay. Specificity of DNA binding complex was evaluated by competition with excess unlabeled NF-B oligonucleotide. Lane 1, control; lane 2, IL-1ß; lane 3, IL-1ß+100x excess unlabeled NF-B, which completely displaced labeled NF-B binding. Data are representative of four separate experiments. C, Chronic hypoxia blocks IL-1ß–induced NF-B activity. Myocytes were treated with IL-1ß (10 ng/mL) under hypoxic (1% O2) or normoxic conditions for 48 hours. Nuclear extracts were incubated as described in "Methods." Lane 1, control; lane 2, IL-1ß; lane 3, hypoxia alone; lane 4, IL-1ß in combination with chronic hypoxia, which blocked IL-1ß–induced NF-B activity. Data are representative of five separate experiments.

IL-1RA and IL-1Rs
IL-1RA release in the culture medium was determined by quantitative ELISA. We found that neither hypoxia, IL-1ß, nor their combination induced IL-1RA. All values were in the range of background (<14 pg/mL, n=6). Cardiac myocyte IL-1Rs were investigated by use of the radioligand [¹²⁵I]IL-1ß. We found that cardiac myocytes express high-affinity functional IL-1Rs (dissociation constant, 10 pmol/L). Fig 7 shows the density of IL-1Rs in cardiac myocyte membranes. We found that incubation with IL-1ß (10 ng/mL) for 48 hours upregulated its own receptor and that chronic hypoxia alone also significantly enhanced the density of binding sites but to a lesser extent than IL-1ß. During hypoxia, however, IL-1ß did not upregulate the density of IL-1Rs compared with control values. Receptor affinity did not differ significantly among the four treatment groups.

View larger version (29K): [in this window] [in a new window]   Figure 7. Effects of IL-1ß and chronic hypoxia on IL-1R binding. Maximal receptor density (Bmax) for IL-1Rs determined by radioligand binding competition curves. For details see "Methods." Control; IL-1ß 10 ng/mL for 48 hours; hypoxia 1% O2 for 48 hours; IL-1ß+hypoxia: combination of IL-1ß and hypoxia for 48 hours. One-way ANOVA was significant (F=13.74, P<.0001). *P<.05 by post hoc testing vs control (Student-Newman-Keuls). Results are from six separate experiments. IL-1ß induces an increase in receptor density; hypoxia also induces an increase in receptor density, but in the presence of hypoxia, IL-1ß no longer stimulates its own receptor. Agonist affinity values for each of the four conditions did not differ significantly.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The principal new finding of this study is that induction of iNOS by the inflammatory cytokine interleukin IL-1ß is modulated by chronic hypoxia in cardiac myocytes. The results indicate that IL-1ß is capable of inducing iNOS mRNA production, the appearance of iNOS protein, and subsequent generation of NO. Our data also show that activation of the transcription factor NF-B may constitute one potential mechanism of IL-1ß regulation of the iNOS gene in myocytes, a finding similar to that seen by others in macrophages^{28 44} and myocytes.⁴⁵ This possibility is emphasized by the novel observation that chronic hypoxia, which inhibits iNOS expression at the gene and protein levels, abolishes IL-1ß activation of NF-B DNA binding.

The molecular mechanism by which IL-1ß induces expression of iNOS in cardiac myocytes has not been clearly established. A recent report from our laboratory⁴⁶ showed that IL-1ß differentially regulates cardiac myocyte and fibroblast growth in culture and implicated a tyrosine kinase second messenger pathway in the regulation of IL-1ß–induced myocyte growth. However, the NO pathway does not participate in the IL-1ß–stimulated growth response in cardiac myocytes.^{46 47} In other studies, PKC-mediated pathways have been involved in IL-1ß–induced iNOS in macrophages, hepatocytes, and mesangial cells.^{38 39 48} The tyrosine kinase inhibitor genistein prevented NO production, which suggests that tyrosine kinase signaling pathways are involved in the regulation of NOS expression and activity induced by IL-1ß. Consistent with our results is a recent report that genistein blocks iNOS mRNA and NO production induced by IL-1ß in mesangial cells.⁴⁹ Moreover, in ventricular myocytes, Tsujino et al³⁵ showed that the PKC inhibitor calphostin did not prevent the effects of IL-1ß, suggesting that PKC was not involved in iNOS induction. Our data, taken together with these recent reports, make it unlikely that PKC pathways participate in iNOS induction in cardiac myocytes.

A direct effect of chronic hypoxia on iNOS gene expression is excluded by the failure of hypoxia alone to stimulate iNOS mRNA production, iNOS protein formation, or NO release. However, coincubation of myocytes with IL-1ß in the presence of chronic hypoxia downregulates iNOS expression and activity stimulated by IL-1ß. Our observations are in contrast to reports of an enhancement of interferon-–induced iNOS transcription by hypoxia in a macrophage cell line⁵⁰ or modulation of NO partition without a change in iNOS mRNA expression elicited by lipopolysaccharide during 24 hours of hypoxia in mesangial cells.²⁶ It was also found that short-term (30 minutes) hypoxia increased NO synthase activity stimulated by bradykinin and acetylcholine in adult myocytes,⁵¹ but no experiments examining the effects of long-term hypoxia on NO or iNOS in cardiac cells have previously been reported. The discrepancy between these studies and our data could be explained either by the specificity of cellular responses, the stimuli investigated and distinct signal pathways implicated, or the duration and severity of hypoxia.

Cytokine-inducible NOS is believed to be tightly controlled at several stages in its synthesis, and there is substantial evidence for transcriptional activation of the iNOS gene in the mouse macrophage.⁵² Recent work has identified the importance of NF-B/Rel proteins in iNOS induction.^{28 44 45 53} NF-B is an important transcription factor that is involved in the transmission of the signal from cytoplasm to the nucleus. Our study showed induction of NF-B binding activity by IL-1ß in cardiac myocytes, an effect that was attenuated by the tyrosine kinase inhibitor genistein. Our observation that chronic hypoxia also attenuated the IL-1ß–stimulated NF-B signal suggests that NF-B may be an important transcriptional activator of iNOS in cardiac myocytes.

We recognize that additional promoter analysis experiments would yield complementary information regarding the role of NF-B in cardiac myocyte iNOS gene induction. However, studies in which both murine and human iNOS promoter constructs containing mutated NF-B DNA motifs have been transfected into several different cell lines have already been reported to abolish iNOS induction, confirming the importance of this transcription factor.^{28 53} Moreover, data from other studies also support the key role of NF-B in iNOS regulation. For example, it has been reported that pyrrolidine dithiocarbamate, an inhibitor of NF-B activation, potently suppresses IL-1ß–induced iNOS in several different cell types, including cardiac myocytes.⁴⁵ It is thus well established that IL-1ß uses the NF-B pathway to trigger iNOS transcription. Although caution should be taken in extrapolating these studies because of the specificity of cell responses, our data taken together with these reports are consistent with the hypothesis that the attenuation of IL-1ß–induced NF-B by chronic hypoxia may be at least one mechanism by which chronic hypoxia inhibits iNOS gene and protein expression.

There is considerable evidence that iNOS is also regulated at the posttranscriptional level, and mRNA stability is a major control point in the regulation of NOS with both negative and positive features.^{52 54} iNOS protein is also regulated at the posttranslational level, which may influence the equilibrium between inactive and active forms.⁵² Our data showing virtually complete abolition of NO production despite partial preservation of iNOS mRNA and NF-B binding suggest that posttranslational mechanisms may be operative in hypoxia, eg, limitation of substrates or cofactors required for NO production such as biopterin, L-arginine, and heme.^{52 55} However, posttranscriptional or posttranslational modulation of iNOS or NO in chronic hypoxia was not directly addressed in the present study.

Sensitivity to IL-1 may also be altered by regulation of receptor number or release of the naturally occurring receptor antagonist (IL-1RA). It is well known that IL-1ß binds to specific receptors (IL-1RI and IL-1RII). However, the main IL-1R appears to be type 1 (IL-1RI), which mediates signal transduction in many cells, whereas the type II receptor (IL-1RII) is expressed only in a few cell types, in which it functions as a decoy receptor.⁵⁶ In addition, IL-1RA is a naturally occurring antagonist of IL-1 that inhibits the action of IL-1 by competitively binding to IL-1Rs and demonstrates no agonist activity.⁵⁷ To better understand the mechanism of the hypoxic blunting of the IL-1ß response, we asked whether either the ILs were downregulated or IL-1RA was released during hypoxia. Either of these phenomena could be an alternative explanation of our observations.

We found that hypoxia did not induce IL-1RA release, which makes it unlikely that IL-1RA is responsible for the decrease in the IL-1ß response. Using radioligand binding analysis, we report for the first time that IL-1Rs are expressed in cardiac myocytes and that IL-1ß treatment for 48 hours increases the density of IL-1Rs. Our data are in agreement with those of Ilyin and Plata-Salamar,⁵⁸ who found that IL-1ß increased IL-1Rs in vivo. Similar results were reported by Colotta et al,⁵⁹ who found that cytokines induced protein and mRNA transcripts of IL-1R. We also found that chronic hypoxia enhanced receptor density to a lesser extent than IL-1ß. The functional relevance of hypoxia-induced IL-1R binding sites is uncertain, however, because hypoxia alone did not induce NO synthesis. Interestingly, during IL-1ß stimulation in the presence of hypoxia, receptor density was unchanged compared with control. Assuming that upregulation of IL-1R observed during IL-1ß treatment could participate in IL-1ß–induced iNOS and NO production, which is maximal at 48 hours, it is possible that one mechanism by which hypoxia blunts IL-1ß–induced NO synthesis is by counteracting the upregulation of IL-1Rs.

In response to tissue injury, as happens during ischemia/reperfusion, cytokines induce both immune and nonimmune cells (eg, cardiac myocytes) to produce significant amounts of NO. This molecule and its oxidation products are toxic and can cause tissue injury, as described in both in vivo and in vitro models.^{17 18} Our experiments raise the possibility that sustained hypoxia, which is a principal component of ischemia, may protect against NO-mediated tissue damage by decreasing the ability of cytokines to induce iNOS gene transcription, possibly by inhibition of NF-B activation. Whether this occurs indirectly through the autocrine/paracrine production of growth factors or other agents that are potent inhibitors of NO production remains a subject for further investigation.

In conclusion, this study shows transcriptional regulation of iNOS expression by the proinflammatory cytokine IL-1ß in cardiac myocytes and that chronic hypoxia decreases iNOS expression at least in part by impairing IL-1ß signal transduction pathways that involve IL-1R binding and activity of the transcription factor NF-B.

	Selected Abbreviations and Acronyms

 

EMSA = electrophoretic mobility shift assay IL-1ß = interleukin-1ß IL-1R = interleukin-1 receptor IL-1RA = interleukin-1 receptor antagonist iNOS = inducible nitric oxide synthase L-NMMA = NG-monomethyl-L-arginine NF-B = (transcription factor) nuclear factor-B PKC = protein kinase C RT-PCR = reverse transcriptase–polymerase chain reaction

	Acknowledgments

 
This work was supported by Program Project Grant HL-25847 from the National Heart, Lung, and Blood Institute and the Research Service, Department of Veterans Affairs. We thank Norman Honbo and Trini Miguel for technical assistance.

Received January 15, 1997; revision received March 24, 1997; accepted March 26, 1997.

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