INTRODUCTION

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PROINFLAMMATORY CYTOKINES are a class of secretory polypeptides that are synthesized and released locally by macrophages, leukocytes, and endothelial cells in response to injury (1). Nitric oxide (NO) has been reported to play an important role as an effector molecule in cytokine signal transduction in a variety of cell types (5, 7). NO is formed from the amino acid L-arginine by a distinct family of NO synthases (NOS) (11). Two distinct constitutive isoforms of NOS have been cloned and sequenced from brain and endothelium (3, 8). Proinflammatory cytokines have been shown to induce a third isoform of this enzyme (iNOS) in a variety of other cell types, including cardiac myocytes (CM) (2). Potential modulatory effects of the autonomic nervous system on these immunologically mediated responses have not been adequately explored.

Norepinephrine (NE) is well known to activate - and -adrenergic receptors (6). These receptors have seven transmembrane domains and couple to multiple G proteins to modulate several distinct signal transduction pathways (10). Agonist binding to these receptors results in activation of protein kinase C (10, 19), protein kinase A (10), and mitogen-activated protein (MAP) kinase (19). We previously reported that interleukin-1 (IL-1) alone induced the transcription of iNOS mRNA, iNOS protein, and NO₂ production by isolated neonatal rat CM in culture (13). We now report that NE enhanced IL-1-induced NO₂ production by activation of - and -adrenergic receptors through a novel MAP kinase-dependent mechanism.

	MATERIALS AND METHODS

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All animal experiments were performed in compliance with the guidelines of the National Institutes of Health and the Animal Care and Use Committee of the Robert C. Byrd Health Sciences Center of West Virginia University.

Materials. All reagents were purchased from Sigma Chemical (St. Louis, MO) unless otherwise indicated.

Isolation of CM. Myocytes were prepared from the ventricles of 1- to 2-day-old rat pups, as previously described (12). Briefly, the ventricles of 30-50 hearts were minced in Ca²⁺- and Mg²⁺-free Hanks' balanced salt solution and digested for 15-min periods in 10 ml of a solution containing 0.1% trypsin (GIBCO BRL), 15 U/ml collagenase, and 0.1 mg/ml deoxyribonuclease (Worthington Biochemical, Freehold, NJ) in Hanks' balanced salt solution. Digestion was stopped by addition of 10 ml of DMEM-Ham's F-12 (DMEM-F-12; GIBCO BRL) containing 5% calf serum. Cycles were repeated until all the tissue was digested. The myocytes were cultured in DMEM-F-12 culture medium supplemented with 5% calf serum, penicillin (50 U/ml), and streptomycin (50 µg/ml). Cells were seeded at a density of 1.25 × 10⁵ cells/cm² on various dishes (Falcon Plastics, Cockeysville, MD; Costar, Cambridge, MA) according to the experimental requirements. Culture medium was changed to fresh serum-free DMEM-F-12 containing insulin, transferrin, selenium, and BSA 48 h after plating. Myocytes formed confluent monolayers of spontaneously beating cells 24 h later. These cells were washed, and fresh serum-free DMEM-F-12 was added. IL-1 (Genzyme, Boston, MA), N^G-methyl-L-arginine, and NE were added at this time and incubated as indicated.

Northern blot analysis. After exposure of cells (2.5 × 10⁶ cells in 60-mm dish) to experimental conditions, total RNA was extracted using Tri Reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer's instructions. A 10-µg sample of total RNA per lane was subjected to electrophoresis on 1.2% agarose gels containing 2.2 M formaldehyde. RNAs were transferred onto Zeta-probe blotting membranes (Bio-Rad Laboratories, Hercules, CA) using Vacuum Blotter (model 785, Bio-Rad Laboratories) and ultraviolet auto-cross-linked (GS gene linker, Bio-Rad Laboratories). Membranes were hybridized for 16 h at 62°C with HS-114 hybridization solution (Molecular Research Center, Cincinnati, OH) containing murine iNOS (Alexis, San Diego, CA) and human GAPDH4 (Cayman Chemical, Ann Arbor, MI) cDNA probes labeled with deoxy-[-³²P]CTP (3,000 Ci/mM; Amersham, Arlington Heights, IL) by random priming (Megaprime DNA labeling system; Amersham). The hybridized membranes were serially washed at 55°C using 1× sodium citrate-NaCl and 1% SDS solution and exposed to Kodak XAR-5 film overnight at 70°C with an intensifying screen.

Western blot analysis. CM were lysed directly in each plate (1.25 × 10⁶ cells in 30-mm plate) by application of a buffer containing 10 mM Tris · HCl (pH 7.4), 150 mM NaCl, 2 mM EGTA, 2 mM dithiothreitol, 1 mM sodium orthovanadate, 100 µg/ml phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. Protein concentrations were determined by Bradford assay. The samples were treated with 2× Laemmli loading buffer and boiled for 5 min. Equal amounts (20 µg) of the denatured proteins per lane were subjected to 12% SDS-PAGE, transferred to a nitrocellulose membrane, and reversibly stained with Ponceau red to verify equal loading. The blots were probed with a 1:2,000 dilution of mouse monoclonal antibodies specific for iNOS (Alexis). The iNOS was detected using the Amersham enhanced chemiluminescence system.

MAP kinase assay. MAP kinase activity was measured using the Biotrak kit (Amersham). Briefly, CM were lysed as mentioned above, and the samples were centrifuged at 12,000 g, 4°C for 20 min. The protein concentrations of supernatants were determined by Bradford assay. Protein (5 µg) from each sample was used for the MAP kinase assay. MAP kinase activity was determined by measuring the transfer of -³²P from ATP to the threonine on the synthetic peptide substrate (KRELVEPLTPAGEAPNQALLR).

MAP kinase in-gel assay. A myelin basic protein in-gel kinase assay was performed exactly as described by Duff et al. (4). MAP kinase activity was quantified by densitometry using the Optimas software program run on a Gateway 2000 personal computer (Optimas, Bothell, WA).

Assay for NO₂ production. NO₂ assays on neonatal rat CM supernatants were performed as described previously (13). Briefly, the stable metabolic end product of NO synthesis, NO₂, was used as a measure of NO production. Cell culture supernatants from 48-well plates were mixed with an equal volume of Griess reagent for 1 h. The absorbance at 550 nm was measured with a microplate reader (Molecular Devices). We previously demonstrated that the ratio of NO₂ to total NO₂ + NO₃ did not significantly change throughout the various experiments. Thus the NO₂ levels accurately reflected the total amount of NO production.

Statistical methods. Values are means ± SE of 12 different determinations derived from 3 wells each from 4 separate myocyte preparations of 30-50 hearts/preparation. ANOVA and the Student-Newman-Keuls tests were used for multigroup comparisons. P < 0.05 was considered statistically significant.

	RESULTS

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Preliminary experiments were conducted using various concentrations of NE (10⁷-10⁵ M) at various time intervals (2, 6, 12, 24, and 48 h) alone and in combination with cytokines. Optimal concentrations and time intervals were chosen to facilitate exploration and delineation of distinct signaling pathways not previously associated with iNOS regulation. The potential physiological consequences of micromolar concentrations of NE may be most relevant to local effects achieved at autonomic nerve endings. Exposure of CM to IL-1 alone (500 U/ml) resulted in a significant increase in NO₂ production at 48 h, as we previously reported (14) (Fig. 1A). Exposure of CM to maximal concentrations of NE (10 µM) alone had no effect on NO₂ production over vehicle alone. The addition of NE to IL-1 resulted in a statistically significant increase in NO₂ production compared with IL-1 alone.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   A: effects of vehicle (VEH), interleukin 1 (IL-1, 500 U/ml), norepinephrine (NE, 10 µM), IL-1 + NE, IL-1 + NE + prazosin (PZ, 1 µM), IL-1 + NE + propranolol (PRO, 1 µM), and IL-1 + NE + PD-98059 [20 µM, a selective mitogen-activated protein (MAP) kinase kinase inhibitor] on NO2 production by neonatal rat cardiac myocytes (CM). * P < 0.01 vs. vehicle. ** P < 0.01 vs. IL-1. B: effects of IL-1 (500 U/ml), NE (10 µM), and adenylate cyclase stimulator forskolin (Forsk) on NO2 production by CM. * P < 0.01 vs. IL-1. ** P < 0.01 vs. IL-1 + forskolin. Values are means ± SE of 12 different determinations derived from 3 wells each from 4 separate CM preparations.

The addition of the ₁-adrenergic receptor antagonist prazosin alone or the -adrenergic receptor antagonist propranolol alone partially reduced the NE-mediated increase in NO₂ production stimulated by IL-1 (Fig. 1A). The addition of prazosin and propranolol together completely blocked the NE-induced increase in IL-1-stimulated NO₂ production. This effect of NE appears to be mediated through MAP kinase, since PD-98059 (20 µM), a selective MAP kinase kinase inhibitor (21), also completely abolished the NE-induced increase in IL-1-stimulated NO₂ production.

Additional experiments were conducted to confirm that NE-mediated NO production involves - and -adrenergic receptors. The addition of increasing concentrations of the adenylate cyclase stimulator forskolin to IL-1 resulted in a statistically significant increase in NO₂ production compared with IL-1 alone. NE further increased NO₂ production in the presence of maximal concentrations of forskolin (Fig. 1B). The addition of the -adrenergic agonist phenylephrine to IL-1 also resulted in a significant increase in NO₂ production compared with IL-1 alone (6.6 ± 0.7 vs. 3.4 ± 0.08 µmol · 1.25 × 10⁵ cells¹ · 48 h¹, respectively, P < 0.05, n = 6). However, this increase was less than that achieved with NE (9.6 ± 0.7 µmol · 1.25 × 10⁵ cells¹ · 48 h¹, P < 0.05, n = 6). This effect of -adrenergic stimulation with phenylephrine was similar to the -adrenergic effect of NE observed in the presence -adrenergic inhibition with propranolol (Fig. 1A). Phenylephrine alone had no effect on NO production compared with vehicle-treated controls (0.5 ± 0.1 vs. 0.46 ± 0.15 µmol · 1.25 × 10⁵ cells¹ · 48 h¹, respectively, P = NS, n = 6).

The role of MAP kinase in NO production was further explored by measuring enzyme activity. IL-1 alone did not significantly increase MAP kinase activity above control levels (Fig. 2A). NE alone significantly increased MAP kinase activity, which peaked at 10 min (0.54 ± 0.05, 0.73 ± 0.05, 1.0 ± 0.06, 0.83 ± 0.06, and 0.77 ± 0.05 pmol P_i · min¹ · 5 µg protein¹ for control and 5, 10, 20, and 120 min, respectively). NE + IL-1-stimulated MAP kinase activity was only partially reduced by the addition of prazosin alone or propranolol alone but was completely blocked by the addition of prazosin and propranolol together. The addition of the MAP kinase kinase inhibitor PD-98059 also completely blocked NE + IL-1-stimulated MAP kinase activity. These results were further confirmed by the MAP kinase in-gel assay (Fig. 2B).

View larger version (32K): [in this window] [in a new window]   Fig. 2.   A: effects of vehicle, IL-1 (500 U/ml), NE (10 µM), IL-1 + NE, IL-1 + NE + prazosin (1 µM), IL-1 + NE + propranolol (1 µM), IL-1 + NE + prazosin + propranolol, and IL-1 + NE + PD-98059 (20 µM, a MAP kinase kinase inhibitor) on MAP kinase activity in CM at 10 min. Values are means ± SE of 9 different determinations derived from 3 separate CM preparations. * P < 0.01 vs. vehicle. ** P < 0.01 vs. IL-1. PD-98059 (20 µM) alone (0.45 ± 0.1 pmol Pi · min1 · 5 µg protein1) did not change basal MAP kinase activity (0.48 ± 0.07 pmol Pi · min1 · 5 µg protein1). B: representative MAP kinase in-gel assay illustrating MAP kinase activation by IL-1 (500 U/ml) + NE (10 µM) and inhibition of this effect by PD-98059 (20 µM) and prazosin (1 µM) + propranolol (1 µM). Experiment was repeated 3 times with identical results. MAP kinase activity was determined by densitometry and expressed as percentage of control (from left to right: 0, 15, 112, 42, 39, 25, 17, and 0%). Molecular mass markers indicated on left.

The effects of NE on iNOS mRNA expression and protein synthesis were studied using Northern and Western analyses (Fig. 3). The addition of maximal concentrations of NE alone to CM induced negligible iNOS mRNA expression with no iNOS protein synthesis. The addition of NE to IL-1 considerably enhanced iNOS mRNA expression and iNOS protein synthesis compared with IL-1 alone (Fig. 3). The addition of prazosin alone or propranolol alone only partially decreased NE-enhanced IL-1-stimulated iNOS mRNA expression (Fig. 4). The addition of prazosin and propranolol together reduced IL-1 + NE-induced iNOS mRNA to the same level as IL-1 alone (Fig. 4). PD-98059 alone did not reduce IL-1-stimulated iNOS mRNA levels. However, PD-98059 greatly reduced the enhancement by NE of IL-1-stimulated iNOS mRNA expression.

View larger version (37K): [in this window] [in a new window]   Fig. 3.   A: representative Northern blot analysis of inducible NO synthase (iNOS) mRNA expression in CM exposed to NE, IL-1, and IL-1 + NE. NE alone induced negligible levels of iNOS mRNA after 12 h of treatment. However, NE greatly enhanced IL-1-stimulated iNOS mRNA expression. Experiment was conducted 3 times with 3 separate CM preparations with identical results. Amount of iNOS mRNA expression was determined by densitometry and expressed as percentage of glyceraldehyde 3-phosphate dehydrogenase (GAPDH; from left to right: 0, 1, 4, 4, 6, 12, 29, 51, 47, 48, 13, 47, 65, 64, and 73%). B: representative Western blot analysis revealing that NE alone did not increase iNOS protein synthesis but demonstrably enhanced effect of IL-1 on iNOS protein synthesis. Experiment was conducted 3 times with 3 separate CM preparations with identical results. Molecular mass marker indicated on left.

View larger version (49K): [in this window] [in a new window]   Fig. 4.   Representative Northern blots illustrating effects of adrenergic receptor antagonists prazosin (1 µM) and propranolol (1 µM) and MAP kinase kinase inhibitor PD-98059 (20 µM) on iNOS mRNA expression in CM exposed to IL-1 (500 U/ml) + NE (10 µM). Total RNA was extracted from CM at 6 h after treatment. Experiment was conducted 3 times with identical results. Amount of iNOS mRNA expression was determined by densitometry and expressed as percentage of GAPDH (from left to right: 1, 21, 59, 39, 42, 25, 22, and 27%).

	DISCUSSION

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The present study provides evidence that NE enhances IL-1-stimulated NO production by activation of - and -adrenergic receptors through a novel MAP kinase-dependent mechanism in neonatal rat CM. Exposure of CM to NE alone failed to result in detectable NO₂ levels in the supernatant. However, the addition of NE together with IL-1 to CM significantly enhanced NO₂ production over IL-1 alone (Fig. 1A). A considerable increase in iNOS mRNA was apparent by 6 h of treatment and persisted for 48 h (Fig. 3A). This increase in iNOS mRNA was associated with a similar increase in iNOS protein (Fig. 3B). These data reveal that activation of NE signaling pathways alone is not sufficient to induce functional iNOS protein by CM. Activation of NE signaling pathways only augments the effect of IL-1 on NO production in CM. The failure of NE alone to induce iNOS is particularly interesting in view of a previous report of enhanced NO release from CM after exposure to NE (9). NE increased NO release from 149 ± 11 to 767 ± 83 nM in CM, as detected by a highly sensitive porphyrinic microsensor. The detection limit for this NO electrode is 5 nM. These levels are below the micromolar range of detectability of the Griess reagent used to detect NO₂ levels in the present study. In addition, the millisecond response time for this electrode limits its applicability to episodic signals of brief duration. It will only provide a continuous upward baseline drift in response to the continuous high output of NO from IL-1-induced iNOS expression in CM. These technical differences explain the apparent disparity between these two studies. Combining the results of these two studies leads to the conclusion that NE does modulate constitutive and inducible NOS in CM. The physiological and pathophysiological consequences of this regulation remain to be determined.

It is well established that CM possess - and -adrenergic receptors (17). NE exerts its biologic effects by activation of - and/or -adrenergic receptors (6). Maximal concentrations of prazosin or propranolol each only partially reduced NE-mediated NO₂ production (Fig. 1A). This suggests that - and -adrenergic receptors are involved in NE-mediated NO₂ production. It is apparent that ₁-adrenergic receptors are involved in NE-enhanced NO production stimulated by IL-1, since prazosin is an ₁-adrenergic receptor antagonist. This was further supported by the observation that the addition of both prazosin and propranolol completely abolished NE-mediated NO₂ production (Fig. 1A). In addition, the ₁-agonist phenylephrine also increased NO₂ production in the presence of IL-1. The effect of phenylephrine was similar to the -adrenergic effect of NE observed in the presence of propranolol, a -adrenergic receptor antagonist. Additional experiments with ₂-agonists and antagonists would be necessary to definitively exclude any contribution of this receptor subtype.

We previously showed that protein kinase A activation is necessary, but not sufficient, for iNOS mRNA stability, protein formation, and NO₂ production (14). We also showed that the adenylate cyclase stimulator forskolin significantly increased IL-1-stimulated NO production (13). NE-mediated enhancement of IL-1-stimulated NO production could similarly result exclusively from stimulating adenylate cyclase through activation of -adrenergic receptors. This is unlikely, since NE further increased NO production in the presence of maximal concentrations of forskolin. Higher concentrations of forskolin did not further increase NO production (Fig. 1B). It is also unlikely that NE mediates its effects on IL-1-stimulated NO production exclusively through -adrenergic receptors. The addition of maximal concentrations of the ₁-adrenergic agonist phenylephrine to IL-1 enhanced NO production to a lesser extent than NE (6.6 ± 0.7 vs. 9.6 ± 0.7 µmol · 1.25 × 10⁵ cells¹ · 48 h¹, respectively, P < 0.05, n = 6). This effect of ₁-adrenergic stimulation with phenylephrine is similar to the effect of NE in the presence of -adrenergic inhibition with propranolol (Fig. 1A). These data further support our finding that NE enhances IL-1-stimulated NO production through ₁- and -adrenergic receptors.

- And -adrenergic receptors are known to be coupled to different signal transduction pathways (10). NE has been reported to increase MAP kinase activity in CM by activation of ₁- and -adrenergic receptors (22). PD-98059 has been reported to selectively depress MAP kinase activity by inhibition of MAP kinase kinase in other cell types (21). The addition of PD-98059 to IL-1 + NE completely mimicked the effects of combined ₁- and -adrenergic blockade on NO₂ production (Fig. 1A). The addition of both prazosin and propranolol also completely blocked the additional MAP kinase activity produced by IL-1 + NE above IL-1 alone (Fig. 2A). The identical effect was seen by the addition of the selective MAP kinase kinase inhibitor PD-98059 to IL-1 + NE. Enhancement of IL-1-stimulated iNOS mRNA expression by MAP kinases has previously been reported in adult rat ventricular myocytes (18). Effects on iNOS protein or NO₂ production were not reported, however. It is unclear whether enhanced iNOS mRNA expression in adult myocytes is also associated with a corresponding increase in iNOS enzyme activity. Such comparisons between neonatal and adult myocyte iNOS signaling pathways have not been performed.

The increase in MAP kinase activity observed at 10 min after NE administration appears to be the critical event in NE enhancement of IL-1-stimulated NO production by CM. This is consistent with the known effects of this class of kinases (4, 16). Activation of MAP kinase is an early response to a wide variety of stimuli resulting in gene transcription (4). NE enhancement of IL-1-stimulated iNOS mRNA expression can be attributed to increased MAP kinase activity. This was supported by our finding that prazosin, propranolol, and PD-98059 each similarly inhibited NE-stimulated MAP kinase activity and reduced NE-mediated iNOS mRNA expression (Fig. 4).

NE may increase IL-1-stimulated iNOS mRNA expression through transcription rate and message stability. MAP kinase activation has been shown to be an early step in the activation of gene transcription (4). We also previously reported that cAMP enhances iNOS mRNA stability (13). Further efforts could be directed to determine the relative contributions of transcription rate and message stability with nuclear run-on and actinomycin D experiments.

Elevated circulating levels of NE have been associated with a worse prognosis in patients with congestive heart failure (CHF) (20). Recent evidence in the clinical literature has associated NO production in CM with apoptosis in patients with CHF (23). It is intriguing to speculate that NE-induced enhancement of NO production in CM contributes to apoptosis and worsening CHF in patients. Considerably more work in experimental models is warranted to determine the concentrations and durations of exposure to IL-1 and NE required to achieve these effects in vivo and in vitro.

The clinical relevance of these observations to CHF is supported by the results of a multicentered clinical trial with use of a combined - and -adrenergic inhibitor, carvedilol (15). The addition of carvedilol to standard CHF treatment resulted in a 65% reduction in all-cause mortality (P < 0.001) (15). The mechanisms responsible for the beneficial effects of this combined - and -adrenergic blocker in CHF warrant further investigation. The results of this study provide a new and exciting direction for further basic investigations into the relationship between cytokine-stimulated NO production, adrenergic signaling, and CHF.