Role of reactive oxygen species and NAD(P)H oxidase in alpha 1-adrenoceptor signaling in adult rat cardiac myocytes

Lei Xiao, David R. Pimentel, Jing Wang, Krishna Singh, Wilson S. Colucci, and Douglas B. Sawyer

Myocardial Biology Unit, Cardiovascular Division, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, 02118


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We recently reported that alpha 1-adrenoceptor (alpha 1-AR) stimulation induces hypertrophy via activation of the mitogen/extracellular signal-regulated kinase (MEK) 1/2-extracellular signal-regulated kinase (ERK) 1/2 pathway and generates reactive oxygen species (ROS) in adult rat ventricular myocytes (ARVM). Here we investigate the intracellular source of ROS in ARVM and the mechanism by which ROS activate hypertrophic signaling after alpha 1-AR stimulation. Pretreatment of ARVM with the ROS scavenger Mn(III)terakis(1-methyl-4-pyridyl) porphyrin pentachloride (MnTMPyP) completely inhibited the alpha 1-AR-stimulated activation of Ras-MEK1/2-ERK1/2. Direct addition of H2O2 or the superoxide generator menadione activated ERK1/2, which is also prevented by MnTMPyP pretreatment. We found that ARVM express gp91phox, p22phox, p67phox, and p47phox, four major components of NAD(P)H oxidase, and that alpha 1-AR-stimulated ERK1/2 activation was blocked by four structurally unrelated inhibitors of NAD(P)H oxidase [diphenyleneiodonium, phenylarsine oxide, 4-(2-aminoethyl)benzenesulfonyl fluoride, and cadmium]. Conversely, inhibitors for other potential ROS-producing systems, including mitochondrial electron transport chain, nitric oxide synthase, xanthine oxidase, and cyclooxygenase, had no effect on alpha 1-AR-stimulated ERK1/2 activation. Taken together, our results show that ventricular myocytes express components of an NAD(P)H oxidase that appear to be involved in alpha 1-AR-stimulated hypertrophic signaling via ROS-mediated activation of Ras-MEK1/2-ERK1/2.

myocardial hypertrophy; norepinephrine; mitogen/extracellular signal-regulated kinase 1/2-extracellular signal-regulated kinase 1/2; Ras; NAP(P)H oxidase expression


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

REACTIVE OXYGEN SPECIES (ROS) are now recognized as critical regulators of intracellular signaling cascades (14, 20) and play a role in mediating growth of cardiac myocytes (2, 27, 32, 42). We recently reported that alpha 1-adrenergic receptor (alpha 1-AR) activation causes generation of ROS and hypertrophy in adult rat ventricular myocytes (ARVM; see Ref. 2). We have further shown that alpha 1-AR-stimulated hypertrophy in ARVM is mediated via activation of the Ras, mitogen/extracellular signal-regulated kinase (MEK) 1/2, and extracellular signal-regulated kinase (ERK) 1/2 signaling pathway (40). Kinase cascades in the mitogen-activated protein kinase (MAPK) family have emerged as central intracellular signaling intermediates regulating myocyte hypertrophy (15), and the sensitivity of these signaling cascades to ROS (6) makes it likely that they play a role in ROS-mediated myocyte hypertrophy (1). In particular, the ERK1/2 pathway can be activated by sources of ROS in the intact heart and isolated myocytes (42). The ROS-dependent activation of myocyte hypertrophy after alpha 1-AR stimulation might therefore occur through this ROS-sensitive system.

The enzymatic source of ROS involved in triggering hypertrophic growth in ventricular myocytes remains unknown. Several ROS-producing systems, including NAD(P)H oxidase (17), mitochondrial electron transport chain (12), xanthine oxidase (13), nitric oxide synthase (NOS; see Ref. 39), and cyclooxygenase (Cox; see Ref. 38) have been identified in many cell types, and each of these has been implicated in the activation of intracellular signaling cascades leading to changes in cell structure and function (37). The NAD(P)H oxidases are membrane-associated, multisubunit enzyme complexes that catalyze the single-electron reduction of oxygen using NADH or NAD(P)H as the electron donor (18). NAD(P)H oxidase consists of at least the following four major subunits: p22phox, gp91phox [or nox-1 in vascular smooth muscle cells (VSMC)], p67phox, and p47phox. These enzymes have been well studied in phagocytes and appear to be the primary source of ROS responsible for ANG-stimulated proliferation and hypertrophy in VSMC and endothelial cells, where cytosolic O<UP><SUB>2</SUB><SUP>−</SUP></UP> generated by the NAD(P)H oxidase complex acts as an intracellular signaling molecule resulting in the activation of a Ras-Raf-MEK1/2-ERK1/2 (18, 26). However, the expression of an NAD(P)H oxidase has not been demonstrated in ventricular myocytes.

The purpose of this study was to determine 1) whether alpha 1-AR stimulation in ARVM induces ROS-dependent activation of the Ras-Raf-MEK1/2-ERK1/2 pathway and 2) whether NAD(P)H oxidase might be the source of ROS involved in alpha 1-AR activation of hypertrophic signaling. We show that alpha 1-AR activation of Ras-Raf-MEK1/2-ERK1/2 in ventricular myocytes is ROS dependent. We further show that transcripts of several components of the NAD(P)H oxidase complex are expressed in ARVM and that inhibitors of NAD(P)H oxidase, but not other known ROS-generating systems, prevent alpha 1-AR stimulation of ERK1/2, thereby implicating the NAD(P)H oxidase in cardiac myocyte hypertrophic signaling.


    MATERIALS AND METHODS
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INTRODUCTION
MATERIALS AND METHODS
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Materials. L-Norepinephrine (NE), DL-propranolol (Pro), Nomega -nitro-L-arginine, myxothiazol, thenoyltrifluoroacetone (TTFA), CdSO4, CdCl2, H2O2, nimesulide, NS-398, indomethacin, aspirin, allopurinol, oxypurinol, BSA, L-carnitine, creatine, taurine, and Ponceau S solution were from Sigma (St. Louis, MO). Mn(III)terakis(1-methyl-4-pyridyl)porphyrin pentachloride (MnTMPyP), nitro-L-arginine methyl ester, NG-monomethyl-L-arginine, diphenyleneiodonium (DPI) and phenylarsine oxide (PAO), 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), rotenone, DIDS, antimycin A, and menadione were from Calbiochem (San Diego, CA). Euk-8 was from Eukarion (Bedford, MA). The Ras activation assay kit was purchased from Upstate Biotechnology (Lake Placid, NY). The p44/42 MAP kinase assay kit and anti-phospho-specific antibodies for ERK1/2 and MEK1/2 kinases were purchased from New England Biolabs (Beverly, MA). The cDNA probe for rabbit p67phox was kindly provided by Dr. Patrick J. Pagano at Henry Fort Hospital (Detroit, MI). The antibody for p67phox was purchased from BD Transduction Laboratories (Lexington, KY). SuperScript II reverse transcriptase and Taq DNA polymerase were purchased from GIBCO-BRL (Rockville, MD). The TOPO TA cloning kit was purchased from Invitrogen (Carlsbad, CA).

Cell isolation and culture. The animal protocol used in this study was approved by the Institutional Animal Care and Use Committee at Boston University Medical Center. Ventricular myocytes were isolated from hearts of adult male Sprague-Dawley rats (250-275 g) as previously described (2, 40). Briefly, rats were anesthetized with pentobarbital sodium (50 mg/kg ip) and heparinized (1,000 USP/kg iv). Hearts were removed and perfused retrograde with Krebs-Henseleit bicarbonate (KHB) buffer for 5 min. The perfusion buffer was changed to nominally Ca2+-free KHB buffer for 2-3 min until spontaneous beating stopped. Hearts were then perfused with KHB buffer containing 0.04% collagenase type II for 20 min. After removing atria and great vessels, the hearts were minced in the same buffer containing trypsin (0.02 mg/ml) and DNA (0.02 mg/ml). The cell mixture was filtered, and the cells were sedimented two times through a 6% BSA cushion to remove nonmyocyte cells. The cell pellet was resuspended and plated in DMEM, supplemented with BSA (2 g/l), L-carnitine (2 mM), creatine (5 mM), taurine (5 mM), and 0.1% penicillin-streptomycin. Cells were plated on laminin (1 µg/cm2)-coated dishes at a density of 100 cells/mm2 and kept at 37°C for 24 h before treatment. There were ~95% rod-shaped cells at the treatment time. ARVM were kept in the above-defined serum-free medium throughout all experiments.

Western blot analysis. The method is similar to that described previously (40). Briefly, ARVM were lysed in lysis buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris, pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.4 mM phenylmethylsulfonyl fluoride, and 0.5% Nonidet P-40) and sonicated for 2 s to shear DNA. Soluble lysates were separated by microcentrifugation, and volumes representing equal amounts of proteins with Laemmli sample buffer (Bio-Rad) were boiled at 95°C for 5 min before being resolved by 10% SDS-PAGE. The proteins were transferred to a polyvinylidene difluoride membrane (Amersham Pharmacia Biotech), blocked with 5% nonfat dry milk for 1 h at room temperature, and then incubated with primary antibody at 4°C overnight. Protein was detected with horseradish peroxidase-conjugated secondary antibody (Santa Cruz) and SuperSignal chemiluminescent substrate solution (Pierce). The protein loading of each sample was verified by staining the membrane with 0.1% Ponceau S solution.

Ras activation assay. Activated Ras was detected by a Ras activation assay kit according to the manufacture's instruction. Briefly, cell lysates (500 µg) were immunoprecipitated at 4°C overnight with an agarose conjugate Ras-GTP affinity probe [Raf-1 Ras-binding domain (RBD)] corresponding to the human RBD of Raf-1. The immunoprecipitates were resolved by 10% SDS-PAGE and detected by Western blot analysis using anti-Ras antibody.

ERK1/2 kinase assay. ERK1/2 kinase assays were conducted according to the manufacturer's instruction using a p44/42 MAP kinase assay kit. Briefly, cell lysates (200 µg) were immunoprecipitated with immobilized phospho-p44/42 MAP kinase monoclonal antibody at 4°C overnight. The immunoprecipitates were washed two times with lysis buffer and kinase buffer before being resuspended in kinase assay buffer containing the specific ERK substrate Elk-1 fusion proteins (2 µg). The reaction was carried out at 30°C for 30 min in the presence of 200 µM ATP in vitro. The reaction was terminated by addition of SDS sample buffer. Phosphorylation of Elk-1 fusion proteins was analyzed by 10% SDS-PAGE and Western blot analysis using phospho-Elk-1 specific antibody.

Amplification of partial cDNA fragments of rat NAD(P)H oxidase from ARVM by RT-PCR. The cDNA sequences of the cloned rat p22phox and p47phox and mouse gp91phox were used as the basis for the designing of the upstream or sense and downstream or antisense oligonucleotide primers (Table 1). Each pair of primers complementary to the cDNA sequence of the cloned NAD(P)H oxidase subunit was then used to amplify the counterpart cDNA fragment from total RNA isolated from ARVM. The RT and PCR reactions were performed according to the manufacturer's protocol (GIBCO-BRL) using SuperScript II reverse transcriptase and Taq DNA polymerase. The RT reaction was performed under the following conditions: 10 min at 70°C, 52 min at 42°C, and 15 min at 70°C using oligo(dT) and total RNA isolated from cultured ARVM. The cDNA fragments were amplified by PCR using the above primers under the following conditions: 3 min at 94°C; followed by 40 s at 94°C, 1 min at 60°C, and 1 min at 72°C, for 35 cycles; and a final incubation for 10 min at 72°C. To confirm the sequence identities of the above RT-PCR products, the amplified cDNA fragments were then purified and inserted in TOPO TA cloning vector pCRII-TOPO (Invitrogen) for cycle sequencing with M-13 forward and reverse primers. Sequences were determined by use of a DNA Sequencer (ABI model 373; Applied Biosystems, Foster City, CA). Sequences were validated by sequencing RT-PCR products from three separate RT-PCR reactions.

                              
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Table 1.   Sequences, GenBank accession number, and species specificity of oligonucleotide primers

Northern blot analysis. Total RNA was isolated from ARVM by the method of Chomczynski and Sacchi (4) as previously described (40, 41). Approximately 15 µg of total RNA were separated in 1% formaldehyde/agarose gel and transferred to a nylon membrane (Genescreen Plus; NEN). After sequencing to confirm identity, the above cloned cDNA fragments of p22phox, gp91phox, and p47phox were labeled with the [alpha -32P]dCTP as oligonucleotide probes by the random hexamer priming method. A previously reported cDNA probe (714 bp) for rabbit p67phox (30) was also labeled with the [alpha -32P]dCTP by the same method. Hybridization was carried out with the alpha -32P-labeled probes in 5× SSC, 5× Denhardt's solution, 50% formamide, 1% SDS, and 100 µg/ml of salmon sperm DNA at 42°C for 18 h. The membrane was washed two times for 15 min at 42°C in 0.2× SSC and 0.1% SDS followed by two additional washes for 15 min at 68°C in 0.1× SSC and 0.1% SDS. The blots were analyzed by autoradiography and were developed after exposure at -70°C for 1-2 days. All blots were reprobed with an [alpha -32P]CTP-labeled oligonucleotide complimentary to 18S rRNA. All mRNA levels were normalized to the level of 18S rRNA to correct for potential variations in the amount of RNA loaded or transferred.

Statistical analysis. All data are reported as means ± SE. Significance tests (Student's unpaired t-test or ANOVA) were performed using the InStat program (GraphPAD Software). A P value <0.05 was considered to be significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ROS dependence of alpha 1-AR stimulated activation of Ras-MEK1/2-ERK1/2 in ARVM. As we have reported (40), alpha 1-AR stimulation with NE (1 µM) in the presence of propranolol (2 µM) caused a marked activation of Ras-MEK1/2-ERK1/2, which is evident within 5 min and remains activated for up to 48 h (ERK1/2). Also, as we have previously shown (40), Ras-MEK1/2-ERK1/2 activation was completely abolished by the alpha 1-AR-selective antagonist prazosin (100 nM, 30 min; Fig. 1). Pretreatment with the superoxide dismutase and catalase mimetic (8) MnTMPyP (50 µM, 30 min), which we have found prevents alpha 1-AR induced hypertrophy in ARVM (2), completely prevented the alpha 1-AR-stimulated activation of Ras, MEK1/2, and ERK1/2 and phosphorylation of the ERK1/2 substrate Elk-1 (Fig. 1, A and B). A similar result was obtained using another structurally unrelated ROS scavenger, Euk-8. Euk-8 (100 µM, 30 min) pretreatment significantly prevented the alpha 1-AR-stimulated ERK1/2 activation (data not shown) and hypertrophy in ARVM (2). These results suggested that ROS in the alpha 1-AR stimulated activation of the Ras-MEK1/2-ERK1/2 pathway.


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Fig. 1.   Mn(III)terakis(1-methyl-4-pyridyl)porphyrin pentachloride (MnTMPyP) inhibits alpha 1-adrenoceptor (AR)-stimulated activation of Ras-mitogen/extracellular signal-regulated kinase (MEK) 1/2-extracellular signal-regulated kinase (ERK) 1/2 in adult rat ventricular myocytes (ARVM). ARVM were cultured for 24 h in defined media and then treated with 1 µM norepinephrine (NE) in the presence of 2 µM DL-propranolol (Pro) for 5-120 min. A: activated Ras were detected in cell lysates using the Ras Activation Assay described in MATERIALS AND METHODS. Phosphorylation of MEK1/2 was measured by Western blot analysis using anti-phospho-specific MEK1/2 antibody. Phosphorylation of ERK1/2 was determined using anti-phospho-specific ERK1/2 antibodies (top) and immune complex kinase assay using Elk-1 fusion proteins as substrate (bottom). Activation of Ras and phosphorylation of MEK1/2, ERK1/2, and Elk-1 were completely inhibited by the pretreatment of ARVM with the alpha 1-AR selective antagonist prazosin (100 nM, 30 min) or ROS scavenger MnTMPyP (50 µM, 30 min). B: time courses for alpha 1-AR-stimulated activation of Ras, MEK1/2, and ERK1/2. Phosphorylation of ERK1/2 was completely inhibited by the pretreatment of ARVM with MnTMPyP (50 µM, 30 min). The data for MEK1/2 and ERK1/2 are from 3 independent experiments, and the data for Ras are from 2 independent experiments.

To confirm that ROS are upstream of the ERK1/2 pathway in ARVM, we examined the effects of H2O2 and the O<UP><SUB>2</SUB><SUP>−</SUP></UP> generator menadione on ERK activation. Both H2O2 and menadione activated ERK1/2 phosphorylation in a concentration-dependent manner that approaches the level of alpha 1-AR-stimulated ERK1/2 activation (Fig. 2). Pretreatment of ARVM with MnTMPyP (50 µM, 30 min) completely prevented the activation of ERK1/2 by H2O2 or a lower concentration of the superoxide generator menadione (0.2 and 2 µM) and partially inhibited the ERK1/2 activation by menadione at a higher concentration (20 µM, 39 ± 6% inhibition; Fig. 2). This result demonstrates the ability of ROS to activate the ERK1/2 pathway in ARVM and confirms that MnTMPyP acts as an ROS scavenger in this system.


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Fig. 2.   Direct application of reactive oxygen species (ROS) in ARVM activates ERK1/2 that is prevented by MnTMPyP. ARVM were pretreated with the ROS scavenger MnTMPyP (50 µM) for 30 min before treatment with NE/Pro, H2O2, or menadione for 5 min. A: H2O2 caused concentration-dependent (0.1, 0.25, and 0.5 mM) phosphorylation of ERK1/2 that is completely prevented by MnTMPyP. B: menadione caused concentration-dependent (0.2, 2, and 20 µM) phosphorylation of ERK1/2. MnTMPyP completely prevented ERK1/2 activation by a lower concentration menadione (0.2 and 2 µM) and partially inhibited the ERK1/2 activation by menadione at a higher concentration (20 µM) in ARVM (n = 3 independent experiments). * P < 0.01 vs. control. #P < 0.01 vs. the group that was treated with 20 µM menadione alone.

Evidence of NAD(P)H oxidase expression in ARVM. NAD(P)H oxidase is a membrane-bound enzyme complex that is a source of cytosolic O<UP><SUB>2</SUB><SUP>−</SUP></UP> and is composed of at least four subunits, including two important membrane-bound subunits p22phox and gp91phox and two cytosolic subunits p47phox and p67phox. However, the existence and function of NAD(P)H oxidase have never been demonstrated in ventricular myocytes. We used oligonucleotide primers complementary to the cDNA sequence of the cloned rat p22phox, p47phox, and mouse gp91phox to amplify the counterpart cDNA fragments from the total RNA isolated from ARVM by RT-PCR (Table 1). As shown in Fig. 3A, top, the sizes of the amplified cDNA fragments by RT-PCR from ARVM are identical to the predicted sizes of the cloned rat p22phox (457 bp), p47phox (570 bp), and the recently reported rat gp91phox (614 bp; GenBank accession number AJ295950). The identities of these RT-PCR products were confirmed by sequencing. To confirm that these RT-PCR products are amplified from ARVM instead of contaminating nonmyocytes that could potentially provide template signals for RT, Northern blot analysis using the above cloned cDNA fragments as probes was performed on total RNA isolated from cultured ARVM. Results from Northern blot analysis show the expression of mRNA of p22phox, p47phox, and gp91phox in total RNA isolated from cultured ARVM (Fig. 3A, bottom). Using a cDNA probe for rabbit p67phox, we also detected the expression of mRNA of p67phox in ARVM by Northern blot assay (Fig. 3B). Likewise, we detected p67phox protein in ARVM by Western blot (Fig. 3B). Thus the four major components of a functional NAD(P)H oxidase are present in cardiac myocytes.


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Fig. 3.   Expression of components of NAD(P)H oxidase in ARVM. A, top: RT-PCR result for NAD(P)H oxidase subunits of total RNA isolated from ARVM. Lane 1, 100-bp DNA ladder; lane 2, p22phox (457 bp); lane 3, gp91phox (614 bp); lane 4, p47phox (570 bp); lane 5, reagent control. A, bottom: Northern blot results of NAD(P)H oxidase subunits of total RNA isolated from ARVM. B, bottom: Northern blot result of p67phox of total RNA isolated from ARVM. B, bottom: Western blot result of p67phox of cell lysates from ARVM and HL-60 cells (positive control). Data are representative of 3 independent experiments.

NAD(P)H oxidase inhibitors prevented alpha 1-AR-stimulated activation of ERK1/2 in ARVM. To test the potential role of NAD(P)H oxidase in the alpha 1-AR signaling pathway, four structurally unrelated pharmacological inhibitors of NAD(P)H oxidase (DPI, PAO, AEBSF, and cadmium) were used. DPI concentration dependently inhibited alpha 1-AR-stimulated activation of ERK1/2 with complete (94 ± 8%, P < 0.01, n = 3) inhibition at 50 µM (Fig. 4A). PAO (1 µM) also completely (93 ± 8% inhibition, P < 0.01, n = 3) prevented ERK1/2 activation induced by alpha 1-AR stimulation (Fig. 4B). Both AEBSF and cadmium ions (CdSO4 and CdCl2) concentration dependently inhibited alpha 1-AR-stimulated activation of ERK1/2, with 63 ± 9% (P < 0.01, n = 3) inhibition by 2 mM AEBSF (Fig. 4B) and 83 ± 8% (P < 0.01, n = 3) inhibition by 1 mM cadmium ions (both CdSO4 and CdCl2; Fig. 4C).


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Fig. 4.   NAD(P)H oxidase inhibitors prevent alpha 1-AR-stimulated ERK1/2 activation in ARVM. ARVM were incubated with four NAD(P)H oxidase inhibitors for 30 min at 37°C before the treatment with NE/Pro for 5 min. A: diphenyleneiodonium (DPI) caused concentration-dependent (10, 50, and 100 µM) inhibition of the alpha 1-AR-stimulated ERK1/2 activation with complete inhibition at 50 µM. B: phenylarsine oxide (PAO, 1 µM) also completely prevented the alpha 1-AR-stimulated ERK1/2 activation, whereas 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF, 2 mM) significantly (63 ± 9% inhibition, P < 0.01) inhibited alpha 1-AR-stimulated ERK1/2 activation. C: cadmium ions, either CdSO4 or CdCl2 at 1 mM, significantly inhibited the alpha 1-AR-stimulated ERK1/2 activation with 83 ± 8% inhibition (P < 0.01; n = 3 independent experiments). * P < 0.01 vs. control. #P < 0.01 vs. positive control that was treated with NE/Pro alone.

Mitochondria are another important source of intracellular ROS that have been implicated in activation of signaling pathways in cardiac myocytes (12), and DPI inhibits other flavin-containing oxidase enzymes, including those in the mitochondrial electron transport chain. We therefore used inhibitors of the mitochondrial electron transport chain to test whether ROS derived from mitochondria are involved in alpha 1-AR-stimulated activation of ERK1/2. Five structurally unrelated compounds that act at distinct sites to inhibit the electron transport chain were used, including 10 µM rotenone, 50 µM TTFA, 10 µM antimycin A, and 4 µM myxothiazol (12, 36). DIDS (200 µM), an inhibitor of the mitochondrial outer member anion channel, was also used as this has been shown to inhibit leakage of O<UP><SUB>2</SUB><SUP>−</SUP></UP> from mitochondria (36). ARVM were pretreated with these inhibitors for 30 min at 37°C before alpha 1-AR stimulation. In all cases, there was no effect of mitochondrial electron transport inhibition on alpha 1-AR-stimulated ERK1/2 activation or baseline ERK activation (Fig. 5), suggesting that mitochondrial ROS are not involved in the signaling pathway of alpha 1-AR-stimulated hypertrophy.


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Fig. 5.   Inhibitors of mitochondrial electron transport chain have no effect on alpha 1-AR-stimulated activation of ERK1/2 in ARVM. ARVM were treated with inhibitors of the mitochondrial electron transport chain [10 µM rotenone, 50 µM thenoyltrifluoroacetone (TTFA), 10 µM antimycin A, 4 µM myxothiazol, and 200 µM DIDS] for 30 min before treatment with NE/Pro for 5 min. A: alpha 1-AR-stimulated ERK1/2 activation was not affected by pretreatment with any of the 5 inhibitors of the mitochondrial electron transport chain in ARVM. B: pretreatment with the 5 compounds in A (30 min) alone did not alter ERK1/2 phosphorylation in ARVM (n = 3 independent experiments). * P < 0.01 vs. control.

Other flavin-containing enzymes that might be affected by the NAD(P)H oxidase inhibitor DPI include xanthine oxidase, NOS, and Cox, each of which has been shown to produce ROS in various cell types, including VSMC and neonatal rat ventricular myocytes (NRVM; see Refs. 13 and 37). Specific inhibitors of these enzymes were used to determine if any of these were involved in alpha 1-AR-stimulated ERK1/2 activation. Pretreatment of ARVM with either allopurinol or oxypurinol (selective inhibitors of xanthine oxidase) had no inhibitory effect on alpha 1-AR-stimulated ERK1/2 activation (Fig. 6A). Similarly, three L-arginine analogs used as NOS inhibitors had no inhibitory effect on alpha 1-AR-stimulated ERK1/2 activation (Fig. 6B). Finally, neither the nonselective Cox inhibitor indomethacin nor the Cox-2 selective inhibitors NS-398 and nimesulide inhibited alpha 1-AR-stimulated ERK1/2 activation (Fig. 6C). Thus xanthine oxidase, NOS, and Cox do not appear to be involved in alpha 1-AR-stimulated activation of ERK1/2 and hypertrophy in ARVM.


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Fig. 6.   Inhibitors of xanthine oxidase, nitric oxide synthase (NOS), or cyclooxygenase (Cox) have no effect on alpha 1-AR-stimulated activation of ERK1/2 in ARVM. ARVM were pretreated for 30 min with inhibitors of the following enzymes: xanthine oxidase (0.1 mM allopurinol or 0.3 mM oxypurinol; A); NOS [5 mM Nomega -monomethyl-L-arginine (L-NMMA), Nomega -nitro-L-arginine methyl ester (L-NAME), or Nomega -nitro-L-arginine (L-NNA); B]; or Cox (10 µM NS-398, 1 µM nimesulide, or 20 mM indomethacin; C). There was no effect of any inhibitor either on baseline or alpha 1-AR-stimulated ERK1/2 activity in ARVM (n = 3 independent experiments). * P < 0.01 vs. control.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We previously showed that ROS are involved in alpha 1-AR-induced hypertrophy in ARVM (2). Furthermore, we found that the alpha 1-AR-stimulated hypertrophy in ARVM is mediated via activation of Ras-MEK1/2-ERK1/2 (40). Here we demonstrate that alpha 1-AR activation of the Ras-MEK1/2-ERK1/2 hypertrophic pathway is redox sensitive, with the redox-sensitive step occurring at, or proximal to, the level of Ras activation. Moreover, we provide molecular evidence that an NAD(P)H oxidase system is expressed in cardiac myocytes and pharmacological evidence that this oxidase mediates alpha 1-AR-stimulated ERK1/2 activation.

Role of ROS in Gq-coupled receptor activation of hypertrophy. Recently, the role of ROS as an intracellular signaling molecule has been recognized in many biological systems, including myocyte hypertrophy (2, 27, 32, 42). ROS-mediated signaling has been implicated in the hypertrophic effects of several extracellular growth signals, including ANG, endothelin, and tumor necrosis factor-alpha in both VSMC and NRVM (27, 35). Similar to receptors for endothelin and ANG, alpha 1-AR are members of the G protein-coupled receptor family that couple to Gq. In VSMC, activation of Gq protein-coupled receptors, i.e., ANG II receptor, stimulates ROS generation through an NAD(P)H oxidase, leading to activation of MAPK pathways (34). Our present alpha 1-AR signaling data in ARVM suggest that coupling via NAD(P)H oxidase could be a common feature of Gq-mediated growth signaling in the cardiovascular system.

Role of ROS in activation of the alpha 1-AR-stimulated signaling pathway. The mechanism by which intracellular ROS activate the ERK1/2 pathway is currently unknown. Our studies provide new information on the relationship between ROS and the alpha 1-AR-stimulated ERK1/2 pathway. alpha 1-AR stimulation is known to directly activate phospholipase C-beta (PLC-beta ) and results in formation of inositol phosphates and diacylglycerol that cause the downstream effects (43). We previously found that MnTMPyP completely inhibited alpha 1-AR-stimulated hypertrophy in ARVM but had no effect on alpha 1-AR-stimulated formation of total intracellular inositol phosphates (2). Thus PLC and its upstream signaling molecules in the alpha 1-AR signaling pathway are not redox sensitive. Therefore, the redox-sensitive step in this pathway is likely downstream from PLC-beta . The finding, in the present study, that MnTMPyP completely inhibited the alpha 1-AR-stimulated activation of Ras, MEK1/2, and ERK1/2 suggests that the redox-sensitive step in the alpha 1-AR signaling pathway in ARVM is located at, or proximal to, Ras.

These findings in ARVM are consistent with recent reports in other cells. Mukhin et al. (26) showed that ROS, possibly from an NAD(P)H oxidase-like enzyme, mediates the Gi-coupled 5-hydroxytryptamine1A receptor-stimulated ERK1/2 activation in transfected Chinese hamster ovary fibroblasts. The redox-sensitive step in this pathway is downstream of Gibeta gamma subunits and upstream of, or at the level of, Src (26). In addition, ROS have been identified as central mediators in the signaling events of several G protein-coupled receptors in cardiac myocytes, and the Gialpha and Goalpha subunits were identified as the critical targets of ROS for the activation of the ERK1/2 pathway in NRVM (28). Thus ROS appear to be important upstream intermediates in G protein-coupled receptor-stimulated ERK1/2 activation, and the target proteins of ROS are located at early steps in these signaling pathways.

The precise location and mechanism for the redox-sensitive signaling pathway remains to be determined. In Jurkat T cells, the reactive free radical nitric oxide activates ERK1/2 directly by nitrosylating a cysteine residue on the upstream enzyme Ras (23). Although our data do not support a role for NOS/nitric oxide in alpha 1-AR-stimulated activation of Ras, reactive free radicals, including H2O2, can also activate Ras (24), likely through direct modifications of redox-sensitive cysteine residues. Other redox-dependent steps potentially involved in mediating alpha 1-AR-stimulated activation of the ERK1/2 signaling pathway (9) include Src, calcium-calmodulin, and proline-rich nonreceptor tyrosine kinase 2 (1, 16, 29).

Role of NAD(P)H oxidase in alpha 1-AR signaling. NAD(P)H oxidase is a membrane-associated, multisubunit enzyme complex that was first described as an important generator of ROS and reactive nitrogen species as part of the immune response in phagocytes (3). The neutrophil oxidase consists of at least four major subunits, including two membrane-spanning components (p22phox and gp91phox) and two cytosolic components (p67phox and p47phox; see Ref. 18). This oxidase system has now been identified in other cell types and has recently been shown to be the primary source of ROS that mediates ANG-stimulated proliferation and hypertrophy in VSMC (35). Although the cardiovascular NAD(P)H oxidases retain a similar enzyme complex structure to neutrophil oxidase with four subunits, their component structures and biochemical characteristics are considerably different (18). Using RT-PCR, Northern blot, and Western blot analysis, we have demonstrated that these four major subunits of NAD(P)H oxidase (p22phox, gp91phox, p67phox, and p47phox) are expressed in cardiac myocytes. These results provide the first direct evidence for the existence of an NAD(P)H oxidase system in cardiac myocytes.

Our pharmacological data implicate an NAD(P)H oxidase system as the source of ROS linking alpha 1-AR to activation of the Ras-MEK1/2-ERK1/2 cascade. Four known inhibitors of NAD(P)H oxidases, each structurally unique and acting on a distinct site of the NAD(P)H oxidase, significantly inhibited alpha 1-AR stimulated ERK1/2 activation in ARVM. As discussed below, several of these inhibitors lack specificity for NAD(P)H oxidase. However, taken together with the negative data using inhibitors of other oxidase enzymes, these studies support the conclusion that an NAD(P)H oxidase is the source of ROS linking alpha 1-AR stimulation to ERK1/2 activation in ARVM.

DPI is a known inhibitor for NAD(P)H oxidase and exerts its effect by blocking the binding of FAD to a flavin site of the oxidase (7). Although DPI also inhibits the activity of other flavin-containing enzymes, we found no effect of selective inhibitors of mitochondrial respiration, xanthine oxidase, NOS, or Cox on alpha 1-AR stimulated ERK1/2 activation. Therefore, the inhibitory effect of DPI on alpha 1-AR-stimulated ERK1/2 activation is not likely occurring via the blockade of these flavin-containing enzymes.

PAO is reported to selectively bind to the beta -subunit (gp91phox) of NAD(P)H oxidase and to preferentially inhibit NAD(P)H oxidase activation at a low concentration (1 µM; 11 and 25). Cadmium has been shown to reversibly block the proton channel-associated NAD(P)H complex (21) and thereby inhibit oxidase function (19, 22). AEBSF has been shown to prevent the assembly of the NAD(P)H oxidase complex and oxidase activity (10, 26). Like DPI, these inhibitors also have other effects that need to be considered in interpreting these data. PAO can inhibit tyrosine phosphatase, though at higher concentrations than used here (IC50 = 18 µM; see Ref. 31). Moreover, PAO inhibits, rather than potentiates, ERK1/2 signaling. Thus the tyrosine phosphatase inhibitory properties of PAO do not appear to confound interpretation of the present data. AEBSF is an irreversible inhibitor of serine proteases (5). However, we found no effect of the serine protease inhibitor phenylmethylsulfonyl fluoride on alpha 1-AR-stimulated ERK1/2 activation (data not shown), suggesting that the protease inhibitory action of AEBSF does not confound the interpretation of these results.

In summary, our results show that 1) ROS mediate alpha 1-AR-stimulated activation of the Ras-MEK1/2-ERK1/2 cascade in ARVM; 2) the redox-sensitive step in the alpha 1-AR signaling pathway in ARVM is located at, or proximal to, Ras; and 3) NAD(P)H oxidase is expressed in cardiac myocytes and appears to be the intracellular source of ROS in response to alpha 1-AR stimulation in ARVM. These results shed new light on the coupling between adrenergic and hypertrophic signaling in ventricular myocytes and implicate new targets in the treatment of heart diseases that involve myocardial hypertrophy and remodeling.


    NOTE ADDED IN PROOF

Since the completion and original submission of this work, a report has appeared from Tanaka et al. (33) demonstrating that alpha 1-AR-stimulated hypertrophy in adult rat cardiac myocytes is inhibited by antioxidants and that alpha 1-AR-stimulated ROS production as measured by DCF fluorescence is inhibited by DPI.


    ACKNOWLEDGEMENTS

This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-07224 (L. Xiao), HL-42539 and HL-61639 (W. S. Colucci), HL-057947 (K. Singh), and HL-03878 (D. B. Sawyer); a Scientist Development Grant from the American Heart Association (AHA) National Center (L. Xiao), a Grant-in-Aid from the AHA Massachusetts Affiliate (D. B. Sawyer and K. Singh); and a Merit grant from the Department of Veterans Affairs (K. Singh).


    FOOTNOTES

Address for reprint requests and other correspondence: D. B. Sawyer, Cardiovascular Division, Dept. of Medicine, Boston Univ. Medical Center, X-704, 650 Albany St., Boston MA 02118 (E-mail: Douglas.sawyer{at}bmc.org).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpcell.00254.2001

Received 8 June 2001; accepted in final form 4 December 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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