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Nitric oxide attenuates insulin- or IGF-I-stimulated aortic smooth muscle cell motility by decreasing H₂O₂ levels: essential role of cGMP

Department of Physiology and Vascular Biology Center, University of Tennessee Health Science Center, Memphis, Tennessee 38163

Submitted 24 November 2003 ; accepted in final form 23 January 2004

	ABSTRACT

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Insulin and insulin-like growth factor I (IGF-I) both play important roles in vascular remodeling. Moreover, nitric oxide (NO) is well established as a counterregulatory agent that opposes the actions of several vascular agonists, in part by decreasing smooth muscle motility. We tested the hypothesis that NO blocks insulin or IGF-I-induced rat aortic smooth muscle cell motility via a mechanism involving the attenuation of agonist-induced elevation of hydrogen peroxide levels and cGMP as mediator. Insulin or IGF-I induced an increase of hydrogen peroxide levels and cell motility. Both effects were blocked by catalase or diphenyleneiodonium, indicating that hydrogen peroxide elevation is necessary for induction of cell motility. Two NO donors mimicked the effects of catalase, indicating that NO decreases cell motility by suppressing agonist-induced elevation of hydrogen peroxide. A cGMP analogue mimicked the effect of NO, whereas a guanyl cyclase inhibitor blocked the effect of NO on hydrogen peroxide levels, indicating that elevation of cGMP is both necessary and sufficient to account for the reduction of hydrogen peroxide levels. A NO donor as well as a cGMP analogue attenuated insulin-stimulated NADPH activity, indicating that NO decreases hydrogen peroxide levels by inhibiting the generation of superoxide, via a cGMP-mediated mechanism. Finally, exogenous hydrogen peroxide increased cell motility and reversed the inhibitory effect of cGMP. These results support the view that NO plays an antioxidant role via reduction of hydrogen peroxide in cultured rat aortic smooth muscle cells and that this effect is both necessary and sufficient to account for its capacity to decrease cell motility.

vascular remodeling; antioxidant; Akt; NAD(P)H oxidase

A functional role of insulin-like growth factor I (IGF-I) in neointima formation has also been proposed based on the decrease of neointima formation in rats treated with an IGF-I antagonist and the increase of neointima formation in transgenic mice, overexpressing IGF-I specifically in blood vessels (49, 50). In addition, IGF-I gene expression is upregulated in medial vascular smooth muscle cells after balloon injury, at a time corresponding to increased DNA synthesis (21).

Several polypeptide growth factors increase the levels of hydrogen peroxide in vascular smooth muscle cells, and this effect appears to be necessary for induction of cell proliferation and migration (1, 43). The capacity of growth factors to increase hydrogen peroxide is linked primarily to activation of NAD(P)H oxidase, which generates superoxide anion, followed by enzymatic conversion of superoxide to hydrogen peroxide by superoxide dismutase (15). Hydrogen peroxide is then degraded by catalase and glutathione peroxidase to water. NAD(P)H oxidase is upregulated after vascular injury, and this effect may play an important role in the generation of a neointima (40, 45).

Antioxidants have the capacity to attenuate neointima formation, supporting the view that reactive oxygen species are important in the pathogenesis of vascular disease (38). In cultured smooth muscle cells, hydrogen peroxide induces tyrosine phosphorylation of growth factor receptor kinases and activation of downstream effectors, such as Ras or mitogen-activated protein (MAP) kinase, consistent with a putative role of this oxidant in regulation of mechanisms thought to be associated with vascular remodeling (33). Catalase blocks PDGF-induced hydrogen peroxide generation, MAP kinase activation and proliferation in cultured vascular smooth muscle cells, consistent with an essential role of hydrogen peroxide in signaling and proliferation (43). Moreover, the inhibitory effect of catalase in vivo supports the view that hydrogen peroxide itself plays an important role in regulation of vascular remodeling (3).

Exogenous NO or NO generated by the endothelium or by vascular smooth muscle cells decreases vascular smooth muscle cell motility and proliferation as well as neointima formation in various models of vascular injury (37, 47). However, the mechanisms underlying the antimitogenic and antimotogenic effects of NO are incompletely understood. Previous studies (23, 39) have reported that NO can attenuate signal transduction pathways involving MAP kinases or the cell cycle. We have reported that NO upregulates the activity of a tyrosine phosphatase, PTP1B, that targets insulin-stimulated signal transduction and cell motility in cultured vascular smooth muscle cells (18). Because hydrogen peroxide appears to be a second messenger that regulates cell motility, it seems likely that NO may also target mechanisms regulating hydrogen peroxide generation. Indeed, recent studies (7, 12) have found that NO can attenuate the capacity of neutrophils to generate superoxide by directly interacting with NAD(P)H oxidase. NO can also reduce superoxide levels by directly reacting with superoxide and forming peroxynitrite (36).

The aforementioned findings prompted us to test several hypotheses related to the motogenic effect of insulin or IGF-I, as well as the antimotogenic effect of NO in cultured rat aortic smooth muscle cells. First, we tested the hypothesis that insulin and IGF-I have the capacity to increase hydrogen peroxide levels and that this effect is necessary for induction of cell motility. Second, we tested the hypothesis that NO attenuates insulin- or IGF-I-induced hydrogen peroxide elevation via a cGMP-mediated mechanism that induces inhibition of NAD(P)H oxidase activity. Third, we tested the hypothesis that the capacity of NO to decrease insulin receptor (IR) or IGF-I receptor tyrosine phosphorylation levels can be explained by attenuation of agonist-induced increase of hydrogen peroxide levels. Finally, we tested the hypothesis that the capacity of NO to attenuate hydrogen peroxide levels is sufficient and necessary to explain NO-induced inhibition of cell motility.

	MATERIALS AND METHODS

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Materials. Lactating female rats of the Sprague-Dawley strain and their pups were purchased from Charles River Laboratories (Wilmington, MA), or pups of the same strain were bred in the University of Tennessee vivarium. Primaria tissue culture plates were from Falcon/Becton Dickinson (Oxnard, CA). Type I collagenase, soybean trypsin inhibitor, fetal bovine serum, bovine serum albumin (fraction V), catalase, 1H-[1,2,4]oxadiozolo[4,3-a]quinoxalin-1-one (ODQ), NADPH, diphenylene iodonium (DPI), or lucigenin were from Sigma (St. Louis, MO). 2,2-(Hydroxynitrosohydrazino) bis-ethanamine (DETANO) and S-nitroso-N-acetyl-penicillamine (SNAP) were obtained from Alexis Biochemicals (Carlsbad, CA). DMEM-Ham's F-12 (1:1) was from GIBCO (Grand Island, NY). Porcine pancreatic elastase, insulin, transferrin, and selenous acid were from Collaborative Research (Lexington, MA). Antibodies against phosphotyrosine [RC20H Anti-PTyr-horseradish peroxidase (HRP)] were from Transduction Laboratories (Lexington, KY). Antibodies against IR or IGF-I receptor were from Santa Cruz Biotechnology (Santa Cruz, CA), whereas the antibody against Akt was from Cell Signaling (Beverly, MA). All other reagents were of the highest quality available and were generally obtained from Sigma (St. Louis, MO) or Baxter (Edison, NJ), unless stated otherwise.

Cell culture. Smooth muscle cells were obtained from the thoracic aortas of newborn Sprague-Dawley rats (6–9 days old) and cultured as previously described (42). All experiments in this study were performed using primary cultures; moreover, each individual experiment represents results from one such cell isolate, generally obtained from two newborn rat litters. All experiments were done using confluent cultures that were serum starved for 24–48 h before experiments. The current studies were performed via a protocol approved by the Animal Care and Use Committee, University of Tennessee Health Science Center, in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 86-23, Revised 1996).

Measurement of intracellular hydrogen peroxide levels. Intracellular hydrogen peroxide levels were detected by measurement of the fluorescence of oxidized DCF, as described (1). Most experiments were done by preincubation of serum-starved primary cultures with various antagonists (30 µM DETANO or 50 µM SNAP for 30 min, 1 mM N-acetylcysteine for 90 min, or 0.3 µM catalase for 3 h), followed by incubation with agonists (insulin for 5 min, IGF-I for 30 s). DCF was loaded by incubation of cells for the last 10 min of the preincubation period with 10 µM DCF/diacetate. Excess DCF was removed by being washed three times with Hanks' balanced salt solution, followed by measurement of epifluorescence levels in a Nikon microscope equipped with an FITC filter (excitation wavelength 488 nm, emission wavelength 515–540 nm) and a Coolsnap digital camera (Photometrics; Tucson, AZ). Fluorescence images were collected via a single rapid scan (5–10 s) to avoid photobleaching and quantitation was performed via the MetaMorph software (Universal Imaging; West Chester, PA). Care was taken to maintain camera settings and acquisition times constant for all treatment categories. All values had an appropriate background subtracted before analysis of DCF fluorescence.

Immunoprecipitation and immunoblotting. Cells were lysed in RIPA buffer (150 mM NaCl, 1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, and 50 mM Tris, pH 7.2) containing 2 mM sodium vanadate, 1 mM PMSF, 5 µg/ml aprotinin, and 5 µg/ml leupeptin. Protein concentration in lysates was determined by the bicinchoninic acid method. Immunoprecipitation was performed by incubation of cell lysates (2 mg protein equivalent) with antibodies directed against IR- subunit or IGF-IR (4 µg protein equivalent), for 3 h at 4°C, followed by the addition of protein G-Sepharose beads (Pharmacia) and further incubation for 2 h at 4°C. Sepharose beads were then washed twice with the above-mentioned buffer and once with 50 mM Tris buffer (pH 7.2) containing 150 mM NaCl, 1 mM sodium vanadate, and 1 mM PMSF, followed by boiling of beads in Laemmli sample buffer and loading of supernatant onto gels for SDS-PAGE. Proteins were transferred onto polyvinylidene difluoride membranes (Immobilon PVDF, Millipore), followed by a probe with anti-phosphotyrosine (RC20H Anti-PTyr-HRP) antibodies (1:5,000). The blots were then stripped and reprobed with primary antibodies directed against IR- (1:500), IGF-IR (1:500), or Akt (1:1,000), followed by HRP-coupled secondary antibodies with appropriate specificity. Immunoreactive bands were visualized using Western Lightning Chemiluminescence Reagents Plus (Perkin-Elmer).

Measurement of NADPH oxidase activity via chemiluminescence assay. NADPH oxidase activity was measured via lucigenin chemiluminescence assay, based on the finding that relatively low concentrations of lucigenin, of the order of 5–10 µM, allow specific measurement of superoxide levels (28, 41). Cells were treated with experimental agents, followed by a gentle washing with ice-cold PBS, scraping, and trituration with the use of a 26-gauge needle, based on a procedure described by Parinandi et al. (30). The cell suspension was kept on ice. An aliquot of cell lysate (200 µl, corresponding to 10⁶ cells) was added to prewarmed PBS containing 10 µM lucigenin and with or without 50 µM NADPH, containing or lacking the NADPH oxidase inhibitor DPI and chemiluminescence was measured in a Packard scintillation counter in which the coincidence circuit was turned off. Chemiluminescence was measured for 3 min and NADPH oxidase activity was expressed as counts per minute per million cells.

Measurement of cell motility. Cell motility was measured via a monolayer wounding assay, involving migration of cells into the breach, as described by Chang et al. (5). All experiments were done in the presence of hydroxyurea, to prevent cell proliferation, as described previously (5). The motility index was expressed as the distance migrated in 24 h.

Data analysis. Data are expressed as means ± SE and statistically evaluated using Student's paired t-test, one- or two-way ANOVA of the raw data, followed by Fisher's test. P < 0.05 was considered to be significant.

	RESULTS

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Insulin stimulates rat aortic smooth muscle cell motility via a mechanism independent of IGF-I receptor activation. In a previous study (42), we showed that insulin has the capacity to induce cell motility in cultured aortic smooth muscle cells isolated from newborn rats. In the current study, we show that IGF-I also has the capacity to stimulate cell motility, to about the same extent as insulin (Fig. 1). This raises the issue of whether the effect of insulin is mediated via the IGF-I receptor (IGF-IR) rather than the IR. To address this possibility, we used an antibody that has been reported to block the IGF-I receptor in rat aortic smooth muscle cells in selective fashion (2). As shown in Fig. 1, anti-IGF-IR failed to alter the motility-stimulatory effect of insulin, although it attenuated IGF-I-induced motility by >80%. This indicates that the motility-stimulatory effect of insulin is unlikely to occur via stimulation of the IGF-IR.

View larger version (24K): [in this window] [in a new window]   Fig. 1. Blocking antibody (ab) directed against insulin-like growth factor I (IGF-I) receptor (IGF-IRab) attenuates IGF-I-induced aortic smooth muscle cell motility but not insulin-induced motility. Cells were treated with 100 nM insulin or 3.3 nM IGF-I for 24 h, in the presence or absence of antibody (in 1 ml culture medium) directed against the IGF-I receptor. Results are means ± SE of three experiments. *P < 0.05 compared with control; **P > 0.05 compared with treatment with 5 µg IGFRab.

View larger version (16K): [in this window] [in a new window]   Fig. 2. Time course of increase of hydrogen peroxide levels induced by insulin or IGF-I. A: effect of insulin: cells were treated with 100 nM insulin. Altered hydrogen peroxide levels were determined via measurement of oxidized 2',7'-dichlorohydrofluorescein (DCF) fluorescence. B: effect of IGF-I: cells were treated with 3.3 nM IGF-I. Results are means ± SE of three experiments. *P < 0.05 compared with no-treatment control at each time point.

View larger version (13K): [in this window] [in a new window]   Fig. 3. Catalase or N-acetylcysteine blocks insulin-induced increase of hydrogen peroxide levels. Cells were pretreated with catalase (0.3 µM; 3 h) or N-acetylcysteine (1 mM; 90 min), followed by treatment with 100 nM insulin for 5 min. Altered hydrogen peroxide levels were measured via oxidized DCF fluorescence. Results are the means ± SE of three independent experiments. *P < 0.05 compared with control.

View larger version (26K): [in this window] [in a new window]   Fig. 4. Nitric oxide (NO) donors or cGMP analog blocks insulin- or IGF-I-induced increase of hydrogen peroxide levels. A: microscopic view of fluorescence in cells. Cells were preincubated for 10 min with 30 µM 8-(4-chlorophenylthio)-cGMP (8-pCPT)-cGMP or for 30 min with 30 µM 2,2-(hydroxynitrosohydrazino)bis-ethanamine (DETANO), followed by incubation for 5 min with 100 nM insulin and measurement of hydrogen peroxide levels via oxidized DCF fluorescence. Similar results were obtained via the use of the NO donor S-nitroso-N-acetyl-penicillamine (SNAP; not shown). B: summary of experiments with insulin as the agonist. Cells were pretreated with 30 µM DETANO, 50 µM SNAP, or 30 µM 8-p-CPT-cGMP for 30 min, followed by treatment with 100 nM insulin for 5 min and measurement of hydrogen peroxide levels. Results are the means ± SE of three independent experiments. C: summary of experiments with IGF-I as the agonist. Cells were pretreated with 30 µM DETANO for 30 min, followed by treatment with 3.3 nM IGF-I for 30 s and measurement of hydrogen peroxide levels. Catalase blocked oxidation of DCF induced by IGF-I (not shown, two experiments). Results are the means ± SE of three independent experiments. *P < 0.05 compared with control.

An antagonist of guanyl cyclase, ODQ, reverses the inhibitory effect of NO, relative to insulin-induced hydrogen peroxide elevation. To complement the aforementioned experiments, we tested the hypothesis that increased cGMP is necessary to mediate the inhibitory effect of NO on insulin-induced increase of hydrogen peroxide levels. ODQ is considered to be a selective inhibitor of NO-stimulated guanyl cyclase activity (14). Treatment of cells with insulin or NO donor, in the absence or presence of ODQ, demonstrated that the latter agent could reverse the inhibitory effect of NO, without inducing an effect of its own (Fig. 5). These results support the hypothesis that elevation of cGMP induced by NO is necessary to mediate its capacity to block the increase of hydrogen peroxide levels.

View larger version (17K): [in this window] [in a new window]   Fig. 5. The guanyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3a]quinoxalin-1-one (ODQ) reverses the inhibitory effect of NO donor on insulin-induced increase of hydrogen peroxide levels. Cells were pretreated with 30 µM DETANO and/or 2.5 µM ODQ for 30 min, before treatment with 100 nM insulin for 5 min, followed by measurement of hydrogen peroxide levels via oxidized DCF fluorescence. Results are means ± SE of three independent experiments. *P < 0.05 compared with control.

View larger version (14K): [in this window] [in a new window]   Fig. 6. cGMP agonists attenuate insulin-induced activation of NADPH oxidase activity. Cells were pretreated with 30 µM 8-p-CPT-cGMP for 10 min or 30 µM DETANO for 30 min, before exposure to 100 nM insulin or 20 ng/ml PDGF, for 5 min. Cell lysates were prepared and NADPH activity was determined via measurement of lucigenin chemiluminescence. Results are the means ± SE of three independent experiments. *P < 0.05 compared with control.

View larger version (23K): [in this window] [in a new window]   Fig. 7. NO donor, cGMP, or catalase blocks insulin-induced tyrosine phosphorylation of insulin receptor. Cells were pretreated with 30 µM DETANO for 30 min, 30 µM 8-p-CPT-cGMP for 10 min or 0.3 µM catalase for 180 min, followed by treatment with 100 nM insulin for 5 min. Insulin receptor was then immunoprecipitated, and the levels of phosphotyrosine and insulin receptor were determined by sequential Western blot analyses. A: the top blot shows phosphotyrosine levels in receptor protein, whereas the bottom blot shows insulin receptor protein levels, via reprobe of blot shown at top. B: quantitative summary of four independent Western blot experiments. Results in B are given as the ratio of phosphotyrosine to insulin receptor levels (means ± SE). *P < 0.05 compared with control.

View larger version (23K): [in this window] [in a new window]   Fig. 8. NO donor, cGMP, or catalase block IGF-I-induced tyrosine phosphorylation of IGF-I receptor (IGF-IR). Cells were pretreated with 30 µM DETANO for 30 min, 30 µM 8-p-CPT-cGMP for 10 min, or 0.3 µM catalase for 180 min, followed by treatment with 3.3 nM IGF-I for 5 min. IGF-IR was then immunoprecipitated and the levels of phosphotyrosine and IGF-IR were determined by sequential Western blot analyses. A: the top blot shows phosphotyrosine levels in receptor protein, whereas the bottom blot shows IGF-I receptor protein levels, via reprobe of the blot shown at top.B: quantitative summary of four Western blot experiments. Results are given as the ratio of phosphotyrosine to IGF-IR levels (means ± SE). * P < 0.05 compared with control.

View larger version (23K): [in this window] [in a new window]   Fig. 9. NAD(P)H inhibitor diphenylene iodonium (DPI) blocks insulin-induced tyrosine phosphorylation of insulin receptor. Cells were pretreated with 10 µM DPI for 60 min, followed by 100 nM insulin for 5 min. Insulin receptor was then immunoprecipitated, and the levels of phosphotyrosine and insulin receptor were determined by sequential Western blot analyses. A, top: phosphotyrosine levels in receptor protein. Bottom, insulin receptor protein levels via reprobe of the top blot are shown. B: quantitative summary of three Western blot experiments. Results in B are given as the ratio of phosphotyrosine to insulin receptor levels (means ± SE). *P < 0.05 compared with control.

View larger version (22K): [in this window] [in a new window]   Fig. 10. NO donors attenuate insulin-induced Akt tyrosine phosphorylation. Cells were pretreated with 30 µM DETANO or 50 µM SNAP for 30 min, followed by treatment with 100 nM insulin for 5 min. Akt was then immunoprecipitated, and the levels of phosphotyrosine and Akt protein were determined by sequential Western blot analyses. A, top: phosphotyrosine levels in Akt. Bottom, Akt protein levels via a reprobe of the blot shown at top. B: quantitative summary of three Western blot experiments. Results in B are given as the ratio of phosphotyrosine to Akt protein levels (means ± SE). *P < 0.05 compared with control; **P < 0.05 compared with insulin treatment.

View larger version (18K): [in this window] [in a new window]   Fig. 11. Catalase attenuates insulin- or IGF-I-induced cell motility. Cells were pretreated with 0.3 µM catalase or 0.3 µM boiled catalase for 180 min, followed by incubation with 100 nM insulin, or 3.3 nM IGF-I for 24 h. Cell motility was measured with the use of NIH Image software. Results are the means ± SE of three experiments. *P < 0.05 compared with control.

View larger version (10K): [in this window] [in a new window]   Fig. 12. NAD(P)H oxidase inhibitor DPI attenuates insulin-induced cell motility. Cells were preincubated with 10 µM DPI for 60 min, followed by incubation with 100 nM insulin for 24 h. Results are the means ± SE of three experiments. *P < 0.05 compared with control.

View larger version (14K): [in this window] [in a new window]   Fig. 13. cGMP agonists attenuate insulin- or IGF-I-induced cell motility. Cells were incubated with DETANO (30 µM), 8-p-CPT-cGMP (30 µM), insulin (100 nM) (A) or SNAP (50 µM) or IGF-I (3.3 nM) (B) for 24 h, before measurement of cell motility. Results are the means ± SE of three independent experiments. *P < 0.05 compared with control.

View larger version (24K): [in this window] [in a new window]   Fig. 14. Hydrogen peroxide induces cell motility and reverses the inhibitory effect of cGMP. Cells were incubated with 100 nM insulin, 30 µM 8-p-CPT-cGMP, or various concentrations of hydrogen peroxide for 24 h. Numerical values in legend refer to hydrogen peroxide concentration (in µM). Results are the means ± SE of three experiments. *P < 0.05 compared with control.

	DISCUSSION

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The main purpose of this work was to test the overall hypothesis that the capacity of NO to block insulin- or IGF-I-induced hydrogen peroxide elevation in cultured vascular smooth muscle cells can explain NO-induced inhibition of cell motility. The study was performed in primary cultures of vascular smooth muscle cells isolated from newborn rats for two reasons. First, these cells express relatively high levels of the presumed downstream cGMP effector PKG, compared with repetitively subcultured cells, which have a deficiency or lack of PKG (results not shown). Second, the cells express a cytoskeletal phenotype that resembles that found in neointimal cells (27).

Our study is the first to show that NO targets one (or more) NAD(P)H oxidase(s) via a cGMP-mediated mechanism, followed by decrease of hydrogen peroxide levels, rather than via a direct effect, as reported in neutrophils (7, 12, 35). The capacity of cGMP analog to inhibit insulin-stimulated NADPH oxidase activity strongly suggests that the decrease of hydrogen peroxide levels by cGMP agonists can be explained by inhibition of NADPH oxidase activity. It should also be noted, however, that our experiments do not identify a specific NADPH oxidase as the target of the cGMP pathway, inasmuch as several different enzymes have the capacity to generate superoxide and DPI is not a selective inhibitor of the plasma membrane NAD(P)H oxidase. Thus it is clear that additional work will be required to identify the specific enzyme(s) involved in this process. We also report the novel finding that the increase of hydrogen peroxide induced by either insulin or IGF-I is both sufficient and necessary to explain the mitogenic effect of these agents. Furthermore, the reduction of hydrogen peroxide is both necessary and sufficient to explain the capacity of NO to decrease insulin- or IGF-I-induced motility in cultured rat aortic smooth muscle cells.

Interestingly, the time course of hydrogen peroxide elevation induced by insulin was transient, whereas that induced by IGF-I was more sustained. The reasons for this difference are unknown, but they are consistent with the view that hydrogen peroxide induces upregulation of IGF-I mRNA levels, which could in turn stimulate an IGF-I-induced feed-forward mechanism involving the intermediacy of hydrogen peroxide (10). Moreover, the transiency of insulin's effect indicates that a relatively short period of hydrogen peroxide elevation is sufficient to induce aortic smooth muscle cell motility.

Recent studies (29) have demonstrated unequivocally the presence of NAD(P)H oxidase in intact blood vessels. Moreover, NAD(P)H oxidase is recognized as the predominant system that generates superoxide in vascular smooth muscle (15, 32). The NAD(P)H oxidase system has been reported to be upregulated in balloon injury or atherosclerosis and the activity of the enzyme has been linked to growth factor-induced cell proliferation (40, 43, 45). Consistent with these data are findings that antioxidants have the capacity to attenuate neointima formation (38).

Previous studies have reported that NO can decrease superoxide generation in neutrophils via a direct interaction (7, 12, 35), whereas the current results indicate a requirement for the cGMP pathway. Events donwstream from cGMP may include activation of PKG and reduction of cytoplasmic Ca²⁺, but this remains to be documented. Speculatively, a reduction of cytoplasmic Ca²⁺ by NO via the cGMP/PKG pathway (8, 17) can explain the current results because there is evidence that implicates Ca²⁺ as a regulator of NADPH oxidase (6, 9). Moreover, reduced Ca²⁺ is likely to be associated with reduced PKC activity and PKC has been reported to be involved in regulation of NADPH activity (6).

Other investigators have found that NO can directly interact with superoxide, forming peroxynitrite, a strong oxidant that can also induce oxidation of the reporter dye DCF (22). However, it is unlikely that addition of NO to our cells would generate significant amounts of peroxynitrite, because this would have increased, not decreased, the levels of oxidized DCF, as reported previously (22). Moreover, the capacity of cGMP to block hydrogen peroxide elevation and motogenesis, and that of ODQ to reverse the inhibitory effect of NO, argues against an involvement of peroxynitrite in explaining our results.

An emerging link between reactive oxygen species and cellular regulation is the inhibitory effect of hydrogen peroxide on protein tyrosine phosphatase activity. Thus elevation of hydrogen peroxide by insulin or EGF is associated with decreased activity of at least one important phosphatase, namely PTP1B (24). A recently proposed mechanism that can account for the phosphatase inhibitory effect of hydrogen peroxide is oxidation of a catalytically essential cysteine residue, present in most if not all such phosphatases (34). We have reported that NO attenuates the phosphorylation of the IR by inducing activation of PTP1B (42), and in the current study we have found similar results relative to IGF-IR in IGF-I-stimulated cells. Because PTP1B can directly target the insulin and IGF-I receptors (4, 48), it seems likely that inhibition of PTP1B by hydrogen peroxide contributes to increased tyrosine phosphorylation of the transmembrane insulin or IGF-I receptors; reciprocally, the decrease of hydrogen peroxide levels by NO would then contribute to increasing PTP1B activity and maintaining the phosphorylation of receptors at low levels. These events would then contribute to attenuating insulin/IGF-I-induced signal transduction, as shown by the capacity of NO to attenuate Akt phosphorylation (current study) and MAP kinase activity (42).

In summary, we report novel data indicating that NO targets the NAD(P)H oxidase, via a cGMP-dependent mechanism that attenuates the capacity of insulin or IGF-I to increase hydrogen peroxide levels. The suppression of hydrogen peroxide is both necessary and sufficient to explain the antimotogenic effect of NO in differentiated rat aortic smooth muscle cells. Finally, it is interesting to note the existence of an inverse relationship between NO and superoxide levels in human vascular tissues (16). Taken together, these studies raise the possibility that NO could act as agonist of a counterregulatory pathway that decreases the generation of reactive oxygen species, thus functioning as endogenous antioxidant in vascular smooth muscle. The mechanisms described in our report are likely to be of relevance to in vivo events and the testing of this hypothesis is now in progress.

	GRANTS

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This work was supported by National Heart, Lung, and Blood Institute Grants HL-63886, HL-64165, and HL-64981 and by the Vascular Biology Center, University of Tennessee Health Science Center (Memphis, TN).

	ACKNOWLEDGMENTS

 
Present address for Y. Lin: Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, 825 NE 13th St., Oklahoma City, OK 73104.

Present address for M. Dixit: Institut für Kardiovasculäre Physiologie, Klinikum der J.W. Goethe-Universität, Theodor-Stern-Kai 7, Frankfurt am Main, Germany.

	FOOTNOTES

 

Address for reprint requests and other correspondence: A. Hassid, Dept. of Physiology, Univ. of Tennessee Heath Science Center, Memphis, TN 38103 (E-mail: ahassid{at}tennessee.edu).

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.

¹ We have used the term "NADPH oxidase" to refer to enzyme activity specifically determined in assays using NADPH as the reductant; on the other hand, to indicate oxidase activity in intact cells, we have used the term "NAD(P)H oxidase" to reflect the indeterminate nature of the reductant cofactor.

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