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(Hypertension. 2003;42:569.)
© 2003 American Heart Association, Inc.

Scientific Contributions

Insulin-Stimulated Hydrogen Peroxide Increases Guanylate Cyclase Activity in Vascular Smooth Muscle

From the Division of Renal Diseases and Hypertension, Department of Medicine, The University of Texas Health Science Center, Houston, Tex.

Correspondence to Andrew M. Kahn, MD, The University of Texas Health Science Center, Houston, Medical School, 6431 Fannin St, MSB 4.148, Houston, TX 77030. E-mail Andrew.M.Kahn{at}uth.tmc.edu

	Abstract

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Insulin resistance is associated with vascular disease. Physiological concentrations of insulin inhibit cultured vascular smooth muscle cell (VSMC) contraction and migration by increasing nitric oxide (NO)-stimulated cGMP accumulation. The failure to do so in insulin-resistant states may aggravate vascular disease. We sought to determine the mechanism of insulin’s increase in cGMP accumulation. Isobutylmethylxanthine, an inhibitor of phosphodiesterase activity, inhibited the decline in cGMP levels measured by immunoassay in cGMP-loaded cultured rat aortic VSMCs, but 1 nmol insulin did not. Thus, insulin’s increase in cGMP accumulation is due to stimulated production, not inhibited hydrolysis and/or efflux. Insulin, which increases the NADH/NAD⁺ ratio in these cells, stimulated superoxide anion (O₂^-) accumulation measured by lucigenin luminescence to 256±25% (P<0.05) by a process that was blocked by the NADH oxidase inhibitor diphenyliodonium (DPI) and enhanced by the superoxide dismutase inhibitor diethyldithiocarbonate (DETCA). Insulin also stimulated hydrogen peroxide (H₂O₂) accumulation measured by horseradish peroxidase/luminol luminescence to 221±22% (P<0.05) by a DETCA-sensitive mechanism. H₂O₂ (100 µmol/L) in the absence of insulin increased NO-stimulated cGMP accumulation to 151±11% (P<0.05). Insulin alone increased NO-stimulated cGMP accumulation to 183±17% (P<0.05), and this was blocked by either DPI or DETCA. We conclude that insulin increases NADH oxidase-derived O₂^- production in cultured rat VSMCs. This did not cause the expected scavenging of NO resulting in the reduction of NO-stimulated guanylate cyclase activity, but enough O₂^- was metabolized to H₂O₂ to increase overall NO-stimulated cGMP production.

Key Words: muscle, smooth, vascular • insulin • cyclic GMP • nitric oxide

	Introduction

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Atherosclerosis and restenosis after balloon catheter angioplasty are increased in insulin-resistant states.^1–3 Some studies support the view that hyperinsulinemia, which is present in these conditions, stimulates vascular smooth muscle cell (VSMC) migration and proliferation, thereby accelerating the pathological process.^4,5 Other data show that insulin can stimulate cGMP accumulation in normal cultured VSMCs^6,7 and that cGMP inhibits migration and proliferation of those cells.^8–10 The latter data are consistent with the notion that insulin normally inhibits atherosclerosis and restenosis in vivo and that the functional lack of insulin in insulin-resistant states contributes to vascular disease.

We have shown that insulin alone does not affect cGMP accumulation in cultured VSMCs, but when cGMP production is stimulated by a permissive amount of nitric oxide (NO), physiological concentrations of insulin increase the accumulation of cGMP.⁶ We found that insulin stimulates glucose uptake and aerobic glycolysis in these cells and that the resultant increase in lactate is responsible for insulin’s increase in NO-stimulated cGMP accumulation.^6,11,12 Increased lactate in VSMCs has been reported to increase superoxide anion (O₂^-) production.¹³ O₂^- is well known to scavenge NO, thereby decreasing VSMC guanylate cyclase activity.¹⁴ It was unclear how to reconcile these phenomenon with our previous finding that insulin-stimulated lactate increases NO-stimulated cGMP accumulation.^6,12 The present study was designed to explain these discrepant findings and determine the mechanism of insulin’s increase in NO-stimulated cGMP accumulation. Since reactive oxygen species have been linked to decreased phosphodiesterase activity,^15,16 the first goal of these studies was to determine whether insulin increases NO-stimulated cGMP generation or inhibits its hydrolysis and/or efflux.

Omar et al¹³ have shown that lactate increases cGMP-mediated relaxation of endothelium-denuded vascular smooth muscle tissue from calf pulmonary artery. They demonstrated that lactate’s increase in NADH, caused by the action of lactate dehydrogenase, stimulates the activity of NADH oxidase that yields O₂^-. In other experiments with endothelium-denuded bovine pulmonary artery, they showed that hydrogen peroxide (H₂O₂), the product of O₂^- and superoxide dismutase (SOD), reacts with catalase and that the resultant intermediate form of the catalase enzyme (compound I) stimulates soluble guanylate cyclase activity.¹⁷ Others have reported that H₂O₂ stimulates guanylate cyclase activity in guinea pig aorta,¹⁸ rabbit urethral and cavernosal smooth muscle,¹⁹ and human platelets.²⁰ Thus, it was possible that insulin’s increase in lactate in cultured VSMCs caused increased O₂^- and H₂O₂ production and the latter increased guanylate cyclase activity. The second goal of this study was to test this hypothesis.

	Methods

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Cell Culture
Twelve male Sprague Dawley rats per cell culture preparation were killed with intraperitoneal pentobarbital sodium, and the thoracic aortas were dissected free. Endothelia and adventitia were stripped away; the media of the arteries were minced, incubated at 37°C in a solution containing elastase (type V, Sigma) and collagenase (type I, Worthington Biochemical), and cultured VSMCs were prepared in 10% FBS as previously described.⁹ Confluent cultured cells of passages 3 to 10 in 35-mm plastic dishes after 14 hours without FBS were used in experiments. Since a permissive amount of NO is necessary for insulin to stimulate cGMP accumulation in cultured VSMCs, some experiments were performed with cells that had been induced to express iNOS with lipopolysaccharide and interleukin-1ß as previously described.²¹

Cyclic GMP Assay
Dishes of VSMCs were incubated for 30 minutes at 37°C with the desired agents in physiological salt solution (PSS) containing (in mmol/L) 136 NaCl, 4 KCl, 5 glucose, 1.8 CaCl₂, 0.8 MgSO4, 10 HEPES-Tris, pH 7.4, plus 0.1% BSA. cGMP content of acetylated cell lysates was determined with a direct cGMP enzyme immunoassay kit (Assay Designs).

Superoxide Anion Assay
Dishes of VSMC were incubated for 30 minutes at 37°C with the desired agents in PSS; the cells were washed with Hanks balanced salt solution (HBSS) and released from the dishes by incubating them with collagenase (1 mg/mL), BSA (2 mg/mL), and soybean trypsin inhibitor (1 mg/mL) in HBSS at 37°C for 5 minutes. Cells were collected by centrifugation (200g for 5 minutes at 4°C), and the cell pellet was resuspended in HBSS; 20 µL of 2.5 mmol/L dark-adapted lucigenin in HBSS was added to 80 µL of cell suspension (40 µg protein) at room temperature, and photon emission was measured every 15 seconds for 10 minutes in a luminometer (model 20/20, Turner Designs) as previously described.²² Readings stabilized by 3 minutes, and that value minus a blank value obtained in the absence of cells was recorded. O₂^- content of samples was obtained from a standard curve generated with the use of xanthine/xanthine oxidase, as previously described.²³

Hydrogen Peroxide Assay
Dishes of cells were washed and incubated with the desired reagents in 2 mL of PSS at 37°C for 30 minutes. H₂O₂ content of 200 µL incubation solution was measured with the Peroxil Luminol Hydrogen Peroxide Determination Kit (World Precision Instruments). In this assay, horseradish peroxidase reacts with H₂O₂ to form reactive intermediates that oxidize luminol. The chemiluminescence emitted by luminol was measured in a luminometer, and H₂O₂ content of samples minus a blank was obtained from a standard curve with the use of authentic H₂O₂ in PSS.

Values for cGMP, O₂^-, and H₂O₂ varied from experiment to experiment, but the relative effects of experimental perturbations were highly reproducible among different experiments. Thus, data are expressed as a percentage of values obtained under control conditions, and the mean absolute control values are stated in the figure legends. Statistical analysis was performed on paired data by use of Student t test and ANOVA, with multiple comparisons using the Newman-Keuls test. Statistical significance was taken as a value of P<0.05.

Bovine insulin, cGMP, IBMX, BSA, diphenyliodonium (DPI), diethyldithiocarbonate (DETCA), and H₂O₂ were obtained from Sigma, and S-nitroso-N-acetylpenicillamine was from Alexis. Protein was measured by the method of Bradford.

	Results

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Insulin and cGMP Hydrolysis
We have reported that insulin increases NO-stimulated cGMP accumulation in cultured VSMCs.⁶ A potential explanation for this finding is that insulin inhibits phosphodiesterase activity, thereby inhibiting the hydrolysis of NO-stimulated cGMP and/or inhibits cGMP efflux from the cell. To test this possibility, VSMCs were preloaded with cGMP, washed, incubated without NO in the presence and absence of 1 nmol insulin, and the decline in cell cGMP was measured over 1 hour. The permeable analogs of cGMP, 8-Br-cGMP and dibutyryl cGMP, are resistant to hydrolysis by phosphodiesterases, so cells were loaded with authentic cGMP, for which there is modest cell permeability.²⁴ As shown in Figure 1, cell cGMP fell by 39% after 1 hour, and insulin did not inhibit its decline. To rule out the possibility that the observed fall in cGMP merely represented the release of extracellularly bound cGMP and to serve as a positive control, cGMP-loaded cells were also incubated with the phosphodiesterase inhibitor IBMX. As also shown in Figure 1, IBMX inhibited the fall of cGMP by 75%, indicating that most of the decline of cGMP under control conditions represented the hydrolysis of cGMP. Since cGMP levels in preloaded cells were approximately equal to insulin-stimulated values, insulin’s increase in NO-stimulated cGMP accumulation was not due to inhibition of cGMP hydrolysis and/or efflux.

View larger version (25K): [in this window] [in a new window]   Figure 1. Effect of insulin and IBMX on cGMP hydrolysis. Confluent dishes of VSMCs were preincubated in PSS plus 10 µmol/L cGMP for 1 hour at 37°C. Cells were washed 6 times with 1 mL PSS at 37°C and incubated for 1 hour in 1 mL PSS plus 0.1% BSA with or without 1 mmol/L IBMX or 1 nmol insulin, and cGMP was measured. Data are expressed as percentage ±SEM of cGMP content of cells after washing (time zero), which averaged 9.1 pmol/mg protein, and are from 4 separate experiments. *P<0.05 vs control.

Effect of Insulin on O₂^-
Insulin increases lactate content and the lactate-to-pyruvate ratio in these cells 3-fold, as we have previously reported.¹² The concomitant increase in the NADH/NAD⁺ ratio would be expected to increase O₂^- production by NADH oxidase, as has been described in other vascular smooth muscle tissue.¹³ As shown in Figure 2, 30-minute exposure to 1 nmol insulin increased O₂^- levels in these cells. DPI, an inhibitor of NADH oxidase, did not affect basal O₂^- but completely blocked insulin stimulation of O₂^- levels. DETCA, an inhibitor of SOD, increased both basal and insulin-stimulated O₂^- levels. These data indicate that insulin stimulates O₂^- production by NADH oxidase and that SOD is active in these cells.

View larger version (26K): [in this window] [in a new window]   Figure 2. Effect of insulin, DPI, and DETCA on O2-. Confluent dishes of VSMCs were incubated in PSS plus 0.1% BSA with or without 1 nmol insulin, 10 µmol/L DPI, or 10 µmol/L DETCA for 30 minutes at 37°C, the cells were harvested, and O2- levels were measured by lucigenin luminescence. Data are expressed as percentage ±SEM of control, which averaged 1.1 nmol/mg protein, and are from 4 separate experiments. *P<0.05 vs control. **P<0.05 vs DETCA.

O₂^- Metabolism and cGMP
If insulin-stimulated O₂^- production leads to an increase of insulin in NO-stimulated cGMP production, this effect of insulin should be blocked by DPI. As shown in Figure 3A, insulin alone had no effect on cGMP. SNAP, an NO donor, increased cGMP and insulin stimulated cGMP further, as we have previously described.⁶ As also shown in Figure 3A, DPI did not affect basal or SNAP-stimulated cGMP, but it blocked the further stimulation of cGMP by insulin. These data support the possibility that insulin-stimulated O₂^- production leads to the increase of this hormone in cGMP production. Figure 3B shows that blocking SOD with DETCA did not affect basal cGMP levels, but it partially inhibited SNAP-stimulated cGMP levels and it completely blocked the additional stimulation of cGMP by insulin. These data indicate that the metabolism of insulin-stimulated O₂^- by SOD to H₂O₂ mediates insulin’s increase in NO-stimulated cGMP production.

View larger version (36K): [in this window] [in a new window]   Figure 3. Effect of SNAP, insulin, DPI, and DETCA on cGMP production. A, Confluent dishes of VSMCs were incubated with PSS plus 0.1% BSA and 0.5 mmol/L IBMX with and without 1 nmol insulin and/or 0.1 µmol/L SNAP and/or 10 µmol/L DPI for 30 minutes at 37°C, and cGMP was measured. Data are expressed as percentage ±SEM of cGMP under control conditions, which averaged 2.5 pmol/mg protein from 4 separate experiments. *P<0.05 vs control. **P<0.05 vs SNAP. B, Confluent dishes of VSMCs were incubated in PSS plus 0.1% BSA and 0.5 mmol/L IBMX with and without 1 nmol insulin and/or 0.1 µmol/L SNAP and/or 10 µmol/L DETCA for 30 minutes at 37°C, and cGMP was measured. Data are expressed as a percentage ±SEM of cGMP under control conditions, which averaged 2.1 pmol/mg protein from 4 separate experiments. *P<0.05 vs control. **P<0.05 vs SNAP.

H₂O₂ and cGMP
We then tested whether insulin increases H₂O₂ and whether the latter stimulates guanylate cyclase activity. As shown in Figure 4A, insulin increased H₂O₂ levels in VSMCs. As expected, inhibiting SOD activity with DETCA decreased basal and insulin-stimulated H₂O₂ levels. Figure 4B shows that H₂O₂ alone did not affect cGMP levels in these VSMCs, but when cGMP production was stimulated by NO from iNOS, H₂O₂ increased cGMP levels further. Since H₂O₂ was present in the incubation buffer, NO was provided by iNOS instead of SNAP in this experiment because oxidizing agents retard NO release from SNAP.²⁵ H₂O₂ per se did not affect iNOS activity (data not shown). These data indicate that the insulin’s increase in H₂O₂ increases NO-stimulated cGMP production.

View larger version (31K): [in this window] [in a new window]   Figure 4. Insulin increases H2O2, which increases cGMP. A, Confluent dishes of VSMCs were preincubated in PSS with or without 10 µmol/L DETCA for 30 minutes at 37°C, washed 10 times with 1 mL PSS, and incubated in PSS plus 0.1% BSA with or without 1 nmol insulin. Data are expressed as percentage ±SEM of H2O2 under control conditions, which are averaged 108 pmol/mg protein from 4 separate experiments. *P<0.05 vs control. B, Confluent dishes of cells were or were not induced to express iNOS by overnight preincubation with LPS and IL-1ß and incubated in PSS plus 0.5 mmol/L IBMX with or without 100 µmol/L H2O2 for 30 minutes at 37°C, and cGMP was measured. Data are expressed as a percentage ±SEM of cGMP under control conditions, which averaged 1.9 pmol/mg protein from 4 separate experiments. *P<0.05 vs control. **P<0.05 vs iNOS.

	Discussion

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Cyclic GMP inhibits cultured VSMC migration and proliferation,^8–10 and we have shown that physiological concentrations of insulin increase cGMP accumulation in these cells, but only if NO is present.⁶ The present study indicates that insulin does not inhibit the hydrolysis and/or efflux of cGMP once its formation has been stimulated by NO, since insulin did not inhibit the decline of cGMP levels in preloaded cells. It is conceivable that insulin simultaneously stimulates or inhibits cGMP hydrolysis while inhibiting or stimulating cGMP efflux from the cell such that insulin has no net effect on the decline of cGMP. Nevertheless, the present data indicate that insulin increases NO-stimulated guanylate cyclase activity rather than retarding cGMP disappearance.

The mechanism of insulin-stimulated cGMP production is unknown, but we previously showed that insulin increases lactate accumulation in cultured VSMCs.¹² If this effect of insulin was blocked, insulin failed to increase NO-stimulated cGMP levels. Raising lactate with specific metabolic substrates in the absence of insulin also increased NO-stimulated cGMP accumulation.¹² We concluded that the insulin increase in lactate increased NO-stimulated cGMP accumulation in these cells. Increased lactate increases NADH by the lactate dehydrogenase reaction, which drives NADH oxidase toward O₂^- in VSMCs.¹³ This should scavenge NO, thereby decreasing, not increasing, guanylate cyclase activity stimulated by a finite amount of NO.

Burke et al¹⁷ showed that H₂O₂ reacts with catalase, which stimulates guanylate cyclase activity in VSMCs in the apparent absence of NO. This occurred immediately, and the response decayed rapidly with time. In the present study, we showed in cultured VSMCs that insulin, which increases lactate, increased O₂^- levels. O₂^- was converted to H₂O₂ by SOD and H₂O₂ increased cGMP over a 30-minute period in the permissive presence of NO. The relatively prolonged effect of H₂O₂ on cGMP accumulation in the present study may have been due to the presence of IBMX in the incubation buffer. When O₂^- production was inhibited by blocking NADH oxidase with DPI, or when H₂O₂ production was inhibited by blocking SOD with DETCA, insulin failed to stimulate cGMP production. Thus, the present studies confirm the findings of Burke et al and extend those findings by showing that insulin stimulates guanylate cyclase activity in cultured VSMCs by the same series of reactions.

In previous studies, we showed that insulin inhibits cultured VSMC contraction in a dose-dependent manner and that this effect is cGMP-dependent^26,27; 1 nmol insulin, the concentration used in the present study, is in the physiological range in rat.²⁸ We did not test whether maximal H₂O₂-stimulated cGMP production in the presence of NO was stimulated further by insulin. If it did, insulin would have increased guanylate cyclase activity by an additional mechanism.

It is unknown why cultured rat VSMCs require the permissive presence of NO for H₂O₂ to stimulate guanylate cyclase activity. This was also reported for rabbit urethral and cavernosal smooth muscle,¹⁹ but this was not the case for bovine vascular smooth muscle tissue¹⁷ or human platelets.²⁰ Additional studies are necessary to answer this question.

We postulate that the increase of insulin in O₂^-derived H₂O₂ predominated over insulin increase in O₂^- scavenging of NO such that NO-stimulated cGMP production was overall increased by insulin. Even though the rate constant for the reaction of O₂^- with NO (7x10⁹ mol/s) exceeds that for O₂^- with SOD (2x10⁹ mol/s),²⁹ under normal conditions, the relatively high abundance of SOD ensures that the latter reaction occurs preferentially.¹⁴ It is noteworthy that DETCA partially inhibited SNAP-stimulated cGMP production (Figure 3B). This may be explicable since DETCA increased basal O₂^- levels (Figure 2), which would be expected to increase scavenging of NO, thereby lowering overall guanylate cyclase activity in the face of inhibited H₂O₂ production. Gutpe et al³⁰ found that lactate decreased NO-induced relaxation in endothelium-removed bovine pulmonary artery in the presence of DETCA, presumably because of scavenging of NO by O₂^-. In the present study, insulin, which increases lactate in cultured VSMCs, did not inhibit SNAP-stimulated cGMP production in the presence of DETCA (Figure 3B). The reason for this is unknown, but DETCA may not have completely blocked insulin-stimulated H₂O₂ production.

Perspectives
The present studies may have pathophysiological significance. Insulin-resistant states are associated with accelerated vascular disease.^1–3 The failure of insulin to stimulate guanylate cyclase in the vascular smooth muscle of these individuals could contribute to their vascular pathology. Alternatively, it has been proposed that the hyperinsulinemia associated with those conditions can aggravate it.^4,5 Although H₂O₂ is known to stimulate guanylate cyclase activity in several tissues, including VSM,^17–20 O₂^-, H₂O₂, and other oxidants are associated with accelerated vascular disease.³¹ In the present study, insulin increased O₂^- and H₂O₂, and the latter stimulated cGMP production. This should ameliorate vascular disease. On the basis of these studies, insulin might be expected to have beneficial or deleterious vascular effects, and it needs to be determined if the final effect of insulin on vascular pathology depends on various factors that tip the net effect of insulin one way or the other.

	Acknowledgments

 
This study was supported by grant HL-50660 from the National Institutes of Health. The authors acknowledge the excellent secretarial support of Wendy Williams.

Received June 2, 2003; first decision June 9, 2003; accepted August 14, 2003.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Stout RW. Insulin and atheroma: 20-yr perspective. Diabetes Care. 1990; 13: 631–654.[Abstract]

2. Asakura Suzuki M, Nonogi H, Haze K, Sato A, Inada H, Okida Y, Yamashita K, Harano Y. Restenosis after percutaneous transluminal coronary angioplasty in patients with non-insulin dependent diabetes mellitus (NIDDM). J Cardiovasc Risk. 1998; 5: 331–334.[CrossRef][Medline] [Order article via Infotrieve]

3. Osani H. Kanayama H, Miyazaki Y, Fukushima A, Shinoda M, Ito T. Usefulness of enhanced insulin secretion during an oral glucose tolerance test as a predictor of restenosis after direct percutaneous transluminal coronary angioplasty during acute myocardial infarction in patients without diabetes mellitus. Am J Cardiol. 1998; 81: 698–701.[CrossRef][Medline] [Order article via Infotrieve]

4. Reaven GM, Chen YDI. Insulin resistance, its consequences, and coronary heart disease: must we choose one culprit? Circulation. 1996; 93: 1780–1783.[Free Full Text]

5. Sowers JR. Insulin and insulin-like growth factor in normal and pathological cardiovascular physiology. Hypertension. 1997; 29: 691–699.[Free Full Text]

6. Kahn AM, Allen JC, Siedel CL, Lichtenberg DS, Zhang S. Insulin increases NO- stimulated guanylate cyclase activity in cultured VSMC while raising redox potential. Am J Physiol. 2000; 278: E627–E633.

7. Sandu OA, Ito M, Begum N. Signal transduction in smooth muscle selected contribution: insulin utilizes NO/cGMP pathway to activate myosin phosphatase via Rho inhibition in vascular smooth muscle. J Appl Physiol. 2001; 91: 1475–1482.[Abstract/Free Full Text]

8. Dubey RK, Jackson EK, Lusher TF. Nitric oxide inhibits angiotensin II-induced migration of rat aortic smooth muscle cell: role of cyclic-nucleotides and angiotensin₁ receptor. J Clin Invest. 1995; 96: 141–149.[Medline] [Order article via Infotrieve]

9. Zhang S, Yang Y, Kone BC, Allen JC, Kahn AM. Insulin-stimulated cyclic guanosine monophosphate inhibits vascular smooth muscle cell migration by inhibiting Ca/Calmodulin-dependent protein kinase II. Circulation. 2003; 107: 1539–1544.[Abstract/Free Full Text]

10. Garg UC, Hasssid A. Nitric oxide-generating vasodilators and 8-bromo-cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells. J Clin Invest. 1989; 83: 1774–1777.[Medline] [Order article via Infotrieve]

11. Kahn AM, Lichtenberg RA, Allen JC, Seidel CL, Song T. Insulin-stimulated glucose transport inhibits Ca²⁺ influx and contraction in vascular smooth muscle. Circulation. 1995; 92: 1597–1603.[Abstract/Free Full Text]

12. Kahn AM, Allen JC, Zhang S. Insulin increases NADH/NAD⁺ redox state, which stimulates guanylate cyclase in vascular smooth muscle. Am J Hypertens. 2002; 15: 273–279.[CrossRef][Medline] [Order article via Infotrieve]

13. Omar HA, Mohazzab-H KM, Mortelliti MP, Wolin MS. O₂^- dependent modulation of calf pulmonary artery tone by lactate: potential role of H₂O₂ and cGMP. Am J Physiol. 1993; 264: L141–L145.[Medline] [Order article via Infotrieve]

14. McIntyre M, Bohr DF, Dominiczak AF. Endothelial function in hypertension, the role of superoxide anion. Hypertension. 1999; 34: 539–545.[Abstract/Free Full Text]

15. Spoto G, Di Giulio C, Contento A, Di Stillo M. Hypoxic and hyperoxic effect on blood phosphodiesterase activity in young and old rats. Life Sci. 1998; 63: PL349–PL353.[Medline] [Order article via Infotrieve]

16. Maclean MR, Johnston ED, Mcculloch KM, Pooley L, Houslay MD, Sweeney G. Phosphodiesterase isoforms in the pulmonary arterial circulation of the rat: changes in pulmonary hypertension. Pharmacol Exp Ther. 1997; 283: 619–624.[Abstract/Free Full Text]

17. Burke TM, Wolin MS. Hydrogen peroxide elicits pulmonary arterial relaxation and guanylate cyclase activation. Am J Physiol. 1987; 252: H721–H732.[Medline] [Order article via Infotrieve]

18. Fujimoto S, Mori M, Tsushima H. Mechanisms underlying the hydrogen peroxide-induced, endothelium-independent relaxation of the norepinephrine- contraction in guinea-pig aorta. Eur J Pharmacol. 2003; 459: 65–73.[CrossRef][Medline] [Order article via Infotrieve]

19. Naseem KM, Mumtaz FH, Thompson CS, Sullivan ME, Khan MA, Morgan RJ, Mikhailidis DP, Bruckdorfer KR. Relaxation of rabbit lower urinary tract smooth muscle by nitric oxide and carbon monoxide: modulation by hydrogen peroxide. Eur J Pharmacol. 2000; 387: 329–335.[Medline] [Order article via Infotrieve]

20. Wu C, Kuo S, Lee F, Teng C. YC-1 potentiated the antiplatelet effect of hydrogen peroxide via sensitization of soluble guanylate cyclase. Eur J Pharmacol. 1999; 381: 185–191.[CrossRef][Medline] [Order article via Infotrieve]

21. Kahn AM, Allen JC, Seidel CL, Zhang S. Insulin inhibits migration of vascular smooth muscle cells with inducible nitric oxide synthase. Hypertension. 2000; 35: 303–306.[Abstract/Free Full Text]

22. Griending KK, Minieri CA, Ollerenshaw JD, Alexander RW. Angiotensin II stimulates NADH and NADPH oxidase activity in cultured vascular smooth muscle cells. Circ Res. 1994; 74: 1141–1148.[Abstract/Free Full Text]

23. Ohara Y, Peterson TE, Harrison DG. Hypercholesterolemia increases endothelial superoxide anion production. J Clin Invest. 1993; 91: 2546–2551.[Medline] [Order article via Infotrieve]

24. Cusson JR, Thembly J, LaRochelle P, Schifferin EL, Gutkowska J, Hamet P. Clinical relationships of cyclic GMP. In: Murad F, ed. Cyclic GMP, Synthesis, Metabolism and Function. San Diego, Calif: Academic Press; 1994: 305–319.

25. Ioannidis I, Batz M, Paul T, Korth HG, Sustmann R, DeGroot H. Enhanced release of nitric oxide causes increased cytotoxicity of S-nitroso-N-acetyl-DL- penicillamine and sodium nitroprusside under hypoxic conditions. Biochem J. 1996; 318: 789–795.[Medline] [Order article via Infotrieve]

26. Kahn AM, Seidel CL, Allen JC, O’Neil RG, Shelat H, Song T. Insulin reduces contraction and intracellular calcium concentration in vascular smooth muscle. Hypertension. 1993; 22: 735–742.[Abstract/Free Full Text]

27. Kahn AM, Husid A, Allen JC, Seidel CL, Song T. Insulin acutely inhibits cultured vascular smooth muscle cell contraction by a nitric oxide synthase-dependent pathway. Hypertension. 1997; 30: 928–933.[Abstract/Free Full Text]

28. Brands MW, Lee WF, Keen HL, Alonso-Galacia M, Zappe DH, Hall JE. Cardiac output and renal function during insulin hypertension in Sprague-Dawley rats. Am J Physiol. 1996; 271: R276–R281.[Medline] [Order article via Infotrieve]

29. Pryor WA, Squadrito GL. The chemistry of peroxynitrite: a product from the reaction of nitric oxide with superoxide. Am J Physiol. 1995; 268: L699–L722.[Medline] [Order article via Infotrieve]

30. Gutpe S, Rupawalla T, Mohazzab-H KM, Wolin MS. Regulation of NO-elicited pulmonary artery relaxation and guanylate cyclase activation by NADH oxidase and SOD. Am J Physiol. 1999; 276: H1535–H1542.[Medline] [Order article via Infotrieve]

31. Maytin M, Leoplold J, Loscalzo J. Oxidant stress in the vasculature. Curr Atheroscler Rep. 1999; 156–164.

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