This Article Abstract Full Text (PDF) Data Supplement Data Supplement All Versions of this Article: 52/3/507    most recent HYPERTENSIONAHA.108.118216v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kinoshita, H. Articles by Hatano, Y. Search for Related Content PubMed PubMed Citation Articles by Kinoshita, H. Articles by Hatano, Y. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB GLUCOSE Related Collections Cell signalling/signal transduction Ion channels/membrane transport Type 1 diabetes Type 2 diabetes Other diabetes Glucose intolerance Other Vascular biology

(Hypertension. 2008;52:507.)
© 2008 American Heart Association, Inc.

Original Articles

Roles of Phosphatidylinositol 3-Kinase-Akt and NADPH Oxidase in Adenosine 5'-Triphosphate–Sensitive K⁺ Channel Function Impaired by High Glucose in the Human Artery

Hiroyuki Kinoshita; Naoyuki Matsuda; Hikari Kaba; Noboru Hatakeyama; Toshiharu Azma; Katsutoshi Nakahata; Yasuhiro Kuroda; Kazuaki Tange; Hiroshi Iranami; Yoshio Hatano

From the Department of Anesthesiology (H. Kinoshita, K.N., K.T., Y.H.), Wakayama Medical University, Wakayama; Departments of Primary Care and Emergency Medicine (N.M.), Graduate School of Medicine, Kyoto University, Kyoto; Departments of Molecular Medical Pharmacology (H. Kaba) and Anesthesiology (N.H.), Toyama University School of Medicine, Toyama; Department of Anesthesiology (T.A.), Saitama Medical University, Moroyama; Department of Emergency Medical Center (Y.K.), Kagawa University Hospital, Miki-cho; Department of Anesthesia (H.I.), Japanese Red Cross Society, Wakayama Medical Center, Wakayama, Japan.

Correspondence to Hiroyuki Kinoshita, Department of Anesthesiology, Wakayama Medical University, 811-1 Kimiidera, Wakayama 641-0012, Japan. E-mail hkinoshi{at}pd5.so-net.ne.jp

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
The present study was designed to examine roles of the phosphatidylinositol 3-kinase-Akt pathway and reduced nicotinamide-adenine dinucleotide phosphate oxidases in the reduced ATP-sensitive K⁺ channel function via superoxide produced by high glucose in the human artery. We evaluated the activity of the phosphatidylinositol 3-kinase-Akt pathway, as well as reduced nicotinamide-adenine dinucleotide phosphate oxidases, the intracellular levels of superoxide and ATP-sensitive K⁺ channel function in the human omental artery without endothelium. Levels of the p85- subunit and reduced nicotinamide-adenine dinucleotide phosphate oxidase subunits, including p47phox, p22phox, and Rac-1, increased in the membrane fraction from arteries treated with D-glucose (20 mmol/L) accompanied by increased intracellular superoxide production. High glucose simultaneously augmented Akt phosphorylation at Ser 473, as well as Thr 308 in the human vascular smooth muscle cells. A phosphatidylinositol 3-kinase inhibitor LY294002, as well as tiron and apocynin, restored vasorelaxation and hyperpolarization in response to an ATP-sensitive K⁺ channel opener levcromakalim. Therefore, it can be concluded that the activation of the phosphatidylinositol 3-kinase-Akt pathway, in combination with the translocation of p47phox, p22phox, and Rac-1, contributes to the superoxide production induced by high glucose, resulting in the impairment of ATP-sensitive K⁺ channel function in the human visceral artery.

Key Words: ATP-sensitive K⁺ channels • human artery • hyperglycemia • NADPH oxidase • phosphatidylinositol 3-kinase

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays a key role as a vascular smooth muscle regulator in addition to its function on endothelial cells.¹ Previous studies on animals and humans demonstrated that high glucose, as well as diabetes mellitus, enhances the PI3K activity in vascular smooth muscle cells.^2–4 Vascular smooth muscle cells contain several sources of reactive oxygen species, among which the reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases are predominant. ⁵ Indeed, these enzymes mediate many pathophysiological processes in vascular smooth muscle cells, including vascular malfunction resulting from diabetes mellitus or long-term exposure toward high glucose.^5–8 However, the roles of NADPH oxidases in acute exposure, such as 60 minutes to high glucose, remain to be determined. In addition, the relationship between PI3K and NADPH oxidases in the superoxide production induced by high glucose in the human vascular smooth muscle has not been studied.

Hyperglycemia, as well as diabetes mellitus, impairs vasodilation mediated by ATP-sensitive K⁺ channels in human vascular smooth muscle cells.^9–11 In addition, these pathophysiological conditions have been shown to produce increased levels of superoxide in the vasculature.^6,10,12 However, the mechanism of impaired ATP-sensitive K⁺ channel function induced by superoxide resulting from exposure of human blood vessels to high glucose has been still unknown.

Therefore, the present study was designed to examine the role of the PI3K-Akt pathway in relation to NADPH oxidases in the reduced ATP-sensitive K⁺ channel function via superoxide produced by high glucose in the intact human artery.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
All of the experiments were performed using human omental arteries without endothelium in the presence of D-glucose (5.5 mmol/L). For details on Western immunoblotting analysis,^13–15 measurements of in situ superoxide production,^16,17 and organ chamber and electrophysiological experiments,^10,11 please see the data supplement available online at http://hyper.ahajournals.org.

Statistical Analysis
The data are expressed as means±SDs. Statistical analysis was performed using repeated-measures ANOVA, followed by the Student-Newman-Keuls test for multiple comparisons. Differences were considered to be statistically significant when the P value was <0.05.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Levels of PI3K Subtypes and Akt Phosphorylation
Levels of p85- subunit increased in the membrane fraction from arteries treated with D-glucose (20 mmol/L) for 60 minutes, whereas this enhancement was abolished by the treatment with D-glucose (20 mmol/L) in combination with a PI3K antagonist LY294002 (Figure 1). The addition of D-glucose (20 mmol/L) did not alter the levels of other subtypes, including p85-β, p110-, and p110- subunits. Expression of dually phosphorylated Akt at Ser 473 and Thr 308 was augmented by the treatment of arteries with D-glucose (20 mmol/L) for 60 minutes, whereas LY294002 completely inhibited this augmentation (Figure 2).

View larger version (24K): [in this window] [in a new window]   Figure 1. The membrane translocation of PI3K subunits, including p85-, p85-β, p110-, and p110- subunits, in the human omental artery. In the top trace of each panel, representative Western blots of p85- (A), p85-β (B), p110- (C), and p110- (D) subunits in the memebrane fraction (top) and the total fraction (bottom) after 60 minutes of incubation with control solution in combination with D-glucose (20 mmol/L) are shown. In the bar graph, the cumulative immunoblot data are shown. *P<0.05 vs control; #P<0.05 vs D-glucose.

View larger version (16K): [in this window] [in a new window]   Figure 2. Akt phosphorylation at Ser 473 and Thr 308 in the human omental artery. In the top trace of each panel, representative Western blots of the total Akt (A), the phosphorylated Akt at Ser 473 (B), and the phosphorylated Akt at Thr 308 (C) after 60 minutes of incubation with control solution in combination with D-glucose (20 mmol/L) are shown. In the bar graph, the cumulative immunoblot data are shown. *P<0.05 vs control.

Levels of NADPH Oxidase Subunits
The addition of D-glucose (20 mmol/L) did not alter the membrane levels of Nox1, Nox2, and Nox4 (Figure 3). Protein levels of p22phox, p47phox, and Rac-1 in the membrane fraction were augmented by the treatment with D-glucose (20 mmol/L) for 60 minutes, whereas the increase was inhibited by LY294002 (10^–5 mol/L).

View larger version (28K): [in this window] [in a new window]   Figure 3. Protein expressions of NADPH oxidase subunits, including Nox1 (A), Nox2 (B), Nox4 (C), p22phox (D), p47phox (E), and Rac-1 (F) in the membrane (top) and the cytosolic (bottom) fractions from human omental arteries, after 60 minutes of incubation with control solution in combination with D-glucose (20 mmol/L) are shown. In the bar graph, the cumulative immunoblot data are shown. *P<0.05 vs control.

Measurements of In Situ Superoxide Production
D-Glucose (20 mmol/L for 60 minutes) enhanced ethidium bromide fluorescence, which was reduced to the intensity seen in the artery exposed to L-glucose (20 mmol/L) by the treatment with LY294002 (10^–5 mol/L), apocynin (1 mmol/L), or tiron (10 mmol/L; Figure 4).

View larger version (20K): [in this window] [in a new window]   Figure 4. A through E, Representative images of in situ superoxide production. Gray dots indicate margins of human omental arteries without endothelium. F, Relative superoxide production in the omental arteries treated with the addition of L-glucose (20 mmol/L), D-glucose (20 mmol/L), and D-glucose (20 mmol/L) in combination with LY294002 (10–5 mol/L), apocynin (1 mmol/L), or tiron (10 mmol/L). *Difference between the arteries treated with D-glucose and the arteries treated with L-glucose and that between the arteries treated with D-glucose and the arteries treated with D-glucose in combination with LY294002, apocynin, or tiron are statistically significant (P<0.05).

Organ Chamber and Electrophysiological Experiments
A selective ATP-sensitive K⁺ channel antagonist, glibenclamide (10^–6 mol/L), abolished the vasorelaxation induced by a selective ATP-sensitive K⁺ channel opener levcromakalim during contraction to U46619, whereas it did not affect the basal tone. Incubation with D-glucose (20 mmol/L for 60 minutes) impaired levcromakalim-induced vasorelaxation (Figure 5). LY294002 (10^–5 mol/L), as well as tiron (10 mmol/L) or apocynin (1 mmol/L), restored vasorelaxation in response to levcromakalim in arteries treated with D-glucose (20 mmol/L) (Figure 5). These inhibitors did not affect the vasorelaxation produced by levcromakalim in arteries incubated with L-glucose (20 mmol/L) (Figure 5). LY294002 (10^–5 mol/L) and apocynin (1 mmol/L) did not alter relaxation induced by diltiazem, as well as basal tone in arteries treated with the high concentration of D-glucose (Figure S2).

View larger version (25K): [in this window] [in a new window]   Figure 5. A, Levcromakalim-induced vasodilation in the absence or in the presence of L-glucose, D-glucose, and/or glibenclamide. Difference between rings treated with L-glucose or D-glucose and rings treated with glibenclamide (*; P<0.05) and that between rings treated with L-glucose and rings treated with D-glucose are statistically significant (#; P<0.05). Data are expressed as percent of maximal vasorelaxation induced by papaverine (3x10–4 mol/L; 100%=1.9±0.8 g [n=6], 2.1±1.2 g [n=6] and 2.2±0.3 g [n=6] for rings treated with L-glucose, D-glucose, or L-glucose plus glibenclamide, respectively [NS]). B, Levcromakalim-induced vasodilation in the absence or in the presence of L-glucose, D-glucose, and/or LY294002 (10–5 mol/L). *Differences between rings treated with D-glucose and rings treated with L-glucose or LY294002 are statistically significant (P<0.05). Data are expressed as percent of maximal vasorelaxation induced by papaverine (3x10–4 mol/L; 100%=2.0±0.6 g [n=6], 2.3±1.2 g [n=6]), 1.9±0.9 g [n=6] and 2.2±0.9 g [n=6] for rings treated with L-glucose, D-glucose, L-glucose plus LY294002, or D-glucose plus LY294002, respectively [NS]). C, Levcromakalim-induced vasodilation in the absence or in the presence of L-glucose, D-glucose, and/or Tiron (10 mmol/L). *Differences between rings treated with D-glucose and rings treated with L-glucose or Tiron are statistically significant (P<0.05). Data are expressed as percent of maximal vasorelaxation induced by papaverine (3x10–4 mol/L; 100%=2.4±0.8 g [n=5], 2.2±1.1 g [n=5]), 2.1±0.7 g [n=5] and 2.3±0.5 g [n=5] for rings treated with L-glucose, D-glucose, L-glucose plus Tiron, or D-glucose plus Tiron, respectively [NS]). D, Levcromakalim-induced vasodilation in the absence or in the presence of L-glucose, D-glucose, and/or apocynin (1 mmol/L). *Differences between rings treated with D-glucose and rings treated with L-glucose or apocynin are statistically significant (P<0.05). Data are expressed as percent of maximal vasorelaxation induced by papaverine (3x10–4 mol/L; 100%=2.5±1.1 g [n=5], 2.6±1.1 g [n=5], 3.1±1.3 g [n=5] and 3.0±1.3 g [n=5] for rings treated with L-glucose, D-glucose, L-glucose plus apocynin, or D-glucose plus apocynin, respectively [NS]).

Levcromakalim (3x10^–6 mol/L) induced hyperpolarization in the omental artery treated with L-glucose (20 mmol/L), which was abolished by glibenclamide. D-Glucose (20 mmol/L) reduced levcromakalim-induced hyperpolarization. LY294002 and apocynin restored hyperpolarization in response to levcromakalim in arteries treated with D-glucose (20 mmol/L), whereas the addition of LY294002 to apocynin did not further augment the hyperpolarization (Figure S3).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The PI3K signaling pathway plays a key role as a vascular smooth muscle regulator.¹ In the membrane fraction from human arteries without endothelium exposed to high glucose (459 mg/dL; 60 minutes), the level of the p85- subunit, but not those of the p85-β, p110- and p110- subunits, increased, whereas this enhancement was abolished by a selective PI3K antagonist LY294002. This antagonist also inhibited increased levels of intracellular superoxide induced by high glucose. These results suggest that a p85- subunit solely contributes to the increased production of superoxide induced by acute high glucose in the human vascular smooth muscle cells. In animals, reduced expression of the p85- subunit improved insulin signaling and ameliorated type 2 diabetes, indicating that the modulation of this subunit may provide a therapeutic role in the treatment of hyperglycemic or diabetic derangements in the vasculature.¹⁸ However, our results are in contrast to a previous study whereby the incubation with glucose (25 mmol/L) for 18 hours potentiated chemotaxis in human vascular smooth muscle cells exposed to serum factors in the β-subunit–dependent fashion.³ In diabetic rat aortas, the -subunit activation by chronic exposure to high glucose has been reported.² The above conflicting results may be because of the differences in duration of incubation with glucose or in models for evaluation, although we did not observe the time course for the activation of PI3K induced by high glucose. Cumulative findings documented that Akt is located down stream of PI3K.¹⁹ We have confirmed that high glucose augments vascular Akt phosphorylation at Ser 473 and Thr 308 and that LY294002 abolished this enhancement, supporting a role of the PI3K-Akt signaling pathway in the superoxide production induced by high glucose in the human arterial smooth muscle.

We have first evaluated the intracellular translocation of NADPH oxidase subunits induced by acute high glucose in the human vasculature. Our experiments included membrane-bound subunits Nox1, Nox2, Nox4, and p22phox and cytosolic subunits p47phox and Rac-1, because the existence has been documented in the human vascular smooth muscle cells.^20–25 Western blot analysis has revealed that 60 minutes of exposure to high glucose augments membrane levels of p22phox, p47phox, and Rac-1. It is crucial to note in the vascular smooth muscle cells that the membrane translocation of Rac-1 is critical for Nox1 or Nox2 activation and that p47phox solely supports Nox2 activity.^5,26 Taken together with our results demonstrating unchanged expression of Nox1, Nox2, and Nox4 after D-glucose exposure, it is most likely that, in the human vascular smooth muscle cells, high glucose augments Nox2 function via the enhanced membrane levels of p22phox, p47phox, and Rac-1 without altering Nox2 expression. This conclusion is consistent with the following previous studies in the human vascular smooth muscle cells showing oxidative stress induced by something other than high glucose. Angiotensin II rapidly induced membrane translocation of p47phox, resulting in superoxide production in the human subcutaneous arterial smooth muscle cells.^24,25 In the more prolonged exposure 24 hours, angiotensin II or thrombin enhanced p22phox expression and p47phox translocation, respectively, in the cultured human vascular smooth muscle cells.^23,24 Membrane expression of p22phox and p47phox increased in the human coronary arteries from explanted hearts of patients with coronary artery disease.²² These results suggest important roles of the NADPH subunits mentioned earlier, which favor Nox2 function, in the increased oxidative stress induced by different stimuli in the human vascular smooth muscle cells.

It is important to note that, in the current study, LY294002 inhibited the increase in membrane levels of NADPH oxidase subunits, resulting in the reduction of superoxide production. These results strongly indicate a possible role of the PI3K-Akt pathway as an upstream signaling cascade before the activation of NADPH oxidase induced by high glucose in human blood vessels. The involvement of PI3K in the production of oxygen-derived free radicals mediated by NADPH oxidase activated by vasoactive substances has been suggested in the vascular smooth muscle cells from animals.^27,28 The interrelation between PI3K and NADPH oxidase subunits, including p47phox and Rac-1, in the production of oxygen-derived free radicals has been demonstrated in human tissues other than blood vessels, supporting the tight connection of these signaling cascades in the superoxide production in humans.^29–31

As shown in our previous studies, glibenclamide abolished vasorelaxation, as well as hyperpolarization, in response to levcromakalim in the human omental artery, and, therefore, we are capable of evaluating human vascular function mediated by ATP-sensitive K⁺ channels using this model.^10,11 In this study, we used this model to evaluate the possibility of whether the regulation of the PI3K-Akt pathway may ameliorate K⁺ channel function aggravated by superoxide produced by acute high glucose, because previous studies in humans and animals demonstrated that hyperglycemia, as well as diabetes mellitus, enhances superoxide production, resulting in the inhibition of vascular ATP-sensitive K⁺ channel activity.^{9,10,12,32,33} LY294002, similar to superoxide inhibitors tiron and apocynin, completely recovered vasorelaxation and hyperpolarization via ATP-sensitive K⁺ channels in the human artery exposed to high glucose, indicating a crucial role of PI3K activity in this K⁺ channel malfunction.^34,35 Therefore, the regulation of the PI3K-Akt pathway in the vascular smooth muscle cells may contribute as a therapeutic intervention to restore ATP-sensitive K⁺ channel function impaired by oxidative stress produced by hyperglycemia. However, it is currently unknown how superoxide produced by high glucose inactivates ATP-sensitive K⁺ channels, although previous studies using endothelium-intact arterioles exposed to high glucose indicate that the channel protein nitration induced by peroxynitrite is a plausible candidate to the inhibition of voltage-gated K⁺ channels produced by high glucose.³⁶

It has been known that high glucose stimulates protein kinase C in the vascular smooth muscle cells.^6,10 Previous studies demonstrated that blockade of protein kinase C reduces phosphorylation of c-src, resulting in the inhibition of this kinase.³⁷ C-src has been shown to activate NADPH oxidase, whereas the inhibition of this cascade reduces the intracellular superoxide production in the vascular smooth muscle cells.^24,38 Importantly, previous studies also suggest that PI3K activation lies downstream of c-src.^24,37,38 Taken together with these findings and ours, it is most likely that high glucose is capable of producing superoxide by NADPH oxidase via PI3K activation, resulting from activation of c-src induced by protein kinase C.

Perspectives
This is the first study examining the relationship between PI3K and NADPH oxidases in the superoxide production induced by high glucose in the human vascular smooth muscle cells. Considering the involvement of NADPH oxidase in the increased oxidative stress by high glucose, this enzyme should be a target for intervention strategies based on reversing vascular malfunction in hyperglycemia, as well as diabetes mellitus. More importantly, our results with a PI3K antagonist demonstrated the possibility that inhibitors limited to PI3K in the vascular smooth muscle cells may play a role as an antioxidant by the inhibition of NADPH oxidase in variable diseased states, including insulin tolerance, although the endothelial PI3K-Akt pathway contributes to beneficial vascular functions, including the production of endothelial NO.¹³ In the current study, the impaired activity of ATP-sensitive K⁺ channels in the human omental artery is accompanied by the activation of both the PI3K-Akt pathway and NADPH oxidase subunits. Acidosis corresponding with ischemia causes visceral vasodilation via activation of ATP-sensitive K⁺ channels, indicating a crucial role of these channels as a regulator in visceral circulation.³⁹ Also, it is possible to administer ATP-sensitive K⁺ channel openers, such as nicorandil, to patients with glucose intolerance.⁴⁰ Therefore, it can be concluded that PI3K, as well as NADPH oxidase antagonism in the vascular smooth muscle cells, may ameliorate the malfunction of ATP-sensitive K⁺ channels induced by the conditions with acute glucose intolerance.

	Acknowledgments

 
Sources of Funding

This work was supported in part by Grant-in-Aid 19390409 (H. Kinoshita), 18659462 (H. Kinoshita), 18689038 (K.N.), and 17390432 (Y.H.) for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Tokyo, Japan).

Disclosures

None.

	Footnotes

 
This work was presented in part at the annual meeting of the American Society of Anesthesiologists, San Francisco, Calif, October 13–17, 2007.

Received June 15, 2008; first decision July 1, 2008; accepted July 8, 2008.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Sata M, Nagai R. Phosphatidylinositol 3-kinase: a key regulator of vascular tone? Circ Res. 2002; 91: 273–275.[Free Full Text]

2. Kobayashi T, Hayashi Y, Taguchi K, Matsumoto T, Kamata K. ANG II enhances contractile responses via PI3-kinase p110 pathway in aortas from diabetic rats with systemic hyperinsulinemia. Am J Physiol. 2006; 291: H846–H853.

3. Campbell M, Allen WE, Sawyer C, Vanhaesebroeck B, Trimble ER. Glucose-potentiated chemotaxis in human vascular smooth muscle is dependent on cross-talk between the PI3K and MAPK signaling pathways. Circ Res. 2004; 95: 380–388.[Abstract/Free Full Text]

4. Campbell M, Allen WE, Silversides JA, Trimble ER. Glucose-induced phosphatidylinositol 3-kinase and mitogen-activated protein kinase-dependent upregulation of the platelet-derived growth factor-β receptor potentiates vascular smooth muscle cell chemotaxis. Diabetes. 2003; 52: 519–526.[Abstract/Free Full Text]

5. Clempus RE, Griendling KK. Reactive oxygen species signaling in vascular smooth muscle cells. Cardiovasc Res. 2006; 71: 216–225.[Abstract/Free Full Text]

6. Inoguchi T, Li P, Umeda F, Yu HY, Kakimoto M, Imamura M, Aoki T, Etoh T, Hashimoto T, Naruse M, Sano H, Utsumi H, Nawata H. High glucose level and free fatty acid stimulate reactive oxygen species production through protein kinase C-dependent activation of NAD(P)H oxidase in cultured vascular cells. Diabetes. 2000; 49: 1939–1945.[Abstract]

7. Liu S, Ma X, Gong M, Shi L, Lincoln T, Wang S. Glucose down-regulation of cGMP-dependent protein kinase I expression in vascular smooth muscle cells involves NAD(P)H oxidase-derived reactive oxygen species. Free Radical Biol Med. 2007; 42: 852–863.[CrossRef][Medline] [Order article via Infotrieve]

8. Newsholme P, Haber EP, Hirabara SM, Rebelato ELO, Procopio J, Morgan D, Oliveira-Emilio HC, Carpinelli AR, Curi R. Diabetes associated cell stress and dysfunction: role of mitochondrial and non-mitochondrial ROS production and activity. J Physiol. 2007; 583: 9–24.[Abstract/Free Full Text]

9. Miura H, Wachtel RE, Loberiza FR Jr, Saito T, Miura M, Nicolosi AC, Gutterman DD. Diabetes mellitus impairs vasodilation to hypoxia in human coronary arterioles: reduced activity of ATP-sensitive potassium channels. Circ Res. 2003; 92: 151–158.[Abstract/Free Full Text]

10. Kinoshita H, Azma T, Nakahata K, Iranami H, Kimoto Y, Dojo M, Yuge O, Hatano Y. Inhibitory effect of high concentration of glucose on relaxations to activation of ATP-sensitive K⁺ channels in human omental artery. Arterioscler Thromb Vasc Biol. 2004; 24: 1–6.[Free Full Text]

11. Kinoshita H, Azma T, Iranami H, Nakahata K, Kimoto Y, Dojo M, Yuge O, Hatano Y. Synthetic peroxisome proliferator-activated receptor- agonists restore impaired vasorelaxation via ATP-sensitive K⁺ channels by high glucose. J Pharmacol Exp Ther. 2006; 318: 1–7.[Abstract/Free Full Text]

12. Liu Y, Gutterman DD. The coronary circulation in diabetes: influence of reactive oxygen species on K⁺ channel-mediated vasodilation. Vascular Pharmacol. 2002; 38: 43–49.[CrossRef]

13. Matsuda N, Hayashi Y, Takahashi Y, Hattori Y. Phosphorylation of endothelial nitric-oxide synthase is diminished in mesenteric arteries from septic rabbits depending on the altered phosphatidylinositol 3-kinase/Akt pathway: reversal effect of fluvastatin therapy. J Pharmacol Exp Ther. 2006; 319: 1348–1354.[Abstract/Free Full Text]

14. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951; 193: 265–275.[Free Full Text]

15. Carpenter CL, Duckworth BC, Auger KR, Cohen B, Schaffhausen BS, Cantleye LC. Purification and characterization of phosphoinositide 3-kinase from rat liver. J Biol Chem. 1990; 32: 19704–19711.

16. Miller FJ, Gutterman DD, Rios CD, Heistad DD, Davidson BL. Superoxide production in vascular smooth muscle contributes to oxidative stress and impaired relaxation in atherosclerosis. Circ Res. 1998; 82: 1298–1305.[Abstract/Free Full Text]

17. Nakahata K, Kinoshita H, Azma T, Matsuda N, Hama-Tomioka K, Haba M, Hatano Y. Propofol restores brain microvascular function impaired by high glucose via the decrease in oxidative stress. Anesthesiology. 2008; 108: 269–275.[Medline] [Order article via Infotrieve]

18. Mauvais-Jarvis F, Ueki K, Fruman DA, Hirshman MF, Sakamoto K, Goodyear LJ, Iannacone M, Accili D, Cantley LC, Kahn CR. Reduced expression of the murine p85 subunit of phosphoinositide 3-kinase improves insulin signaling and ameliorates diabetes. J Clin Invest. 2002; 109: 141–149.[CrossRef][Medline] [Order article via Infotrieve]

19. Datta K, Bellacosa A, Chan TO, Tsichlis PN. Akt is a direct target of the phosphatidylinositol 3-kinase: activation by growth factors, v-src and v-Ha-ras, in Sf9 and mammalian cells. J Biol Chem. 1996; 271: 30835–30839.[Abstract/Free Full Text]

20. Hilenski LL, Clempus RE, Quinn MT, Lambeth JD, Griendling KK. Distinct subcellular localizations of Nox1 and Nox4 in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 2004; 24: 677–683.[Abstract/Free Full Text]

21. Guzik TJ, Sadowski J, Kapelak B, Jopek A, Rudzinski P, Pillai R, Korbut R, Channon KM. Systemic regulation of vascular NAD(P)H oxidase activity and Nox isoform expression in human arteries and veins. Arterioscler Thromb Vasc Biol. 2004; 24: 1614–1620.[Abstract/Free Full Text]

22. Guzik TJ, Sadowski J, Guzik B, Jopek A, Kapelak B, Przybylowski P, Wierzbicki K, Korbut R, Harrison DG, Channon KM. Coronary artery superoxide production and Nox isoform expression in human coronary artery disease. Arterioscler Thromb Vasc Biol. 2006; 26: 333–339.[Abstract/Free Full Text]

23. Patterson C, Ruef J, Madamanchi NR, Barry-Lane P, Hu Z, Horaist C, Ballinger CA, Brasier AR, Bode C, Runge MS. Stimulation of a vascular smooth muscle cell NAD(P)H oxidase by thrombin: evidence that p47phox may participate in forming this oxidase in vitro and in vivo. J Biol Chem. 1999; 274: 19814–19822.[Abstract/Free Full Text]

24. Touyz RM, Yao G, Schiffrin EL. c-Src induces phosphorylation and translocation of p47phox; role in superoxide generation by angiotensin II in human vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 2003; 23: 981–987.[Abstract/Free Full Text]

25. Touyz RM, Yao G, Quinn MT, Pagano PJ, Schiffrin EL. p47phox associates with the cytoskeleton through cortactin in human vascular smooth muscle cells: Role in NAD(P)H oxidase regulation by angiotensin II. Arterioscler Thromb Vasc Biol. 2005; 25: 512–518.[Abstract/Free Full Text]

26. Bedard K, Krause K-H. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol Rev. 2007; 87: 245–313.[Abstract/Free Full Text]

27. Krieg T, Landsberger M, Alexeyev MF, Felix SB, Cohen MV, Downey JM. Activation of Akt is essential for acetylcholine to trigger generation of oxygen free radicals. Cardiovasc Res. 2003; 58: 196–202.[Abstract/Free Full Text]

28. Seshiah PN, Weber DS, Rocic P, Valppu L, Taniyama Y, Griendling KK. Angiotensin II stimulation of NAD(P)H oxidase activity: Upstream mediators. Circ Res. 2002; 91: 406–413.[Abstract/Free Full Text]

29. Ceolotto G, Bevilacqua M, Papparella I, Baritono E, Franco L, Corvaja C, Mazzoni M, Semplicini A, Avogaro A. Insulin generates free radicals by an NAD(P)H, phosphatidylinositol 3'-kinase-dependent mechanism in human skin fibroblasts ex vivo. Diabetes. 2004; 53: 1344–1351.[Abstract/Free Full Text]

30. Park HS, Lee SH, Park D, Lee JS, Ryu SH, Lee WJ, Rhee SG, Bae YS. Sequential activation of phosphatidylinositol 3-kinase, βPix, Rac-1, and Nox1 in growth factor-induced production of H₂O₂. Mol Cell Biol. 2004; 24: 4384–4394.[Abstract/Free Full Text]

31. Lee SB, Bae IH, Bae YS, Um H-D. Link between mitochondria and NADPH oxidase 1 isozyme for the sustained production of reactive oxygen species and cell death. J Biol Chem. 281; 47: 36228–36235.

32. Gutterman DD, Miura H, Liu Y. Redox modulation of vascular tone: Focus of potassium channel mechanisms of dilation. Arterioscler Thromb Vasc Biol. 2005; 25: 671–678.[Abstract/Free Full Text]

33. Erdös B, Simandle SA, Snipes JA, Miller AW, Busija DW. Potassium channel dysfunction in cerebral arteries of insulin-resistant rats is mediated by reactive oxygen species. Stroke. 2004; 35: 964–969.[Abstract/Free Full Text]

34. Heumüller S, Wind S, Barbosa-Sicard E, Schmidt HHHW, Busse R, Schröder K, Brandes RP. Apocynin is not an inhibitor of vascular NADPH oxidases but an antioxidant. Hypertension. 2008; 51: 211–217.[Abstract/Free Full Text]

35. Motley ED, Eguchi K, Patterson MM, Palmer PD, Suzuki H, Eguchi S. Mechanism of endothelial nitric oxide synthase phosphorylation and activation by thrombin. Hypertension. 2007; 49: 577–583.[Abstract/Free Full Text]

36. Li H, Gutterman DD, Rusch NJ, Bubolz A, Liu Y. Nitration and function loss of voltage-gated K⁺ channels in rat coronary microvessels exposed to high glucose. Diabetes. 2004; 53: 2436–2442.[Abstract/Free Full Text]

37. Jackson EK, Gao L, Zhu C. Mechanism of the vascular angiotensin II/₂-adrenoceptor interaction. J Pharmacol Exp Ther. 2005; 314: 1109–1116.[Abstract/Free Full Text]

38. Jackson EK, Gillespie DG, Zhu C, Ren J, Zacharia LC, Mi Z. ₂-Adrenoceptors enhance angiotensin II-induced renal vasoconstriction: role for NADPH oxidase and Rho A. Hypertension. 2008; 51: 719–726.[Abstract/Free Full Text]

39. Wang X, Wu J, Li L, Chen F, Wang R, Jiang C. Hypercapnic acidosis activates K_ATP channels in vascular smooth muscle. Circ Res. 2003; 92: 1225–1232.[Abstract/Free Full Text]

40. Mannhold R. K_ATP channel openers: structure-activity relationships and therapeutic potential. Med Res Rev. 2004; 24: 213–266.[CrossRef][Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				K. Hama-Tomioka, H. Kinoshita, T. Azma, K. Nakahata, N. Matsuda, N. Hatakeyama, H. Kikuchi, and Y. Hatano The Role of 20-Hydroxyeicosatetraenoic Acid in Cerebral Arteriolar Constriction and the Inhibitory Effect of Propofol Anesth. Analg., December 1, 2009; 109(6): 1935 - 1942. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Data Supplement Data Supplement All Versions of this Article: 52/3/507    most recent HYPERTENSIONAHA.108.118216v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kinoshita, H. Articles by Hatano, Y. Search for Related Content PubMed PubMed Citation Articles by Kinoshita, H. Articles by Hatano, Y. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB GLUCOSE Related Collections Cell signalling/signal transduction Ion channels/membrane transport Type 1 diabetes Type 2 diabetes Other diabetes Glucose intolerance Other Vascular biology