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Am J Physiol Heart Circ Physiol 294: H2845-H2854, 2008. First published April 25, 2008; doi:10.1152/ajpheart.91422.2007
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Role of oxidative stress in high glucose-induced decreased expression of Gi{alpha} proteins and adenylyl cyclase signaling in vascular smooth muscle cells

Yuan Li, Magda Descorbeth, and Madhu B. Anand-Srivastava

Department of Physiology, Faculty of Medicine, University of Montreal, Montreal, Quebec, Canada

Submitted 7 December 2007 ; accepted in final form 23 April 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We have recently shown that aorta from streptozotocin (STZ)-induced diabetic rats and A10 vascular smooth muscle cells (VSMCs) exposed to high glucose exhibited decreased levels of inhibitory guanine nucleotide regulatory protein (Gi){alpha} proteins. In the present studies, we investigated the implication of oxidative stress in the hyperglycemia/diabetes-induced decreased expression of the Gi{alpha} protein and adenylyl cyclase signaling in VSMCs by using antioxidants. The levels of Gi{alpha} proteins were significantly decreased in A10 VSMCs exposed to high glucose and in aortic VSMCs from STZ-diabetic rats compared with control cells and were restored to control levels by antioxidants. In addition, 111Mn-tetralis(benzoic acid porphyrin) and uric acid, scavengers of peroxynitrite, and NG-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthase but not catalase, also restored the high glucose-induced decreased expression of Gi{alpha} proteins to the control levels in A10 VSMCs. Furthermore, the enhanced production of superoxide anion (O2–) and increased activity of NADPH oxidase in these cells were also restored to control levels by diphenyleneiodonium, an inhibitor of NADPH oxidase. In addition, the diminished inhibition of adenylyl cyclase activity by inhibitory hormones and forskolin-stimulated adenylyl cyclase activity by low concentrations of GTP{gamma}S as well as the enhanced stimulation of adenylyl cyclase by stimulatory agonists in hyperglycemic cells were restored to control levels by antioxidant treatments. These results suggest that high glucose-induced decreased levels of Gi{alpha} proteins and associated signaling in A10 VSMCs may be attributed to the enhanced oxidative stress due to augmented levels of peroxynitrite and not to H2O2.

G proteins; antioxidants; inhibitory guanine nucleotide regulatory protein

VARIOUS complications, including contractility and increased cell proliferation, are the most common complications with diabetes, and chronic hyperglycemia seems to be an important contributing factor in this process (16, 23, 33). Various signaling mechanisms such as adenylyl cyclase/cyclic (c)AMP, phosphatidyl inositol turnover, and mitogen-activated protein kinase have been shown to be implicated in the regulation of vascular tone and cell proliferation, and an aberration of these mechanisms may contribute to vascular complications observed in diabetes/hyperglycemia.

The adenylyl cyclase/cAMP is one of the signal transduction systems implicated in the regulation of cardiovascular functions, including arterial tone, reactivity, and cell proliferation. The hormone-sensitive adenylyl cyclase system is composed of three components, receptor, catalytic subunit, and G proteins, grouped as stimulatory guanine nucleotide regulatory protein (Gs) and inhibitory guanine nucleotide regulatory protein (Gi), which mediate the stimulatory and inhibitory responses of hormones on adenylyl cyclase, respectively (20, 40). G proteins are heterotrimeric proteins composed of {alpha}-, β-, and {gamma}-subunits. Molecular cloning has revealed four different forms of Gs{alpha} resulting from the differential splicing of one gene (12) and three distinct forms of Gi{alpha} (Gi{alpha}-1, Gi{alpha}-2, and Gi{alpha}-3) encoded by three distinct genes (30). All three forms of Gi{alpha} have been shown to be implicated in adenylyl cyclase inhibition (54) and the activation of atrial acetylcholine-K+ channels (55).

Several abnormalities in the expression of G proteins and adenylyl cyclase regulation have been demonstrated in various pathophysiological conditions, such as heart failure and hypertension (1, 3, 19). Mice deficient in Gi{alpha}-2 have been shown to exhibit a phenotype of insulin resistance (39). In addition, recent studies showing that the overexpression of Gi{alpha}-2 ameliorates streptozotocin (STZ)-diabetes further suggest the involvement of the Gi{alpha}-2 protein in the pathogenesis of diabetes (56). Diabetes-induced alterations in G protein and adenylyl cyclase activity and its responsiveness to various hormones have been demonstrated in several tissues (21, 45, 51). We have recently shown that aorta from an STZ-induced diabetic rat exhibited a decreased expression of Gi{alpha} proteins and associated functions (26). The decrease in the expression of the Gi{alpha} protein was dependent on the severity of diabetes. We have further shown that aorta or vascular smooth muscle cells (VSMCs) exposed to high glucose (hyperglycemic conditions) exhibited a decreased expression of Gi{alpha} proteins and associated adenylyl cyclase signaling, whereas the levels of Gs{alpha} were not affected (25).

The increased oxidative stress has been reported in hypertension and other cardiovascular diseases including diabetes (10, 14). The enhanced activity/levels of protein kinase C (PKC) and diacylglycerol (DAG) induced by hyperglycemia in VSMCs has been shown to be mediated by increased oxidative stress (34) because an intraperitoneal injection of antioxidant {alpha}-tocopherol to diabetic animals or the incubation of VSMCs with {alpha}-tocopherol prevented the increase in the levels of DAG and PKC due to diabetes and hyperglycemia, respectively (34). Taken together, it may be possible that hyperglycemia-induced increased oxidative stress may also be a contributing factor in decreasing the expression of Gi{alpha} proteins in VSMCs. To examine this possibility, the present studies were undertaken to determine the effect of antioxidant treatment on the hyperglycemia-induced decreased expression of Gi{alpha} proteins and associated adenylyl cyclase signaling in A10 VSMCs. This rat embryonal thoracic aorta cell line has been shown to demonstrate characteristics similar to those of VSMCs (29) and has been a useful model to study vascular cellular processes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Adenosine 5'-triphosphate (ATP), cAMP, isoproterenol, forskolin (FSK), glucagon, oxotremorine, and diphenyleneiodonium (DPI) were purchased from Sigma (St. Louis, MO). Creatine kinase, myokinase, and guanosine 5'-O-(3 thiotriphosphate) (GTP{gamma}S) were purchased from Boehringer Mannheim (Montreal, Quebec, Canada). [{alpha}-32P]ATP was from Amersham (Ontario, Canada). [Des(Glu18,Ser19,Glu20,Leu21,Cly22)atrial natriuretic peptide4–23-NH2] (C-ANP4–23) was purchased from Peninsula (Belmont, CA). AS/7 and EC/2 antibodies were from Dupont (Mississauga, Ontario, Canada), whereas RM/1 antibodies were purchased from Dupont (Mississauga, Ontario, Canada) and Santa Cruz.

Animal preparation. Male Sprague-Dawley rats (200 g and 6–8-wk-old) were maintained on standard rat chow and tap water ad libitum with 12-h:12-h light-dark cycles in a quiet environment. Diabetes was induced by an intraperitoneal injection of STZ (60 mg/kg body wt) dissolved in sodium citrate buffer (pH 4.5) as described previously (26). Age-matched control rats were injected with an equal volume of buffer solution. Blood glucose levels were monitored from day 1 to 5 after the injection using a dextrometer (Ames). STZ-injected rats with blood glucose levels in excess of 26 mM were considered to be diabetic rats (STZ) and used in the study. The blood glucose level of control rats was 5.5 mM. The rats, after 5 days of treatment, were euthanized, and the aorta were dissected out and used for cell culture. All of the protocols used in the present study were approved by the Comité de Déontologie de L'expérimenttation Sur les Animaux (Canada).

Cell culture and incubation. The A10 cells line from the embryonic thoracic aorta of rats was obtained from American Type Culture Collection. VSMCs from control and diabetic aorta were cultured as described previously (6). The cells were cultured in Dulbecco's modified Eagle's medium containing normal glucose (5.5 mM), 10% FBS, and 1% antibiotic-antimycotic (containing penicillin, streptomycin, and amphotericin B) at 37°C in 95% room air-5% CO2 as described previously (25). The cells were passaged upon reaching confluence with 0.5% trypsin containing 0.2% EDTA and utilized between passages 5 and 15. The confluent cells were incubated in a medium containing 2% FBS for 24 h for growth arrest. After 24 h, the cells were exposed to high glucose (26 mM or as otherwise indicated) for 72 h (or as otherwise indicated) in the presence of 2% FBS. This treatment maintains the cells in a quiescent state without cell death as determined by the trypan blue exclusion technique (about 90–95% cells were viable). The antioxidants N-acetyl-L-cysteine (NAC; 20 mM), DPI (10 µM), 111Mn-tetralis(benzoic acid porphyrin) (MnTBAP; 20 µM), uric acid (100 µM), and NG-nitro-L-arginine methyl ester (L-NAME; 10 µM) were added after 48 h of glucose treatment. Cells growing in normal glucose were used as control. Mannitol (20.5 mM) was used as a control for osmolarity. The cells were harvested using a rubber cell scraper and were homogenized in a glass/Teflon homogenizer containing 10 mM Tris·HCl buffer containing 1 mM EDTA (pH 7.5). The homogenate was centrifuged at 1,000 g for 10 min. The supernatant was discarded, and the pellet was resuspended in the 10 mM Tris·HCl buffer containing 1 mM EDTA and used for immunoblotting and adenylyl cyclase assay.

Adenylyl cyclase activity determination. Adenylyl cyclase activity was determined by measuring [{alpha}-32P]cAMP formation from [{alpha}-32P] as described previously (25). The assay medium contained 50 mM glycylglycine (pH 7.5), 0.5 mM MgATP, [{alpha}-32P]ATP [(1–1.5) x 106 counts per minute], 5 mM MgCl2 (in excess of the ATP concentration), 100 mM NaCl, 0.5 mM cAMP, 1 mM 3-isobutyl-methylxanthine, 0.1 µM EGTA, 10 µM GTP{gamma}S (or as otherwise indicated), and an ATP regenerating system consisting of 2 mM phosphocreatine, 0.1 mg of creatine kinase/ml, and 0.1 mg of myokinase/ml in a final volume of 200 µl. Incubation was initiated by the addition of the membrane preparation (30–70 µg) to the reaction mixture, which had been thermally equilibrated for 2 min at 37°C. The reactions, conducted in triplicate for 10 min at 37°C, were terminated by the addition of 0.6 ml of 120 mM zinc acetate. cAMP was purified by the coprecipitation of other nucleotides with ZnCO3, by the addition of 0.5 ml of 144 mM Na2CO3, and subsequent chromatography by the double-column system, as described previously (25).

Immunoblotting. Immunoblotting of the G protein was performed as described earlier (25). After SDS-PAGE, the separated proteins were electrophoretically transferred to nitrocellulose paper (Schleicher and Schuell) with a mini transfer apparatus (Bio-Rad) at 100 V for 1 h or a semidry transblot apparatus (Bio-Rad) at 15 V for 45 min. After transfer, the membranes were washed twice in phosphate-buffered saline (PBS) and were incubated in PBS containing 3% BSA at room temperature for 2 h. The blots were then incubated with antisera against G proteins in PBS containing 1% BSA and 0.1% Tween 20 at room temperature for 2 h. The antigen-antibody complexes were detected by incubating the blots with goat anti-rabbit IgG (Bio-Rad) conjugated with horseradish peroxidase for 2 h at room temperature. The blots were washed three times with PBS before the reaction with enhanced chemiluminescence (ECL). Western blot analysis detection reagents were from Amersham. A quantitative analysis of the G proteins was performed by densitometric scanning of the autoradiographs employing the enhanced laser densitometer (LKB Ultrascan XL) and quantified using the gel Scan XL evaluation software (version 2.1) from Pharmacia (Quebec, Canada).

Superoxide anion measurements. Basal superoxide anion production in VSMCs was measured using the lucigenin-ECL method with a low concentration (5 µmol/l) of lucigenin as described previously (35). The cells, after treatment with DPI (10 µM), were washed in oxygenated Krebs-HEPES buffer, scraped, and placed in scintillation vials containing lucigenin solution, and the emitted luminescence was measured with a liquid scintillation counter (Wallac 1409; Perkin Elmer Life Science, St. Laurent, Quebec, Canada) for 5 min. The average luminescence value was estimated as the background value subtracted, and the result was divided by the total weight of proteins in each sample.

Statistical analysis. Results are expressed as means ± SE. Comparisons between groups were made with ANOVA in conjunction with the Newman-Keuls test. Results were considered significant at a value of P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Superoxide anion production in VSMCs. The results shown in Fig. 1 demonstrate that O2– production and NADPH oxidase activity were significantly augmented in hyperglycemic A10 VSMCs compared with the control cells. DPI, an antioxidant that inhibits NADPH oxidase activity, significantly restored the enhanced levels of O2– and NADPH oxidase activity toward control levels in hyperglycemic A10 VSMCs. These results suggest that hyperglycemia increases the production of O2–, which may be attributed to increased levels of NADPH oxidase.


Figure 1
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  		Fig. 1. Effect of diphenyleneiodonium (DPI) on high glucose-induced enhanced superoxide anion (O2–) production and NADPH oxidase activity in A10 vascular smooth muscle cells (VSMCs; A10 cells). A10 cells were incubated in the presence of 5.5 mM [control (CTL)] or 26 mM glucose (hyperglycemia) for 72 h. DPI (10 µM) was added after 48 h of glucose treatment, and O2– production and NADPH oxidase activity were determined as described in MATERIALS AND METHODS. The results are expressed as percentages of CTL, taken as 100%. Values are means ± SE of 3 separate experiments. **P < 0.01 vs. CTL (5.5 mM); ##P < 0.01; ###P < 0.001. cpm, Counts per minute.

 
Effect of antioxidants on Gi protein levels. We have recently demonstrated that A10 VSMCs exposed to high glucose for 72 h decreased the levels of Gi{alpha}-2 and Gi{alpha}-3 proteins, whereas the levels of Gs{alpha} proteins were not altered. Since high glucose and aortic VSMCs from STZ-diabetic rats exhibit enhanced production of the O2– anion, it was of interest to examine whether hyperglycemia and diabetes-induced decreased expression of Gi{alpha} proteins are attributed to the enhanced oxidative stress. To test this, we studied the effect of antioxidants on the levels of Gi{alpha} proteins, which were determined by immunoblotting techniques using specific antibodies against Gi{alpha} proteins. As shown in Fig. 2, the levels of immunodetectable Gi{alpha}-2 (Fig. 2, left) and Gi{alpha}-3 (Fig. 2, right) as detected by antibody AS/7 and antibody EC/2, respectively, were significantly decreased by about 30–50% in hyperglycemic cells compared with control cells as determined by densitometric scanning (Fig. 2, bottom), whereas the treatment of VSMCs with mannitol did not alter the levels of Gi{alpha} proteins (data not shown). The prior treatment of the cells with antioxidants tocopherol (Fig. 2A), NAC (Fig. 2B), and DPI (Fig. 2C) restored the decreased expression of the Gi{alpha}-2 and Gi{alpha}-3 proteins to control levels. In addition, VSMCs from STZ-diabetic rats also exhibited decreased expression of Gi{alpha}-2 (Fig. 3A) and Gi{alpha}-3 proteins (Fig. 3B) compared with that of the control rats, and this decreased expression of Gi{alpha} proteins was reversed to control levels by DPI and NAC.


Figure 2
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  		Fig. 2. Effect of antioxidants on the expression of inhibitory guanine nucleotide regulatory (Gi) proteins in A10 VSMCs (A10 cells). A10 cells were incubated in the presence of 5.5 mM (CTL) or 26 mM glucose (hyperglycemia) for 72 h. {alpha}-Tocophorol (10 µM; A), 10 mM N-acetyl-L-cysteine (NAC; B), and 10 µM DPI (C) were added after 48 h of glucose treatment. The cell lysates were prepared and subjected to Western blot analysis using specific antibodies for Gi{alpha}-2 (left, top) and Gi{alpha}-3 (right, top) as described previously (Ref. 25). The proteins were quantified by densitometric scanning (bottom). The results are expressed as percentages of CTL, taken as 100%. Values are means ± SE of 3 separate experiments. *P < 0.05; **P < 0.01.

 

Figure 3
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  		Fig. 3. Effect of antioxidants on the expression of Gi proteins in VSMCs from CTL and streptozotocin (STZ)-diabetic rats. VMSCs from CTL and STZ-diabetic rats were incubated in the absence or presence of 10 mM NAC or 10 µM DPI for 24 h. The cell lysates were prepared and subjected to Western blot analysis using specific antibodies for Gi{alpha}-2 (A, top) and Gi{alpha}-3 (B, top) as described previously (Ref. 25). The proteins were quantified by densitometric scanning (bottom). The results are expressed as percentages of VSMCs from CTL, taken as 100%. Values are means ± SE of 3 separate experiments. *P < 0.05; **P < 0.01.

 
Implication of peroxynitrate in hyperglycemic-induced decreased expression of Gi{alpha} protein. O2– formed by NADPH oxidase is converted to H2O2 by superoxide dismutase (SOD), which by the action of catalase is converted to H2O. On the other hand, O2– can also interact with nitric oxide (NO) and form peroxynitrite (ONOO–). The availability of NO has been reported to be decreased in diabetes and under hyperglycemic conditions (11, 21, 36), which may be due to the formation of ONOO–. We have recently shown that ONOO– decreases the expression of Gi{alpha} proteins in VSMCs (9). Taken together, it may be possible that the high glucose-induced decreased expression of Gi{alpha} proteins may be attributed to the increased levels of ONOO–. To investigate this possibility, the effect of MnTBAP and uric acid, the scavengers of ONOO–, on the high glucose-induced decreased expression of Gi{alpha} was examined, and the results are shown in Fig. 4. Treatment of A10 VSMCs with high glucose decreased the expression of Gi{alpha}-2 and Gi{alpha}-3 proteins by about 40% and 30%, respectively, which was restored to control levels by MnTBAP (Fig. 4, A and B) and uric acid (Fig. 4, C and D), whereas catalase treatment was without effect in restoring hyperglycemia-induced decreased levels of Gi{alpha} proteins (data not shown). In addition, the treatment of hyperglycemic cells with L-NAME, which decreases the levels of NO and thereby inhibits the formation of ONOO–, also restored hyperglycemia-induced decreased levels of Gi{alpha} proteins to control levels (Fig. 4, E and F).


Figure 4
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  		Fig. 4. Effect of uric acid, 111Mn-tetralis(benzoic acid porphyrin) (MnTBAP), and NG-nitro-L-arginine methyl ester (L-NAME) on the expression of Gi proteins in A10 VSMCs (A10 cells). A10 cells were incubated in the presence of 5.5 mM (CTL) or 26 mM glucose (hyperglycemia) for 72 h. Uric acid (100 µM; A and B), 20 µM MnTBAP (C and D), or 10 µM L-NAME (E and F) were added after 48 h of glucose treatment as described in MATERIALS AND METHODS. The cell lysates were prepared and subjected to Western blot analysis using specific antibodies for Gi{alpha}-2 (A, C, and E) and Gi{alpha}-3 (B, D, and F) as described previously (Ref. 25). The proteins were quantified by densitometric scanning. The results are expressed as percentages of control, taken as 100%. Values are means ± SE of 3 separate experiments. *P < 0.05; **P < 0.01.

 
Effect of antioxidants on receptor-independent function of Gi{alpha}. Since antioxidants restored the high glucose-induced decreased levels of Gi{alpha}-2 and Gi{alpha}-3 proteins toward control levels, it was of interest to examine whether the restoration of the decreased levels of Gi{alpha} proteins by antioxidants is also reflected in the restoration of decreased Gi{alpha} functions. To investigate this, the effect of DPI on receptor-independent and receptor-dependent functions was examined in hyperglycemic A10 cells. For the receptor-independent functions of Gi, the effect of DPI was investigated on the inhibitory effect of GTP{gamma}S (10–12 to 10–8M) on FSK-stimulated adenylyl cyclase activity in control and hyperglycemic A10 cells. The results shown in Fig. 5 indicate that the attenuated inhibition of FSK-stimulated adenylyl cyclase activity by different concentrations of GTP{gamma}S in hyperglycemic A10 cells that was attributed to the decreased levels of Gi{alpha} proteins compared with those of untreated control cells was reversed toward control levels by DPI. However, DPI did not have any effect on the GTP{gamma}S-mediated inhibition of FSK-stimulated adenylyl cyclase activity in control cells (data not shown).


Figure 5
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  		Fig. 5. Effect of DPI on GTP{gamma}S-mediated inhibition of forskolin (FSK)-stimulated adenylyl cyclase activity in A10 VSMCs (A10 cells) exposed to high glucose. A10 cells were incubated in the presence of 5.5 mM glucose (CTL) or 26 mM glucose (hyperglycemia) for 72 h. DPI (10 µM) was added after 48 h of glucose treatment. Membranes were prepared as described previously (Ref. 25). Adenylyl cyclase activity was determined in these membranes in the presence of 100 µM FSK alone, taken as 100%, and in the presence of various concentrations of GTP{gamma}S (10–12 to 10–8 M) as described previously (Ref. 25). Values are means ± SE of 3 separate experiments. *P < 0.05; **P < 0.01 vs. CTL (5.5 mM glucose).

 
Effect of antioxidants on receptor-dependent functions. To investigate the effect of the antioxidant on the receptor-dependent functions of Gi{alpha} proteins, the effect of DPI, {alpha}-tocopherol, and NAC on ANG II, C-ANP4–23, and oxotremorine, which inhibit adenylyl cyclase through Gi{alpha} proteins (2, 4, 5, 32), was examined on adenylyl cyclase activity in control and hyperglycemic A10 cells. Figure 6 shows that ANG II-, C-ANP4–23-, and oxotremorine-mediated inhibitions of adenylyl cyclase that were significantly attenuated in hyperglycemic A10 cells were restored to control levels by antioxidant treatments.


Figure 6
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  		Fig. 6. Effect of antioxidants on hormonal inhibition of adenylyl cyclase activity in A10 VSMCs (A10 cells). A10 cells were incubated in the presence of 5.5 mM glucose (CTL) or 26 mM glucose (hyperglycemia) for 72 h. {alpha}-Tocophorol (10 µM), 10 mM NAC, or 10 µM DPI were added after 48 h of glucose treatment. Membranes were prepared as described in MATERIALS AND METHODS. Adenylyl cyclase activity was determined in the presence of 10 µM GTP{gamma}S alone, taken as 100%, or in combination with 10 µM angiotensin II (ANG II), 0.1 µM [des(Glu18,Ser19,Glu20,Leu21,Cly22)atrial natriuretic peptitde4–23-NH2] (C-ANP4–23), or 50 µM oxotremorine as described previously (Ref. 25). Values are means ± SE of 3 separate experiments. *P < 0.05 vs. CTL (5.5 mM glucose); §P < 0.05 vs. high glucose (26 mM).

 
Effect of antioxidants on Gs{alpha}-mediated stimulation of adenylyl cyclase activity. We have previously shown that the treatment of A10 VSMCs with high glucose for 72 h augmented the Gs{alpha}-mediated functions. To examine whether DPI could also reverse the hyperglycemia-induced enhanced effects of Gs{alpha} proteins, the effect of DPI on the GTP{gamma}S-mediated stimulation of adenylyl cyclase was investigated in hyperglycemic A10 cells, and the results are shown in Fig. 7. GTP{gamma}S stimulated adenylyl cyclase activity in a concentration-dependent manner in control and hyperglycemic A10 VSMCs; however, as reported earlier (25), the extent of the stimulation was significantly increased in hyperglycemic VSMCs compared with control cells. For example, GTP{gamma}S at 10–4 M augmented the adenylyl cyclase activity by sixfold in control cells compared with ~3.5-fold in hyperglycemic A10 cells, and the treatment of hyperglycemic cells with 10 µM DPI restored the enhanced stimulation of adenylyl cyclase toward control levels; however, DPI did not have any effect on the GTP{gamma}S-mediated stimulation of adenylyl cyclase in control cells (data not shown).


Figure 7
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  		Fig. 7. Effect of DPI on GTP{gamma}S-mediated stimulation of adenylyl cyclase activity in A10 VSMCs (A10 cells) exposed to high glucose. A10 cells were incubated in the presence of 5.5 mM glucose (CTL) or 26 mM glucose (hyperglycemia) for 72 h. DPI (10 µM) was added after 48 h of glucose treatment. Membranes were prepared as described in MATERIALS AND METHODS. Adenylyl cyclase activity in the membranes was determined in the absence, taken as 100%, or presence of increasing concentrations of GTP{gamma}S (10–7 to 10–4 M) as described previously (Ref. 25). Values are means ± SE of 3 separate experiments. *P < 0.05; **P < 0.01; ***P < 0.001 vs. CTL (5.5 mM glucose); §P < 0.05; §§P < 0.01 vs. high glucose (26 mM).

 
Effect of antioxidants on hormonal stimulation of adenylyl cyclase activity. To investigate whether the antioxidant could also modulate the high glucose-induced increased stimulation of adenylyl cyclase by stimulatory hormones, the effect of DPI on both isoproterenol- and glucagon-stimulated adenylyl cyclase activity and was examined in hyperglycemic A10 cells. As shown in Fig. 8A, both isoproterenol and glucagon stimulated adenylyl cyclase activity to various degrees in A10 VSMCs; however, as reported earlier (25), the extent of the stimulation was significantly augmented by about 60% and 40%, respectively, in hyperglycemic cells compared with control cells, and this increase was restored to control levels by DPI treatment. In addition, the enhanced stimulation of adenylyl cyclase by sodium fluoride (NaF; ~30%) and FSK (~60%), which stimulate the enzyme activity by a receptor-independent mechanism in high glucose-treated cells, was also restored to control levels by DPI treatment (Fig. 8B).


Figure 8
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  		Fig. 8. Effect of DPI on hormonal stimulation of adenylyl cyclase activity in A10 VSMCs (A10 cells) exposed to high glucose. A10 cells were incubated in the presence of 5.5 mM glucose (CTL) or 26 mM glucose (hyperglycemia) for 72 h. DPI (10 µM) was added after 48 h of glucose treatment. Membranes were prepared as described in MATERIALS AND METHODS. Adenylyl cyclase activity in the membranes was determined in the presence of 10 µM GTP alone or in combination with 50 µM isoproterenol (Iso) or 1 µM glucagon, in the absence or presence of 10 mM NaF or 50 µM FSK as described previously (Ref. 25). Values are means ± SE of 3 separate experiments. *P < 0.05; ***P < 0.001 vs. CTL (5.5 mM glucose); §P < 0.05; §§§P < 0.001 vs. high glucose (26 mM).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We have previously reported that aorta from 5-day STZ-diabetic rats exhibited decreased expression of Gi{alpha}-2 and Gi{alpha}-3 proteins and associated functions (26). A significant decrease in Gi{alpha} proteins was observed on day 3 of the STZ injection, when the blood glucose level was increased to 20 mM, and below that concentration of blood glucose, no significant decrease in Gi{alpha} proteins was detected. We have further shown that aorta as well as A10 VSMCs exposed to high glucose (26 mM) that simulate the diabetic state also exhibited decreased levels of Gi{alpha}-2 and Gi{alpha}-3 proteins, whereas the levels of Gs{alpha} were not altered (25). The decreased expression of Gi{alpha} proteins was concentration and time dependent. A significant decrease was observed at 20 mM glucose, and below that concentration, the levels of Gi{alpha} proteins were not altered. These data indicate a correlation between the levels of glucose (in vivo and in vitro) and decreased expression of Gi{alpha} proteins and suggest that hyperglycemia may be a contributing factor in the diabetes-induced decreased expression of Gi{alpha} proteins.

In the present studies, we report that aortic VSMCs from STZ-diabetic rats, like diabetic aorta (25), also exhibit decreased expression of Gi{alpha} proteins, suggesting that aortic VSMCs cultured from STZ-diabetic rats retained the diabetic phenotype. We also report that A10 VSMCs exposed to high glucose exhibit an enhanced activity of NADPH oxidase and augmented production of O2–, which contributes to the decreased expression of Gi{alpha} proteins in aortic VSMCs from STZ-diabetic rats and A10 cells exposed to high glucose. In this regard, hyperglycemia-induced enhanced oxidative stress has also been reported earlier in cultured VSMCs, endothelial cells, and different tissues from STZ-diabetic rats (10, 14). Multiple cellular sources of O2– have been documented, which include NADH/NADPH and xanthine oxidases, the mitochondrial respiratory chain, the arachidonic acid cascades (including lipoxygenase and cyclooxygenase), and microsomal enzymes (13, 17). The contribution of mitochondrial O2– production induced by high glucose has been reported in endothelial cells (41, 48), which plays an important role in the pathogenesis of diabetes-associated endothelial dysfunctions. Whether the high glucose-induced enhanced production of O2– in A10 VSMCs is attributed to mitochondria needs to be investigated. However, Liu et al. (38) have recently shown that the high glucose-induced enhanced production of O2– in VSMCs was abolished by O2–, scavenger Tempol, or apocynin, a specific inhibitor of NADPH oxidase, and unaffected by rotenone, an inhibitor of mitochondrial respiratory chain complex 1, L-NAME, an inhibitor of NO synthase (NOS), or oxypurinol, an inhibitor of xanthine oxidase, which suggests that NADPH oxidase is primarily responsible for high glucose-induced O2– production in VSMCs. The contribution of DAG-PKC, which activates NADPH oxidase in the enhanced production of reactive oxygen species (ROS), has been reported in diabetic tissues (29) and in cultured endothelial as well as in aortic VSMCs exposed to high glucose (27). In addition, the expression of NADPH oxidase components was shown to be upregulated in vascular tissues and the kidney from animal models of diabetes as well as in micro- and macrovascular tissues in patients with diabetes and obese subjects (27–29). We have also reported that the treatment of A10 VMSCs with high glucose augmented the levels of p47phox and p22phox proteins, the subunits of NADPH (18). Taken together, it may be suggested that increased levels of NADPH oxidase may be responsible for the enhanced levels of O2– and NADPH oxidase activity. In support of this are the studies of Liu et al. (38), who have recently reported that the transfection of VSMCs with small-interfering RNA-p47phox abolished the hyperglycemia-induced enhanced production of O2–. The increased levels of various vasoactive peptides including ANG II and endothelin in diabetes and under hyperglycemic conditions (24, 46, 49) that have been shown to increase oxidative stress by activating the NADPH oxidase (53) may contribute to the enhanced oxidative stress induced by high glucose in VSMCs. In this regard, we have recently reported that the ANG II treatment of VSMCs increased the production of O2– and the expression of Nox4 and p47phox (37). Furthermore, the ANG II type 1 receptor blocker candesartan and angiotensin-converting enzyme inhibitor quinapril have been shown to attenuate the enhanced expression of p47phox in the kidney from STZ-diabetic rats, suggesting the implication of ANG II in diabetes-induced increased oxidative stress (42).

Although a role of oxidative stress in the enhanced activity of PKC in diabetes and hyperglycemia has been well established (27–29), evidence for a direct role of oxidative stress in the high glucose/diabetes-induced decreased expression of Gi{alpha} proteins and associated signaling is lacking. Our results showing that antioxidants such as {alpha}-tocopherol, NAC, scavengers of O2–, and DPI, an inhibitor of NADPH oxidase, that restored the enhanced levels of O2– induced by hyperglycemia also restored the hyperglycemia-induced decreased expression of Gi{alpha}-2 and Gi{alpha}-3 to control levels suggest the implication of NADPH oxidase/O2– in the hyperglycemia-evoked decreased expression of Gi{alpha} proteins. However, we have earlier shown that enhanced oxidative stress contributes to the enhanced expression of Gi{alpha} proteins in VSMCs from spontaneously hypertensive rats (35) and in A10 VSMCs exposed to ANG II (37). Therefore, to clarify these discrepancies, we investigated the contribution of different ROS and reactive nitrogen species in the hyperglycemia-induced decreased expression of Gi{alpha} proteins. Hyperglycemia, through the activation of NF-{kappa}B, has been shown to augment the expression of inducible NOS (iNOS), which increases the generation of NO (50). O2– formed by NADPH oxidase activation is converted to H2O2 by SOD and also reacts with NO to form a potent cytotoxin ONOO–, which may be responsible for the decreased expression of Gi{alpha} proteins in hyperglycemia.

We showed that the hyperglycemia-induced decreased expression of Gi{alpha} protein may be attributed to the increased levels of ONOO– because scavengers of ONOO–, uric acid and MnTBAP, as well as L-NAME, which inhibits the production of NO and thereby the formation of ONOO–, were able to restore the hyperglycemia-induced decreased expression of Gi{alpha} proteins to control levels. The implication of ONOO– in the NO-induced decreased expression of Gi{alpha} proteins in aortic and A10 VSMCs has recently been shown (7). In addition, we have recently shown that the treatment of VSMCs with ONOO– increased the levels of cGMP and decreased the expression of Gi{alpha} proteins (9). cGMP has also been shown to decrease the levels of Gi{alpha} proteins in VSMCs (8); however, the ONOO–-induced decreased expression of Gi{alpha} proteins was not mediated through cGMP because 1H[1,2,4]oxadiazole[4,3-a]quinoxaline-1-one, an inhibitor of soluble guanylyl cyclase, was unable to restore the decrease expression of Gi{alpha} proteins to the control level (9).

There is accumulating evidence that supports the hypothesis that diabetes is associated with increased nitrosative stress and ONOO– formation in several tissues both in experimental animals and humans (44). ONOO– attacks various biomolecules in the vascular endothelium, vascular smooth muscle, and myocardium, leading to cardiovascular dysfunction (44). ONOO– has also been reported to damage DNA and thereby triggers the activation of Poly(ADP-ribose)polymerase-1 (PARP-1), a nuclear enzyme (43). The activation of PARP-1 depletes the intracellular concentration of its substrate NAD+ by inhibiting the rate of glycolysis, electron transport, and ATP formation, produces the ADP-ribosylation of GAPDH, and results in cardiovascular dysfunction (43). The increased levels of nitrotyrosine, a relatively specific marker of ONOO– formation, have been shown in different tissues from STZ-diabetic rats and subjects with diabetes (44). For example, increased nitrotyrosine plasma levels were shown in patients with Type 2 diabetes (15), and iNOS-dependent ONOO– production was shown to be increased in platelets from individuals with diabetes (52). In addition, hyperglycemia has also been reported to induce increased nitrotyrosine formation in the artery wall of monkeys (47). Taken together, it may be possible that hyperglycemia-induced increased levels of ONOO–, formed by the interaction of NO and O2–, may contribute to the hyperglycemia-induced decreased expression of Gi{alpha} proteins in VSMCs.

We also showed that antioxidants that result in the restoration of hyperglycemia-induced decreased expression of Gi{alpha} proteins to control levels also restored to the control levels the decreased Gi-mediated functions (receptor dependent and independent), as demonstrated by the restoration of decreased inhibition of adenylyl cyclase by ANG II, C-ANP4–23, and oxotremorine to control levels. In addition, the GTP{gamma}S-mediated decreased inhibition of FSK-stimulated adenylyl cyclase activity (receptor-independent functions of Gi{alpha} proteins) in hyperglycemic cells was also restored to control levels by DPI. Furthermore, the hyperglycemia-induced enhanced stimulation of adenylyl cyclase by GTP{gamma}S and stimulatory hormones such as isoproterenol and glucagon was also restored to control levels by DPI. This may be attributed to the Gi{alpha} and not to Gs{alpha} proteins because hyperglycemia was unable to alter the levels of Gs{alpha} proteins in these cells. In this regard, the interaction between Gi and Gs{alpha} proteins has been well established. This is further substantiated by our studies showing that the restoration of decreased levels of Gi{alpha} proteins to control levels by the antioxidant also restored the Gs{alpha}-mediated augmented hormonal and GTP{gamma}S-induced stimulation of adenylyl cyclase to control levels.

In conclusion, we have provided the first evidence that the diabetes/hyperglycemia-induced decreased expression of Gi{alpha} proteins and associated adenylyl cyclase signaling may be attributed to the augmented levels of O2– and ONOO– (Fig. 9). The treatment with antioxidants reversed the hyperglycemia-induced decreased expression of Gi{alpha} proteins and adenylyl cyclase signaling to control levels. In this regard, the overexpression of constitutively activated Gi{alpha}-2 has also been shown to improve STZ-induced diabetes in rats (56). Thus, taken together, it may be suggested that antioxidants, by augmenting the decreased levels of Gi{alpha} proteins induced by high glucose, may have beneficial effects in improving the cardiovascular complications of diabetes.


Figure 9
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  		Fig. 9. Schematic diagram summarizing the possible mechanisms by which hyperglycemia/diabetes decreases the expression of Gi{alpha} proteins and adenylyl cyclase signaling. NO, nitric oxide.

 

   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by a grant from the Canadian Institutes of Health Research.


   ACKNOWLEDGMENTS
 
We thank Christiane Laurier for valuable secretarial help.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. B. Anand-Srivastava, Dept. of Physiology, Univ. of Montreal, C.P. 6128, Succ. Centre-ville, Montreal, QC, Canada H3C 3J7 (e-mail: madhu.anand-srivastava{at}umontreal.ca)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Anand-Srivastava MB, de Champlain J, Thibault C. DOCA-salt hypertensive rat hearts exhibit altered expression of G-proteins. Am J Hypertens 6: 72–75, 1993.[Web of Science][Medline]
  2. Anand-Srivastava MB, Srivastava AK, Cantin M. Pertussis toxin attenuates atrial natriuretic factor-mediated inhibition of adenylyl cyclase. Involvement of inhibitory guanine nucleotide regulatory protein. Biochem J 262: 4931–4934, 1987.
  3. Anand-Srivastava MB. Enhanced expression of inhibitory guanine nucleotide regulatory protein in spontaneously hypertensive rats: relationship to adenylate cyclase inhibition. Biochem J 288: 79–85, 1992.[Web of Science][Medline]
  4. Anand-Srivastava MB. Angiotensin II receptors are negatively coupled to adenylyl cyclase in rat myocardial sarcolemma. Involvement of inhibitory guanine nucleotide regulatory protein. Biochem Pharmacol 38: 489–496, 1989.[CrossRef][Web of Science][Medline]
  5. Anand-Srivastava MB. Angiotensin II receptors are negatively coupled to adenylyl cyclase in rat aorta. Biochem Biophys Res Commun 117: 420–428, 1983.[CrossRef][Web of Science][Medline]
  6. Anand-Srivastava MB, Franks DJ, Cantin M, Genest J. Presence of "Ra" and "P"-site receptors for adenosine coupled to adenylate cyclase in cultured vascular smooth muscle cells. Biochem Biophys Res Commun 108: 213–219, 1982.[CrossRef][Web of Science][Medline]
  7. Bassil M, Anand-Srivastava MB. Nitric oxide modulates Gi-protein expression and adenylyl cyclase signaling in vascular smooth muscle cells. Free Radic Biol Med 7: 1162–1173, 2006.
  8. Bassil M, Anand-Srivastava MB. Cyclic GMP modulates the expression of Gi protein and adenylyl cyclase activity in vascular smooth muscle cells. Cell Biochem Biophys 47: 99–108, 2007.[CrossRef][Web of Science][Medline]
  9. Bassil M, Anand-Srivastava MB. Peroxynitrite inhibits the expression of Gi{alpha} protein and adenylyl cyclase signaling in vascular smooth muscle cells. Am J Physiol Heart Circ Physiol 294: H775–H784, 2008.[Abstract/Free Full Text]
  10. Baynes JW, Thorpe SR. Role of oxidative stress in diabetic complications: a new perspective on an old paradigm. Diabetes 48: 1–9, 1999.[Abstract]
  11. Bohlen JM, Nase GP. Arteriolar nitric oxide concentration is decreased during hyperglycemia-induced βII PKC activation. Am J Physiol Heart Circ Physiol 280: H621–H627, 2001.[Abstract/Free Full Text]
  12. Bray P, Caster A, Simons C, Guo V, Puckett C, Kamholz J, Spiegel A, Nirenberg M. Human cDNA clones for four species of Gs{alpha} signal transduction protein. Proc Natl Acad Sci USA 83: 8893–8897, 1986.[Abstract/Free Full Text]
  13. Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature 14: 813–820, 2001.
  14. Cai H, Harrison DG. Endothelial dysfunction in cardiovascular diseases: the role of oxidative stress. Circ Res 87: 840–844, 2000.[Abstract/Free Full Text]
  15. Ceriello A, Mercuri F, Quagliaro L, Assaloni R, Motz E, Tonutti L, Taboga C. Detection of nitrotyrosine in the diabetic plasma: evidence of oxidative stress. Diabetologia 44: 834–838, 2001.[CrossRef][Web of Science][Medline]
  16. Cowell JA. Vascular thrombosis in type II diabetes mellitus. Diabetes 32: 8–11, 1993.
  17. De Vriese AS, Verbeuren TJ, Van de Voorde J, Lameire NH, Vanhoute PM. Endothelial dysfunction in diabetes. Br J Pharmacol 130: 963–974, 2000.[CrossRef][Web of Science][Medline]
  18. Descorbeth M, Anand-Srivastava MB. High glucose augmented G{alpha}q-mediated cell signaling in vascular smooth muscle cells (VSMC): role of oxidative stress (Abstract). FASEB J 20: A664, 2006.[Free Full Text]
  19. Feldman AM, Cates AE, Veazey WB, Hershberger RE, Bristow MR, Baughman KL, Baumgartner WA, Van Dop C. Increase of the 40,000 mol wt pertussis toxin substrate (G protein) in the failing human heart. J Clin Invest 82: 189–197, 1988.[Web of Science][Medline]
  20. Fleming JW, Wisler PL, Watanbe AM. Signal transduction by G-proteins in cardiac tissues. Circulation 85: 420–433, 1992.[Abstract/Free Full Text]
  21. Gawler D, Milligan G, Spiegel AM, Unson CG, Houslay MD. Abolition of expression of inhibitory guanine nucleotide regulatory protein Gi activity in diabetes. Nature 327: 229–232, 1987.[CrossRef][Medline]
  22. Giugliano D, Marfella R, Coppola L, Verazzo G, Acampora R, Giunta R, Nappo F, Lucarelli C, D'Onofrio F. Vascular effects of acute hyperglycemia in humans are reversed by L-arginine. Evidence for reduced availability of nitric oxide during hyperglycemia. Circulation 95: 1783–1790, 1997.[Abstract/Free Full Text]
  23. Giugliano D, Ceriello A, Paolisso G. Oxidative stress and diabetic vascular complications. Diabetes Care 19: 257–267, 1996.[Abstract]
  24. Hargrove GM, Dufresne J, Whiteside C, Muruve DA, Wong NC. Diabetes mellitus increases endothelin-1 gene transcription in rat kidney. Kidney Int 58: 1534–1545, 2000.[CrossRef][Web of Science][Medline]
  25. Hashim S, Li Y, Nagakura A, Takeo S, Anand-Srivastava MB. Modulation of G-protein expression and adenylyl cyclase signaling by high glucose in vascular smooth muscle. Cardiovasc Res 63: 709–718, 2004.[Abstract/Free Full Text]
  26. Hashim S, Liu YY, Wang R, Anand-Srivastava MB. Streptozotocin-induced diabetes impairs G-protein linked signal transduction in vascular smooth muscle. Mol Cell Biochem 240: 57–65, 2002.[CrossRef][Web of Science][Medline]
  27. 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 49: 1939–1945, 2000.[Abstract]
  28. Inoguchi T, Nawata H. NAD(P)H oxidase activation: a potential target mechanism for diabetic vascular complications, progressive beta-cell dysfunction and metabolic syndrome. Curr Drug Targets 6: 495–501, 2005.[CrossRef][Web of Science][Medline]
  29. Inoguchi T, Sonta T, Tsubouchi H, Etoh T, Kakimoto M, Sonoda N, Sato N, Sekiguchi N, Kobayashi K, Sumimoto H, Utsumi H, Nawata H. Protein kinase C-dependent increase in reactive oxygen species (ROS) production in vascular tissues of diabetes: role of vascular NAD(P)H oxidase. J Am Soc Nephrol 14, Suppl 3: S227–S232, 2003.[Abstract/Free Full Text]
  30. Itoh H, Toyama R, Kozasa T, Tsukamoto T, Matsuoka M, Kaziro Y. Presence of three distinct molecular species of Gi protein: a subunit structure of rat cDNA and human genomic DNAs. J Biol Chem 263: 6656–6664, 1988.[Abstract/Free Full Text]
  31. Kimes BW, Brandt BL. Characterization of two putative smooth muscle cell lines from rat thoracic aorta. Exp Cell Res 98: 349–366, 1976.[CrossRef][Web of Science][Medline]
  32. Kostenis E, Zeng FY, Wess J. Structure-function analysis of muscarinic receptors and their associated G proteins. Life Sci 64: 355–362, 1999.[CrossRef][Web of Science][Medline]
  33. Koya D, King GL. Protein kinase C activation and the development of diabetic complications. Diabetes 47: 859–866, 1998.[Abstract]
  34. Kunisaki M, Hursell SE, Umeda F, Nawata H, King G. Normalization of diacyglycerol-protein kinase C activation by vitamin E in aorta of diabetic rats and cultured rat smooth muscle cells exposed to elevated glucose levels. Diabetes 43: 1372–1377, 1997.[CrossRef]
  35. Lappas G, Bou Daou G, Anand-Srivastava MB. Oxidative stress contributes to the enhanced expression of Gi{alpha} protein and adenylyl cyclase signaling in vascular smooth muscle cells from spontaneously hypertensive rats. J Hypertens 23: 2251–2261, 2005.[Web of Science][Medline]
  36. Lash JM, Nase GP, Bohlen HG. Acute hyperglycemia depresses arteriolar NO formation in skeletal muscle. Am J Physiol Heart Circ Physiol 277: H1513–H1520, 1999.[Abstract/Free Full Text]
  37. Li Y, Lappas G, Anand-Srivastava MB. Role of oxidative stress in angiotensin II-induced enhanced expression of Gi{alpha} proteins and adenylyl cyclase signaling in A10 vascular smooth muscle cells. Am J Physiol Heart Circ Physiol 292: H1922–H1930, 2007.[Abstract/Free Full Text]
  38. 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 Radic Biol Med 42: 852–863, 2007.[CrossRef][Web of Science][Medline]
  39. Moxham CM, Malbon CC. Insulin action impaired by deficiency of the G-protein subunit Gi alpha 2. Nature 379: 840–844, 1996.[CrossRef][Medline]
  40. Neer EJ. Heterotrimeric G proteins. Organizers of transmembrane signals. Cell 80: 249–257, 1995.[CrossRef][Web of Science][Medline]
  41. Nishikawa T, Edelstein D, Dux L, Yamagishi SI, Matsumura T, Kaneda Y, Yorek MA, Beebe D, Oates PJ, Hammes HP, Gilardino I, Brownlee M. Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage. Nature 404: 787–790, 2000.[CrossRef][Medline]
  42. Onozato ML, Tojo A, Goto A, Fujita T, Wilcox CS. Oxidative stress and nitric oxide synthase in rat diabetic nephropathy: effects of ACEI and ARB. Kidney Int 61: 186–194, 2002.[CrossRef][Web of Science][Medline]
  43. Pacher P, Liaudet I, Garcia Sorjano F, Mabley JG, Szabo E, Szabo C. The role of poly (ADP-ribose) polymerase activation in the development of myocardial and endothelial dysfunction in diabetes. Diabetes 51: 514–521, 2002.[Abstract/Free Full Text]
  44. Pacher P, Obrosova IG, Mabley JG, Szabo C. Role of nitrosative stress and peroxynitrite in the pathogenesis of diabetic complications. Emerging new therapeutical strategies. Curr Med Chem 12: 267–275, 2005.[Web of Science][Medline]
  45. Palmer GC, Wilson GL, Chronister RB. Streptozotocin-induced diabetes produces alterations in adenylate cyclase in rat cerebrum, cerebral microvessels and retina. Life Sci 24: 365–374, 1983.
  46. Park JY, Takahara N, Gabriele A, Chou E, Naruse K, Suzuma K, Yamauchi T, Ha SW, Meier M, Rhodes CJ, King GL. Induction of endothelin-1 expression by glucose: an effect of protein kinase C activation. Diabetes 49: 1239–1248, 2000.[Abstract]
  47. Pennathur S, Wagner JD, Leeuwenburgh C, Litwak KN, Heinecke JW. A hydroxyl radical-like species oxidizes cynomolgus monkey artery wall proteins in early diabetic vascular disease. J Clin Invest 107: 853–860, 2001.[Web of Science][Medline]
  48. Rajesh M, Mukhopadhyay P, Batkai S, Hasko G, Liaudet L, Drel VR, Obrusova IG, Packer P. Cannabidiol attenuates high glucose-induced endothelial cell inflammatory response and barrier disruption. Am J Physiol Heart Circ Physiol 293: H610–H619, 2007.[Abstract/Free Full Text]
  49. Sodhi CP, Kanwar YS, Sahai A. Hypoxia and high glucose upregulate AT1 receptor expression and potentiate ANG II-induced proliferation in VSM cells. Am J Physiol Heart Circ Physiol 284: H846–H852, 2003.[Abstract/Free Full Text]
  50. Spitaler MM, Graier WF. Vascular targets of redox signaling in diabetes mellitus. Diabetologia 45: 476–494, 2002.[CrossRef][Web of Science][Medline]
  51. Strassheim D, Palmer T, Houslay MD. Diabetes abolishes the GTP-dependent, but not the receptor-dependent inhibitory function of the inhibitory guanine-nucleotide-binding regulatory protein (Gi) on adipocyte adenylate cyclase activity. Biochem J 266: 521–526, 1990.[Web of Science][Medline]
  52. Tannous M, Rabini RA, Vignini A, Moretti N, Fumelli P, Zielinski B, Mazzanti L, Mutus B. Evidence for iNOS-dependent peroxynitrite production in diabetic platelets. Diabetologia 42: 539–544, 1999.[CrossRef][Web of Science][Medline]
  53. Touyz RM, Yao G, Viel E, Amiri F, Schiffrin EL. Angiotensin II and endothelin-1 regulate MAP kinases through different redox-dependent mechanisms in human vascular smooth muscle cells. J Hypertens 22: 1141–1149, 2004.[CrossRef][Web of Science][Medline]
  54. Wong YH, Conklin BB, Bourne HR. Gi-mediated hormonal inhibition of cyclic AMP accumulation. Science 255: 339–342, 1992.[Abstract/Free Full Text]
  55. Yatani A, Mattera R, Codina J. The G protein-gated atrial K+ channel is stimulated by three distinct Gi{alpha} subunits. Nature 336: 680–682, 1988.[CrossRef][Medline]
  56. Zheng XL, Guo J, Wang HY, Malbon CC. Expression of constitutively activated Gialpha2 in vivo ameliorates streptozotocin-induced diabetes. J Biol Chem 273: 23649–23651, 1998.[Abstract/Free Full Text]




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