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Am J Physiol Heart Circ Physiol 292: H1922-H1930, 2007. First published December 8, 2006; doi:10.1152/ajpheart.01166.2006
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Role of oxidative stress in angiotensin II-induced enhanced expression of Gi{alpha} proteins and adenylyl cyclase signaling in A10 vascular smooth muscle cells

Yuan Li, Georgios Lappas, and Madhu B. Anand-Srivastava

Department of Physiology and Groupe de recherche sur le système nerveux autonome, Faculty of Medicine, University of Montreal, Montréal, Québec, Canada

Submitted 23 October 2006 ; accepted in final form 29 November 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We have previously reported that angiotensin II (ANG II) treatment of A10 vascular smooth muscle cells (VSMCs) increased inhibitory G proteins (Gi protein) expression and associated adenylyl cyclase signaling which was attributed to the enhanced MAP kinase activity. Since ANG II has been shown to increase oxidative stress, we investigated the role of oxidative stress in ANG II-induced enhanced expression of Gi{alpha} proteins and examined the effects of antioxidants on ANG II-induced enhanced expression of Gi{alpha} proteins and associated adenylyl cyclase signaling in A10 VSMCs. ANG II treatment of A10 VSMCs enhanced the production of O2– and the expression of Nox4 and P47phox, different subunits of NADPH oxidase, which were attenuated toward control levels by diphenyleneiodonium (DPI). In addition, ANG II augmented the expression of Gi{alpha}-2 and Gi{alpha}-3 proteins in a concentration- and time-dependent manner; the maximal increase in the expression of Gi{alpha} was observed at 1 to 2 h and at 0.1–1.0 µM. The enhanced expression of Gi{alpha}-2 and Gi{alpha}-3 proteins was restored to control levels by antioxidants such as N-acetyl-L-cysteine, {alpha}-tocopherol, DPI, and apocynin. In addition, ANG II also enhanced the ERK1/2 phosphorylation that was restored to control levels by DPI. Furthermore, the inhibition of forskolin-stimulated adenylyl cyclase activity by low concentrations of 5'-O-(3-triotriphosphate) (receptor-independent Gi functions) and ANG II-, des(Glu18,Ser19,Glu20,Leu21,Gly22)atrial natriuretic peptide4-23-NH2 (natriuretic peptide receptor-C agonist), and oxotremorine-mediated inhibitions of adenylyl cyclase (receptor-dependent functions) that were augmented in ANG II-treated VSMCs was also restored to control levels by antioxidant treatments. In addition, Gs{alpha}-mediated diminished stimulation of adenylyl cyclase by stimulatory hormones in ANG II-treated cells was also restored to control levels by DPI. These results suggest that ANG II-induced enhanced levels of Gi{alpha} proteins and associated functions in VSMCs may be attributed to the ANG II-induced enhanced oxidative stress, which exerts its effects through mitogen-activated protein kinase signaling pathway.

G protein; mitogen-activated protein kinase; adenylyl cyclase

ANGIOTENSIN II (ANG II), a vasoactive peptide and key component of the renin-angiotensin system, regulates a variety of physiological functions including blood pressure, cell proliferation, cell differentiation, and apoptosis (27, 31, 33). ANG II elicits its physiological effects by interacting with two distinct receptor subtypes designated as ANG II types 1 and 2 (AT1 and AT2, respectivey) receptors (35). The presence of AT1 receptor subtype has been shown in rat vascular tissues; however, a small proportion of AT2 receptors are also present in rat aorta (9, 39). Most of the physiological effects of ANG II are mediated through the activation of AT1 receptors that are coupled to several signaling pathways including adenylyl cyclase/cAMP inhibition through Gi{alpha} proteins (4, 5, 28), mitogen-activated protein kinases (MAPKs) (10, 24, 38), and phosphatidylinositol turnover (15).

The adenylyl cyclase/cAMP system is composed of three components: receptor, catalytic subunit, and guanine nucleotide regulatory proteins (G proteins). The G proteins act as transducers and, in the presence of guanine nucleotides, transmit the signal from the hormone-occupied receptor to the catalytic subunit. The agonist-mediated stimulation and inhibition of adenylyl cyclase are mediated through stimulatory (Gs) and inhibitory (Gi) guanine nucleotide protein, respectively (29, 32), resulting in the increased or decreased formation of cAMP, respectively. G proteins are heterotrimeric, consisting of {alpha}-, beta-, and {gamma}-subunits. The {alpha}-subunits bind and hydrolyze GTP and confer specificity in receptor and effector interactions. Four different isoforms of Gs have been identified, which appear to be products of an alternate splicing of a single gene (8, 19), whereas three different isoforms of Gi{alpha} (Gi{alpha}-1, Gi{alpha}-2, and Gi{alpha}-3) have been identified and are shown to be the products of three different genes (17, 18).

Alterations in the levels of Gi proteins that result in the impaired cellular functions lead to various pathological states such as hypertension. We have recently shown an increased expression of Gi proteins and Gi protein mRNA in hearts and aortas from spontaneously hypertensive rats (SHR) and in hearts from experimental hypertensive rats including deoxycorticosterone acetate (DOCA)-salt hypertensive rats and 1 kidney 1 clip (1K1C [PDB] ) rats with established hypertension (3, 6, 12, 13, 34). In addition, we have further shown that vascular smooth muscle cells (VSMCs) from SHR also exhibit enhanced levels of Gi{alpha} protein compared with Wistar-Kyoto rats, which were restored to control levels by antioxidants (21), suggesting a role of oxidative stress in enhanced levels of Gi{alpha} proteins in SHR. The levels of various vasoactive peptides, including ANG II, that have been shown to be enhanced in hypertension may be responsible for the enhanced expression of Gi{alpha} proteins. This was supported by studies showing that treatment of hypertensive rats with captopril, an antihypertensive drug that acts by blocking the ANG II-converting enzyme, and losartan; an AT1 receptor-antagonist decreased the blood pressure and the enhanced levels of Gi proteins (16, 26). Furthermore, ANG II has been shown to increase the levels of Gi proteins in A10 smooth muscle cells (25), which was attenuated by losartan and a mitogen-activated protein/extracellular signal-regulated (ERK) kinase inhibitor (1, 11). ANG II also increases oxidative stress by activating NADPH oxidase (14). Taken together, it may be possible that ANG II-induced enhanced expression of Gi{alpha} proteins in A10 cells is also attributed to the enhanced oxidative stress. To examine this possibility, the present studies were therefore undertaken to investigate the effect of antioxidant treatment on ANG II-induced enhanced expression of Gi{alpha} proteins and adenylyl cyclase signaling in A10 VSMCs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. 3-Isobutyl-1-methylxanthine (IBMX) was purchased from Aldrich Chemical (Milwaukee, WI). N-acetyl-L-cysteine (NAC), diphenyleneiodonium (DPI), apocynin, and {alpha}-tocopherol were from British Drug House (Toronto, Ontario, Canada). [{alpha}-32P]ATP was purchased from Amersham (Oakville, Ontario, Canada). Antibodies L-5, C-10, and monoclonal phosphospecific-tyrosine204 ERK1/2, NADPH oxidase subunit antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Myokinase (EC 2.7.4.3 [EC] ) and all other chemicals used in the experiments were purchased from Sigma Chemical (St. Louis, MO).

Cell culture and incubation. A-10 cell line from embryonic thoracic aorta of rat was obtained from American Type Culture Collection (Rockville, MA). The cells were plated in 75-cm2 flasks and incubated at 37°C in a 95% air-5% CO2-humidified atmosphere in Dulbecco's modified Eagle's medium (DMEM) (with glucose, L-glutamine, and sodium bicarbonate) containing antibiotics and 10% heat-inactivated fetal bovine serum (FBS) as described previously (21, 25, 26). Confluent cell cultures were starved by incubation for 3 h in DMEM without FBS at 37°C. These cells were then incubated with ANG II (10–7 M) for different time periods at 37°C. To examine the effect of antioxidants on ANG II-induced responses, the cells were pretreated with or without antioxidants for 24 h and then challenged with ANG II (10–7 M) for 1 h. After incubation, cells were washed twice with ice-cold homogenization buffer (10 mM Tris·HCl, pH 7.5, containing 1 mM EDTA). The cells were scraped into ice-cold homogenization buffer using a rubber policeman and collected by centrifugation at 4°C for 10 min at 600 g. The cells were then homogenized in a Dounce homogenizer (10 strokes), and the homogenate was used for adenylyl cyclase assay and immunoblotting.

Adenylyl cyclase activity determination. Adenylyl cyclase activity was determined by measuring [32P]cAMP formation from [{alpha}-32P]ATP, as previously described (3, 6). Briefly, the assay medium contained 50 mM glycylglycine (pH 7.5), 0.5 mM MgATP, [{alpha}-32 P]ATP (1.5 x 106 counts/min), 5 mM MgCl2 (in excess of the ATP concentration), 100 mM NaCl, 0.5 mM cAMP, 1 mM IBMX, 0.1 mM EGTA, 10 µM guanosine 5'-O-(3-thiotriphosphate) (GTP{gamma}S), and an ATP-regenerating system (consisting of 2 mM phosphocreatine, 0.1 mg/ml creatine kinase, and 0.1 mg/ml myokinase) in a final volume of 200 µl. Incubations were initiated by the addition of the membrane preparations (20–30 µg) to the reaction mixture, which had been thermally equilibrated for 2 min at 37°C. The reactions were conducted in triplicate for 10 min at 37°C and were terminated by the addition of 0.6 ml of 120 mM zinc acetate. cAMP was purified by coprecipitation of other nucleotides with ZnCO3, an addition of 0.5 ml of 144 mM Na2CO3, and subsequent chromatography by the double-column system, as described by Salomon et al. (30). Under the assay conditions used, adenylyl cyclase activity was linear with respect to protein concentrations and time of incubation. Protein was determined essentially as described by Lowry et al. (22) with bovine serum albumin as standard.

Immunoblotting. Immunoblotting of G proteins, Nox4, p47phox (NADPH oxidase subunits), and ERK1/2 was performed by using specific antibodies as described previously (3, 21). After SDS-PAGE, the separated proteins were electrophoretically transferred to nitrocellulose paper (Schleicher and Schuell, Keene, NH) with a semidry transblot apparatus (Bio-Rad, Mississauga, Ontario, Canada) at 15 V for 45 min. After transfer, the membranes were washed twice in phosphate-buffered saline (PBS) and were incubated in PBS containing 8% dehydrated milk at room temperature for 2 h. The blots were then incubated with respective primary antibodies: L-5 for Gi{alpha}-2, C-10 for Gi{alpha}-3, monoclonal phosphospecific Tyr204 ERK1/2 antibody for p-ERK1/2, N-15 for Nox4, and C-20 for p47phox antibodies in PBS containing 3% dehydrated milk and 0.1% Tween 20 at room temperature for 2 h. The antibody-antigen complexes were detected by second antibody, and protein bands were visualized by enhanced-chemiluminescence Western-blotting detection reagents from Amersham as described previously (3, 21). Quantitative analysis of specific bands was performed by densitometric scanning of the autoradiographs with an enhanced laser densitometer (LKB Ultroscan XL, Pharmacia, Dorval, Quebec, Canada) and quantified by using gel-scan XL evaluation software (version 2.1) from Pharmacia.

Superoxide anion measurements. Basal superoxide anion production was measured by using the lucigenin-enhanced chemiluminescence with low concentration (5 µM) of lucigenin as described previously (21). The cells after treatment with ANG II (10–6 M) or DPI (10 µM) alone or in combination 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 Sciences, St. Laurent, Quebec, Canada) for 5 min. The average luminescence value was estimated, the background value substracted, 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 analysis of variance in conjunction with Newman-Keuls tests. Results were considered significant at a value of P < 0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of ANG II on Gi{alpha} protein expression in A10 VSMCs. We have previously reported that exposure of A10 VSMCs to ANG II for 24 h increased the expression of Gi{alpha} proteins; however, it was of interest to examine whether the short-term treatment of VSMCs with ANG II also resulted in the enhanced levels of Gi{alpha} proteins. Figure 1 shows the temporal relationship between ANG II treatment and the levels of Gi{alpha}-2 and Gi{alpha}-3 proteins. ANG II treatment (10–7 M) of the cells increased the expression of both Gi{alpha}-2 (Fig. 1A) and Gi{alpha}-3 (Fig. 1B) proteins in a time-dependent manner. The levels of Gi{alpha}-2 and Gi{alpha}-3 started increasing as early as 30 min and peaked off at about 125–135% of control at 1 to 2 h and remained elevated up to 24 h as determined by densitometric scanning. On the other hand, the levels of Gs{alpha} were not affected by such treatment (data not shown).


Figure 1
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  		Fig. 1. Effect of angiotensin II (ANG II) pretreatment on the levels of Gi{alpha}-2 and Gi{alpha}-3 proteins in A10 vascular smooth muscle cells (VSMCs). Top: A10 VSMCs were incubated in the absence (control) or presence of 10–7 M ANG II for different time periods as described in MATERIALS AND METHODS. Membranes were prepared and used for immunoblotting. Membrane proteins (30 µg) were resolved by SDS-PAGE and transferred to nitrocellulose, which was then immunoblotted using L-5 antibody for Gi{alpha}-2 (A) or C-10 antibody for Gi{alpha}-3 (B) as described previously (21). Immunoblots are representative of 3 or 4 separate experiments. Bottom: quantification of protein bands by densitometric scanning. The results are expressed as percentage of control (CTL) taken as 100%. Values are means ± SE of 3 or 4 separate experiments. *P < 0.05 and **P < 0.01 vs. CTL.

 
Figure 2 shows the effect of various concentrations of ANG II on the expression of Gi{alpha}-2 (Fig. 2A) and Gi{alpha}-3 (Fig. 2B) proteins in A10 VSMCs. ANG II treatment for 1 h increased the levels of Gi{alpha}-2 and Gi{alpha}-3 in a concentration-dependent manner. The maximal increase of about 170% in Gi{alpha}-2 and Gi{alpha}-3 protein was observed at 10–5 and 10–7 M, respectively.


Figure 2
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  		Fig. 2. Effect of various concentrations of ANG II pretreatment on the levels of Gi{alpha}-2 and Gi{alpha}-3 proteins in A10 VSMCs. Top: A10 VSMCs were incubated in the absence (CTL) or presence of 10–9–10–5M ANG II for 1 h as described in MATERIALS AND METHODS. Membranes were prepared and used for immunoblotting. Membrane proteins (30 µg) were revolved by SDS-PAGE and transferred to nitrocellulose, which was then immunoblotted using L-5 antibody for Gi{alpha}-2 (A) or C-10 antibody for Gi{alpha}-3 (B) as described previously (21). Immunoblots are representative of 3 or 4 separate experiments. Bottom: quantification of protein bands by densitometric scanning. The results are expressed as percentage of CTL taken as 100%. Values are means ± SE of 3 or 4 separate experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. CTL.

 
Effect of ANG II on superoxide anion production in A10 VSMCs. ANG II has been shown to increase oxidative stress by activating NADPH oxidase (14). A role of ANG II in enhanced production of superoxide anion in VSMCs from SHR has been reported (21). To investigate whether ANG II treatment of A10 VSMCs also resulted in enhanced production of superoxide anion, the levels of superoxide anion (O2–) were measured by lucigenin in A10 VSMCs. The results shown in Fig. 3 show that treatment of cells with ANG II for 24 h increased the production of O2– by sixfold, which was significantly restored toward control levels by DPI, an inhibitor of NADPH oxidase.


Figure 3
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  		Fig. 3. Effect of diphenyleneiodonium (DPI) on superoxide anion (O2–) production in A10 VSMCs. A10 VSMCs were pretreated without (CTL) or with 10–6 M DPI for 24 h and challenged with 10–6 M ANG II for 1 h, and O2– production was determined as described in MATERIALS AND METHODS. Values are means ± SE of 3 separate experiments. ***P < 0.001 vs. CTL.

 
Effect of ANG II on the expression of NADPH oxidase subunits. To investigate whether ANG II-induced enhanced O2– production is attributed to its ability to increase the expression of different subunits of NADPH oxidase, we determined the effect of ANG II on the levels of Nox4 and p47phox in A10 VSMCs. The results shown in Fig. 4 indicate that ANG II (10–7 M) increased the levels of Nox4 (Fig. 4A) and p47phox (Fig. 4B) by about 75%, which were restored toward control levels by DPI.


Figure 4
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  		Fig. 4. Effect of DPI on the expression of Nox4 and p47phox protein expression in control and ANG II-treated A10 VSMCs. A10 VSMCs were pretreated without (CTL) or with 10–6 M DPI for 24 h and challenged with 10–7 M ANG II for 1 h. Membrane proteins (30 µg) were separated and transferred to nitrocellulose, which was then immunoblotted using specific antibodies against Nox4 (N-15, A) and p47phox (C-20, B) as described in MATERIALS AND METHODS. The beta-actin was used to assess the loading of the protein. Values are means ± SE of 3 separate experiments. ***P < 0.001 vs. CTL; ##P < 0.01 vs. ANG II-treated groups.

 
Effect of antioxidants on ANG II-induced increased expression of Gi{alpha} proteins. Since ANG II increases reactive oxygen species (ROS) production, it was of interest to investigate whether ANG II-induced increased ROS production in these cells contributes to the increased expression of Gi{alpha} proteins. To test this, we examined the effect of various antioxidants on ANG II-induced increased expression of Gi{alpha} proteins, and the results are shown in Fig. 5. Treatment of cells with ANG II (10–7 M) for 1 h increased the levels of Gi{alpha}-2 and Gi{alpha}-3 by about 50%, which were restored to control levels by apocynin (Fig. 5A); NADPH oxidase inhibitor, NAC (Fig. 5B); scavenger of O2–, {alpha}-tocopherol (Fig. 5C); and DPI (Fig. 5D), NADPH oxidase inhibitor, suggesting the implication of ROS in ANG II-induced enhanced expression of Gi{alpha} proteins in A10 VSMCs.


Figure 5
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  		Fig. 5. Effect of different antioxidants on the levels of Gi{alpha}-2 and Gi{alpha}-3 proteins in A10 VSMCs. A10 VSMCs were pretreated without (CTL) or with apocynin (10 µM) (A), N-acetyl-L-cysteine (NAC, 5 mM, B), a-tocopherol (10 µM, C), and DPI (1 µM, D) for 24 h and then challenged with 10–7 M ANG II for 1 h. Membrane proteins (30 µg) were separated and transferred to nitrocellulose, which was then immunoblotted with specific antibodies against Gi{alpha}-2 (left) and Gi{alpha}-3 (right) as described in MATERIALS AND METHODS. Values are means ± SE of 3 separate experiments. *P < 0.05 and **P < 0.01 vs. CTL; #P < 0.05 and ##P < 0.001 vs. ANG II-treated groups.

 
Implication of ROS in ANG II-induced enhanced ERK1/2 phosphorylation. We have previously shown that ANG II-induced enhanced expression of Gi{alpha} proteins was inhibited by mitogen extracellular signal-regulated kinase inhibitor PD-98059 in A10 VSMCs (11), suggesting the implication of MAPK signaling in ANG II-induced enhanced expression of Gi{alpha} proteins. To investigate whether ANG II could also increase the ERK1/2 phosphorylation in these cells and whether it is attributed to the increased oxidative stress, we examined the effect of DPI on ANG II-induced ERK1/2 phosphorylation in A10 cells. The results shown in Fig. 6 indicate that ANG II (10–7 M) increased the ERK1/2 phosphorylation by about 50%, which was completely abolished by DPI treatment.


Figure 6
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  		Fig. 6. Effect of DPI on ANG II-induced ERK1/2 phosphorylation (p-ERK1/2) in A10 VSMCs. A10 VSMCs were pretreated without (CTL) or with 10–6 M DPI for 24 h and challenged with 10–7 M ANG II for 1 h. Membrane proteins (30 µg) were separated and transferred to nitrocellulose, which was then immunoblotted with specific antibodies against phospho-specific-Tyr204-ERK1/2 (E-4) or ERK1/2 (C-14) as described in MATERIALS AND METHODS. ERK1/2 (C-14) was used to assess the loading of the protein. Data presented as means ± SE of 3 separated experiment. ***P < 0.001 vs. CTL.

 
Effect of antioxidants on receptor-independent function of Gi{alpha}. Since antioxidants restored the ANG II-induced enhanced levels of Gi{alpha}-2 and Gi{alpha}-3 proteins toward control levels, it was of interest to examine whether the restoration of the enhanced levels of Gi{alpha} proteins by antioxidants is also reflected in the restoration of increased Gi{alpha} functions. To investigate this, the effect of DPI on receptor-independent and -dependent functions was examined in ANG II-treated cells. For the receptor-independent functions of Gi, the effect of DPI was investigated on the inhibitory effect of GTP{gamma}S (10–12–10–8 M) on forskolin (FSK)-stimulated adenylyl cyclase activity in control and ANG II-treated A10 cells. The results shown in Fig. 7 indicate that the enhanced inhibition of FSK-stimulated adenylyl cyclase activity by different concentrations of GTP{gamma}S in ANG II-treated cells which was attributed to the enhanced levels of Gi{alpha} proteins compared with untreated control cells was reversed to control levels by DPI.


Figure 7
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  		Fig. 7. Effect of DPI on 5'-O-(3-triotriphosphate) (GTP{gamma}S)-mediated inhibition of forskolin (FSK)-stimulated adenylyl cyclase activity in control and ANG II-treated A10 VSMCs. A10 VSMCs were pretreated without (CTL) or with 10–6 M DPI for 24 h and challenged with 10–7 M ANG II for 1 h. Membranes were prepared as described in MATERIALS AND METHODS, and adenylyl cyclase activity from these membranes was determined in the presence of 50 µM FSK alone, taken as 100%, and in the presence of various concentrations of GTP{gamma}S (10–12–10–7 M). Values are means ± SE of 3 separate experiments. *P < 0.05 and **P < 0.01 vs. CTL; §P < 0.05 and §§P < 0.01 vs. ANG II-treated group.

 
Effect of antioxidants on receptor-dependent functions. To investigate the effect of antioxidant on the receptor-dependent functions of Gi{alpha} proteins, the effect of DPI was examined on the hormonal inhibitions of adenylyl cyclase activity in control and ANG II-treated A10 cells. For this, the effect of DPI on ANG II, des(Glu18,Ser19,Glu20,Leu21,Gly22)atrial natriuretic peptide4-23-NH2 (C-ANP4-23), and oxotremorine that inhibit adenylyl cyclase through Gi{alpha} proteins (2, 4, 5, 9, 23) was examined on adenylyl cyclase activity in control and ANG II-treated cells. Figure 8 shows that ANG II-, C-ANP4-23-, and oxotremorine-mediated inhibitions of adenylyl cyclase which were significantly augmented in ANG II-treated cells were restored to control levels by DPI treatment. Figure 9 shows the effect of DPI on concentration-dependent inhibition of adenylyl cyclase by C-ANP4-23. C-ANP4-23 inhibited adenylyl cyclase activity in a concentration-dependent manner, with an apparent Ki of about 0.5 nM. The maximal inhibition observed was about 25%. However, as reported earlier (25), the inhibition was greater in ANG II-treated cells compared with untreated control cells. For example, C-ANP4-23 at 10–6 M inhibited adenylyl cyclase by about 25% in control cells and about 45% in ANG II-treated cells. DPI, on the other hand, completely abolished the C-ANP4-23-mediated enhanced inhibition of adenylyl cyclase activity in ANG-II treated cells.


Figure 8
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  		Fig. 8. Effect of DPI on hormonal inhibition of adenylyl cyclase activity in CTL and ANG II-treated A10 VSMCs. A10 VSMCs were pretreated without (CTL) or with 10–6 M DPI for 24 h and challenged with 10–7 M ANG II for 1 h. Membranes were prepared as described in MATERIALS AND METHODS. The adenylyl cyclase activity in these membranes was determined in the 10 µM GTP{gamma}S alone, taken as 100% (basal) or in combination with 10–5 M ANG II, 10–7 M des(Glu18,Ser19,Glu20,Leu21,Gly22)atrial natriuretic peptide4-23-NH2 (C-ANP4-23), or 5 µM oxotremorine (Oxo). Values are means ± SE of 3 separate experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. CTL.

 

Figure 9
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  		Fig. 9. Effect of DPI on C-ANP4-23-mediated inhibition of adenylyl cyclase activity in CTL and ANG II-treated A10 VSMCs. A10 VSMCs were pretreated without (CTL) or with 10–6 M DPI for 24 h and challenged with 10–7 M ANG II for 1 h. Membranes were prepared as described in MATERIALS AND METHODS, and adenylyl cyclase activity in these membranes was determined in the presence 10 µM GTP{gamma}S alone, taken as 100% (basal), and in the presence of various concentrations of C-ANP4-23 (10–12–10–6 M). Values are means ± SE of 3 separate experiments. **P < 0.01 vs. CTL; §§§P < 0.001 vs. ANG II-treated group.

 
Effect of antioxidants on Gs{alpha} -mediated stimulation of adenylyl cyclase activity. The interaction between Gi{alpha} and Gs{alpha} has been well established. We have previously shown that treatment of A10 VSMCs with ANG II for 24 h attenuated the Gs{alpha}-mediated functions (25). Therefore, it was of interest to examine whether DPI could also reverse the ANG II-mediated Gs{alpha} effects. For this, the effect DPI on concentration-dependent stimulation of adenylyl cyclase by GTP{gamma}S was investigated in ANG II-treated cells, and the results are shown in Fig. 10. GTP{gamma}S stimulated adenylyl cyclase activity in a concentration-dependent manner in control and ANG II-treated A10 VSMCs; however, as reported earlier (25), the extent of stimulation was significantly decreased in ANG II-treated VSMCs compared with untreated control cells. For example, at 10–4 M, GTP{gamma}S augmented the adenylyl cyclase activity by fourfold in control cells compared with twofold in ANG II-treated A10 cells, and the treatment of ANG II-treated cells with 10 µM DPI restored the decreased stimulation of adenylyl cyclase to control levels.


Figure 10
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  		Fig. 10. Effect of DPI on GTP{gamma}S-mediated stimulation of adenylyl cyclase activity in CTL and ANG II-treated A10 VSMCs. A10 VSMCs were pretreated without (CTL) or with 10–6 M DPI for 24 h and challenged with 10–7 M ANG II for 1 h. Membranes were prepared as described in MATERIALS AND METHODS, and adenylyl cyclase activity in these membranes was determined in the absence (taken as 100%) and in the presence of various concentrations of GTP{gamma}S (10–8–10–4 M). Values are means ± SE of 3 separate experiments. **P < 0.01 and ***P < 0.001 vs. CTL; §§P < 0.01 and §§§P < 0.001 vs. ANG II-treated group.

 
Effect of antioxidants on hormonal stimulation of adenylyl cyclase activity. To investigate whether antioxidant could also modulate ANG II-induced decreased stimulation of adenylyl cyclase by stimulatory hormones (25), the effect of DPI on isoproterenol and glucagon-stimulated adenylyl cyclase activity was examined in ANG II-treated A10 cells. As shown in Fig. 11, both isoproterenol and glucagon stimulated adenylyl cyclase activity to various degrees in A10 VSMCs; however, as reported earlier (25), the extent of stimulation was significantly decreased by about 30% and 40%, respectively, in ANG II-treated cells compared with control cells, and this decrease was restored to control levels by DPI treatment. In addition, the decreased stimulation of adenylyl cyclase by sodium fluoride (~30%) and FSK (~60%) that stimulate the enzyme activity by a receptor-independent mechanism in ANG II-treated cells was also restored to control levels by DPI treatment.


Figure 11
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  		Fig. 11. Effect of DPI on hormonal stimulation of adenylyl cyclase activity in CTL and ANG II-treated A10 VSMCs. A10 VSMCs were pretreated without (CTL) or with 10–6 M DPI for 24 h and challenged with 10–7 M ANG II for 1 h. Membranes were prepared as described in MATERIALS AND METHODS, and adenylyl cyclase activity in these membranes was determined in the presence of adenosine deaminase and 10 µM GTP alone (basal) or in combination with 1 µM glucagon, 50 µM isoproterenol (Iso), 10 µM 5'(N-ethylcarboxamido)adenosine, or 100 µM FSK. Values are means ± SE of 3 separate experiments. *P < 0.05 and ***P < 0.001 vs. CTL.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We have previously reported that SHR and 1K1C hypertensive rats, which have high levels of ANG II, exhibited enhanced expression of Gi{alpha}-2 and Gi{alpha}-3 protein mRNA in the heart and aorta, whereas the levels of Gs{alpha} were unaltered (3, 13). The enhanced expression of Gi{alpha}-2 and Gi{alpha}-3 were restored toward control levels by captopril treatment (13, 26), suggesting that ANG II contributes to the augmented levels of Gi{alpha} proteins in SHR and 1K1C [PDB] hypertensive rats. This was further supported by our recent studies demonstrating that ANG II treatment of A10 VSMCs resulted in enhanced expression of Gi{alpha} at the protein and mRNA levels which was restored to control levels by losartan (1). In addition, the ANG II-evoked increased expression of Gi{alpha}-2 and Gi{alpha}-3 was inhibited by actinomycin treatment, suggesting that the ANG II-mediated increases in Gi proteins are at the transcriptional level (25).

In the present studies, we report that A10 VSMCs exposed to ANG II exhibit enhanced production of superoxide anion which contributes to the enhanced expression of Gi{alpha} protein in these cells. In this regard, ANG II-induced enhanced oxidative stress has also been reported earlier in rat VSMCs (3) and human internal mammary arteries (7). In addition, we have also shown recently the implication of ANG II in enhanced production of O2– in aortic VSMCs from SHR (6). ANG II-evoked increased formation of O2– is attributed to the activation of NADPH oxidase, which is a membrane-bound enzyme and is composed of multiple subunits such as p22phox, gp91phox/Nox1/Nox4, p47phox, p67phox, and p40phox (36). Our results showing that ANG II increased the expression of Nox4 and p47phox in A10 VSMCs, which was restored to the control levels by DPI, suggest that ANG II-induced increased formation of O2– may be attributed to the increased levels of Nox 4 and p47phox, critical subunits involved in NADPH oxidase activation. Our results are in accordance with the studies of other investigators who have also shown that ANG II increased the expression of p47phox and p22phox in human mesenteric VSMCs (37).

Although a role of oxidative stress in ANG II-mediated cell signaling has been well established (37), evidence for a direct role of oxidative stress in ANG II-mediated increased expression of Gi{alpha} protein and associated adenylyl cyclase is lacking. Our results showing that antioxidants such as apocynin, {alpha}-tocopherol, NAC, and DPI restored ANG II-evoked enhanced expression of Gi{alpha}-2 and Gi{alpha}-3 to control levels suggest the implication of oxidative stress in ANG II-induced enhanced levels of Gi{alpha} proteins in smooth muscle cells. In support of this are our earlier findings showing that enhanced oxidative stress caused by enhanced levels of ANG II contributes to the enhanced expression of Gi{alpha} proteins in SHR (21). We have also shown that antioxidants that result in the restoration of ANG II-induced enhanced expression of Gi{alpha} proteins to control levels also restored to the control levels the enhanced inhibition of adenylyl cyclase by ANG II, C-ANP4-23, and oxotremorine as well as GTP{gamma}S-mediated enhanced inhibition of FSK-stimulated adenylyl cyclase activity, the receptor-dependent and -independent functions of Gi{alpha} proteins, respectively.

In addition, ANG II-induced diminished stimulation of adenylyl cyclase by GTP{gamma}S and stimulatory hormones such as isoproterenol and glucagon were also restored to control levels by DPI. This may be attributed to the Gi{alpha} proteins and not to Gs{alpha} proteins, because ANG II was unable to alter the levels of Gs{alpha} proteins in these cells. This is further substantiated by our studies showing that restoration of the enhanced levels of Gi{alpha} proteins to control levels by antioxidant treatments also restored the GTP{gamma}S-mediated diminished stimulation of adenylyl cyclase to control levels. In addition, ANG II-evoked enhanced levels of Gi{alpha} proteins may also be responsible for the diminished stimulation of adenylyl cyclase by glucagon, isoproterenol, FSK, and sodium fluoride in ANG II-treated cells, because the restoration of the ANG II-induced enhanced levels of Gi{alpha} proteins to control levels was also able to restore the ability of isoproterenol, glucagon, sodium fluoride, and FSK to stimulate adenylyl cyclase activity toward control levels. Our results are consistent with our studies performed in SHR, showing that the restoration of enhanced levels of Gi{alpha} proteins by antioxidants also resulted in the restoration of enhanced Gi functions and diminished Gs-mediated functions to control levels (21). In addition, the restoration of enhanced levels of Gi{alpha} proteins toward control levels by captopril (13, 26) or losartan has also been reported to restore the enhanced Gi{alpha} functions to control levels (1, 14, 16).

ANG II has been shown to activate various signaling pathways including MAPK (24, 28, 38). We have shown previously the implication of MAPK signaling in ANG II-induced enhanced expression of Gi{alpha} proteins in A10 VSMCs (11). We also reported that the enhanced expression of ERK1/2 phosphorylation in VSMCs from SHR was attenuated to control levels by antioxidants, suggesting a role of oxidative stress in enhanced ERK1/2 phosphorylation (21). Thus it can be suggested that oxidative stress by increasing MAPK signaling may be responsible for the enhanced expression of Gi{alpha} proteins. This notion is supported by our findings showing that ANG II-induced increased ERK1/2 phosphorylation is also attenuated by DPI in A10 VSMCs. Taken together, it may be suggested that ANG II-induced enhanced oxidative stress caused by O2– production may be responsible for the activation of MAPK signaling which then results in the enhanced expression of Gi{alpha} proteins in VSMCs.

In conclusion, we have provided the first evidence that ANG II-induced increased oxidative stress through increased MAPK activity may be responsible for the enhanced expression of Gi{alpha} proteins in A10 VSMCs. The increased expression of Gi{alpha} proteins that results in decreased levels of cAMP and thereby increased vascular resistance may be one of the contributing factors in the pathogenesis of hypertension. Thus it may be suggested that antioxidant treatment by decreasing oxidative stress, and thereby MAPK activity, results in the attenuation of ANG II-induced enhanced levels of Gi{alpha} proteins and increased levels of cAMP, which by decreasing vascular resistance may attenuate the development of high blood pressure.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This study was supported by Canadian Institutes of Health Research Grant MOP-53074.


   ACKNOWLEDGMENTS
 
We thank Christiane Laurier for valuable secretarial help.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. B. Anand-Srivastava, Dept. of Physiology, Faculty of Medicine, Univ. of Montreal, C. P. 6128, Succ. Centre-ville, Montréal, Québec, H3C 3J7, Canada (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
 

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Y. Li, M. Descorbeth, and M. B. Anand-Srivastava
Role of oxidative stress in high glucose-induced decreased expression of Gi{alpha} proteins and adenylyl cyclase signaling in vascular smooth muscle cells
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