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Am J Physiol Heart Circ Physiol 294: H775-H784, 2008. First published November 30, 2007; doi:10.1152/ajpheart.00841.2007
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Peroxynitrite inhibits the expression of Gi{alpha} protein and adenylyl cyclase signaling in vascular smooth muscle cells

Marcel Bassil, Yuan Li, and Madhu B. Anand-Srivastava

Department of Physiology, Faculty of Medicine, and Groupe de Recherche sur le Système Nerveux Autonome, University of Montreal, Montreal, Quebec, Canada

Submitted 18 July 2007 ; accepted in final form 20 November 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We previously showed that S-nitroso-N-acetylpenicillamine, a nitric oxide donor, decreased the levels and functions of Gi{alpha} proteins by formation of peroxynitrite (ONOO–) in vascular smooth muscle cells (VSMC). The present studies were undertaken to investigate whether ONOO– can modulate the expression of Gi{alpha} protein and associated adenylyl cyclase signaling in VSMC. Treatment of A-10 and aortic VSMC with ONOO– for 24 h decreased the expression of Gi{alpha}-2 and Gi{alpha}-3, but not Gs{alpha}, protein in a concentration-dependent manner; expression was restored toward control levels by 111Mn-tetralis(benzoic acid porphyrin) and uric acid, but not by 1H[1,2,4]oxadiazole[4,3-a]quinoxaline-1-one (ODQ) and KT-5823. cGMP levels were increased by ~50% and 150% by 0.1 and 0.5 mM ONOO–, respectively, and attenuated toward control levels by ODQ. In addition, 0.5 mM ONOO– attenuated the inhibition of adenylyl cyclase by ANG II and C-type atrial natriuretic peptide (C-ANP4–23), as well as the inhibition of forskolin-stimulated adenylyl cyclase activity by GTP{gamma}S, whereas, the Gs-mediated stimulations were augmented. In addition, 0.5 mM ONOO– decreased phosphorylation of ERK1/2 and p38 MAP kinase and enhanced JNK phosphorylation but did not affect AKT1/3 phosphorylation. These results suggest that ONOO– decreased the expression of Gi proteins and associated functions in VSMC through a cGMP-independent mechanism and may involve the MAP kinase signaling pathway.

G protein; mitogen-activated protein kinase

GUANINE NUCLEOTIDE REGULATORY (G) proteins, a family of GTP-binding proteins, play an important role in the regulation of a variety of signal transduction systems, including the adenylyl cyclase-cAMP system. The hormone-sensitive adenylyl cyclase system consists of receptor, catalytic subunit, and G proteins. The stimulatory and inhibitory responses of the hormones to adenylyl cyclase are mediated by stimulatory (Gs) and inhibitory (Gi) G proteins, respectively (46). G proteins exist as heterotrimers of {alpha}-, β-, and {gamma}-subunits. Molecular cloning has revealed four different isoforms of Gs{alpha}, resulting from differential splicing of one gene (45), and three distinct isoforms of Gi{alpha} (Gi{alpha}-1, Gi{alpha}-2, and Gi{alpha}-3), encoded by three different genes (32). All three forms of Gi{alpha} are implicated in the regulation of adenylyl cyclase inhibition (32) and the activation of atrial K+ channels (58).

Gi protein and associated adenylyl cyclase signaling has been shown to be implicated in a variety of cellular functions, including vascular smooth muscle tone (58), cell proliferation (27), salt and water transport (18, 35), and catecholamine release (54), all of which play a key role in the regulation of blood pressure. Alterations in Gi protein and cAMP levels that result in the impaired cellular functions lead to various pathological states, including hypertension. Gi protein and Gi mRNA levels have been shown to be upregulated in hearts and aortas from spontaneously hypertensive rats and various experimental models of hypertensive rats, including N{omega}-nitro-L-arginine methyl ester-hypertensive rats (3, 5, 16, 23, 53). Increased expression of Gi{alpha} proteins in hypertensive rat models may be attributed to the augmented levels of vasoactive peptides. We previously showed that ANG II, endothelin-1, and arginine vaspressin increased the levels of Gi{alpha} proteins in vascular smooth muscle cells (VSMC), whereas atrial natriuretric peptides (ANP) and nitric oxide (NO), which increase cGMP levels, decreased Gi{alpha} protein expression in the cells (9, 10, 13, 14, 48).

Peroxynitrite (ONOO–), which has been shown to be a powerful oxidant, exerts a prolonged vasorelaxant action under physiological conditions (11, 17). ONOO– produces dilatation in several vascular beds, including coronary (37), mesenteric (12), and cerebral (57). The relaxant action of ONOO– has been shown to have many characteristics in common with NO (44). The relaxant effect of ONOO– is attributed to the nitrosylation of tissue thiols, nitration of tyrosine, or inhibition of Ca2+-activated K+ channels (38) and activation of ATP-sensitive K+ channels in cat cerebral arteries (57). Increasing evidence suggests that physiological levels of ONOO– may act as a regulator of several physiological functions (19, 29).

We recently showed that NO decreases Gi{alpha} protein expression in VSMC, which was restored to control levels by 111Mn-tetralis(benzoic acid porphyrin) (MnTBAP) and uric acid, and suggested that intracellular ONOO–, formed by the interaction of NO· and O2–, may be responsible for NO·-mediated decrease of Gi{alpha} protein expression in VSMC. The present studies were undertaken to investigate whether ONOO– can also modulate expression of Gi{alpha} proteins and adenylyl cyclase signaling in VSMC.

We have provided the first evidence that treatment of VSMC with ONOO– decreased the expression of Gi{alpha} proteins by a cGMP-independent mechanism. The decreased levels of Gi{alpha} and resultant increases in cAMP levels by ONOO– may be one of the mechanisms through which ONOO– exhibits the vasorelaxant effect.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials

C-type ANP (C-ANP4–23) was obtained from Peninsula Laboratories (Belmont, CA); T-12 and C-10, antibodies directed against specific COOH-terminal sequences of Gi{alpha}-2 and Gi{alpha}-3, respectively, were from Santa Cruz Biotechnology (Santa Cruz, CA); other antibodies and 1H[1,2,4]oxadiazole [4,3-a]quinoxaline-1-one (ODQ) were from Sigma (St. Louis, MO); KT-5823 and ONOO– from Calbiochem (La Jolla, CA); and all other chemicals were from commercial sources, as described previously (40, 42).

Methods

Cell culture and incubation. VSMC from rat aorta were cultured as described previously (6). A-10 cells, from embryonic thoracic aorta of rats (American Type Culture Collection, Rockville, MD), were plated in 75-cm2 flasks and incubated at 37°C in a 95% air-5% CO2 humidified atmosphere in DMEM (with glucose, L-glutamine, and sodium bicarbonate) containing 1% antibiotics and 10% heat-inactivated FCS. The cells were passaged on reaching confluence with 0.5% trypsin containing 0.2% EDTA and used between passages 5 and 15, as described previously (6, 47). Confluent cell cultures were starved by incubation for 3 h in DMEM without FCS at 37°C to reduce the interference by growth factors in the serum. These cells were then incubated with 0.5 mM OONO– (or as otherwise indicated) for 24 h at 37°C. After incubation, the cells were washed twice with ice-cold homogenization buffer [10 mmol/l Tris·HCl (pH 7.5) containing 1 mmol/l EDTA] and homogenized with 10 strokes in a Dounce homogenizer (Wheaton, Millville, NJ). The homogenate was used for adenylyl cyclase assay and immunoblotting. Cell viability was checked with the trypan blue exclusion technique.

Determination of adenylyl cyclase activity. Adenylyl cyclase activity was determined by measurement of [32P]cAMP formation from [{alpha}-32P]ATP, as described previously (40, 42). Typical assay medium contained 50 mmol/l glycylglycine (pH 7.5), 0.5 mmol/l MgATP, [{alpha}-32P]ATP (1.5 x 106 cpm), 5 mmol/l MgCl2 (in excess of ATP concentration), 100 mmol/l NaCl, 0.5 mmol/l cAMP, 1 mmol/l IBMX, 0.1 mmol/l EGTA, 10 µmol/l GTP{gamma}S, and an ATP-regenerating system consisting of 2 mmol/l creatine phosphate, 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 conducted in triplicate for 10 min at 37°C were terminated by the addition of 0.6 ml of 120 mmol/l zinc acetate containing 0.5 mmol/l unlabeled cAMP. cAMP was purified by coprecipitation of other nucleotides with ZnCO3 by addition of 0.5 ml of 144 mmol/l Na2CO3 and subsequent chromatography by the double-column system, as described previously (52). Protein concentration was determined by the method of Lowry et al. (39), with BSA as standard. All the animal procedures used in the present studies were approved by the Comité de Déontoligie de l'Experimentation sur les Animaux of the University of Montreal (No. 99050).

Western blotting. Western blotting of Gi protein and natriuretic peptide receptor type C (NPR-C)- and type 1 angiotensin receptor (AT1R)-containing proteins was performed as described previously (10, 40, 42). After separation by SDS-PAGE, the proteins were electrophoretically transferred to a nitrocellulose membrane (Schleicher and Schuell) with a semidry transblot apparatus (Bio-Rad) at 15 V for 45 min. The proteins on the membrane were stained with Ponceau S to confirm that an equivalent amount of protein was loaded into each well. The membranes were then blocked with 5% BSA, washed twice in PBS, and incubated in PBS containing 2% BSA overnight. The blots were then incubated in PBS containing 0.05% Tween 20 at 4°C overnight with antibodies as follows: T-12 against Gi{alpha}-1 and Gi{alpha}-2, C-10 against Gi{alpha}-3, K-20 against Gs{alpha}, N-20 against NPR-C, C-20 against p38 MAP kinase, D-8 against phosphorylated p38 MAP kinase, E-4 against phosphorylated ERK1/2, C-14 against ERK1/2, F-3 against JNK kinase, G-7 against phosphorylated JNK, H-136 against AKT1/2, Ser473 against phosphorylated AKT1/2/3, 74-1 against dynein, and β-actin antibody against β-actin. The antibody-antigen complexes were detected by incubation of the membranes with goat anti-rabbit IgG (Bio-Rad) conjugated with horseradish peroxidase in PBS containing 5% dehydrated milk and 0.05% Tween for 1 h at room temperature. The blots were washed three times with PBS before reaction with enhanced chemiluminescence Western blotting detection reagents (Amersham). The blots were quantified by densitometric scanning using an enhanced laser densitometer (Alpha Innotech) and gel scan evaluation software (AlphaImager version 5.5, Alpha Innotech). One-dimensional scanning covered the entire area of protein bands in autoradiograms.

Determination of intracellular cGMP levels. Intracellular cGMP levels were determined as described previously (34). The cells were incubated with 1 mM IBMX for 15 min and then with 20 µM ODQ for 30 min at 37°C, stimulated in the absence or presence of ONOO– for 30 min, and washed twice with PBS. The reaction was terminated by rapid aspiration and addition of ice-cold 1 M HCl. cGMP levels were determined by RIA using an RIA kit (Biomedical Technologies, Stoughton, MA).

Statistical Analysis

Values are means ± SE. Comparisons between groups were made using ANOVA in conjunction with the Newman-Keuls multiple comparison tests. Differences between groups were considered statistically significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of ONOO– on G Protein Levels in VSMC

We previously showed that treatment of VSMC with S-nitroso-N-acetylpenicillamine (SNAP) for 24 h decreased the expression of Gi{alpha} protein; this response was attributed to the formation of intracellular ONOO–. We further investigated whether treatment of VSMC with ONOO– for 24 h could also decrease expression of Gi{alpha} proteins. As shown in Fig. 1, T-12 antibody recognized a single 40-kDa protein, referred to as Gi{alpha}-2 (Gi{alpha}-1 is absent in aorta) (33), that was significantly inhibited by ONOO– in A-10 cells. About 20% inhibition was observed at 0.1 mM ONOO– and ~30% inhibition was observed at 0.5 mM, as determined by densitometric scanning. Similarly, C-10 antibody recognized a single 41-kDa protein, referred to as Gi{alpha}-3; however, the relative amount of immunodetectable Gi{alpha}-3 as determined by densitometric scanning was also decreased by ONOO–. The maximal inhibition of ~35% was observed at 0.5 mM. In addition, ONOO– also decreased Gi{alpha}-2 and Gi{alpha}-3 protein in aortic VSMC by ~35% and 30%, respectively (data not shown). On the other hand, K-20 antibody recognized three isoforms of Gs{alpha}, Gs{alpha}45, Gs{alpha}47, and Gs{alpha}52, in control and treated A-10 and aortic VSMC; however, no significant difference in the relative amounts of immunodetectable Gs{alpha}45, Gs{alpha}47, and Gs{alpha}52 was observed in either group (data not shown). The decreased expression of Gi{alpha}-2 and Gi{alpha}-3 by ONOO– was restored toward control levels by MnTBAP and uric acid, scavengers of ONOO– (Fig. 2).


Figure 1
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  		Fig. 1. Effect of peroxynitrite (ONOO–) on Gi{alpha}-2 and Gi{alpha}-3 levels in A-10 vascular smooth muscle cells (VSMC). A-10 cells were incubated in the absence or presence of ONOO– for 24 h. Membranes were prepared and used for immunoblotting. Membrane proteins (20 or 30 µg) from control and treated cells were resolved by SDS-PAGE and transferred to nitrocellulose, which was then immunoblotted using T-12 antibody for Gi{alpha}-2, C-10 antibody for Gi{alpha}-3, and dynein antibody as a loading control. Blots are representative of 3 or 4 separate experiments. Graphs show quantification of protein bands by densitometric scanning. Results are expressed as percentage of control, taken as 100%. Values are means ± SE of 3 or 4 separate experiments. **P < 0.01; ***P < 0.001.

 

Figure 2
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  		Fig. 2. Effect of 111Mn-tetralis(benzoic acid porphyrin) (MnTBAP) and uric acid on ONOO–-induced decreased expression of Gi{alpha} proteins in VSMC. Aortic VSMC were incubated in the absence or presence of 50 µM MnTBAP or 100 µM uric acid for 30 min before treatment with 0.5 mM ONOO– for 24 h. Membranes were prepared and used for immunoblotting. Membrane proteins (20 or 30 µg) from control and treated cells were resolved by SDS-PAGE and transferred to nitrocellulose, which was then immunoblotted using T-12 antibody for Gi{alpha}-2 and C-10 antibody for Gi{alpha}-3. Blots are representative of 3 or 4 separate experiments. Graphs show quantification of protein bands by densitometric scanning. Results are expressed as described in Fig. 1 legend. Values are means ± SE of 3 or 4 separate experiments. *P < 0.05; **P < 0.01.

 
Effect of ONOO– on cGMP levels in VSMC

We previously showed that NO increased cGMP levels in aortic VSMC (10). We further investigated whether ONOO– could also increase cGMP levels in these cells. ONOO– increased the levels of cGMP by ~50% at 0.1 mM and ~150% at 0.5 mM (Fig. 3). ODQ, an inhibitor of soluble guanylyl cyclase, at 20 µM restored completely to control levels the increased levels of cGMP induced by 0.1 mM ONOO–, whereas the increased levels of cGMP evoked by 0.5 mM ONOO– were restored toward control levels by ~50%.


Figure 3
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  		Fig. 3. Effect of ONOO– on cGMP levels in A-10 VSMC. A-10 VSMC were preincubated with 1 mM IBMX for 15 min and further incubated with 20 µM 1H[1,2,4]oxadiazole[4,3-a]quinoxaline-1-one (ODQ) for 30 min at 37°C. Cells were then stimulated with or without 0.5 mM ONOO– for 30 min. cGMP levels were determined by RIA. Basal cGMP levels in control cells were 461.5 ± 35.9 pmol cGMP/mg protein. Values are means ± SE of 3 separate experiments performed in duplicate. *P < 0.05; **P < 0.01; ***P < 0.001.

 
Effect of ODQ and KT-5823 on ONOO–-Induced Decrease of Gi{alpha} Protein Expression

We previously showed that cGMP decreased Gi{alpha} protein expression in VSMC (9). Since ONOO– increases cGMP levels in these cells, it was of interest to examine whether the ONOO–-induced decrease of Gi{alpha} protein expression is attributed to increased cGMP levels. To investigate this, the effect of ODQ was studied on Gi{alpha} protein expression in control and ONOO–-treated A-10 VSMC. ODQ was unable to restore the ONOO–-induced decrease in Gi{alpha}-2, as well as Gi{alpha}-3, protein levels in these cells (Fig. 4), suggesting that ONOO– decreased Gi{alpha} protein levels by a cGMP-independent mechanism.


Figure 4
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  		Fig. 4. Effect of ODQ on ONOO–-induced decrease of Gi{alpha}-2 and Gi{alpha}-3 protein expression in A-10 VSMC. A-10 VSMC were incubated in the absence or presence of 0.5 mM ONOO– for 24 h. ODQ (20 µM) was added 30 min before the treatment and was present throughout treatment. Membranes were prepared and used for immunoblotting. Membrane proteins (20 or 30 µg) from control and treated cells were resolved by SDS-PAGE and transferred to nitrocellulose, which was then immunoblotted using T-12 antibody for Gi{alpha}-2 and C-10 antibody for Gi{alpha}-3. Blots are representative of 4 or 5 separate experiments. Graphs show quantification of protein bands by densitometric scanning. Results are expressed as described in Fig. 1 legend. Values are means ± SE of 4 or 5 separate experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

 
To further confirm our findings, we tested the effect of a protein kinase G inhibitor, KT-5823, on the ONOO–-induced decrease of Gi{alpha} protein expression in A-10 VSMC. Similar to ODQ, KT-5823 was unable to reverse the ONOO–-induced decrease of Gi{alpha}-2 and Gi{alpha}-3 protein expression in A-10 VSMC (Fig. 5). These data strongly suggest that the ONOO–-induced decrease of Gi{alpha} protein expression occurs through a cGMP/PKG-independent mechanism.


Figure 5
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  		Fig. 5. Effect of KT-5823 on ONOO–-induced decrease of Gi{alpha}-2 and Gi{alpha}-3 protein expression in A-10 VSMC. A-10 VSMC were incubated in the absence or presence of 0.5 mM ONOO– for 24 h. KT-5823 (1 µM) was added 30 min before treatment and was present throughout treatment. Membranes were prepared and used for immunoblotting. Membrane proteins (20 or 30 µg) from control and treated cells were resolved by SDS-PAGE and transferred to nitrocellulose, which was then immunoblotted using T-12 antibody for Gi{alpha}-2 and C-10 antibody for Gi{alpha}-3. Blots are representative of 4 or 5 separate experiments. Graphs show quantification of protein bands by densitometric scanning. Results are expressed as described in Fig. 1 legend. Values are means ± SE of 4 or 5 separate experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

 
Effect of ONOO– on ERK1/2, p38 MAP Kinase, JNK, and AKT Signaling

Our previous findings imply that MAP kinases and phosphatidylinositol 3-kinase (PI3-kinase) pathways enhance ANG II-induced Gi protein expression in A-10 VSMC (21). Since NO has also been shown to modulate the activity of MAP kinases (34, 51), we examined whether ONOO– also could alter MAP kinase and PI3-kinase signaling in aortic VSMC. We investigated the effects of ONOO– on the phosphorylation of ERK1/2, p38 MAP kinase, JNK, and AKT. ONOO– decreased phosphorylation of ERK1/2 and p38 MAP kinase by ~30 and 35%, respectively, enhanced JNK phosphorylation by ~65%, and did not alter AKT phosphorylation (Fig. 6).


Figure 6
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  		Fig. 6. Effect of ONOO– on MAP kinases and phosphatidylinositol 3-kinase in VSMC. Aortic VSMC were incubated in the absence or presence of 0.5 mM ONOO–. Membranes were prepared and used for immunoblotting. Membrane proteins (20 or 30 µg) from control and treated cells were resolved by SDS-PAGE and transferred to nitrocellulose, which was then immunoblotted using specific antibodies for phosphorylated and total ERK1/2, phosphorylated and total p38, phosphorylated and total JNK, and phosphorylated and total AKT. Blots are representative of 3 or 4 separate experiments. Graphs show quantification of protein bands by densitometric scanning. Results are expressed as described in Fig. 1 legend. Values are means ± SE of 3 or 4 separate experiments. **P < 0.01; ***P < 0.001.

 
Effect of ONOO– on Gi Functions

To investigate whether the ONOO–-induced decrease of Gi{alpha} levels was also reflected in decreased Gi functions, the effects of ONOO– on receptor-independent and -dependent functions were examined in aortic VSMC. The receptor-independent functions of Gi{alpha} were investigated by studying the effect of low concentrations of GTP{gamma}S on FSK-stimulated adenylyl cyclase activity. GTP{gamma}S inhibited FSK-stimulated adenylyl cyclase activity in a concentration-dependent manner in control cells (Fig. 7); however, the extent of inhibition was significantly attenuated in ONOO–-treated cells.


Figure 7
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  		Fig. 7. Effect of GTP{gamma}S on forskolin (FSK)-stimulated adenylyl cyclase activity in control and ONOO–-treated VSMC. VSMC were incubated in the absence or presence of 0.5 mM ONOO– for 24 h. Membranes were prepared, and adenylyl cyclase activity was determined in the absence or presence of 100 µM FSK alone or in combination with various concentrations of GTP{gamma}S. Basal enzyme activities in the absence and presence of FSK were 39.15 ± 4.68 and 2,113.82 ± 230.53 pmol cAMP·mg protein–1·10 min–1, respectively, in control cells and 26.11 ± 3.3 and 1,976.77 ± 206.87 pmol cAMP·mg protein–1·10 min–1, respectively, in ONOO–-treated cells. Values are means ± SE of 3 separate experiments. **P < 0.01.

 
The receptor-dependent functions of Gi{alpha} proteins were examined by studying the effect of ONOO– on hormonal inhibition of adenylyl cyclase. C-ANP4–23 and ANG II inhibited adenylyl cyclase activity in VSMC by ~24% and 34%, respectively, in control cells (Fig. 8). However, ONOO– completely attenuated the C-ANP4–23- and ANG II-mediated inhibition of adenylyl cyclase activity in VSMC.


Figure 8
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  		Fig. 8. Effect of ONOO– on receptor-dependent functions of Gi proteins in VSMC. VSMC were incubated in the absence or presence of 0.5 mM ONOO– for 24 h. Membranes were prepared, and adenylyl cyclase activity was determined in the presence of 10 µM GTP{gamma}S alone or in combination with 10–7 M C-type atrial natriuretic peptide (C-ANP4–23) or 10–5 M ANG II. Adenylyl cyclase activity in the presence of 10 µM GTP{gamma}S was 1,064.03 ± 107.26 and 1,094.14 ± 112.13 pmol cAMP·mg protein–1·10 min–1 in control and ONOO–-treated cells, respectively. Values are means ± SE of 3 separate experiments performed in triplicate. *P < 0.05; **P < 0.01.

 
Effect of ONOO– on NPR-C and AT1 Receptor Expression

To investigate whether receptor downregulation also contributes to the complete attenuation of C-ANP4–23- and ANG II-mediated inhibition of adenylyl cyclase in ONOO–-treated cells, the effect of ONOO– on NPR-C and AT1 receptor expression was examined by an immunoblotting technique using specific antibody against NPR-C and AT1 receptor in control and treated cells. NPR-C and AT-1 receptor antibody recognized 66-kDa NPR-C and 60-kDa AT-1, respectively, in control and treated cells (Fig. 9); however, densitometric scanning showed that ONOO– significantly decreased NPR-C levels by ~40% but did not affect AT1 receptor levels.


Figure 9
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  		Fig. 9. Effect of ONOO– on NPR-C and AT1 receptor (AT1R) protein expression in VSMC. VSMC were treated with 0.5 mM ONOO– for 24 h. Membranes (50 µg) from control and treated cells were prepared and resolved by SDS-PAGE using specific antibodies against NPR-C and AT1 receptor. Blots are representative of 3 or 4 separate experiments. Graphs show quantification of protein bands by densitometric scanning. Results are expressed as described in Fig. 1 legend. Values are means ± SE of 3 or 4 separate experiments. ***P < 0.001.

 
Effect of ONOO– on Gs{alpha}-Mediated Functions

The interaction of Gi{alpha} and Gs{alpha} proteins is well established. Since ONOO– decreased Gi{alpha} protein levels without altering Gs{alpha} protein levels, it was of interest to test whether Gs{alpha}-mediated functions were affected by ONOO–. Therefore, we examined the effect of isoproterenol and GTP{gamma}S on adenylyl cyclase activity in control and ONOO–-treated cells. Isoproterenol and GTP{gamma}S stimulated adenylyl cyclase activity in control and ONOO–-treated cells to various degrees (Fig. 10); however, the extent of stimulation was significantly enhanced by ~40% and 100%, respectively, in treated VSMC.


Figure 10
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  		Fig. 10. Effect of ONOO– on agonist-mediated stimulation of adenylyl cyclase in VSMC. VSMC were incubated in the absence or presence of 0.5 mM ONOO– for 24 h. Membranes were prepared, and adenylyl cyclase activity was determined in the presence of 10 µM GTP{gamma}S, 10 µM GTP alone or in combination with 50 µM isoproterenol (ISO), or in the absence or presence of 10 mM sodium fluoride (NaF) or 50 µM FSK. Basal adenylyl cyclase activity was 49.25 ± 5.26 and 27.07 ± 5.57 pmol cAMP·mg protein–1·10 min–1 in control and ONOO–-treated cells, respectively, in the absence of GTP and 232.36 ± 21.9 and 207.85 ± 30.55 pmol cAMP·mg protein–1·10 min–1 in the presence of 10 µM GTP. Values are means ± SE of 3 separate experiments performed in triplicate. *P < 0.05; **P < 0.01.

 
The effect of ONOO– on NaF- and FSK-stimulated adenylyl cyclase activity was also examined (Fig. 10, C and D). NaF and FSK stimulated enzyme activity to various degrees in control and treated cells; however, the stimulation exerted by ONOO– was significantly augmented by ~1.5- to 2.5-fold in treated cells. On the other hand, basal adenylyl cyclase activity was decreased in ONOO–-treated cells (49.3 ± 5.3 and 27.1 ± 5.6 pmol cAMP·mg protein–1·10 min–1 in control and ONOO–-treated cells, respectively).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
ONOO–, a vasorelaxant, has been shown to modulate epidermal growth factor receptor signaling in Caco-2 cells (55) and activate protein kinase C{alpha} in pulmonary endothelial cells (15). ONOO– was also reported to upregulate tyrosine phosphorylation signaling by activating kinases of the Src family in human erythrocytes and bovine aortic endothelial cells (43, 59). Using MnTBAP and uric acid, scavengers of ONOO–, we previously showed that the SNAP-induced decrease of Gi{alpha} protein expression in VSMC was attributed to the formation of intracellular ONOO– (10). However, the present studies demonstrate for the first time that treatment of VSMC with ONOO– for 24 h decreased the expression of Gi{alpha} protein and associated functions without affecting the levels of Gs{alpha} proteins. The decreased expression of Gi{alpha} proteins was not due to the increased formation of cGMP by ONOO–, because ODQ, an inhibitor of soluble guanylyl cyclase, which attenuated the ONOO–-induced increase of cGMP toward control levels, and KT-5823, an inhibitor of protein kinase G, were ineffective in restoring the decreased expression of Gi{alpha} proteins to control levels. Our results are consistent with our earlier studies showing that NO·-induced decreased expression of Gi{alpha} proteins in VSMC was also not mediated through increased cGMP levels (10). Several other studies have also shown that ONOO–-mediated inhibition of tyrosine phosphatases and modulation of fibroblast growth factor occurred by a cGMP-independent mechanism involving cysteine oxidation (8). In addition, ONOO– has also been shown to inhibit inducible NO synthase activation by a cGMP-independent mechanism (31).

We also showed that ONOO– decreased the phosphorylation of ERK1/2 and p38 MAP kinase and augmented JNK phosphorylation in aortic VSMC, and we suggest that an ONOO–-induced decrease of MAP kinase activity may be responsible for the decreased expression of Gi{alpha} proteins in these cells. MAP kinase signaling in ANG II-evoked enhanced expression of Gi{alpha} proteins has been implied (22). Our results are consistent with the studies of other investigators who also reported the ONOO–-induced reduced phosphorylation of p38 MAP kinase in preeclampsia of the placenta and in kidneys of βs sickle cell mice (36, 56) and increased JNK phosphorylation in endothelial cells from bovine thoracic aortas (25). On the other hand, our results are in contrast to the studies of Raines et al. (51) and Pesse et al. (50), who showed increased ERK1/2 activity in cells expressing neuronal NO synthase and in H9C2 cardiomyocytes, respectively. The apparent discrepancies may be attributed to the different cell types, i.e., human embryonic neuronal NO synthase-transfected kidney 293 cells/H9C2 cardiomyocytes vs. VSMC, and the time of treatment, i.e., 10–15 min vs. 24 h. Our results showing the inability of ONOO– to alter the phosphorylation of AKT are consistent with the results of other investigators, who were also unable to show the modulation of PI3-kinase downstream signaling, protein kinase B (PKB/Akt), by ONOO– in adipocytes (26). It should be noted that the decreased expression of Gi protein and associated signaling, as well as MAP kinase phosphorylation, by ONOO– may not be attributed to apoptosis, because our cell viability results from trypan blue exclusion indicated that >90–95% cells were viable.

Our results showing that the ONOO–-evoked decrease of Gi{alpha} protein expression was also reflected in the decreased functions of Gi{alpha} proteins are consistent with the results of studies showing that ONOO– attenuated adenosine A1 receptor-mediated signaling by functional uncoupling of the receptor from the Gi protein. However, the levels of the receptor and/or G protein were not examined (24). In addition, a correlation between the decreased levels of Gi{alpha} proteins and decreased/attenuated Gi functions has been reported (4, 41). Furthermore, the observation that ONOO– treatment of the cells also decreased the levels of NPR-C further suggests that ONOO–-induced downregulation of NPR-C, in addition to the decreased levels of Gi{alpha} proteins, may be responsible for the attenuated receptor-mediated inhibition of adenylyl cyclase by C-ANP4–23. On the other hand, ANG II-induced attenuation of adenylyl cyclase inhibition by ONOO– may not be attributed to decreased expression of the AT1 receptor, because AT1 receptor expression was not affected by ONOO– and may solely be attributed to downregulation of Gi{alpha} protein expression. On the other hand, the decreased basal adenylyl cyclase activity in VSMC exposed to ONOO– may not be attributed to the decreased expression of Gi{alpha} protein, because the basal activity is in the native state and is not under the influence of Gi protein and may be attributed to the decreased expression of adenylyl cyclase. In this regard, NO·-induced downregulation of V and VI isoforms of adenylyl cyclase has recently been shown (30); however, whether ONOO– also decreases the levels of adenylyl cyclase needs to be investigated.

We have also shown that treatment of VSMC with ONOO– resulted in augmented responsiveness of adenylyl cyclase to GTP{gamma}S and isoproterenol stimulation, which may be attributed to the decreased expression of Gi{alpha} proteins, increased levels of Gs{alpha} proteins, or impaired catalytic subunit or upregulation of β-adrenergic receptor, respectively. Since no alterations in the levels of Gs{alpha} proteins were observed in response to ONOO–, it may be suggested that decreased levels of Gi{alpha} proteins due to impaired catalytic subunit by ONOO– may be responsible for augmented stimulation of adenylyl cyclase by GTP{gamma}S and isoproterenol. A relationship between decreased levels of Gi protein and augmented stimulation of adenylyl cyclase by stimulatory hormones and GTP{gamma}S has been shown in various studies (2, 4, 41). Our results showing that ONOO– enhanced the stimulation of adenylyl cyclase by FSK and NaF are consistent with the studies of Fisch et al. (20), who showed that 3-morpholinosydnonimine, a donor of ONOO–, increased the levels of cAMP in the presence of FSK in platelets (20) and may be attributed to ONOO–-induced decreased levels of Gi{alpha} protein and decreased basal adenylyl cyclase activity. An increased stimulation of adenylyl cyclase by NaF and FSK in various pathological states exhibiting decreased expression of Gi{alpha} proteins has been reported (4, 41). In addition, several agents that decreased Gi{alpha} protein expression have been shown to augment the sensitivity of adenylyl cyclase to FSK or NaF stimulation (1, 2, 7, 10, 28).

In conclusion, we have provided the first evidence to demonstrate that ONOO– treatment of VSMC decreased the levels of Gi{alpha} proteins and associated functions and increased the levels of cGMP. The decrease of Gi{alpha} protein levels was cGMP independent and may involve MAP kinase signaling. It may thus be suggested that ONOO–-induced decreased levels of Gi and resultant increased levels of cAMP may be a potential mechanism through which ONOO– exerts a vasorelaxant and antiproliferative effect.


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


   ACKNOWLEDGMENTS
 
We thank Claude Gauthier for valuable help in graphics design.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. B. Anand-Srivastava, Dept. of Physiology, Faculty of Medicine, Univ. of Montreal, CP 6128, Succ. Centreville, 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. Amiloride interacts with guanine nucleotide regulatory proteins and attenuates the hormonal inhibition of adenylate cyclase. J Biol Chem 264: 9491–9496, 1989.[Abstract/Free Full Text]
  2. Anand-Srivastava MB. Downregulation of atrial natriuretic peptide ANP-C receptor is associated with alterations in G-protein expression in A10 smooth muscle cells. Biochemistry 39: 6503–6513, 2000.[CrossRef][Web of Science][Medline]
  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. Platelets from spontaneously hypertensive rats exhibit decreased expression of inhibitory guanine nucleotide regulatory protein. Relation with adenylyl cyclase activity. Circ Res 73: 1032–1039, 1993.[Abstract/Free Full Text]
  5. 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]
  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. Anand-Srivastava MB, Srivastava AK, Cantin M. Pertussis toxin attenuates atrial natriuretic factor-mediated inhibition of adenylate cyclase. Involvement of inhibitory guanine nucleotide regulatory protein. J Biol Chem 262: 4931–4934, 1987.[Abstract/Free Full Text]
  8. Bagnasco P, MacMillan-Crow LA, Greendorfer JS, Young CJ, Andrews L, Thompson JA. Peroxynitrite modulates acidic fibroblast growth factor (FGF-1) activity. Arch Biochem Biophys 419: 178–189, 2003.[CrossRef][Web of Science][Medline]
  9. Bassil M, Anand-Srivastava MB. Cyclic GMP modulates the expression of Gi protein and adenylyl cyclase signaling in vascular smooth muscle cells. Cell Biochem Biophys 47: 99–108, 2007.[CrossRef][Web of Science][Medline]
  10. Bassil M, Anand-Srivastava MB. Nitric oxide modulates Gi-protein expression and adenylyl cyclase signaling in vascular smooth muscle cells. Free Radic Biol Med 41: 1162–1173, 2006.[CrossRef][Web of Science][Medline]
  11. Beckman JS, Beckman TW, Chen J, Marshall PA, Freeman BA. Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci USA 87: 1620–1624, 1990.[Abstract/Free Full Text]
  12. Benkusky NA, Lewis SJ, Kooy NW. Attenuation of vascular relaxation after development of tachyphylaxis to peroxynitrite in vivo. Am J Physiol Heart Circ Physiol 275: H501–H508, 1998.[Abstract/Free Full Text]
  13. Boumati M, Li Y, Anand-Srivastava MB. Modulation of ANP-C receptor signaling by arginine-vasopressin in A-10 vascular smooth muscle cells: role of protein kinase C. Arch Biochem Biophys 415: 193–202, 2003.[CrossRef][Web of Science][Medline]
  14. Boumati M, Li Y, Anand-Srivastava MB. Modulation of ANP-C receptor signaling by endothelin-1 in A-10 smooth muscle cells. Arch Biochem Biophys 401: 178–186, 2002.[CrossRef][Web of Science][Medline]
  15. Chakraborti T, Das S, Chakraborti S. Proteolytic activation of protein kinase C{alpha} by peroxynitrite in stimulating cytosolic phospholipase A2 in pulmonary endothelium: involvement of a pertussis toxin sensitive protein. Biochemistry 44: 5246–5257, 2005.[CrossRef][Web of Science][Medline]
  16. Di Fusco F, Anand-Srivastava MB. Nitric oxide synthase inhibition by N{omega}-nitro-L-arginine methyl ester modulates G-protein expression and adenylyl cyclase activity in rat heart. Am J Hypertens 10: 471–475, 1997.[Web of Science][Medline]
  17. Dowell FJ, Martin W. The effects of peroxynitrite on rat aorta: interaction with glucose and related substances. Eur J Pharmacol 338: 43–53, 1997.[CrossRef][Web of Science][Medline]
  18. Feraille E, Doucet A. Sodium-potassium-adenosinetriphosphatase-dependent sodium transport in the kidney: hormonal control. Physiol Rev 81: 345–418, 2001.[Abstract/Free Full Text]
  19. Ferdinandy P, Schulz R. Peroxynitrite: toxic or protective in the heart? Circ Res 88: E12–E13, 2001.[Web of Science][Medline]
  20. Fisch A, Michael-Hepp J, Meyer J, Darius H. Synergistic interaction of adenylate cyclase activators and nitric oxide donor SIN-1 on platelet cyclic AMP. Eur J Pharmacol 289: 455–461, 1995.[CrossRef][Web of Science][Medline]
  21. Ge C, Anand-Srivastava MB. Involvement of phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways in AII-mediated enhanced expression of Gi proteins in vascular smooth muscle cells. Biochem Biophys Res Commun 251: 570–575, 1998.[CrossRef][Web of Science][Medline]
  22. Ge C, Garcia R, Anand-Srivastava MB. Altered expression of Gi-protein and adenylyl cyclase activity in hearts from one kidney one clip hypertensive rats: effect of captopril. J Hypertens 17: 1617–1626, 1999.[Web of Science][Medline]
  23. Ge C, Garcia R, Anand-Srivastava MB. Enhanced expression of Gi{alpha} protein and adenylyl cyclase signaling in aortas from 1 kidney 1 clip hypertensive rats. Can J Physiol Pharmacol 84: 739–746, 2006.[CrossRef][Web of Science][Medline]
  24. Giuntini J, Giusti L, Lucacchini A, Mazzoni MR. Modulation of A1 adenosine receptor signaling by peroxynitrite. Biochem Pharmacol 67: 375–383, 2004.[CrossRef][Web of Science][Medline]
  25. Go YM, Patel RP, Maland MC, Park H, Beckman JS, Darley-Usmar VM, Jo H. Evidence for peroxynitrite as a signaling molecule in flow-dependent activation of c-Jun NH2-terminal kinase. Am J Physiol Heart Circ Physiol 277: H1647–H1653, 1999.[Abstract/Free Full Text]
  26. Guzman-Grenfell AM, Garcia-Macedo R, Gonzalez-Martinez MT, Hicks JJ, Medina-Navarro R. Peroxynitrite activates glucose uptake in 3T3-L1 adipocytes through a PI3-K-dependent mechanism. Front Biosci 10: 47–53, 2005.[CrossRef][Web of Science][Medline]
  27. Hashim S, Li Y, Anand-Srivastava MB. Small cytoplasmic domain peptides of natriuretic peptide receptor-C attenuate cell proliferation through Gi{alpha} protein/MAP kinase/PI3-kinase/AKT pathways. Am J Physiol Heart Circ Physiol 291: H3144–H3153, 2006.[Abstract/Free Full Text]
  28. 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]
  29. Herold S, Fago A. Reactions of peroxynitrite with globin proteins and their possible physiological role. Comp Biochem Physiol A 142: 124–129, 2005.[CrossRef][Medline]
  30. Hill J, Howlett A, Klein C. Nitric oxide selectively inhibits adenylyl cyclase isoforms 5 and 6. Cell Signal 12: 233–237, 2000.[CrossRef][Web of Science][Medline]
  31. Huhmer AF, Nishida CR, Ortiz de Montellano PR, Schoneich C. Inactivation of the inducible nitric oxide synthase by peroxynitrite. Chem Res Toxicol 10: 618–626, 1997.[CrossRef][Web of Science][Medline]
  32. Itoh H, Toyama R, Kozasa T, Tsukamoto T, Matsuoka M, Kaziro Y. Presence of three distinct molecular species of Gi protein {alpha}-subunit. Structure of rat cDNAs and human genomic DNAs. J Biol Chem 263: 6656–6664, 1988.[Abstract/Free Full Text]
  33. Jones DT, Reed RR. Molecular cloning of five GTP-binding protein cDNA species from rat olfactory neuroepithelium. J Biol Chem 262: 14241–14249, 1987.[Abstract/Free Full Text]
  34. Kim H, Shim J, Han PL, Choi EJ. Nitric oxide modulates the c-Jun N-terminal kinase/stress-activated protein kinase activity through activating c-Jun N-terminal kinase kinase. Biochemistry 36: 13677–13681, 1997.[CrossRef][Web of Science][Medline]
  35. Kinoshita S, Sidhu A, Felder RA. Defective dopamine-1 receptor adenylate cyclase coupling in the proximal convoluted tubule from the spontaneously hypertensive rat. J Clin Invest 84: 1849–1856, 1989.[Web of Science][Medline]
  36. Kiroycheva M, Ahmed F, Anthony GM, Szabo C, Southan GJ, Bank N. Mitogen-activated protein kinase phosphorylation in kidneys of βs sickle cell mice. J Am Soc Nephrol 11: 1026–1032, 2000.[Abstract/Free Full Text]
  37. Liu S, Beckman JS, Ku DD. Peroxynitrite, a product of superoxide and nitric oxide, produces coronary vasorelaxation in dogs. J Pharmacol Exp Ther 268: 1114–1121, 1994.[Abstract/Free Full Text]
  38. Liu Y, Terata K, Chai Q, Li H, Kleinman LH, Gutterman DD. Peroxynitrite inhibits Ca2+-activated K+ channel activity in smooth muscle of human coronary arterioles. Circ Res 91: 1070–1076, 2002.[Abstract/Free Full Text]
  39. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265–275, 1951.[Free Full Text]
  40. Marcil J, de Champlain J, Anand-Srivastava MB. Overexpression of Gi-proteins precedes the development of DOCA-salt-induced hypertension: relationship with adenylyl cyclase. Cardiovasc Res 39: 492–505, 1998.[Abstract/Free Full Text]
  41. Marcil J, Schiffrin EL, Anand-Srivastava MB. Aberrant adenylyl cyclase/cAMP signal transduction and G protein levels in platelets from hypertensive patients improve with antihypertensive drug therapy. Hypertension 28: 83–90, 1996.[Abstract/Free Full Text]
  42. Marcil J, Thibault C, Anand-Srivastava MB. Enhanced expression of Gi-protein precedes the development of blood pressure in spontaneously hypertensive rats. J Mol Cell Cardiol 29: 1009–1022, 1997.[CrossRef][Web of Science][Medline]
  43. Minetti M, Mallozzi C, Di Stasi AM. Peroxynitrite activates kinases of the Src family and upregulates tyrosine phosphorylation signaling. Free Radic Biol Med 33: 744–754, 2002.[CrossRef][Web of Science][Medline]
  44. Moro MA, Darley-Usmar VM, Lizasoain I, Su Y, Knowles RG, Radomski MW, Moncada S. The formation of nitric oxide donors from peroxynitrite. Br J Pharmacol 116: 1999–2004, 1995.[Web of Science][Medline]
  45. Murakami T, Yasuda H. Rat heart cell membranes contain three substrates for cholera toxin-catalyzed ADP-ribosylation and a single substrate for pertussis toxin-catalyzed ADP-ribosylation. Biochem Biophys Res Commun 138: 1355–1361, 1986.[CrossRef][Web of Science][Medline]
  46. Neer EJ. Heterotrimeric G proteins: organizers of transmembrane signals. Cell 80: 249–257, 1995.[CrossRef][Web of Science][Medline]
  47. Palaparti A, Anand-Srivastava MB. Angiotensin II modulates ANP-R2/ANP-C receptor-mediated inhibition of adenylyl cyclase in vascular smooth muscle cells: role of protein kinase C. J Mol Cell Cardiol 30: 1471–1482, 1998.[CrossRef][Web of Science][Medline]
  48. Palaparti A, Ge C, Anand-Srivastava MB. Angiotensin II enhances the expression of Gi{alpha} in A10 cells (smooth muscle): relationship with adenylyl cyclase activity. Arch Biochem Biophys 365: 113–122, 1999.[CrossRef][Web of Science][Medline]
  49. Palaparti A, Li Y, Anand-Srivastava MB. Inhibition of atrial natriuretic peptide (ANP) C receptor expression by antisense oligodeoxynucleotides in A10 vascular smooth-muscle cells is associated with attenuation of ANP-C-receptor-mediated inhibition of adenylyl cyclase. Biochem J 346: 313–320, 2000.[CrossRef][Web of Science][Medline]
  50. Pesse B, Levrand S, Feihl F, Waeber B, Gavillet B, Pacher P, Liaudet L. Peroxynitrite activates ERK via Raf-1 and MEK, independently from EGF receptor and p21Ras in H9C2 cardiomyocytes. J Mol Cell Cardiol 38: 765–775, 2005.[CrossRef][Web of Science][Medline]
  51. Raines KW, Cao GL, Porsuphatana S, Tsai P, Rosen GM, Shapiro P. Nitric oxide inhibition of ERK1/2 activity in cells expressing neuronal nitric-oxide synthase. J Biol Chem 279: 3933–3940, 2004.[Abstract/Free Full Text]
  52. Salomon Y, Londos C, Rodbell M. A highly sensitive adenylate cyclase assay. Anal Biochem 58: 541–548, 1974.[CrossRef][Web of Science][Medline]
  53. Thibault C, Anand-Srivastava MB. Altered expression of G-protein mRNA in spontaneously hypertensive rats. FEBS Lett 313: 160–164, 1992.[CrossRef][Web of Science][Medline]
  54. Tsuda K, Tsuda S, Nishio I, Goldstein M, Masuyama Y. Modulation of [3H]dopamine release by neuropeptide Y in rat striatal slices. Eur J Pharmacol 321: 5–11, 1997.[CrossRef][Web of Science][Medline]
  55. Uc A, Kooy NW, Conklin JL, Bishop WP. Peroxynitrite inhibits epidermal growth factor receptor signaling in Caco-2 cells. Dig Dis Sci 48: 2353–2359, 2003.[CrossRef][Web of Science][Medline]
  56. Webster RP, Brockman D, Myatt L. Nitration of p38 MAPK in the placenta: association of nitration with reduced catalytic activity of p38 MAPK in pre-eclampsia. Mol Hum Reprod 12: 677–685, 2006.[Abstract/Free Full Text]
  57. Wei EP, Kontos HA, Beckman JS. Antioxidants inhibit ATP-sensitive potassium channels in cerebral arterioles. Stroke 29: 817–823, 1998.[Abstract/Free Full Text]
  58. Yatani A, Mattera R, Codina J, Graf R, Okabe K, Padrell E, Iyengar R, Brown AM, Birnbaumer L. The G protein-gated atrial K+ channel is stimulated by three distinct Gi {alpha}-subunits. Nature 336: 680–682, 1988.[CrossRef][Medline]
  59. Zou MH, Hou XY, Shi CM, Kirkpatick S, Liu F, Goldman MH, Cohen RA. Activation of 5'-AMP-activated kinase is mediated through c-Src and phosphoinositide 3-kinase activity during hypoxia-reoxygenation of bovine aortic endothelial cells. Role of peroxynitrite. J Biol Chem 278: 34003–34010, 2003.[Abstract/Free Full Text]



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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
Am J Physiol Heart Circ Physiol, June 1, 2008; 294(6): H2845 - H2854.
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