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Cardiovascular Research 2005 66(3):503-511; doi:10.1016/j.cardiores.2005.02.005
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Copyright © 2005, European Society of Cardiology

Angiotensin II-evoked enhanced expression of RGS2 attenuates Gi-mediated adenylyl cyclase signaling in A10 cells

Yuan Li, Shehla Hashim and Madhu B. Anand-Srivastava*

Department of Physiology, Faculty of Medicine, University of Montreal, and Groupe de recherche sur le système nerveux autonome (GRSNA), Canada

* Corresponding author. Department of Physiology, Faculty of Medicine, University of Montreal, C.P. 6128, Succ. Centre-ville, Montreal, Quebec, Canada, H3C 3J7. Tel.: +1 514 343 2091; fax: +1 514 343 2111. Email address: anandsrm{at}physio.umontreal.ca

Received 16 September 2004; revised 3 February 2005; accepted 7 February 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: We have recently shown that pretreatment of A10 vascular smooth muscle cells (VSMC) with angiotensin II (Ang II) for 24 h enhanced the expression of Gi{alpha}-proteins, however, Gi{alpha}-mediated adenylyl cyclase signaling was attenuated. Since regulators of G-protein signaling (RGS) have been shown to negatively regulate the G{alpha}-protein, we investigated the role of RGS2, in Ang II-induced attenuation of Gi{alpha}-mediated signaling in A10 vascular smooth muscle cells (VSMC).

Methods: A10 VSMC were incubated with Ang II (10–7 M) for different periods of time at 37 °C. The levels of G-proteins and RGS2 protein were determined by immunoblotting using specific antibodies. Adenylyl cyclase activity stimulated or inhibited by agonists was determined to examine the functions of G-proteins.

Results: Ang II treatment of A10 VSMC enhanced the expression of Gi{alpha} and RGS2 proteins in a time-dependent manner, the maximal expression of Gi{alpha}-proteins was observed at 1 h that remained elevated up to 24 h, whereas enhanced expression of RGS2 in these cells was detected at 1 h, peaked at 2 h and returned to base line level by 8 h. The increased expression of RGS2 by Ang II was inhibited by actinomycin D. The increased expression of Gi{alpha} at 30 min when the levels of RGS2 were not augmented was reflected in increased Gi functions as was demonstrated by increased inhibition of adenylyl cyclase by inhibitory hormones (receptor-dependent functions) and increased inhibition of forskolin (FSK)-stimulated adenylyl cyclase by GTP{gamma}S (receptor-independent functions). However, with increased expression of RGS2, the Gi-mediated functions started declining and were completely abolished at 2 h of treatment when the levels of RGS2 were maximally augmented. Furthermore, prior treatment of cells with RGS2 antibody, that completely attenuated Ang II-induced expression of RGS2, prevented the GTP{gamma}S-induced attenuation of FSK-stimulated adenylyl cyclase activity. In addition, treatment of the cells with Ang II for 30 min that increased the levels of Gi{alpha}-protein and not of RGS2 protein resulted in the attenuation of Gs{alpha}-mediated isoproterenol-induced stimulation of adenylyl cyclase and this attenuation was restored to control level at 1 h and remained unchanged thereafter.

Conclusion: These results suggest that RGS2 may be involved in short-term regulation of Ang II-induced Gi-mediated adenylyl cyclase signalling.

KEYWORDS RGS2 protein; G-protein; Adenylyl cyclase; Angiotensin II; VSMC

Abbreviations: C-ANP4–23, [des(Glu18, Ser19, Glu20, Leuc21, Gly22) ANP4–23-NH2] • VSMC, vascular smooth muscle cells • RGS2, regulator of G-protein signaling • Ang II, angiotensin II • Gi, inhibitory guanine nucleotide regulatory protein • Gs, stimulatory guanine nucleotide regulatory protein • GTP{gamma}S, guanosine 5'-{gamma}-thiotriphosphate


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Angiotensin II (Ang II), a growth promoting factor and a key component of the renin–angiotensin system, elicits a wide variety of biological responses, including vasoconstriction, stimulation of aldosterone secretion and renal sodium reabsorption [1–3]. Ang II elicits its physiological effects by interacting with two distinct receptor subtypes designated as AT1 and AT2 [4]. The presence of AT1 receptor subtype has been shown in rat vascular tissues, however, a small proportion of AT2 receptors is also present in rat aorta [5,6]. Most of the physiological effects of Ang II are mediated by AT1 receptors. AT1 receptors are coupled to several second messenger systems including inhibition of adenylyl cyclase/cAMP through Gi{alpha}-proteins [7–9].

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 the stimulatory (Gs) and inhibitory (Gi) guanine nucleotide protein respectively [10,11] resulting in the increased or decreased formation of cAMP respectively. G-proteins are heterotrimeric consisting of {alpha}, β and {gamma}-subunits. The {alpha}-subunits bind and hydrolyze GTP and confer specificity in receptor and effector interactions [11]. Four different isoforms of Gs have been identified which appear to be products of alternate splicing of a common precursor [12,13]. On the other hand, three distinct forms of Gi{alpha}; Gi{alpha}-1, Gi{alpha}-2 and Gi{alpha}-3 have been identified and shown to be the products of three different genes [14,15].

A family of proteins, regulators of G-protein signaling (RGS) that displays GTPase-activating proteins (GAPS) activity toward heterotrimeric G-protein {alpha}-subunits and directly modulates G-protein activity has recently been shown [16]. All RGS contain a highly conserved carboxy terminal domain of ~130 amino acids (RGS domain or box) which has been shown to be responsible for binding of RGS to the G{alpha}-subunits to accelerate GTP hydrolysis and to decrease the life time of the active G{alpha}-GTP and free Gβ{gamma}-subunits and thereby turning off the signaling pathways [16–18]. Several recent studies have shown that RGS protein, in addition to the negative regulator of G{alpha}-subunits, may also interact with β{gamma}-subunits [19]. Vascular smooth muscle cells (VSMC) from rat aorta have been reported to express six different RGS proteins [20] of which RGS2 has been shown to attenuate Gi and Gs-mediated signalling [21] and thereby regulate blood pressure. The expression of RGS2 mRNA has been shown to be regulated by various modulators [16,22].

Ang II has also been shown to increase RGS2 mRNA levels in VSMC from rat aorta [20]. However, the relationship between RGS2 and Ang II as well as other Gi-mediated adenylyl cyclase signaling has not been investigated. We have recently shown that Ang II treatment of A10 VSMC for 24 h augmented the levels of Gi{alpha}-2 and Gi{alpha}-3 and their mRNA, however, the functions of Gi{alpha}-proteins were not augmented but were completely abolished [23]. Taken together, it may be possible that Ang II-mediated attenuation of Gi-mediated signaling in A10 VSMC may be attributed to Ang II-induced enhanced expression of RGS2 in these cells. The present studies were undertaken to investigate the role of RGS2 in Ang II-induced attenuation of Gi-mediated adenylyl cyclase signaling. We have provided the first evidence that the Ang II-evoked overexpression of RGS2 may contribute to the attenuation of Gi-mediated adenylyl cyclase signaling in A10 vascular smooth muscle cells.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
C-ANP4–23 was purchased from Peninsula Laboratories (CA, USA). [{alpha}32P]ATP was purchased from Amersham (Oakville, Ontario, Canada). AS/7 and EC antibodies directed against specific C-terminus sequences of Gi{alpha}-2 and Gi{alpha}-3 respectively, were purchased from Dupont Canada. Polyclonal antibody N-16 against RGS2 obtained from Santa Cruz Biotechnology was raised against a peptide mapping at the amino terminus of RGS2 of human origin and was highly specific for RGS2. All other chemicals were obtained from commercial sources as described previously [23,24].

2.1. Cell culture and incubation
A10 cell line from embryonic thoracic aorta of rat was obtained from American Type Culture Collection, Rockville, MA, and USA. The cells were plated in 7.5 cm2 flasks and incubated at 37 °C in 95% air and 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 [23,24]. 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. 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 x g. The cells were then homogenized in a Dounce homogenizer (10 strokes) and the homogenate was used for adenylyl cyclase assay and immunoblotting.

2.2. Cell permeabilization and antibody treatment
The cells after washing once with streptolysin 0 (SLO) buffer, containing 200 mM Hepes, 50 mM NaCl, 140 mM KCl, 5 mM MgCl2 and 50 mM EGTA (pH 7.4), were incubated with SLO (0.8 U/ml) at 37 °C for 10 min. The cells were washed with DMEM without FBS. The permeabilized cells were incubated in the absence or presence of RGS2 antibody (40 µg/ml) for 30 min and were challenged with Ang II for 2 h. The cells were harvested and used for adenylyl cyclase activity assay and Western blotting.

2.3. Adenylyl cyclase activity determination
Adenylyl cyclase activity was determined by measuring [32P]cAMP formation from [{alpha}-32P]ATP, as described previously [23,24].

2.4. Immunoblotting
Immunoblotting of G-proteins and RGS2 was performed using specific antibodies as described previously [23].

2.5. Statistical analysis
Data are expressed as mean ± S.E.M. and were analyzed by ANOVA in conjunction with Newman–Keuls test where applicable. Comparisons between groups (control and Ang II-treated cells) were made with Student's t-test for unpaired samples. Difference between groups was considered statistically significant at P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1. Regulation of Gi{alpha}-protein expression and functions by Ang II in A10 VSMC
We have recently shown that pretreatment of A10 VSMC with Ang II for 24 h enhanced the expression of Gi{alpha}-2 and Gi{alpha}-3 proteins but not functions [23,24], which was shown to be attributed to the phosphorylation and thereby inactivation of Gi proteins. In order to investigate if short-term treatment of cells with Ang II also results in the overexpression of Gi proteins, the cells were treated with Ang II for 30 min to 24 h. As shown in Fig. 1, Ang II (10–7 M) treatment of the cells, enhanced the expression of Gi{alpha}-2 (A) and Gi{alpha}-3 (B) in a time-dependent manner. The level Gi{alpha}-2 and Gi{alpha}-3 started increasing at as early as 30 min and peaked at about 125–150% of control at 1–2 h and remained elevated up to 24 h as determined by densitometric scanning (lower panels). On the other hand, the levels of Gs{alpha}-protein were not affected by Ang II treatment (data not shown).


Figure 1
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  		Fig. 1 Effect of Ang II pre-treatment on the levels of Gi{alpha}-2 and Gi{alpha}-3 proteins in A10 vascular smooth muscle cells (VSMC). Upper panels: A10 VSMC 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 (20 µg) were revolved by SDS-PAGE and transferred to nitrocellulose, which was then immunoblotted using AS/7 antibody for Gi{alpha}-2 (A) or EC/2 antibody for Gi{alpha}-3 (B) as described previously [23]. Autoradiograms are representative of 3 or 4 separate experiments. Lower panels: the graph shows quantification of protein bands by densitometric scanning. The results are expressed as percentage of control taken as 100%. Values are mean ± S.E.M. of 3 or 4 separate experiments. *P<0.05, **P<0.01.

 
We have previously shown that treatment of A10 cells with Ang II for 24 h that increased the expression of Gi{alpha}-protein resulted in the attenuation of receptor-independent and -dependent functions of Gi protein [23]. In order to investigate if short-term treatment of cells with Ang II also resulted in similar attenuation of Gi functions, we examined the effect of low concentrations of GTP{gamma}S on FSK-stimulated adenylyl cyclase activity (receptor-independent functions of Gi) in cells treated with Ang II for 30 min. The results shown in Fig. 2, indicate that GTP{gamma}S inhibited FSK-stimulated adenylyl cyclase activity in a concentration-dependent manner in control as well as in Ang II-treated cells, however, the extent of inhibition was significantly augmented in Ang II-treated cells. For example at 10–8 M, GTP{gamma}S inhibited FSK-stimulated activity by about 20% in control cells and by about 40% in Ang II-treated cells. These data suggest that Ang II-induced increased expression of Gi{alpha}-proteins at 30 min was also reflected in enhanced Gi functions.


Figure 2
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  		Fig. 2 Effect of GTP{gamma}S on FSK-stimulated adenylyl cyclase activity in control and Ang II-treated A10 vascular smooth muscle cells (VSMC). A10 VSMC were incubated in the absence or presence of 10–7 M Ang II for 30 min as described in "Materials and methods". Membranes were prepared and adenylyl cyclase activity was determined in the presence of 100 µM FSK alone or in combination with various concentrations of GTP{gamma}S. Basal enzyme activities in the absence or presence of 100 µM FSK were 65.1 ± 1.2 and 2561.5 ± 61.6 pmol cAMP/mg protein/10 min respectively in control cells and 76.5 ± 10 and 2906.5 ± 122.8 pmol cAMP/mg protein/10 min respectively in Ang II-treated cells. Values are means ± S.E.M. of three separate experiments. *P<0.05, **P<0.01.

 
Fig. 3 shows the temporal relationship between Ang II-pretreatment and receptor-independent and receptor-dependent functions of Gi{alpha}-proteins. As shown in Fig. 2, GTP{gamma}S (10–8 M) inhibited FSK-stimulated adenylyl cyclase activity by about 20% in control untreated cells, however, this inhibition was significantly enhanced by about 75% (20% vs. 35%) in cells pretreated with Ang II for 30 min. Thereafter, the inhibition started declining and reached below basal level at 4 h of treatment (Fig. 3A). In addition, receptor-dependent functions of Gi{alpha} were examined by studying AT1 and ANP-C receptor-mediated inhibition of adenylyl cyclase. As shown in Fig. 3B and C, Ang II and C-ANP4–23; a ring deleted analog of ANP that specifically interacts with ANP-C receptor and inhibits adenylyl cyclase through Gi{alpha}-protein [10,25] inhibited adenylyl cyclase activity that was stimulated by 10 µM GTP{gamma}S in control untreated cells by about 25 and 30% respectively. However, Ang II treatment exerted biphasic effect: 30 min of treatment with Ang II increased the Ang II- and C-ANP4–23-mediated inhibitions by about 60% (from 25 to 40% and 30 to 48% respectively), which were declined rapidly thereafter and were almost completely abolished at 4 h of treatment (Fig. 3B,C). These results indicate that enhanced levels of Gi{alpha}-2 and Gi{alpha}-3 proteins induced by Ang II were reflected in maximal enhanced functions only at 30 min and thereafter the functions were reduced/abolished, suggesting that up to 30 min–1 h of Ang II treatment, the Gi{alpha}-2 and Gi{alpha}-3 proteins were functional.


Figure 3
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  		Fig. 3 Effect of Ang II pre-treatment on receptor-independent and receptor-dependent functions of Gi{alpha}-proteins in A10 vascular smooth muscle cells (VSMC). A10 VSMC 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 adenylyl cyclase activity was determined in the presence of 100 µM FSK alone or in combination with 10–8 M GTP{gamma}S (A). The results are expressed as GTP{gamma}S-mediated inhibition of FSK-stimulated adenylyl cyclase activity. Adenylyl cyclase activity in control cells in the presence of 100 µM FSK was 2872.8 ± 182.0 and FSK+GTP{gamma}S was 2285.1 ± 33.6. Adenylyl cyclase activity in control and Ang II-treated membranes was determined in the presence of 10 µM GTP{gamma}S alone or in combination with 10–5 M Ang II (B) or 10–7 M C-ANP4–23 (C). The results are expressed as C-ANP4–23 or Ang II-mediated inhibition of adenylyl cyclase activity. Adenylyl cyclase activity in control cells in the presence of 10 µM GTP{gamma}S alone was 152.4 ± 12.8 and in combination with 10–7 M C-ANP4–23 or 10–5 M Ang II was 105.5 ± 5.1 or 111.9 ± 3.8 pmol cAMP/mg protein/10 min. Values are means ± S.E.M. of three separate experiments performed in triplicates. *P<0.05, **P<0.01, ***P<0.001.

 
3.2. Effect of Ang II treatment on RGS2 protein expression
Ang II has been shown to increase the expression of RGS2 mRNA in cultured vascular smooth muscle cells from thoracic aorta [23]. In order to investigate if Ang II-treatment also increased the expression of RGS2 proteins in A10 VSMC that may be responsible for the inactivation of Ang II-induced enhanced expression of Gi protein, the effect of Ang II treatment on RGS2 protein expression was examined. The results shown in Fig. 4 indicate that Ang II (10–7 M) increased the expression of RGS2 (24 kDa) in a time dependent manner. Ang II-induced increased expression (50 ± 5% over control) was observed at 1 h which peaked at ~200 ± 15.6% of control at 2 h and started declining (~150 ± 21.0%) at 4 h and by 24 h of Ang II treatment, RGS2 protein levels were significantly decreased from control levels.


Figure 4
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  		Fig. 4 Effect of Ang II pre-treatment on RGS2 proteins expression in A10 vascular smooth muscle cells (VSMC). Upper panel: A10 VSMC 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 membrane proteins (50 µg) were resolved by SDS-PAGE and transferred to nitrocellulose, which was then immunoblotted using antibody N-16 for RGS2 as described previously [23]. Autoradiograms are representative of 3 separate experiments. Lower panel: the graph shows quantification of protein bands by densitometric scanning. The results are expressed as percentage of control taken as 100%. Values are mean ± S.E.M. of 3 or 4 separate experiments. *P<0.05, **P<0.01.

 
We have investigated further the effect of actinomycin D (an inhibitor of RNA synthesis) on the Ang II-induced increased levels of RGS2 protein. The results shown in Fig. 5, indicate that actinomycin D attenuated the Ang II-induced enhanced levels of RGS2 proteins to control levels, suggesting that Ang II-induced increased levels of RGS2 proteins may be due to an increase in RNA synthesis.


Figure 5
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  		Fig. 5 Effect of actinomycin D on Ang II-evoked RGS2 protein levels in A10 vascular smooth muscle cells (VSMC). Upper panels: A10 VSMC were pretreated without or with actinomycin D (AD; 5 µM) for 24 h and were further incubated in the absence or presence of 10–7 M Ang II for 1 h. Membranes were prepared and membrane proteins (50 µg) were resolved by SDS-PAGE and transferred to nitrocellulose, which was then immunoblotted using N-16 antibody for RGS2 as described previously [23]. Autoradiograms are representative of 3 or 4 separate experiments. Lower panels: the graph shows quantification of proteins bands by densitometric scanning. The results are expressed as percentage of control taken as 100%. Values are means ± S.E.M. of 3 or 4 separate experiments. *P<0.05.

 
3.3. Effect of RGS2 antibody treatment on Ang II-induced attenuation of Gi functions
In order to investigate if Ang II-induced enhanced expression of RGS2 is responsible for attenuation of Gi functions, the effect of RGS2 antibody on GTP{gamma}S-induced inhibition of FSK-stimulated adenylyl cyclase activity was examined in Ang II-treated vascular smooth muscle cells. The results shown in Fig. 6A indicate that GTP{gamma}S (10–8 M) inhibited FSK-stimulated adenylyl cyclase activity by about 30% in control cells. The treatment of cells with Ang II for 2 h attenuated the inhibition by about 65%. However, pre-treatment of the cells with RGS2 antibody prevented this attenuation. Similarly, prior treatment of the cells with RGS2 antibody prevented the Ang II-induced enhanced expression of RGS2 (Fig. 6B), whereas, RGS4 antibody did not affect the levels of RGS2 protein augmented by Ang II treatment. These results suggest that Ang II-induced increased expression of RGS2 may be responsible for the attenuation of Gi{alpha}-mediated functions.


Figure 6
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  		Fig. 6 Effect of RGS2 and RGS4 antibody treatments on Ang II-induced attenuation of Gi functions in A10 vascular smooth muscle cells (VSMC). (A) The permeabilized A10 cells were incubated in the absence (control) or presence of 10–7 M Ang II alone or in combination with RGS2 or RGS4 antibody. Membranes were prepared and adenylyl cyclase activity was determined in the presence of 100 µM FSK alone or in combination with 10–8 M GTP{gamma}S. The results are expressed as GTP{gamma}S-mediated inhibition of FSK-stimulated adenylyl cyclase activity. Adenylyl cyclase activity in control cells in the presence of 100 µM FSK was 2341.5 ± 160.7 and FSK+GTP{gamma}S was 1810.0 ± 90.0 pmol cAMP/mg protein/10 min. *P<0.05. (B) The permeabilized A10 cells were incubated in the absence or presence of RGS2 or RGS4 antibody for 30 min and were challenged with 10–7 M Ang II for 2 h. Membranes were prepared and membrane proteins (50 µg) from various treatment groups were resolved by SDS-PAGE, and transferred to nitrocellulose, which was then immunoblotted using antibody for RGS2 and RGS4 or antibody β-actin as control. Autoradiograms are representative of 3 or 4 separate experiments. The results are expressed as percentage of control taken as 100%. **P<0. 01.

 
3.4. Effect of Ang II-induced enhanced expression of RGS2 on Gs{alpha}-mediated stimulation of adenylyl cyclase
Since RGS2 has recently been shown to inhibit odorant-stimulated cAMP levels in olfactory epithelium, it was of interest to investigate if Ang II-induced overexpression of RGS2 could also modulate Gs-mediated stimulation of adenylyl cyclase. Fig. 7 shows the time course of Ang II treatment on isoproterenol-stimulated adenylyl cyclase activity. Isoproterenol (50 µM) stimulated adenylyl cyclase activity by about 2-fold (~100) in control cells, however, this stimulation was significantly attenuated by treatment of cells with Ang II for 30 min.


Figure 7
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  		Fig. 7 Effect of Ang II treatment on isoproterenol-mediated stimulation of adenylyl cyclase activity in A10 vascular smooth muscle cells (VSMC). A10 VSMC were incubated in the absence (control) or presence of 10–7 M Ang II for various time periods as described in "Materials and methods". The membranes were prepared and adenylyl cyclase activity was determined in the presence of 10 µM GTP alone or in combination with 50 µM isoproterenol (ISO). Adenylyl cyclase activity in control cells in the presence of 10 µM GTP was 155.7 ± 13.1 and GTP+ISO was 324.8 ± 10.4 pmol cAMP/mg protein/10 min. Results are expressed as isoproterenol-mediated stimulation of adenylyl cyclase activity. The percentage stimulation in control cells were taken as 100%. Values are mean ± S.E.M. of 3 separate experiments performed in triplicates. *P<0.05.

 
Thereafter, the stimulation was reversed to the same level as that of control cells and remained at the same level up to 4 h of treatment.

3.5. Effect of Ang II-induced enhanced expression of RGS2 on forskolin (FSK)-stimulated adenylyl cyclase activity
FSK stimulates adenylyl cyclase activity by interacting directly with catalytic subunit of adenylyl cyclase. In order to investigate if Ang II-induced overexpression of RGS2 could also modulate the activity of catalytic subunit of adenylyl cyclase, the effect of ANG II treatment for 1 h that increased the expression of RGS2 was examined on FSK-stimulated adenylyl cyclase activity. The results shown in Table 1 indicate that FSK-mediated stimulation of adenylyl cyclase was significantly enhanced in RGS2 overexpressed cells, and this stimulation was attenuated by the prior treatment of the cells with RGS2 specific antibody. However, these treatments did not alter the basal adenylyl cyclase activity.


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  		Table 1 Effect of RGS2 antibody on FSK-stimulated adenylyl cyclase activity in A10 vascular smooth muscle cells (VSMC)

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
We have previously demonstrated that Ang II treatment of A10 VSMC for 24 h augmented the expression of Gi{alpha}-2 and Gi{alpha}-3 proteins and their mRNA, however, the Gi functions were not increased but attenuated [23,24]. This attenuation was shown to be attributed partly to the phosphorylation of Gi{alpha}-protein which uncouples the receptor from adenylyl cyclase [24]. In the present studies we demonstrate for the first time that Ang II-induced overexpression of RGS2 protein may also be responsible for short-term regulation of Gi-mediated adenylyl cyclase signalling in A10 VSMC.

Ang II treatment of A10 VSMC increased the expression of both Gi (Gi{alpha}-2 and Gi{alpha}-3) and RGS2 proteins in a time dependent manner, however, Ang II-induced increased expression of Gi{alpha}-2 and Gi{alpha}-3 remained elevated up to 24 h of treatment, whereas, Ang II exerted biphasic effect on the expression of RGS2 protein. The maximal increase was observed at 2 h which started declining thereafter and reached basal level at 8 h. The increased expression of RGS2 was at transcriptional level because actinomycin D, an inhibitor of RNA synthesis attenuated the enhanced expression of RGS2 to control levels. A similar time-dependent profile of increased expression of RGS2 mRNA by Ang II in VSMC has also been reported previously [20], however, these investigators did not examine the relationship between increased levels of RGS2 and adenylyl cyclase signalling.

We have shown that Ang II-evoked enhanced expression of Gi{alpha}-protein was reflected in enhanced receptor-independent and -dependent function of Gi{alpha}-protein only at 30 min of Ang II treatment, when RGS2 levels were not elevated. However, the overexpression of RGS2 by prolonged treatment with Ang II resulted in complete attenuation of Gi functions. For example, GTP{gamma}S-mediated inhibition of FSK-stimulated adenylyl cyclase activity that was increased at 30 min of Ang II treatment started declining thereafter and reached below control level at 2 h of Ang II treatment. Similarly, Ang II- and C-ANP4–23 inhibited adenylyl cyclase activity to maximal extent at 30 min of Ang II treatment and thereafter the inhibitions started decreasing and at 4 h, these were completely abolished. These results suggest that Ang II-induced overexpression of RGS2 may be implicated in attenuation of Gi-mediated adenylyl cyclase signalling. This notion is further substantiated by our results showing that prior treatment of the cells with antibody of RGS2 and not with antibody of RGS4 that blocked the Ang II-induced increased expression of RGS2 also prevented the attenuation of GTP{gamma}S-mediated inhibition of FSK-stimulated adenylyl cyclase activity. However, the chronic treatment of the cells (24 h) that has been shown to result in the attenuation of the Gi-mediated signalling [23] may not be attributed to the RGS2 protein because the levels of RGS2 proteins were not augmented but declined after 4 h of Ang II treatment and may involve some other mechanisms. In this regard, the phosphorylation of Gi{alpha}-protein by Ang II treatment of VSMC for 24 h has been reported previously [24] which may be responsible for the attenuation of Gi-mediated signalling. In support of this, Katada et al. [26] have reported the phosphorylation and thereby inactivation of Gi{alpha}-proteins and uncoupling of inhibitory hormone receptors from adenylyl cyclase by protein kinase C (PKC), the other signalling pathway to which AT1 receptors are coupled. In addition, downregulation of the receptors may also be one of the mechanisms that contribute to the attenuation of receptor-dependent functions of Gi protein. In this regard, the downregulation of Ang II and ANP-C receptor by Ang II treatment in VSMC has been reported previously [27,28]. Thus, taken together, it may be suggested that overexpression of RGS2 may be involved in the short-term regulation of Gi-mediated functions in response to Ang II, whereas, other mechanisms such as phosphorylation of Gi protein and receptor downregulation may be implicated in long-term (chronic) regulation of Gi-mediated signalling by Ang II.

A role of RGS2 in the attenuation of Gi-mediated adenylyl cyclase signalling may be important in the regulation of various cellular functions including vascular permeability, salt and water transport, vascular resistance and catecholamine release, all of which play a key role in the regulation of blood pressure. Alterations in the levels of Gi{alpha}-proteins, RGS2 and cAMP that result in the impaired cellular functions lead to various pathological states including hypertension. We have recently shown that inactivation of enhanced expression of Gi{alpha}-protein by pertussis toxin treatment that resulted in the enhanced levels of cAMP attenuated the development of high blood pressure in spontaneously hypertensive rats [29]. Recently, RGS2 knockout mice have been shown to be hypertensive and exhibited increased Gq{alpha} signaling [21], however, these investigators did not examine Gi-mediated adeylyl cyclase signaling in these mice. Taken together it may be suggested that the increased Gi-mediated adenylyl cyclase signaling in addition to Gq{alpha}-mediated signaling may contribute to the development of hypertension in RGS2 knock out mice.

In the present studies, we have also shown that treatment of A10 cells with Ang II for 30 min decreased isoproterenol-mediated stimulation of adenylyl cyclase, which may not be attributed to the overexpression of RGS2, because of the fact that the levels of RGS2 at 30 min of Ang II treatment were not significantly elevated. However, the overexpression of RGS2 at later time points (1 h to 4 h) resulted in the restoration of diminished stimulation of adenylyl cyclase by isoproterenol to control levels and not in the increased Gs-mediated stimulation of adenylyl cyclase. These results suggest that RGS2 may not be implicated in Gs{alpha}-mediated adenylyl cyclase signaling in VSMC. On the other hand, the inhibition of isoproterenol-mediated stimulation of adenylyl cyclase at 30 min of Ang II treatment may be attributed to the Ang II-evoked increased expression of Gi{alpha}-proteins which were functional and were not deactivated by RGS2. In this regard, the interaction between Gi{alpha} and Gs{alpha} has been well established. The increased expression of Gi{alpha} has been shown to decrease Gs{alpha}-mediated stimulation of adenylyl cyclase in heart and aorta from spontaneously hypertensive rats (SHR) as well as in other models of hypertension [30–32].

Furthermore, the augmentation of FSK-stimulated adenylyl cyclase activity by Ang II-evoked overexpression of RGS2 may be attributed to the deactivation of Gi{alpha}-protein or/and the activation of catalytic subunit of adenylyl cyclase. The inactivation of Gi{alpha}-proteins by pertussis toxin [29] or the decreased expression of Gi{alpha}-protein in platelets from SHR and hypertensive subjects has been shown to augment FSK-stimulated adenylyl cyclase activity [33,34]. In addition, the decreased expression of Gi{alpha}-protein by C-ANP4–23 has also been reported to augment FSK-stimulated adenylyl cyclase activity in A10 VSMC [35]. However, our results are in contrast to the studies of other investigators who have shown that RGS2 decreased the activity of adenylyl cyclase type III in olfactory neurons and activity of type V and VI in Sf9 membranes [36]. The apparent discrepancies may be attributed to the difference in the cell system and the methodology of overexpression of RGS2 (Ang II vs. recombinant RGS2).

In conclusion, we have provided the first evidence to demonstrate that overexpression of RGS2 by Ang II in vascular smooth muscle cells attenuates Gi-mediated adenylyl cyclase signaling. It may be suggested that the RGS2-mediated attenuation of Gi-mediated signaling and resultant increased levels of cAMP by decreasing vascular resistance may play an important role in the regulation of blood pressure.


   Acknowledgement
 
We would like to thank Christiane Laurier for her valuable secretarial help.


   Notes
 
* This study was supported by a grant from Canadian Institutes of Health Research (MOP 53074).

Time for primary review 31 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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