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Cardiovascular Research 1998 39(2):492-505; doi:10.1016/S0008-6363(98)00111-4
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Overexpression of Gi-proteins precedes the development of DOCA-salt-induced hypertension: relationship with adenylyl cyclase

Josée Marcil, Jacques de Champlain and Madhu B Anand-Srivastava*

Department of Physiology, Faculty of Medicine, Groupe de Recherche sur le Système Nerveux Autonome, University of Montreal, Montréal, Québec, Canada H3C 3J7

* Corresponding author: Tel.: +1 (514) 343 2091; Fax: +1 (514) 343 2111

Received 17 June 1997; accepted 24 February 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: In the present studies, we have investigated if aorta, like heart from deoxycorticosterone acetate (DOCA)-salt hypertensive rats, (HR) also exhibit enhanced expression of G-protein levels and if these alterations occur before or after the development of blood pressure. Methods: Sprague–Dawley rats treated with DOCA-salt or vehicle for 1, 2, 3 and 4 weeks were used for these studies. The levels of inhibitory guanine nucleotide regulatory proteins (Gi{alpha}-2, Gi{alpha}-3) and Gβ proteins were determined by immunoblotting, whereas the levels of Gi{alpha}-2 and Gi{alpha}-3 and adenylyl cyclase type V enzyme mRNA were determined by Northern-blotting techniques. Results: The blood pressure was significantly increased in DOCA-salt-treated rats as compared to sham-operated rats after 2 to 4 weeks of treatment; whereas no change in blood pressure was observed after 1 week of treatment (prehypertensive state). However, the levels of Gi{alpha}-2, Gi{alpha}-3 and Gβ proteins and Gi{alpha}-2 and Gi{alpha}-3 mRNA were significantly enhanced in hearts and aorta from DOCA-salt treated rats after 1 week of treatment and remained elevated up to 4 weeks of treatment. In addition, the Gi-mediated inhibitions of adenylyl cyclase by Angiotensin II (Ang II) and C-ANF4-23 were also greater in DOCA-salt-treated rats as compared to sham-operated rats after 1 week and longer periods of treatments (2 to 4 weeks). On the other hand, the levels of Gs{alpha} were not altered up to 2 weeks of DOCA-salt treatment but significantly decreased in rats treated for 3 and 4 weeks. Furthermore, the stimulatory effects of guanine 5'-[{gamma}-thio]triphosphate (GTP{gamma}S), isoproterenol and forskolin on adenylyl cyclase were decreased in both hearts and aorta from DOCA-salt-treated rats after 1 to 4 weeks of treatment as compared to sham-operated rats. The mRNA levels of adenylyl cyclase, type V enzyme in hearts from DOCA-salt treated rats were significantly decreased after 3 and 4 weeks of DOCA-salt treatment but not in rats treated for 1 or 2 weeks. Conclusions: These results indicate that the enhanced expression of Gi{alpha}-2 and Gi{alpha}-3 precedes the development of blood pressure in DOCA-salt-induced hypertension. It can thus be suggested that the increased levels of Gi proteins and resulting decreased levels of cAMP may be one of the factors that contribute to the impaired cardiac contractility and increased vascular tone in DOCA-salt hypertension.

KEYWORDS C-ANF4–23, a ring-deleted analog of atrial natriuretic factor, C-ANF4–23 (des[Gln18, Ser19, Gln20, Leu21, Gly22] ANF4–23-NH2]; FSK, forskolin; ADA, adenosine desaminase; GTP{gamma}S, guanosine 5'-[{gamma}-thio]triphosphate; Gs, stimulatory guanine nucleotide regulatory protein; Gi, inhibitory guanine nucleotide regulatory protein; WKY, Wistar-Kyoto rats; SHR, spontaneously hypertensive rats


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Guanine nucleotide regulatory proteins (G-proteins) are a family of GTP-binding proteins that play an important role in the regulation of a variety of signal transduction systems including adenylyl cyclase/cAMP system. The adenylyl cyclase system is composed of three components: receptor, catalytic subunit and stimulatory (Gs) and inhibitory (Gi) guanine nucleotide regulatory proteins [1, 2]. The stimulation and inhibition of adenylyl cyclase by hormones are mediated by two distinct G-proteins, Gs and Gi respectively that couple the receptor to the catalytic subunit. The G-proteins are heterotrimeric and are composed of {alpha}, β and {gamma} subunits. The specificity of G-proteins is attributed to {alpha} subunits. Molecular cloning has revealed four different forms of Gs{alpha} resulting from the differential splicing of one gene [3–5]and three distinct forms of Gi{alpha}; Gi{alpha}-1, Gi{alpha}-2 and Gi{alpha}-3 encoded by three distinct genes [6–8]. All three forms of Gi-{alpha} (Gi{alpha}1-3) have been reported to be implicated in adenylyl cyclase inhibition [8]and activation of atrial K+ channels [9]. In addition, five different β subunits of 35–36 kDa and seven {gamma} subunits of 8–10 kDa have been identified by molecular cloning [10, 11]. Several functions of the complex β{gamma} subunit have been reported such as anchoring the G-protein to the membrane [10], stimulation of type II and IV enzyme [12], inhibition of Ca2+/calmodulin or Gs-activated type I enzyme and activation of muscarinic-gated atrial K+ channels [13]. Molecular cloning has also revealed eight different types of adenylyl cyclases but only types V and VI have been identified in heart and aorta [14–16].

The adenylyl cyclase/cAMP system has been implicated in both the control of heart contractility [17, 18]and vascular smooth muscle tone [19, 20]. Several abnormalities in adenylyl cyclase activity and cAMP levels, which may be involved in the elevation of blood pressure, have been reported in cardiovascular tissues from genetic (spontaneously hypertensive rats (SHR)) and different models of experimentally-induced hypertensive rats [21–27]. An increased expression of Gi-protein and Gi protein mRNA in heart and aorta from SHR and hearts from DOCA-salt hypertensive rats with established hypertension has been reported [21–23, 26]. On the other hand, the levels of Gs{alpha} were shown to be unaltered in SHR but were decreased in DOCA-salt HR [21–23]. The increased expression of Gi{alpha}-2 has also been reported to occur before the onset of hypertension in SHR [28]. The present studies were undertaken to investigate if similar alterations in G-protein expression and their functions observed in heart [23]are also observed in aorta from DOCA-salt HR and if these changes precede the development of blood pressure or are associated with hypertension.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
Adenosine triphosphate, cyclic AMP and isoproterenol were purchased from Sigma (St. Louis, MO, USA). Creatine kinase, myokinase (EC 2.7.4.3 [EC] ), guanosine 5'- [3'-thio] triphosphate (GTP{gamma}S), guanosine triphosphate (GTP) and adenosine deaminase (EC 3.5.4.4) were purchased from Boehringer-Mannheim (Montreal, Quebec, Canada). [{alpha}-32P] ATP, [{alpha}-32P]dCTP (3000 Ci/mmol) and [{gamma}-32P]ATP (3000 Ci/mmol) and Western-blotting detection kit were from Amersham (Oakville, Ontario, Canada). AS/7, EC/2, RM/1 and SW/1 antibodies were from Dupont (Mississauga, Ontario, Canada). cDNAs of G-proteins were kindly obtained from Dr. Randall Reed (John Hopkins University) and Hiroshi Itoh (University of Tokyo). Chemicals necessary for total RNA extraction and Northern-blot analysis were obtained from Sigma (St. Louis, MO, USA), except guanidinium thiocyanate which was from Research Organics (Cleveland, OH, USA), and glyoxal from BDH (St. Laurent, Québec, Canada).

2.1 Experimental animals
DOCA-salt hypertension was induced in Sprague–Dawley rats by weekly injections of a suspension of deoxycorticosterone (10 mg) subcutaneously to uninephrectomized rats, as described earlier [29]. The DOCA-salt treated rats were given free access of 0.9% saline in drinking water, whereas control animals were given tap water to drink. Four groups of rats were used in the present studies. Each group has control and DOCA-salt-treated rats and received DOCA or DOCA-salt respectively for 1 or 2 or 3 or 4 weeks. At the end of each period of treatment, DOCA-salt-treated and their respective control rats were sacrificed by decapitation after measuring the blood pressure by the tail cuff method (without anesthesia). Hearts and aortae from control and DOCA-salt treated rats were dissected out and frozen immediately in liquid nitrogen and stored at –80°C.

2.2 Isolation of heart sarcolemma
The heart sarcolemma, from control and DOCA-salt-treated rats, was isolated essentially by a method described elsewhere [21, 27]. The ventricles were washed thoroughly, cut into small pieces, and homogenized in a virtis blender for 30 s in 10 volumes of 10 mmol/l Tris–HCl buffer (pH 7.4) containing 1 mmol/l of EDTA. The homogenate was filtered through cheesecloth and centrifuged at 1000g for 10 min. The sediment was suspended in 20–25 volumes of 10 mmol/l of Tris–HCl buffer (pH 7.4), stirred in a cold-room for 30 min and recentrifuged at 1000g for 10 min. This process was repeated twice, first by suspending the sediment in 10 mmol/l of Tris–HCl buffer at pH 8.0, and then in the same buffer at pH 7.4. The sediment was again suspended in 20–25 volume of 10 mmol/l Tris–HCl (pH 7.4), extracted with 0.4 mol/l LiBr for 45 min, and centrifuged at 1000g for 10 min. It was then suspended in 10 mmol/l Tris–HCl (pH 7.4), stirred for 15–20 min, and centrifuged at 1000g for 10 min. The sarcolemmal fraction thus obtained was suspended in a buffer containing 10 mmol/l Tris–HCl and 1 mmol/l EDTA (pH 7.4), and was used for the determination of adenylyl cyclase activity and immunoblotting studies.

2.3 Preparation of washed aorta particle
Washed aorta particles were prepared as described previously [21]. The dissected aortae were quickly frozen in liquid nitrogen and stored at –80°C until assayed. The frozen aortae were pulverized to a fine powder in a mortar cooled in liquid nitrogen. The aorta powder was homogenized in a Teflon/glass homogenizer in a buffer containing 10 mmol/l Tris–HCl and 1 mmol/l EDTA, pH 7.5. The homogenate was centrifuged at 16 000g for 15 min at 4°C. The supernatant was discarded and the pellet was suspended in the Tris–EDTA buffer, pH 7.5 and used for determination of adenylyl cyclase activity and immunoblotting studies.

2.4 Adenylyl cyclase activity determination
Adenylyl cyclase activity was determined by measuring [32P]cAMP formation from [{alpha}32-P]ATP, as described previously [21]. Typical assay medium contained 50 mmol/l glycylglycine pH 7.5, 0.5 mmol/l MgATP, 5 mmol/l MgCl2, 0.5 mmol/l cAMP, 100 mmol/l NaCl, 1 mmol/l 3-isobutyl-1-methylxanthine (or otherwise as indicated), 0.1 mmol/l EGTA, 10 µmol/l GTP (or otherwise as indicated), [{alpha}32P]ATP (1–1.5x106 CPM) 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 reaction mixture to the membranes which have been thermally equilibrated for 2 min at 37°C. The reactions conducted in triplicate for 10 min were terminated by the addition of 0.6 ml of 120 mmol/l zinc acetate containing 0.5 mmol/l unlabelled cAMP. cAMP was purified by coprecipitation of other nucleotides with ZnCO3 and by the addition of 0.5 ml of 144 mmol/l Na2CO3 and subsequent chromatography by the double column system as described by Salomon et al. [30]. The unlabelled cAMP served to monitor the recovery of the [32P]cAMP by measuring absorbance at 259 nm. Under the assay conditions used, adenylyl cyclase activity was linear with respect to protein concentration and time of incubation. Protein concentration was determined as described by Lowry et al. [31]with crystalline bovine serum albumin (BSA) as standard.

2.5 Immunoblotting
After SDS–PAGE, the separated proteins were transferred to a nitrocellulose paper (Schleicher and Schuell) using a semidry transblot apparatus (Bio-Rad) at 15 volt for 45 min as described previously [21]. After transfer, the membranes were washed twice in phosphate-buffered saline (PBS) and were incubated in PBS containing 3% dehydrated milk at room temperature for 2 h. The blots were then incubated with antisera against G proteins in PBS containing 1.5% dehydrated milk and 0.1% Tween 20 at room temperature for overnight. The antigen–antibody complexes were detected by incubating the blots with goat anti-rabbit IgG (Bio-Rad) conjugated with horseradish peroxidase for 3 h at room temperature. The blots were washed three times with PBS before reacting with enhanced-chemiluminescence (ECL) Western-blotting detection reagents from Amersham.

The autoradiograms were quantified by densitometric scanning using an enhanced laser densitometer (LKB Ultroscan XL, Pharmacia, Quebec, Canada) and gel scan XL evolution software (version 2.1, Pharmacia). The scanning was one dimensional and scanned the entire area of protein bands in autoradiograms.

2.6 Extraction of total RNA and Northern-blot analysis
Total RNA was isolated as described previously [26]by the guanidinium thiocyanate–phenol–chloroform method described by Chomczynski and Sacchi [32]. Briefly, frozen heart ventricles were homogenized in a denaturating solution (solution D) containing 4 mol/l guanidinium thiocyanate, 25 mmol/l sodium citrate pH 7.0, 0.5% sarkosyl and 0.1 mol/l 2-mercaptoethanol. The homogenates were extracted once with 1 volume of phenol and 0.2 volumes of chloroform–isoamylalcohol (49:1) in the presence of 0.2 mol/l sodium acetate, pH 4.0, and once with 1 volume of chloroform–isoamylalcohol (49:1). Total RNA was then precipitated with isopropanol. Following a second precipitation in solution D and isopropanol (v:v), total RNA was washed in 70% ethanol and resuspended in water.

2.7 Radiolabelling of the probes and Northern analysis
DMSO/glyoxal treated total RNAs (5–10µg) were resolved on 1% agarose gel electrophoresis and transferred to Hybond-N-filters (Amersham). The filters were prehybridized in a hybridyzation solution containing 600 mmol/l NaCl, 8 mmol/l EDTA, 120 mmol/l Tris–HCl pH 7.4, 0.1% sodium pyrophosphate, 0.2% SDS and 500 U/ml heparin at 65°C for 6 h before the addition of cDNA probe. The probes were radiolabelled with ({alpha}–32P) dCTP (3000 Ci/mmol) by random priming essentially as described by Feinberg et al. [33]. Filters were hybridized at 65°C for 16 h in hybridization solution containing 10% dextran sulfate and cDNA probe at 1–3x106 cpm/ml. Filters were then rinsed twice for 30 min at 65°C with a solution containing 300 mmol/l NaCl, 4 mmol/l EDTA, 60 mmol/l Tris–HCl pH 7.4, and 0.2% and one time for 30 min at 65°C with a solution containing 150 mmol/l NaCl, 2 mmol/l EDTA, 30 mmol/l Tris–HCl pH 7.4 and 0.1% SDS. Autoradiography was performed with X-ray films at –70°C for 24 to 48 h. In order to assess the possibility of any variation in the amounts of total RNA in individual samples applied to the gel, each filter was hybridized with a 32-mer oligonucleotide recognizing a highly conserved region in the 28S ribosomal RNA donated by Dr Yoshihiro Ishikawa (Lederle laboratory, New York). The blots which had been probed with the G-protein cDNA were dehybridized by washing for 2 h at 65°C in 50% formamide, 300 mmol/l NaCl, 4 mmol/l EDTA and 60 mmol/l Tris–HCl pH 7.4, and rehybridized overnight at room temperature with the oligonucleotide. The 32-mer oligonucleotide recognizing the 28 S rRNA was end-labelled with [{gamma}-32P]ATP using T4 polynucleotide kinase as described by Sambrook et al. [34]. RNA was directly quantified by densitometric scanning using an enhanced laser densitometer (LKB Ultroscan XL, Pharmacia, Québec, Canada) and gel scan XL evolution software (version 2.1, Pharmacia). The scanning was one dimensional and scanned the entire area of mRNA in the autoradiograms.

2.8 Analysis of data
Data are presented as mean±SEM. Comparisons between groups (control DOCA and DOCA-salt-treated rats) were made using Student's t-test for unpaired samples, whereas ANOVA followed by post-hoc student's t-test was performed for the dose–response curve of isoproterenol-stimulated adenylyl cyclase activity. The results were considered significantly different if P<0.05.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
As shown in Table 1, the systolic blood pressure as well as heart/body weight ratio was not different in rats treated with DOCA-salt for 1 week as compared to sham-operated rats, however it was significantly increased after 2,3 and 4 weeks of treatment.


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  		Table 1 Body weight, heart weight, body weight to heart weight ratio and blood pressure of DOCA-salt and their control after various duration of treatment

 
3.1 G-proteins levels
G-proteins play an important role in the regulation of adenylyl cyclase activity. We have recently shown an altered expression of G-proteins in hearts from DOCA-salt hypertensive rats after 2 and 4 weeks of DOCA-salt treatment [23]. To investigate if aorta also exhibits similar alterations in G-protein levels and whether these changes occur before the onset of hypertension, we determined the levels of G-proteins in heart and aorta from rats treated with DOCA-salt for 1 week and longer periods by immunoblotting techniques using specific antibodies AS/7 against Gi{alpha}-1 and Gi{alpha}-2, EC/2 against Gi{alpha}-3, RM/1 against Gs{alpha} and SW/1 against Gβ. Fig. 1A shows that RM/1 antibody recognized three isoforms of Gs{alpha}; Gs{alpha}45, Gs{alpha}47 and Gs{alpha}52 in heart and aorta from both control and DOCA-salt-treated rats after 1 to 4 weeks of treatment. However, the relative amount of Gs{alpha}45 and not of Gs{alpha}47 and Gs{alpha}52 in heart was slightly but significantly decreased by 16.1±2.1% (n=5); 37.3±6.2% (n=5) in rats treated with DOCA-salt for 3 and 4 weeks respectively and was not altered after 1 and 2 weeks of DOCA-salt treatment as compared to their sham-operated rats. On the other hand, aorta from DOCA-salt treated rats (1–4 weeks) did not exhibit any significant alterations in the levels of Gs{alpha}45, Gs{alpha}47 and Gs{alpha}52 as compared to their sham-operated control rats. In addition, AS/7 antibodies recognized a single protein of 40 kDa referred to as Gi{alpha}-2 (Gi{alpha}-1 has been shown to be absent in heart and aorta [35, 36]) from both control and rats treated with DOCA-salt for various time periods, however the relative amount of immunodetectable Gi{alpha}-2 in heart was significantly increased in DOCA-salt-treated rats after 1, 2, 3 and 4 weeks of treatments compared to their sham-operated rats (Fig. 1B). The increase in the levels of Gi{alpha}-2 protein, as determined by densitometric scanning, in hearts from rats treated with DOCA-salt for 1, 2, 3 and 4 weeks respectively as compared to their respective control rats was 59.3±4.3% (n=6); 112.7±7.6% (n=6); 257.8±17.3% (n=6); 172.0±8% (n=7) (Fig. 3A) and in aorta was 158.4±9.6% (n=5), 136.3±13.3% (n=5), 188.0±4% (n=4) and 251.0±5% (n=4) after 1, 2, 3 and 4 weeks of treatment respectively (Fig. 3B). Similarly, EC/2 antibody recognized a single protein of 41 kDa referred to as Gi{alpha}-3 in heart and aorta in both control and rats treated with DOCA-salt for 1 to 4 weeks (Fig. 1C), however like Gi{alpha}-2, the relative amount of immunodetectable Gi{alpha}-3 in heart, as determined by densitometric scanning, was also significantly increased in DOCA-salt rats as compared to their sham-operated rats by 27.3±1.6% (n=6); 133.6±13.7% (n=6); 207.3±13.4% (n=6); 298±6% (n=7) after 1, 2, 3 and 4 weeks of DOCA-salt treatment, respectively (Fig. 3A). In addition, aorta from DOCA-salt-treated rats also exhibited enhanced levels of Gi{alpha}-3 as compared to their respective controls. The increases were 137.2±10.4% (n=6), 131.3±7.3% (n=6), 165.0±3.2% (n=4), and 156.0±7.1% (n=4) after 1, 2, 3 and 4 weeks of DOCA-salt treatment (Fig. 3A). Furthermore, the relative amount of immunodetectable Gβ recognized by SW/1 antibody was significantly increased in hearts from rats treated with DOCA-salt for 1, 2, 3 and 4 weeks by 15.3±5.2% (n=5); 11.4±1.5% (n=5); 20.3±0.7% (n=5); 25.7±2.1% (n=5) respectively and in aorta by 15.3±2.4% (n=5) after 1 week, 134.0±14% (n=6) after 2 weeks 194.0±9.5% (n=4) after 3 weeks and 268.0±15% (n=4) after 4 weeks of DOCA-salt treatment as compared to their sham-operated rats (Fig. 1D).


Figure 1
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  		Fig. 1 Quantification of G-proteins by immunoblotting in heart sarcolemma from control and 1- to 4-week DOCA-salt-treated rats. The membrane protein (50 µg) from the four different groups were separated on SDS–PAGE and transferred to nitrocellulose, which was then immunoblotted using RM/1 antibody specific for Gs{alpha} (A), AS/7 specific for Gi{alpha}-2 (B) and EC/2 specific for Gi{alpha}-3 (C) and SW/1 specific for β subunit (D) as described under Section 2. The autoradiogram is representative of 6 separate experiments. The detection of the different G-proteins was performed by using the chemiluminescence (ECL) Western-blotting detection reagents from Amersham. Detection of levels of Gs{alpha}, Gi{alpha}-2, Gi{alpha}-3 and β subunit was also done in aorta from 1- to 4-week DOCA-salt-treated rats by the same technique as described under Section 2. The autoradiogram is representative of 4–5 separate experiments.

 

Figure 3
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  		Fig. 3 Densitometric scanning of the Gi{alpha}-2 and Gi{alpha}-3 protein (A,C) or mRNA (B,D) from DOCA-salt-treated rats. The results are expressed as the increase in G-protein or mRNA levels in DOCA-salt-treated rats over their respective control rats, which have been taken as 100%. Values are means±SEM of 4 (B), 6 (A,C) or 3 (D) separate experiments. *P<0.05, **P<0.01, ***P<0.001.

 
We extended our studies further to investigate if mRNA levels change concomitantly with protein levels and examined the mRNA levels by Northern-blot analysis using specific cDNA probes encoding for Gs{alpha}, Gi{alpha}-2 and Gi{alpha}-3. Fig. 2A and C show the mRNA expression of Gi{alpha}-2 in heart and aorta respectively, from DOCA-salt HR after different times of treatment. The Gi{alpha}-2 cDNA probe detected a message of 2.3 kilobases in hearts and aorta from control and DOCA-salt-treated rats, however, the amount of Gi{alpha}–2 mRNA, determined by densitometric scanning was significantly increased by 52.1±3.2% (n=4); 122.4±0.8% (n=4); 144.2±0.7% (n=4); 115.5±3.4% (n=4) in hearts from rats treated with DOCA-salt for 1, 2, 3 and 4 weeks respectively (Fig. 3B) and by 47.9±3.5% (n=3) 107.8±5.7% (n=3) and 165.2±9.6% in aorta (Fig. 3D) as compared to their sham-operated rats. Similarly, Gi{alpha}-3 cDNA probe detected a message of 3.5 kilobase in hearts and aorta from control and DOCA-salt-treated rats (Fig. 2B and D); however, the mRNA levels in hearts were significantly increased parallel to the time of DOCA-salt treatment by 137.6±0.6% (n=4); 232.1±3.4% (n=4); 259.9±9.8% (n=4); 334.2±13.6% (n=4) after 1, 2, 3 and 4 weeks of DOCA-salt treatment respectively (Fig. 3B) and by 127.6±5.4, 102.7±2.1 and 97.0±8.2% in aorta from rats treated with DOCA-salt for 2, 3 and 4 weeks in comparison to their sham-operated rats (Fig. 3D). In addition, the Gs{alpha} cDNA probe detected a message of 1.8 kilobase in hearts from both control and DOCA-salt-treated rats and the levels of Gs{alpha} mRNA, as reported earlier [23], were decreased in DOCA-salt HR by 11.3±0.8% (n=6) and 34.5±6.5% (n=6) after 3 and 4 weeks and not after 1 and 2 weeks of treatments as compared to their control rats (data not shown). The alterations in Gi{alpha}-2 and Gi{alpha}-3 mRNA levels in heart and aorta from DOCA-salt HR may not be attributed to the variation in the amounts of total RNA in individual samples applied to the gels, because of the fact that the hybridization with an oligonucleotide that recognized a highly conserved region of the 28S rRNA, showed a similar amount of 28S rRNA loaded from DOCA-salt HR and their control on the gels.


Figure 2
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  		Fig. 2 Expression of Gi{alpha}-2 and Gi{alpha}-3 mRNA in hearts and aorta from DOCA-salt-treated rats and their controls. Total RNA (10 µg) isolated from the heart and aorta from two groups of rats were separated on 1% agarose and transferred to nylon membrane, which was then hybridized with a full-length cDNA probe encoding for Gi{alpha}-2 (A, C) and Gi{alpha}-3 (B, D) (upper panel). These filters were rehybridized with an oligonucleotide recognizing the 28S rRNA (lower panel) as described under Section 2. The autoradiogram is representative of 4 separate experiments.

 
3.2 Effect of guanine nucleotides on adenylyl cyclase
Since the expression of Gi{alpha} and Gs{alpha} proteins was altered in hearts and aorta from rats treated with DOCA salt after 1 and 3 weeks of treatment respectively, it was of interest to investigate, if these alterations are also reflected in G-protein functions. Table 2 shows the ability of GTP and GTP{gamma}S to stimulate adenylyl cyclase activity in heart and aorta from control and DOCA-salt-treated rats after various periods of treatments. GTP and GTP{gamma}S stimulated adenylyl cyclase activity in control and DOCA-salt treated rats, however, the extent of stimulation was significantly lower in DOCA-salt-treated rats after 1,2,3 and 4 weeks of treatment as compared to control rats (Table 2). Similar results were also observed in aorta from control and DOCA-salt-treated rats and suggest that G-protein function impaired in prehypertensive state (after 1 week of treatment) may be due to the increased levels of Gi{alpha}-2 and Gi{alpha}-3 and not of Gs{alpha}. In addition, adenylyl cyclase activity in the absence (basal) or presence of GTP or GTP{gamma}S was increased parallel to the time of treatment with DOCA (CTL–DOCA) and was significantly greater after 4 weeks of treatment as compared to 1 week of treatment.


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  		Table 2 GTP and GTP{gamma}S-stimulated adenylyl cyclase activity in heart and aorta from DOCA-salt HR and their control sham-operated rats after 1, 2, 3 and 4 weeks of treatment with DOCA

 
3.3 Hormonal regulation of adenylyl cyclase
The interaction of Gs{alpha} and Gi{alpha} has been well established [37, 38]. The increased expression of Gi{alpha} has been shown to result in decreased responsiveness of adenylyl cyclase to hormonal stimulation [21], whereas decreased expression of Gi{alpha} results in an enhanced stimulation of adenylyl cyclase by stimulatory hormones [38]. Since the levels of Gi{alpha} are increased in 1 week DOCA-salt-treated rats without any alterations in Gs{alpha}, it was of interest to examine if the Gs{alpha}-mediated hormonal effects are altered in heart and aorta from DOCA-salt rats after 1 week and longer periods of treatment. Results shown in Fig. 4 indicate that isoproterenol stimulated adenylyl cyclase in heart (Fig. 4A) and aorta (Fig. 4B) from control and DOCA-salt-treated rats (1–4 weeks of treatment) to various degrees, however, the stimulation was slightly but significantly decreased in DOCA-salt treated rats at all the times of DOCA-salt treatment. The decreases were about 20, 30, 30, and 25% respectively in hearts (n=4) and about 30, 40, 35 and 50% in aorta (n=4) from 1, 2, 3 and 4 weeks DOCA-salt-treated rats. Fig. 4C shows the concentration-dependent inhibition of isoproterenol-stimulated adenylyl cyclase actvity in hearts from 1 week DOCA-salt treated and their control rats. The decreased stimulation of adenylyl cyclase by isoproterenol in DOCA-salt treated rats as compared to control rats was associated with decreased Vmax and not of increased Kd ({approx}50 nM in both control and treated rats).


Figure 4
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  		Fig. 4 Effect of isoproterenol on adenylyl cyclase activity in heart sarcolemma and aorta from control and 1- to 4-week DOCA-salt-treated rats. Adenylyl cyclase activity was determined as described under Section 2in the presence of 10 µM of GTP alone (basal) or in combination with 50 µM of isoproterenol in hearts (A) and aorta (B) from 1- to 4-week DOCA-salt-treated rats (Figure 4) and their control ({square}). The values are the mean±SEM of 3 separate experiments. Basal enzyme activity in the presence of GTP in control and DOCA-salt rats are presented in Table 2. (C) Stimulation of adenylyl cylcase activity with a different concentration of isoproterenol in heart from DOCA-salt-treated rats after 1 week of treatment (bullet) and their control ({circ}). Basal enzyme activity in the presence of GTP in control and DOCA-salt HR were 60.89±2.3 and 54.6±2.1 pmol cAMP/(mg protein·10 min–1). Values are the mean±SEM of 4 different experiments. Statistical analysis was performed by 2-way ANOVA followed by post-hoc student's t-test. **P<0.01, ***P<0.001 vs control.

 
Hormonal inhibition of adenylyl cyclase is mediated through Gi{alpha} proteins [39]. Since the levels of Gi{alpha}-2 and Gi{alpha}-3 were increased in DOCA-salt-treated rats as compared to control rats, we investigated the relationship between the enhanced expression of Gi{alpha} proteins and the effect of inhibitory hormones on adenylyl cyclase in control and DOCA-salt treated rats. As shown in Fig. 5A, C-ANF4–23, that inhibits adenylyl cyclase through Gi{alpha} [40, 41], inhibited adenylyl cyclase activity in control and DOCA-salt-treated rats at various times of treatment, however, the extent of inhibition was greater in DOCA-salt-treated rats as compared to the control by about 65, 65, 80 and 60% in hearts (n=5) (Fig. 5A) and by about 30, 45, 80 and 65% in aorta (n=5) (Fig. 5B) in rats treated with DOCA-salt for 1, 2, 3 and 4 weeks, respectively. Similarly AngII that has been shown to inhibit adenylyl cyclase in heart and aorta [42–44]inhibited adenylyl cyclase activity to a greater extent in hearts from DOCA-salt-treated rats as compared to control rats by 50, 45, 40 and 20% (n=5) (5C) and by about 55, 45, 60 and 95% (n=5) (5D) in aorta after 1, 2, 3 and 4 weeks of treatment, respectively.


Figure 5
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  		Fig. 5 Effect of C-ANF4–23 and angiotensin II on adenylyl cyclase activity in heart sarcolemma and aorta from control and 1- to 4-week DOCA-salt-treated rats. Adenylyl cyclase activity was determined as described under Section 2in the presence of 10 µM GTP{gamma}S alone (basal) or in combination with 10–7 M C-ANF4–23 in heart (A) and aorta (B) or with 10–4 M Ang II in heart (C) and aorta (D) from 1- to 4-week DOCA-salt-treated rats (Figure 5) and their control ({square}). Inhibition of adenylyl cylcase activity with ANF in heart (A) and aorta (B) in 1- to 4-week DOCA-salt-treated rats. Values are the mean±SEM of 5 different experiments. Basal enzyme activity in the presence of 10 µM GTP{gamma}S in heart and aorta from control and DOCA-salt HR are presented in Table 2 *P<0.05, **P<0.01, ***P<0.001 vs control.

 
3.4 Forskolin-stimulated adenylyl cyclase activity
Forskolin, which stimulates adenylyl cyclase by interacting with the catalytic subunit of adenylyl cyclase, stimulated the enzyme activity in hearts (Fig. 6A) and aorta (Fig. 6B) from control and DOCA-salt treated rats after 1 week to 4 weeks of treatment, however, the extent of stimulation was diminished in hearts from DOCA-salt-treated rats by about 20, 20, 20 and 15% (n=4) and in aorta by about 40, 28, 25 and 45% (n=4) after 1, 2, 3 and 4 weeks of treatment respectively. In order to investigate if the decreased stimulation is attributed to the decreased levels of catalytic subunit (type V and VI enzyme expressed in heart [45, 46]), the mRNA levels of type V enzyme were determined in hearts by using a cDNA probe encoding type V enzyme. As shown in Fig. 6C, the mRNA levels of type V adenylyl cyclase were not altered in DOCA-salt treated rats after 1 and 2 weeks of treatment, however, these were decreased by 28.1±2.9% (n=4) and 47.7±3.4% (n=4) after 3 and 4 weeks of DOCA-salt treatment respectively, as quantified by densitometric scanning (Fig. 6D).


Figure 6
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  		Fig. 6 Effect of forskolin on adenylyl cyclase activity in heart and aorta from control and 1- to 4-week DOCA-salt-treated rats. Adenylyl cyclase activity was determined in heart sarcolemma (A) and aorta (B) from 1- to 4-week DOCA-salt-treated rats (Figure 6) and their control ({square}) as described under Section 2in the absence (basal) or presence of 50 µM forskolin. GTP and GTP{gamma}S were omitted from the reaction mixture. Basal enzyme activity in the absence of forskolin in control and DOCA-salt-treated rats are presented in Table 2. Values are the mean±SEM of 4 separate experiments *P<0.05, ** P<0.01, ***P<0.001. vs control (C). Expression of the catalytic subunit type V mRNA in heart from their control and DOCA-salt after 1 to 4 weeks of treatment. Total RNA (10 µg) isolated from the heart of the four different groups were separated on a 1% agarose and transferred to a nylon membrane, which was then hybridized with a full-length cDNA probe (upper panel). These filters were rehybridized with an oligonucleotide recognizing the 28S rRNA (lower panel). The autoradiogram is representative of 4 separate experiments. (D) Densitometric scanning of the catalytic subunit type V in heart from 1- to 4-week DOCA-salt-treated rats. The results are expressed as the percentage mRNA in DOCA-salt-treated rats of their control which has been taken as 100%. Values are means±SEM of 4 separate experiments. ***P<0.001.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
In the present studies, we demonstrate that aorta from DOCA-salt HR exhibit altered expression of G proteins (increased Gi and decreased Gs) and consequent changes in G-protein-mediated adenylyl cyclase activity. The increased levels of Gi{alpha}-2 and Gi{alpha}-3 in aorta and heart were observed before the onset of hypertension, whereas decreased levels of Gs{alpha} proteins were associated with the elevation of blood pressure.

The blood pressure was not different in rats treated with DOCA-salt for 1 week as compared to control rats and it started increasing after 2 weeks of DOCA-salt treatment and reached a significantly higher level after 4 weeks of treatment. However, the levels of Gi{alpha}-2 and Gi{alpha}-3 proteins and mRNA, as quantified by immunoblotting and Northern-blotting analyses respectively, were slightly but significantly higher in hearts from rats treated with DOCA salt for 1 week as compared to control rats and were subsequently increased in parallel to the increase in blood pressure. The increased levels of Gi{alpha}-2 and Gi{alpha}-3 mRNA in hearts after 1 week of DOCA-salt treatment suggests that genes for Gi{alpha}-2 and Gi{alpha}-3 are overexpressed in prehypertensive state and may partly be responsible for the observed increase in protein levels. However, a parallel increase in Gi{alpha}-2 and Gi{alpha}-3 protein expression in heart and aorta with blood pressure in rats treated for 2 to 4 weeks with DOCA-salt suggest a relationship between Gi protein expression and elevation of blood pressure, whereas an increase in the levels of Gi{alpha}-2 and Gi{alpha}-3 before the onset of hypertension suggests that the augmentation in Gi{alpha} proteins precedes the development of blood pressure in DOCA-salt hypertension. Whether this increase in Gi{alpha} protein in prehypertensive state may be attributed to the intrinsic humoral factors is not known. The augmented plasma levels of catecholamines and ANF in DOCA-salt hypertensive rats after 4 weeks of DOCA-salt treatment [47, 48]may contribute to the increased levels of Gi{alpha}. However, no studies have been reported to indicate increased levels of these hormones in early stages of development of hypertension. The increased expression of Gi{alpha}-2 and Gi{alpha}-3 proteins as well as mRNA was reflected in Gi-mediated functions. Concurrent to the increased levels of Gi{alpha}-2 and Gi{alpha}-3 in hearts and aorta after 1 week of DOCA-salt treatment, the extent of C-ANF4-23 and Ang II-mediated inhibition of adenylyl cyclase was also increased that may be attributed to the upregulation of receptors or to the exaggerated post-receptor events including Gi{alpha} protein expression. Since ANF receptors are down and not up-regulated in DOCA-salt hypertensive rats after 4 weeks of DOCA-salt treatment [49], it may be possible that an enhanced expression of Gi{alpha}-2 and Gi{alpha}-3 proteins in DOCA-salt HR may contribute to the increased sensitivity of adenylyl cyclase to ANF inhibition. However, on the other hand, the upregulation of Ang II receptors in DOCA-salt HR [50]may also be responsible for the increased inhibition of adenylyl cyclase. Whether ANF and Ang II receptors are down or up-regulated in prehypertensive state after 1 week of DOCA-salt treatment is not known and has to be explored.

Our results on decreased levels of Gs{alpha} protein in hearts from DOCA-salt HR after 2 and 4 weeks of treatment are consistent with the studies reported previously [23]. However, in the present studies, we have shown that unlike Gi{alpha} proteins, the levels of Gs{alpha} were not altered in hearts and aorta in prehypertensive state (after 1 week of DOCA-salt treatment) and suggest that Gs{alpha} protein may not play a role in the development of hypertension and may be associated with hypertension and/or hypertrophy. In this regard, the decreased levels and functions of Gs{alpha} have been shown in compensated left ventricular hypertrophy [51], pressure-overload left ventricular failure [52]and myocardial ischemia [53, 54]. In addition, Asano et al. [55]have also shown the implication of Gs{alpha} and decreased cAMP levels in the attenuated relaxation of feromal arteries from spontaneously hypertensive rats to β-adrenergic agonists.

Since the levels of Gs{alpha} protein and Gs{alpha} mRNA were not altered in heart and aorta from rats treated with DOCA-salt for 1 week, the decreased stimulation of adenylyl cyclase by GTP{gamma}S in these rats may not be attributed to the decreased levels of Gs{alpha}. However, the decreased levels of Gs{alpha} in DOCA-salt-treated rats after 2–4 weeks of treatment may contribute to the diminished responsiveness of adenylyl cyclase to GTP{gamma}S stimulation. In addition, the decreased sensitivity of isoproterenol to stimulate adenylyl cyclase in DOCA-salt-treated rats after 1 week of treatment may be attributed to the down regulation of β-adrenergic receptors, one of the mechanisms responsible for the diminished sensitivity of adenylyl cyclase to hormones in hypertension [56]. Whether the down regulation of β-adrenergic receptors occurs in the prehypertensive state is not known and has to be investigated. Since Gi{alpha} and Gβ{gamma} modulate the functions of Gs{alpha} [37, 38, 57], it may be possible that the enhanced levels of Gi{alpha}-2, Gi{alpha}-3 and Gβ in hearts and aorta from DOCA-salt-treated prehypertensive rats may contribute to the diminished responsiveness of adenylyl cyclase to GTP{gamma}S and isoproterenol stimulation. The decreased sensitivity of isoproterenol to stimulate adenylyl cyclase which results in decreased formation of cAMP may be responsible for the decreased chronotropic and inotropic responses to β-adrenergic stimulation in hypertension [58]as well as for the decreased isometric relaxation rates for papillary muscles in SHR [59]and attenuated relaxation of femoral arteries to β-adrenergic agonist in SHR [55].

The decreased sensitivity of adenylyl cyclase to FSK stimulation in hearts and aorta from rats treated with DOCA-salt for 1 week may be due to the defective catalytic subunit per se or the overexpression of Gi{alpha} or both. However, since the mRNA levels of type V enzyme were not altered in these rats and the levels of type VI enzyme were not determined, it may be possible that the overexpression of Gi{alpha}-2 and Gi{alpha}-3 may be partly responsible for the decreased sensitivity of adenylyl cyclase to FSK stimulation. In this regard, the role of Gi{alpha} in FSK-mediated stimulation of adenylyl cyclase has been reported [40]. In addition, the requirement of Gs{alpha} and guanine nucleotides for FSK activation of adenylyl cyclase has also been shown [60]. Since the levels of Gs{alpha} are decreased in 2 to 4 weeks DOCA-salt treated HR and not in prehypertensive DOCA-salt treated rats, the diminished stimulation of adenylyl cyclase by FSK in prehypertensive DOCA-salt treated rats cannot be attributed to the impaired Gs{alpha}, however, its role in eliciting decreased FSK stimulation of adenylyl cyclase in 2 to 4 weeks DOCA-salt-treated rats may be a possibility.

Taken together, it may be interesting to postulate that the increased and decreased cardiovascular responsiveness of adenylyl cyclase to Ang II and ANF inhibition and isoproterenol and FSK stimulation respectively, that occur in the prehypertensive state and result in decreased formation of cAMP may contribute to the impaired cardiac contractility and increased vascular tone in DOCA-salt hypertension.

In conclusion, we have demonstrated, for the first time, that the enhanced expression of Gi{alpha}-2 and Gi{alpha}-3 in heart and aorta preceeds the development of blood pressure, whereas the levels of Gs{alpha} are decreased after the development of blood pressure.

Time for primary review 17 days.


   Acknowledgements
 
We are grateful to Drs. Randall Reed, Hiroshi Itoh for their kind gift of cDNAs of G-proteins, Dr. Yoshihiro Ishikawa for the kind gift of cDNA of adenylyl cyclase type V enzyme and 32-mer oligonucletoide. We would like to thank Christiane Laurier for her valuable secretarial help, Dr. Réjean Couture and Eric Cellier for their help in statistical analysis of data. This work was supported by grants from Québec Heart Foundation and the Medical Research Council of Canada.


   References
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 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
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