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Cardiovascular Research 2001 49(1):234-243; doi:10.1016/S0008-6363(00)00236-4
© 2001 by European Society of Cardiology
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Copyright © 2000, European Society of Cardiology

Lymphocytes from spontaneously hypertensive rats exhibit enhanced adenylyl cyclase-Gi protein signaling

Josée Marcil and Madhu B. Anand-Srivastava*

Department of Physiology, Faculty of Medicine, Groupe de recherche sur le système nerveux autonome, University of Montreal, C.P. 6128, succ. centre-ville, Montreal, Quebec, Canada H3C 3J7

* Corresponding author. Tel.: +1-514-343-2091; fax: +1-514-343-2111 anandsrm{at}physio.umontreal.ca

Received 12 July 2000; accepted 15 September 2000


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: We have previously demonstrated an augmented activation of Gi{alpha} proteins in heart and aorta from spontaneously hypertensive rats (SHRs), which was attributed to an enhanced expression of Gi{alpha} proteins. Since immortalized lymphoblasts derived from lymphocytes of hypertensive patients have been shown to have enhanced Gi activation, the present studies were undertaken to investigate if lymphocytes from SHRs also exhibit enhanced Gi activation and whether this activation is related to enhanced expression of Gi proteins. Methods: The levels of G-proteins and mRNA were determined by immunoblotting and Northern blotting techniques, using specific antibodies and cDNA probes, respectively. Adenylyl cyclase activity stimulated or inhibited by agonists was determined to examine the functions of G-proteins. Results: The levels of Gi{alpha}-2, Gi{alpha}-3, Gβ but not of Gs{alpha}45 and Gs{alpha}47 were significantly increased in lymphocytes from SHRs as compared to their control Wistar Kyoto (WKY) rats. Similarly the mRNA levels of Gi{alpha}-2 and Gi{alpha}-3 were significantly augmented in SHRs as compared to their age-matched WKYs. The increased levels of Gi{alpha} were reflected in increased functions of Gi in SHRs as indicated by increased inhibition of forskolin-stimulated adenylyl cyclase activity by GTP{gamma}S. The activity of adenylyl cyclase stimulated by GTP{gamma}S, isoproterenol, NECA, NaF and forskolin was significantly decreased in SHRs as compared to their age-matched WKY rats. On the other hand, inhibitory hormones, atrial natriuretic peptide and angiotensin II inhibited adenylyl cyclase activity to a greater extent in SHRs as compared to their age-matched WKY rats. Conclusions: These results indicate that lymphocytes from spontaneously hypertensive rats exhibit enhanced Gi activation (function) which may be attributed to the enhanced expression of Gi proteins. It may be suggested that enhanced Gi expression and associated signaling may be one of the factors responsible for enhanced lymphoblasts proliferation observed in hypertension.

KEYWORDS c-ANP4–23, a ring deleted peptide of atrial natriuretic peptide; C-ANP4–23, (des[Gln18,Ser19,Gln20, Leu21,Gly22]ANP4–23-NH2); FSK, forskolin; GTP{gamma}S, guanosine 5'-[{gamma}-thio]triphosphate; Gs, stimulatory guanine nucleotide regulatory protein; Gi, inhibitory guanine nucleotide regulatory protein, Go, guanine nucleotide protein of unknown function; NECA, N-ethylcarboxamide adenosine; ISO, isoproterenol, Ang II, angiotensin II


   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 which play a key regulatory role as transducer in a variety of signal transduction systems. These include adenylyl cyclase/cAMP system [1], the receptor-mediated activation of phospholipase C and phospholipase A2 [2,3] and a number of hormone and neurotransmitter-regulated ionic channels [4]. The hormone-sensitive adenylyl cyclase system is composed of three components, receptor, catalytic subunit and G-proteins; stimulatory (Gs) and inhibitory (Gi); which mediate the stimulatory and inhibitory responses of hormones on adenylyl cyclase, respectively [5,6]. The G-proteins are heterotrimeric proteins composed of {alpha}, β, {gamma} subunits [6]. The {alpha}-subunits bind and hydrolyze GTP and confer specificity in receptor and effector interactions [6]. The Gs{alpha} and Gi{alpha} subunit are known to be specifically ADP-ribosylated by cholera toxin (CT) and pertussis toxin (PT), respectively, and thereby modify the characteristics of these proteins [7,8]. CT irreversibly activates Gs proteins mediating the stimulation of adenylyl cyclase, whereas PT, in addition to Go, Go1 and Go2, acts on Gi protein and attenuates the GTP-dependent and receptor-mediated inhibition of adenylyl cyclase [8]. Recently, molecular cloning has revealed four different forms of Gs{alpha} resulting from the differential splicing of one gene [9,10], and three distinct forms of Gi{alpha}, Gi{alpha}-1, Gi{alpha}-2 and Gi{alpha}-3 encoded by three distinct genes [11]. All three forms of Gi{alpha} have been shown to be implicated in adenylyl cyclase inhibition [12] and activation of atrial Ach-K+ channels [13] whereas the possible functions of Go{alpha} are PT-sensitive stimulation of PLC [13] inhibition of neuronal Ca2+ channel [14] and interaction with GAP-43 in neuronal growth cones [15], however, Go does not directly interact with adenylyl cyclase [16]. The β{gamma} subunit, on the other hand, has been reported to regulate cardiac K+ channels, phospholipase C-β and adenylyl cyclase [17–19].

The alterations in the expression of Gs{alpha} or Gi{alpha} have been demonstrated in various pathophysiological conditions, such as heart failure, diabetes, and hypothyroidism [20–22]. We have recently shown an enhanced expression of Gi{alpha}2 and Gi{alpha}3 proteins and their mRNA and their relationship with adenylyl cyclase inhibition in heart and aorta from SHRs and other experimentally induced hypertensive rats [23–27], whereas the expression of Gs{alpha} protein and Gs{alpha} mRNA was either unaltered or decreased [25–27]. We have further shown that the enhanced expression of Gi protein occurs before the development of blood pressure suggesting that this may be one of the contributing factors for the pathogenesis of hypertension.

An impairment of Gs functions has been reported in lymphocytes from hypertensive patients [28]. Recently, immortalized lymphoblast derived from lymphocytes from hypertensive patients have been shown to exhibit an enhanced activation of pertussis toxin (PT) sensitive G-protein [29], an enhanced proliferation and enhanced activity of Na+/H+ exchanger isoform (NHE-1) [30], however, the studies on the expression and functions of Gi proteins are lacking in lymphocytes from hypertensive rats or patients. The implication of Gi proteins in the regulation of cell proliferation has been reported [31,32]. Suppression of Gi{alpha}-2 in liver and fat of transgenic mice was shown to be associated with a dramatic reduction in neonatal growth [31]. In addition, hepatocellular carcinoma cells that have increased expression of Gi protein showed increased proliferation [32]. The present studies were therefore undertaken to investigate if lymphocytes from hypertensive rats also exhibit enhanced expression of Gi{alpha} proteins, the abnormality that is frequently observed in hypertension and its relationship with adenylyl cyclase.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
Adenosine triphosphate, cyclic AMP, 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 desaminase (EC 3.5.4.4) were purchased from Boehringer-Mannheim (Montreal, Canada). N-ethylcarboxamide adenosine (NECA) was from Research Biochemicals (Wayland, MA, USA). [{alpha}-32P]ATP and Western blotting detection kit was from Amersham (Oakville, Canada). Hanks' balanced salt solution was purchased from ICN Pharmaceuticals (Montreal, Canada). Ficoll-Paque for in vitro lymphocyte isolation was purchased from Pharmacia (Baie d'Urfée, Québec, Canada). AS/7, EC/2 and RM/1 antibodies were from Dupont (Mississauga, Canada). The cDNAs encoding for Gi{alpha}-2, Gi{alpha}-3 and Gs{alpha} were obtained from Dr. Randall Reed (John Hopkins University) and Hiroshi Itoh (University of Tokyo) and RNA ribosomal 28S was obtained from Dr. Yoshihiro Ishikawa (Lederle Laboratory, New York, USA).

2.1 Isolation of lymphocytes
Lymphocytes were isolated according to the method of Böyum [33]. Male spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto rats (WKYs) 12-week-old purchased from Charles River (St. Constant, Québec, Canada) were anaesthetized and blood was drawn from the carotid artery into a tube containing citrate anticoagulant which consists of: 87 mM sodium citrate, 105 mM dextrose, 11 mM citric acid and 7 mM Na3PO4. A 1-ml portion of anticoagulant was used for each 10 ml of blood. The whole blood was centrifuged at 350 g for 15 min and the ring of lymphocyte was collected and suspended gently in Hanks' balanced salt solution. The suspension was layered on top of the Ficoll-Paque and then centrifuged at 1000 g for 20 min. The lymphocytes on top of the Ficoll-Paque were collected and washed in Hanks' balanced salt solution and centrifuged at 100 g for 10 min at 4°C. The lymphocyte membranes were prepared for the adenylyl cyclase assay or lymphocytes were used for the isolation of total RNA. All the animal procedures used in the present studies were reviewed and approved by the Comité de déontologie de l'expérimentation sur les animaux of the University of Montreal (No. 99050).

2.2 Preparation of lymphocyte membranes
The lymphocytes were homogenized in 10 mM Tris–HCl and 1 mM EDTA, pH 7.5, and centrifuged twice at 16 000 g for 15 min. The supernatant was discarded and the pellet was suspended in a buffer containing 10 mM Tris–HCl and 1 mM EDTA, pH 7.5, and used for adenylyl cyclase determination.

2.3 Immunoblotting
Immunoblotting of G-proteins was performed as described earlier [23]. After SDS–PAGE, the separated proteins were transferred to a nitrocellulose paper (Schleicer and Schuell) using a semidry transblot apparatus (Bio-Rad) at 15 V for 45 min. After transfer, the membranes were washed twice in phosphate-buffered saline (PBS) and were incubated in PBS containing 3% 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 2 h. The antigen–antibody complexes were detected by incubating the blots with goat anti-rabbit IgG (Bio-Rad) conjugated with horseradish peroxidase for 2 h at room temperature. The blots were washed three times 10 min 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.

2.4 Isolation of total RNA from lymphocyte
The total RNA was isolated as described previously [24] by the guanidium thiocyanate method described by Chomczynski and Sacchi [34]. DMSO–glyoxal treated total RNAs (5–10 µg) were resolved on 1% agarose gel electrophoresis and transferred to Hybond-N-filters (Amersham). The filters were prehybridized in a hybridization solution containing 600 nM NaCl, 8 mM EDTA, 120 mM 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 radiolabeled with ({alpha}-32P) dCTP (3000 Ci/mmol) by random priming as described previously [24]. Filters were hybridized at 65°C for 16 h in hybridization solution containing 10% of dextran sulfate and cDNA probe (1–3x106 cpm/ml). Filters were then rinsed twice for 30 min with a solution containing 300 mM NaCl, 4 mM EDTA, 60 mM Tris–HCl, pH 7.4, and 0.2% SDS and once for 30 min at 65°C in a solution containing 150 mM NaCl, 2 mM EDTA, 30 mM Tris–HCl, pH 7.4 and 0.1% SDS. Autoradiography was performed with X-ray films at –70°C for 24–48 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. The blots which had been probed with G-protein cDNA were dehybridized by washing for 2 h in 50% formamide, 300 mM NaCl, 4 mM EDTA and 60 mM Tris–HCl, pH 7.4, and rehybridized overnight at room temperature with the oligonucleotide. The 32-mer oligonucleotide recognizing the 28S RNA was end-labeled with [{gamma}-32P] ATP using T4 polynucleotide kinase as described previously [24]. RNA was directly quantified by densitometric scanning using an enhanced laser densitometer (LKB Ultroscan XL) 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.

2.5 Adenylyl cyclase activity determination
Adenylyl cyclase activity was determined by measuring [32P]cAMP formation from [{alpha}32-P]ATP, as described previously [23]. Typical assay medium contained 50 mM glycylglycine, pH 7.5, 0.5 mM MgATP, 5 mM MgCl2, 0.5 mM cAMP, 100 mM NaCl, 1 mM 3-isobutyl-1-methylxanthine (or otherwise as indicated), 0.1 mM EGTA, 10 µM GTP (or otherwise as indicated), 1–1.5x106 CPM ({alpha}-32P) and an ATP-regenerating system consisting of 2 mM 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 had been thermally equilibrated for 2 min at 37°C. The reactions conducted in triplicate for 10 min at 37°C were terminated by the addition of 0.6 ml of 120 mM zinc acetate containing 0.5 mM unlabeled cAMP. cAMP was purified by coprecipitation of the nucleotides with ZnCO3 and by the addition of 0.5 ml of 144 mM Na2CO3 and subsequent chromatography by the double-column system as described previously [33]. The unlabeled 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.

2.6 Analysis of data
Data are presented as mean±SEM. Comparison between groups were made using either Student's t-test or analysis of variance (ANOVA) where appropriate. The results were considered significantly different if P<0.05.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Experimental animals
The systolic blood pressure was significantly higher in 12-week-old SHRs compared to age-matched WKYs (WKYs 117.9±4.6, SHRs; 181.3±4.8, n = 20), however, the heart weight/body weight (mg/g) ratio was not significantly different in the two groups — WKYs; 3.43±0.17 (n = 20) and SHRs; 3.56±0.38 (n = 20).

3.2 G-protein levels
Since immortalized lymphoblast derived from lymphocytes from hypertensive patients exhibited increased activation of Gi proteins [29], it was of interest to investigate if lymphocytes from SHRs exhibit increased expression of Gi proteins which may be responsible for the enhanced Gi activation. For this reason, we determined the levels of G-proteins by immunoblotting experiments 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.

As shown in Fig. 1, AS/7 antibody recognized a single protein of 40 kDa referred to as Gi{alpha}-2 in lymphocytes from WKYs and SHRs (Fig. 1A); however, the relative amount of immunodetectable Gi{alpha}-2 as determined by densitometric scanning was significantly increased by about 20.0±1.2% (n = 3). Similarly, EC/2 antibodies recognized a single protein of 41 kDa referred to as Gi{alpha}-3 in lymphocytes from both WKYs and SHRs (Fig. 1B), however, the relative amount of immunodetectable Gi{alpha}-3 was also significantly increased by about 25.0±1.0% (n = 3) in SHRs as compared to their age-matched WKYs as determined by densitometric scanning. In addition, SW/1 antibody recognized a single protein of approximately 35 kDa in WKYs and SHRs (Fig. 1C); however, the relative amount of immunodetectable Gβ was significantly increased by about 195.0±2.1% (n = 3) in SHRs as compared to their age-matched WKYs (Fig. 1C). On the other hand, RM/1 antibody recognized two isoforms of Gs{alpha}; Gs{alpha}45, and Gs{alpha}52 in both WKYs and SHRs (Fig. 1D), however, the relative amounts of immunodetectable Gs{alpha}45 and Gs{alpha}52 were not significantly different in both groups as determined by densitometric scanning.


Figure 1
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  		Fig. 1 Quantification of G-proteins (Gs{alpha}, Gi{alpha}-2, Gi{alpha}-3 and Gβ) in lymphocyte from 12-week-old WKYs and their age-matched SHRs by immunoblotting. The lymphocyte membrane proteins (30 µg) from WKYs and SHRs were resolved by SDS–PAGE and transferred to nitrocellulose paper, which was then immunoblotted with antibody RM/1 for Gs{alpha} (A), AS/7 for Gi{alpha}-2 (B), EC/2 for Gi{alpha}-3 (C) and SW/1 for Gβ (D) as described under Methods. The detection of the different G-proteins was performed by using the chemiluminescence (ECL) Western blotting detection reagents from Amersham. The autoradiographs are representative of three separate experiments.

 
We extended our studies further to investigate if mRNA levels change concomitantly with protein levels and therefore examined the mRNA level by Northern blot analysis using specific cDNA probes encoding for Gs{alpha}, Gi{alpha}-2 and Gi{alpha}-3 (Fig. 2). The Gs{alpha} cDNA probe detected a message of 1.8 Kb in lymphocyte from WKYs and SHRs (Fig. 2A); and the amount of mRNA was not different in SHRs than WKYs. Similarly, the Gi{alpha}-2 cDNA probe detected a message of 2.3 Kb in lymphocyte from both WKYs and SHRs, however, the amount of Gi{alpha}-2 mRNA was significantly increased by about 45.0±2.6% (n = 3) in SHRs as compared to WKYs (Fig. 2B). In addition, the Gi{alpha}-3 cDNA probe detected a message of 3.5 Kb in lymphocyte from WKYs and SHRs and the Gi{alpha}-3 mRNA levels were also significantly increased by about 125.0±3.2% (n = 3) in SHRs as compared to WKYs (Fig. 2C). The alteration in Gi{alpha}-2 and Gi{alpha}-3 mRNA levels in lymphocytes from SHRs may not be attributed to the variation in the amounts of total RNA in individual samples applied to the gels, due to the fact that the hybridization with an oligonucleotide that recognizes a highly conserved region of the 28S rRNA showed a similar amount of 28S rRNA loaded from WKYs and SHRs on the gels (lower panels).


Figure 2
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  		Fig. 2 mRNA expression of Gs{alpha} (A), Gi{alpha}-2 (B) and Gi{alpha}-3 (C) in lymphocytes from 12-week-old WKYs and their age-matched SHRs. Total RNA (5 µg) isolated from the lymphocytes of WKYs and SHRs was subjected to 1% agarose gel electrophoresis and transferred to nylon membranes, the blots were then hybridized with full length radiolabeled cDNA probe encoding Gs{alpha} (A), Gi{alpha}-2 (B) and Gi{alpha}3 (C) (upper panels) and further rehybridized with an oligonucleotide recognizing the 28S rRNA (lower panel) as described in Methods. The autoradiogram is representative of three separate experiments.

 
3.3 Effect of guanine nucleotide on adenylyl cyclase
In order to investigate if impaired response of guanine nucleotides to adenylyl cyclase exists in lymphocytes, the ability of GTP{gamma}S to stimulate adenylyl cyclase activity was examined in lymphocytes from SHR and WKY rats. As shown in Fig. 3, GTP{gamma}S stimulated adenylyl cyclase activity in a concentration-dependent manner in lymphocytes from both WKYs and SHRs, however, the extent of stimulation was significantly decreased in SHRs as compared to their control WKY rats. GTP{gamma}S at 10 µM stimulated adenylyl cyclase activity by about 1100% in lymphocytes from WKYs and about 600% in lymphocytes from SHRs.


Figure 3
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  		Fig. 3 Effect of GTP{gamma}S on adenylyl cyclase activity in lymphocyte membranes from 12-week-old WKYs and age-matched SHRs. Adenylyl cyclase activity was determined in the absence or presence of various concentrations of GTP{gamma}S in WKYs ({circ}) and SHRs (bullet) as described in Methods. Basal enzyme activities in WKYs and SHRs were 288.7±10.4 and 240.3±25.6 pmol cAMP (mg protein, 10 min)–1, respectively. Values are mean±SEM of six separate experiments. *, P<0.05, **, P<0.01 and ***, P<0.001.

 
3.4 Hormonal stimulation of adenylyl cyclase activity
To examine whether Gs-mediated hormonal responsiveness was also decreased in lymphocytes from SHRs, we studied the effect of isoproterenol, a β-adrenergic agonist, and N-ethylcarboxamide adenosine (NECA) which interacts with adenosine A2 receptors on adenylyl cyclase activity in lymphocytes from WKY and SHR rats. Isoproterenol (50 µM) and NECA (10 µM) stimulated adenylyl cyclase activity from both WKYs and SHRs to various degrees, however, the extent of stimulation was significantly decreased in SHRs (Fig. 4A and B). Isoproterenol at 50 µM stimulated adenylyl cyclase activity by about 200% in WKYs and 70% in SHRs (4A) and NECA (10 µM), stimulated the enzyme activity by 150±5% in WKYs and 110±2% in SHRs (4B). Fig. 4C shows the concentration-dependent inhibition of isoproterenol-stimulated adenylyl cyclase activity in lymphocytes from WKYs and SHRs. The decreased stimulation of adenylyl cyclase in SHRs as compared to WKYs was associated with decreased Vmax and not of increased IC50 (~0.5 µM in both WKYs and SHRs).


Figure 4
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  		Fig. 4 Effect of isoproterenol and NECA on adenylyl cyclase activity in lymphocyte membranes from 12-week-old WKYs and age-matched SHRs. Adenylyl cyclase activity was determined in the presence of 10 µM GTP alone or in combination with 50 µM isoproterenol (A) or 10 µM NECA (B) or different concentrations of isoproterenol (C) in WKYs ({circ}) or SHRs (bullet) as described in Methods. 3-Isobutyl-1-methylxanthine was replaced by Ro 20-1724 (Roche). Enzyme activities in the presence of 10 µM of GTP in WKYs and SHRs were 264.8±13.4 and 257.0±7.4 pmol cAMP (mg protein10 min)–1, respectively. Values are mean±SEM of four separate experiments. **, P<0.01.

 
3.5 Hormonal inhibition of adenylyl cyclase activity
Since Gi{alpha} proteins are implicated in the hormonal inhibition of adenylyl cyclase, we investigated the inhibitory effects of Ang II and C-ANP4–23 on adenylyl cyclase activity that are mediated through Gi{alpha} protein [35,36] in lymphocytes from WKYs and SHRs. Fig. 5 shows that C-ANP4–23 and Ang II inhibited adenylyl cyclase activity slightly but significantly by about 15% in lymphocytes from WKYs, however, these inhibitions were augmented by about 2-fold in lymphocytes from SHRs (Fig. 5A and B). Fig. 5C shows that C-ANP4–23 inhibited adenylyl cyclase activity in a concentration-dependent manner in both WKYs and SHRs, however, the extent of inhibition was significantly greater in SHRs.


Figure 5
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  		Fig. 5 Effect of Ang II and C-ANP4–23 on adenylyl cyclase activity in lymphocyte membranes from 12-week-old WKY and age-matched SHR rats. Adenylyl cyclase activity was determined in the presence of 10 µM GTP{gamma}S alone or in the presence of 10 µM Ang II (A) or 0.1 µM C-ANP4–23 (B) or different concentrations of C-ANP4–23 (C) in WKYs ({circ}) or SHRs (bullet) as described in Methods. Enzyme activities in the presence of 10 µM GTP{gamma}S in WKYs and SHRs were 2289.8±40.5 and 2578.2±14.5 pmol cAMP (mg protein10 min)–1, respectively. Values are mean±SEM of six experiments. * P<0.05, **, P<0.01 and ***, P<0.001.

 
To investigate if the enhanced expression of Gi{alpha} in SHRs is also reflected in Gi functions, the effect of low concentrations of GTP{gamma}S on FSK-stimulated adenylyl cyclase activity was examined in lymphocytes from SHR and WKY rats. The results shown in Fig. 6 indicate that GTP{gamma}S at low concentrations inhibited FSK-stimulated adenylyl cyclase activity in a concentration-dependent manner in both WKYs and SHRs, however, the inhibition was significantly augmented in lymphocytes from SHRs as compared to WKYs. At 0.1 nM, GTP{gamma}S inhibited FSK-stimulated enzyme activity by about 15% in WKYs and by about 50% in SHRs.


Figure 6
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  		Fig. 6 Effect of GTP{gamma}S on forskolin (FSK)-stimulated adenylyl cyclase activity in lymphocyte membranes from 12 weeks-old WKYs and their age-matched SHRs. Adenylyl cyclase activity was determined in the presence of 100 µM forskolin alone or in combination with various concentrations of GTP{gamma}S in WKYs ({circ}) or SHRs (bullet) as described in Methods. Values are mean±SEM of three separate experiments. *, P<0.05 and **, P<0.01.

 
3.6 FSK- and NaF-stimulated adenylyl cyclase activity
Fig. 7 shows the stimulatory effects of FSK and NaF on adenylyl cyclase in lymphocytes from SHR and WKY rats. Forskolin and NaF stimulated adenylyl cyclase to various degrees in both WKYs and SHRs, however, the percent stimulations were significantly decreased in lymphocytes from SHRs as compared to WKYs.


Figure 7
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  		Fig. 7 Effect of forskolin (FSK) and NaF on adenylyl cyclase activity in lymphocyte membranes from 12-week-old WKYs and their age-matched SHRs. Adenylyl cyclase activity was determined in the absence or presence of 50 µM forskolin (A) or 10 mM NaF (B) as described in Methods. GTP or GTP{gamma}S was omitted from the reaction mixture. Basal enzyme activities in WKYs and SHRs were 263.1±19.3 and 230.7±11.6 pmol cAMP (mg protein10 min)–1. Values are mean±SEM of six separate experiments. *, P<0.05 and **, P<0.01.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
We and others have previously reported an enhanced expression of Gi{alpha} proteins in heart and aorta and decreased expression in platelets from SHRs and different models of hypertension [23–27,37,38] which was associated with altered sensitivity of adenylyl cyclase to hormonal stimulation/inhibition. In the present studies, we demonstrate for the first time that lymphocytes from SHRs showed an enhanced expression of Gi{alpha}-2, Gi{alpha}-3 and Gβ proteins as compared to WKYs, whereas the levels of Gs{alpha} proteins were unaltered. An increased activation of PT-sensitive G-proteins has recently been reported in immortalized lymphoblasts derived from hypertensive subjects [29], however, these investigators were unable to observe any changes in the levels of PT-sensitive G-proteins. In addition, the expression of β subunit Gβ2 and Gβ4 was also unaltered in lymphoblasts from hypertensive patients [39]. These apparent discrepancies may be attributed to the different models used in the present studies (rat vs. human). The levels of Gi{alpha}-2 and Gi{alpha}-3 mRNA were also increased in SHR lymphocytes which may be responsible for the observed increase in Gi{alpha} protein levels. Whether the increase in G-protein expression in SHRs is attributed to the intrinsic hormonal factors is not known. However, increased plasma levels of catecholamines and angiotensin II in SHRs may contribute to the enhanced expression of Gi{alpha} protein. In this regard, angiotensin II treatment has been shown to increase the expression of Gi{alpha}-2 and Gi{alpha}-3 in vascular smooth muscle cells [40]. Similarly, β-adrenergic receptor stimulation has also been reported to enhance the expression of Gi{alpha} proteins in rat heart [41].

In the present studies, we demonstrate that, like lymphoblasts from hypertensive patients [29], the lymphocytes from SHRs also express enhanced Gi activation (functions) as demonstrated by increased inhibition of FSK-stimulated adenylyl cyclase activity by GTP{gamma}S as compared to the lymphocytes from WKYs. In addition, the receptor-mediated functions of Gi were also enhanced in hypertensive lymphocytes as was shown by increased inhibition of adenylyl cyclase by C-ANP4–23 and Ang II, which may be attributed to the upregulation of receptors or to the exaggerated postreceptor events including enhanced Gi{alpha} protein expression. Since Ang II and ANP receptors have been shown to be unaltered or decreased in hypertensive tissues [42,43], it may be possible that an enhanced expression of Gi{alpha}-2 and Gi{alpha}-3 proteins in hypertensive lymphocytes may contribute to the increased sensitivity of adenylyl cyclase to hormonal inhibition.

Our results showing an unaltered expression of Gs{alpha} protein and Gs{alpha} mRNA in lymphocytes from SHRs are in agreement with the studies of Feldman et al. [28] who were unable to observe any changes in the expression of Gs{alpha} protein in lymphocytes from younger hypertensive patients. Taken together, it may be suggested that a decreased stimulation of adenylyl cyclase by GTP{gamma}S in hypertensive lymphocytes may not be attributed to Gs{alpha} protein and may be due to the increased expression of Gi{alpha}-2, Gi{alpha}-3 and Gβ in SHRs. On the other hand, the decreased sensitivity of adenylyl cyclase to isoproterenol and NECA stimulation in hypertensive lymphocytes are in accordance with other studies [28,44] and may be attributed to the down regulation of receptors [45], impaired receptor G-protein coupling [28] increased activity and/or expression of G-protein coupled receptor kinase (GRK) that phosphorylates agonist-coupled G-protein-linked receptors [46,47] or to the increased expression of Gi{alpha} proteins. However, Yoshikawa et al. [44] did not observe any changes in the concentration and affinity of β-adrenergic receptors in lymphocytes from hypertensive patients as compared to the normotensive subjects. Taken together, it may be possible that the enhanced levels of Gi{alpha}-2 and Gi{alpha}-3 as well as of GRK may be responsible for the diminished responsiveness of adenylyl cyclase to NECA and isoproterenol in lymphocytes from SHRs.

The decreased stimulation of adenylyl cyclase by NaF and forskolin in lymphocytes from SHRs as compared to WKYs is in agreement with the studies performed in heart and aorta from SHRs [23] and other models of hypertensive rats [25–27] as well as in lymphocytes from hypertensive patients [28] and may be attributed to the defective catalytic subunit per se or to the overexpression of Gi{alpha} or both. In this regard, the implication of Gi in FSK-mediated stimulation of adenylyl cyclase has been reported [48]. In addition, the requirement of Gs{alpha} and guanine nucleotide for FSK activation of adenylyl cyclase has also been shown [49]. However, since the levels of Gs{alpha} were not altered in the present studies, the diminished stimulation of adenylyl cyclase by FSK in SHRs cannot be attributed to impaired Gs{alpha}.

In conclusion, we have demonstrated for the first time that lymphocytes from SHRs, like lymphoblasts derived from lymphocytes from hypertensive patients, exhibit an enhanced activation (functions) of Gi{alpha} proteins which may be attributed to the enhanced expression of Gi{alpha}-2 and Gi{alpha}-3 genes and translated proteins. From these results, it can be suggested that the enhanced expression and activation of Gi-protein and associated signaling may be one of the factors responsible for enhanced lymphoblast proliferation in hypertension.

Time for primary review 27 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 32-mer oligonucleotide. We would also like to thank Christiane Laurier for her valuable secretarial help. This work was supported by a grant from the Medical Research Council of Canada (MT 15046).


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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