Volume overload cardiac hypertrophy exhibits decreased expression of Gsalpha and not of Gialpha in heart

Francesco Di Fusco, Shehla Hashim, and Madhu B. Anand-Srivastava

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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We have recently reported enhanced levels of Gialpha proteins in genetic and other experimentally induced models of hypertension, whereas the levels of Gsalpha were decreased in hypertensive rats expressing cardiac hypertrophy. The present studies were undertaken to investigate whether the decreased levels of Gsalpha are associated with cardiac hypertrophy per se and used an aortocaval fistula (AV shunt; volume overload) rat model that exclusively expresses cardiac hypertrophy. Cardiac hypertrophy in Sprague-Dawley rats (200-250 g) was induced under anesthesia, and, after a period of 10 days, the hearts were used for adenylyl cyclase activity determination, protein quantification, and mRNA level determination. A temporal relationship between the expression of Gsalpha proteins and cardiac hypertrophy was also examined on days 2, 3, 7, and 10 after induction of AV shunt in the rat. The heart-to-body-weight ratio (mg/g) was significantly increased in AV shunt rats after 3, 7, and 10 days of induction of AV shunt compared with sham-operated controls, whereas arterial blood pressure was not different between the two groups. Guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S) stimulated adenylyl cyclase activity in a concentration-dependent manner in heart membranes from both groups; however, the degree of stimulation was significantly decreased in AV shunt rats. In addition, the stimulatory effects of isoproterenol were also diminished in AV shunt rats compared with control rats, whereas glucagon-stimulated adenylyl cyclase activity was not different in the two groups. The inhibitory effects of oxotremorine (receptor-dependent Gi functions) and low concentrations of GTPgamma S on forskolin-stimulated adenylyl cyclase activity (receptor-independent Gi functions) were not different in the two groups. In addition forskolin and NaF also stimulated adenylyl cyclase activity to a lesser degree in AV shunt rats compared with control rats. The levels of Gialpha -2 and Gialpha -3 proteins and mRNA, as determined by immunoblotting and Northern blotting, respectively, were not different in both groups; however, the levels of Gsalpha 45 and Gsalpha 47, and not of Gsalpha 52, proteins were significantly decreased in AV shunt rats by days 7 and 10 compared with control rats, whereas no change was observed on days 2 and 3 after induction of AV shunt. These results suggest that the decreased expression of Gsalpha proteins may not be the cause but the effect of hypertrophy and that the diminished responsiveness of adenylyl cyclase to GTPgamma S, isoproterenol, NaF, and forskolin in hearts from AV shunt rats may partly be due to the decreased expression of Gsalpha . It can be concluded from these studies that the decreased expression of Gsalpha may be associated with cardiac hypertrophy and not with arterial hypertension.

G protein; adenylyl cyclase; aortocaval fistula; AV shunt


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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THE ADENYLYL CYCLASE/cAMP signal transduction system has been implicated in the regulation of various physiological functions such as vascular tone and reactivity, cardiac functions, platelet functions, etc. (49). The adenylyl cyclase system consists of three distinct components: receptor, catalytic subunit, and guanine nucleotide regulatory proteins (G proteins). The stimulatory and inhibitory responses of hormones on adenylyl cyclase are mediated via the stimulatory (Gs) and inhibitory (Gi) proteins, respectively (20, 41, 42). G proteins are heterotrimeric proteins composed of alpha -, beta -, and gamma -subunits, and the specificity of G proteins is attributed to the alpha -subunit (42). Molecular cloning has revealed four different isoforms of Gsalpha resulting from the differential splicing of one gene (8, 34, 36) and three distinct isoforms of Gialpha : Gialpha -1, Gialpha -2, and Gialpha -3 encoded by three different genes (23-25). All three forms of Gialpha have been shown to be implicated in adenylyl cyclase inhibition (51) and the activation of ACh-K+ channels (9). Five different beta -subunits of 35-36 kDa and seven gamma -subunits of 8-10 kDa have been identified by molecular cloning (10, 40). The Gbeta gamma -subunit has been shown to regulate various effectors including adenylyl cyclase, phospholipase Cbeta , and K+ channels (40, 45, 50). Of the eight types of adenylyl cyclase that have been cloned and expressed (14), only two types, namely, types V and VI, have been identified in the heart, aorta, and brain (26, 35). Adenylyl cyclase types II and IV are activated by Gbeta gamma in the presence of Gsalpha ; type I is inhibited by Gbeta gamma ; and types III, V, and VI do not appear to be directly regulated by Gbeta gamma (46, 48).

Alterations in G protein levels and adenylyl cyclase activity and its responsiveness to various hormones have been documented in cardiovascular tissues from genetic (spontaneously hypertensive rats, SHR) and various experimental models of hypertension (1, 2-4, 7, 30). We have recently shown an increased expression of Gialpha -2 and Gialpha -3 at protein and mRNA levels and an altered hormonal inhibition and stimulation of adenylyl cyclase in heart and aorta from SHR as well as in DOCA-salt hypertensive rats (2, 4, 5, 7, 30), whereas an unaltered expression of Gialpha and Gsalpha proteins has also been reported in hearts from SHR and other models of hypertension (32, 33). However, due to the expression of cardiac hypertrophy with hypertension, it is not known whether these changes are due to the expressed hypertrophy or hypertension. We have recently shown that Nomega -nitro-L-arginine methyl ester (L-NAME) hypertensive rats that do not express cardiac hypertrophy (6) exhibited an increased expression of Gi levels with unaltered Gs levels (15). Taken together, it may be possible that decreased levels of Gsalpha may be associated with cardiac hypertrophy and not with arterial hypertension. To investigate this possibility, we have used volume-overload (VO) cardiac hypertrophied rats that express cardiac hypertrophy but not arterial hypertension (18) and examined the G protein expression (Gialpha and Gsalpha ) and regulation of adenylyl cyclase by various modulators in VO hypertrophied rat hearts. We have shown that aortocaval fistula (AV shunt) rats exhibit decreased expression of Gsalpha protein and mRNA, which occurs only after the development of cardiac hypertrophy.


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Materials. Plasmids containing rat cDNAs encoding Gialpha -2, Gialpha -3, Gsalpha , and the adenylyl cyclase catalytic subunit V were kindly obtained from Dr. Randall Reed from the John Hopkins University and Dr. Hiroshi Itoh from the University of Tokyo. The 32-mer oligonucleotide that recognizes a highly conserved region of the 28S rRNA was kindly donated by Dr. Yoshihiro Ishiwaka from Lederle Laboratory of New York. Chemicals necessary for total RNA extraction and Northern blot analysis were obtained from Sigma Chemical (St. Louis, MO), except guanidinium thiocyanate, which was from Research Organics (St. Laurent, PQ, Canada). Enzymes used for radiolabeling of cDNA probes were obtained from BRL (Burlington, Ontario, Canada), and other chemicals were from Pharmacia (Baie D'Urfee, PQ, Canada). Nylon filter (Hybond-N), [alpha -32P]dCTP (3,000 Ci/mmol) and [gamma -32P]ATP (3,000 Ci/mmol) were purchased from Amersham (Oakville, Ontario, Canada).

Induction of VO hypertrophy by AV shunt. Male Sprague-Dawley rats (200-250 g) were purchased from Charles River Canada (St. Constant, Quebec, Canada). Cardiac hypertrophy was induced in rats by the method described by Garcia and Diebold (18). Briefly, each rat was anesthetized with pentobarbital sodium (60 mg/kg body wt), and an excision was made along the abdominal cavity and the visceral organs displaced. The descending aorta was isolated and ligated caudal to the renal artery and cephalic to the aortic bifurcation. The aorta was punctured through the adjacent wall and into the inferior vena cava with a 20-gauge needle. The needle was removed fully, and the initial aortic entry puncture point was sealed with a drop of cyanoacrylate glue. The ligation was removed after 10-20 s to insure proper drying, and the patency of the shunt was verified visually by mixing of arterial and venous blood. The entire surgical procedure took <10 min. The sham-operated control protocol was identical to AV shunt induction, except no aortic puncture was performed. After 10 days, the blood pressure was measured by the tail-cuff method, and the rats were killed by decapitation. Some rats were killed after 2, 3, and 7 days after the induction of AV shunt. The hearts were removed for adenylyl cyclase activity determination, mRNA levels, and G protein quantification.

This method of induction of VO cardiac hypertrophy has been shown to produce an "eccentric" form of cardiac hypertrophy that is characterized by normal wall thickness, a disproportionately large increase in heart chamber volume, and the serial addition of sarcomeres (19, 37).

Preparation of heart particulate fraction. Heart particulate fraction was prepared as described previously (31). The dissected hearts were quickly frozen in liquid N2 and pulverized to a fine powder using a mortar and pestle cooled in liquid N2. The powder was stored at -80°C until assayed. The powder was homogenized (12 stokes) in a teflon glass homogenizer, in a buffer containing 10 mM Tris · HCl, 1 mM EDTA (pH 7.5). The homogenate was centrifuged at 1,000 g for 10 min. The supernatant was discarded, and the pellet was finally suspended in the above buffer and used for adenylyl cyclase activity determination and immunoblotting studies.

Immunoblotting. Immunoblotting of G proteins was performed as described previously (3). After SDS-PAGE, the separated proteins were electrophoretically transferred to a nitrocellulose sheet (Schleicher & Schuell) with 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% skim milk at room temperature for 2 h. The blots were then incubated with antibodies against G proteins in PBS containing 1.5% skim milk and 0.1% Tween-20 at room temperature overnight. 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 then washed three times with PBS before reaction with enhanced chemiluminescence Western blotting detection reagents (Amersham).

Adenylyl cyclase activity determination. Adenylyl cyclase activity was determined by measuring [32P]cAMP formation from [alpha -32P]ATP, as described previously (2, 3). The assay medium containing 50 mM glycylglycine, pH 7.5, 0.5 mM MgATP, [alpha -32P]ATP (1-1.5 × 106 counts/min), 5 mM MgCl2, 100 mM NaCl, 0.5 mM cAMP, 1 mM IBMX, 0.1 mM EGTA, 10 µM guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S; or otherwise as indicated), and an ATP-regenerating system consisting of 2 mM phosphocreatine, 0.1 mg creatine kinase/ml, and 0.1 mg myokinase/ml in a final volume of 200 µl. Incubations were initiated by addition of the membrane preparation (50-100 µg) to the reaction mixture, which had been thermally equilibrated for 2 min at 37°C. The reactions, conducted in triplicate for 10 min at 37°C, were terminated by the addition of 0.6 ml of 120 mM zinc acetate. cAMP was purified by coprecipitation of other nucleotides with ZnCO3, by addition of 0.5 ml of 144 mM Na2CO3, and subsequent chromatography by the double-column system, as described by Salomon et al. (38). Under the assay conditions used, adenylyl cyclase activity was linear with respect to protein concentration and time of incubation.

Protein was determined essentially as described by Lowry et al. (29), with crystalline BSA as standard.

Na+-K+-ATPase activity determination. Na+-K+-ATPase activity was determined as described previously (43). Briefly, the reaction mixture containing 50 mM Tris-EDTA, 4 mM MgCl2, 100 mM NaCl, 20 mM KCl, and 4 mM Tris-ATP was preincubated at 37°C for 5 min. The reaction was initiated by addition of the membrane protein (50 µg) and was further incubated for 10 min. The reaction was terminated by the addition of 1 ml of cold 12% (TCA), and the Pi in the supernatant was determined by the method of Taussky and Shorr (47).

Northern analysis. Total RNA was isolated by the guanidinium thiocyanate-phenol-chloroform method described by Chomczynski and Sacchi (12). cDNA inserts encoding for Gi alpha -2, Gialpha -3, and Gsalpha were radiolabeled with [alpha -32P]dCTP by random priming essentially described by Feinberg and Vogelstein (16).

DMSO/glyoxal-treated total RNA was resolved on 1% agarose gels and transferred to nylon membrane as described by Sambrook et al. (39). Filters, after prehybridization at 65°C for 6 h in hybridization solution (600 mM NaCl, 8 mM EDTA, 120 mM Tris, pH 7.4) containing 0.1% sodium pyrophosphate, 0.2% SDS, and heparin (500 U/ml), were hybridized overnight in hybridization solution containing 10% dextran sulfate (wt/vol) and the cDNA probe at 4-5 × 107 counts · min-1 · ml-1. Filters were then rinsed at 65°C twice for 30 min in 300 mM NaCl, 4 mM EDTA, 60 mM Tris, pH 7.4, and 0.1% SDS. Autoradiography was performed with X-ray films at -70°C. To assess the possibility of any variations in the amounts of total RNA in individual samples applied to the gel, each filter was hybridized with the 32P end-labeled oligonucleotide, which recognizes a highly conserved region of 28S rRNA. The blots that had been probed with the G protein cDNA were dehybridized by washing for 1 h at 65°C in 50% formamide, 300 mM NaCl, 4 mM EDTA, and 60 mM Tris, pH 7.4, and rehybridized overnight at room temperature with the oligonucleotide. Quantitative analysis of the hybridization of probes bound was performed by densitometric scanning of the autoradiographic film employing the enhanced laser densitometer (LKB Utroscan XL) and quantified using the gel scan XL evaluation software (version 2.1) from Pharmacia.

Data analysis. Results are expressed as means ± SE. Comparisons between groups (control and AV shunt rats) were made with Student's t-test for unpaired samples. Results were considered significant at a value of P < 0.05.


    RESULTS
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RESULTS
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Blood pressure and indexes of cardiac hypertrophy. The heart-to-body-weight ratio in AV shunt rats was significantly increased compared with the control sham-operated rats (P < 0.001), whereas no change in arterial blood pressure was detected (Table 1), suggesting that the AV shunt rat model exhibits cardiac hypertrophy and no hypertension. There was no significant difference in the heart-to-body-weight ratio at 2 days of induction of AV shunt; however, it was significantly increased by 15 ± 0.5% and 20 ± 2% at 3 and 7 days, respectively, relative to sham-operated control rats (P < 0.001, n = 4-6 for each group). In addition, no significant difference in the activity of Na+-K+-ATPase as a membrane marker was observed in AV shunt rats compared with control rats (control rats; 18.7 ± 0.07 µmol Pi · mg protein-1 · h-1; n = 4), and AV shunt rats (19.4 ± 0.25 µmol Pi · mg protein-1 · h-1; n = 4). The data presented for adenylyl cyclase activity are related to milligrams membrane protein and not to the amount of protein measured in the hypertrophied myocardium, because the protein content per heart was increased in AV shunt rats (305.0 ± 10 vs. 241.2 ± 12 mg/heart) due to an elevated mass of cardiac proteins; however, protein content (mg/g heart) was not different in AV shunt rats compared with control rats (control, 254.8 mg/g; AV shunt rats, 258.1 mg/g).

                              
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Table 1.   Effect of an aortocaval fistula on arterial blood pressure and heart-to-body-weight ratio

G protein levels. To investigate whether the altered levels of G proteins (enhanced Gi and decreased Gsalpha ) observed in hypertensive rats associated with hypertrophy (4) were attributed to hypertension or to hypertrophy, the levels of Gi and Gs proteins were determined in hearts for AV shunt rats by immunoblotting techniques using specific antibodies AS/7 against Gialpha -1 and Gialpha -2, EC/2 against Gialpha -3, RM/1 against Gsalpha , and SW/1 against the common beta -subunit of Gbeta gamma . The results depicted in Fig. 1, show that the AS/7, EC/2, and SW/1 antibodies recognized a single protein of relative molecular mass 40 kDa referring to Gialpha -2, [Gialpha -1 has been shown to be absent in heart (25)], 41 kDa referring to Gialpha -3, and 35 kDa referring to the common beta -subunit of Gbeta , respectively, in both AV shunt and sham-operated rats; however, no differences in the amounts of immunodetectable Gialpha -2 and Gialpha -3 and Gbeta were detected in both groups. In addition, RM/1 antibody recognized three isoforms of Gsalpha : Gsalpha 45, Gsalpha 47, and Gsalpha 52 in hearts from control and AV rats; however, the amounts of immunodetectable Gsalpha 45 and Gsalpha 47 and not of Gsalpha 52 were significantly decreased in AV shunt rats compared with control rats.


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Fig. 1.   Quantification of G proteins in hearts from control (CTL) and aortocaval fistula (AV shunt; VO) rats. The heart membrane (particulate fraction) proteins (50 µg) from both groups were separated on SDS-PAGE and transferred to nitrocellulose, which was then immunoblotted using AS/7 antibody specific for Gialpha -1 and Gialpha -2, EC/2 antibody specific for Gialpha -3, RM/1 antibody specific for Gsalpha , and SW/1 antibody specific for Gbeta as described in MATERIALS AND METHODS. The detection of different G proteins was performed by using the chemiluminescence Western blotting detection reagents from Amersham. The autoradiogram is representative of 3-4 separate experiments.

We extended our studies further to investigate whether mRNA levels of G proteins change concomitantly with protein levels and determined the mRNA levels of G proteins by Northern-blot analysis using specific cDNA probes encoding Gsalpha , Gialpha -2, and Gialpha -3. The results depicted in Fig. 2 demonstrate that the cDNA probes for Gsalpha (A), Gialpha -2 (B), and Gialpha -3 (C) detected messages of 1.8, 2.3, and 3.5 kb, respectively, in both AV shunt rats and their sham-operated controls; however, the amounts of Gialpha -2 mRNA and Gialpha -3 mRNA were not different in the two groups. On the other hand, the levels of Gs mRNA were significantly decreased by ~35% ± 10 (n = 4) in AV shunt rats compared with control rats as determined by densitometric scanning.


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Fig. 2.   Expression of Gsalpha , Gialpha -2, and Gialpha -3 mRNA in hearts from control and AV shunt rats. Total RNA (10 µg) isolated from heart ventricles from 2 groups of rats was separated on 1% agarose and transferred to a nylon membrane, which was then hybridized with full-length cDNA probes encoding for Gsalpha (A, left), Gialpha -2 (B), and Gialpha -3 (C, top) as described in MATERIALS AND METHODS. The filters were rehybridized with an oligonucleotide recognizing the 28S rRNA (bottom) as described in MATERIALS AND METHODS. The autoradiogram is representative of 4 separate experiments. Densitometric scanning of the Gsalpha mRNA (A, right) from control and AV shunt rats. The results are expressed as percent mRNA levels compared with their control rats (taken as 100%). Values are means ± SE of 4 separate experiments. *P < 0.05.

To investigate whether the decreased expression of Gsalpha in hearts from AV shunt rats is a cause or effect of hypertrophy, we determined the levels of Gsalpha proteins at various days of the development of hypertrophy. The results shown in Fig. 3 indicate that the levels of Gsalpha 45 and Gsalpha 47 and not of Gsalpha 52 proteins were decreased by 7.0 ± 0.23% and 30.3 ± l.34%, respectively, at 7 days and by 10.7 ± 0.74% and 36.7 ± 6.3%, respectively, at 10 days of induction of AV shunt as determined by densitometric scanning, whereas no significant change was observed at 2 or 3 days of induction of AV shunt.


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Fig. 3.   A: quantification of Gsalpha proteins in hearts from control sham-operated rats and from rats at 2, 3, 7, and 10 days post-AV shunt. The heart membrane (particulate fraction) proteins (50 µg) from different groups (control and AV shunt) were separated on SDS-PAGE and transferred to nitrocellulose, which was then immunoblotted using RM/1 antibody specific for Gsalpha . The detection of Gsalpha protein was performed by using the chemiluminescence Western-blotting detection reagents from Amersham. The autoradiograms are representative of 3-4 separate experiments. B and C: densitometric scanning of Gsalpha 45 and Gsalpha 47 proteins from control and AV shunt rats. The results are expressed as percentage of controls (taken as 100%). Values are means ± SE of 4 separate experiments.

GTPgamma S-stimulated adenylyl cyclase activity. To investigate whether the decreased levels of Gsalpha in AV shunt rats also resulted in decreased Gsalpha -mediated functions, the effect of GTPgamma S, on adenylyl cyclase activity was examined, and the results are shown in Fig. 4. GTPgamma S stimulated adenylyl cyclase in a concentration-dependent manner in hearts from both groups; however, the extent of stimulation was significantly decreased in AV shunt rats. For example, GTPgamma S at 5 µM increased the adenylyl cyclase activity by ~650% in the control rats and 580% in AV shunt rats.


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Fig. 4.   Effect of guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S) on adenylyl cyclase activity in heart particulate fraction from control and AV shunt rats. Adenylyl cyclase activity was determined in the presence and absence of increasing concentrations of GTPgamma S in control and AV shunt rats as described in MATERIALS AND METHODS. The basal adenylyl cyclase activities in sham-operated controls and AV shunt rats were 60.6 ± 1.5 and 58.4 ± 0.62 pmol cAMP · mg protein-1 · 10 min-1, respectively. Values are means ± SE of 3 separate experiments. *P < 0.05.

Hormonal regulation of adenylyl cyclase. Because G proteins couple the hormone receptors to adenylyl cyclase and mediate the stimulatory and inhibitory responses of hormones on adenylyl cyclase, it was interesting to determine whether the altered expression of Gsalpha is reflected in the hormonal regulation of adenylyl cyclase. For this reason, the effect of some hormones that stimulate or inhibit adenylyl cyclase through Gs or Gi, respectively, on adenylyl cyclase activity was examined in hearts from AV shunt rats. Figure 5 shows that isoproterenol and glucagon stimulated adenylyl cyclase activity in hearts from control and AV shunt rats to various degrees; however, the extent of isoproterenol-mediated stimulation was significantly diminished in AV shunt rats compared with control rats (580% vs. 410%), whereas the stimulatory effect of glucagon was not different in the two groups of rats.


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Fig. 5.   Effect of stimulatory hormones on adenylyl cyclase activity in heart particulate fraction from control and AV shunt rats. Adenylyl cyclase activity was determined in the presence of 10 µM GTP alone (basal) or in combination with 50 µM isoproterenol (ISO) or 1 µM glucagon (GLUC) as described in MATERIALS AND METHODS. The basal adenylyl cyclase activities in sham-operated control and AV shunt rats were 58.2 ± 1.3 and 52.7 ± 2.1 pmol cAMP · mg protein-1 · 10 min-1, respectively. Values are means ± SE of 3 separate experiments. *P < 0.05.

To examine whether the unaltered expression of Gialpha -2 and Gialpha -3 in AV shunt rats compared with control rats was also reflected in unaltered Gi functions, the receptor-independent and receptor-dependent functions of Gi were examined. Figure 6A shows the effect of low concentration of GTPgamma S on forskolin-stimulated adenylyl cyclase activity (receptor-independent Gi functions). GTPgamma S inhibited forskolin-stimulated adenylyl cyclase activity in a concentration-dependent manner in control and AV shunt rats; however, the extent of inhibition was not different in both groups. Similarly, oxotremorine-mediated inhibition (receptor-dependent Gi functions) of adenylyl cyclase was also not different in hearts from AV shunt rats compared with sham-operated controls (Fig. 6B).


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Fig. 6.   A: effect of GTPgamma S on forskolin-stimulated adenylyl cyclase activity in heart particulate fraction from control and AV shunt rats. Adenylyl cyclase activity was determined in the absence (basal) or presence of increasing concentrations of GTPgamma S as described in MATERIALS AND METHODS. Basal adenylyl cyclase activities in sham-operated controls and AV shunt rats were 49.9 ± 2.7 and 47.8 ± 3.4 pmol cAMP · mg protein-1 · 10 min-1, respectively. Values are means ± SE of 3 separate experiments. B: effect of oxotremorine on adenylyl cyclase activity in heart particulate fraction from control and AV shunt rats. Adenylyl cyclase activity was determined in the absence (basal) or presence of increasing concentrations of oxotremorine as described in MATERIALS AND METHODS. Basal adenylyl cyclase activities in sham-operated controls and AV shunt rats in the presence of 10 µM GTPgamma S were 363.7 ± 20.5 and 433.8 ± 1.3 pmol cAMP · mg protein-1 · 10 min-1, respectively. Values are means ± SE of 3 separate experiments.

Forskolin and NaF-stimulated adenylyl cyclase activity. Forskolin stimulates adenylyl cyclase by interacting directly with the catalytic subunit of adenylyl cyclase. To explore whether the catalytic subunit of adenylyl cyclase is impaired in AV shunt rats, the effect of forskolin on adenylyl cyclase was determined in hearts from control and AV shunt rats. Figure 7 shows that forskolin stimulated adenylyl cyclase activity in hearts from both VO and sham-operated controls; however, the extent of stimulation was significantly decreased by ~55% in VO rats. Similarly, NaF, which stimulates adenylyl cyclase by a receptor-independent mechanism but requires Gs proteins, also stimulated adenylyl cyclase to a lower extent in VO rats compared with sham-operated control rats.


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Fig. 7.   Effect of agonists on adenylyl cyclase activity in heart particulate fraction from control and AV shunt rats. Adenylyl cyclase activity was determined in the absence (basal) or presence of 10 mM NaF or 50 µM forskolin (FSK) as described in MATERIALS AND METHODS. The basal adenylyl cyclase activities in sham-operated control and AV shunt rats were 52.8 ± 2.5 and 50.2 ± 1.4 pmol cAMP · mg protein-1 · 10 min-1, respectively. Values are means ± SE of 3 separate experiments. **P < 0.05.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have previously reported an increased expression of Gialpha protein and mRNA in hearts and aorta from SHR and DOCA-salt hypertensive rats compared with their respective controls (2, 4). In addition, we have shown recently that L-NAME hypertensive rats, which do not exhibit cardiac hypertrophy, also exhibited an increased expression of Gialpha proteins (6). However, the levels of Gsalpha were unaltered in L-NAME hypertensive rats (6) and SHR (2) but decreased in DOCA-salt hypertensive rats (4). These results suggest that the decreased expression of Gsalpha may be associated with cardiac hypertrophy and not with arterial hypertension per se. In the present studies, we report that the AV shunt rat model that expressed eccentric cardiac hypertrophy and no arterial hypertension exhibited a decreased expression of Gsalpha protein and mRNA, whereas the expression of Gialpha -2 and Gialpha -3 was not altered. Our results are in agreement with the studies reported in pigs after chronic VO cardiac hypertrophy, where the amounts of Gsalpha as measured by reconstitution assays were decreased in hearts and the levels of pertussis toxin substrates were unaltered (21). The decreased levels and functions of Gsalpha protein have been shown in compensated left ventricular hypertrophy (11), pressure overload left ventricular failure (28), and myocardial ischemia (44). The increased levels of catecholamines have been reported in VO-induced cardiac hypertrophy in rats (13) as well as in pigs (21), which may be responsible for eliciting a decreased expression of Gsalpha proteins in the current studies. In this regard, isoproterenol treatment of the rats has been shown to decrease the levels of Gsalpha proteins without changing the levels of Gialpha protein in ventricular myocardium (27). The alteration in Gsalpha mRNA in AV shunt rats may not be attributable to the variations in the amounts of total RNA applied to the gels, because hybridization with a 32-mer oligonucleotide that recognizes a highly conserved region of 28S rRNA showed a similar amount of 28S rRNA loaded from control and AV shunt rats onto the gels.

We have also shown that the responsiveness of adenylyl cyclase to GTPgamma S stimulation was decreased in the hearts from AV shunt rats compared with control rats, which may be due to the decreased levels of Gsalpha protein or increased levels of Gialpha protein. Because the levels of Gialpha were not altered in AV shunt rats, the decreased stimulation of adenylyl cyclase by GTPgamma S can be explained by the decreased expression of Gsalpha protein in these rats. A relationship between decreased levels of Gsalpha protein and a decreased stimulation of adenylyl cyclase by guanine nucleotides in DOCA-salt hypertensive rats has been shown previously (4). Furthermore, attenuated responsiveness of adenylyl cyclase to isoproterenol in AV shunt rats compared with control rats may be due to the decreased levels of Gsalpha and/or decreased number of beta -adrenergic receptors or impaired catalytic subunit, or a combination of all the three components. In this regard, a downregulation of beta -adrenergic receptors due to increased levels of catecholamines and reduced sensitivity to the chronotropic effect of isoproterenol has been reported in pigs with VO hypertrophy (21). In addition, since catalytic subunit of adenylyl cyclase was also impaired in AV shunt rats as was demonstrated by decreased stimulation of adenylyl cyclase by forskolin, it may be suggested that the decreased expression of Gsalpha , impaired catalytic subunit of adenylyl cyclase, and downregulation of beta -adrenergic receptors may all be responsible for the decreased responsiveness of adenylyl cyclase to isoproterenol stimulation in AV shunt rats. However, the reason for unaltered glucagon-mediated stimulation of adenylyl cyclase in AV shunt rats is not clear and needs to be investigated. It may be possible that Gsalpha 52 protein that is not altered in AV shunt rats may be implicated in the coupling of glucagon receptors to adenylyl cyclase.

The fact that GTPgamma S inhibited forskolin-stimulated enzyme activity to the same extent in hearts from control and AV shunt rats suggests that Gi functions were not altered in volume hypertrophied rats. These results correlate very well with the levels of Gi proteins, which were also not changed in these rats. Our results are in agreement with the studies of Hammond et al. (21) who did not observe any changes in the levels of pertussis toxin substrates in pigs after chronic VO hypertrophy. Furthermore, unaltered responsiveness of adenylyl cyclase to oxotremorine inhibition in hearts from AV shunt rats may also be due to the unaltered levels and functions of Gi proteins that couple these receptors to the adenylyl cyclase system and/or may be due to the possibility that muscarinic receptors are not downregulated in these rats.

The decreased sensitivity of adenylyl cyclase to forskolin stimulation in AV shunt rats may be due to the defective catalytic subunit of adenylyl cyclase and/or to the decreased levels of Gsalpha proteins. In this regard, the requirement of Gsalpha and guanine nucleotides for forskolin activation has been reported (22). In addition, a diminished stimulation of adenylyl cyclase by NaF in AV shunt rats may also be attributed to the decreased levels of Gsalpha proteins. Similar decreases in forskolin and NaF stimulation of adenylyl cyclase have also been reported in hearts from DOCA-salt hypertensive rats with established hypertrophy (4, 30) and Syrian hamster with dilated cardiomyopathy (17), myocardial ischemia (42), and VO hypertrophy (21).

In conclusion, we have shown that the expression of Gsalpha 45 and Gssalpha 47 but not of Gsalpha 52 was decreased in hearts of VO-hypertrophied rats, whereas the levels of Gialpha were unaltered. The decreased expression of Gsalpha proteins was observed only after the development of cardiac hypertrophy. The attenuated responsiveness of adenylyl cyclase to isoproterenol, forskolin, and NaF stimulation in AV shunt rats may partly be attributed to the decreased levels of Gsalpha proteins in this model of hypertrophy. From these results, it is suggested that the decreased expression of Gsalpha in AV shunt rats may be associated with cardiac hypertrophy and not with arterial hypertension.


    ACKNOWLEDGEMENTS

We are grateful to Drs. Randall Reed and Hiroshi Itoh for their kind gift of cDNAs of G proteins. We thank Matteo Pagano for help in performing some experiments and Christiane Laurier for her valuable secretarial help.


    FOOTNOTES

This work was supported by grants from the Quebec Heart Foundation and the Medical Research Council of Canada.

M. B. Anand-Srivastava was a recipient of the Medical Research Council Scientist Award, from the Medical Research Council of Canada, during the course of these studies.

Address for reprint requests and other correspondence: M. B. Anand-Srivastava, Dept. of Physiology, Faculty of Medicine, Univ. of Montreal, C.P. 6128, Succ. Centre-ville, Montreal, Quebec, Canada H3C 3J7 (E-mail: anandsrm{at}physio.umontreal.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 12 August 1999; accepted in final form 6 April 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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