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Properties of the Ventricular Adrenergic Signal Transduction System During Ontogeny of Spontaneous Hypertension in Rats

Bazan, Ariane; Van de Velde, Eric; de Paepe, Boel; Fraeyman, Norbert

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Journal of Cardiovascular Pharmacology: April 2000 - Volume 35 - Issue 4 - p 653-663
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Abstract

Alterations in cardiac function have been implicated in the development of essential hypertension, and adrenergic signal transduction has been repeatedly implicated in most of these alterations, both at the level of altered inotropic responsiveness [for review, see (1)] and in the ventricular hypertrophic process (2). One important mechanism involved in the regulation of myocardial force of contraction is formation of cyclic adenosine monophosphate (cAMP) (3). The cellular content of cAMP is under dual control: its synthesis is induced mainly by catecholamines through interaction at β-adrenoceptors. These receptors stimulate adenylate cyclase (AC) through the activation of stimulatory guanine-nucleotide-binding proteins Gs(4). AC is also under inhibitory control: inhibitory guanine-nucleotide-binding proteins Gi(4) couple cardiac M2-muscarinic (5) and A1-adenosine (6) receptors to AC. Another mechanism involved in inotropic heart responses is transduced through α1-adrenoceptors, which are coupled to multiple signal transduction pathways, including the Gq/11 protein-mediated (7) stimulation of phospholipase C and formation of inositol phosphates [for review, see (8)]. Additionally, the α1-adrenergic signal transduction has been implicated in myocardial growth responses (9). In failing human heart (10) and in various models of hypertension, including the spontaneously hypertensive rat (SHR) (1), desensitization of AC and reduced inotropic responsiveness to β-adrenergic stimulation are reported. Moreover, a crucial role for α1-adrenergic signal transduction in the cardiac hypertrophic process of SHRs was demonstrated (2).

Although these observations clearly implicate the adrenergic signal transduction in the pathogenesis of hypertension, results of biochemical analysis display remarkable controversies [for review, see (1,11)]. As far as G proteins are concerned, a number of technical considerations have repeatedly hampered conclusive Gα protein measurements, as indicated by several authors (12-14). In a recent article, we showed how inclusion of an internal standard protein can make Western blotting and immunostaining techniques reliable tools for comparative protein measurements (15). In addition, most authors reported only partial results with respect to the stage of the hypertensive syndrome considered or the components of the transduction pathway investigated. We therefore systematically investigated all components of the adrenergic pathway in ventricular tissue, including β-and α1-adrenoceptors as well as G, G, and Gq/11α proteins at three different stages of the hypertensive syndrome in SHRs: the prehypertensive stage (rats aged 3.5 weeks), and the established hypertensive stage in young and older adult rats (rats aged 3 and 8 months, respectively). It was found that the differences in properties of the adrenergic signal transduction system between SHRs and WKY rats are exclusively observable before and at onset of the overt hypertension.

MATERIALS AND METHODS

Animals

Male SHRs from the Okamoto-Aoki strain, aged 3.5 weeks, 3 months, and 8 months, and sex- and age-matched normotensive Wistar-Kyoto (WKY) control rats were obtained from Harlan (Zeist, the Netherlands). They had free access to food (normal rat food) and tap water. Systolic arterial blood pressure was assessed in triplicate on at least two different occasions by the tail-cuff method (16); the last assessment was performed the day preceding tissue preparation. Body and ventricle weights were determined immediately before tissue preparation. Animals were treated according to ethical guidelines, and all animal experiments were performed in accordance with Belgian law on animal experimentation. Rat characteristics are summarized in Table 1.

TABLE 1
TABLE 1:
Characteristics of the rats

Tissue preparation

Animals were killed by decapitation, and the heart ventricles were homogenized in homogenization buffer (154 mM NaCl, 10 mM Tris-HCl, 2 mM EGTA, pH 7.4, for ligand binding or 50 mM Tris-HCl, 2 mM EDTA, 0.5 mM phenylmethylsulfonylfluoride, 0.001 mM leupeptin, pH 7.4, for immunoblotting experiments). The homogenate was centrifuged for 10 min at 3,000 g, and the supernatant was subsequently centrifuged for 20 min at 27,000 g. The pellet was washed twice in homogenization buffer and resuspended in assay buffer (50 mM Tris-HCl, 20 mM MgCl2, 2 mM EGTA, pH 7.4), yielding a crude membrane preparation used in ligand-binding experiments. The protein concentration was determined with the dye-binding method of Macart and Gerbaut (17) using bovine serum albumin as a standard. For immunoblotting experiments, the final pellet was resuspended in Laemmli (18) final sample buffer (62.5 mM Tris-HCl, pH 6.8, containing 5% mercaptoethanol, 2% sodium dodecylsulfate (SDS), 10% glycerol, and 0.001% bromophenol blue). Protein concentration was determined by the method of Lowry et al. (19) as modified by Chang (20), allowing determination of proteins in SDS-containing buffers.

Radioligand-binding experiments

Saturation and competition binding experiments were performed as described before (21). In brief, β-receptor density was determined by saturation binding with a concentration range of 1 to 300 pM [125Iodo](−)-cyanopindolol (ICYP). Nonspecific binding was calculated from a parallel incubation including 100 μM isoproterenol (in the presence of 0.1% ascorbic acid). It amounted to 7.9 ± 0.7%, 24.4 ± 2.4%, and 12.2 ± 1.5% of total binding for cardiac membranes of SHRs and WKY rats, aged 3.5 weeks, 3 months, and 8 months, respectively, at a ligand concentration equivalent to the dissociation constant (n = 16 in each group); nonspecific binding was equivalent in both rat strains. In competition binding experiments, approximately 100 pM ICYP was incubated with the nonselective β-agonist isoproterenol (from 10−3 to 10−10M) with or without guanylylimidobisphosphate (GppNHp; 250 μM) or with a β-subtype-selective antagonist (from 10−4 to 10−11M). CGP 20,712A and ICI 89,406 were used as β1-selective antagonists, and ICI 118,551 as a β2-selective antagonist. The α1-receptor density was determined by saturation binding with a concentration range of 10-1,000 pM [3H]prazosin hydrochloride. Nonspecific binding was calculated from a parallel incubation including 10−5M of the α1-antagonist phentolamine and amounted, at the value of the dissociation constant, to 28.0 ± 2.4%, 35.3 ± 3.5%, and 29.0 ± 2.2% of the total binding at 3.5 weeks, 3 months, and 8 months of age, respectively (n = 12 in each group). Determination of the nonspecific binding with 80 nM cold prazosin hydrochloride produced similar results. In competition binding experiments, ∼100 pM [3H]prazosin hydrochloride was incubated with the α1A-selective antagonist WB 4,101 (from 10−6 to 10−11M). Approximately 40 μg protein and 40-70 μg protein was used for each point in β- and α1-ligand-binding experiments, respectively; total assay volume was 250 μl. After 60 min of incubation (22), bound ligand was separated from free ligand by filtration through a Whatman GF/C fiberglass filter (Skatron Cell Harvester Analis, Namur, Belgium). Radioactivity was counted in a Packard gamma counter or with a Beckman Liquid Scintillation counter for the iodinated or the tritiated ligand, respectively. The α1-subtype determination with the α1B-alkylating agent chloroethylclonidine (CEC) was based on the method of Han et al. (23). In brief, ventricle membranes were incubated with increasing concentrations of CEC (between 10−3 and 10−8M) for 10 min at 37°C. The reaction was stopped by adding ice-cold assay buffer and centrifugation at 20,000 g for 10 min. The membrane pellet was resuspended in assay buffer and used for the saturation experiments as described earlier.

Immunoblotting experiments

All methods have been described in detail before (21). Antibodies against G and G proteins were raised according to standard methods; the selected amino acid sequences were TPEPGEDPRVTRAKY (amino acids 325-339) for G and SKFEDLNKRKDT (amino acids 306-317) for G(24). Rabbit anti-Gq/11α antiserum was purchased from Rockland Laboratories (SanverTECH, Boechout, Belgium). When the antibody against G is used, two clearly discernible bands with molecular masses of 52 kDa (Gsαlong, Gsα1) and 45 kDa (Gsαshort, Gsαs) are seen. G protein and Gq/11α protein are recognized on immunoblots as one single band at a molecular mass of 40-42 kDa. Reactivity of the antisera was tested by using a conventional enzyme-linked immunosorbent assay (ELISA), in which immunoplates were coated with the synthetic peptides, and found to be high and specific for the corresponding antigens. The antisera were, moreover, shown to label all splice variants in two-dimensional gel electrophoresis experiments of the G and G proteins, respectively. Protein samples were diluted to 2 (G and Gq/11α assays) or 4 mg/ml (G assays); 1.5 μg/sample of elastase, used as an internal standard protein (15), was added to the tissue dilution, and 50 μl of this final sample was loaded on the gel. Each Gα assay type consisted of a parallel run of four gels, which were blotted simultaneously. Before immunostaining, the blots underwent an additional quenching step (overnight at 37°C) with goat preimmune serum (diluted 1:5), which eliminated a 48-kDa band resulting from an aspecific reaction of the secondary antibody with ventricle tissue [goat anti-rabbit immunoglobulin Gs (IgGs)]. Preliminary experiments demonstrated that accurate and linear dose-response curves could be obtained; optimal results were obtained at a staining time of ∼10 min. Stained blots were scanned with the Image Master DTS scanner (Pharmacia, LKB, Piscataway, NJ, U.S.A.), and staining intensity was integrated over the band surface and recorded by using PDI software (Pharmacia, LKB). These figures were corrected for the protein level of the applied sample (as checked by a second determination after dilution) and for the internal protein standard. The results, given in arbitrary units per milligram protein, were used as estimates for the concentration of Gα proteins in the membrane fraction. The absolute concentrations of G and G proteins were estimated in a preliminary series of ELISA experiments (25); the antigenic peptide was used as a standard. G and G protein concentration in our myocardial membrane preparations amounted to ∼75 and 100 pmol/mg membrane proteins, respectively.

Materials

ICYP and [3H]prazosin hydrochloride (specific activities, 2,000 and 25 Ci/mmol, respectively) were obtained from Amersham (Amersham, Roosendaal, The Netherlands). (−)-Isoproterenol hydrochloride was obtained from Sigma (Poole, U.K.). GppNHp and elastase (from porcine pancreas) were purchased from Boehringer-Mannheim (Brussels, Belgium). CGP 20,712A {(±)-(2-(3-carbamoyl-4-hydroxyphenoxy)-ethylamino)-3-[4-(1-methyl-4-trifluoro-methyl-2-imidazolyl)-phenoxy]-2-propanolmethane sulphonate} was a gift from Ciba Geigy (Groot-Bijgaarden, Belgium). ICI 118,551 {erythro-(±)-1-(7-methylindan-4-yloxy)-3-isopropylaminobutan-2-ol} and ICI 89,406 {1-(2-cyanophenoxy)-3-β-(3-phenylureido)-ethylamino-2-propanol} were gifts from ICI (Destelbergen, Belgium). Chloroethylclonidine (CEC) dihydrochloride and WB 4,101 {2-(2,6-dimethoxy-phenoxyethyl)aminoethyl-1,4-benzodioxane hydrochloride} were obtained from Research Biochemical Inc. (Natick, MA, U.S.A.) Prazosin hydrochloride was obtained from Pfizer (Brussels, Belgium). Goat anti-rabbit affinity-purified IgGs labeled with peroxidase were obtained from Sigma. Rabbit anti-elastase and rabbit anti-Gq/11α antisera were purchased from Rockland Laboratories (Sanver TECH, Boechout, Belgium).

Statistics

Values are expressed as mean ± SEM. For the ligand-binding experiments (and the Gsαlong/Gsαshort ratio), all data were initially compared in a two-way analysis of variance (ANOVA) with interaction of both parameters Age and Strain. When significance was obtained, the influence of the hypertensive status was calculated separately in each age group by means of an unpaired two-tailed Student's t test, and the age-related differences were calculated for each rat strain in a one-way ANOVA. When this one-way ANOVA was significant, unpaired two-tailed t tests were used to compare the age groups. When the initial two-way ANOVA was not significant, it was concluded that the Age effects were independent of the Strain, and that the Strain effects were independent of the Age group. Strain effects were calculated between the pooled data of all ages in each strain; age effects over the pooled data of both strains were calculated in a similar way. For Gα protein assays, SHR and WKY data were initially compared in a one-way ANOVA. When this ANOVA was significant, unpaired two-tailed t tests were used to compare SHR and WKY in each age group. The t values were corrected for multiple comparisons according to Bonferroni; p < 0.05 was considered statistically significant.

RESULTS

Radioligand-binding experiments

β-Adrenoceptor characteristics. The data on the β-adrenoceptor characteristics are summarized in Table 2. Representative examples of saturation binding curves are shown in Fig. 1; mean competition binding curves with isoproterenol as displacer are shown in Fig. 2. Specific ICYP binding was monophasic and saturable. Scatchard analysis revealed one class of binding sites. The number of β-adrenoceptors was ∼30% higher in 3.5-week-old SHRs as compared with age-matched controls. β-Adrenoceptor density decreased significantly between 3.5 weeks and 3 months in both SHRs and WKY rats (with ∼70% and ∼50%, respectively). Finally, similar β-adrenoceptor densities were found in SHRs and WKY rats at 3 and 8 months (32-35 fmol/mg protein). The equilibrium dissociation constant of ICYP (Kd in pmol/L) was comparable in both SHRs and WKY rats of all age groups. The percentage of high-affinity binding sites for isoproterenol is higher (∼9%) in SHRs than in WKY rats throughout all age groups (p < 0.01 over pooled data); this difference is most pronounced at the age of 3.5 weeks, where it amounts to ∼14%. The equilibrium inhibition constants of the high-affinity binding sites are similar in SHRs and WKY rats, increase between 3.5 weeks and 3 months, and decrease thereafter. The equilibrium inhibition constants of the low-affinity binding sites are similar in SHRs and WKY rats of all ages, and are comparable with the inhibition constant for the low-affinity binding sites in the presence of GppNHp. Based on both saturation and competition data, Bmax × %HA in fmol/mg protein was calculated as an estimation of the absolute high-affinity β-receptor population. These data are given in Table 2 and illustrated in Fig. 3. It is now seen that there is about half more β-adrenoceptors with agonist high affinity in prehypertensive SHRs than in age-matched controls. After this age, a gradual decrease is seen in both strains, with similar numbers at 3 and 8 months.

TABLE 2
TABLE 2:
β-Adrenoceptor characteristics
FIG. 1
FIG. 1:
[125Iodo](−)-cyanopindolol (ICYP) saturation in ventricles of spontaneously hypertensive rats (SHRs) and Wistar-Kyoto (WKY) rats. Representative examples of saturation binding with ICYP as ligand in ventricle plasma membranes of SHRs (solid symbols) and WKY control rats (open symbols), aged 3.5 weeks (A), 3 months (B), and 8 months (C). The amount of bound ligand is expressed in fmol/mg protein.
FIG. 2
FIG. 2:
Isoproterenol displacement of [125Iodo](−)-cyanopindolol (ICYP) in ventricles of spontaneously hypertensive rats (SHRs) and Wistar-Kyoto (WKY) rats. Mean competition binding curves with ICYP as ligand and the nonselective β-agonist (−)isoproterenol as displacer in ventricle plasma membranes of SHRs (A-C) and WKY control rats (D-F), aged 3.5 weeks, 3 months, and 8 months, respectively. The binding at the lowest and highest concentrations of competitor is set at 100% and 0, respectively. Solid symbols indicate competition binding without guanylylimidobisphosphate (GppNHp); open symbols indicate competition binding with 250 μM GppNHp. Curves are the mean of eight experiments in duplicate.
FIG. 3
FIG. 3:
High-affinity β-receptor density. Mean absolute high-affinity β-receptor density (Bmax × %HA) in fmol/mg protein in ventricle plasma membranes of spontaneously hypertensive rats (SHRs) and Wistar-Kyoto (WKY) control rats, aged 3.5 weeks, 3 months, and 8 months, respectively.

The results on the β-adrenoceptor subtypes are given in Table 3. By using the β1-selective antagonists, the percentage of high-affinity binding sites is comparable in SHRs and WKY rats of all age groups (∼55%), and remains constant during aging. The equilibrium inhibition constants of the high-affinity binding sites for CGP 20,712A do not vary with age in any of the strains, but are significantly lower in SHRs as compared with WKY rats (p < 0.05 over pooled data). With the β2-selective antagonist, neither the percentage of high-affinity binding sites (∼30%) nor the inhibition constants vary between rat strains or age groups. Note that there is a slight discrepancy in the β12-distribution as calculated through displacement with either β1- or β2-antagonists. This is because ICYP has a slight β2 selectivity, which leads to an underestimation of the β1 population in competition with β1-selective antagonists and to an overestimation of the β2 population in competition with β2-selective antagonists (26).

TABLE 3
TABLE 3:
β-adrenoceptor subtype determination

α1-Adrenoceptor characteristics. The results on the α1-adrenoceptor characteristics are summarized in Table 4. Specific prazosin binding was monophasic and saturable, and Scatchard analysis revealed one class of binding sites. α1-Adrenoceptor density was similar in both rat strains, and decreased by ∼40% between 3.5 weeks and 3 months of age. The equilibrium dissociation constant did not vary significantly with age in either rat strain. With the α1A-selective antagonist WB 4,101, the percentage of high-affinity binding sites is found to be similar (∼16.4%) with similar inhibition constants in SHRs and WKY rats of all age groups. Preincubation of the ventricle membranes with increasing concentrations of CEC resulted in a concentration-dependent decrease in adrenoceptor density (data not shown). Preincubation with 100 μM CEC induced a ∼85% decrease of the [3H]prazosin-sensitive α1-adrenoceptor density in both SHRs and WKY rats of all ages.

TABLE 4
TABLE 4:
α1-Adrenoceptor characteristics

Immunoblotting experiments

Representative examples of immunoblot experiments for G, G, and Gq/11α assessment in ventricles of SHRs (S) and WKY rats (W) are depicted in Fig. 4A-C, respectively; the corresponding average values are shown in Fig. 5A-C, respectively. Data on the Gsα1/Gsαs ratio are given in Table 5.

FIG. 4
FIG. 4:
G, G, and Gq/11α immunoblots in ventricles of spontaneously hypertensive rats (SHRs) and Wistar-Kyoto (WKY) rats. Representative examples of a G (A), G (B), and Gq/11α (C) immunoblot experiment in ventricular membranes of SHRs (S) and age-matched WKY control rats (W), aged 3.5 weeks, 3 months, and 8 months; molecular-weight markers are as indicated.
FIG. 5
FIG. 5:
G, G, and Gq/11α concentrations in ventricles of spontaneously hypertensive rats (SHRs) and Wistar-Kyoto (WKY) rats. Corresponding mean Gsα1 (l; A), Gsαs (s; A), G (B), and Gq/11α (C) levels in arbitrary units/mg protein in ventricular membranes of SHRs (hatched) and WKY controls (blank). Values are expressed as mean ± SEM; n = 5. *p < 0.05 and **p < 0.01 versus normotensive controls; *p < 0.005 and **p < 0.001 versus older rats of the same strain.
TABLE 5
TABLE 5:
Gsαlong/Gsαshort ratio

Gsαprotein levels. No major strain differences are seen in the concentration of either G protein isoform in any of the age groups considered. In both SHRs and WKY rats, a decline of the total G protein concentration of ∼30% between 3.5 weeks and 3 months is observed, and this is due to a selective decline of the large isoform (of ∼40%). In 8-month-old rats of both strains, the total G protein concentration is further reduced to approximately half the initial concentration, resulting from a further decline of the large isoform. The small isoform also tends to decrease with age in both strains, but this tendency is not significant. As a result, the Gsα1/Gsαs ratio diminishes with age in both rat strains: in 3.5-week-old rats, a majority of the large-molecular-weight form is found (∼15% more than the small form), whereas at 3 months, almost as much of both isoforms is observed, and at 8 months, the ratio reverses in favor of the small-molecular-weight form (∼15% more than the large form).

Giαprotein levels. In 3.5-week-old SHRs, ∼35% more G proteins are found than in WKY controls. At 3 months, the G concentration is still higher in SHRs than in WKY rats (with ∼27%), but this difference is not significant (p = 0.1). At 8 months, similar G concentrations in both SHR and WKY are found. In both strains, the G concentration diminishes between 3.5 weeks and 3 months, and remains stable thereafter.

Gq/11αprotein levels. Whereas in 3.5-week-old SHRs, the Gq/11α concentration is ∼20% lower than the concentration in WKY rats, in 3-month-old SHRs significantly (∼50%) more Gq/11α proteins are observed than in WKY rats; no difference is found at 8 months. There is an age-related decrease in Gq/11α concentration in both strains between 3.5 weeks and 3 months. In SHRs, the Gq/11 concentration further declines between 3 and 8 months, whereas in WKY rats, no further decrease in 8-month-old animals is seen.

DISCUSSION

We present the results of a detailed investigation of the characteristics of the adrenergic signal transduction system in ventricles of SHRs. The β- and α1-adrenoceptor characteristics (including receptor density, coupling, and subtype distribution) as well as G, G, and Gq/11α protein levels were assayed in SHRs and WKY rats of three age groups (3.5 weeks, 3 months, and 8 months). A summary of the results is given in Table 6.

TABLE 6
TABLE 6:
Summary of principal results

There is general agreement that in prehypertensive SHRs, the plasma noradrenaline levels are significantly increased (27-29), leading to desensitization of the β-adrenergic signal transduction. This results in a diminished production of cAMP on β-adrenergic stimulation in isolated membrane preparations (1). Our data, however, indicate an upregulation of β-adrenoceptors together with an increase in the proportion of the high-affinity-binding β population. This finding is in agreement with Mochizuki and Ogawa (30), whereas Castellano et al. (31) observed increased β1-mRNA levels in ventricle myocytes of prehypertensive SHRs. Matsumori et al. (32) showed that it was the distribution of the β-adrenoceptor population over surface versus intracellular locations, rather than the total density, which was altered in SHRs. By using the lipophilic ligand ICYP on a vesicular membrane preparation, both populations were accessible in our experimental setup. The higher degree of coupling in the prehypertensive SHRs, together with the absolute increase in β population, might suggest a mechanism of β-adrenoceptor-G protein co-internalization, as was proposed by Milligan (33,34). Indeed, it has been suggested that β1 receptors could cluster in caveolae, retaining an association with the plasma membrane (35). Because there is, moreover, a large body of evidence associating β-adrenergic signal transduction and enrichment of G proteins with caveolae (35), this is indicative for a possible mechanism of β1-adrenoceptor-G protein co-internalization. Finally, as in previous reports (for review see 36), the β12-subtype distribution of the β population was found similar in 3-week-old rats of both strains.

As G levels were found increased in prehypertensive SHRs, our data support the idea of heterologous G upregulation (37,38) as a possible cause for desensitization. Anand-Srivastava (39) similarly reported elevated G protein levels in 2- to 4-week-old SHRs. Both a diminished β-adrenergic-stimulated cAMP production and enhanced Gi levels have also been reported in other conditions of naturally increased catecholamine levels, such as aging (21) or heart failure (10).

In vivo sympathetic stimulation of the heart is exerted through the neurotransmitter noradrenaline, which is a mixed β- and α-agonist. Our data do not indicate any desensitizing influence on the α1-adrenoceptor population, because similar densities are observed in rats of both strains. This is in accordance with the results of Yamada et al. (40) in prehypertensive SHRs. Again, the reduced Gq/11α levels in these SHRs indicate that desensitization might have taken place at the level of the G proteins. Indeed, as has been described before (41,42), Gq/11α proteins are subject to desensitization in response to activation of phospholipase C-coupled receptors. Gq/11α proteins in ventricle membrane are coupled to α1-mediated positive inotropic and to hypertrophic pathways (43-45). A few authors have studied these inotropic responses (developed tension) in these very young prehypertensive SHRs and reported mainly unchanged or decreased responses on norepinephrine or phenylephrine stimulation (46,47), which is compatible with our findings. However, hypertrophic mechanisms are active and most probably involve α1-mediated mechanisms from the prehypertensive stage on (48,49); these observations therefore seem less compatible with our findings. As we did no experiments on physiological outcomes, we cannot link our receptor-G protein data to specific effects. However, it is known that G signal transduction can also induce hypertrophic effects (50,51); moreover, α1-adrenoceptors have sometimes been shown to couple with G proteins (52). Because G levels are increased in this study, this pathway is a possible alternative for the observed (α1-induced) hypertrophic effects in prehypertensive SHRs.

In young established hypertensive SHRs, elevated (27,53,54) and unchanged (28,55,56) noradrenaline levels have been reported. In isolated ventricle membrane preparations of these SHRs, inotropic responsiveness and cAMP production on β-adrenergic stimulation are diminished (1). Our data indicate that differences in absolute β-adrenoceptor density are not implicated in this desensitization, because β densities are similar in both SHRs and WKY rats. This is in keeping with most literature data (for review, see 36). Similarly, no differences in ventricular G protein levels between SHRs and WKY rats are detected, which is in agreement with previous results (14,39,57). As in prehypertensive SHRs, it is suggested that G upregulation (37,38) accounts for the desensitizing influence, although the observed increase in G concentrations (by 25%) does not reach significance (p = 0.1). Similarly, other reports indicated increased G protein levels in young established hypertensive SHRs (27,58,59). Considering the age-related effects for the β-signal transduction chain, we observed a decrease both in β-adrenoceptor population and in G and G protein levels in rat heart, with maturation from 3.5 weeks to 3 months. This is in agreement with previous studies concerning β-adrenoceptor density (54,60,61), G protein (62), and G protein levels (63). Moreover, we observed a maturational decrease in the Gslong/Gsshort ratio, shifting from a majority of the long isoforms in juvenile rats to a majority of the short isoform in adult animals. There is some evidence for the unidentical behavior of both splice variants [e.g., it was found that the larger species has a greater ability to support hormone-stimulated adenylate cyclase activity and that there is a modest difference in the rate of GDP dissociation between the two variants (for summary see 64, 65)]. Given their unidentical behavior, the relative abundance of both G splice variants might affect signal transduction efficiency during rat heart development. A maturational decrease in Gslong/Gsshort ratio also was seen in other tissues (e.g., kidney, Fraeyman et al., personal communication) and could therefore typically accompany maturational tissue processes. Alternatively, it might constitute another element contributing to the decreased β-mediated cAMP generation, as is observed with maturation in rats (66-68). In the latter case, our results would suggest a lesser signal transduction efficiency for the short isoform as compared with the large one, which is in line with the mentioned reports (64,65).

The in vitro ventricular inotropic responsiveness on phenylephrine stimulation is reported unchanged in young established hypertensive SHRs (69). Accordingly, unchanged α1-adrenoceptor densities were observed, which is in keeping with some literature data, although both increased and decreased densities have been reported (36). Remarkably, a substantial strain-related increase in Gq/11α concentration also is observed in these young SHRs. Michel et al. (14) reported unchanged Gq/11α levels in 17-week-old SHRs, which is in apparent contradiction to our results in young established hypertensive SHRs. However, this could be explained by either the difference in age of the rats or, more probably, by the different specificities of the antisera used: our anti-G antiserum recognizes both αq and α11, whereas the antiserum used by Michel et al. (14) was specific for αq. Because both the in vitro responsiveness on α stimulation (69) and the in vivo cardiac output are normalized in 3-month-old SHRs (70,71), the observed increase in Gq/11α proteins is not likely to participate to an increased α1-adrenergic inotropy. However, at this stage of the etiology, an important level of cardiac hypertrophy also is observed (this study, 72,73). The elevation in Gq/11α proteins might therefore rather be implicated in hypertrophic responses, implying the α1-adrenergic signal transduction as well as other signal transduction pathways. The Gq/11α-Ang II pathway has indeed also been shown to be linked with hypertrophic responses (74). Moreover, it also was demonstrated that stimulation of the angiotensin II (Ang II) pathway can induce the inhibition of the β-adrenergic response (73,75). It is clear that further investigations will be required to unravel the mechanisms that induce the observed increase in Gq/11α concentration. Summarizing the age-related effects for the α1-signal transduction chain, we observed a decrease both in α1-adrenoceptor population and in G protein levels in rat heart with maturation from 3.5 weeks to 3 months. This is in agreement with previous studies concerning α1-adrenoceptor density (40,76) and Gq protein levels (77). The inotropic responsiveness of ventricular strips to α1-adrenergic stimulation also was shown to decline in rats aged between 2 weeks and 3 months (78). Moreover, it seems logical that these signal transduction components (participating in hypertrophic mechanisms) would be expressed to a greater extent during periods of development characterized by rapid cardiac growth. Biochemical α1-transduction components and physiologic effect are therefore straightforward in rat heart during maturation.

In well-established hypertensive, 8-month-old SHRs, catecholamine levels are normalized (53,54). Both the β-and α1 (phenylephrine)-adrenergic inotropic responses in hypertrophic hearts of nonfailing SHRs are comparable to those of age-matched WKY rats (79,80). The ensuing hypertrophic process is now solely induced by the increased afterload, and is therefore independent of any neurogenic stimulation. Accordingly, all adrenergic signal transduction characteristics were found similar to those of age-matched WKY rats. As for the β-adrenoceptor and the G and G protein levels, these results are confirmed by previous reports (30,32,81); literature data on Gq/11α levels in SHRs of this age group could not be found. Both in SHRs and WKY rats, the β- and α1-adrenoceptor densities are similar at 3 and 8 months; this age-related pattern is similar to that described before (21,76,82-84).

In conclusion, our data indicate that differences in properties of the adrenergic signal transduction system between SHRs and WKY rats are exclusively present before and at onset of the overt hypertension. Moreover, the hypertensive genotype apparently affects G proteins more readily than adrenoceptors.

Acknowledgment: The financial support of the Belgian NFWO (grants 3.9006.87 and 7.0080.91) is acknowledged. A. Bazan was supported with a specialization grant from the Flemish Institute for the Promotion of Scientific-Technological Research in Industry (IWT). We are grateful to Prof. Dr. W. De Potter for the use of the Image Master DTS scanner. The technical expertise of Marc Goethals for the synthesis of the peptides and of Erik Vandevelde for the camera work is acknowledged. We thank Prof. Dr. R. Lefebvre for his critical reading of the text.

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Keywords:

Adrenergic receptors; G proteins; Hypertension; Spontaneously hypertensive rat (SHR); Heart-Signal transduction

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