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(Hypertension. 1998;31:747-754.)
© 1998 American Heart Association, Inc.

Scientific Contributions

Effects of Angiotensin II Type 1 Receptor Blockade and Angiotensin-Converting Enzyme Inhibition on Cardiac ß-Adrenergic Signal Transduction

Michael Böhm; Oliver Zolk; Markus Flesch; Frank Schiffer; Petra Schnabel; Johannes-Peter Stasch; ; Andreas Knorr

From the Klinik III für Innere Medizin (M.B, O.Z., M.F., F.S., P.S.), Universität zu Köln, Köln, Germany, and Bayer AG (J.P.S., A.K.), Wuppertal, Germany.

Correspondence to Prof Dr Michael Böhm, Klinik III für Innere Medizin, Universität zu Köln, Joseph-Stelzmann-Straße 9, 50924 Köln, Germany. E-mail michael.boehm{at}medizin.uni-koeln.de

	Abstract

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Abstract—Inhibition of the renin-angiotensin system has been shown to improve symptoms and prognosis in heart failure. We compared the effects of inhibition of angiotensin-converting enzyme or blockade of angiotensin II type 1 (AT₁) receptors in a model with renin-induced hypertension that is known to exhibit similar changes in sympathetic activation and ß-adrenergic desensitization, as observed in heart failure. Treatment with captopril (100 mg/kg of feed) or the AT₁-antagonist Bay 10–6734 (100 mg/kg of feed) was performed in transgenic rats harboring the mouse renin 2^d gene [TG(mREN2)27]. Neuropeptide Y and angiotensin II levels, adenylyl cyclase activity, ß-adrenergic receptors, G_s, and G_i were investigated. TG(mREN2)27 showed a depletion of myocardial neuropeptide Y stores and an increase in myocardial angiotensin II concentrations. Isoprenaline- and guanylylimidodiphosphate-stimulated adenylyl cyclase activities and ß-adrenergic receptor density were reduced, whereas the catalyst and G_s-function were unchanged. G_i protein and mRNA concentrations were increased. All alterations were normalized by both treatments. Systolic left ventricular pressures, plasma atrial natriuretic peptide, and myocardial steady state atrial natriuretic peptide mRNA concentrations and heart weights were similarly reduced by both treatments. Sympathetic neuroeffector defects are similarly reversed by angiotensin-converting enzyme inhibition or AT₁ antagonism. The data support the concept that pharmacological interventions in the myocardial renin-angiotensin system significantly reverse local sympathetic neuroeffector defects. This could be important for the beneficial effects of these agents.

Key Words: hypertrophy • angiotensin receptor subtypes • catecholamines • adrenoceptors • G proteins

	Introduction

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ß-Adrenergic desensitization is one pathophysiologically important alteration of the failing heart that contributes to contractile dysfunction and reduced exercise tolerance.¹ The mechanisms involved are a local and a systemic activation of the sympathetic nervous system.^{2 3} In addition, the RAS is also activated in the failing heart, presumably leading to an enhanced local formation of angiotensin II.⁴ Because the two systems are closely related to each other,⁵ it is not clear whether local activation of the RAS or the sympathetic nervous system is the predominator of contractile dysfunction and poor prognosis in this malignant condition. In patients with heart failure, ACE inhibitors have been shown to reduce sympathetic drive and partly restore the number of ß-adrenergic receptors.⁶ In addition, treatment with ACE inhibitors in hypertensive cardiac hypertrophy reduced sympathetic activity and restored ß-adrenergic signal transduction defects at a dose that did not prevent cardiac hypertrophy, and the increase in blood pressure was unaffected.^{7 8} Recently, AT₁ receptor antagonists were developed for the treatment of hypertension and heart failure. However, there is still controversy regarding whether this class of agents has effects that are different from of ACE inhibitors.

In the present study, transgenic rats [TG(mREN2)27] harboring the mouse ren 2^d gene of renin were used.⁹ This model exhibits an activation of the cardiac tissue RAS as well as an activation of the sympathetic nervous system within the heart.¹⁰ These mechanisms lead to similar ß-adrenergic signal transduction defects,¹⁰ as occur in cardiomyopathic human hearts with terminal heart failure (ie, downregulation of ß-adrenergic receptors^{11 12 13} and an increase in inhibitory G protein subunits [G_i]).^{14 15 16} Although TG(mREN2)27 rats are not in heart failure and might not represent all pathobiochemical alterations observed in this condition, it can be taken as valuable model to study drug effects on ß-adrenergic signal transduction. To address the question of whether AT₁ receptor antagonists or ACE inhibitors exhibit differential alterations of ß-adrenergic neuroeffector mechanisms, TG(mREN2)27 rats were treated with the ACE inhibitor captopril or the AT₁ receptor antagonist Bay 10–6734. Doses were chosen that reduced blood pressure and cardiac hypertrophy in TG(mREN2)27 rats to values comparable to those measured in control animals.

	Methods

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Transgenic Animals
Transgenic animals [TG(mREN2)27] were bred, housed, and treated in the animal laboratory of Bayer AG (Wuppertal, Germany). SD control rats were the animals into which the transgene was originally introduced. The inbred strain has been held for 15 generations. Animals were housed according to the guidelines of animal care of the State of Nordrhein-Westfalen (Germany). Only male rats were used. The animals were maintained on a standard laboratory animal diet (Ssnift) and tap water ad libitum. Treatment was started at the age of 8 weeks for 8 weeks, at which time hypertension was fully established. Bay 10–6734 or captopril was milled into the pellets at a concentration of 100 mg/kg each; this is equivalent to an average daily dose of 6.5 mg/kg. The rats were exposed to alternating 12-hour dark/light cycles at 20° to 22°C. Twelve rats were treated in each group. Before death, the animals were thoracotomized, and left ventricular systolic pressure was determined as measure of aortic pressure. This procedure was performed with the animals under ether anesthesia, so blood pressure values were somewhat lower than those reported in the literature.^{9 10} Animals were killed by decapitation, and the hearts were rapidly removed. After decapitation, animals were bled from the carotid arteries. Serum was prepared from the sampled blood, frozen in liquid nitrogen, and stored at -80°C until the humoral measurements were performed. Hearts were quickly removed; the left ventricle was immediately prepared, split in the three pieces from the base to the apex, and immediately frozen in liquid nitrogen. These techniques allowed us to study adenylyl cyclase activity, G proteins, receptors, and respective mRNA steady state levels in uniform regions of the hearts. Hearts of TG(mREN2)27 exhibited concentric hypertrophy but no dilatation or any signs of heart failure such as excessive scarring. The hearts were evaluated macroscopically; microscopic tissue examination was not performed.

Adenylyl Cyclase Determinations
Adenylyl cyclase activity was determined as according to Salomon et al¹⁷ with modifications as described recently.¹⁸ The activity of adenylyl cyclase was determined in a reaction mixture containing 50 µmol/L [-³²P]ATP (0.3 µCi/100 µL), 50 mmol/L triethanolamine-HCl, 5 mmol/L MgCl₂, 100 µmol/L EGTA, 1 mmol/L 3-isobutyl-1-methylxanthine, 5 mmol/L creatine phosphate, 0.4 mg/mL creatine kinase, and 0.1 mmol/L cAMP, pH 7.4, in a final volume of 100 µL. The reaction was started by the addition of the membrane suspension. The mixture was preincubated for 5 minutes at 37°C. The incubation time was 20 minutes at the same temperature. Reactions were stopped by the addition of 500 µL of 120 mmol/L zinc acetate. Catalyst studies were performed in which MnCl₂ was substituted for MgCl₂ (5 mmol/L) in the reaction mixture.

Membrane Preparation for Receptor and G Protein Determinations
Myocardial tissue was chilled in 30 mL of ice-cold homogenization buffer (10 mmol/L Tris · HCl, 1 mmol/L EDTA, 1 mmol/L dithiothreitol, pH 7.4). Connective tissue was trimmed away; myocardial tissue was minced with scissors, and membranes were prepared with a motor-driven glass/Teflon homogenizer for 1 minute. Afterward, the membrane preparation was homogenized by hand for 1 minute with a glass/glass homogenizer. The homogenate was spun at 484g (Beckman JA 20) for 10 minutes. The supernatant was filtered through two layers of cheese cloth, diluted with an equal volume of ice-cold 1 mol/L KCl, and stored on ice for 10 minutes. This suspension was centrifuged at 100 000g for 30 minutes. For radioligand binding experiments, the pellet was resuspended in 50 vol of incubation buffer (50 mmol/L Tris · HCl, 10 mmol/L MgCl₂, pH 7.4) and homogenized for 1 minute with a glass/glass homogenizer. This suspension was recentrifuged at 100 000g for 45 minutes. The final pellet was resuspended in incubation buffer (50 vol) and was stored at -80°C. All preparation steps were performed on ice in a cool room at 4°C. Centrifugation steps were also performed at 4°C. Previous experiments in our laboratory have shown that storage for as long as 3 years at -70° to 80°C does not alter adenylyl cyclase activity and G protein or receptor content.

Radioligand Binding Studies
The assays were performed in a total volume of 250 µL of incubation buffer (composition given above). The incubation was carried out at 37°C for 60 minutes. Myocardial ß-adrenoceptors were studied using ¹²⁵I-cyanopindolol as previously described.¹⁴

Pertussis Toxin–Induced [³²P]ADP-Ribosylation
[³²P]ADP-ribosylation of G_i by pertussis toxin was performed for 12 hours at 4°C in a volume of 50 µL containing 100 mmol/L Tris · HCl, pH 8.0, at 20°C, 25 mmol/L dithiothreitol, 2 mmol/L ATP, 1 mmol/L GTP, 50 nmol/L [³²P]NAD (800 Ci/mmol), and 20 µg/mL pertussis toxin that had been activated through incubation with 50 mmol/L dithiothreitol for 1 hour at 20°C before the labeling reaction as described previously.^{14 18}

Immunoblotting Techniques
Immunoblotting techniques were performed as described previously.¹⁴ The polyclonal antiserum (MB 1) was raised in rabbits against the carboxyl-terminal decapeptide of retinal transduction (KENLKDCGLF) coupled to keyhole limpet hemocyanine as described by Goldsmith et al.¹⁹ The antiserum recognized G_i1 and G_i2 but not G_o and G_i3²⁰ (not shown). Blots were stained with an alkaline phosphatase–labeled goat anti-IgG antiserum.

Reconstitution of Myocardial G_s Into S49 cyc^- Membranes
Reconstitution assays were performed according to Sternweis et al²¹ as described previously.¹⁸

Isolation of Total RNA
Total RNA from frozen left ventricular tissue samples was prepared according to the protocol of Chomczynkski and Sacchi.²² Typically, between 50 µg and 100 µg of total RNA was obtained from 150 mg of tissue. The amount of RNA was determined by UV absorption. The absorbance ratio of A_260nm/A_280nm was 1.8 to 2.0 in all cases.

Northern Blot Analysis
Next, 10 µg of total RNA was separated in a 6% formaldehyde/1.2% agarose gel, blotted onto nylon membranes (Schleicher & Schuell) through overnight capillary blotting, and fixed through UV irradiation. Complete mRNA transfer to the nylon membrane had been confirmed by ethidium bromide staining of the gel. After fixation, the blots were prehybridized in 50% formamide solution (5x SSC, 5x Denhardt's solution, 50% formamide, 1% SDS, 50 mmol/L sodium phosphate, pH 6.8, 10% dextran sulfate, and 0.1 mg/mL salmon sperm DNA). Hybridization was performed in 50% formamide solution at 42°C for 16 hours. A 680-bp cDNA fragment (PstI/PstI) encoding for human ANP²³ and a 1700-bp fragment (KpnI/EcoRI) encoding for rat G_i2 were kindly donated by J. Reed (Durham, NC).²⁴ The fragments were cut out from the plasmid vector with the appropriate restriction enzymes, separated from the vector DNA on a 1% low- melt agarose gel, and labeled with -³²P-dCTP (Amersham Buchler Ltd) through random prime labeling by using the Multiprime DNA labeling kit from Amersham. The concentration of the respective labeled probe in the hybridization solution was 1x10⁶ cpm/mL. After hybridization at 42°C overnight, the membrane was washed twice in 2x SSC/0.1% SDS at room temperature for 15 minutes and twice in 0.2x SSC/0.1% SDS at 68°C for 45 minutes. Standardization was performed through hybridization of the same membrane using a 40-base single-stranded synthetic oligonucleotide probe for glyceraldehyde-3-phosphate-dehydrogenase (Dianova). Hybridization conditions were the same as described above. Stringency washes were performed briefly with 2x SSC/0.1% SDS at room temperature, for 30 minute with 2x SSC/0.1% SDS at 65°C, and twice for 5 minutes with 2x SSC/0.1% SDS at room temperature. Membranes were exposed to Kodak films (Kodak X-OMAT). Quantification of the signals was performed through densitometric analysis with the Image Quant Densitometric System (Molecular Dynamics). Before the final experiments, care was taken to ensure that RNA content loaded onto the gel was the same for all the samples. The exposure time of the photographic film was optimized for obtaining hybridization signals that were linear with increasing exposure time.

Neuropeptide Y Determinations
For neuropeptide Y measurements, tissue samples were homogenized with a Polytron homogenizer (Brinkmann Instruments) in 0.1 mol/L Tris · HCl, pH 7.4. Neuropeptide Y was determined with a commercially available radioimmunoassay (Amersham).

Angiotensin II Determinations
For angiotensin II measurements, tissue samples were homogenized with a motor-driven glass/polytetrafluoethylene homogenizer in ice-cold homogenization buffer (10 mmol/L Tris · HCl, 1 mmol/L EDTA, and 1 mmol/L DTT, pH 7.4) that included the protease inhibitors soybean trypsin inhibitor (2 µg/mL), benzamidine (3 µmol/L), pepstatin (1 µmol/L), leupeptin (1 µmol/L), and phenylmethylsulfonyl fluoride (100 µmol/L). After centrifugation (484g for 15 minutes), the supernatant was diluted with 2 mL of ice-cold 1 mol/L KCl and centrifuged at 100 000g for 30 minutes. No extraction or concentration procedure was performed to avoid differential concentration in the individual groups. The resulting supernatant was purified with Sep-Pak C₁₈ columns. The eluates were lyophilized, and the dry residues were dissolved in 1.2 mL of Tris · HCl reconstitution buffer, pH 7.4. Angiotensin II was determined with a commercially available radioimmunoassay according to the instructions of the manufacturer (Biermann). The sensitivity of the radioimmunoassay was 0.7 pg/mL, and the angiotensin II antibody had a cross-reactivity of 0.14% for angiotensin I.

ANP Determinations
ANP in plasma was measured after extraction using Sep-Pak C₁₈ cartridges (Bond Elut; Varian) and a specific and sensitive radioimmunoassay kit according to the manufacturer's instruction (Biotrend).

Miscellaneous
Protein concentrations were determined according to Lowry et al²⁵ using bovine serum albumin as standard. SDS-polyacrylamide gel electrophoresis was performed as described by Läemmli.²⁶

Materials
Forskolin was donated by Hoechst AG. GTP, Gpp(NH)p, ATP, creatine phosphate, and creatine kinase were purchased from Boehringer-Mannheim, and 3-isobutyl-1-methylxanthine was from EGA-Chemie. The ligand [¹²⁵I]iodocyanopindolol was from Amersham. Dithiothreitol was from Serva. Pertussis toxin was from List Biological Laboratories. All other compounds used were of analytical or best grade commercially available. Only deionized and double distilled water was used throughout the experiments. The pharmacological properties of Bay 10–6734 (6-n-butyl-4-methoxycarbonyl-2-oxo-1[2'-(1H-tetrazol-5yl-3-fluor-biphenyl-4-yl)methyl]1,2-dihydropyridine), a selective AT₁ receptor antagonist, have been recently described.^{27 28}

Statistics
The data shown are mean±SEM. Statistical significance was estimated with Student's t test for unpaired observations and analysis of variance according to Wallenstein et al.²⁹ A value of P<.05 was considered significant. K_D values were determined graphically in each individual experiment.

	Results

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Blood Pressure and Heart Weights
Treatment of TG(mREN2)27 with the AT₁ receptor antagonist Bay 10–6734 or the ACE inhibitor captopril resulted in a similar reduction in blood pressure to values observed in controls. The relative heart weights were significantly reduced in the Bay 10–6734–and captopril-treated TG(mREN2)27 animals to values comparable to those observed in controls (Table). Treatment with either compound did not change body weights.

View this table: [in this window] [in a new window]   Table 1. Body Weights, Relative Heart Weights, and Systolic Blood Pressure and Neuroendocrine Parameters in SD Rats, Untreated TG(mREN2)27 Rats, and TG(mREN2)27 Rats Treated With Captopril or Bay 10-6734

Neuroendocrine Activation
ANP mRNA and ANP Concentrations
To investigate whether neuroendocrine activation occurs in TG(mREN2)27 and whether it can be reversed by pharmacological inhibition of the RAS, we studied plasma ANP concentrations. Plasma ANP concentrations were increased by 100% in TG(mREN2)27. Treatment with Bay 10–6734 or captopril similarly reduced plasma concentration of ANP. No significant difference was observed between treated TG(mREN2)27 and nontreated control rats. Furthermore, we studied myocardial ANP mRNA expression in left ventricular tissue. There was a specific hybridization signal at 18S observed in untreated TG(mREN2)27, whereas no signal or traces of ANP mRNA were detected in controls (not shown). Treatment with captopril or Bay 10–6734 markedly reduced ANP mRNA expression. The data are summarized in the Table. There was a 40-fold increase in relative ANP mRNA in TG(mREN2)27 compared with controls. This increase in ANP mRNA was reversed by 80% after treatment of TG(mREN2)27 with captopril or Bay 10–6734.

Myocardial Angiotensin II and Neuropeptide Y Concentrations
Because TG(mREN2)27 animals exhibit an activation of the tissue RAS,⁹ we determined whether angiotensin II concentrations were increased in the heart. Angiotensin II concentrations were significantly elevated by 70% in the left ventricles of TG(mREN2)27 (Table). Antihypertensive treatment with Bay 10–6734 or captopril reduced angiotensin II concentrations to values similar to those detected in controls. Angiotensin II has previously been shown to facilitate norepinephrine release from sympathetic nerve endings, thereby producing a local sympathetic activation in the heart.^{30 31} To determine local sympathetic activation, we investigated myocardial neuropeptide Y concentrations. Neuropeptide Y is coreleased with norepinephrine from sympathetic nerves,³² and its steady state concentrations are reported to be less sensitive to acute stress than norepinephrine concentrations.^{33 34} As shown in Fig 1A, there was a reduction in myocardial neuropeptide Y concentrations by 70% in nontreated transgenic animals compared with control rats, reflecting an increase of local sympathetic activation. Treatment with captopril or Bay 10–6734 increased myocardial neuropeptide Y concentrations by 60% versus nontreated TG(mREN2)27 (Fig 1A).

View larger version (34K): [in this window] [in a new window]   Figure 1. Left ventricular neuropeptide Y (NPY) concentrations (A) and ß-adrenoceptor density (B) in SD control rats, untreated TG(mREN2)27 rats, and TG(mREN2)27 rats treated with Bay 10–6734 (Bay) or captopril. Ordinates, Left ventricular immunoreactive NPY (pg/g wet wt) (n=6 to 8) (A) or ß-adrenoceptor density (Bmax) (fmol of [125I]iodocyanopindolol bound/mg of protein) (n=7 to 10) (B).

ß-Adrenergic Signal Transduction
ß-Adrenergic Receptors
To address the question of whether the local sympathetic activation leads to defects of ß-adrenergic signal transduction, we studied ß-adrenergic receptors through the use of saturation radioligand binding experiments. The density of myocardial ß-adrenergic receptors was significantly reduced by 30% in TG(mREN2)27 (Fig 1B). Consistent with the reduction in local sympathetic activation, the number of ß-adrenergic receptors completely recovered after pharmacological treatment with both captopril and Bay 10–6734. The antagonist affinity of [¹²⁵I]iodocyanopindolol, as judged on the basis of the K_D values, did not differ between the studied groups (not shown).

Adenylyl Cyclase Activity
To study whether local angiotensin II elevation, sympathetic activation, and decline in the number of ß-adrenergic receptors as well as the effects of pharmacological treatment on these parameters have a functional effect on the formation of cAMP, adenylyl cyclase activity was determined. Fig 2A shows adenylyl cyclase activity in left ventricular membranes under basal conditions or after stimulation with isoprenaline, Gpp(NH)p, or forskolin. The effects of isoprenaline, Gpp(NH)p, and forskolin on adenylyl cyclase activity were reduced in TG(mREN2)27 compared with control animals. Treatment with Bay 10–6734 as well as with captopril restored the adenylyl cyclase activities in TG(mREN2)27. The effects of Bay 10–6734 and captopril on adenylyl cyclase activities were similar.

View larger version (39K): [in this window] [in a new window]   Figure 2. Basal and isoprenaline-, Gpp(NH)p-, and forskolin-stimulated adenylyl cyclase activities (A) and MnCl2- and MnCl2-plus-forskolin–stimulated adenylyl cyclase activities (B) in left ventricular membranes from SD control rats, untreated TG(mREN2)27 rats, or TG(mREN2)27 rats treated with Bay 10–6734 (Bay) or captopril. *P<.05 versus SD (n=7). A, After treatment of TG(mREN2)27, the activity of adenylyl cyclase was not significantly different from that of SD but significantly (P<.05) increased compared with that of control TG(mREN2)27. B, Activity of the catalyst did not differ significantly between SD or treated or untreated TG(mREN2)27 (n=7).

Catalyst Activity
A reduction in the forskolin effects on adenylyl cyclase could be an indication of a defect of the catalyst.³⁵ Because forskolin effects are also dependent on GTP-activated G proteins,³⁵ the effects of forskolin were studied in the presence of manganese ions, which have been reported to uncouple the catalyst from the influence of G proteins.³⁶ The data are summarized in Fig 2B. MnCl₂ and MnCl₂ plus forskolin similarly stimulated adenylyl cyclase activity in all groups. Taken together, there was no evidence that the catalyst is altered in TG(mREN2)27 compared with controls or that ACE inhibitor or AT₁ antagonist treatment has an effect on catalyst activity.

Stimulatory G Protein Subunits (G_s)
The reduction in the Gpp(NH)p effect on adenylyl cyclase activity in the presence of an unchanged catalyst activity could be explained by a reduced activity of G_s in TG(mREN2)27. To investigate whether G_s was functionally impaired, reconstitution experiments were performed. G_s was solubilized from left ventricular membranes and reconstituted into S49 cyc^- cell membranes, which genetically lack G_s. In native S49 cyc^- membranes, no stimulation with Gpp(NH)p or isoprenaline was observed. After reconstitution of G_s from rat left ventricles, isoprenaline and Gpp(NH)p stimulation was restored (not shown). Fig 3 summarizes the effects of reconstitution of G_s on basal and Gpp(NH)p-stimulated adenylyl cyclase activity. There was neither an impairment in TG(mREN2)27 nor an effect of pharmacological treatment on G_s function.

View larger version (39K): [in this window] [in a new window]   Figure 3. Basal and Gpp(NH)p-stimulated adenylyl cyclase activities in S49 cyc- mouse lymphoma cell membranes reconstituted with Gs solubilized from left ventricular membranes of SD control rats, untreated TG(mREN2)27 rats, or TG(mREN2)27 rats treated with Bay 10–6734 (Bay) or captopril. There was no significant difference among the three groups (n=6).

Inhibitory G Protein Subunits (G_i)
One important and well known mechanism of adenylyl cyclase desensitization is an increase in G_i in heart failure and cardiac hypertrophy. In TG(mREN2)27, an increase of pertussis toxin substrates and a restoration of adenylyl cyclase after treatment of membranes with pertussis toxin have been observed previously.¹⁰ Consistently, pertussis toxin substrates (Fig 4), immunochemically detectable G_i (Fig 5A), and steady state mRNA levels of G_i2 (Fig 5B) were increased by 31%, 42%, and 48%, respectively. Treatment of TG(mREN2)27 with the AT₁ antagonist Bay 10–6734 or the ACE inhibitor captopril led to a complete reversal of the elevated concentrations of pertussis toxin substrate, G_i protein, or G_i mRNA.

View larger version (41K): [in this window] [in a new window]   Figure 4. Gi-related pertussis toxin substrates (40 kD) in left ventricular membranes from SD control rats, untreated TG(mREN2)27 rats, or TG(mREN2)27 treated with Bay 10–6734 (Bay) or captopril. Top, Selected autoradiograms. Values are mean for pertussis toxin substrates as determined by detecting the Cerenkow radiation of the excised relevant Gi-related bands (40 kD). Left ventricular membranes were treated with pertussis toxin plus [32P]NAD as substrate. Then, 25 µg of treated left ventricular membranes was separated electrophoretically on each lane before autoradiography. Film exposure time was 4 hours. Ordinate, Radioactivity (cpm). *P<.05 vs control (n=8 or 9).

View larger version (33K): [in this window] [in a new window]   Figure 5. Immunoblot analysis of Gi (40 kD) in left ventricular myocardial membranes (A) and Northern blot analysis of Gi mRNA (B) from SD control rats, untreated TG(mREN2)27 rats, or TG(mREN2)27 rats treated with Bay 10- 6734 (Bay) or captopril. *P<.05 vs control (n=6).

	Discussion

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The presented findings clearly indicate that AT₁ receptor antagonism is equally effective as ACE inhibition in normalizing ß-adrenergic signal transduction defects such as reduced adenylyl cyclase activity, ß-adrenergic receptor desensitization, and increase in G_i2 mRNA and protein content. The observations are consistent with a reduction in local sympathetic activation as determined by the restoration of the depleted myocardial neuropeptide Y stores in TG(mREN2)27 animals.

Effects of Treatment on Sympathetic Signal Transduction
ß-Adrenergic desensitization has been suggested to represent an important mechanism of contractile dysfunction in heart failure.^{11 12 13 14 15 16} The diminished formation of the second-messenger cAMP after stimulation of cardiac ß-adrenergic receptors is due to a decline in the number of ß-adrenergic receptors,^{11 12 13} an uncoupling of ß-adrenergic receptors, and an increase in inhibitory G protein subunits.^{14 15 16} The underlying mechanism inducing these desensitization processes is an activation of the sympathetic nervous system and, in particular, sympathetic activation in the heart itself.² Several reports indicate that ß-adrenergic neuroeffector defects occur not only in terminal heart failure but also in hypertensive heart disease.³⁷ These data have been obtained in rat models of hypertensive heart disease (eg, spontaneously hypertensive rats^{38 39} ), rat models of acquired forms of hypertension (eg, reduced renal mass,⁴⁰ renal artery banding,^{40 41} and deoxycorticosterone treatment^{40 42} ), Dahl rats with salt-sensitive hypertension,⁴³ and the transgenic rat strain TG(mREN2)27.¹⁰ ß-Adrenergic dysregulation has been suggested to represent an important pathogenetic mechanism contributing to the progression from compensated hypertrophy to overt failure¹⁰ because adenylyl cyclase desensitization has already developed in the compensated stage of cardiac hypertrophy and this condition is considered to be an independent risk factor for the development of heart failure.

Previously, we examined the ß-adrenergic alterations of transgenic rats with renin-induced hypertension TG(mREN2)27.¹⁰ These rats have been shown to exhibit a similarity to the neuroeffector alterations occurring in the human heart and thus appear to represent a suitable model with which to study the effects of experimental treatment regimens on the cellular alterations in sympathetic signal transduction. In addition, these rats show increased activity of the tissue RAS in several organs, including the heart, as shown by the increased myocardial angiotensin II concentrations. Increased myocardial concentrations of angiotensin II could represent an important regulatory mechanism to increase sympathetic activity of the heart.^{30 31} Presynaptic angiotensin II receptors facilitate the release of norepinephrine from cardiac sympathetic nerve terminals.⁴⁴ Angiotensin II has been reported to increase circulating norepinephrine concentrations in rats.⁴⁵ This effect was sensitive to the AT₁ receptor antagonist losartan.⁴⁵ In rats, infusion of angiotensin II produced multifocal myocardial necrosis, the development of which could be antagonized by losartan and ß-adrenergic receptor blockade.⁴⁵ Consistently, the reduction in angiotensin II formation by ACE inhibitors has been shown to attenuate ß-adrenergic subsensitivity and restore the number of ß-adrenergic receptors^{7 46 47} and to reduce elevated G_i.^{8 46} Interestingly, these effects have been shown to occur at lower dosages than those are necessary to normalize blood pressure and reverse cardiac hypertrophy.⁴⁶

In the present study, isoprenaline- and guanine nucleotide–stimulated adenylyl cyclase activity was depressed in TG(mREN2)27. This was accompanied by a reduction in ß-adrenergic receptors and an increase in G_i on the functional (pertussis toxin labeling) as well as the protein and mRNA levels. Experiments with forskolin plus manganese ions as well as functional reconstitution of G_s into S49 cyc^- cell membranes, which genetically lack G_s, provided evidence for unchanged bioactivity of G_s and the catalyst of adenylyl cyclase. Consistent with the notion that sympathetic activation in the heart is involved in the desensitization process, the myocardial neuropeptide Y concentrations were markedly reduced. Sympathetic activation was completely reversed through treatment of TG(mREN2)27 with Bay 10–6734 or captopril. This indicates that with respect to the reversal of sympathetic activation, no difference exists between ACE inhibition or AT₁ receptor antagonism.

After the stimulation of ß-adrenergic receptors with agonists, the number of receptors declines.⁴⁸ When rats are treated with high concentrations of ß-adrenoceptor agonists, there is an increase in the levels of inhibitory G protein subunits at the protein and mRNA levels.⁴⁹ This is due to an increase in the transcription rate of the G_i2 gene, as shown in nuclear run-on assays in rats treated with isoprenaline.⁵⁰ Depleted myocardial neuropeptide Y stores are indicative of sympathetic activation, which leads to ß-adrenergic desensitization. Reduction in angiotensin II effects by AT₁ blockade or ACE inhibition restores ß-adrenergic desensitization by reducing sympathetic drive to the heart. Therefore, local angiotensin II formation could be involved in sympathetic activation in the heart.

It is important to note that in the rat heart, ACE contributes 70% to angiotensin II formation, whereas in the human heart, the contribution of ACE amounts to only 15%.^{51 52} In this respect, it is noteworthy that chymase has not been demonstrated in the rat heart. However, in a recent study, the high contribution was questioned of alternative angiotensin II–forming pathways in the human heart.⁵² These investigators reported that angiotensin II formation was 85% sensitive to enalaprilate in vivo and in human heart homogenates in vitro.⁵³ If alternative angiotensin II–forming pathways were relevant in the heart,⁵¹ one would expect different effects of ACE inhibition and AT₁ receptor antagonism. The similar effects of AT₁ antagonism and ACE inhibition have been demonstrated previously in a model of anterior myocardial infarction. As the effect on ß-adrenergic signal transduction, both treatments similarly affected compensatory hypertrophy in noninfarcted areas, coronary flow reserve, and interstitial fibrosis.⁵⁴ The lack of difference between ACE inhibition and AT₁ receptor antagonism in this study argues against a role for accumulation of biogenic peptides like bradykinin and other peptide mediators in ß-adrenergic desensitization, the degradation of which is impaired after ACE inhibition.⁵⁵

In conclusion, in hypertension due to overexpression of renin in various tissues, ACE inhibition and AT₁ antagonist treatment equally restored myocardial angiotensin II and neuropeptide Y concentrations, presumably leading to a normalized ß-adrenergic neuroeffector signal transduction. Because pharmacological inhibition of angiotensin II effects completely reverses ß-adrenergic neuroeffector defects through normalization of the levels of ß-adrenergic receptors and inhibitory G protein subunits, the restoration of ß-adrenergic balance in the heart might be a relevant mechanism by which ACE inhibitors and AT₁ receptor antagonists exert beneficial effects in cardiac failure and hypertrophy.

	Selected Abbreviations and Acronyms

 

ACE = angiotensin-converting enzyme ANP = atrial natriuretic peptide AT1 = angiotensin II type 1 Gpp(NH)p = guanosine-5'-(ß,-imido)triphosphate RAS = renin-angiotensin system SD = Sprague-Dawley SSC = standard saline citrate

	Acknowledgments

 
This work was supported by the Deutsche Forschungsgemeinschaft. Dr Böhm is a recipient of the Gerhard Hess program of the Deutsche Forschungsgemeinschaft. The expert assistance of Bodo Cremers is gratefully acknowledged.

Received July 23, 1997; first decision August 20, 1997; accepted October 24, 1997.

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