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(Circulation. 1995;92:3006-3013.)
© 1995 American Heart Association, Inc.

Articles

Dose-Dependent Dissociation of ACE-Inhibitor Effects on Blood Pressure, Cardiac Hypertrophy, and ß-Adrenergic Signal Transduction

Michael Böhm, MD; Maurizio Castellano, MD; Enrico Agabiti-Rosei, MD; Markus Flesch, MD; Martin Paul, MD; Erland Erdmann, MD

From the Klinik III für Innere Medizin, Universität zu Köln, Germany (M.B., M.F., E.E.); Scienze Mediche, Universitá degli Studi di Brescia, Italy (M.C., E.A.R.); and the Freie Universität Berlin, Berlin, Germany (M.P.).

Correspondence to Michael Böhm, Klinik III für Innere Medizin, Universität zu Köln, Joseph-Stelzmann Str 9, 50924 Köln, Germany.

	Abstract

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Background Dose-dependent effects of ACE inhibitors on blood pressure, cardiac hypertrophy, and ß-adrenergic signal transduction were examined in an animal model with ß-adrenergic desensitization, which has been identified in failing hearts and in hypertensive cardiac hypertrophy. It is unknown whether beneficial ACE-inhibitor effects are due to an unloading of the failing heart or a reduction of neuroendocrine activation with ß-adrenergic resensitization.

Methods and Results Low-dose (LD, 1 mg/kg) and high-dose (HD, 25 mg/kg) fosinopril treatment was performed in spontaneously hypertensive rats (SHR) and control (WKY) rats. Myocardial norepinephrine concentrations, adenylyl cyclase activity, ß-adrenergic receptors (radioligand binding), G_s (functional reconstitution), and G_i (pertussis toxin labeling) were determined. Ventricular weights and blood pressures were measured. HD but not LD reduced blood pressure and left ventricular weights in SHR. Isoprenaline- and guanylylimidodiphosphate-stimulated adenylyl cyclase activities as well as ß₁-adrenergic receptors were reduced in SHR. The catalyst and G_s were unchanged, but G_i and norepinephrine concentrations were increased. Both LD and HD treatments restored ß-adrenergic alteration.

Conclusions LD treatment with ACE inhibitors restored ß-adrenergic signal transduction defects independently of regression of cardiac hypertrophy. This could contribute to the effects of ACE inhibitors in patients, who are often treated with nonhypotensive doses.

Key Words: angiotensin • enzymes • blood pressure • hypertrophy • receptors, adrenergic, beta

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
ß-Adrenergic desensitization has been well characterized as a key alteration of the failing human heart leading to contractile dysfunction and to reduced exercise tolerance.¹ The cellular mechanisms are a downregulation of ß₁-adrenergic receptors^{2 3 4} and an increased activity of G_i proteins.^{5 6 7} Recently, several reports provided evidence that not only in the failing human heart but also in hypertensive cardiac hypertrophy, a heterologous desensitization of the adenylyl cyclase system occurs,^{8 9 10 11 12 13 14 15 16 17 18} which involves upregulation of G_i proteins^{11 14 15 16 17 18} and downregulation of ß-adrenergic receptors.^{8 9 11 16 19 20} Since myocardial hypertrophy represents an adaptional process after pressure load of the myocardium²¹ and is regarded as one initial step in the development of heart failure,²² it has been suggested that adenylyl cyclase desensitization already occurs during cardiac hypertrophy and can be regarded as one of the early changes leading to the progression to heart failure.^{11 14 15 16} In the present study, SHR and controls were studied as a model for hypertensive cardiac hypertrophy, with cellular alterations of sympathetic neuroeffector mechanisms comparable to those in failing myocardium.^{14 15 16} Since cardiac hypertrophy caused by hypertension occurs in SHR, these animals represent a suitable model to investigate whether treatment with ACE inhibitors is able to restore normal ß-adrenergic neuroeffector mechanisms independently of a reduction of blood pressure and myocardial hypertrophy. This question is of great clinical importance because many patients with heart failure are treated with low, nonantihypertensive doses of ACE inhibitors. In these patients, the mechanisms of the beneficial effect on outcome are as yet unknown.

	Methods

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Animals and Treatment
Experiments were conducted on SHR and WKY rats obtained from Charles River Laboratory, Calco, Italy. The rats were held in a temperature-controlled room on a 12-hour light/dark cycle. Rat chow and tap water were available ad libitum. Two groups of SHR (n=10 each) received fosinopril in their drinking water from 6 to 12 weeks of age at a dose of 25 mg · kg^-1 · d^-1 ("high dose") or 1 mg · kg^-1 · d^-1 ("low dose"). These doses were selected in accordance with the results of previous experiments with similar ACE inhibitors and of a preliminary trial with fosinopril, indicating that the high dose was expected to reduce blood pressure to levels almost superimposable on those of WKY rats, whereas the low dose had no significant antihypertensive effect. WKY control rats were treated in the same way. The concentration of fosinopril in the water was adjusted to the water consumption. The treated groups were compared with untreated SHR (n=10) and untreated WKY rats (n=10). Systolic blood pressure and heart rate were measured with the tail-cuff method (IITC Life Science Instruments). At the end of week 12, the animals were killed by decapitation. The hearts were immediately excised, dissected into right and left ventricles, weighed, frozen in liquid nitrogen, and stored at -80°C.

Membrane Preparations for Radioligand Binding Studies and G-Protein Studies
Myocardial tissue was chilled in 30 mL ice-cold homogenization buffer (in mmol/L: Tris-HCl 10, Na₂-EDTA 1, and DTT 1; 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. After that, the membrane preparation was homogenized by hand for 1 minute with a glass-glass homogenizer. The homogenate was spun at 484g (rotor, Beckman JA 20) for 10 minutes. The supernatant was filtered through two layers of cheesecloth, 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 volumes 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 -70°C. As determined in independent experiments on rat and human myocardial membranes, the storage of the preparations up to 1.5 years did not alter the recovery of ß-adrenergic receptors (determined in previous experiments).

Radioligand Binding Studies
The assays were performed in a total volume of 250 µL incubation buffer. The incubation was carried out at 37°C for 60 minutes. These conditions allowed complete equilibration of the receptors with the radioligand. The reaction was terminated by rapid vacuum filtration through Whatman GF/C filters, and the filters were immediately washed three times with 6 mL each of ice-cold incubation buffer. All experiments were performed in triplicate. Radioactivity was determined in a gamma counter (LKB Wallac, 1272 Clinigamma). Myocardial ß-adrenergic receptors were studied with [¹²⁵I]iodocyanopindolol as radiolabeled ligand as described previously.¹⁶ Specific activity was 2000 Ci/mmol. (-)-Propranolol 1 µmol/L was used to determine nonspecific binding. In a second series of experiments, ß₁- and ß₂-adrenergic receptor subtypes were determined in competition experiments by use of the ß₁-selective antagonist CGP 207.12A and the ß₂-selective antagonist ICI 118.551; the ratio of ß₁- to ß₂-adrenergic receptors was determined from competition of 25 pmol/L [¹²⁵I]iodocyanopindolol binding (approximate K_d value) by 50 nmol/L ICI 118.551 or 300 nmol/L CGP 207.12A. These concentrations of the selective antagonists completely antagonized binding to the ß₁- and ß₂-adrenergic receptor subtypes, as judged from complete competition curves analyzed according to the method of DeLean et al²³ (data not shown). From the ratio of ß₁- to ß₂-adrenergic receptors, the density of each subtype was calculated from B_max values obtained in saturation experiments. B_max values had to be determined and competition experiments with ICI 118.551 and CGP 207.12A performed on different days, and densities of the ß₁- and ß₂-adrenergic receptors were calculated in each individual heart. The experiments on WKY rats and SHR were conducted on the same day in parallel.

Adenylyl Cyclase Determinations
Adenylyl cyclase was determined according to Salomon et al²⁴ with some modifications, as described elsewhere.²⁵ In brief, washed membrane fractions (10 000g sediment) were prepared from homogenates of rat hearts. 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 at pH 7.4 in a final volume of 100 µL. 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. Next, the zinc acetate was neutralized by 600 µL Na₂CO₃ (144 mmol/L). After centrifugation for 5 minutes at 10 000g, 0.8 mL of the supernatant was applied on neutral alumina columns equilibrated with 0.1 mmol/L Tris/HCl, pH 7.5. The effluent was collected, and [³²P]cAMP was determined by measuring radioactivity in a liquid scintillation spectrometer (LKB Wallac 1272 Clinigamma).

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 DTT; 2 mmol/L ATP; 1 mmol/L GTP; 50 nmol/L [³²P]NAD (800 Ci/mmol); Lubrol PX 0.5% (vol/vol); and 20 µg/mL pertussis toxin that had been activated by incubation with 50 mmol/L DTT for 1 hour at 20°C before the labeling reaction. The experimental details have been described earlier.^{25 26} Samples were subjected to SDS-PAGE (10% wt/vol acrylamide, 16 cm total gel length). Gels were stained with Coomassie blue and dried before autoradiography was performed.

Treatment of Membranes With Pertussis Toxin Plus NAD
Pertussis toxin treatment was performed under the same incubation conditions as used for [³²P]ADP ribosylation, except that [³²P]NAD was replaced by 3 mmol/L NAD in the reaction. After two washings, membranes were subjected to [³²P]ADP ribosylation or determination of adenylyl cyclase activity. Control membranes were subjected to the same incubation conditions, except that pertussis toxin was omitted from the medium. The same results were obtained when heat-inactivated pertussis toxin was used. Experimental details have been described previously.²⁵

S49 Lymphoma cyc^- Cells
S49 lymphoma cyc^- cells were grown in suspension culture in RPMI 1640 medium supplemented with 10% (vol/vol) FCS (culture volume, 100 mL) or 10% (vol/vol) horse serum (culture volume, 100 mL), NaHCO₃ (44 mmol/L), glucose (5.5 mmol/L), L-glutamine (5 mmol/L), nonessential amino acids (5 mmol/L), sodium pyruvate (1 mmol/L), penicillin (50 U/mL), and streptomycin (50 µg/mL) in a humidified atmosphere of 90% air/10% CO₂. The cell density was maintained at 1x10⁶ cells/mL. Cells (1x10¹⁰ to 2x10¹⁰ cells in 10 to 20 L medium) were harvested by centrifugation in a Beckman type JA-10 rotor at 1000g for 20 minutes at 4°C. The pellets were resuspended in 50 mL triethanolamine/HCl (10 mmol/L) (pH 7.4 at 20°C). The final pellet was resuspended in 100 to 150 mL of lysis buffer containing sucrose (0.25 mol/L), Tris/HCl (20 mmol/L, pH 7.5 at 20°C), MgCl₂ (1.5 mmol/L), ATP (1 mmol/L), benzamidine (3 mmol/L), leupeptin (1 µmol/L), PMSF (1 mmol/L), and soybean trypsin inhibitor (2 µg/mL). Cells were homogenized by nitrogen cavitation. The cavitate was centrifuged in a JA-20 rotor (Beckman) at 1500g for 45 seconds at 4°C to remove unbroken cells and nuclei and filtered through two layers of cheesecloth. A crude membrane fraction was isolated from the resulting supernatant by centrifugation in a JA-20 rotor at 5000g for 20 minutes at 4°C. The membranes were washed three times with a buffer containing (in mmol/L) Tris/HCl 20 (pH 7.5 at 20°C), EDTA 1, DTT 1, benzamidine 3, and PMSF 1; leupeptin 10 µmol/L; and soybean trypsin inhibitor 2 µg/mL; resuspended in 10 mg of protein/mL with this buffer; and stored at -80°C. The yield of membrane protein was 100 mg/10¹⁰ cells.

Reconstitution of Myocardial G_s Into S49 cyc^- Membranes
Reconstitution assays were performed according to Sternweis et al.²⁷

Norepinephrine Determinations
For norepinephrine measurements, tissue samples were homogenized with a Polytron in 0.1 mol/L Tris/HCl (pH 7.4). After centrifugation (10 000g, 30 minutes), norepinephrine was extracted with alumina columns and determined by high-performance liquid chromatography with electrochemical detection as described by Castelleno et al.²⁰

Miscellaneous
Protein was determined according to the method of Lowry et al,²⁸ with BSA used as standard. SDS-PAGE was performed as described by Lämmli.²⁹ 5'-Nucleotidase activity was analyzed by the method of Dixon and Purdom.³⁰

Materials
Forskolin was donated by Hoechst AG, Dr Metzger (Frankfurt, Germany). GTP, Gpp(NH)p, ATP, creatine phosphate, and creatine kinase were purchased from Boehringer-Mannheim and isobutylmethylxanthine from EGA-Chemie. [³²P]ATP was from Amersham-Buchler. DTT was from Serva. Pertussis toxin was from List Biological Laboratories.

Statistics
The data shown are mean±SEM. Statistical significance was estimated with Student's t test for unpaired observations and ANOVA according to Wallenstein et al.³¹ A value of P<.05 was considered significant. K_d values were determined graphically in each individual experiment.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Blood Pressure and Cardiac Weights
As summarized in the Table, treatment of SHR with a high dose of fosinopril significantly reduced blood pressure to values similar to those observed in WKY control rats. Low-dose fosinopril treatment failed to significantly reduce systolic blood pressure in SHR. In WKY rats, no significant effects on blood pressure were observed after high-dose or low-dose treatment with fosinopril. Heart rate was significantly higher in SHR controls compared with WKY controls (P<.001), but treatment with fosinopril had no effect on heart rate in either group (not shown). Fig 1 shows the wet weights of the right and left ventricles of SHR or control rats under control conditions or after treatment with fosinopril. In SHR, left ventricular weight was significantly higher than in WKY rats. No significant differences could be observed in the right ventricular weights between the groups. In SHR, high doses of fosinopril treatment reduced left ventricular weight significantly, whereas no significant effects on left ventricular hypertrophy were observed in animals on low-dose fosinopril treatment. Fosinopril treatment at low and high doses had no effect on the weight of the nonhypertrophied right ventricles in SHR.

View this table: [in this window] [in a new window]   Table 1. Systolic Blood Pressure in SHR and WKY Rats After Oral Treatment With a Low Dose or High Dose of Fosinopril

View larger version (25K): [in this window] [in a new window]   Figure 1. Bar graphs showing the weights of the right ventricles (top) and the left ventricles (bottom) in SHR and WKY rats under control conditions (control) and after oral treatment with a low (LD) or a high (HD) dose of fosinopril.

Adenylyl Cyclase Activity
To study the effect of treatment on signal transduction defects of adenylyl cyclase, we first set out to investigate the alterations of the ß-adrenergic receptor–adenylyl cyclase system in the strain of SHR we used. Fig 2 shows adenylyl cyclase activity in rat myocardial membranes after stimulation with isoprenaline, Gpp(NH)p, or forskolin. The effect of isoprenaline was reduced significantly, by 60%, in membranes from SHR compared with WKY rats. To determine postreceptor events, the effects of the metabolically stable guanine nucleotide derivative Gpp(NH)p as well as of the diterpen derivative forskolin were studied. As with isoprenaline, the effects of forskolin and Gpp(NH)p were reduced in myocardial membranes from SHR compared with WKY rats. These data provided evidence for an impaired ß-adrenergic signal transduction, altered G-protein function, a depressed activity of the catalyst of the adenylyl cyclase, or a combination thereof.

View larger version (20K): [in this window] [in a new window]   Figure 2. Bar graph showing isoprenaline-, Gpp(NH)p-, and forskolin-stimulated adenylyl cyclase activity in myocardial membranes from SHR and WKY rats (Ctr). Ordinate shows increase of adenylyl cyclase activity in pmol cAMP per mg proteinx20 minutes.

To assess whether or not the function of the catalyst is impaired, the effects of forskolin and forskolin plus Gpp(NH)p were studied alone and in the presence of MnCl₂. MnCl₂ is known to uncouple the catalyst from the influence of GTP-activated G-protein -subunits.^{32 33} The effects of forskolin and forskolin plus Gpp(NH)p were reduced in SHR compared with control rats in the absence of MnCl₂ (Fig 3, left). In the presence of 5 mmol/L MnCl₂, adenylyl cyclase activity was not different between SHR and WKY rats. The lack of effect of Gpp(NH)p in the presence of forskolin and MnCl₂ (Fig 3, right) shows that under the experimental conditions used, the effects of forskolin are independent of Gpp(NH)p-stimulated G-protein -subunits. Taken together, the activity of the catalyst appears to be unchanged.

View larger version (20K): [in this window] [in a new window]   Figure 3. Bar graph showing adenylyl cyclase activity after stimulation with forskolin (30 µmol/L) and forskolin plus Gpp(NH)p (100 µmol/L) alone or in the presence of 5 mmol/L MnCl2. MgCl2 was withheld from the assay medium. Ordinate shows adenylyl cyclase activity in pmol cAMP per mg proteinx20 minutes. Ctr indicates control (WKY) rats.

The reduced effects of Gpp(NH)p (Fig 2, middle) could be due to an altered function or content of G proteins. To investigate the function of G_s, reconstitution experiments were performed in S49 cyc^- mouse lymphoma cell membranes. High G_s was solubilized from membranes of SHR and control rats and reconstituted into S49 cyc^- membranes. Reconstitution of G_s from SHR and control membranes increased basal adenylyl cyclase activity similarly, by about 110%, in S49 cyc^- membranes. In reconstituted S49 cyc^- membranes, isoprenaline and Gpp(NH)p stimulated adenylyl cyclase to similar levels when G_s from SHR or control animal membranes were investigated. Thus, these experiments show that the activity of myocardial G_s was similar in SHR compared with control rats.

The reduced effects of Gpp(NH)p on adenylyl cyclase in SHR could also be explained by an increased activity of G_i in this condition. To address this question, myocardial membranes were treated with pertussis toxin plus NAD or heat-inactivated pertussis toxin plus NAD. Fig 5 shows an autoradiograph of [³²P]ADP ribosylation by pertussis toxin in native membranes and membranes modified by pertussis toxin plus NAD. Incorporation of radioactivity into the 40-kD G_i-related protein was markedly attenuated. This demonstrated that the vast majority of G-protein -subunits was covalently modified by this procedure. In treated and control membranes, adenylyl cyclase was determined. As shown in Fig 6, basal and Gpp(NH)p-stimulated adenylyl cyclase activities were significantly depressed in membranes from SHR compared with control animals. The difference was abolished in membranes after treatment with pertussis toxin plus NAD. The effect of pertussis toxin to increase adenylyl cyclase activity was significant only in membranes from SHR but not in membranes from WKY rats. Taken together, these experiments show that an increased activity of G_i is present in membranes from SHR and contributes to adenylyl cyclase activity in this condition.

View larger version (16K): [in this window] [in a new window]   Figure 5. Representative autoradiograph of [32P]ADP ribosylation (40 kD) by pertussis toxin (PT) of the inhibitory G-protein -subunits in myocardial membranes from one WKY control rat treated with activated pertussis toxin plus NAD or with inactivated pertussis toxin plus NAD. Membranes were separated by SDS-PAGE (10%) before autoradiography. Each lane contained 25 µg membrane protein. The exposure time of the film was 30 minutes.

View larger version (21K): [in this window] [in a new window]   Figure 6. Bar graph showing basal (left) and Gpp(NH)p-stimulated (right) adenylyl cyclase activity in control and pertussis toxin plus NAD–treated rat myocardial membranes from the left ventricles of SHR and WKY rats. Ordinate shows adenylyl cyclase activity in pmol cAMP per mg protein x20 minutes. Each column gives the mean of four or five independent determinations. Note that the difference in adenylyl cyclase activity between SHR and WKY rats was abolished after treatment with pertussis toxin plus NAD. Only in SHR but not in WKY rats, pertussis toxin produced a significant increase in basal or Gpp(NH)p-stimulated adenylyl cyclase activity.

Effects of Fosinopril Treatment
To quantify G_i proteins and to investigate the effects of high- and low-dose treatment on G_i levels, we studied G_i with [³²P]ADP ribosylation catalyzed by pertussis toxin. Fig 7 shows pertussis toxin–catalyzed [³²P]ADP ribosylation in membranes of SHR compared with WKY rats. Pertussis toxin substrates of rat myocardial membranes comigrated with purified G_i/G_o -subunits from bovine brain. Incorporation of radioactivity into the 40-kD membrane protein was increased in SHR compared with WKY rats. After treatment with fosinopril, incorporation of [³²P]ADP ribose was reduced. In this experiment, [³²P]ADP ribosylation was similar in the high-dose fosinopril-treated SHR compared with the WKY control. The data are summarized in Fig 8. In SHR, G_i proteins, as measured with the pertussis toxin–catalyzed [³²P]ADP ribosylation technique, were increased significantly, by 35%, in SHR compared with WKY rats. Treatment with high- and low-dose fosinopril reduced G_i proteins in SHR but not in WKY rats. The reduction of G_i was similar after high- and low-dose treatment. The data were similar when related to 5'-nucleotidase activity as myocardial membrane marker (not shown).

View larger version (31K): [in this window] [in a new window]   Figure 7. Representative autoradiograph of [32P]ADP ribosylation (40 kD) by pertussis toxin of the inhibitory G-protein -subunits in myocardial membranes from one WKY control rat and from SHR under control conditions and after oral treatment with a low (LD) or a high (HD) dose of fosinopril. Membranes were separated by SDS-PAGE (10%) before autoradiography. Each lane contained 25 µg of membrane protein. The exposure time of the film was 90 minutes. Gi/Go standard (0.5 µg) purified from bovine brain is shown for comparison.

View larger version (29K): [in this window] [in a new window]   Figure 8. Bar graph showing incorporation of radioactivity by pertussis toxin–catalyzed [32P]ADP ribosylation into an 40-kD protein corresponding to inhibitory G-protein -subunits of myocardial membranes from SHR and WKY rats under control conditions and after oral treatment with a low (LD) or a high (HD) dose of fosinopril.

Myocardial ß-Adrenergic Receptors
In cardiac hypertrophy of SHR, a small but significant reduction of ß-adrenergic receptors has been observed. Fig 9A demonstrates that total numbers of ß-adrenergic receptors were reduced significantly, by 22%, in SHR compared with WKY rats. The reduction was merely due to a decline of the number of ß₁-adrenergic receptors (Fig 9B), whereas no significant change was observed with ß₂-adrenergic receptors (Fig 9C). After treatment with fosinopril at high and low doses, no significant difference could be observed between WKY rats and SHR.

View larger version (29K): [in this window] [in a new window]   Figure 9. Bar graphs showing ß-adrenergic receptors (A), ß1-adrenergic receptors (B), and ß2-adrenergic receptors (C) in myocardial membranes from the left ventricles of SHR and WKY control rats under control conditions (Controls) and after oral treatment with a low (LD) or a high (HD) dose of fosinopril.

Myocardial Norepinephrine Concentrations
One potential mechanism for adenylyl cyclase desensitization is an excessive action of catecholamines on the myocardium. Therefore, we investigated myocardial norepinephrine concentrations in SHR and WKY rats and the effects of fosinopril treatment.

Fig 10 shows that myocardial norepinephrine concentrations were markedly increased in SHR compared with WKY rats. Fosinopril at high or low doses did not change norepinephrine concentration in myocardium of WKY rats. In SHR, myocardial norepinephrine concentrations were significantly reduced after oral treatment with high or low doses of fosinopril. There was no difference between SHR on high- or low-dose fosinopril.

View larger version (28K): [in this window] [in a new window]   Figure 10. Bar graph showing the norepinephrine concentrations in left ventricular tissue from SHR and WKY rats under control conditions (Control) and after oral treatment with a low (LD) or a high (HD) dose of fosinopril.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study shows that heterologous desensitization of adenylyl cyclase occurs in SHR compared with age-matched control WKY rats. Desensitization is produced by a small decline of ß-adrenergic receptors and of the ß₁-adrenergic receptor subtype, in particular by an increase of G_i. The direct experimental evidence for the latter suggestion is that depressed basal and Gpp(NH)p-stimulated adenylyl cyclase activities are completely restored after treatment of membranes with pertussis toxin to inactivate G_i. Fosinopril treatment at low doses did not reduce blood pressure or cardiac hypertrophy but reduced G_i proteins and restored myocardial ß₁-adrenergic receptors. High-dose treatment with fosinopril reduced blood pressure, cardiac hypertrophy, and signal transduction defects of adenylyl cyclase. Thus, there appears to be a clear dose-dependent dissociation between the effects of ACE inhibitors on blood pressure, cardiac structure, and ß-adrenergic signal transduction.

ß-Adrenergic desensitization has been shown to occur in end-stage human heart failure. It is regarded as one important alteration leading to contractile dysfunction and to impaired exercise tolerance.¹ In this condition, a downregulation of myocardial ß-adrenergic receptors^{2 3 4} and an increase of G_i proteins^{5 6 7} have been identified as underlying alterations of the myocardial cells. Heterologous adenylyl cyclase desensitization has also been observed in various models of cardiac hypertrophy due to the secondary^{14 19} or genetic forms^{9 11 15 16 19} of hypertension. Since, according to the Framingham study,³⁴ chronic pressure overload is one leading cause of chronic heart failure, it has been suggested that the decrease of ß-adrenergic effects on adenylyl cyclase observed in hypertensive cardiac hypertrophy could be one pathophysiological factor contributing to the progression of contractile dysfunction in the hypertrophied heart to overt heart failure.^{11 14 15 16} Since similar cellular alterations occur in the failing human heart and in various animal models of hypertension, the latter condition appears to be an appropriate tool to study drug effects on ß-adrenergic signal transduction alterations.

The beneficial effects of medical treatment with ACE inhibitors is well established in prevention³⁵ and treatment³⁶ of heart failure. However, it is not clear which of the potentially relevant mechanisms, ie, reduction of sympathetic tone or inhibition of myocardial cell growth due to afterload reduction, is responsible for the improved prognosis of patients with heart failure after treatment with ACE inhibitors. Because of the blood pressure–lowering effects of these agents, a lower dose is often used in patients with heart failure than in patients with hypertension. At present, data on myocardial effects produced by different doses of ACE inhibitors are not available.

It is interesting to note that changes of ß-adrenergic signal transduction similar to those in heart failure also occur in myocardial hypertrophy in the absence of failure. Presynaptic angiotensin II receptors facilitate the release of norepinephrine from sympathetic nerve terminals.³⁷ In the human heart, low concentrations of angiotensin II facilitate norepinephrine release,³⁸ whereas higher concentrations block the uptake of norepinephrine in rabbit heart.³⁹ Thus, it is likely that a reduction of angiotensin II effects by ACE inhibition could inhibit the local adrenergic drive of the myocardium directly and independently from myocardial hypertrophy and blood pressure. These findings raise the question of whether or not the beneficial effects of ACE inhibitors could be due to an attenuation of myocardial hypertrophy processes, to a restoration of ß-adrenergic signal transduction defects, or to both mechanisms. In the present study, this issue was addressed by use of a nonantihypertensive and an antihypertensive dose of fosinopril. After treatment of SHR with the high and the low doses, ß-adrenergic receptors and G_i proteins were not significantly different compared with the control animals. In the WKY rat controls, none of the treatment regimens had a significant influence on the density of ß-adrenergic receptors or the amount of myocardial G_i proteins. At the low dose of fosinopril, there was no significant reduction of heart weight, relative heart weight, left ventricular weight, or blood pressure. Thus, it is concluded that even at low doses of ACE inhibitors, a normalization of cellular components of the ß-adrenergic signal transduction pathway can be expected, even though there is no effect on cardiac hypertrophy or blood pressure. The findings presented provide an explanation why beneficial effects are observed even at low doses of ACE inhibitor. The use of rather high dosages of captopril in the SAVE study⁴⁰ has often been a matter of debate, because many patients are treated with much lower dosages in clinical practice. Although beneficial effects of low-dose ACE inhibition on prognosis are as yet unclear, the present results are in favor of this suggestion, because one key feature of failing myocardium, namely, cellular alterations leading to ß-adrenergic desensitization, is normalized.

In SHR, an increase of myocardial norepinephrine stores has been observed in this and in previous studies.^{41 42 43} The increase in norepinephrine concentrations has also been observed in the myocardium of prehypertensive SHR^{20 43} and in the blood vessels of hypertensive animals.⁴¹ Low- and high-dose fosinopril treatment reduced the myocardial norepinephrine concentrations. Since ß-adrenergic activation has been shown to represent one alteration to downregulate ß-adrenergic receptors and to increase G_i,⁴⁴ the reduction of local catecholamine effects in the heart by a reduction of catecholamine content could be one mechanism involved in the reduction of G_i and the increase of ß-adrenergic receptors. However, an increase in myocardial norepinephrine content is a rather unusual reflection of an increase of the activity of the sympathetic nervous system. In the heart with failing myocardium, the myocardial norepinephrine concentrations are reduced.⁴⁵ The increased activity of cardiac sympathetic nerves^{46 47} and also a reduction of norepinephrine uptake 1 carrier sites⁴⁸ have been suggested to contribute to this phenomenon. In transgenic rats that harbor the mouse renin ren-2 gene, which develop severe arterial hypertension and cardiac hypertrophy in the absence of heart failure, myocardial norepinephrine concentrations have also been reported to be reduced.¹⁶ Thus, the increase of myocardial norepinephrine is not a general phenomenon of hypertensive cardiac hypertrophy rather than a peculiarity of SHR. The mechanism of the reduction of norepinephrine concentrations after ACE inhibition is also not clear. Stimulation of presynaptic angiotensin II receptors facilitates the release of norepinephrine from sympathetic nerve terminals.³⁸ Thus, one would expect a reduced release, ie, increased myocardial norepinephrine stores after ACE inhibition, but not the opposite. As shown by the present data, the mechanism of the reduction of elevated norepinephrine stores needs further investigation.

In conclusion, a heterologous desensitization of adenylyl cyclase occurs in the myocardium of SHR, which is due to an increase of the activity of G_i proteins and to a small reduction of the number of ß-adrenergic receptors. ACE-inhibitor treatment is able to completely normalize the cellular alterations even at low, nonantihypertensive doses. Since a reduction of myocardial hypertrophy did not occur at low doses, these findings provide evidence that the beneficial effects in patients with heart failure, who are often treated with low doses of ACE ihibitors, could be due to the restoration of ß-adrenergic neuroeffector mechanisms and could occur independent of a reduction of myocardial hypertrophy processes.

	Selected Abbreviations and Acronyms

 

ACE = angiotensin-converting enzyme DTT = dithiothreitol Gpp(NH)p = guanylylimidodiphosphate SHR = spontaneously hypertensive rats WKY = Wistar-Kyoto

View larger version (16K): [in this window] [in a new window]   Figure 4. Bar graph showing basal (left), isoprenaline-stimulated (10 µmol/L, middle), and Gpp(NH)p-stimulated (100 µmol/L, right) adenylyl cyclase activity in S49 cyc- mouse lymphoma membranes reconstituted with Gs solubilized from myocardial membranes of SHR and age-matched WKY rats as controls (Ctr). Ordinate shows adenylyl cyclase activity in pmol cAMP per mg protein x20 minutes.

	Acknowledgments

 
Experimental work was supported by the Deutsche Forschungsgemeinschaft and by a grant from Bristol-Myers Squibb, Munich, Germany. Dr Böhm is supported by the Gerhard-Hess and Heisenberg programs of the Deutsche Forschungsgemeinschaft. We thank Evelyn Behrendt and Judith Sabo for excellent technical assistance.

Received March 14, 1995; revision received May 31, 1995; accepted June 23, 1995.

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