Transgenic TGR(mREN2)27 rats (TGRs), carrying the mouse ren-2 renin gene, have been developed as monogenetic animal models of hypertension (1). These rats are characterized by a fulminant hypertension with typical secondary complications such as nephrosclerosis (2,3), myocardial hypertrophy (4), and vascular damage (2,5). In a previous study, we were able to demonstrate that adult TGRs show circadian rhythms in blood pressure (BP) that are inverse to those in heart rate and locomotor activity (6). In human secondary hypertension due to endocrine and renal diseases, BP rhythmicity is frequently abolished or reversed (7), comparable to our observations in TGRs, which, therefore, could serve as an animal model of human secondary hypertension.
It has repeatedly been observed that a reduced day-night variation in BP in hypertensive patients is associated with a higher degree of myocardial hypertrophy (8,9) and renal functional impairment (10), the major secondary complications of hypertension. Therefore, pharmacologic treatment of these patients aims at the restoration of a physiologic nocturnal BP decrease in addition to a decrease in the overall BP level. It has, however, not been shown that a pharmacologically induced BP decrease at night is indeed able to reduce the severity of end-organ damage in hypertensive subjects. Data in patients with cerebrovascular events demonstrated that antihypertensive-treated patients with nocturnal BP fall had even a greater risk of recurrent cerebral ischemia than did those without nocturnal decreases and untreated subjects with physiological BP decreases at night (11). Based on these observations, we performed a pilot study in TGRs and investigated the effects of two different amlodipine treatment schedules on BP pattern and on myocardial hypertrophy and β-adrenergic signal transduction in comparison with untreated normotensive and hypertensive rats. The calcium channel blocker amlodipine was used in this study, because the effects of BP and its circadian profile on myocardial hypertrophy and β-adrenergic signaling should be studied, excluding direct drug interactions with the renin-angiotensin and sympathetic nervous systems. Amlodipine was administered once daily in the morning to reduce increased BP levels in the resting period of the rats or twice daily in the morning and at night to achieve a continuous reduction in BP throughout 24 h. Effects of treatment on β-adrenergic signaling were investigated, because TGRs show a reduced β-adrenergic stimulation of adenylyl cyclase (12,13), which could either be a consequence of myocardial hypertrophy per se or could reflect the facilitation of sympathetic neurotransmission by increased cardiac angiotensin II levels, leading to desensitization of β-adrenoceptors. By comparing amlodipine effects on cardiac hypertrophy with those on β-adrenergic signaling, it should be possible to draw conclusions regarding the mechanisms involved in disturbed β-adrenoceptor function in this animal model.
Normotensive male Sprague-Dawley rats (SPRDs, strain Mol:SPRD Han; n = 12) and transgenic hypertensive rats (TGRs, strain: TGR(mREN2)27 MolGene; n = 42) were obtained from Moellegaard Breeding & Research Centre (Skensved, Denmark) at age 8 weeks. Rats were housed singly in plastic cages (380 × 220 × 150 mm), three cages per shelf in a cabinet, under controlled environmental conditions at an ambient temperature of 23 ± 1 °C, and with food and water ad libitum. The animals were synchronized to a light-dark schedule of 12:12 h with lights on at 8.00 h. At age 10 weeks, radiotransmitters for continuous monitoring of BP, heart rate, and locomotor activity (TA11PA-C40; Data Sciences International, St. Paul, MN, U.S.A.) were implanted in 30 rats (six SPRDs and 24 TGRs) as described (6).
In six TGRs, the effects of amlodipine on BP and heart rate were studied after acute i.p. injection of 1, 3, and 10 mg/kg body weight either at 8:00 or at 20:00 h. Effects were calculated in relation to the preceding control day as area under the drug effect-time curve (AUC) from the time of injection until 12 h later. Animals used in the acute dose-response experiments were not included in the subsequent treatment study.
Treatment study protocol
After 3 weeks of monitoring under untreated conditions, the animals were divided into four groups: SPRDs, untreated TGRs, and TGRs receiving amlodipine once daily and twice daily. In animals of the once-daily treatment group, amlodipine, 5 mg/kg, was injected i.p. at 8:00 h (i.e., at the beginning of the resting period) and vehicle (distilled water) at 20:00 h. In the twice-daily treatment group, rats received amlodipine, 2.5 mg/kg, at 8:00 and at 20:00 h. BP, heart rate, and locomotor activity were monitored telemetrically throughout the whole study and analyzed (14). After 5 weeks of treatment, rats were killed between 10:00 and 13:00 h, heart ventricles were dissected, freed from fat and connective tissue, and immediately frozen in liquid nitrogen. On the day of killing, rats did not receive amlodipine.
Single ventricles were weighed and immediately homogenized in ice-cold assay buffer (TRIS, 50 mM; MgCl2, 10 mM; pH 7.4 at 37°C) by using an Ultra-Turrax-homogenizer (IKA, Staufen, Germany) at 20,000 r/min. The resulting suspension was divided into three subsets for determination of β-adrenoceptor density (suspension A), adenylyl cyclase activity (suspension B), and 5-nucleotidase activity (suspension C). Suspension A was homogenized a second time by using a Potter S glass-homogenizer (Braun, Melsungen, Germany). After 10-min centrifugation at 25,000 g, the supernatants of suspensions A, B, and C were discarded. Pellets A and B were resuspended in assay buffer. Pellet C was homogenized in assay buffer containing 0.3% Triton-X 100 for solubilization of membrane-bound 5-nucleotidase. After a second centrifugation (10 min, 25,000 g), the resulting supernatant was used for photometric determination of 5-nucleotidase activity.
Radioligand binding studies
Cardiac β-adrenoceptor densities (Bmax) were determined in saturation experiments by using the nonselective β-adrenoceptor antagonist [3H](−)-CGP-12177 (0.125-4 nM) as described (15). For determination of β-adrenoceptor subtypes, displacement experiments were performed by using increasing concentrations of the β1-adrenoceptor antagonist CGP-20712A in the presence of [3H](−)-CGP-12177 (2 nM).
Adenylyl cyclase assay
The formation of cyclic adenosine monophosphate (cAMP) was determined in the presence of 3-isobutyl-1-methyl-xanthine (IBMX) and an adenosine triphosphate (ATP)-regenerating system (16). In brief, 0.3 ml of membrane suspension was added to 1.2 ml of prewarmed assay buffer (37°C, pH 7.4) containing IBMX, 1 mM; ATP, 0.5 mM; phosphocreatine, 10 mM; and creatine phosphokinase, 0.1 mg/ml. The reaction was stopped after 8 min by heating the tubes at 120°C; cAMP formed was measured in the supernatant by radioassay (TRK 432; Amersham Buchler, Braunschweig, Germany).
Subtype-specific β-adrenergic stimulation in the presence of guanosine triphosphate (GTP), 10 μM, was assessed in functional competition experiments (17). Adenylyl cyclase activity was determined in the presence of a fixed concentration of isoprenaline (1 μM) with increasing concentrations of the β1-adrenoceptor antagonist CGP-20712A (100 pM-100 μM). Concentration-response curves for isoprenaline (10 nM-100 μM) were determined to calculate the maximal increase in cAMP formation after stimulation of both β-adrenoceptor subtypes.
The activity of solubilized 5-nucleotidase was measured photometrically by using a commercially available kit (265-UV, Sigma, Deisenhofen, Germany).
The protein content of the membrane fractions A and B was determined by the method of Lowry et al. (18) with minor modifications. Solubilized protein from suspension C was measured by using the Coomassie Plus assay (Pierce, oud-Beijerland, Holland). Bovine serum albumin was used as standard and dissolved in the respective assay buffer.
Saturation curves for determination of maximal specific binding (Bmax), competition curves for calculation of the β-adrenoceptor subtype distribution, and concentration-response curves were fitted to the experimental data by using PHARMFIT (19). Differences between treatment groups in cardiovascular and biochemical parameters were tested by analysis of variance (ANOVA) followed by Scheffé's test for multiple pairwise comparisons. Bivariate ANOVA was used with data from the dose-response study to test for influence of dose and time of injection. The program package BIAS (20) was used. Data are expressed as means ± SD, unless otherwise indicated.
Acute i.p. injection of amlodipine dose-dependently reduced systolic and diastolic BP to a comparable degree after morning and evening dosing (ANOVA dose, p < 0.01, time not significant). The increase in heart rate depended on both the dose of amlodipine and the time of dosing (ANOVA dose, p < 0.01; time, p < 0.05) and was greater after injection of amlodipine at 8:00 h despite a similar reduction in BP as after evening dosing (Fig. 1).
Cardiovascular parameters. Under control conditions, SPRDs showed normotensive BP values with 24-h means in systolic and diastolic BP of 125 and 90 mm Hg, respectively (Table 1). Day-night differences were −6.4 mm Hg (systolic) and −5.9 mm Hg (diastolic), indicating a physiological BP decrease in the daily resting period (Table 2). The three groups of TGRs were clearly hypertensive under pretreatment conditions, with 24-h means of ∼205 mm Hg in systolic BP and 150 mm Hg in diastolic BP (Table 1). Positive day-night differences were observed in BP in all groups of TGRs, demonstrating the inverse circadian BP pattern (Table 2). No significant differences were observed between the three groups of TGRs before treatment. Circadian patterns in cardiovascular parameters in untreated SPRDs and TGRs are shown in Figs. 2 and 3.
Administration of amlodipine, 5 mg/kg/day, at 8:00 h led to a significant reduction in systolic BP by ∼50 mm Hg and diastolic BP by ∼35 mm Hg (Table 1). The decrease in BP by amlodipine occurred immediately after injection and was reproducible throughout the total treatment period of 5 weeks. Day-night differences in BP became negative during once-daily amlodipine treatment, indicating that BP was reduced predominantly during the daily resting period. The 24-h mean in heart rate was not affected by once-daily injection of amlodipine, but the day-night difference was markedly decreased (Table 2). The resulting circadian patterns in BP and heart rate during once-daily amlodipine treatment are illustrated in Fig. 4, demonstrating the rather irregular shape of the BP curve, characterized by a marked drug-induced BP decrease and a continuous increase during the following 24 h. The circadian pattern in heart rate was obscured by a pronounced increase after morning dosing of amlodipine, which presumably reflects the BP reduction.
In TGRs, twice-daily dosing of amlodipine, 2.5 mg/kg, was less effective than morning dosing of 5 mg/kg and resulted in overall BP reductions of 30 mm Hg (systolic) and 15 mm Hg (diastolic). Day-night differences became negative under this treatment, indicating a more pronounced BP reduction during the daily resting period of the rats (Table 2). In heart rate, neither the 24-h mean nor the day-night difference was significantly changed by amlodipine, twice daily. The circadian BP profile during amlodipine treatment was characterized by two peaks and troughs within 24 h, resulting from twicedaily injections of the drug (Fig. 5). As observed with once-daily injection of amlodipine, twice-daily dosing resulted in immediate reductions in BP, followed by continuous increases, which could be found day-by-day throughout the whole treatment period. Day-night variation in heart rate was preserved but showed two drug induced increases within 24 h, reflecting the reductions in BP.
Myocardial hypertrophy. Untreated TGRs had significantly higher ventricular weight ratio between ventricular and body weight as well as 5-nucleotidase activity than did normotensive SPRD controls (Table 3). Treatment with amlodipine once and twice daily resulted in decreases in the different markers of myocardial hypertrophy. Ventricular weights were significantly lower than those observed in untreated TGRs but were still higher than those in normotensive SPRD controls. In TGRs receiving amlodipine, 5 mg/kg/day at 8:00 h, relative ventricular weight and 5-nucleotidase activity did not differ from normotensive SPRDs, whereas in TGRs receiving amlodipine 2.5 mg/kg twice daily, only 5-nucleotidase activity returned to control values.
β-Adrenergic signaling. Densities of cardiac β-adrenoceptors and their subtypes were not different between SPRDs, untreated, and amlodipine-treated TGRs. In contrast, stimulation of cardiac adenylyl cyclase by β1-adrenoceptors showed significant differences between the four groups of rats studies (Table 4), with a 25% reduction of β1-adrenergic stimulation of adenylyl cyclase in TGRs compared with SPRDs. Treatment with amlodipine had no effects on the functional efficacy of β-adrenoceptor subtypes.
This study demonstrates that repeated injections of amlodipine were able to reduce long-term BP levels in TGRs and, depending on the treatment schedule, differently to influence the disturbed day-night pattern. As a consequence of the antihypertensive effect, amlodipine-treated TGRs showed reduced signs of myocardial hypertrophy, whereas cardiac β-adrenergic signaling was not affected by treatment with the calcium channel blocker.
Sustained reductions in BP of TGRs by long-term drug treatment have been achieved with converting enzyme inhibitors (21) and angiotensin II-receptor antagonists (22), demonstrating the importance of an activated renin-angiotensin system in this animal model of hypertension. In this study, the calcium channel blocker amlodipine was used as antihypertensive agent, because amlodipine does not directly interfere with the renin-angiotensin system. However, it has been shown that amlodipine in oral doses of ≥15 mg/kg induced marked increases in plasma renin activity and renal renin messenger RNA (mRNA) in normotensive rats and to a smaller degree in two kidney-one clip rats (23). In our study, plasma renin activity and concentration were not different in untreated and amlodipine-treated TGRs (data not shown). Thus it was possible to study the effects of BP reductions on myocardial hypertrophy, excluding direct drug effects on angiotensin II-dependent myocardial growth processes. In normotensive Wistar rats, amlodipine was found to reduce BP more effectively after i.p. injection in the evening than in the morning (24). In agreement with these cardiovascular data, Fujimura et al. (25) reported that diuretic effects of amlodipine and peak plasma concentrations of the drug were greater after oral dosing in the evening than in the morning. However, in our dose-response study in TGRs, i.p. injection of amlodipine reduced BP equally effectively during the activity and resting period of the animals (Fig. 1), which allowed us to investigate once- versus twice-daily dosing, excluding differences in time-dependent pharmacodynamics of amlodipine itself in the TGR strain. The discrepancy between normotensive Wistar rats and hypertensive TGRs in time-dependent efficacy of amlodipine cannot easily be explained. One could speculate that the inverse circadian BP profile in TGRs, with peak values during the daily resting period, leads to increased sensitivity to antihypertensive agents, which may compensate for a possibly slower absorption and lower peak plasma concentration after morning dosing.
In hypertensive patients, calcium channel blockers have repeatedly been found to reduce signs of myocardial hypertrophy (26,27). In spontaneously hypertensive rats (SHRs), which are widely used as animal model of human primary hypertension, 3 weeks' treatment with amlodipine, 10 mg/kg/day, has been found to reduce left ventricular mass, myocardial protein, and collagen content (28). However, another study in SHRs did not show regression of myocardial hypertrophy by amlodipine, whereas the converting enzyme inhibitor lisinopril caused a marked reduction in left ventricular mass (29). Kim et al. (30) compared the effects of the converting enzyme inhibitor enalapril, the angiotensin II-receptor antagonist losartan, and amlodipine on left ventricular hypertrophy in stroke-prone SHRs and observed that, despite comparable decreases in BP, regression of left ventricular hypertrophy was greater with losartan than with amlodipine. These findings could indicate that myocardial hypertrophy is greatly dependent on the trophic effects of angiotensin II. A meta-analysis of clinical studies in hypertensive patients indeed demonstrated a superior effect of converting enzyme inhibitors on regression of left ventricular hypertrophy in comparison with other antihypertensive drugs (26). In a more recent study in mildly hypertensive subjects, however, the most pronounced reductions in left ventricular mass were observed with chlorthalidone treatment (27), although diuretic treatment leads to activation of the renin-angiotensin system. Thus the role of the renin-angiotensin system in the development and maintenance of myocardial hypertrophy has not finally been defined.
TGRs, which are characterized by an overactivation of the renin-angiotensin system, could be an appropriate model for investigating the contribution of angiotensin II to myocardial hypertensive damage. In these animals, the angiotensin II-receptor antagonist telmisartan led to significant and dose-dependent reductions of cardiac hypertrophy (22). Regression of hypertrophy could be obtained with a nonantihypertensive dose of telmisartan, but higher doses, which were able to decrease BP in TGRs, resulted in greater reductions in myocardial mass. Ohta et al. (31) reported that, in TGRs treated with different antihypertensive drugs for 6 weeks, only the angiotensin II-receptor antagonist TCV-116 was able to normalize left ventricular weight despite comparable BP reductions in groups treated with a combination of atenolol and doxazosin or atenolol and manidipin, a dihydropyridine calcium channel blocker. In that study, however, BP was measured in anesthetized rats by the tail-cuff method, whereas, in our study, long-term BP levels were monitored telemetrically in conscious, freely moving animals. Our observation that amlodipine was able markedly to reduce ventricular weight in TGRs without direct drug effects on the renin-angiotensin system indicates that the level of BP per se must be of greater importance in ventricular hypertrophy than trophic effects of angiotensin II. Thus even in rats with an activated renin-angiotensin system, BP reduction alone induces a regression of cardiac hypertrophy.
Interestingly, the disturbed function of β1-adrenoceptors in ventricular tissue from TGRs could not be reversed by amlodipine treatment despite reductions in BP and myocardial hypertrophy. It may be argued that reflexively induced increases in sympathetic tone after the amlodipine-induced BP reductions could have prevented a resensitization of cardiac β-adrenoceptors. However, on the day of death, rats did not receive amlodipine to exclude a direct drug effect on the parameters monitored. The interval between the last dose of amlodipine and tissue procurement was ≥14 h in twice-daily and 26 h in once-daily treated rats. However, we cannot rule out that the repeated, immediate reductions in BP by amlodipine may have influenced baroreflex sensitivity and, thus, sympathetic tone. Prolonged administration of losartan has been found to reverse alterations in the TGR cardiac β-adrenoceptor-adenylyl cyclase system (32). The different effects of amlodipine and losartan on the disturbed β-adrenergic signal transduction indicate that alterations in the renin-angiotensin system of TGRs play a greater role in desensitization of myocardial β-adrenoceptors than do hypertension and cardiac hypertrophy. Based on these observations, it seems plausible that in TGRs, elevated cardiac concentrations of angiotensin II enhance sympathetic neurotransmission and thereby promote agonist-dependent desensitization of cardiac β-adrenoceptors. Therefore in hypertension caused by an activated renin-angiotensin system, BP reduction alone could prevent some but not all cardiac secondary complications, and specific inhibitors of the renin-angiotensin system may be the drugs of choice.
In addition to general effects of amlodipine on cardiovascular parameters, this study focused on the influence of the circadian BP pattern on myocardial hypertensive damage by comparison of two different amlodipine-treatment schedules. Once-daily administration of the calcium channel blocker aimed to normalize the inverse circadian BP profile in TGRs, whereas twice-daily dosing of half of the single dose was thought more smoothly to shift the disturbed BP profile to a lower level. Unfortunately, these goals could not completely be achieved in this study. Once-daily dosing of amlodipine, indeed, decreased BP predominantly during the daily resting period of the rats, but the resulting circadian profile looked rather irregular, with a trough soon after drug application in the morning, followed by a continuous increase until the next drug injection 24 h later. Twice-daily injection of the drug induced a similar pattern in 12-h intervals. Even after 5 weeks of treatment, typical circadian profiles in BP could not be obtained. Moreover, twice-daily dosing was less effective with regard to overall BP reduction. Although amlodipine injected once daily at the beginning of the resting period had more pronounced effects on myocardial hypertrophy in TGRs, definite conclusions concerning the role of the circadian BP profile cannot be drawn because of the differences in 24-h mean BP between the treatment groups. The irregular BP profiles observed during 5 weeks of treatment with amlodipine clearly demonstrate that, in rats, the half-life of amlodipine must be rather short, which is in agreement with pharmacokinetic data in Wistar rats, in which the plasma half-life of amlodipine after i.v. injection did not exceed 250 min (unpublished results). These findings are in contrast to data in hypertensive patients showing an antihypertensive effect of amlodipine throughout 24 h after dosing and an undisturbed circadian BP pattern after once-daily morning or evening administration (33).
In conclusion, this study demonstrates that myocardial hypertrophy in transgenic hypertensive TGR(mREN2)27 rats can be reduced by amlodipine treatment, whereas disturbed cardiac β-adrenergic signaling was not affected. The role of the circadian BP profile on hypertensive damage to the heart remains to be elucidated in future studies.
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