The Influence of Concomitant Administration of Simvastatin at Different Doses with Metoprolol on Heart Rate and Blood Pressure in Normocholesterolemic

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1 original papers Adv Clin Exp Med 2011, 20, 1, ISSN X Copyright by Wroclaw Medical University Jacek Owczarek, Magdalena Jasińska, Urszula Kurczewska, Daria Orszulak-Michalak The Influence of Concomitant Administration of Simvastatin at Different Doses with Metoprolol on Heart Rate and Blood Pressure in Normocholesterolemic and Hypercholesterolemic Rats* Wpływ łącznego podania simwastatyny w różnych dawkach z metoprololem na częstość rytmu serca oraz ciśnienie tętnicze u szczurów z zaburzeniami i bez zaburzeń lipidowych Department of Biopharmacy, Medical University of Lodz, Poland Abstract Background. β 1 -adrenergic blocking drugs and inhibitors of HMGCo-A reductase (statins) are widely used by a lot of patients in the prevention of cardiovascular events. Previous studies have shown that the administration of statins influences β adrenergic signaling. Objectives. The aim of the study was to evaluate the influence of a chronic 4-week administration of simvastatin given at different doses with concomitant metoprolol on heart rate and blood pressure. Material and Methods. The experiments were performed in normotensive Wistar rats. The animals were divided into 2 groups: normocholesterolemic and hypercholesterolemic. During the 4 weeks, hypercholesterolemic rats received a diet with 5% cholesterol and 2,5% cholic acid. Drugs: metoprolol 30 mg kg 1 b.w.; simvastatin 1 mg kg 1 b.w.; simvastatin 20 mg kg 1 b.w.; simvastatin 1 mg kg 1 b.w. + metoprolol 30 mg kg 1 b.w., or simvastatin 20 mg kg 1 b.w. + metoprolol 30 mg kg 1 b.w. were given intragastrically over the 4-week period. For the evaluation of heart rate and blood pressure, the signals were provided by an Isotec pressure transducer. Results. Concomitant administration of simvastatin at 1 and 20 mg kg 1 b.w with metoprolol in normocholesterolemic rats statistically decreased heart rate as compared to the control group and the group receiving simvastatin alone. No statistical differences between the groups receiving simvastatin at dose 1 mg/kg 1 with metoprolol and simvastatin at dose 20 mg/kg 1 with metoprolol ( ± 7.90 vs ± 5,15 1/min p = 0.05) were observed. In the hypercholesterolemic animals, the heart rate evaluation after concomitant administration of simvastatin given at different doses with metoprolol showed comparable statistical implications as compared to normocholesterolemic rats. Concomitant administration of simvastatin in different doses on normo- and hypercholesterolemic rats did not significantly influence blood pressure. Conclusions. No significant impact of chronic administration of simvastatin given at different doses to normoand hypercholesterolemic Wistar rats receiving metoprolol on the heart rate was observed (Adv Clin Exp Med 2011, 20, 1, 39 45). Key words: rats, simvastatin, metoprolol, heart rate. Streszczenie Wprowadzenie. Leki blokujące receptory β 1 -adrenergiczne oraz inhibitory reduktazy 3-hydroksy 3-metyloglutarylo-koenzymu A (statyny) są powszechnie stosowane u pacjentów w zapobieganiu incydentom sercowo-naczyniowym. Wcześniejsze badania wykazały, że podawanie statyn wpływa na sygnalizację β-adrenergiczną. Cel pracy. Ocena wpływu podawania simwastatyny w różnych dawkach na wartości rytmu serca i ciśnienia tętniczego po łącznym 4-tygodniowym podawaniu z metoprololem. Materiał i metody. Badanie zostało przeprowadzone na szczurach Wistar o prawidłowym ciśnieniu krwi. Zwierzęta *The study was supported by Medical University of Lodz; grant ; ;

2 40 J. Owczarek et al. scheduled prior to the beginning of the experiment. After the adaptation period, the animals were divided into 2 groups: normocholesterolemic and hypercholesterolemic. Normocholesterolemic rats received a normal diet during the 4 weeks. Hypercholesterolemic rats received a normal diet with 5% cholesterol and 2,5% cholic acid during the 4 weeks. After this period, each group was divided into 6 subgroups, rats receiving, respectively: group I: placebo (0.2% methylcellulose), group II: metoprolol 30 mg kg 1 b.w.; group III: simvastatin 1 mg kg 1 b.w.; group IV: simvastatin 20 mg kg 1 b.w.; group V: simvastatin 1 mg kg 1 b.w. + metoprolol 30 mg kg 1 b.w.; group VI: simvastatin 20 mg kg 1 b.w. + metoprolol 30 mg kg 1 b.w. The drugs were given intragastrically (i.g.) over the four-week period. Animals had free access to diet (normal or hypercholesterolemic) and water. After the drug administration period, heart rate and hemodynamic parameters were measured. Surgery was performed 24 hours after administration of the last drug dose and 10 hours after the last food supply. For the subsequent surgical procedures, anesthesia was initiated by an intraperitoneal (i.p.) dose of pentobarbital sodium at 60 mg kg 1 b.w. The anesthesia was maintained by intraperitoneal bolus injections of pentobarbital sodium at 10 mg kg 1 b.w. as needed. For the measurement of the heart rate (HR) and arterial blood pressure (mean (MAP), systolic (SAP) and diastolic (DAP)), a catheter was implanted into the right carotid artery. The signals were provided by an Isotec pressure transducer connected to a direct current bridge amplifier (Hugo Sachs Elektronik). Analog signals were amplified and recorded on a computer via an A/D converter (HSE Haemodyn software for Microsoft Windows 95/98/NT) and were evaluated according to the algorithms. The values of MAP, SAP and DAP were calculated automatically by the software during the measurebyły podzielone na 2 grupy: z prawidłowym stężeniem cholesterolu i podwyższonym stężeniem cholesterolu. Szczury z grupy z podwyższonym stężeniem cholesterolu otrzymywały dietę wzbogaconą 5% zawartością cholesterolu i 2,5% zawartością kwasu cholowego. Leki: metoprolol 30 mg kg 1 m.c.; simwastatyna 1 mg kg 1 m.c.; simwastatyna 20 mg kg 1 m.c.; simwastatyna 1 mg kg 1 m.c. + metoprolol 30 mg kg 1 m.c., simwastatyna 20 mg kg 1 m.c. + metoprolol 30 mg kg 1 m.c. były podawane dożołądkowo przez okres 4 tygodni. Wyniki. Łączne podanie simwastatyny w dawkach: 1 i 20 mg kg 1 m.c. z metoprololem szczurom z grupy z prawidłowym stężeniem cholesterolu znacząco statystycznie zwalniało rytm serca w porównaniu z grupą kontrolną oraz grupy otrzymującej samą simwastatynę. Nie odnotowano istotnej statystycznie różnicy w zakresie rytmu serca między osobnikami z grup z prawidłowym stężeniem cholesterolu otrzymującymi różne dawki simwastatyny (1 i 20 mg kg 1 m.c.) łącznie z metoprololem (381,64 ± 7.90 vs 388,21 ± 5,15 1/min p = 0,05). W grupie zwierząt z hipercholesterolemią ocena wartości rytmu serca po łącznym podaniu różnych dawek simwastatyny (1 i 20 mg kg 1 m.c.) łącznie z metoprololem wykazywała podobną zależność statystyczną. Łączne podanie różnych dawek simwastatyny łącznie z metoprololem nie wpłynęło istotnie statystycznie na wartości ciśnienia tętniczego. Wnioski. Nie zaobserwowano wpływu przewlekłego podawania simwastatyny w różnych dawkach na zwolnienie rytmu serca u szczurów z prawidłowym i podwyższonym stężeniem cholesterolu otrzymujących metoprolol (Adv Clin Exp Med 2011, 20, 1, 39 45). Słowa kluczowe: szczury, simwastatyna, metoprolol, rytm serca. Cholesterol-lowering drugs such as inhibitors of 3-hydroxy 3-methylglutaryl-coenzyme A reductase (HMG-CoA, statins), are widely used by a lot of patients in the prevention of cardiovascular events [1, 2]. It is connected with the LDL-cholesterol lowering activities as well as the pleiotropic effects of statins [3, 4]. Lowering criteria including both LDL-cholesterol and total cholesterol blood level in patients with cardiovascular disease requires higher doses of statins for optimization of hypolipemic therapy. β 1 -adrenergic blocking drugs are recommended for reduction of myocardial ischemia and prevention of angina pectoris. They have been shown to decrease mortality and sudden death particularly by reduction resting heart rate and in this case by reduction of myocardial oxygen consumption in patients with ischemic heart disease and heart failure [5 8]. Previous studies have shown that statins might influence β-adrenergic signaling [9] although in our studies, two-week administration of simvastatin in different doses to normocholesterolemic rats did not modify metoprolol injection-induced depressed heart rate and does not modify metoprolol s impact on blood pressure [10, 11]. The aim of the study was to evaluate the influence of simvastatin given at different doses on heart rate and blood pressure with concomitant chronic 4-week administration of metoprolol. Material and Methods Animals The study was approved by the Ethics Committee of the Medical University of Lodz (Poland) 43/ŁB405/2008. The experiments were performed on 101, anesthetized Wistar rats, outbred males. A several-day adaptation period was

3 Dose-dependent Interaction of Simvastatin and Metoprolol in Rats 41 ment procedure. For further statistical analysis, the output values of the parameters were calculated as the mean from 3-min periods. After heart rate and blood pressure assessment, 0.25 ml of blood samples were taken for a further lipid profile examination. Surgical procedures and recording of heart rate and blood pressure were provided as described previously [10, 11]. Statistics All data were presented as means ± SD (standard deviation). Statistical comparisons between baseline conditions and metoprolol injection were done by paired Student s t-test. Comparisons between the groups were performed using ANOVA. Post-hoc comparisons were performed using an LSD test. Normal distribution of parameters was checked by means of a Shapiro-Wilks test. If data were not normally distributed or the values of variance were different, ANOVA with Kruscal-Wallis and Mann-Whitney s U tests were used. All parameters were considered statistically significantly different if p < The statistical analysis of the heart rate and hemodynamic parameters was performed using Statgraphics 5.0 plus software. Results Four-week administration of simvastatin alone or concomitant administration with metoprolol to normocholesterolemic rats significantly decreased LDL cholesterol and increased HDL cholesterol level as compared to the control group. A decrease of LDL cholesterol level after administration of simvastatin alone or concomitant administration with metoprolol was observed in hypercholesterolemic rats. The changes in animal lipid profiles are shown in Table 1. Table 1. Total cholesterol (TCH), HDL cholesterol, LDL cholesterol(ldl), triglycerides (TG) (mean ± SD) in rats fed normo- and hypercholesterolemic diet [mmol/l] Tabela 1. Cholesterol całkowity (TCH), HDL cholesterol, LDL cholesterol (LDL), triglicerydy (TG) (średnie ± SD) u szczurów na diecie normalnej i hipercholesterolowej [mmol/l] TCH HDL LDL TG K_N (N = 10) 1.48 ± ± / ± 0.13 M_N (N = 9) 1.14 ± ± / ± 0.08 S1_N (N = 10) 1.34 ± ± 0.05* 0.56 ± 0.14* 0.46 ± 0.14 S20_N (N = 7) 1.39 ± ± 0.14* 0.44 ± 0.09* 0.56 ± 0.18 S1_M_N (N = 6) 1.51 ± ± 0.13* 0.62 ± 0.09* 0.58 ± 0.26 S20_M_N (N = 8) 1.32 ± ± 0.18* 0.56 ± 0.10* 0.43 ± 0.15 K_H (N = 10) 8.09 ± ± ± ± 0.62 M_H (N = 9) 7.65 ± ± ± ± 0.15 S1_H (N = 8) 6.35 ± ± 0.12* 4.45 ± 0.21* 2.82 ± 0.57 S20_H (N = 9) 2.01 ± 0.16* 0.42 ± ± 0.92* 0.65 ± 0.64* S1_M_H (N = 8) 7.23 ± ± ± 0.33* 3.47 ± 0.79 S20_M_H (N = 7) 1.32 ± 0.18* 0.33 ± ± 0.03* 0.89 ± 0.23* K_N normocholesterolemic control group, K_H hypercholesterolemic control group, M_H hypercholesterolemic group receiving metoprolol, M_N normocholesterolemic group receiving metoprolol, S1_N normocholesterolemic group receiving simvastatin at dose 1 mg/kg b.w., S1_H hypercholesterolemic group receiving simvastatin at dose 1 mg/ kg b.w., S20_N normocholesterolemic group receiving simvastatin at dose 20 mg/kg b.w., S20_H hypercholesterolemic group receiving simvastatin at dose 20 mg/kg b.w., S1_M_N normocholesterolemic group receiving metoprolol and simvastatin at dose 1 mg/kg b.w., S20_M_N normocholesterolemic group receiving metoprolol and simvastatin at dose 20 mg/kg b.w., S1_M_H hypercholesterolemic group receiving metoprolol and simvastatin at dose 1 mg/kg b.w., S20_M_H hypercholesterolemic group receiving metoprolol and simvastatin at dose 20 mg/kg b.w. (*) P < 0.05 as compared to the control group. K_N grupa kontrolna normocholesterolowa, K_H grupa kontrolna hipercholesterolowa, M_H grupa hipercholesterolowa otrzymująca metoprolol, M_N grupa normocholesterolowa otrzymująca metoprolol, S1_N grupa normocholesterolowa otrzymująca simwastatynę w dawce 1 mg/kg m.c., S1_H grupa hipercholesterolowa otrzymująca simwastatynę w dawce 1 mg/kg m.c., S20_N grupa normocholesterolowa otrzymująca simwastatynę w dawce 20 mg/kg m.c., S20_H grupa hipercholesterolowa otrzymująca simwastatynę w dawce 20 mg/kg m.c., S1_M_N grupa normocholesterolowa otrzymująca metoprolol i simwastatynę w dawce 1 mg/kg m.c., S20_M_N grupa normocholesterolowa otrzymująca metoprolol i simwastatynę w dawce 20 mg/kg m.c., S1_M_H grupa hiperchlesterolowa otrzymująca metoprolol i simwastatynę w dawce 1 mg/kg m.c., S20_M_H grupa hipercholesterolowa otrzymująca metoprolol i simwastatynę w dawce 20 mg/kg m.c. (*) P < 0.05 w porównaniu z grupą kontrolną.

4 42 J. Owczarek et al. Fig. 1. Mean heart rate [beats/min] in Wistar rats fed normo- and hypercholesterolemic diet K_N normocholesterolemic control group, K_H hypercholesterolemic control group, M_H hypercholesterolemic group receiving metoprolol, M_N normocholesterolemic group receiving metoprolol, S1_N normocholesterolemic group receiving simvastatin at dose 1 mg/kg b.w., S1_H hypercholesterolemic group receiving simvastatin at dose 1 mg/kg b.w., S20_N normocholesterolemic group receiving simvastatin at dose 20 mg/kg b.w., S20_H hypercholesterolemic group receiving simvastatin at dose 20 mg/ kg b.w., S1_M_N normocholesterolemic group receiving metoprolol and simvastatin at dose 1 mg/kg b.w., S20_M_N - normocholesterolemic group receiving metoprolol and simvastatin at dose 20 mg/kg b.w., S1_M_H hypercholesterolemic group receiving metoprolol and simvastatin at dose 1 mg/kg b.w., S20_M_H hypercholesterolemic group receiving metoprolol and simvastatin at dose 20 mg/kg b.w. (*) P < 0.05 as compared to the control group. (a) p < 0.05 as compared to the group receiving simvastatin alone Ryc. 1. Średnie wartości rytmu serca [liczba uderzeń/min] u szczurów Wistar na diecie normo- i hipercholesterolowych K_N grupa kontrolna normocholesterolowa, K_H grupa kontrolna hipercholesterolowa, M_H grupa hipercholesterolowa otrzymująca metoprolol, M_N grupa normocholesterolowa otrzymująca metoprolol, S1_N grupa normocholesterolowa otrzymująca simwastatynę w dawce 1 mg/ kg m.c., S1_H grupa hipercholesterolowa otrzymująca simwastatynę w dawce 1 mg/kg m.c., S20_N grupa normocholesterolowa otrzymująca simwastatynę w dawce 20 mg/kg m.c., S20_H grupa hipercholesterolowa otrzymująca simwastatynę w dawce 20 mg/kg m.c., S1_M_N grupa normocholesterolowa otrzymująca metoprolol i simwastatynę w dawce 1 mg/kg m.c., S20_M_N grupa normocholesterolowa otrzymująca metoprolol i simwastatynę w dawce 20 mg/kg m.c., S1_M_H grupa hiperchlesterolowa otrzymująca metoprolol i simwastatynę w dawce 1 mg/kg m.c., S20_M_H - grupa hipercholesterolowa otrzymująca metoprolol i simwastatynę w dawce 20 mg/kg m.c. (*) P < 0.05 w porównaniu z grupą kontrolną. (a) p < 0,05 w porównaniu z grupą otrzymującą simwastatynę The heart rate (HR) in the control groups receiving a normo- and hypercholesterolemic diet was ± 22.6 and ± beats/min (p = 0.05) respectively. The four-week administration of metoprolol to normo and hypercholesterolemic rats significantly decreased HR as compared to the control groups ( ± 20.5 and ± 21,8 1/min). In groups receiving simvastatin at 1 and 20 mg kg 1 b.w. during the 4 weeks, the HR was comparable to the control rats. No differences between groups receiving simvastatin at 1 and 20 mg/kg 1 were observed, as well. After concomitant administration of simvastatin at 1 and 20 mg kg 1 b.w. with metoprolol to normocholesterolemic rats, the HR statistically decreased as compared to the control rats and the group receiving simvastatin alone. No statistical differences between groups receiving simvastatin at 1 mg/kg 1 with metoprolol and simvastatin at 20 mg/kg 1 with metoprolol ( ± 7.90 vs ± ± 5.15 beats/min; p = 0.05) were denoted. The HR after concomitant administration of simvastatin at 1 and 20 mg kg 1 b.w. with metoprolol to hypercholesterolemic rats showed the same statistical implications as compared to normocholesterolemic rats. No statistical differences between hypercholesterolemic groups receiving simvastatin at dose 1 mg/kg 1 with metoprolol and simvastatin at dose 20 mg/kg 1 with metoprolol were shown, as well ( ± vs ± ± beats/min) (Fig. 1). Concomitant administration of simvastatin at 1 and 20 mg kg 1 b.w. with metoprolol in normoand hypercholesterolemic rats did not significantly influence arterial blood pressure. The results of MAP, SAP and DAP are presented in Tab. 2. Discussion In our study, concomitant administration of simvastatin given at different doses with metoprolol did not influence the heart rate as compared to rats receiving metoprolol alone. Similar effects were observed in our previous studies [10, 11]. Conversely, some reports on the influence of

5 Dose-dependent Interaction of Simvastatin and Metoprolol in Rats 43 Table 2. Summary statistics (mean ± SD) for arterial blood pressure [mm Hg] in normo- and hypercholesterolemic rats Tabela 2. Statystyka opisowa (średnia ± SD) dla wartości ciśnienia tętniczego [mm Hg] u szczurów normo- i hipercholesterolowych N Systolic arterial pressure (SAP) [mm Hg] Mean arterial pressure (MAP) [mm Hg] Diastolic arterial pressure (DAP) [mm Hg] K_H ± ± ± 3.85 K_N ± ± ± 6.18 M_H ± ± ± 5.80 M_N ± ± ± 4.22 S1_H ± ± ± 5.87 S1_M_H ± ± ± 4.98 S1_M_N ± ± ± 4.19 S1_N ± ± ± 4.18 S20_H ± ± 3.62 S20_M_H ± ± ± 4.36 S20_M_N ± ± ± 5.79 S20_N ± ± ± 6.64 K_N normocholesterolemic control group, K_H hypercholesterolemic control group, M_H hypercholesterolemic group receiving metoprolol, M_N normocholesterolemic group receiving metoprolol, S1_N normocholesterolemic group receiving simvastatin at dose 1 mg/kg b.w., S1_H hypercholesterolemic group receiving simvastatin at dose 1 mg/ /kg b.w., S20_N normocholesterolemic group receiving simvastatin at dose 20 mg/kg b.w., S20_H hypercholesterolemic group receiving simvastatin at dose 20 mg/kg b.w., S1_M_N normocholesterolemic group receiving metoprolol and simvastatin at dose 1 mg/kg b.w., S20_M_N normocholesterolemic group receiving metoprolol and simvastatin at dose 20 /mg/kg b.w., S1_M_H hypercholesterolemic group receiving metoprolol and simvastatin at dose 1 mg/kg b.w., S20_M_H hypercholesterolemic group receiving metoprolol and simvastatin at dose 20 mg/kg b.w. K_N grupa kontrolna normocholesterolowa, K_H grupa kontrolna hipercholesterolowa, M_H grupa hipercholesterolowa otrzymująca metoprolol, M_N grupa normocholesterolowa otrzymująca metoprolol, S1_N grupa normocholesterolowa otrzymująca simwastatynę w dawce 1 mg/kg m.c., S1_H grupa hipercholesterolowa otrzymująca simwastatynę w dawce 1 mg/ /kg m.c., S20_N grupa normocholesterolowa otrzymująca simwastatynę w dawce 20 mg/kg m.c., S20_H grupa hipercholesterolowa otrzymująca simwastatynę w dawce 20 mg/kg m.c., S1_M_N grupa normocholesterolowa otrzymująca metoprolol i simwastatynę w dawce 1 mg/kg m.c., S20_M_N grupa normocholesterolowa otrzymująca metoprolol i simwastatynę w dawce 20 mg/kg m.c., S1_M_H grupa hiperchlesterolowa otrzymująca metoprolol i simwastatynę w dawce 1 mg/kg m.c., S20_M_H grupa hipercholesterolowa otrzymująca metoprolol i simwastatynę w dawce 20 mg/kg m.c. HMG-CoA reductase inhibitors on β-adrenergic receptor activity have been described. It has been shown that statins, by reducing the production of important isoprenoids like farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), could increase β-adrenergic receptor density [9]. The isoprenylation process could influence the membrane association and function of heterotrimeric G-proteins by interfering with γ- subunit (Gγ) isoprenylation [12, 13], especially the isoprenylation of signaling molecules, including the monomeric GTPases of the Rho and Ras families. Moreover, the isoprenylation of Gγ has been found to be essential for membrane attachment of Gγ as well as Gβ [14]. Previous studies demonstrated that treatment of cardiac myocytes with a statin reduced camp and induced a significant increase in β-adrenergic receptor density and, in this way, statins might have an impact on heart rate. The dose dependent influence of statins on beta1- adrenergic signaling was studied as well [9]. The clinical manifestations of the interaction of statins with β 1 -adrenergic blockers on heart rate have not been elucidated yet. Their concomitant usage does not seem to impact heart rate negatively, nor blood pressure. Concomitant administration of simvastatin at 1 and 20 mg kg 1 b.w. with metoprolol in normo- and hypercholesterolemic rats did not significantly influence blood pressure. Even so, clinical observations in the UCSD Statin Study and the Anglo-Scandinavian Cardiac Outcomes Trial- Lipid Lowering Arm (ASCOT-LLA) suggest that HMG-CoA reductase inhibitors might have some blood-pressure-lowering properties in addition to their effect on lipids [15, 16]. On the other hand, the CARE study showed no significant reduction of BP with statin therapy [2]. Different results between the CARE and ASCOT studies may result from the degree of LDL-C reduction achieved in the above trials. The laboratory studies showed the

6 44 J. Owczarek et al. link between statins therapy and vascular function and this could explain the beneficial pharmacological effect of statins in patients with hypertension. Statins improve endothelial function and decrease oxidative stress and inflammation [17 19]. They were shown to reduce the production of reactive oxygen species, such as superoxide anion, and hydroxyl radicals, as shown in experimental studies. This action may also contribute to the vasodilatory effects of statins. It has already been established that statins increase the endothelial production of NO and this effect is correlated with the upregulation of endothelial NO synthase expression [20]. It has suggested that the effect described above might be intensified by the simultaneous inhibition of G proteins with reduced endothelial NO synthase mrna degradation and, thus, increased NO bioavailability [21, 22]. Vascular statin activity may result from drug impact on the decrease of vasoconstrictor endothelin-1 level [21]. Another possible mechanism of statin s BP-lowering effect involves the downregulation of the angiotensin IItype 1 receptor. The angiotensin II type 1 receptor is overexpressed in hypercholesterolemic patients and it may be improved by the administration of statins, which were also shown to markedly reduce the vasoconstrictor response to angiotensin II infusion [24]. Reduction of large artery stiffness and blood pressure in normolipidemic patients with isolated systolic hypertension statins could improve systemic arterial compliance [25]. The authors concluded that administration of simvastatin in different doses not decrease slowing heart rate after chronic concomitant administration of metoprolol in normo- and hypercholesterolemic rats. 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