Time-Dependent Effects of Individual and Combined Treatments With Nebivolol,Lisinopril,and Valsartan on Blood Pressure and Vascular Reactivity to Angiotensin II and Norepinephrine

Abstract

Clinical guidelines suggest the combination of 2 drugs as a strategy to treat hypertension. However, some antihypertensive combinations have been shown to be ineffective. Therefore, it is necessary to determine whether differences exist between the results of monotherapy and combination therapy by temporal monitoring of the responses to angiotensin II and norepinephrine, which are vasoconstrictors involved in the development of hypertension. Thus, the purpose of this work was to determine the vascular reactivity to angiotensin II and norepinephrine in spontaneously hypertensive rat (SHR) aortic rings after treatment with valsartan, lisinopril, nebivolol, nebivolol-lisinopril, and nebivolol-valsartan for different periods of time. In this study, male SHR and Wistar Kyoto normotensive (WKY) rats were divided into 7 groups treated for 1, 2, and 4 weeks: (1) WKY + vehicle, (2) SHR + vehicle; (3) SHR + nebivolol; (4) SHR + lisinopril; (5) SHR + valsartan; (6) SHR + nebivolol-lisinopril; and (7) SHR + nebivolol-valsartan. Blood pressure was measured by the tail-cuff method, and vascular reactivity was determined from the concentration-response curve to angiotensin II and norepinephrine in aortic rings. The results showed that the combined and individual treatments reduced mean blood pressure at all times evaluated. All treatments decreased vascular reactivity to angiotensin II; however, in the case of lisinopril and nebivolol-lisinopril, the effect observed was significant up to 2 weeks. All treatments decreased the reactivity to norepinephrine up to week 4. These results show a time-dependent difference in vascular reactivity between the pharmacological treatments, with nebivolol-valsartan and nebivolol-lisinopril being both effective combinations. Additionally, the results suggest crosstalk between the renin-angiotensin and sympathetic nervous systems to reduce blood pressure and to improve treatment efficacy.

Keywords

vascular reactivity combination therapy nebivolol-valsartan nebivolol-lisinopril

Introduction

Hypertension is one of the leading causes of morbidity and mortality worldwide. This disease is treated with different antihypertensive agents to reduce blood pressure (BP).^1,2 However, monotherapy with antihypertensive agents has failed to maintain BP levels at 120/80 mmHg in approximately 70% of patients.³ Long-term prospective studies have shown that some hypertensive patients are not controlled with monotherapy and require combination therapy to obtain a better therapeutic response, to reduce adverse effects, and possibly to improve patient adherence.^4

-7 However, more studies are necessary to understand how to use antihypertensive polytherapy.⁸

Within this context, the combination of antihypertensive drugs of 2 different classes results in a further BP reduction of a larger magnitude than double the dose of either drug alone.⁹ Besides, combining drugs with complementary mechanisms of action could provide benefits beyond BP lowering, such as improving tolerability.¹⁰ Since both the renin-angiotensin system (RAS) and the sympathetic nervous system (SNS) are implicated in the pathophysiology of hypertension, it is reasonable to propose that the administration of drugs that selectively inhibit either system may improve the antihypertensive effect.¹¹ Some efficacious combinations for hypertension treatment involve the use of β-blockers and diuretics or AT1 receptor antagonists, like nebivolol with valsartan, to reduce blood pressure.¹² Furthermore, the combination of nebivolol with an RAS-modulating drug, such as valsartan, should be studied because some alternative and counter-regulatory pathways are modified by this treatment.¹³ This therapy may be able to prevent or reverse functional changes at the vascular level.¹⁴ However, studies on the time course of the antihypertensive effect induced by such drug combinations are scarce. The question is whether or not the antihypertensive effect could be explained through time-dependent NE- and Ang II-induced vascular reactivity reduction. Thus, the purpose of this work was to study the effect of nebivolol-lisinopril and nebivolol-valsartan on BP and vascular reactivity to angiotensin II (Ang II) and norepinephrine (NE) using SHR rat aortic rings to show the time-dependent interaction between the RAS and the adrenergic system.

Methods

One hundred and eight male spontaneously hypertensive rats (SHR) with a mean blood pressure (MBP) of 135.62 ± 2.3 mmHg, and 18 male Wistar Kyoto (WKY) rats with a MBP of 93.25 ± 0.7 mmHg as normotensive control were obtained from the animal facilities of the Cinvestav Southern Unit (IPN). The animals (6 months old and body weight of 250 g) received rat chow and water ad libitum and were housed in acrylic boxes under standard laboratory conditions. The experiments followed the Official Mexican Norm NOM-062-ZOO-1999, and the Institutional Committee for the Care and Use of Experimental Animals approved the experimental protocol. Carcasses were handled in compliance with the Official Mexican Norm NOM-087-ECOL-SSA1-2002.

The rats were divided into 7 experimental groups (n = 6 per group) and 3 times of treatment (1, 2, and 4 weeks): (1) control normotensive WKY treated with vehicle (isotonic saline solution); (2) control SHR treated with vehicle; (3) SHR + lisinopril (1 mg/kg); (4) SHR + valsartan (1 mg/kg); (5) SHR + nebivolol (0.8 mg/kg); (6) SHR + nebivolol-lisinopril (0.56-0.9 mg/kg); and (7) SHR + nebivolol-valsartan (0.53-0.72 mg/kg). All treatments were administered at same time as single dose via i.m. once a day. The monotherapy (0.25, 0.5, 0.75, 1, 1.25 mg/kg), and combination (0.1 + 0.125 = 0.225, 0.2 + 0.25 = 0.45, 0.4 + 0.5 = 0.9, 0.8 + 1 = 1.8 mg/kg for nebivolol and lisinopril or valsartan, respectively) doses were obtained from dose-response curves using SHR rats determining systolic and diastolic blood pressure to achieve 120/80 mmHg. The combination doses were determined according isobolographic method to perform the dose-response curve. The rats were trained during a week for the tail-cuff method to avoid animal stress and obtain repeated measurements. Blood pressure was determined by the tail-cuff method before and at the end of the antihypertensive treatment using SPAM equipment (INC-ICh, Mexico) and analyzed with the Sievart 1 software before and after application of the treatments. The MBP was calculated as follows: MBP = diastolic BP + 1/3 (systolic BP − diastolic BP).¹⁵ The drugs were administered in a volume of 0.05 ml/i.m. for 1, 2, and 4 weeks. Then, following euthanasia by CO₂ chamber, the thoracic aorta was dissected for the isolated tissue bath assay.

Drugs

Nebivolol, lisinopril, valsartan, NE and Ang II were obtained from Sigma-Aldrich (St. Louis, MO, USA). Nebivolol, lisinopril and valsartan were dissolved in physiological saline solution and the concentration of each drug was adjusted to correspond to the dose administered (mg/kg) in a final volume of 0.05 ml. Ang II and NE were prepared on the same day as the assay and dissolved in deionized water at a concentration of 0.01 M.

Isolated Organ Bath Experiments

The thoracic aorta was quickly excised and placed in a freshly prepared Krebs-Henseleit solution containing 118 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄-7H₂O, 2.5 mM CaCl₂-2H₂O, 25 mM NaHCO₃, 11.7 mM dextrose, and 0.026 mM calcium disodium EDTA, continuously oxygenated with a mixture of 95% O₂/5% CO₂. The aorta’s connective and adherent adipose tissues were removed and cut into rings (≃3 mm long). Each ring was transferred to 10-ml isolated-tissue chambers containing Krebs-Henseleit solution at 37° C and pH 7.4 and continuously aerated with a mixture of 95% O₂/5% CO₂. The rings were suspended between 2 wire hooks (Nubryte wire) to record the development of semi-isometric force; one of them was fixed to the bottom of the chamber and the other to a force transducer (BIOPAC TSD125C) connected to a BIOPAC MP100A-CE system (BIOPAC Systems Inc., Santa Barbara, CA, USA) using the Acqknowledge 3.8.1 software. The initial tension applied to each aortic ring was adjusted to 3 g. The integrity of the endothelium was evaluated in 10⁻⁶ M phenylephrine-treated aortic rings by adding 10⁻⁵ M acetylcholine, and a vasodilator response ≥ 50% was defined for inclusion of the aortic ring in the experiment.

Statistical Analysis

The results of the temporal evolution of BP values (n = 6 per group) are expressed using the mean blood pressure in mmHg ± standard error (SEM), while the NE and Ang II concentration-response curves (18 aortic rings from 6 rats per group), are expressed using the mean aortic contraction in g ± standard error (SEM) and the mean area under the curve (AUC) ± SEM. The differences in the AUC data were determined using 1-way ANOVA and Tukey’s test. Two-way ANOVA and Sidak’s post hoc test were used to evaluate the differences in the concentration-response curve data. In all cases, a P value <.05 was considered statistically significant.

Results

Blood Pressure

Figure 1 shows the time course and AUC of the MBP (panels A and C) and heart rate (panels B and D) over 1, 2, and 4 weeks of treatment with the vehicle, each drug, or their combinations. All therapies, both individually and combined, significantly reduced the MBP and AUC at the different times compared to the control SHR group (MBP data of 4 weeks in mmHg: 101.7 ± 3 for lisinopril, 100.6 ± 3.5 for valsartan, 97.8 ± 2.4 for nebivolol, 102.3 ± 2.5 for nebivolol-lisinopril, 100.8 ± 3.1 for nebivolol-valsartan, 92.5 ± 2.5 for the vehicle in WKY rats, and 141.8 ± 5.5 for the vehicle in SHR). There were no significant changes in heart rate or AUC values according to treatment (heart rate data in bpm: 342.8 ± 19.3 for lisinopril, 372.6 ± 17.6 for valsartan, 329.6 ± 14.6 for nebivolol, 413.7 ± 14.3 for nebivolol-lisinopril, 382.1 ± 13 for nebivolol-valsartan, 345.4 ± 14 for the vehicle in WKY rats, and 406 ± 4.8 for the vehicle in SHR).

Figure 1.

(A) Mean blood pressure values (mmHg × week) and (C) area under the curve and (B) heart rate values (bpm × week) and (D) area under the curve of rats treated for 1, 2, and 4 weeks with lisinopril, valsartan, nebivolol, nebivolol-lisinopril and nebivolol-valsartan, WKY vehicle (normotensive control), and SHR vehicle (hypertensive control). The results are reported as mean values ± SEM from n = 6 rats per group and time of treatment. *P < .05 vs SHR.

Concentration-Response Curves

Figure 2 shows the vascular reactivity to Ang II (panels A, B, and C) and the AUC of Ang II-induced contraction (panels C, D and E) in aortic rings of treated and untreated WKY and SHR rats. The data summarized in the figure show that Ang II-induced vascular reactivity increased in the hypertensive group compared to the normotensive WKY group. In this respect, except for the EC50 values that remained unchanged, the magnitudes of Ang II-induced aortic contraction, Emax values, and the corresponding AUCs were higher in SHR than in the WKY normotensive group, and this effect persisted throughout the experiment (Table 1). In the first week of pharmacological treatment, compared to the SHR group and except for the lisinopril treatment, monotherapy and polytherapy reduced the Ang II-induced vascular reactivity and AUC. Concerning the valsartan, nebivolol, and nebivolol-valsartan treatments, such effects were associated with a reduction in Emax values; in the case of the lisinopril treatment, the effect was associated with an EC50 reduction. The lisinopril and nebivolol-lisinopril treatment reduced Ang II-induced vascular contraction at 2 weeks, and the nebivolol-valsartan combination-induced reduction mentioned above was higher than in the first week. The results obtained after 2 and 4 weeks show that all pharmacological treatments reduced the Ang II Emax values, and this reduction persisted until the end of week 4. In all cases, the minimal contractility obtained with Ang II at the end of 4 weeks of treatment was even less than that observed in the normotensive WKY group.

Figure 2.

Cumulative concentration-response curve to angiotensin II (Ang II) (A, B, and C) in aorta from rats treated for 1 week (A), 2 weeks (B), and 4 weeks (C) with lisinopril (▴), valsartan (Δ), nebivolol (▪), nebivolol-lisinopril (□) and nebivolol-valsartan (♦), WKY vehicle (ˆ), and SHR vehicle (•). The AUC to Ang II (log [Ang II] × g) is given on the right side of each curve (D, E, and F). The results are reported as mean values ± SEM from n = 6 rats per group and time of treatment. *P < .05 vs SHR.

Table 1.

Efficacy (Emax) and Effective Concentration 50 (EC₅₀) to Ang II During the Treatments for 1 Week, 2 Weeks, and 4 Weeks in the Different Groups.

Group	1-week		2-week		4-week
Group	Emax	EC₅₀ (nM)	Emax	EC₅₀ (nM)	Emax	EC₅₀ (nM)
WKY (Vehicle)	0.35 ± 0.05*	7.8 ± 0.3	0.39 ± 0.08*	8.1 ± 0.19	0.37 ± 0.06*	7.6 ± 0.24
SHR (Vehicle)	0.81 ± 0.05	6.5 ± 0.4	0.84 ± 0.07	6.2 ± 0.3	0.79 ± 0.03	6.0 ± 0.05
SHR-Lisinopril	0.58 ± 0.13	2.2 ± 0.1*	0.22 ± 0.02*	8.0 ± 0.2	0.19 ± 0.04*	1. 6± 0.67*
SHR-Valsartan	0.2 ± 0.03*	8.2 ± 0.5	0.27 ± 0.02*	1.3 ± 0.4*	0.11 ± 0.01*	4.0 ± 0.5
SHR-Nebivolol	0.16 ± 0.02*	3.2 ± 0.6	0.46 ± 0.04*	3.9 ± 0.25	0.19 ± 0.02*	2.4 ± 0.4*
SHR-Neb + Lisinopril	0.54 ± 0.18*	2.8 ± 0.2	0.16 ± 0.01*	0.8 ± 0.03*	0.15 ± 0.01*	1.4 ± 0.38*
SHR-Neb + Valsartan	0.28 ± 0.04*	5.6 ± 0.4	0.31 ± 0.03*	6.1 ± 0.2	0.1 ± 0.01*	4.0 ± 0.65

The results are showed as mean values ± SEM from n = 6 rats. *P < .05 vs SHR.

Figure 3 shows the concentration-response curves to NE (panels A, B, and C) and the AUC of NE-induced contraction (panels C, D and E) in aortic rings of SHR treated for 1 week, 2 weeks, and 4 weeks with the different drugs. Only treatments with nebivolol-valsartan, nebivolol-lisinopril and nebivolol significantly reduced vascular reactivity to NE compared to the SHR group in the first week, and the response was time-dependent and persisted until week 4. Treatment with valsartan and lisinopril decreased the response to NE at 2 weeks. In addition, all treatments reduced Emax and increased EC50 values at 4 weeks (Table 2). Except for lisinopril and valsartan, the individual and combination therapies decreased the AUC of NE at 1 week and such reduction was higher with all the treatments at 4 weeks. It is important to mention that all treatments diminished Ang II- and NE-induced vascular reactivity and AUC at 4 weeks.

Figure 3.

Cumulative concentration-response curve to norepinephrine (NE) (A, B, and C) in aorta from rats treated for 1 week (A), 2 weeks (B), and 4 weeks (C) with lisinopril (▴), valsartan (Δ), nebivolol (▪), nebivolol-lisinopril (□) and nebivolol-valsartan (♦), WKY vehicle (ˆ), and SHR vehicle (•). The AUC to NE (log [NE] × g) is given on the right side of each curve (D, E, and F). The results are reported as mean values ± SEM from n = 6 rats per group and time of treatment. *P < .05 vs SHR.

Table 2.

Efficacy (Emax) and Effective Concentration 50 (EC₅₀) to NE During the Treatments for 1 Week, 2 Weeks, and 4 Weeks in the Different Groups.

Group	1-week		2-week		4-week
Group	Emax	EC₅₀ (µM)	Emax	EC₅₀ (µM)	Emax	EC₅₀ (µM)
WKY (Vehicle)	1.6 ± 0.12	0.59 ± 0.01*	1.59 ± 0.15	0.6 ± 0.05*	1.7 ± 0.12	0.62 ± 0.1*
SHR (Vehicle)	1.5 ± 0.08	0.12 ± 0.01	1.53 ± 0.07	0.12 ± 0.03	1.6 ± 0.05	0.12 ± 0.07
SHR-Lisinopril	1.6 ± 0.14	0.31 ± 0.02	1.52 ± 0.05	0.77 ± 0.01*	1.14 ± 0.08*	0.58 ± 0.01*
SHR-Valsartan	1.19 ± 0.07	0.17 ± 0.01	1.2 ± 0.05	0.87 ± 0.01*	1.15 ± 0.04*	0.83 ± 0.01*
SHR-Nebivolol	1.42 ± 0.1	0.7 ± 0.01*	1.54 ± 0.03	0.75 ± 0.01*	1.26 ± 0.04	0.85 ± 0.01*
SHR-Neb + Lisinopril	1.79 ± 0.1	1.0 ± 0.09*	1.61 ± 0.02	0.79 ± 0.01*	1.07 ± 0.07*	0.98 ± 0.01*
SHR-Neb +Valsartan	1.94 ± 0.06*	0.59 ± 0.01*	1.16 ± 0.04	0.15 ± 0.01	1.13 ± 0.07*	1.09 ± 0.11*

The results are showed as mean values ± SEM from n = 6 rats. *P < .05 vs SHR.

Discussion

This paper shows that combined treatments with nebivolol-lisinopril and nebivolol-valsartan for 4 weeks significantly reduced the MBP and in a time-dependent manner the vascular reactivity to Ang II and NE, as demonstrated by the reduction of Emax and AUC, and changes in EC50 values in the SHR animal model.

Effects of Combined Treatment on BP and HR

In this work, the single treatment with lisinopril, valsartan and nebivolol as combined treatments (nebivolol + lisinopril and nebivolol + valsartan) were able to reduce experimental hypertension, as described in other studies.^16

-21 It is important to note that, in the present work, none of the treatments changed the heart rate at the doses used. In this case, although nebivolol is a β-adrenergic blocker, it had a minor effect on the heart rate compared to carvedilol,²² which agrees with our results.

The combined therapy has been used to increase the antihypertensive efficacy, reduce the adverse effects, and the doses of single therapy. In this sense, Burnier et al (2015) proposed that, in combination therapy like irbesartan with hydrochlorothiazide, the BP-lowering effect of each drug is enhanced, while each drug has the potential to neutralize counter-regulatory mechanisms and to reduce side effects.¹⁴ Accordingly, in the present work a lower dose of each drug was used in the combined treatments compared to the individual treatments, in order to reduce the blood pressure up to normotensive value.

It is well known that RAS has an important role on the regulation of BP and extracellular fluid volume.²³ Dysfunction of the RAS is associated with the development of cardiovascular diseases such as hypertension.²⁴ In this sense, the Ang II plasma and renal tissue levels in SHR rat are significantly higher than those in WKY rat, and the ACEi treatment reduces the Ang II plasma levels in SHR.^25

-28 Additionally, hypertension-induced alterations might be related to changes in the structure and function of the artery wall.^29,30 The functional alterations of blood vessels in vascular reactivity contribute to the development and persistence of hypertension.³¹

On the other hand, the SNS regulates BP through the release of catecholamines such as norepinephrine, and an increase in this neurotransmitter is associated with hypertension in humans and animal models.^32,33 Besides, 6-month-old SHR plasma noradrenaline was significantly higher than in WKY which suggests that peripheral sympathetic activity play an important role in the development of hypertension in SHR.^34,35

Besides, RAS and SNS may interact through pathophysiological mechanisms contributing to the development of hypertension by increasing vascular contractility and arterial remodeling, affecting vascular smooth muscle function.³⁶ In this sense, studies indicate that both sympathetic and RAS hyperreactivity are a generalized phenomenon in hypertension, and their activation augments BP values and leads to the development of organ damage and renal or cardiac disease.^37,38 These data are in agreement with our results due to the fact that the Ang II- and NE-induced contractility was increased in SHR (Figures 2 and 3, Tables 1 and 2).

Effects of Single Nebivolol and Valsartan Treatment on Vascular Reactivity

Studies shown a relationship between RAS and SNS. In this sense, it has been demonstrated that the activation of atypical AT1 prejunctional receptors in rat left ventricle and canine pulmonary artery is associated to NE release.³⁹ Also, it has been reported that Ang II through AT1 receptor (AT1 R) induces transcription and expression of α_1b- and α_1d-adrenergic receptors in rat smooth vascular muscle cells culture.⁴⁰

In this sense, Ang II increases sympathetic nerve activity, and the activation of the sympathetic nervous system increases RAS expression in the circulatory system. These 2 mechanisms exacerbate one another, resulting in the development of hypertension.^33,41 However, there are no data about the temporal occurrence of these mechanisms, and the present work show that those are time-dependent crosstalk regulatory mechanisms between SNS and RAS, because pharmacological treatment with the adrenergic β-blocker nebivolol reduced vascular reactivity to Ang II (Figure 2), and the AT1 antagonist valsartan decreased vascular reactivity to NE (Figure 3). Accordingly, Ang II increases the potassium-evoked NE release from the hypothalamus and parietal cortex slices, the magnitude of this effect is higher in SHR than in normotensive rats and the effect can be blocked by AT1 receptor antagonist.^42,43 It is important to mention that the changes on Ang II- and NE-induced vascular reactivity reduction were time-dependent. In this context, valsartan treatment reduced the Ang II reactivity starting from the first week of treatment (Figure 2) and decreased the NE-induced vascular reactivity starting from 2 weeks of treatment (Figure 3). These changes are mainly associated to a Emax reduction. In the case of Ang II, the EC50 value decreased only at 2 weeks (Table 1). With respect to NE, an increase in EC50 value was observed at 2 weeks (Table 2). Then, these results suggest that the reduction in Ang II contractility by valsartan could be a consequence of a receptors density reduction and time-dependent affinity increase due to compensatory mechanisms. These results are evidence of the crosstalk between RAS and SNS which tend to normalize the BP in hypertension (Figure 1). In this sense, it is known that the prejunctional AT1 stimulation by Ang II has a stimulatory effect on sympathetic ganglia and sympathetic nerve endings, and this is counteracted by AT1 antagonist like valsartan.⁴⁴ The valsartan-induced crosstalk mechanism on NE vascular response could be related with a reduction in receptors density, NE affinity or NE levels, as seen as changes in Emax and EC50 values (Table 2). However, the NE reduction level could explain the valsartan-induced changes on NE reactivity. Accordingly, it is known that a reduction in vascular sensitivity to NE could be explained due to a decrease in NE release and increasing clearance.³⁹ Additionally, a study demonstrated that the blockage of AT1 receptors with candesartan or losartan indirectly stimulated the prejunctional α₂-adrenergic receptors and increased the NE reuptake using pithed rats.⁴⁵

Concerning nebivolol, it reduced the Ang II vascular reactivity (Figure 2) and Emax value (Table 1) in the first week and decreased the response and Emax to NE in the fourth week (Table 2). These changes induced by nebivolol suggest a time-dependent reduction of receptors density and affinity increase. In agreement with our results, Wang et al (2012) found that nebivolol (8 mg/kg/day, i.g.) administered to SHR rat for 8 weeks reduced Ang II and NE vasoconstriction in the femoral and renal arteries.⁴⁶ In addition, nebivolol (a β1 antagonist and β3 agonist) decreases SNS activity¹⁹ due to catecholamine release inhibition by the adrenal glands.⁴⁷ This fact may contribute to the decrease in the response to NE in our study. Regarding time-dependent β-adrenergic receptors density changes induced by nebivolol, there are no evidence about it, and it is necessary to perform more studies.

Furthermore, nebivolol-valsartan combination reduced Ang II-induced vascular reactivity (Figure 2) and BP in a way that depends on treatment time (Figure 1). The results of the present work suggest that the mechanisms involved in the effect of nebivolol-valsartan treatment could be related to a decrease in the density of AT1 receptors because only the Ang II Emax values were reduced with this treatment (Table 1). However, additional studies are needed to verify it. Concerning NE-induced contractility, the combined treatment reduced the efficacy of NE in the fourth week of treatment (Figure 3), like Ang II reactivity this effect could be related to a α_1D-adrenergic receptor density decrease.⁴⁰ Regarding nebivolol-valsartan results, Barki-Harrington et al, (2003) showed that the β-adrenergic receptor (βAR) blockade inhibits signaling of AT1Rs, whereas selective AT1 R blockade inhibits downstream signaling of βARs.⁴⁸ Nevertheless, the temporal changes were not analyzed in those studies. Additionally, Sander et al (2016) suggested that the combination of nebivolol with valsartan offers a clinical benefit by combining β1-adrenoceptor and AT1 receptor blockade with β3 receptor activation, resulting in an increase in NO and vasodilation.⁴⁹

Effects of Nebivolol and Lisinopril Treatment on Vascular Reactivity

Lisinopril decreased the vascular reactivity (Figure 3) and Emax and increased EC50 values to NE at 4 weeks (Table 2). These results suggest a time-dependent decrease in α_1D-adrenergic receptor density and affinity. However, additional studies about receptor density are needed. These results could be explained due to a decrease in NE levels. Accordingly, an in vitro study showed that captopril significantly inhibited the electric stimulation-evoked NE release in rat hypothalamus and medulla oblongata slices,⁵⁰ as well as in peripheral tissues such as rat mesenteric artery.⁵¹ Therefore, the inhibition of NE release by an ACE inhibitor could be partially due to a reduction in Ang II formation in the CNS; thus, this mechanism might reduce the sympathetic outflow to the periphery and contribute in part to the BP-lowering efficacy in both human and experimental hypertension.⁵² Besides, inhibitors of the RAS, including ACE inhibitors and angiotensin receptor blockers, might suppress sympathetic hyperactivity in the brain.⁵² Another study demonstrated a time-dependent effect induced by lisinopril in which the oral administration of this drug (10 mg/kg) resulted in delayed but long-lasting inhibition of ACE activity in the SHR brain.⁵³ However, there is not enough data to understand how the central RAS may contribute to long-term antihypertensive action.

On the other hand, the effects observed with nebivolol-lisinopril on to Ang II- and NE- induced vascular reactivity decrease (Figures 2 and 3) through the time-dependent changes in Emax value at 4 weeks to NE and from 1-week to Ang II, and EC50 values to Ang II and NE at 1 week (Tables 1 and 2). In contrast with Ang II, the nebivolol-lisinopril induce an increase in NE affinity. These results might be a consequence of the reduction in Ang II and NE receptor density and affinity changes. However, there are not evidences that help us to explain the time-dependent receptor density changes.

The differences in time-dependent vascular responses might be related to tissue RAS changes induced by chronic pharmacological treatment, due to the fact that the circulating RAS has been associated with acute effects.^46,54 Within this context, the combination of RAS inhibitors with β-adrenergic blockers like lisinopril, valsartan and nebivolol could offer a complementary mechanism through different systems for better antihypertensive therapy. In addition, the vascular antiadrenergic properties could contribute to the time-dependent efficacy of ACEI and ARB in subjects with hypertension.

In conclusion, this work showed that valsartan and the combination therapy with nebivolol-lisinopril and nebivolol-valsartan provide time-dependent beneficial effects by decreasing BP and vascular reactivity. Furthermore, the reduction in response to Ang II may be the cause of the subsequent decrease in the reactivity to NE during nebivolol and RAS inhibitors treatment. Besides, monotherapy and combined therapy with nebivolol-valsartan showed crosstalk related to time-dependent changes in the affinity to Ang II and NE. These time-dependent changes may help us to understand the effects produced by the combined administration of drugs and how this is associated with treatment effectiveness.

Footnotes

Acknowledgments

To MVZ. Juan Martinez Parente and C. Ramon Martinez Gomez, CINVESTAV Unidad Sur.

Author Contributions

Diego Lezama-Martinez contributed in drafting the manuscript, experiments, data collection, analysis, and authoring; Maria Elena Hernandez-Campos and Jazmin Flores-Monroy contributed in idea, analysis, and authoring; and Luisa Martinez-Aguilar and Ignacio Valencia-Hernandez contributed in idea, drafting the manuscript, data collection, analysis, and authoring.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Supported by grants from DGAPA-UNAM PAPIIT IA205119, UNAM PIAPI-1828 FES Cuautitlán, Universidad Nacional Autónoma de México, CONACYT A1-S-8958, Secretaría de Investigación y Posgrado (SIP: 20195123), Comisión de Operación y Fomento de Actividades Académicas, and Programa Institucional de Estímulo al Desempeño de los Investigadores del Instituto Politécnico Nacional.

ORCID iDs

Diego Lezama-Martinez

Maria Elena Hernandez-Campos

References

Whelton

Carey

Aronow

, et al. 2017 ACC/AHA/AAPA/ABC/ACPM/AGS/APhA/ASH/ASPC/NMA/PCNA Guideline for the prevention, detection, evaluation, and management of high blood pressure in adults: executive summary: a report of the American College of Cardiology/American Heart Association Task Force on clinical practice guidelines. Hypertension. 2018;71(6):1269–1324. doi:10.1161/hyp.0000000000000066

Weber

Schiffrin

White

, et al. Clinical practice guidelines for the management of hypertension in the community: a statement by the American Society of Hypertension and the International Society of Hypertension. J Clin Hypertens (Greenwich). 2014;16(1):14–26. doi:10.1111/jch.12237

Guerrero-Garcia

Rubio-Guerra

. Combination therapy in the treatment of hypertension. Drugs Context. 2018;7:212531. doi:10.7573/dic.212531

Kalra

Agrawal

. Combination therapy in hypertension: an update. Diabetol Metab Syndr. 2010;2(1):44. doi:10.1186/1758-5996-2-44

Mancia

Fagard

Narkiewicz

, et al. 2013 ESH/ESC Guidelines for the management of arterial hypertension: the Task Force for the Management of Arterial Hypertension of the European Society of Hypertension (ESH) and of the European Society of Cardiology (ESC). J Hypertens. 2013;31(7):1281–1357. doi:10.1097/01.hjh.0000431740.32696.cc

Cowart

Taylor

. Should two-drug initial therapy for hypertension be recommended for all patients? Curr Hypertens Rep. 2012;14(4):324–332. doi:10.1007/s11906-012-0280-9

Mancia

Rea

Corrao

Grassi

. Two-drug combinations as first-step antihypertensive treatment. Circ Res. 2019;124(7):1113–1123. doi:10.1161/circresaha.118.313294

James

Oparil

Carter

, et al. 2014 evidence-based guideline for the management of high blood pressure in adults: report from the panel members appointed to the Eighth Joint National Committee (JNC 8). JAMA. 2014;311(5):507–520. doi:10.1001/jama.2013.284427

Wald

Law

Morris

Bestwick

Wald

. Combination therapy versus monotherapy in reducing blood pressure: meta-analysis on 11,000 participants from 42 trials. Am J Med. 2009;122(3):290–300. doi:10.1016/j.amjmed.2008.09.038

10.

Waeber

Feihl

Ruilope

. Fixed-dose combinations as initial therapy for hypertension: a review of approved agents and a guide to patient selection. Drugs. 2009;69(13):1761–1776. doi:10.2165/11316710-000000000-00000

11.

Igreja

Pires

Bonifácio

, et al. Blood pressure-decreasing effect of etamicastat alone and in combination with antihypertensive drugs in the spontaneously hypertensive rat. Hypertens Res. 2015;38(1):30–38. doi:10.1038/hr.2014.143

12.

Cicero

AFG

Kuwabara

Borghi

. A critical review of nebivolol and its fixed-dose combinations in the treatment of hypertension. Drugs. 2018;78(17):1783–1790. doi:10.1007/s40265-018-0999-y

13.

Battise

Boland

Nuzum

. Nebivolol/Valsartan: a novel antihypertensive fixed-dose combination tablet. Ann Pharmacother. 2019;53(4):402–412. doi:10.1177/1060028018813575

14.

Burnier

. Antihypertensive combination treatment: state of the art. Curr Hypertens Rep. 2015;17(7):51. doi:10.1007/s11906-015-0562-0

15.

Sainas

Milia

Palazzolo

, et al. Mean blood pressure assessment during post-exercise: result from two different methods of calculation. J Sports Sci Med. 2016;15(3):424–433.

16.

Gomez

Cirillo

Moncloa

. The clinical pharmacology of lisinopril. J Cardiovasc Pharmacol. 1987;9(suppl 3):S27–S34. doi:10.1097/00005344-198700003-00008

17.

Pool

Glazer

Chiang

Gatlin

. Dose-response efficacy of valsartan, a new angiotensin II receptor blocker. J Hum Hypertens. 1999;13(4):275–281. doi:10.1038/sj.jhh.1000788

18.

Belz

Breithaupt-Grogler

Butzer

Fuchs

Hausdorf

Mang

. The pharmacological potency of various AT(1) antagonists assessed by Schild regression technique in man. J Renin Angiotensin Aldosterone Syst. 2000;1(4):336–341. doi:10.3317/jraas.2000.063

19.

Maffei

Lembo

. Nitric oxide mechanisms of nebivolol. Ther Adv Cardiovasc Dis. 2009;3(4):317–327. doi:10.1177/1753944709104496

20.

Neutel

Giles

Punzi

Weiss

Finck

. Long-term safety of nebivolol and valsartan combination therapy in patients with hypertension: an open-label, single-arm, multicenter study. J Am Soc Hypertens. 2014;8(12):915–920. doi:10.1016/j.jash.2014.09.017

21.

Weber

Basile

Stapff

Khan

Zhou

. Blood pressure effects of combined β-blocker and angiotensin-converting enzyme inhibitor therapy compared with the individual agents: a placebo-controlled study with nebivolol and lisinopril. J Clin Hypertens (Greenwich). 2012;14(9):588–592. doi:10.1111/j.1751-7176.2012.00666.x

22.

Studinger

Tabak

Chen

, et al. The effect of low-dose carvedilol, nebivolol, and metoprolol on central arterial pressure and its determinants: a randomized clinical trial. J Clin Hypertens (Greenwich). 2013;15(12):910–917. doi:10.1111/jch.12210

23.

Yim

Yoo

. Renin-Angiotensin system—considerations for hypertension and kidney. Electrolyte Blood Press. 2008;6(1):42–50. doi:10.5049/ebp.2008.6.1.42

24.

Paul

Poyan Mehr

Kreutz

. Physiology of local renin-angiotensin systems. Physiol Rev. 2006;86(3):747–803. doi:10.1152/physrev.00036.2005

25.

Fan

Liu

Cui

Huang

Wang

Gao

. Novel mechanism of intra-renal angiotensin II-induced sodium/proton exchanger 3 expression by losartan in spontaneously hypertensive rats. Mol Med Rep. 2014;10(5):2483–2488. doi:10.3892/mmr.2014.2492

26.

Bolterman

Manriquez

Ortiz Ruiz

Juncos

Romero

. Effects of captopril on the renin angiotensin system, oxidative stress, and endothelin in normal and hypertensive rats. Hypertension. 2005;46(4):943–947. doi:10.1161/01.HYP.0000174602.59935.d5

27.

Kohara

Brosnihan

Ferrario

. Angiotensin(1–7) in the spontaneously hypertensive rat. Peptides. 1993;14(5):883–891. doi:https://doi.org/10.1016/0196-9781(93)90063-M

28.

Castro-Moreno

Pardo

Hernández-Muñoz

, et al. Captopril avoids hypertension, the increase in plasma angiotensin II but increases angiotensin 1-7 and angiotensin II-induced perfusion pressure in isolated kidney in SHR. Auton Autacoid Pharmacol. 2012;32(3 Pt 4):61–69. doi:10.1111/aap.12001

29.

Heagerty

Heerkens

Izzard

. Small artery structure and function in hypertension. J Cell Mol Med. 2010;14(5):1037–1043. doi:10.1111/j.1582-4934.2010.01080.x

30.

Thom

Arterial structural modifications in hypertension. Effects of treatment. Eur Heart J. 1997;18(suppl E):E2–E4. doi:10.1016/s0195-668x(97)90001-4

31.

Wilson

Zhang

Buckley

Heathcote

Lee

McCarron

. Increased vascular contractility in hypertension results from impaired endothelial calcium signaling. Hypertension. 2019;74(5):1200–1214. doi:10.1161/hypertensionaha.119.13791

32.

Wyss

. The role of the sympathetic nervous system in hypertension. Curr Opin Nephrol Hypertens. 1993;2(2):265–273. doi:10.1097/00041552-199303000-00014

33.

DiBona

. Sympathetic nervous system and hypertension. Hypertension. 2013;61(3):556–560. doi:10.1161/hypertensionaha.111.00633

34.

Pak

. Plasma adrenaline and noradrenaline concentrations of the spontaneously hypertensive rat. Jpn Heart J. 1981;22(6):987–995. doi:10.1536/ihj.22.987

35.

Berg

Walaas

Roberg

BÅ

Huynh

Jensen

. Plasma norepinephrine in hypertensive rats reflects α(2)-adrenoceptor release control only when re-uptake is inhibited. Front Neurol. 2012;3:160–160. doi:10.3389/fneur.2012.00160

36.

Touyz

Alves-Lopes

Rios

, et al. Vascular smooth muscle contraction in hypertension. Cardiovasc Res. 2018;114(4):529–539. doi:10.1093/cvr/cvy023

37.

Mancia

Grassi

. The autonomic nervous system and hypertension. Circ Res. 2014;114(11):1804–1814. doi:10.1161/CIRCRESAHA.114.302524

38.

Young

Davisson

. Angiotensin-II, the brain, and hypertension: an update. Hypertension. 2015;66(5):920–926. doi:10.1161/hypertensionaha.115.03624

39.

Guimarães

Pinheiro

Tavares

Loio

Moura

. Differential effects of eprosartan and losartan at prejunctional angiotensin II receptors. Naunyn Schmiedeberg’s Arch Pharmacol. 2001;363(5):509–514. doi:10.1007/s002100100396

40.

Long

Gray

Rokosh

Honbo

Karliner

. Cross talk between angiotensin AT1 and alpha 1-adrenergic receptors: angiotensin II downregulates alpha 1a-adrenergic receptor subtype mRNA and density in neonatal rat cardiac myocytes. Circ Res. 1997;81(3):396–403. doi:10.1161/01.res.81.3.396

41.

Dong

Chen

J-W

Tian

L-L

, et al. Role of the renin-angiotensin system, renal sympathetic nerve system, and oxidative stress in chronic foot shock-induced hypertension in rats. Int J Biol Sci. 2015;11(6):652–663. doi:10.7150/ijbs.10250

42.

Huang

Rogers

Henderson

. Effects of angiotensin II on [3 H]noradrenaline release and phosphatidylinositol hydrolysis in the parietal cortex and locus coeruleus of the rat. J Neurochem. 1987;49(5):1541–1549. doi:10.1111/j.1471-4159.1987.tb01025.x

43.

Qadri

Badoer

Stadler

Unger

. Angiotensin II-induced noradrenaline release from anterior hypothalamus in conscious rats: a brain microdialysis study. Brain Res. 1991;563(1-2):137–141. doi:10.1016/0006-8993(91)91526-7

44.

Nap

Balt

Mathy

Van Zwieten

. AT(1)-receptor blockade and sympathetic neurotransmission in cardiovascular disease. Auton Autacoid Pharmacol. 2003;23(5-6):285–296. doi:10.1111/j.1474-8673.2004.00301.x

45.

Raasch

Dominiak

Ziegler

Dendorfer

. Reduction of vascular noradrenaline sensitivity by AT1 antagonists depends on functional sympathetic innervation. Hypertension. 2004;44(3):346–351. doi:10.1161/01.HYP.0000138406.13413.0e

46.

Wang

Zhang

Liu

. Nebivolol treatment improves resistant arterial function and reduces ventricular hypertrophy and angiotensin II in spontaneously hypertension rats. J Renin Angiotensin Aldosterone Syst. 2013;14(2):146–155. doi:10.1177/1470320312470580

47.

Borghi

Acelajado

Gupta

Jain

. Role of nebivolol in the control and management of central aortic blood pressure in hypertensive patients. J Hum Hypertens. 2017;31(10):605–610. doi:10.1038/jhh.2017.26

48.

Barki-Harrington

Luttrell

Rockman

. Dual inhibition of beta-adrenergic and angiotensin II receptors by a single antagonist: a functional role for receptor-receptor interaction in vivo. Circulation. 2003;108(13):1611–1618. doi:10.1161/01.Cir.0000092166.30360.78

49.

Sander

Fernandez

Giles

. Fixed-dose combination therapy of nebivolol and valsartan for the treatment of hypertension. Expert Rev Cardiovasc Ther. 2016;14(5):563–572. doi:10.1586/14779072.2016.1167598

50.

Tsuda

Nishio

Masuyama

Goldstein

. Effects of captopril on [3 H]-norepinephrine release in rat central nervous system. Clin Exp Pharmacol Physiol. 1995;22(9):610–613. doi:10.1111/j.1440-1681.1995.tb02074.x

51.

Tsuda

Shima

Kuchii

Nishio

Masuyama

. Effects of captopril on neurosecretion and vascular responsiveness in hypertension. Clin Exp Hypertens A. 1987;9(2-3):375–379. doi:10.3109/10641968709164200

52.

Tsuda

. Renin-Angiotensin system and sympathetic neurotransmitter release in the central nervous system of hypertension. Int J Hypertens. 2012;2012:474870. doi:10.1155/2012/474870

53.

Cushman

Wang

Fung

Harvey

DeForrest

. Differentiation of angiotensin-converting enzyme (ACE) inhibitors by their selective inhibition of ACE in physiologically important target organs. Am J Hypertens. 1989;2(4):294–306. doi:10.1093/ajh/2.4.294

54.

Campbell

. Clinical relevance of local renin angiotensin systems. Front Endocrinol (Lausanne). 2014;5:113–113. doi:10.3389/fendo.2014.00113