Abstract
The sympathetic nervous system (SNS) plays a fundamental role in the pathophysiology of cardiovascular diseases, including primary arterial hypertension. In this study, we aimed to investigate whether the expression of the rate-limiting enzyme in catecholamine synthesis, tyrosine hydroxylase (TH), and the β2-adrenergic receptor (β2-AR) in immune cells from peripheral blood, reflect central SNS activity in spontaneously hypertensive rats (SHR). TH expression in the lower brainstem and adrenal glands and β2-AR expression in the lower brainstem were analyzed by western blot analyses. In the leukocytes, TH and β2-AR expression was evaluated by flow cytometry before and after chronic treatment with the centrally-acting sympathoinhibitory drug clonidine. Western blot analyses showed increased TH and β2-AR expression in the lower brainstem and increased TH in adrenal glands from SHR compared to normotensive Wistar Kyoto rats (WKY). Lower brainstem from SHR treated with clonidine presented reduced TH and β2-AR levels, and adrenal glands had decreased TH expression compared to SHR treated with vehicle. Flow cytometry showed that the percentage of leukocytes that express β2-AR is higher in SHR than in WKY. However, the percentage of leukocytes that expressed TH was higher in WKY than in SHR. Moreover, chronic treatment with clonidine normalized the levels of TH and β2-AR in leukocytes from SHR to similar levels of those of WKY. Our study demonstrated that the percentage of leukocytes expressing TH and β2-AR was altered in arterial hypertension and can be modulated by central sympathetic inhibition with clonidine treatment.
Brain stem; Sympathetic nervous system; Leukocytes; Spontaneously hypertensive rats; Clonidine
Introduction
Cardiovascular diseases are the leading cause of mortality worldwide, representing approximately 31% of all-cause mortality (11. WHO. Fact Sheets Cardiovascular Diseases 2017; https://www.who.int/news-room/fact-sheets/detail/cardiovascular-diseases-(cvds)
https://www.who.int/news-room/fact-sheet...
). Primary hypertension is a multifactorial disease, and sympathetic nervous system (SNS) hyperactivity plays an important role in its pathophysiology (22. Mancia G. Björn Folkow award lecture. The sympathetic nervous system in hypertension. J Hypertens 1997; 15: 1553–1565, doi: 10.1097/00004872-199715120-00056.
https://doi.org/10.1097/00004872-1997151...
).
Central sympathetic hyperactivity and the resulting activation of the sympathetic outflow to the blood vessels, the heart, and the kidneys is a hallmark in hypertensive patients (22. Mancia G. Björn Folkow award lecture. The sympathetic nervous system in hypertension. J Hypertens 1997; 15: 1553–1565, doi: 10.1097/00004872-199715120-00056.
https://doi.org/10.1097/00004872-1997151...
) and in experimental models of hypertension (33. Wyss JM, Carlson SH. The role of the central nervous system in hypertension. Curr Hypertens Rep 1999; 1: 246–253, doi: 10.1007/s11906-999-0029-2.
https://doi.org/10.1007/s11906-999-0029-...
). Moreover, the increase in sympathetic activity is a mechanism for both initiating and sustaining elevated blood pressure (22. Mancia G. Björn Folkow award lecture. The sympathetic nervous system in hypertension. J Hypertens 1997; 15: 1553–1565, doi: 10.1097/00004872-199715120-00056.
https://doi.org/10.1097/00004872-1997151...
). Additionally, sympathetic overactivity increases cardiovascular risk through the development of left ventricular hypertrophy, contributing to the origin of ventricular arrhythmias and sudden death and reducing the uptake of glucose by skeletal muscle, which can result in insulin resistance and consequent hyperinsulinemia (44. Schlaich M, Straznicky N, Lambert E, Lambert G. Metabolic syndrome: a sympathetic disease? Lancet Diabetes Endocrinol 2015; 3: 148–157, doi: 10.1016/S2213-8587(14)70033-6.
https://doi.org/10.1016/S2213-8587(14)70...
).
Consequently, the assessment of the systemic levels of sympathetic activity appears to be crucial in the management of primary hypertension, allowing for the identification of a subset of hypertensive patients presenting sympathetic overactivity. In these patients, the modulation of central sympathetic activity using centrally acting antihypertensive drugs could be especially beneficial. Human regional sympathetic activity can be studied by microneurography of postganglionic sympathetic efferences directed to the skeletal muscle vasculature and by noradrenaline spillover, which is a measurement of organ-specific catecholamine release into plasma, together with direct measurement of plasma catecholamines and spectral analysis of heart rate signals (55. Esler M. The sympathetic nervous system in hypertension: back to the future? Curr Hypertens Rep 2015; 17: 11, doi: 10.1007/s11906-014-0519-8.
https://doi.org/10.1007/s11906-014-0519-...
). However, these different approaches display important limitations and disadvantages, and some of them are not feasible in daily clinical practice (66. Seravalle G, Dimitriadis K, Dell'Oro R, Grassi G. How to assess sympathetic nervous system activity in clinical practice. Curr Clin Pharmacol 2013; 8: 182–188, doi: 10.2174/1574884711308030003.
https://doi.org/10.2174/1574884711308030...
).
Tyrosine hydroxylase (TH) is a rate-limiting enzyme in catecholamine synthesis and a key component of the central and peripheral SNS (77. Tekin I, Roskoski R Jr, Carkaci-Salli N, Vrana KE. Complex molecular regulation of tyrosine hydroxylase. J Neural Transm (Vienna) 2014; 121: 1451–1481.). The activation of presynaptic β2-adrenergic receptor (β2-AR) by catecholamines leads to noradrenaline release (88. Langer SZ. Presynaptic regulation of the release of catecholamines. Pharmacol Rev 1980; 32: 337–362.) and induces enhanced cardiac output and myocardial contractility, acting on the most abundant β-adrenergic receptor subtype in the heart, β1-AR. α2-Adrenergic receptors (inhibitory auto-receptors) are also present in the noradrenergic nerve endings and are involved in the regulation of noradrenaline release through a negative feedback mechanism mediated by the neurotransmitter itself (88. Langer SZ. Presynaptic regulation of the release of catecholamines. Pharmacol Rev 1980; 32: 337–362.). During sympathetic overactivity, which is the case of several cardiovascular diseases, including arterial hypertension and heart failure, both α- and β-adrenergic receptors are continuously activated. This results in β1-AR desensitization with alterations of down-stream mechanisms (99. Brodde OE, Bruck H, Leineweber K. Cardiac adrenoceptors: physiological and pathophysiological relevance. J Pharmacol Sci 2006; 100: 323–337, doi: 10.1254/jphs.CRJ06001X.
https://doi.org/10.1254/jphs.CRJ06001X...
), resulting in further activation of the β2-AR. Regarding the desensitization of the β2-AR on human leukocytes, it has already been demonstrated that β-AR agonists induce a rapid desensitization of the receptor with an associated redistribution of the receptor into a cellular compartment to which catecholamines have limited access (1010. Motulsky HJ, Cunningham EM, DeBlasi A, Insel PA. Desensitization and redistribution of beta-adrenergic receptors on human mononuclear leukocytes. Am J Physiol 1986; 250: E583–E590.).
The interactions between the autonomic nervous system and the immune system are considered to be bidirectional. Primary and secondary lymphoid organs have rich sympathetic innervations that are responsible for the release of noradrenaline and other neurotransmitters (1111. Kin NW, Sanders VM. It takes nerve to tell T and B cells what to do. J Leukoc Biol 2006; 79: 1093–1104, doi: 10.1189/jlb.1105625.
https://doi.org/10.1189/jlb.1105625...
). Catecholamines are produced by thymic lymphoid and non-lymphoid cells, including epithelial and endothelial cells, platelets, and macrophages (1212. Sorriento D, Santulli G, Del Giudice C, Anastasio A, Trimarco B, Iaccarino G. Endothelial cells are able to synthesize and release catecholamines both in vitro and in vivo. Hypertension 2012; 60: 129–136, doi: 10.1161/HYPERTENSIONAHA.111.189605.
https://doi.org/10.1161/HYPERTENSIONAHA....
,1313. Smith CC, Wilson AP, Prichard BN, Betteridge DJ. Stimulus-induced release of endogenous catecholamines from human washed platelets. Clin Sci (Lond) 1986; 70: 495–500, doi: 10.1042/cs0700495.
https://doi.org/10.1042/cs0700495...
). Moreover, lymphocytes produce catecholamines and express alpha and beta adrenergic receptors (1414. Kohm AP, Sanders VM. Norepinephrine and beta 2-adrenergic receptor stimulation regulate CD4+ T and B lymphocyte function in vitro and in vivo. Pharmacol Rev 2001; 53: 487–525.).
The presence of components of the sympathetic system in leukocytes has been related to central sympathetic hyperactivity (1414. Kohm AP, Sanders VM. Norepinephrine and beta 2-adrenergic receptor stimulation regulate CD4+ T and B lymphocyte function in vitro and in vivo. Pharmacol Rev 2001; 53: 487–525.,1515. Brodde OE, Prywarra A, Daul A, Anlauf M, Bock KD. Correlation between lymphocyte beta 2-adrenoceptor density and mean arterial blood pressure: elevated beta-adrenoceptors in essential hypertension. J Cardiovasc Pharmacol 1984; 6: 678–682, doi: 10.1097/00005344-198407000-00020.
https://doi.org/10.1097/00005344-1984070...
). Furthermore, the detection of components of the sympathetic system in leukocytes has been proposed as a simple strategy to assess the levels of sympathetic activity of the central nervous system (CNS) in the periphery (1414. Kohm AP, Sanders VM. Norepinephrine and beta 2-adrenergic receptor stimulation regulate CD4+ T and B lymphocyte function in vitro and in vivo. Pharmacol Rev 2001; 53: 487–525.–1616. Veglio F, Tayebati SK, Schiavone D, Ricci A, Mulatero P, Bronzetti E, et al. Alpha1-adrenergic receptor subtypes in peripheral blood lymphocytes of essential hypertensives. J Hypertens 2001; 19: 1847–1854, doi: 10.1097/00004872-200110000-00020.
https://doi.org/10.1097/00004872-2001100...
). Human and rat mononuclear cells express TH, and the catecholamines produced by these cells are capable of acting in an autocrine and/or paracrine manner (1717. Qiu YH, Peng YP, Jiang JM, Wang JJ. Expression of tyrosine hydroxylase in lymphocytes and effect of endogenous catecholamines on lymphocyte function. Neuroimmunomodulation 2004; 11: 75–83, doi: 10.1159/000075316.
https://doi.org/10.1159/000075316...
).
The expression of β2-AR is higher in the mononuclear cells of hypertensive patients than in those of normotensive controls; moreover, there is a significant correlation between β2-AR density and mean arterial blood pressure in primary hypertension (1515. Brodde OE, Prywarra A, Daul A, Anlauf M, Bock KD. Correlation between lymphocyte beta 2-adrenoceptor density and mean arterial blood pressure: elevated beta-adrenoceptors in essential hypertension. J Cardiovasc Pharmacol 1984; 6: 678–682, doi: 10.1097/00005344-198407000-00020.
https://doi.org/10.1097/00005344-1984070...
,1818. Middeke M, Remien J, Block LH, Kirzinger S, Landrock A, Holzgreve H. Beta 2-adrenoceptor density on membranes and on intact mononuclear cells in essential hypertension. Res Exp Med (Berl) 1983; 183: 227–232.).
Thus, in the present study, we investigated the existence of an association between TH and β2-AR expression in leukocytes from peripheral blood with the plasma levels of catecholamines, blood pressure, and heart rate. Moreover, the chronic treatment of the animals with the centrally acting sympathoinhibitory drug clonidine was used to test the effects of the negative modulation of the central SNS on TH and β2-AR expression.
Material and Methods
Animals
Male Wistar Kyoto rats (WKY) (from the Animal Breeding Center, CECAL, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil) and spontaneously hypertensive rats (SHR) of the Okamoto-Aoki strain (from the Central Animal Facility of State University of Campinas, Campinas, Brazil), aged 12 to 14 weeks and weighing 200-250 g, were used in this study. The animals were housed at a maximum of 4 individuals per cage, kept in a specific-pathogen-free (SPF) room at controlled conditions of light (12-h light/dark cycle) and temperature (22±1°C), and were provided sterilized water and standard rat chow ad libitum. The experimental procedures were approved by the Board of the Care and Use of Experimental Animals (CEUA, Oswaldo Cruz Foundation, license LW-46/13).
Clonidine treatment
SHR aged between 12 and 14 weeks were treated with 0.1 mg·kg-1·day-1 clonidine (Sigma-Aldrich, USA) dissolved in distilled water once per day for 28 days by gavage. The clonidine dose was chosen according to previous studies from our laboratory, showing that these doses were adequate to inhibit central sympathetic activity and also to normalize arterial pressure in hypertensive animals (1919. Estato V, Nascimento A, Antunes B, Gomes F, Coelho L, Rangel R, et al. Cerebral microvascular dysfunction and inflammation are improved by centrally acting antihypertensive drugs in metabolic syndrome. Metab Syndr Relat Disord 2017; 15: 26–35, doi: 10.1089/met.2016.0085.
https://doi.org/10.1089/met.2016.0085...
). The treatment was performed by the introduction of the solution via a stainless-steel gavage needle (cannula diameter, 1.2 mm) attached to a syringe, and all procedures were performed at the same time of day. The normotensive control group consisted of WKY rats treated with vehicle (WKY+VEH) and SHR were divided into two groups of animals (minimum of 10 animals per group), treated with vehicle (SHR+VEH) or clonidine (SHR+CLO).
Systolic blood pressure and heart rate
Systolic arterial pressure (SAP) and heart rate were measured in conscious animals using a computerized tail-cuff plethysmography system, a noninvasive blood pressure acquisition system (Visitech Blood Pressure Analysis System, model BP-200, USA). The rats were adapted to the pre-warmed tail-cuff apparatus for three consecutive days to acclimatize them and to avoid stress during the experimental procedures. Each session consisted of 15 cycles of measurements; the first 5 cycles were acclimation cycles and the data were not used, whereas the following cycles were used for the calculation of mean values of hemodynamic parameters.
Quantification of total leukocytes
Total blood was collected in microtubes containing EDTA (0.5 mL Vacutube, Biocon, Brazil). The quantification of leukocytes was performed by impedance detection using an automated hematological Poch Analyzer 100-iV DIFF (Sysmex, Japan).
Plasma and organ collection and peripheral blood leukocyte isolation
The animals were sacrificed with an anesthetic overdose (urethane 1.5 mg/kg, ip), the blood was collected by cardiac puncture, and the adrenal glands and the lower brainstem were removed for western blot analysis.
Leukocytes were obtained after centrifugation at 350 g at room temperature for 20 min of the whole blood followed by cycles of red blood cell lysis with a solution containing 0.144 M ammonium chloride and 17 mM Tris-HCl.
Catecholamine measurements by ELISA
The measurement of plasma adrenaline and noradrenaline was performed using 2-CAT ELISA Fast TRACK® (LDN Labor Diagnostika Nord GmbH & Co., Germany) according to the manufacturer's protocol. ELISA assay is widely used in experimental studies and accepted in clinical practice. Moreover, it has already been shown that the ELISA assay is accurate, sensitive, specific, and precise in the evaluation of plasma catecholamines (2020. Westermann J, Hubl W, Kaiser N, Salewski L. Simple, rapid and sensitive determination of epinephrine and norepinephrine in urine and plasma by non-competitive enzyme immunoassay, compared with HPLC method. Clin Lab 2002; 48: 61–71.). Linear regression analysis of adrenaline and noradrenaline concentrations measured with ELISA and with HPLC yield highly significant correlations (2020. Westermann J, Hubl W, Kaiser N, Salewski L. Simple, rapid and sensitive determination of epinephrine and norepinephrine in urine and plasma by non-competitive enzyme immunoassay, compared with HPLC method. Clin Lab 2002; 48: 61–71.).
Flow cytometry
Leukocytes (106) were processed for extracellular β2-AR and intracellular TH analysis. For TH evaluation, permeabilization was performed with 0.1% saponin. Nonspecific binding was blocked with inactivated goat serum at 4°C for 30 min and incubation with primary antibody (rabbit anti-tyrosine hydroxylase at 1:10 dilution, ab137869; Abcam, UK) or rabbit anti-receptor β2-adrenergic at dilution of 1:50 (ab137494; Abcam) for 20 min. After washes with phosphate buffered saline (PBS), the incubation with secondary antibody conjugated to Alexa Fluor 488 at 1:1000 dilution (A-21206; Invitrogen, USA) was performed for 20 min. The samples were fixed with 2% PFA, and analysis was carried out in the Cyan ADP Analyzer system (Beckman Coulter, Inc., USA). Biological samples were analyzed in a minimum of three technical replicates. The data were analyzed with Summit software version 4.3 (Beckman Coulter).
Western blot analysis
Proteins were extracted from 100 mg tissue/mL lysis buffer [25 mM Tris-HCl; 150 mM sodium chloride, 1 mM EDTA; 50 mM Na fluoride; 1 mM Na orthovanadate; 1% triton x-100; 1 mM phenylmethylsulfonyl fluoride; 1/100 protease and phosphatase inhibitors cocktail (Sigma-Aldrich)]. The samples were sonicated twice and centrifuged at 12,000 g for 20 min at 4°C and the supernatant was kept frozen at -80°C. Proteins (50 µg/lane) were separated by SDS/PAGE (12%) and analyzed by immunoblotting with specific primary antibodies [rabbit anti-tyrosine hydroxylase at 1:5000 dilution (58 kDa) (ab137869; Abcam) or rabbit anti-receptor β2-adrenergic at 1:2000 dilution (46 kDa) (ab137494; Abcam)]. The immunogen utilized by the antibody manufacturer was a synthetic peptide encompassing a sequence within aa 340-413, which minimizes the cross-reactivity with different subtypes of β-ARs (β1, β2, and β3). For loading control, we used mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 36 kDa) monoclonal antibody (10R-G109a; Fitzgerald Industries, USA). The membranes were incubated with secondary goat anti-mouse IgG HRP-labeled antibody (cat #31430; Thermo Scientific, Inc., USA) or with secondary goat anti-rabbit IgG HRP-labeled antibody (cat #31460; Thermo Scientific, Inc.) at 1:10000 dilution for 1 h at room temperature, followed by incubation with chemiluminescent kit Supersignal (Thermo Scientific, Inc.) and exposition to X-ray film. Biological samples were analyzed in a minimum of three technical replicates. The X-ray film bands densitometry analysis was performed with the software Image Studio Lite version 4.0 (LI-COR Biotechnology - UK Ltd, United Kingdom) and standardized by the GAPDH density. Considering that a unique band should be visible in western blot assays, it was technically impossible to determine β2-AR expression in the adrenal glands, since an extra band of 20 kDa was observed in the adrenal gland samples. Therefore, these data were not included in the results. Moreover, in the leukocytes, the small protein quantity obtained from the samples precluded the assessment of TH and β2-AR by western blot analysis with the available antibodies.
Statistical analysis
Data are reported as means±SD or median (interquartile range). D'Agostino's K-squared test and Pearson's normality tests were used to evaluate normality of the data. Statistical analyses between two groups with normal distribution were performed using a two-tailed unpaired t-test, and if data did not present a normal distribution, a two-tailed Mann-Whitney test was used. One-way ANOVA followed by Tukey's post hoc test was employed in the analyses of more than two groups with normal distribution. Kruskal-Wallis test followed by Dunn's post-test was used in the evaluations among three groups with abnormal distributions. The Pearson correlation coefficient was used to measure the strength of the linear relationship. Differences with P values of less than 0.05 were considered significant. All analyses were performed using the statistical program GraphPad Prism 5 (GraphPad Software Inc., USA).
Results
Blood pressure, heart rate, and plasma catecholamine levels after chronic treatment with clonidine
As expected, hypertensive animals treated with vehicle (SHR+VEH) presented higher systolic blood pressure and heart rate than did normotensive WKY rats (P<0.001 and P<0.05, respectively; n=11-15, Table 1). Moreover, SHR+VEH showed increased plasma levels of adrenaline and noradrenaline compared with the normotensive animals (P<0.05 and P<0.01, respectively) (Table 1, n=11-15 animals/group).
Compared to SHR+VEH, clonidine treatment reduced systolic pressure to values similar to those obtained in the normotensive group of WKY rats. Reduced heart rate was also observed in the clonidine-treated group, compared with both WKY (P<0.0001) and SHR+VEH (P<0.001). We observed significant reductions in plasma noradrenaline and adrenaline levels after 28 days of clonidine treatment compared with SHR+VEH (P<0.01 and P<0.01, respectively, Table 1).
TH and β2-AR levels in adrenal glands and lower brainstem after clonidine treatment
In the adrenal gland, TH showed higher expression in SHR+VEH (0.85±0.17) than in WKY controls (0.56±0.16, P<0.01). Compared with non-treated SHR (0.85±0.17), clonidine treatment in SHR induced a significant reduction in TH levels in the adrenal glands (0.64±0.15, P<0.05) (Figure 1A).
Protein expression levels of tyrosine hydroxylase (TH) in adrenal glands (A) and of TH (B) and β2-adrenergic receptor (β2-AR) expression (C) in the lower brainstem using western blot analysis. GAPDH was used as an endogenous control for the normalization of relative protein expression. Data are reported as means±SD for n=7-13 animals/group. *P<0.05, **P<0.001, and ***P<0.0001 (one-way ANOVA). WKY: normotensive Wistar Kyoto rats; SHR: spontaneously hypertensive rats; VEH: vehicle; CLO: clonidine.
In the brainstem, we found that TH (0.35±0.17) and β2-AR (0.68±0.13) were more highly expressed in the SHR+VEH group than in the normotensive control groups (0.12±0.09, P<0.001 and 0.33±0.19, P<0.001, respectively). Both protein levels in the brainstem were decreased in the clonidine group (0.21±0.14 and 0.44±0.24, respectively) compared with SHR+VEH (0.35±0.17, P<0.05 and 0.68±0.13, P<0.05, respectively) (Figure 1B and C).
Effects of clonidine treatment on the levels of TH and β2-AR in leukocytes
The total leukocyte count was not different among the groups. The WKY group presented 8.03±3.31×103 leukocytes/mm3, SHR+VEH had 8.14±1.95×103 leukocytes/mm3, and SHR+CLO contained 8.72±2.97×103 leukocytes/mm3 (Table 1).
Flow cytometry analysis revealed significant differences in the percentage of positive measures for TH between WKY rats (37.1±11.3%) and SHR+VEH rats (17.1±14.5%, P<0.001) (Figure 2B, n=10 animals/group). Leukocytes from SHR+CLO presented a higher percentage of positive TH cells (31.1±7.9%) than those from SHR+VEH (17.1±14.5%, P<0.05), similar to the level in WKY rats (Figure 2A and B).
Representative histograms of tyrosine hydroxylase (TH) labeling (A). Quantification of the percentage of leukocytes expressing TH (B) and the median fluorescence intensity (MFI) (C) of TH labeling in total leukocytes from WKY, SHR+VEH, and SHR+CLO groups. Data are reported as means±SD for n=10 animals/group. *P <0.05 and ***P<0.0001 (one-way ANOVA). WKY: normotensive Wistar Kyoto rats; SHR: spontaneously hypertensive rats; VEH: vehicle; CLO: clonidine.
We observed that the median fluorescence intensity of TH was higher in the leukocytes of normotensive WKY rats (5.6±2.1) than in the leukocytes of SHR+VEH (2.8±0.7, P<0.001) (Figure 2C).
A lower percentage of leukocytes positive for β2-AR was observed in WKY rats (6.0±3.9%) than in SHR+VEH (25.7±7.4%, P<0.001). Compared with SHR+VEH (25.7±7.4%, P<0.010), clonidine treatment resulted in a reduction in β2-AR-positive measures in leukocytes (15.0±8.1%). However, clonidine treatment induced a higher percentage of β2-AR-positive cells than was observed in normotensive WKY (6.0±3.9%, P<0.05) (Figure 3A and B).
Representative histograms of β2-adrenergic receptor (β2-AR) labeling (A). Quantification of the percent of leukocytes expressing β2-AR (B) and the median fluorescence intensity (MFI) of the receptor (C) labeling in total leukocytes from WKY, SHR+VEH and SHR+CLO. Data are reported as means±SD for n=10 animals/group. *P<0.05, **P<0.01, and ***P<0.0001 (one-way ANOVA). WKY: normotensive Wistar Kyoto rats; SHR: spontaneously hypertensive rats; VEH: animals treated with vehicle; CLO: animals treated with clonidine.
We found that the median fluorescence intensity of β2-AR was lower in WKY leukocytes (0.94±0.30) than in SHR+VEH leukocytes (1.91±0.64, P<0.001) (Figure 3C).
Correlation analysis
The correlations of TH and β2-AR expression in leukocytes with hemodynamic parameters (Figure 4) and catecholamine levels (Figure 5) were evaluated. We observed inverse correlations between the percentage of leukocytes expressing TH with both systolic pressure (r=-0.4-932; P=0.0048) (Figure 4A) and heart rate (r=-0.3821; P=0.0448) (Figure 4B). There was a direct correlation between β2-AR expression in leukocytes and systolic pressure (r=0.7293; P=0.0001) (Figure 4C), which was not observed between receptor expression and heart rate (Figure 4D).
Pearson's correlation of the percentage of leukocytes positive for tyrosine hydroxylase (TH) (A and B) or β2-adrenergic receptor (β2-AR) (C and D) with heart rate and systolic blood pressure.
Pearson's correlation between the percentage of leukocytes expressing tyrosine hydroxylase (TH) (A and B) or β2-adrenergic receptor (C and D) (β2-AR) and plasmatic catecholamine levels.
There was an inverse linear relationship (r=-0.3775; P=0.0397) between the percentage of leucocytes positive for TH and plasma noradrenaline levels evaluated in normotensive and hypertensive animals after long-term treatment with clonidine or vehicle (Figure 5A). There were also statistically significant correlations when we considered only the group of hypertensive animals as follows: percentage of leucocytes positive for TH vs systolic arterial pressure (r=-0.4571 and P=0.0372) and vs heart rate (r=-0.5176 and P=0.0163); percentage of leucocytes positive for β2-AR vs systolic arterial pressure (r=0.6217 and P=0.0412) and heart rate (r=0.4702 and P=0.1230); percentage of leucocytes positive for TH vs plasma noradrenaline concentrations (r=-0.5226 and P=0.0151).
Discussion
It is recognized that sympathetic hyperactivity is a therapeutic target in the management of arterial hypertension (2121. Scheffers IJ, Kroon AA, Schmidli J, Jordan J, Tordoir JJ, Mohaupt MG, et al. Novel baroreflex activation therapy in resistant hypertension: results of a European multi-center feasibility study. J Am Coll Cardiol 2010; 56: 1254–1258, doi: 10.1016/j.jacc.2010.03.089.
https://doi.org/10.1016/j.jacc.2010.03.0...
), not only through the use of drugs such as beta-blockers or centrally acting antihypertensives but also through interventions such as baroreflex activation (2121. Scheffers IJ, Kroon AA, Schmidli J, Jordan J, Tordoir JJ, Mohaupt MG, et al. Novel baroreflex activation therapy in resistant hypertension: results of a European multi-center feasibility study. J Am Coll Cardiol 2010; 56: 1254–1258, doi: 10.1016/j.jacc.2010.03.089.
https://doi.org/10.1016/j.jacc.2010.03.0...
) and renal denervation (2222. Brinkmann J, Heusser K, Schmidt BM, Menne J, Klein G, Bauersachs J, et al. Catheter-based renal nerve ablation and centrally generated sympathetic activity in difficult-to-control hypertensive patients: prospective case series. Hypertension 2012; 60: 1485–1490, doi: 10.1161/HYPERTENSIONAHA.112.201186.
https://doi.org/10.1161/HYPERTENSIONAHA....
). These latter procedures are somewhat recent, and further studies should be conducted to better elucidate the controversies regarding the efficacy and mechanisms of these interventions.
However, the assessment of sympathetic activity in patients is performed using methods with low sensitivity or that are difficult to implement in clinical practice (2323. Grassi G, Esler M. How to assess sympathetic activity in humans. J Hypertens 1999; 17: 719–734, doi: 10.1097/00004872-199917060-00001.
https://doi.org/10.1097/00004872-1999170...
), including plasma and/or urine catecholamine levels, postganglionic sympathetic neural traffic with microneurography, noradrenaline spillover, or heart rate spectral power analyses (2323. Grassi G, Esler M. How to assess sympathetic activity in humans. J Hypertens 1999; 17: 719–734, doi: 10.1097/00004872-199917060-00001.
https://doi.org/10.1097/00004872-1999170...
,2424. Esler M, Lambert G, Jennings G. Regional norepinephrine turnover in human hypertension. Clin Exp Hypertens A 1989; 11: 75–89, doi: 10.3109/10641968909045414.
https://doi.org/10.3109/1064196890904541...
).
Plasma and urine catecholamine measurements are noninvasive and are the most commonly used techniques to evaluate sympathetic activity in clinical practice. However, these methods depend on the levels of secretion, tissue clearance, and re-uptake of catecholamines. For instance, it has already been demonstrated that plasma noradrenaline levels correspond to noradrenaline concentrations released from presynaptic neurons and, consequently, a sympathetic nerve activity marker, only when norepinephrine re-uptake transporter is blocked (2525. Berg T, Walaas SI, Roberg BA, Huynh TT, Jensen J. Plasma norepinephrine in hypertensive rats reflects alpha(2)-adrenoceptor release control only when re-uptake is inhibited. Front Neurol 2012; 3: 160, doi: 10.3389/fneur.2012.00160.
https://doi.org/10.3389/fneur.2012.00160...
). Moreover, it is important to note that catecholamines found in the plasma are also released by other sites than sympathetic nerves and adrenal glands; actually, catecholamines can also be released by platelets (1313. Smith CC, Wilson AP, Prichard BN, Betteridge DJ. Stimulus-induced release of endogenous catecholamines from human washed platelets. Clin Sci (Lond) 1986; 70: 495–500, doi: 10.1042/cs0700495.
https://doi.org/10.1042/cs0700495...
) and endothelial cells (1212. Sorriento D, Santulli G, Del Giudice C, Anastasio A, Trimarco B, Iaccarino G. Endothelial cells are able to synthesize and release catecholamines both in vitro and in vivo. Hypertension 2012; 60: 129–136, doi: 10.1161/HYPERTENSIONAHA.111.189605.
https://doi.org/10.1161/HYPERTENSIONAHA....
). Finally, the plasma concentrations of dopamine have already been shown to be independent of sympathetic activity in cardiovascular diseases, including arterial hypertension (2626. Franco-Morselli R, Elghozi JL, Joly E, Di Giuilio S, Meyer P. Increased plasma adrenaline concentrations in benign essential hypertension. Br Med J 1977; 2: 1251–1254, doi: 10.1136/bmj.2.6097.1251.
https://doi.org/10.1136/bmj.2.6097.1251...
). Moreover, platelet levels of free-catecholamines are not considered to be a marker of increased sympathetic activity in essential hypertension (2727. Kjeldsen SE, Gjesdal K, Leren P, Eide IK. Decreased platelet-free dopamine and unchanged noradrenaline and adrenaline in essential hypertension. Thromb Haemost 1988; 60: 251–254, doi: 10.1055/s-0038-1647040.
https://doi.org/10.1055/s-0038-1647040...
). In general, dopamine is not taken into account in the evaluation of sympathetic overactivity in arterial hypertension, either in experimental models or in the clinical setting. Thus, we estimated that it was not crucial to evaluate plasma levels of dopamine in the present study.
Microneurography is the recording of postganglionic muscle sympathetic nerve activity, usually studying peroneal or brachial nerves. These local evaluations are invasive and complex to perform in the clinical setting (2828. Delius W, Hagbarth KE, Hongell A, Wallin BG. General characteristics of sympathetic activity in human muscle nerves. Acta Physiol Scand 1972; 84: 65–81, doi: 10.1111/j.1748-1716.1972.tb05157.x.
https://doi.org/10.1111/j.1748-1716.1972...
). Another method to evaluate sympathetic activity is the measurement of the radioactive noradrenaline appearance rate in plasma, which was correlated with muscle sympathetic activity (2929. Wallin BG, Esler M, Dorward P, Eisenhofer G, Ferrier C, Westerman R, et al. Simultaneous measurements of cardiac noradrenaline spillover and sympathetic outflow to skeletal muscle in humans. J Physiol 1992; 453: 45–58, doi: 10.1113/jphysiol.1992.sp019217.
https://doi.org/10.1113/jphysiol.1992.sp...
). Nevertheless, the technique includes radioactivity exposure risk and accesses sympathetic nerves of internal organs (2323. Grassi G, Esler M. How to assess sympathetic activity in humans. J Hypertens 1999; 17: 719–734, doi: 10.1097/00004872-199917060-00001.
https://doi.org/10.1097/00004872-1999170...
).
Therefore, it is important to search for new markers of systemic sympathetic activity. In this context, we analyzed the expression of TH and β2-AR in peripheral blood leukocytes and the relationship between the expression of these proteins in the adrenal gland or lower brainstem and systolic blood pressure.
TH appears to be a key component in the development of hypertension in SHR, since higher TH mRNA levels are detected in the adrenal gland and medulla oblongata of SHR than in those of WKY (3030. Kumai T, Tanaka M, Watanabe M, Nakura H, Tateishi T, Kobayashi S. Elevated tyrosine hydroxylase mRNA levels in medulla oblongata of spontaneously hypertensive rats. Brain Res Mol Brain Res 1996; 36: 197–199, doi: 10.1016/0169-328X(95)00296-5.
https://doi.org/10.1016/0169-328X(95)002...
). Moreover, alterations in the ubiquitin-proteasome system in hypothalamic and brainstem neurons from SHR have already been demonstrated, which could explain the higher levels of TH in these animals (3131. Congo Carbajosa NA, Corradi G, Verrilli MA, Guil MJ, Vatta MS, Gironacci MM. Tyrosine hydroxylase is short-term regulated by the ubiquitin-proteasome system in PC12 cells and hypothalamic and brainstem neurons from spontaneously hypertensive rats: possible implications in hypertension. PLoS One 2015; 10: e0116597, doi: 10.1371/journal.pone.0116597.
https://doi.org/10.1371/journal.pone.011...
).
Additionally, β-AR is present in the CNS, and the intracerebroventricular administration of β-AR nonselective antagonists prevents the increase in renal nerve activity induced by stress in SHR (3232. Koepke JP, DiBona GF. Central beta-adrenergic receptors mediate renal nerve activity during stress in conscious spontaneously hypertensive rats. Hypertension 1985; 7: 350–356, doi: 10.1161/01.HYP.7.3.350.
https://doi.org/10.1161/01.HYP.7.3.350...
). In addition, the intracerebroventricular, intrahypothalamic, or intracisternal administration of a β2-AR selective antagonist also prevented the increase in arterial pressure induced by C1 region electrical stimulation (3333. Ward-Routledge C, Marshall P, Marsden CA. Involvement of central alpha- and beta-adrenoceptors in the pressor response to electrical stimulation of the rostral ventrolateral medulla in rats. Br J Pharmacol 1988; 94: 609–619, doi: 10.1111/j.1476-5381.1988.tb11567.x.
https://doi.org/10.1111/j.1476-5381.1988...
).
Our results confirmed the presence of hyperactivity of the SNS in arterial hypertension, as demonstrated by increased TH and β2-AR expression in the lower brainstem and in the adrenal gland of SHR compared with WKY. Additionally, the chronic treatment of the animals with clonidine, a centrally acting sympathoinhibitory drug, resulted in a reduction in the expression of both TH and β2-AR, thus confirming the association between sympathetic activity and the expression of these proteins. Importantly, there was a positive and significant correlation between the expression of these proteins and systolic blood pressure. Finally, hypertensive animals presented higher adrenaline and noradrenaline plasma levels. Taken together, the above-mentioned results could indicate a down-regulation or desensitization of β2-AR in the leukocytes resulting from the chronic elevation of plasma catecholamine levels in the experimental model of primary arterial hypertension used in the present study (SHR). In this context, it has already been demonstrated that chronic catecholamine elevation induces a decrease of the number of plasma membrane β2-AR in lymphocytes. This phenomenon was shown to be reversible upon normalization of plasma catecholamine levels in patients with pheochromocytoma (3434. Cases A, Bono M, Gaya J, Jimenez W, Calls J, Esforzado N, et al. Reversible decrease of surface beta 2-adrenoceptor number and response in lymphocytes of patients with pheochromocytoma. Clin Exp Hypertens 1995; 17: 537–549, doi: 10.3109/10641969509037423.
https://doi.org/10.3109/1064196950903742...
). Nevertheless, in the present work, we did not evaluate function but only expression of the β2-AR in the leukocytes.
Previous studies demonstrated that mononuclear leukocytes synthesize catecholamines and express β2-AR (3535. Josefsson E, Bergquist J, Ekman R, Tarkowski A. Catecholamines are synthesized by mouse lymphocytes and regulate function of these cells by induction of apoptosis. Immunology 1996; 88: 140–146, doi: 10.1046/j.1365-2567.1996.d01-653.x.
https://doi.org/10.1046/j.1365-2567.1996...
). In the present study, we investigated the expression of TH and β2-AR in total leukocytes using flow cytometry, and showed that leukocytes from both WKY and SHR expressed TH and β2-AR. It is important to note that platelets were eliminated from the analysis in the side and forward scatter parameters of the flow cytometry assays by the different sizes of these cells; moreover, circulating endothelial cells are quite rare in peripheral blood, as described previously (3636. Khan SS, Solomon MA, McCoy JP Jr. Detection of circulating endothelial cells and endothelial progenitor cells by flow cytometry. Cytometry B Clin Cytom 2005; 64: 1–8, doi: 10.1002/cyto.b.20040.
https://doi.org/10.1002/cyto.b.20040...
). Thus, both cell types, which are known to release catecholamines, do not appear to have contributed to our results.
Considering the groups of normotensive and hypertensive animals as a single population, we found an inverse correlation for TH expression and a direct correlation for β2-AR expression in leukocytes with systolic blood pressure. There was also a higher number of leukocytes positive for TH in normotensive animals than in hypertensive animals. On the other hand, a lower percentage of leukocytes presented β2-AR in normotensive animals; therefore, higher levels of leukocytes positive for the receptor were present in animals with higher systolic blood pressure values.
The use of biomarkers with a negative correlation with the biological process has been previously proposed (3737. Martin-Ventura JL, Duran MC, Blanco-Colio LM, Meilhac O, Leclercq A, Michel JB, et al. Identification by a differential proteomic approach of heat shock protein 27 as a potential marker of atherosclerosis. Circulation 2004; 110: 2216–2219, doi: 10.1161/01.CIR.0000136814.87170.B1.
https://doi.org/10.1161/01.CIR.000013681...
). In this context, Demler et al. (3838. Demler OV, Pencina MJ, D'Agostino RB Sr. Impact of correlation on predictive ability of biomarkers. Stat Med 2013; 32: 4196–4210, doi: 10.1002/sim.5824.
https://doi.org/10.1002/sim.5824...
) demonstrated that biomarkers with negative correlations present better power for risk prediction, using area under the receiver operating characteristic curve (ROC) as a measure of evaluating the quality of risk prediction.
Our results corroborate data from the literature (1818. Middeke M, Remien J, Block LH, Kirzinger S, Landrock A, Holzgreve H. Beta 2-adrenoceptor density on membranes and on intact mononuclear cells in essential hypertension. Res Exp Med (Berl) 1983; 183: 227–232.), demonstrating that mononuclear cells from hypertensive patients present higher expression of β2-AR than those from normotensive controls. In that study, a significant correlation of β2-AR density in mononuclear cells with mean arterial pressure was also found. This analysis was performed by grouping, as a single population, the data of normotensive patients together with that of hypertensive patients (1818. Middeke M, Remien J, Block LH, Kirzinger S, Landrock A, Holzgreve H. Beta 2-adrenoceptor density on membranes and on intact mononuclear cells in essential hypertension. Res Exp Med (Berl) 1983; 183: 227–232.). In addition to the alterations in β2-AR receptor expression, alterations in α1-adrenergic receptor expression were observed in peripheral blood lymphocytes from patients with essential hypertension and from the animal model of primary hypertension of the SHR (1616. Veglio F, Tayebati SK, Schiavone D, Ricci A, Mulatero P, Bronzetti E, et al. Alpha1-adrenergic receptor subtypes in peripheral blood lymphocytes of essential hypertensives. J Hypertens 2001; 19: 1847–1854, doi: 10.1097/00004872-200110000-00020.
https://doi.org/10.1097/00004872-2001100...
). It was also observed that patients with heart failure and severe depression had higher β-AR activity in mononuclear cells (3939. Redwine LS, Hong S, Rutledge T, Wentworth B, Pung M, Ziegler MG, et al. Leukocyte ss-adrenergic receptor sensitivity and depression severity in patients with heart failure. Psychosom Med 2014; 76: 726–731, doi: 10.1097/PSY.0000000000000119.
https://doi.org/10.1097/PSY.000000000000...
).
Our results on the expression of leukocytes positive for TH and β2-AR did not exclude a possible direct effect of clonidine on these cells. To evaluate this effect, in vitro analyses of leukocyte treatment with clonidine should be performed. However, the incubation of peripheral blood mononuclear cells with 4 μM clonidine did not alter the density of β-AR without distinction between β1- and β2-AR (4040. Zoukos Y, Thomaides T, Pavitt DV, Leonard JP, Cuzner ML, Mathias CJ. Up-regulation of beta-adrenoceptors on circulating mononuclear cells after reduction of central sympathetic outflow by clonidine in normal subjects. Clin Auton Res 1992; 2: 165–170, doi: 10.1007/BF01818957.
https://doi.org/10.1007/BF01818957...
), indicating that clonidine had no direct effect on these cells.
The causes and the biological significance of the relative reduction of TH expression in the peripheral blood leukocytes of hypertensive animals, compared to the normotensive counterpart, remain to be elucidated. This could be related to a counter-regulatory mechanism resulting from the increased plasma levels of catecholamines in hypertensive animals.
In summary, our results demonstrated that the percentage of leukocytes expressing TH and β2-AR was altered in arterial hypertension and can be modulated by central sympathetic inhibition with clonidine treatment, thus suggesting that the expression of these proteins could be candidates for the development of peripheral markers of the central sympathetic activity in primary arterial hypertension.
Acknowledgments
The authors thank CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), FAPERJ (Fundação de Amparo è Pesquisa do Estado do Rio de Janeiro), and the Oswaldo Cruz Foundation (FIOCRUZ), Rio de Janeiro, Brazil for financial support.
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» https://doi.org/10.1055/s-0038-1647040 -
28Delius W, Hagbarth KE, Hongell A, Wallin BG. General characteristics of sympathetic activity in human muscle nerves. Acta Physiol Scand 1972; 84: 65–81, doi: 10.1111/j.1748-1716.1972.tb05157.x.
» https://doi.org/10.1111/j.1748-1716.1972.tb05157.x -
29Wallin BG, Esler M, Dorward P, Eisenhofer G, Ferrier C, Westerman R, et al. Simultaneous measurements of cardiac noradrenaline spillover and sympathetic outflow to skeletal muscle in humans. J Physiol 1992; 453: 45–58, doi: 10.1113/jphysiol.1992.sp019217.
» https://doi.org/10.1113/jphysiol.1992.sp019217 -
30Kumai T, Tanaka M, Watanabe M, Nakura H, Tateishi T, Kobayashi S. Elevated tyrosine hydroxylase mRNA levels in medulla oblongata of spontaneously hypertensive rats. Brain Res Mol Brain Res 1996; 36: 197–199, doi: 10.1016/0169-328X(95)00296-5.
» https://doi.org/10.1016/0169-328X(95)00296-5 -
31Congo Carbajosa NA, Corradi G, Verrilli MA, Guil MJ, Vatta MS, Gironacci MM. Tyrosine hydroxylase is short-term regulated by the ubiquitin-proteasome system in PC12 cells and hypothalamic and brainstem neurons from spontaneously hypertensive rats: possible implications in hypertension. PLoS One 2015; 10: e0116597, doi: 10.1371/journal.pone.0116597.
» https://doi.org/10.1371/journal.pone.0116597 -
32Koepke JP, DiBona GF. Central beta-adrenergic receptors mediate renal nerve activity during stress in conscious spontaneously hypertensive rats. Hypertension 1985; 7: 350–356, doi: 10.1161/01.HYP.7.3.350.
» https://doi.org/10.1161/01.HYP.7.3.350 -
33Ward-Routledge C, Marshall P, Marsden CA. Involvement of central alpha- and beta-adrenoceptors in the pressor response to electrical stimulation of the rostral ventrolateral medulla in rats. Br J Pharmacol 1988; 94: 609–619, doi: 10.1111/j.1476-5381.1988.tb11567.x.
» https://doi.org/10.1111/j.1476-5381.1988.tb11567.x -
34Cases A, Bono M, Gaya J, Jimenez W, Calls J, Esforzado N, et al. Reversible decrease of surface beta 2-adrenoceptor number and response in lymphocytes of patients with pheochromocytoma. Clin Exp Hypertens 1995; 17: 537–549, doi: 10.3109/10641969509037423.
» https://doi.org/10.3109/10641969509037423 -
35Josefsson E, Bergquist J, Ekman R, Tarkowski A. Catecholamines are synthesized by mouse lymphocytes and regulate function of these cells by induction of apoptosis. Immunology 1996; 88: 140–146, doi: 10.1046/j.1365-2567.1996.d01-653.x.
» https://doi.org/10.1046/j.1365-2567.1996.d01-653.x -
36Khan SS, Solomon MA, McCoy JP Jr. Detection of circulating endothelial cells and endothelial progenitor cells by flow cytometry. Cytometry B Clin Cytom 2005; 64: 1–8, doi: 10.1002/cyto.b.20040.
» https://doi.org/10.1002/cyto.b.20040 -
37Martin-Ventura JL, Duran MC, Blanco-Colio LM, Meilhac O, Leclercq A, Michel JB, et al. Identification by a differential proteomic approach of heat shock protein 27 as a potential marker of atherosclerosis. Circulation 2004; 110: 2216–2219, doi: 10.1161/01.CIR.0000136814.87170.B1.
» https://doi.org/10.1161/01.CIR.0000136814.87170.B1 -
38Demler OV, Pencina MJ, D'Agostino RB Sr. Impact of correlation on predictive ability of biomarkers. Stat Med 2013; 32: 4196–4210, doi: 10.1002/sim.5824.
» https://doi.org/10.1002/sim.5824 -
39Redwine LS, Hong S, Rutledge T, Wentworth B, Pung M, Ziegler MG, et al. Leukocyte ss-adrenergic receptor sensitivity and depression severity in patients with heart failure. Psychosom Med 2014; 76: 726–731, doi: 10.1097/PSY.0000000000000119.
» https://doi.org/10.1097/PSY.0000000000000119 -
40Zoukos Y, Thomaides T, Pavitt DV, Leonard JP, Cuzner ML, Mathias CJ. Up-regulation of beta-adrenoceptors on circulating mononuclear cells after reduction of central sympathetic outflow by clonidine in normal subjects. Clin Auton Res 1992; 2: 165–170, doi: 10.1007/BF01818957.
» https://doi.org/10.1007/BF01818957
Publication Dates
-
Publication in this collection
30 Oct 2020 -
Date of issue
2020
History
-
Received
6 Apr 2020 -
Accepted
27 Aug 2020