1 Institute of Physiology, Cardiovascular Research, University of Zürich, 2 Cardiovascular Center, Cardiology, University of Zürich, Switzerland, 3 Department of Clinical Research, Inselspital, University of Bern, Switzerland and 4 Institute of Clinical Pharmacology, University of Mannheim, Germany
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Abstract |
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Methods. GA, a recognized inhibitor of 11ß-HSD2 was supplemented to the drinking water (3 g/l) of Wistar Kyoto rats over a period of 21 days. From day 8 to 21, spironolactone (5.8±0.6 mg/kg/day), darusentan (45.2±6.5 mg/kg/day), or placebo was added to chow (n=7 per group). After the animals were killed, vascular function of isolated renal artery segments was assessed by isometric tension recording.
Results. Relaxation of pre-constricted renal artery segments in response to acetylcholine (10-10 to 10-5 mol/l) was impaired by GA as compared with controls (12±4% vs 98±5% of norepinephrine 3x10-7 mol/l), whereas endothelium independent relaxations were unaffected. Endothelin receptor antagonism improved renovascular endothelium-dependent relaxation (32±4%, P<0.05 vs placebo) whereas endothelium-dependent relaxation was completely normalized by aldosterone receptor antagonism (85±4%, P<0.01 vs placebo).
Conclusions. In GA-induced hypertension, both aldosterone receptor antagonism and endothelin receptor antagonism normalize blood pressure and improve renovascular function and, thus, may represent a new therapeutic approach in cardiovascular disease associated with impaired 11ß-HSD2 activity.
Keywords: endothelin-1; endothelium; glycyrrhizic acid; liquorice; nitric oxide
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Introduction |
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In patients with hypertension, endothelial dysfunction precedes the rise of blood pressure and predisposes to the development of structural vascular changes [7]. The endothelium releases vasoactive mediators such as nitric oxide (NO) and endothelin (ET)-1, both of which are importantly involved in the regulation of vascular tone [8] and structure [9]. Recently, endothelin receptor blockade has been demonstrated to effectively lower blood pressure in patients with mild to moderate hypertension [10]. In patients with heart failure, the aldosterone receptor antagonist spironolactone has recently been shown to increase NO bioavailability and vascular relaxations [11]. As several studies demonstrate beneficial effects of aldosterone receptor antagonism in heart failure [12,13], a renaissance for aldosterone receptor antagonism has begun. Furthermore, there is good body of evidence for a link between aldosterone and the endothelin system: ET-1 stimulates aldosterone secretion from adrenal cells [14] and has been demonstrated to regulate aldosterone production in patients with chronic congestive heart failure [15]. We recently demonstrated that in GA-induced hypertension, spironolactone is capable of normalizing vascular nitrate and ET-1 levels [16].
As renovascular haemodynamics play a pivotal role in the regulation of systemic blood pressure, the aim of the present study was to evaluate the effects of GA-induced hypertension on renovascular endothelial function. Moreover, we sought to compare the effect of aldosterone and endothelin receptor antagonism, respectively, on renovascular function in this model of hypertension.
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Methods |
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Tissue harvesting
Animals were anaesthetized with pentobarbital (50 mg/kg i.p.) after 3 weeks treatment, and blood samples were collected through puncture of the right ventricle. Aorta and renal arteries were removed, dissected free from adherent tissue, and placed immediately into cold (4°C) modified KrebsRinger bicarbonate solution (mmol/l): NaCl 118.6, KCl 4.7, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25.1, EDTA 0.026, and glucose 10.1. Under a microscope (Leica Wild M3C, Leica), renal arteries were cleaned of adherent tissue and cut into rings 3 mm long. Aortic tissue was snap frozen in liquid nitrogen for assessment of tissue ET-1 and nitrate levels.
Organ chamber experiments
Renal artery rings were suspended to fine tungsten stirrups (diameter 50 µM), placed in an organ bath filled with 25 ml Krebs solution and were connected to force transducers (UTC 2, Gould Statham, CA, USA) for isometric tension recording as described before [18]. After an equilibration period of 60 min, the rings were progressively stretched to their optimal passive tension (2.0±0.2 g) (9). Rings were pre-constricted with norepinephrine (NE, 70% of KCl 100 mmol/l) and relaxations to acetylcholine (Ach 10-10 to 10-5 mol/l) or sodium nitroprusside (SNP 10-11 to 10-5 mol/l) were obtained. Relaxations to Ach were assessed with and without pre-incubation of indomethacin (30 min, 10-7 mol/l) and in the presence or absence of the NO synthase (NOS) inhibitor nitro-L-arginine methylester (L-NAME) (pre-incubation for 30 min, 3x10-4 mol/l). In additional experiments, cumulative concentration-response curves to NE (10-10 to 10-4 mol/l) were obtained in quiescent preparations. Additional concentrations of drugs were added when contractions to the previous concentration were stable. All drugs used in the organ bath were obtained from Sigma Chemical Co. (Buchs, Switzerland) apart from ET-1 and big endothelin which were purchased from Novabiochem/Calbiochem AG (La Jolla, CA, USA). After experiments, vessel rings were blotted dry and weighed.
Calculations and statistical analysis
Relaxations to agonists in isolated arteries are given as per cent pre-contraction in rings pre-contracted with NE to 70% of contraction induced by KCl (100 mmol/l). Contractions were expressed as percentage of 100 mmol/l KCl-induced contractions, which were obtained at the beginning of each experiment. Results are presented as mean±SEM. In all experiments, n equals the number of rats per experiment. For statistical analysis, the sensitivity of the vessels to the drugs was expressed as the negative logarithm of the concentration that caused half-maximal relaxation or contraction (pD2). Maximal relaxation (expressed as a percentage of pre-contraction) or contraction was determined for each individual concentrationresponse curve by non-linear regression analysis with the use of MatLab software. For comparison between two values, the unpaired Student's t test or the non-parametric MannWhitney test was used when appropriate. For multiple comparisons, results were analysed by ANOVA followed by Bonferroni's correction [19]. Pearson's correlation coefficients were calculated by linear regression. A value of P<0.05 was considered significant.
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Results |
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SBP and HR
SBP increased on GA feeding and returned to baseline on either aldosterone receptor antagonism or endothelin receptor antagonism (Table 2) while HR was unchanged during the entire study and was comparable among the treatment groups (Table 3
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Contractions to NE and KCl
Concentration-dependent contractions to NE were unaffected by feeding with GA and aldosterone or endothelin receptor antagonism, respectively (Figure 1). Maximum contractions reached 119±4% of KCl in controls vs 121±5% of KCl on GA feeding vs 122±3% of KCl and 125±2% of KCl on treatment with darusentan and spironolactone, respectively. Contractile responses to 100 mM KCl did not differ between the groups (data not shown).
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Endothelium-dependent relaxations
Native endothelium-dependent relaxations
Endothelium-dependent relaxations of renal artery segments to Ach were blunted by GA feeding (Figure 2A). After having reached a maximum relaxation (62±8%) at a concentration of 3x10-7 mol/l Ach, relaxations in GA-fed animals were reduced at higher concentrations of Ach and finally reached 12±4% at the highest Ach concentration, we evaluated (10-5 mol/l). In comparison, control animals exhibited 99±5% relaxation at the highest Ach concentration (P<0.05 vs GA feeding).
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Endothelium-dependent relaxations in the presence of indomethacin
Reduced renovascular relaxation to higher concentrations of Ach was prevented by pre-incubation with indomethacin (65±2% on GA feeding vs 84±6% and 96±7% for darusentan and spironolactone treatment, respectively) (Figure 2B) but differences between the treatment groups were still present (P<0.05 vs controls for darusentan).
Endothelium-dependent relaxations in the presence of L-NAME
Relaxations to Ach were completely blocked by the NOS inhibitor L-NAME and no differences among the treatment groups were detectable (Figure 2C).
Endothelium-dependent relaxations in the presence of superoxide dismutase
Endothelium-dependent relaxations were unaffected by pre-incubation with superoxide dismutase (SOD): 100±5% for controls vs 14±7% on GA feeding and 29±5% vs 78±6% on treatment with darusentan and spironolactone, respectively (all n.s. in comparison to native endothelium-dependent relaxations).
Endothelium-independent relaxations
Endothelium-independent relaxations to SNP were comparable in all groups (Figure 3). Maximum relaxations reached 125±3% in controls vs 119±4% on GA feeding and 131±6% on darusentan treatment vs 125±4% on spironolactone treatment (all n.s. vs controls).
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Discussion |
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Previously it has been shown that 11ß-HSD2 activity, which regulates corticosterone (cortisol in man) access to its receptors and prevents inappropriate occupancy of type 1 mineralocorticoid receptors by glucocorticoids, is decreased in patients with liquorice-induced as well as salt-sensitive hypertension [6,20]. Interestingly, GA leads to competitive inhibition of 11ß-HSD2 activity [21], which we recently could confirm for the present model of hypertension [22]. We provide here the first evidence that the administration of the 11ß-HSD2 inhibitor GA leads to impairment of renovascular endothelium-dependent relaxation. As renovascular relaxations to Ach were completely blocked by L-NAME and were unaffected by SOD, these data are suggestive for an impairment of endothelial release of NO in GA-induced hypertension. It is of note that in higher concentrations of Ach, relaxations of renal arteries were reduced in a concentration-dependent manner. As this lack of appropriate vasodilatation can be prevented by pre-incubation with indomethacin, it is most likely mediated by vasoconstrictive prostanoids [23]. Taken together, hypertension induced by 11ß-HSD2 inhibition may involve not only glucocorticoid and mineralocorticoid receptor-mediated modulation of renal function but also modulation of the renovascular NO system.
To further elucidate the impact of aldosterone and endothelin receptor antagonism on GA-induced endothelial dysfunction, animals were chronically treated with the aldosterone receptor antagonist spironolactone or the endothelin receptor antagonist darusentan, respectively, which both lead to a normalization of blood pressure.
Both compounds, spironolactone and darusentan, exhibited irreversible receptor blocking, whichin accordance with recent findings [16,24]was represented by sustained improvement of endothelial function in isolated vessels in vitro.
The efficacy of spironolactone in the treatment of heart failure and hypertension in particular in states of sodium retention has been described [25]. In this study we provide the first evidence for complete reversibility of 11ß-HSD2 inhibition-induced renovascular changes by chronic treatment with spironolactone. This is in line with the observation that spironolactone was able to normalize vascular nitrate and ET-1 levels in GA-induced hypertension [16]. It furthermore demonstrates a similar improvement of endothelial function in renal artery as compared with aorta [16]. As darusentan improved renovascular endothelium-dependent relaxation only in part, endothelin ETA receptor-linked pathways may contribute, but not exclusively, to account for mediation of effects initiated by mineralocorticoid receptor activation. The normalization of blood pressure on darusentan treatment despite a certain degree of endothelial dysfunction suggests that other mechanisms than endothelial dysfunctionsuch as related to salt and water metabolismmay be considerable contributors to hypertension in this model.
Recent evidence indicates that elevated aldosterone levels play an important role in the development and progression of myocardial fibrosis and hypertrophy in congestive heart failure [13]. Further data support the hypothesis that sodium retention is not the primary mechanism of cortisol-induced hypertension [3]. These findings may be particularly relevant to the present study as the current data suggest that reduced activity of 11ß-HSD2 could represent an important aldosterone-independent mechanism, through which inappropriate access of glucocorticoids to vascular receptors may influence vascular tone. The fact that aldosterone receptor antagonism has recently proven to decrease mortality in severe heart failure [13], emphasizes the importance of this therapeutic principle.
In conclusion, this study for the first time demonstrates that impairment of renovascular endothelium-dependent relaxation is a feature of hypertension induced by the 11ß-HSD2 inhibitor GA, which can be restored by the aldosterone receptor antagonist spironolactone and, at least in part, by the endothelin ETA receptor antagonist darusentan. Therefore, aldosterone and endothelin receptor antagonism may advance as beneficial treatment options in cardiovascular disease associated with reduced activity of 11ß-HSD2.
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Acknowledgments |
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Notes |
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Correspondence and offprint requests to: Thomas F. Lüscher, MD, FRCP, FACC, Professor and Head of Cardiology, University Hospital, CH-8091 Zürich, Switzerland. Email: cardiotfl{at}gmx.ch
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References |
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