Increase in nitric oxide bioavailability improves endothelial function in endothelin-1 transgenic mice

Thomas Quaschning1, Serkan Koçak1, Christian Bauer2, Hans-Hellmut Neumayer3, Jan Galle1 and Berthold Hocher2,3,

1 Department of Medicine, Division of Nephrology, University Hospital of Würzburg, Würzburg, 2 Institute of Molecular Biology and Biochemistry, Free University of Berlin, Berlin and 3 Department of Nephrology, University Hospital Charité, Humboldt University Berlin, Berlin, Germany



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Endothelin-1 (ET-1) has been described as a very potent vasoconstrictor. Nevertheless, transgenic mice overexpressing ET-1 have been shown to exhibit normal blood pressure. We thus hypothesized that vascular ET-1 effects may be antagonized by increased activity of other regulatory systems, such as the increase in bioavailability of the endothelial counterpart of ET-1, nitric oxide (NO).

Methods. Endothelium-dependent and -independent vascular function was assessed as relaxation/contraction of isolated preconstricted aortic rings to acetylcholine (10-10–10-4 mol/l), sodium nitroprusside (10-10–10-4 mol/l), ET-1 (10-10–10-7 mol/l) and big ET-1 (10-10–10-7 mol/l), respectively, in ET-1 transgenic mice and corresponding controls. To unmask the impact of the NO system, we furthermore analysed vessel rings incubated in vitro with the NO-synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME, 10-4 mol/l).

Results. Maximum endothelium-dependent relaxation was enhanced in ET-1 transgenic mice (93±3% vs 84±4% for wild-type littermates; P<0.05) and was inhibited by preincubation with L-NAME in both ET-transgenic mice and wild-type littermates (11±5% vs 9±4% maximum relaxation, respectively). Endothelium-independent relaxation was similar among all groups. Maximum vascular contraction to ET-1 and big ET-1 was reduced in ET-1 transgenic mice (P<0.05 vs wild-type littermates). Preincubation with L-NAME reduced this difference, indicating the involvement of augmented NO availability. Correspondingly, urinary nitrate/nitrite excretion was significantly elevated in ET-1 transgenic mice.

Conclusions. These data suggest that in transgenic mice overexpressing ET-1, increased NO bioavailability counteracts the contractile potency of elevated ET-1 levels and leads to an improvement of endothelium-dependent relaxation. Thus, in the presence of an activated ET system, up-regulation of NO production may be capable of maintaining vascular tone in a normal range and therefore may prevent the development of hypertension.

Keywords: endothelin; endothelium; hypertension; nitric oxide; relaxation



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The intact endothelium produces a variety of vasoactive substances, such as angiotensin II, thromboxane and the 21-amino-acid peptide endothelin-1 (ET-1). Endothelin-1 is a potent vasoconstrictor and mitogen in vivo and in vitro and exerts its biological effects via activation of specific receptors. While on vascular smooth muscle cells the predominant ET receptor is the ETA receptor subtype [1] contributing to long-lasting vasoconstriction, endothelial cells express the ETB receptor mediating the formation of nitric oxide (NO) and prostacyclin [2]. The ETB receptors are known to promote the pulmonary clearance of circulating ET-1 as well as the reuptake of ET-1 by endothelial cells. Correspondingly, intravenous administration of ET-1 causes a rapid and transient vasodilatation followed by a sustained increase in blood pressure. In addition to the negative feedback loop via ETB receptors, ET-1 production in the vascular wall is inhibited by NO and prostacyclin via cGMP-dependent mechanisms. Therefore, the NO system may be considered as the functional counterpart of the ET system [3], thus contributing to the subtle balance of vascular tone in a wide range of conditions and emphasizing the pivotal role of the endothelium as a regulator of vascular tone.

Since in addition to its direct vasoconstrictor effect ET-1 amplifies the contractile response to other vasoactive agents including norepinephrine and serotonin, ET-1 has been considered to play an important role in several animal models of hypertension [4]. The normalization of blood pressure by ET receptor agonists in various forms of experimental hypertension [5] as well as in human essential hypertension [6] supports its impact on the regulation of vascular tone. However, human ET-1 transgenic mice models do not support the concept that the ET system might play an important role in the pathogenesis of hypertension, since these mice are not hypertensive [7,8]. Counter-regulation of ET-1 activity by vasodilatory systems might account for the normotension of these animals.

We thus hypothesized that enhanced vasodilator capacity may antagonize the cardiovascular effects of elevated endogenous ET production. Since counterregulatory interactions between the NO and ET systems are well established [9], we focused on activation of the NO system accompanying activation of the ET system. Indeed, through enhanced vasodilatation, NO could antagonize vascular ET effects.

Thus, in the present study, we evaluated alterations of the NO system in a mouse model with endogenous overproduction of ET-1. By antagonizing the NO production with the NO synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME), we elucidated the impact of NO bioavailability on endothelial function in states of an activated ET system.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Animals
Male heterozygous human ET-1 transgenic mice (line 856) were generated by microinjection using the human prepro-ET-1 expression cassette as previously described [7]. Genotype was confirmed by polymerase chain reaction of genomic DNA using standard techniques [7]. Due to the small number of resulting homozygous ET-1 transgenic mice, heterozygous mice, which were shown to exhibit a similar phenotype as compared with homozygous mice [7], were used for all experiments. Endothelin-1 transgenic mice exhibited marked elevation of ET plasma levels and ET tissue levels in heart, kidney, liver, spleen, lung and brain as well as in vascular smooth-muscle cells [7]. They furthermore presented with an increase in development of interstitial fibrosis and glomerulosclerosis [7,10].

Transgenic animals and their wild-type littermates were kept under controlled environmental conditions with respect to temperature (20°C), humidity (64%) and a 12 h night/day light cycle. They were fed on standard breeding rodent chow and water ad libitum and were used for experiments at the age of 6 months. The study design and the experimental protocols were conducted according to the local institutional guidelines for the care and use of laboratory animals.

Blood pressure and heart rate
Blood pressure and heart rate were measured by the tail-cuff method (Blood Pressure Monitor BMN-1756; Föhr Medical Instruments, Seeheim, Germany) in unanaesthetized mice that underwent 4 days of extensive training to get used to this procedure. Mean values of five subsequent measurements were calculated.

Tissue harvesting
Mice were anaesthetized with pentobarbital (40 mg/kg body weight, intraperitoneal) and were euthanized by cervical dislocation. The aorta was isolated in no-touch technique, removed and placed immediately into cold (4°C) modified Krebs–Ringer bicarbonate solution (118.6 mmol/l NaCl, 4.7 mmol/l KCl, 2.5 mmol/l CaCl2, 1.2 mmol/l MgSO4, 1.2 mmol/l KH2PO4, 25.1 mmol/l NaHCO3, 0.026 mmol/l EDTA and 10.1 mmol/l glucose). Under a microscope, vessels were rinsed with a cannula to remove residual blood cells, cleaned of adherent tissue and cut into rings 3 mm long.

Organ chamber experiments
Aortic rings were mounted on fine tungsten stir-ups (diameter: 50 µm), placed in an organ bath filled with 10 ml modified Krebs–Ringer bicarbonate solution (37°C, pH 7.4, 95% O2/5% CO2) and were connected to force transducers (Föhr Medical Instruments) for isometric tension recording as described before [11]. After an equilibration period of 60 min, the rings were progressively stretched to their optimal passive tension (2.0±0.2 g) as assessed by the response to 100 mmol/l KCl in modified Krebs–Ringer solution. Rings were preconstricted with norepinephrine (to ~70% of KCl 100 mmol/l-induced tension) and relaxations to acetylcholine (ACH; 10-10–10-5 mol/l) or sodium nitroprusside (SNP; 10-11–10-5 mol/l) were obtained. Relaxations to acetylcholine were assessed with and without preincubation of the NO-synthase inhibitor L-NAME (preincubation for 30 min, 10-4 mol/l). In several rings, preincubation with indomethacine (10-5 mol/l, 30 min) was performed. In additional experiments, cumulative concentration–response curves to ET-1 (10-10–10-7 mol/l) or big ET-1 (10-10–10-7 mol/l), respectively, were obtained in quiescent preparations. All chemical substances and drugs used in this study were purchased from Sigma Aldrich Chemical Co. (Munich, Germany), apart from ET-1 and big ET which were purchased from Calbiochem AG (La Jolla, CA, USA).

Urinary nitrite and nitrate excretion
Animals were kept in metabolic cages on standard rodent chow and nitrate-free water ad libitum and urinary excretion was documented. Urine for determination of total nitrite/nitrate (NOx) levels was collected during the 24 h before sacrifice.

The NOx levels, the stable end-products of NO, were measured by the Griess reaction in urinary samples in triplicate as previously described [12]. In brief, the nitrate content of the sample was reduced to nitrite with nitrate reductase. Samples were determined by spectrophotometric analysis at 540 nm. A standard curve was performed in each experiment. The NOx content of the samples was calculated from the standard curve, which was linear within this range.

Calculations and statistical analysis
Relaxations to agonists in isolated vessels are given as percentage precontraction in rings precontracted with norepinephrine to ~70% of contraction induced by KCl (100 mmol/l). The contractions were expressed as a percentage of 100 mmol/l KCl-induced contractions, which were obtained at the beginning of every experiment. Results are presented as means±SEM. In all experiments, n is the number of mice per experiment. If not otherwise indicated, material of six mice was examined in each 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 precontraction) or contraction was determined for each individual concentration–response curve by non-linear regression analysis with the use of MatLab software (Math Works Inc., Natick, MA, USA). For multiple comparisons, results were analysed by ANOVA followed by Bonferroni's correction [13]. Pearson's correlation coefficients were calculated by linear regression when indicated. Urinary NOx levels were compared by chi-square analysis. A P-value of <0.05 was considered significant.



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Systolic blood pressure and heart rate
Systolic blood pressure, as determined by tail-cuff measurements, did not differ between 6-month-old ET-1 transgenic mice and their wild-type littermates (118±5 vs 116±5 mmHg; NS). The heart rate was similar in ET-1 transgenic mice and their wild-type littermates (578±28 vs 586±25 beats per min; NS).

Endothelium-dependent relaxation
In ET-1 transgenic animals, maximum endothelium-dependent relaxation of preconstricted aortic rings to acetylcholine was significantly elevated in comparison to wild-type littermates (93±3% vs 84±4%, P<0.05; Figure 1Go).



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Fig. 1.  Endothelium-dependent relaxation plotted against increment concentrations of acetylcholine in aortic segments of ET-transgenic mice (tg) and their wild-type littermates (wt) with (squares) and without (circles) in vitro preincubation of vessel rings with the NO-synthase inhibitor L-NAME (10-4 mol/l). Results are given as means±SEM (n=6 per group). *P<0.05 vs wild-type littermates. For symbols without error bars the SEM was less than symbol size.

 
In vitro preincubation with L-NAME (10-4 mol/l) almost completely blunted endothelium-dependent relaxation in both ET-1 transgenic mice and their wild-type littermates (11±5% vs 9±4% maximum relaxation, respectively, NS; Figure 1Go). Endothelium-dependent relaxation was not affected by cyclooxygenase inhibition as performed by in vitro preincubation with indomethacine (10-5 mol/l) (data not shown).

Endothelium-independent relaxation
In contrast to endothelium-dependent relaxation, maximal endothelium-independent relaxation to the NO donor SNP was comparable in all groups (Figure 2Go), thus indicating that the NO-dependent intracellular signal transduction pathway was not affected in this model.



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Fig. 2.  Endothelium-independent relaxation plotted against increment concentrations of sodium nitroprusside in aortic segments of ET-transgenic mice (tg) and their wild-type littermates (wt) with (squares) and without (circles) in vitro preincubation of vessel rings with the NO-synthase inhibitor L-NAME (10-4 mol/l). Results are given as means±SEM (n=6 per group). For symbols without error bars the SEM was less than symbol size.

 

Concentration-dependent contraction to ET-1
Maximum contraction to ET-1 was reduced in ET-1 transgenic mice as compared with their wild-type littermates (27±5% vs 45±6% of 100 mmol/l KCl, P<0.05; Figure 3Go). In vitro preincubation of vessel rings with the NO-synthase inhibitor L-NAME (10-4 mol/l) unmasked the effect of basal NO release and therefore revealed marked elevation in maximum contraction to ET-1 as compared with aortic rings without L-NAME preincubation (P<0.05 vs corresponding rings without preincubation; Figure 3Go). In ET-1 transgenic mice, maximum contraction to ET-1 in the presence of L-NAME was slightly but still significantly lower as compared with their wild-type littermates (88±6% vs 110±6% of 100 mmol/l KCl, respectively, P<0.05; Figure 3Go). However, as related to corresponding vessels without L-NAME preincubation, there was a relatively smaller increase of vascular response to ET-1 in wild-type animals in the presence of L-NAME as compared with a >3-fold increase in ET-transgenic animals (244±28% vs 325±32%; P<0.05), disclosing the up-regulation of NO production in ET-1 transgenic mice. The response to ET-1 was not affected by in vitro preincubation with indomethacine (10-5 mol/l).



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Fig. 3.  Concentration-dependent contraction plotted against ET-1 in aortic segments of ET-transgenic mice (tg) and their wild-type littermates (wt) with (squares) and without (circles) in vitro preincubation of vessel rings with the NO-synthase inhibitor L-NAME (10-4 mol/l). Results are given as means±SEM (n=6 per group). *P<0.05 vs wild-type littermates, **P<0.05 vs corresponding rings without L-NAME preincubation. For symbols without error bars the SEM was less than symbol size.

 

Concentration-dependent contraction to big ET-1
Vascular reactivity to ET-1 was paralleled by contractile response to the ET-1 precursor big ET-1, which was reduced in ET-1 transgenic mice as compared with their wild-type littermates (31±4% vs 44±4% of KCl 100 mmol/l with big ET-1 10-7 mol/l; P<0.05).

Urinary nitrite and nitrate excretion
Excretion of the stable end-products of the NO metabolism, nitrite (NO2-) and nitrate (NO3-), in the urine was elevated in ET-transgenic mice as compared to their wild-type littermates (140.8±12.9 vs 84.5±14.9 µg/day, P<0.05; Figure 4Go), thus indicating enhanced NO formation in ET-1 transgenic animals.



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Fig. 4.  Urinary nitrite and nitrate (NOx) excretion of ET-transgenic mice (tg) and their wild-type littermates (wt). Results are given as means±SEM (n=6 per group). *P<0.05 vs wild-type littermates.

 



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The present study demonstrates that in states of an activated ET system, such as in ET-1 transgenic mice with endogenous overexpression of ET-1, the NO bioavailability is markedly elevated. Increase in NO availability contributed to augmented endothelium-dependent relaxation, whereas endothelium-independent relaxation remained unaltered. The activated vascular NO system in ET-1 transgenic mice reduced the contractile response to ET-1 and big ET-1 and was represented by an increase in urinary nitrate/nitrite excretion. Correspondingly, in vitro preincubation with the NO-synthase inhibitor L-NAME blunted endothelium-dependent relaxation and markedly increased contractile response to ET-1.

Enhanced relaxation to acetylcholine in ET-1 transgenic mice indicates that endothelial function in the aorta is altered in ET-1 transgenic mice as compared with non-transgenic littermates. Furthermore, ET-1 transgenic animals exhibited reduced contraction to ET-1 and its precursor big ET-1. Even though it would be conceivable that the reduced contraction to ET-1 in ET-1 transgenic mice may be due to a down-regulation of vascular ETA receptors and/or up-regulation of ETB receptors, we have previously shown that both ETA and ETB receptor mRNAs are expressed to a similar extent in ET-transgenic and wild-type animals [10]. Furthermore, binding assays for ETA/B receptors revealed comparable sensitivity and specificity for ET-1 in ET-1 transgenic mice and their wild-type littermates [10]. In line with the hypothesis of an up-regulated NO production, comparison of the contractile response to ET-1 in aortic segments with L-NAME preincubation to segments without L-NAME preincubation revealed a marked increase of contractility in the presence of L-NAME. This implies that the decreased response to ET-1 in ET-1 transgenic mice seems to be mainly due to the enhanced vascular NO bioavailability but not to differential regulation of ETA/B receptors. Altered endothelial function was seen without any detectable structural alterations of the aorta [7]. We thus suggest that functional rather than structural alterations of the aorta are responsible for this effect. Furthermore, it is remarkable that altered endothelial function occurs although expression of the transgene is low in the aorta of ET-1 transgenic mice [7], indicating that ET-1 is a very powerful peptide hormone within the network of molecules controlling endothelial function.

An interplay between NO and ET-1 in terms of down-regulation of ET-1 production by NO has been demonstrated before. The functional experiments (Figures 1Go–3GoGo) as well as the determination of the daily urinary nitrate/nitrite excretion indicate that in states of an activated ET system, such as in ET-1 transgenic mice, the NO bioavailability is elevated and attenuates the primary pressor effects of ET-1. This might, at least partially, explain the initially unexpected finding of the absence of hypertension in ET-1 transgenic mice [7] given that an intravenous injection of ET-1 causes a sustained elevation of blood pressure. Meanwhile, Japanese researchers [8] produced a comparable transgenic mouse model with animals overexpressing ET-1, which, similar to our model, do not develop hypertension but renal fibrosis.

The role of the ET system in the pathophysiology of hypertension has been summarized previously [14]. Based on evidence derived from animal models [4], and in particular from treatment studies with ET receptor antagonists [5,15], ET-1 is suggested to play a role in some forms of hypertension, especially in human essential and salt-sensitive hypertension [16]. However, it is also important to consider that ET-1 transgenic mice, as shown by direct blood pressure measurements before [7] and confirmed by colleagues in a comparable model [8], are not hypertensive. Furthermore, ET-2 transgenic rats are also normotensive [17], whereas ETA receptor knockout mice [18] as well as ET-1 deficient mice [19] develop hypertension.

These conflicting findings concerning the relationship between an activated ET system and hypertension demonstrate that an activation of the ET system on its own does not cause hypertension. Our data indicate that the primary activation of the ET system in otherwise healthy animals is compensated by counterregulatory processes, such as an increase in NO availability, resulting in a new vascular balance between NO and ET-1. We would thus suggest that in forms of ET-1 related hypertension, as seen in salt sensitive hypertension, hypertension is most likely due to the inability of the vascular wall to activate counterregulatory pathways, such as an augmented NO production in response to an activated ET system. This concept is in line with the observation of an impaired NO synthesis in DOCA salt-hypertensive rats, which are characterized by an activated vascular ET system [20]. However, our hypothesis that in cases of ET-1 dependent forms of hypertension the impaired relationship between the vascular ET-1 and NO system (but not the local ET-1 concentrations alone) is altered, should be verified in further animal models with an activated vascular ET system.

In conclusion, this study demonstrates that in the presence of a primary activated ET system, NO bioavailability is increased and counter-regulates vascular effects of the potent vasoconstrictor ET-1. Compensatory activation of the NO system improves endothelial function and finally may warrant maintenance of normal blood pressure in ET-1 transgenic mice. It appears that the fragile balance between the NO and the ET system is being reset on a higher level in these mice.



   Acknowledgments
 
This work has been supported by grants from the Deutsche Forschungsgemeinschaft (DFG Ho 1665/5-1 and Ga 431/4-1) to B.H. and J.G.



   Notes
 
Correspondence and offprint requests to: Berthold Hocher, MD, University Hospital Charité, Humboldt University Berlin, Division of Nephrology, Schumannstrasse 20–21, D-10098 Berlin, Germany. Email: berthold.hocher{at}charite.de Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 13. 6.02
Accepted in revised form: 9.10.02