Lack of Endothelial Nitric Oxide Synthase Promotes Endothelin-Induced Hypertension: Lessons from Endothelin-1 Transgenic/Endothelial Nitric Oxide Synthase Knockout Mice : Journal of the American Society of Nephrology

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Hemodynamics and Vascular Regulation

Lack of Endothelial Nitric Oxide Synthase Promotes Endothelin-Induced Hypertension

Lessons from Endothelin-1 Transgenic/Endothelial Nitric Oxide Synthase Knockout Mice

Quaschning, Thomas*; Voss, Florian†, ‡; Relle, Katharina‡, §; Kalk, Philipp‡, §; Vignon-Zellweger, Nicolas; Pfab, Thiemo; Bauer, Christian; Theilig, Franziska**; Bachmann, Sebastian**; Kraemer-Guth, Annette*; Wanner, Christoph; Theuring, Franz; Galle, Jan; Hocher, Berthold

Author Information
Journal of the American Society of Nephrology 18(3):p 730-740, March 2007. | DOI: 10.1681/ASN.2006050541
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The intact endothelium produces a variety of vasoactive substances, such as nitric oxide (NO), thromboxane, and the 21–amino acid peptide endothelin-1 (ET-1). ET-1 is a potent vasoconstrictor (1) and mitogen in vivo and in vitro and exerts its biologic effects via activation of specific receptors: Whereas on vascular smooth muscle cells the predominant ET receptor is the ETA receptor (ETAR) subtype (2,3), contributing to long-lasting vasoconstriction, endothelial cells express the ETB receptor (ETBR), mediating the formation of NO (4) and prostacyclin (5) and featuring the pulmonary clearance of circulating ET-1 (6) as well as the reuptake of ET-1 by endothelial cells (7).

Correspondingly, intravenous administration of ET-1 causes a rapid and transient vasodilation followed by a sustained increase in BP (8). In addition to the negative feedback loop via ETBR, ET-1 production in the vascular wall is inhibited by NO (9) and prostacyclin (10) via cGMP-dependent mechanisms. Therefore, the NO system may be considered as the functional counterpart of the ET system (1116), thus warranting 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. ET-1 has been considered to play an important role in several animal models of hypertension (12,14,15)

The normalization of BP by ET receptor agonists in various forms of experimental hypertension (14,17) as well as in human essential hypertension (18) demonstrates its impact on the regulation of vascular tone. However, two independent ET-1 transgenic mouse models do not support the concept that the ET system might play an important role in the pathogenesis of hypertension, because these ET-1–overproducing mice are not hypertensive (19,20). In line with these data is the finding of normal BP in ET-2 transgenic rats (21,22).

Counterregulatory interactions between the NO and ET systems are well established (9,23). Therefore, we suspected that an activation of the NO system accompanied the activation of the ET system. Indeed, through enhanced vasodilation, NO could antagonize vascular ET effects. Consistent with this hypothesis, we were able to demonstrate an increased NO-dependent vasodilation in ET+/+ mice before (24,25). Considering the short half-life of NO, an increase in NO availability most likely will be due to an increase in NO production. Recently, we were able to demonstrate that the inducible NO synthase (iNOS) is upregulated in ET-1 transgenic mice and contributes to BP regulation (26). However, because endothelial nitric oxide synthase (eNOS) is the quantitatively most important source of NO in blood vessels, the eNOS might likewise contribute to the maintenance of normal BP in ET-1 transgenic mice.

To verify this hypothesis, we generated cross-bred mice of ET+/+ mice and eNOS knockout (eNOS−/−) mice. These ET+/+ eNOS−/− mice combined the attributes of ET-1 overexpression and the lack of relevant eNOS-derived NO production. The aim of this study was to elucidate the impact of reduced NO synthesis on BP and endothelial function in states of an activated ET system.

Materials and Methods


Male heterozygous human ET-1 transgenic mice (line 856) were generated by microinjection using the human prepro-ET-1 expression cassette as described previously (19). Genotype was confirmed by PCR of genomic DNA using standard techniques (19). Because of 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 (19), were used for all experiments. ET-1 transgenic mice exhibited elevation of ET tissue levels in kidney, lung, and brain (19). They furthermore presented with an increase in development of interstitial fibrosis and glomerulosclerosis (19,27).

eNOS−/− mice were a gift from Prof. David Rodman and were generated as described previously (28). Female eNOS−/− mice and male homozygous ET-1 transgenic mice (19) were used for breeding.

Transgenic/knockout mice and their wild-type (WT) littermates were kept under controlled environmental conditions with respect to temperature (20°C), humidity (64%), and a 12-h night-day light cycle. Mice were fed on a standard breeding rodent diet and water ad libitum. Study groups were composed as follows: WT (n = 17), ET transgenic mice (ET+/+; n = 18), eNOS−/− mice (eNOS−/−; n = 25), and cross-bred mice (ET+/+eNOS−/−; n = 22). At the age of at least 4 mo, all mice were killed, organs were weighed, and aortas were harvested for histologic studies. In a subset of each group, aortic segments were used for isometric tension recording in an organ chamber under physiologic conditions (see the Organ Chamber Experiments section). Study design and experimental protocols were conducted according to the local institutional guidelines for the care and use of laboratory animals and are in accordance with the American Heart Association guidelines for research animal use.

Blood Pressure

BP was measured by the tail-cuff method (Blood Pressure Monitor BMN-1756; Föhr Medical Instruments, Seeheim, Germany) in unanesthetized mice that underwent 4 d of extensive training to get used to this procedure (24). For every data point, five measurements were performed and mean values of five subsequent measurements were calculated.

Tissue Harvesting

Mice were anesthetized with pentobarbital (40 mg/kg body wt, intraperitoneally) and were killed by cervical dislocation. The aorta was isolated in no-touch technique as described previously (29), removed, and placed immediately into cold (4°C) modified Krebs-Ringer bicarbonate solution (in mmol/L): 118.6 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25.1 NaHCO3, 0.026 EDTA, and 10.1 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 suspended by fine tungsten stir-ups (diameter 50 μM), placed in an organ bath filled with 10 ml of Krebs solution, and connected to force transducers (Föhr Medical Instruments) for isometric tension recording as described previously (30). After an equilibration period of 60 min, aortic rings were progressively stretched to their optimal passive tension (2.0 ± 0.2g) as assessed by the response to 100 mmol/L KCl in modified Krebs solution (31). Rings were preconstricted with norepinephrine (approximately 70% of KCl 100 mmol/L) and relaxations to acetylcholine (10−10 to 10−5 mol/L) or sodium nitroprusside (10−11 to 10−5 mol/L) were acquired. To rule out the impact of cyclooxygenase products, we performed experiments with and without preincubation with 10−5 mol/L diclofenac for 30 min. In additional experiments, cumulative concentration–response curves to ET-1 (10−10 to 10−7 mol/L) were obtained in quiescent preparations and after preincubation with the selective ETBR antagonist BQ-788 (10−5 mol/L) or the selective ETAR antagonist BQ-123 (10−5 mol/L), respectively. All chemical substances and drugs used in this study were purchased from Sigma Aldrich Chemical Co. (Munich, Germany), apart from ET-1, which was purchased from Calbiochem AG (La Jolla, CA), and BQ-123 and BQ-788, which were obtained from A.G. Scientific (San Diego, CA).

Histologic Evaluation

Computer-aided image analysis was performed as recently described (32). Briefly, for histologic evaluation, aortas were embedded in paraffin, cut in 3-μm sections, and submitted to Elastica–van Gieson staining. Quantitative stereology (i.e., intima/media and lumen area of the aortas) was analyzed using a computer-aided image analysis system. In brief, microscopic pictures of aortas in Elastica–van Gieson staining were transferred from the microscope to a PowerMAC via a Hitachi CCD camera. Then the inner and outer perimeters of aortas were measured using ImageJ, an image-processing software (shareware from the National Institutes of Health); afterward area contents of media and lumen were calculated using standard formulas. Data were expressed as media-lumen ratio serving as marker for aortic wall thickening.

Quantitative Real-Time PCR

Total RNA was extracted from snap-frozen aortic tissue using Trizol reagent (Invitrogen 15596-026, Carlsbad, CA). A DNase treatment was performed using the Nucleospin RNA II extraction kit from Mecherey & Nagel (cat. no. 740955). The RNA concentration was determined by absorbance at 260 nm. RNA was reverse-transcribed to cDNA using the Superscript III reverse transcriptase from Invitrogen (18080-044) according to the manufacturer’s protocol. The specific primers for mouse and human ET-1 and the primers for hypoxanthine guanine phosphoribosyl transferase (HPRT) were designed with the online available computer program Primer 3 (33) using as template the sequence from the Ensembl Database (human ET-1 Ensembl Transcript ID ENST00000075232; murine ET-1 Ensembl Transcript ID ENSMUST00000021796; murine HPRT Ensembl Transcript ID ENSMUST00000026723). The specificity of the primers were verified by sequence analysis using the BLAST program. The other primers used were derived from the literature (Table 1). A total of 5 μl of cDNA 10 μg/ml (50 ng) was used as template for the amplification. A total of 0.5 μl of each specific primer (5 pmol), 6.5 μl of water, and 12.5 μl of SYBR Green PCR Master Mix (4309155; Applied Biosystems, Foster City, CA) were added. All spots were performed in triplicate. The PCR was performed on an Mx3000P thermal cycler (Stratagene, La Jolla, CA). Reaction conditions were 95°C for 10 min followed by 40 cycles of the amplification steps (95°C for 15 s, 60°C for 30 s, and 72°C for 45 s). We obtained the relative gene expression (sQ) of ETAR, ETBR, murine endothelin-1, endothelin-converting enzyme (ECE), eNOS, and iNOS for each genotype in comparison with the WT. The gene expression of the human ET in the cross-bred mice was compared with its expression in human ET-1 transgenic animals. The relative quantity (Q) was normalized to the expression of HPRT using the following formula: nQ = {2[minCt(gene of interest) − Ct(gene of interest)]}/{2[minCt(HPRT) − Ct(HPRT)]}. The normalized quantity (nQ) was then normalized to the expression in WT mice using the formula sQ = nQtest/mQWT, where Ct(gene of interest) and Ct(HPRT) represent the threshold cycle for the gene of interest and for HPRT, respectively; minCt(gene of interest) and minCt(HPRT) are the lower Ct values for the gene of interest and HPRT, respectively; nQtest is the normalized quantity in a given genotype, and mQWT is the mean of the nQ in WT mice.

eNOS/iNOS and ETAR/ETBR Western Blot Analysis

Protein Extraction.

Snap-frozen aorta were pulverized with mortar and pestle under liquid nitrogen. The powder was dissolved in 1 ml of RIPA Buffer (50 mM Tris, 150 mM NaCl, 0.5% sodium deoxycholic acid, 0.1% SDS, and 1% Triton X100) supplemented with a proteinase inhibitor cocktail (Complete; Roche Diagnostic 1697498). The samples were vortexed and sonicated for 5 s for five cycles. The samples were incubated for 10 min at room temperature and then centrifuged for 12 min at 13,000 rpm at 4°C. The supernatants were collected and stored at −20°C. The protein concentration was measured using the BCA assay from Pierce (Rockford, IL; cat. no. 23225).

Western Blotting.

Proteins (25 μg) were loaded on an SDS-PAGE gel 10% acrylamide and separated for 2.5 h at 100 V at 4°C. The proteins were transferred onto a nitrocellulose membrane for 25 min (intensity 2 mA/cm2) using semidry transferring equipment.

The unspecific sites were blocked overnight at 4°C with TBS Tween/5% milk. The anti-ETAR (M60 sc33536; Santa Cruz Biotechnology, Santa Cruz, CA), anti-ETBR (M74 sc33538; Santa Cruz Biotechnology), and anti–β-actin (C11 sc1615; Santa Cruz Biotechnology) antibodies were diluted 1:500 in TBS Tween/4% BSA and incubated for 1 h at room temperature. The anti-eNOS antibody (Alexis ALX-210-509) and the anti-iNOS antibody (gift from J. Pfeilschifter, Institute of Pharmacology, University of Frankfurt, Frankfurt, Germany) were diluted 1:500 in TBS Tween/5% milk and incubated overnight at 4°C. The horseradish peroxidase–coupled anti-rabbit Ig secondary antibody (NA934V; Amersham Biosciences, Buckinghamshire, UK; for ETAR, ETBR, eNOS, and iNOS detection) and the horseradish peroxidase–coupled anti-goat Ig secondary antibody (P0449; DakoCytomation, Glostrup, Denmark; for β-actin detection) were diluted 1:4000 and 1:2000 in TBST Tween/4% BSA, respectively, and incubated for 1 h at room temperature. Between each detection, the membrane was stripped by incubation with glycine 25 mM/SDS 1% (pH 2) for 30 min at 50°C. Every incubation was preceded by three washes with TBS Tween for 10 min.

Immunoreactive sites were visualized using a chemiluminescence detection system (ECL Western Blotting Detection Reagents; RPN2109; Amersham Bioscience). Membranes were exposed to autoradiography film (Kodak BioMax Light Film, Rochester, NY; 8194540), and the films were scanned on a Canon scanner (Canoscan 5000F; Canon, Tokyo, Japan). The bands were analyzed using the AlphaEaseFC software (Alpha Innotech, San Leandro, CA) for densitometry.

ET-1 Tissue Levels

Tissue Preparation.

The probes were stored in liquid nitrogen until further analysis. The frozen tissue was powdered in the presence of liquid nitrogen. The powdered samples were suspended and subsequently homogenized using a motor-driven pestle homogenizer in 2 ml of buffer solution at 48°C (0.14 mol/L NaCl, 2.6 mmol/L KCl, 8 mmol/L Na2HPO4, 1.4 mmol/L KH2PO4, and 1% Triton-X 100 [pH 7.4]). The homogenates were centrifuged at 48°C for 60 min at 100,000 × g. The supernatants were retained for ET-1 ELISA. The recovery rate for ET-1 after preparation of tissue ET-1 as described previously was always between 97.5 and 99%.


The commercially available enzyme immunoassays for ET-1 that were suitable for direct measurement of ET-1 in plasma and after tissue preparation were obtained from Biomedic (Vienna, Austria) and were performed as described previously (15,27).

Statistical Analyses

Relaxations to agonists in isolated vessels are given as percentage of precontraction in rings that were precontracted with norepinephrine to approximately 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 equals the number of mice 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 precontraction) or contraction was determined for each individual concentration–response curve by nonlinear regression analysis with the use of MatLab software (Math Works, Natick, MA). For comparison between two values, the unpaired t test or the nonparametric Mann-Whitney test was used when appropriate. For multiple comparisons, results were analyzed by ANOVA followed by Bonferroni correction (34). Pearson correlation coefficients were calculated by linear regression. For quantitative real-time PCR, values were reported as means ± SEM, and differences between groups were determined using the unpaired t test. P < 0.05 was considered significant.


Systolic BP

Systolic BP was different between the study groups (Figure 1): Compared with WT mice both in eNOS−/− mice and in cross-bred ET+/+ eNOS−/−, a significantly elevated systolic BP was observed (117 ± 4 mmHg for eNOS−/− mice versus 94 ± 6 mmHg in WT mice; P < 0.001), whereas in ET +/+ mice, BP was not different from that in WT mice. Furthermore, BP in cross-bred animals with both attributes was significantly enhanced compared with eNOS−/− alone (130 ± 4 mmHg; P < 0.05 versus eNOS−/−).

Body Weight and Organ Weight

Body weight was comparable in all groups (ET+/+ mice 34 ± 1 g; ET+/+ eNOS−/− mice 34 ± 1 g; eNOS−/− mice 37 ± 1 g; WT controls 37 ± 2 g). In addition, weight of right and left kidney, spleen, lungs, and heart did not differ significantly among the four groups.

Endothelium-Dependent Relaxation

In ET+/+ mice, maximum endothelium-dependent relaxation of preconstricted aortic rings to acetylcholine was significantly elevated in comparison with WT littermates (103 ± 6 versus 87 ± 4% of preconstriction with norepinephrine; P < 0.05; Figure 2). Absence of eNOS completely blunted endothelium-dependent relaxation in both ET+/+ eNOS−/− (−4 ± 4% of preconstriction) and eNOS−/− only mice (−3 ± 4% of preconstriction; each P < 0.01 versus WT controls; Figure 2). Preincubation with diclofenac (10−5 mol/L for 30 min) did not significantly influence endothelium-dependent relaxation (data not shown).

Endothelium-Independent Relaxation

In contrast to endothelium-dependent relaxation, maximal endothelium-independent relaxation to the NO donor sodium nitroprusside was comparable in all groups (Figure 3), indicating that the NO-dependent intracellular signal transduction pathway was not affected in this model. Preincubation with diclofenac (10−5 mol/L for 30 min) did not significantly alter endothelium-independent relaxation (data not shown).

Concentration-Dependent Contraction to ET-1

Maximum contraction to ET-1 was reduced in ET+/+ mice as compared with their WT littermates (30 ± 4 versus 87 ± 8% of 100 mmol/L KCl; P < 0.05; Figure 4). Reduction of ET-1–mediated contraction in ET+/+ eNOS−/− mice was similar (38 ± 5% of 100 mmol/L KCl; P < 0.05 versus WT littermates; Figure 4). Contraction to ET-1 in eNOS−/− mice (90 ± 8% of 100 mmol/L KCl; Figure 4) did not differ from WT controls.

In vitro preincubation of vessel rings with the selective ETAR antagonist BQ-123 (10−5 mol/L) completely blunted contractions to ET-1 in all groups (Figure 5A). In contrast, preincubation of vessel rings with the selective ETBR antagonist BQ-788 (10−5 mol/L) did not significantly affect contractions to ET-1 (Figure 5B).

Histology Results

Media-lumen ratio of aorta was assessed using computer-aided histomorphometry devices: Compared with WT controls, a significantly decreased media-lumen ratio was observed in cross-bred mice, whereas eNOS−/− mice did not differ from controls, and in ET+/+ mice, a trend (P = 0.07) toward higher values was observed. Within the transgenic groups, cross-bred mice exhibited significantly lower media-lumen ratio when compared with both ET+/+ and eNOS−/− mice (Figure 6). When absolute lumen area was compared between the groups, ET+/+ mice (18.8 ± 1.8) exhibited a significantly smaller lumen area than ET+/+eNOS−/− mice (31.9 ± 4.0) or eNOS−/− mice (23.0 ± 1.3) and a trend (P = 0.07) toward lower lumen area compared with WT mice (25.5 ± 3.2).

mRNA Expression

mRNA expression of endogenous murine ET as well as transgenic human ET, ETAR, ETBR, ECE, iNOS, and eNOS was assessed in aortic tissue using quantitative real-time PCR. Results are illustrated in Table 2. Whereas no significant differences regarding all of these parameters between ET+/+ and WT mice were detected, both groups that carried an eNOS knockout (eNOS−/− and ET+/+eNOS−/−) exhibited a significantly enhanced iNOS mRNA expression when compared with WT mice. In cross-bred mice, expression of all parameters related to the endothelin system was markedly upregulated when compared with WT controls: Endogenous murine ET as well as transgenic human ET (compared with ET+/+ mice), ETA, ETB, and ECE (Table 2).

ETA, ETB, iNOS, and eNOS Western Blot Analysis

In ET transgenic mice, ETAR protein was not significantly altered as compared with WT mice (0.79 ± 0.17-fold versus WT; NS). In eNOS−/− mice, ETAR expression was reduced (0.27 ± 0.7-fold versus WT; P < 0.005), whereas in combined ET+/+ eNOS−/− mice, ETAR protein expression was significantly increased (1.61 ± 0.18-fold versus WT; P < 0.05; Figure 7A). In addition, ETBR was markedly overexpressed at the protein level in ET+/+ eNOS−/− mice as compared with WT mice (3.05 ± 0.75-fold; P < 0.05; Figure 7B).

In ET-1 transgenic mice, iNOS protein level was significantly increased (4.68 ± 0.82-fold versus WT; P < 0.05; Figure 7C). Knockout of eNOS did not significantly alter iNOS protein expression (1.32 ± 0.29-fold in eNOS−/− mice versus WT [NS]; 2.58 ± 0.88-fold in ET+/+ eNOS−/− mice versus WT [NS]; Figure 7C). eNOS Western blotting confirmed absence of eNOS protein in eNOS−/− mice, whereas eNOS protein expression in ET+/+ mice was not significantly altered (1.67 ± 0.22; NS; Figure 7D).

ET-1 Tissue Levels

ET-1 concentration in aortic tissue as measured by ELISA was not significantly different among the four groups, independent of whether it referred to wet weight or to milligrams of protein in aortic tissue (Table 3).


In this study, we evaluated the relevance of eNOS activity in states of overproduction of ET-1. We generated cross-bred mice of ET+/+ and eNOS−/− mice. Systolic BP was similar in ET+/+ and WT mice but was significantly elevated in eNOS−/− mice and even more elevated in ET+/+ eNOS−/− cross-bred mice. Endothelium-dependent relaxation was enhanced in ET+/+ mice and was completely blunted in eNOS−/− and ET+/+ eNOS−/− mice, respectively. Therefore, in transgenic mice that overexpressed ET-1, additional knockout of eNOS results in a further enhancement of BP and represents a novel model of hypertension as a result of an imbalance between the vascular ET-1 and NO systems.

An increase in endothelium-dependent relaxation in the aorta of ET+/+ mice has been described for this model before (24), whereas Amiri et al. (35) demonstrated reduced endothelium-dependent relaxation in the resistance vessels in ET-1 overproduction. It is of note that ET-1 concentrations in aortic tissue were not elevated. This is in accordance with a previous description of the model (19), which demonstrated an increase of ET levels in several organ tissues but not in the aorta or in resistance vessels. In our model, ET-1 is mainly expressed in the lung, brain, and kidney (19), whereas Amiri et al. (35) induced ET-1 overproduction selectively in endothelial cells. In any case, our model represents a new model of an imbalance between the aortic ET and NO systems. ET-1 remains constant; the ETAR and ETBR are upregulated, whereas eNOS is not expressed.

Aortic rings from ET+/+ eNOS−/− mice exhibit a similar response to exogenously added ET-1 (10−10 to 10−7 mol/L) alone or in the presence of 10−5 mol/L BQ123 or 10−5 mol/L BQ788, respectively, as compared with pure ET+/+ mice. The response to acetylcholine (10−10 to 10−4 mol/L), however, was completely different.

The direct vascular effects, at least in aortic vessel segments, seem to be mainly mediated by the ETAR (Figure 5). These novel findings represent the proof of concept of our earlier work that suggested that the interaction between the vascular ET and NO systems is critical for BP regulation under the condition of an activated ET system (22,28). In this study, we demonstrate for the first time that a lack of eNOS in the aorta of ET transgenic mice leads to an increase in both vascular ETAR and ETBR expression, resulting in an impaired vascular relationship between the ET and NO systems. As a consequence, BP in these ET+/+ eNOS−/− mice was markedly elevated.

Our experiments concerning aortic contraction by exogenously administered ET-1 can be summarized in two qualitative results: First, contraction in all of our mouse models is ETA dependent, and ETB does not play a contractile role in our setting as indicated by absence of contraction in all mouse models in the presence of an ETA antagonist. Second, the aortic contraction response to exogenously administered ET-1 of both groups that carry the ET transgene seems to be decreased, which is possibly due to occupation of the receptors by endogenous ET-1 in those groups. This finding corresponds well with the absence of enhanced ET-1 levels in the supernatant of aortic tissue in those two groups, because the amount of receptor-bound ET-1 possibly escapes detection by this method. However, we suggest not extending interpretation of contraction experiments from a qualitative to a quantitative level (i.e., anticipating that different levels of ETAR between our groups as indicated by Western blot analysis are automatically translated into corresponding differences in contraction responses). It is widely known that corresponding to differences between mRNA and protein levels, differences between receptor expression and receptor-related response also exist (36,37). To illustrate the complex pathway between ET-1 and its contractile response, it is of note that the latter is in part mediated by protein kinase C (PKC) (38) and can be abolished by experimental downregulation of PKC (39).

Because media-lumen ratio was reduced rather than elevated in hypertensive ET+/+ eNOS−/− mice, there is obviously no linear relationship between hypertension and vascular remodeling in this model. This may at least in part be due to the type of vessel that was used for histomorphometry: Hypertrophy as a result of hypertension is mainly described for small-resistance vessels rather than for the aorta. It is of note that on the other side, vascular hypertrophy in the absence of hypertension has been described in states of ET-1 overexpression (35).

Our results on the mRNA level indicating a stimulation of the ET system in ET+/+ eNOS−/− mice are confirmed to a large extent by investigation on the protein level: We demonstrated a significant increase of ETAR and ETBR protein levels in aortic tissue of ET+/+ eNOS−/− mice by Western blot analysis, and likewise we confirmed the absence of eNOS in both eNOS knockout groups. However, because ET-1 protein levels in aortic tissue as measured by ELISA were not different between the groups, the increase of ET-1 mRNA expression in ET+/+ eNOS−/− mice has not been detected on the protein level.

This discrepancy between the mRNA and the protein level for ET-1 is in line with literature describing a differential regulation of ET-1 mRNA and protein levels (4042) as a result of regulation of ET-1 biosynthesis by ECE and other factors apart from gene transcription. Furthermore, one has to bear in mind that the ETBR was upregulated in ET+/+eNOS−/− mice. It is widely known that the ETBR acts as a clearance receptor for ET-1; therefore, regulation or pharmacologic intervention at the ETBR was shown to have an impact on ET-1 levels (6,7,43,44). Furthermore ET-1 is known to exhibit high affinity to its receptors. This may result on one hand in a reduced contractile response to exogenously added ET-1 (as demonstrated in Figures 4 and 5B) because many ETAR still may be occupied. On the other hand, high receptor affinity may at least in part account for the absence of elevated free ET-1 in aortic tissue as detected by ELISA of the supernatant of aortic tissue homogenates (Table 3).

Regarding iNOS mRNA and protein levels, we also observed a discrepancy between mRNA and protein levels: iNOS mRNA was upregulated in both eNOS knockout groups, whereas on the protein level, the ET+/+ group displayed enhanced iNOS expression only. These findings may also be attributed to the fact that proteins are subjected to regulations apart from gene transcription; for example, iNOS protein levels depend on degradation by the ubiquitin-proteasome pathway (45), which is known to be altered in cardiovascular diseases such as cardiac hypertrophy (46) and atherosclerosis (47). Furthermore, our findings of an upregulation of iNOS on the protein level in ET+/+ mice are supported by previous studies of our group indicating a vital role for iNOS in counterregulating the effects of ET-1 overexpression in ET+/+ mice (26).

Apart from the direct interplay between NO and ET-1, other adaptive mechanisms may be involved in our model. By preincubation of aortic rings with diclofenac, we were able to exclude significant effects of prostaglandins on vasoreactivity. Furthermore, it is known that the endothelium-derived hyperpolarizing factor plays an important role in the relaxation of resistance vessels and may contribute significantly, particularly in the absence of eNOS, to endothelium-dependent vasodilation (48,49). In contrast to resistance vessels, endothelium-derived hyperpolarizing factor does not contribute to relevant vasodilation in the aorta of mice (48). Nevertheless, other mediators may also be involved in adaptive mechanisms; their potential contribution was not evaluated in this study.


This study demonstrates for the first time that in the presence of an activated ET system, additional knockout of eNOS results in further activation of the endothelin production and elevation of BP. The human ET+/+ eNOS−/− mice therefore represent a novel model of hypertension as a result of an imbalance between the vascular ET-1 and NO systems.



Figure 1:
Systolic BP of endothelin (ET) transgenic mice (ET+/+; n = 18), endothelial nitric oxide synthase (eNOS) knockout mice (eNOS−/−; n = 25), cross-bred mice with both attributes (ET+/+ eNOS−/−; n = 22), and their wild-type (WT) littermates (n = 16) before tissue harvesting. Results are means ± SEM. *P < 0.001 versus WT mice and ET+/+ groups; **P < 0.05 versus eNOS−/− mice.
Figure 2:
Endothelium-dependent relaxation to acetylcholine in aortic segments of 12-wk-old ET+/+ mice, eNOS−/− mice, ET+/+ eNOS−/− mice, and their WT littermates. Results are means ± SEM (n = 6 per group). *P < 0.01 versus WT mice; **P < 0.05 versus WT mice.
Figure 3:
Endothelium-independent relaxation to sodium nitroprusside in aortic segments of 12-wk-old ET+/+ mice, eNOS−/− mice, ET+/+ eNOS−/− mice, and their WT littermates. Results are means ± SEM (n = 6 per group).
Figure 4:
Contraction to endothelin-1 in aortic segments of 12-wk-old ET+/+ mice, eNOS−/− mice, ET+/+ eNOS−/− mice, and their WT littermates. Results are means ± SEM (n = 6 per group). *P < 0.05 versus WT mice.
Figure 5:
(A) Contraction to ET-1 in aortic segments of 12-wk-old ET+/+ mice, eNOS−/− mice, ET+/+ eNOS−/− mice, and their WT littermates. Preincubation with the selective ETA receptor antagonist BQ123 10−5 mol/L. Results are means ± SEM (n = 6 per group). (B) Contraction to ET-1 in aortic segments of 12-wk-old ET+/+ mice, eNOS−/− mice, ET+/+ eNOS−/− mice, and their WT littermates. Preincubation with the selective ETB receptor (ETBR) antagonist BQ788 10−5 mol/L. Results are means ± SEM (n = 6 per group). *P < 0.05 versus WT mice.
Figure 6:
(A) Aortic media-lumen ratio of ET+/+ mice (n = 15), eNOS−/− mice (n = 23), ET+/+ eNOS−/− mice (n = 18), and their WT littermates (n = 14). *P < 0.05 versus WT mice; **P < 0.05 versus eNOS−/− mice; P < 0.05 versus ET+/+ mice; § P < 0.001 versus ET+/+ mice. (B) Typical aortic sections in Elastica–Van Gieson staining. (A) WT mice. (B) ET+/+ mice. (C) eNOS−/− mice. (D) ET+/+ eNOS−/− mice.
Figure 7:
Western Blot analysis of ETA (A), ETB (B), iNOS (C), and eNOS (D) in aortic tissue from WT, ET+/+, eNOS−/−, and ET+/+ eNOS−/− mice. Densitometric analysis is shown normalized to the average expression in WT aortas and presented as means ± SEM (n = 4 to 6). *P < 0.05, **P < 0.005 versus WT.
Table 1:
PCR primersa
Table 2:
mRNA levels in aortic tissuea
Table 3:
ET-1 in aortic tissuea

This work was supported by grants from the Deutsche Forschungsgemeinschaft to B.H. (DFG Ho 1665/5-1 and Ho 1665/5-2). P.K. was supported by a grant from the Dr.-Werner-Jackstaedt-Stiftung. This study was also partially supported by the European Commission via the Marie Curie Host Fellowship for Early Stage Reasearch Training (MEST-CT-2005-020268).

The assistance of Jörg Isensee concerning quantitative real-time PCR data processing and the expert advice of Dr. Heiko Funke-Kaiser concerning regulation of gene expression are gratefully acknowledged.

Published online ahead of print. Publication date available at

T.Q. and F.V. contributed equally to this work.


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