Heterozygous Knock-Out of ETB Receptors Induces BQ-123–Sensitive Hypertension in the Mouse
Abstract—Homozygous knock-out of ETA or ETB receptor genes results in lethal developmental phenotypes in the mouse. Such deleterious phenotypes do not occur in heterozygous littermates. However, it remains to be determined whether mice partially defective in ETA or ETB receptors display significant alterations in their responses to exogenous or endogenous endothelin-1 (ET-1). Furthermore, the anesthetized ETB (+/−) knock-out mice showed a significantly higher mean arterial blood pressure than the ETA (+/−) knock-out or their wild-type littermates. The pressor response to ET-1 but not to a selective ETB agonist, IRL-1620, was significantly reduced in the ETA (+/−) knock-out mice. In ETB (+/−) knock-out mice, the pressor effect of IRL-1620 was more markedly altered than those induced by ET-1. In wild-type mice, both ETA and ETB receptors were found to be involved in the pressor effect of ET-1, as confirmed by the significant and specific antagonism induced by either BQ-123 (ETA antagonist) or BQ-788 (ETB antagonist). Also, ETA-selective or mixed ETA/ETB- but not ETB-selective antagonists reversed the hypertensive state of the ETB (+/−) knock-out mice to the level of wild-type littermates. Finally, radiolabeled ET-1 plasmatic clearance was altered in ETB (+/−) but not ETA (+/−) knock-out mice when compared with wild-type animals. Thus, heterozygous knock-out of ETB receptors results in a hypertensive state, suggesting an important physiological role for that particular receptorial entity in opposing the endogenous ET-1–dependent pressor effects in the mouse.
The ETA or ETB receptor homozygous (−/−) knock-out (KO) mice show lethal embryonic defects and a deleterious megacolon phenotype, respectively.1 2 Unlike endothelin receptors, the total deletion of the B2 receptor for bradykinin or AT-1 receptor subtypes for angiotensin II does not induce short-term lethal effects in these genetically modified animals,3 4 albeit the former is sensitive to high salt diets.5 Furthermore, heterozygous knock-out of the B2 or AT-1 receptor does not significantly affect the vasoactive response to bradykinin or angiotensin II (Ang II), respectively,5 6 in the murine model.
Unexpectedly, the heterozygous knock-out of endothelin-1 (ET-1) induced a paradoxical mild yet significant elevation of basal mean arterial blood pressure (MAP) in these animals when compared with wild-type (WT) congeners.7 This hypertensive state may be caused by the adaptation of the ET-1 (+/−) KO mouse through enhanced central and/or peripheral sympathetic influences on the cardiovascular function.8 Whether the same hypertensive state as in ET-1 (+/−) KO occurs after heterozygous knock-out of ETA or ETB receptors remains to be reported.
In the present study, we have therefore explored whether a partial defect in endothelin receptors, as in heterozygous ETA or ETB KO mice, would be sufficient to induce significant phenotypic alterations in the cardiovascular pharmacology of exogenous and endogenous ET-1. However, it was first required to fully identify the respective contribution of the ETA and/or ETB receptors in the vasoactive effects of endothelins in the WT littermates. We have therefore attempted in this study to characterize the pharmacodynamic characteristics of endothelin in the systemic circulation of the WT mouse by the use of the selective ETA antagonist, BQ-123,9 the selective ETB agonist, IRL-1620,10 and antagonist, BQ-788,11 as well as the mixed ETA/ETB antagonist, SB 209670.12 We have also attempted to demonstrate the respective contribution of ETA or ETB receptor types as well as that of endogenous ET-1 in the regulation of blood pressure in both strains of anesthetized KO mice.
Finally, ETB receptors have been reported to be involved in the clearance of endogenous endothelin.13 The effect of heterozygous knock-out of the ETB receptor on the clearance of radiolabeled ET-1 has been analyzed in this report.
Animals Used for In Vivo Studies
C57Bl/6×J129sv WT mice (weighing 25 to 35 g, either sex) served as controls. Also, C57Bl/6×J129sv ETA or ETB (+/−) KO (25 to 35 g, either sex) mice were used. Colonies for each strain of mice (WT or KO) were developed from heterozygous [ETA (+/−) or ETB (+/−) KO] genitor couples that were derived from previously characterized colonies in the laboratory of Dr Masashi Yanagisawa.
In Vivo Experiments
The mice were anesthetized with ketamine/xylazine (74/9.3 mg/kg IP, supplemented as needed). Polyethylene catheters (PE-10) were inserted into the left external jugular vein for drug administration and in the right carotid artery to monitor MAP and heart rate, according to a previously reported method14 ; a cannula (PE-50) was also inserted into the trachea to facilitate breathing. After surgery, the anesthetized animals were allowed to stabilize for 15 to 20 minutes. The pharmacological responses to the various agonists were assessed for ≥20 minutes after the administration of a single bolus dose ranging from 0.01 to 2.5 nmol/kg (ET-1 and Suc-[Glu,9 Ala11 15 ]-ET-1 [8-21] [IRL-1620]) or 5 nmol/kg (norepinephrine, NE); injection volumes never exceeded 40 μL.
Effects of Endothelin Antagonists on ET-1– or IRL-1620–Induced Pressor Response and on Basal MAP in Mice
For some experiments, BQ-123 or BQ-788 (0.01, 0.1, 0.25, and 1 mg/kg) were administered intravenously 5 minutes before a single bolus injection of either agonist. In another series of experiments, the direct hemodynamic effects of BQ-123 (1 and 10 mg/kg IP), BQ-788 (10 mg/kg IP), or the metabolically stable ETA/ETB antagonist SB 209670 (10 mg/kg IP) were monitored for 90 minutes in WT or ETB (+/−) KO mice.
Plasmatic Clearance Studies
In a first series of experiments, radiolabeled [125I]-ET-1 (0.073 pmol/mouse, subthreshold dose) or [125I]-Ang II (0.11 pmol/mouse, subthreshold dose) were injected in the left jugular vein of WT, ETA, or ETB (+/−) KO mice. Simultaneously, blood samples were collected through a cannulated carotid artery at 3-second time intervals for 2 minutes. Subsequently, radioactivity in each blood sample was measured (counts per minute) with a γ-emission counter (1470 Wizard Gamma Counter Wallac).
In a second series of experiments, BQ-123 (1 mg/kg) or BQ-788 (1 mg/kg) was administered through the left jugular vein of WT mice 5 minutes before the injection of radiolabeled [125I]-ET-1 (0.073 pmol/mouse). Blood samples were collected and radioactivity levels were measured as described above.
ET-1 and IRL-1620 were purchased from American Peptide Co. BQ-123 was synthesized in our laboratory. BQ-788 was purchased from Peptides International. NE was purchased from Sigma. SB 209670 was a generous gift from SmithKline Beecham. [125I]-ET-1 and [125I]-Ang II were purchased from Peninsula Laboratories. All agents were prepared and administered in PBS (pH 7.4, Sigma), except for BQ-123 and BQ-788, which were first dissolved in PBS+20% dimethylsulfoxide (DMSO) to obtain 10 mg/mL stock solutions. DMSO was used because these antagonists are insoluble in aqueous solution at that concentration. [125I]-ET-1 and [125I]-Ang II were dissolved in distilled water. Finally, NE was prepared in an ascorbic acid solution (Baker).
Data used in the text and figures are expressed as mean±SEM of the number of observations. Student’s t or Mann-Whitney U tests (when recommended by the Statistical Program Graph Pad Instat) were used for parametric grouped data. Probability values of ≤0.05 were considered significant.
The apparent affinities (ED50) for ET-1 or IRL-1620 were calculated by linear regression analysis of the full dose-response curve for in vivo experiments. The apparent affinities for antagonists (ID50) were obtained by monitoring the response of ET-1 or IRL-1620 (at doses generally selected within the ED50 range) in the presence of increasing doses (0.01 to 1 mg/kg) of either BQ-123 or BQ-788. ED50 or ID50 values were interpolated by linear regression of the dose-response curve in a 0% to 100% limit (no values rejected on the curve) with the Quattro Pro Program for Windows (Version 5.00). Furthermore, maximal responses (Emax) were systematically attained for all peptidic agonists.
The care of the animals and all research protocols conform to the guiding principles for animal experimentation as enunciated by the Canadian Council on Animal Care and approved by the Ethics Committee on Animal Research of the Université de Sherbrooke Medical School.
The basal MAP (in mm Hg) of mice under ketamine/xylazine anesthesia was averaged for WT (70.1±0.7, male; 70.2±0.6, female, n=118), for ETA (+/−) KO (71.6±1.7, male; 69.7±1.5, female, n=23), or for ETB (+/−) KO (93.2±1.1, male; 91.9±2.1, female, n=53), the latter strain showing a significantly higher blood pressure (P<0.05) when compared with ETA (+/−) KO or WT animals. No gender-dependent differences in MAP were depicted. Furthermore, basal heart rate was not significantly different between WT (164.9±11.9 bpm, n=14), and ETA (+/−) KO (154.8±14.9 bpm, n=10) or in ETB (+/−) KO mice (180.2±18.3 bpm, n=12) (data not shown).
Effects of ET-1 and IRL-1620 on MAP of Mouse in Presence or Absence of ETA- or ETB-Selective Antagonists
ET-1 (0.1 nmol/kg) was more efficient to increase MAP (25.5±1.2 mm Hg) than IRL-1620 (0.1 nmol/kg, 12.1±1.0; 0.5 nmol/kg, 21.3±2.2 mm Hg). A 5-minute treatment with BQ-123 or BQ-788 (0.01 to 1 mg/kg IV, 5 minutes) dose-dependently reduced ET-1–induced vasopressor response (ID50 for BQ-123, 0.22 mg/kg; for BQ-788, 0.22 mg/kg) (Figure 1⇓, A and B). The IRL-1620–induced pressor response was also dose-dependently reduced by BQ-788 (ID50, 0.10 mg/kg) but not by BQ-123 (Figure 1⇓, A and B). In contrast, NE-induced increase in MAP was not altered by treatment with BQ-123 or BQ-788 even at the highest dose of antagonists (1 mg/kg) (NE before BQ-123 or BQ-788: 30.4±1.7 mm Hg; after BQ-123, 35±3.2 mm Hg; after BQ-788, 33.6±3.5 mm Hg) (data not shown). In another series of experiments, BQ-123 and BQ-788 showed the same apparent affinities against ET-1 in ETA (+/−) KO (0.22 mg/kg) and ETB (+/−) KO (0.21 mg/kg) mice, respectively, when compared with WT littermates (Figure 1⇓, A and B).
Treatment With ETA-Selective Antagonist Reverses Hypertensive State of ETB (+/−) KO Mice
Intravenous administration of the highest dose of BQ-123 or BQ-788 (10 mg/kg each) was avoided because of a significant depressor effect of the vehicle (PBS+DMSO, 20%). In contrast, the same vehicle did not significantly affect MAP when administered intraperitoneally in WT mice (0 minutes, 72.1±4.1 mm Hg; 10 minutes, 69.1±3.5 mm Hg; 30 minutes, 69.1±3.2 mm Hg; and 90 minutes, 71.7±3.6 mm Hg after administration). Furthermore, BQ-123 and BQ-788 (1 mg IP) significantly reduced the pressor response to ET-1 (0.1 nmol/kg) (control, 25.8±1.2; +BQ-123, 12.5±1.5 mm Hg, P<0.05) and IRL-1620 (0.5 nmol/kg) (control, 21.3±2.2; +BQ-788, 8.6±0.2 mm Hg, P<0.05), respectively, 90 minutes after administration of the antagonists.
BQ-123 (1 mg/kg IP) had no influence on the basal MAP of WT mice. In contrast, the same dose of the ETA antagonist induced a significant reduction in MAP of ETB (+/−) KO mice. The hypotensive response to BQ-123 (1 mg/kg IP) reached significance 20 minutes after administration. However, ETB (+/−) KO mice treated for 90 minutes still displayed significantly higher MAP (P<0.05 at all time points) than the BQ-123–treated WT littermates (Figure 2A⇓).
Figure 2B⇑ shows that BQ-123 (10 mg/kg) administered intraperitoneally significantly reduced (by ≈10 mm Hg) basal MAP of WT mice after 1 or 2 minutes after injection of the antagonist. This reduction in MAP was maintained for ≥90 minutes. Interestingly, the same treatment also reduced by ≈30 mm Hg the MAP of ETB (+/−) KO mice, bringing it back to the level of the BQ-123–treated WT counterparts. BQ-123 (10 mg/kg) administered intraperitoneally for 60 minutes in WT mice had no significant effect on the pressor response to NE (5 nmol/kg) (control, 29.6±3.2 mm Hg; in presence of BQ-123, 30.2±3.0 mm Hg). On the other hand, a treatment with BQ-788 (10 mg/kg) administered intraperitoneally had no effect on basal MAP either in WT or in ETB (+/−) KO mice (Figure 2D⇑). Finally, SB 209670 (10 mg/kg IP) induced a significant hypotensive response (starting at time point 1 minute with a maximal hypotension of ≥18 mm Hg) in ETB (+/−) KO but not in WT animals (Figure 2C⇑).
Effect of Partial KO of ETB Receptors or of BQ-123 or BQ-788 on Plasmatic Clearance of Exogenously Applied [125 I]-ET-1
Figure 3A⇓ illustrates the increase in [125I]-ET-1 (expressed in counts per minute) in blood samples collected from ETB (+/−) KO mice when compared with WT animals. Our results show a reduced clearance of [125I]ET-1 in ETB (+/−) KO (Figure 3A⇓) but not in ETA (+/−) KO mice (Figure 3B⇓) when compared with the WT littermates (n=8, P<0.05) starting at the 9-second time point. In contrast, no differences were found in the clearance of [125I]-Ang II in blood samples of ETB (+/−) KO mice when compared with WT mice (Figure 3C⇓) (n=8). In a fashion similar to the partial KO of the ETB receptors, a treatment with an ETB antagonist, BQ-788, but not with BQ-123 induced an increase in [125I]-ET-1 in the blood of WT mice (n=6, P<0.05) (Figure 4⇓, A and B).
We have shown that intravenously administered ET-1 and IRL-1620 both induced a dose-dependent increase in MAP of the anesthetized mouse, illustrating the significant contribution of both ETA and ETB receptors on systemic resistance in that species. Those results have been confirmed by the use of the selective ETA or ETB antagonists BQ-123 and BQ-788. With either of these antagonists, a significant reduction of ET-1–induced pressor response was observed, and the two receptor blockers show similar apparent affinity against the response to ET-1. Curiously, unlike the observations reported in many other animal species, such as the rabbit,14 the rat,11 the dog,15 and humans,16 selective blockade of ETB receptors with BQ-788 did not potentiate the pressor responses to ET-1 in the mouse. On the other hand, in WT mice, ETA and ETB receptor activation accounts for ≈70% and 30%, respectively, of the maximal pressor responses induced by ET-1, as shown in the present study (Table⇓). ETA but not ETB receptors for endothelin appear to be importantly involved in the vasoconstrictive properties of endogenous endothelin in the human brachial arteries.16 In addition, it has been shown that blockade of ETB receptors per se in both the rabbit and humans promotes an enhancement of vascular resistance, suggesting a predominant role for this receptor type in the modulation of endogenous endothelin-dependent constriction.14 16 The above-mentioned considerations illustrate that the mouse model is quite different from the majority of other species studied, as far as the contribution of ETA and ETB receptors is concerned in the vasoactive effects of endothelins.
The important contribution of ETB receptors in the pressor and constrictive effects of ET-1 in the murine model confirms the observations by Giller et al17 in nonlethal piebald mice, in which only 25% of normal mRNA for the ETB receptor has been reported. In this model, it was shown that another selective ETB agonist, sarafotoxin S6C, was devoid of initial hypotensive effect albeit it induced a marked pressor response.17 In the piebald lethal mice, in which the ETB receptor is fully mutated and functionally null, the response to sarafotoxin S6C was abolished when compared with the nonlethal piebald mice.17
Albeit not demonstrated in the present report, heterozygous knock-out of the receptor gene for ETA or ETB receptors results in a significant reduction (40% to 50%) in the mRNA and protein in the genetically manipulated mice2 (M. Yanagisawa, personal communication). In our hands, this partial knock-out of either gene resulted in a significant alteration in the cardiovascular properties of endothelins in the mouse (see Table⇑). Heterozygous knock-out of the ETB receptor, in contrast to that of the ETA receptor gene, resulted in a significant increase in MAP of nearly 25 mm Hg, as also previously reported in heterozygous KO mice for the ET-1 gene.7 Albeit the hypertensive state occurring in ET-1 KO mice was demonstrated to involve cardioreflex dysregulation,7 8 one can postulate that partial knock-out of the ETB receptor will preferably result in a modification of the ETB-dependent clearance mechanisms initially reported by Fukuroda et al.13 We suggest that partial knock-out of the ETB receptor may first affect the high-affinity ETB clearance receptors that have been reported on the endothelium.18 Such alterations in ETB receptor clearance mechanisms would favor a significant increase in luminal and basolateral ET-1 concentrations, as recently reported by Ohuchi et al.19 In support of that hypothesis, Giller et al17 suggested a role for increased ET-1 plasma levels in the hypertensive state occurring in piebald lethal mice. In conditions in which the ETB receptor is fully repressed, as in ETB KO (−/−) mice, this particular phenomenon (ie, reduction in ETB receptor clearance) is compensated by the complete loss of this receptorial entity at the vascular smooth muscle level. The significant contribution of ETB receptors in resistance vessels is substantiated by the fact that in ETB heterozygous KO mice, the maximal pressor response to ET-1 is unaltered, unlike that of the selective ETB agonist IRL-1620 (Table⇑).
The pivotal role of increased ET-1 levels in the hypertensive state of ETB KO (+/−) mice is further substantiated by the fact that we were able to significantly reverse the hypertensive state of these animals with the ETA-selective and specific antagonist BQ-123 as well as with the orally available ETA/ETB antagonist SB 209670 but not with the ETB blocker BQ-788. We also demonstrated an increase in exogenously applied [125I]-ET-1 in blood samples of ETB (+/−) KO mice when compared with WT animals. This suggests an altered ETB-dependent clearance mechanism in mice partially deficient in ETB receptors. Furthermore, a treatment with the ETB blocker BQ-788 but not with the ETA blocker BQ-123 also significantly increased radiolabeled ET-1 in the blood of WT mice.
On the other hand, one should note that the interpretation of our results should be limited to the condition of anesthesia prevailing in the studied animals. Whether this hypertensive state occurs in physiological situations, such as in conscious ETB (+/−) KO mice, remains to be investigated.
It is concluded that heterozygous knock-out of ETA or ETB receptors is sufficient to alter the pharmacodynamic properties of ET-1. Furthermore, ETB KO (+/−) mice display an ETA antagonist–sensitive hypertension suggested to be related to the impaired clearance of endogenous ET-1.
Interestingly, a significant correlation has been reported between the mutation of the ETB receptor gene locus and the occurrence of Hirschprung disease.20 Thus, it may be of interest to monitor the parents of these patients for a possibly higher prevalence of hypertensive states.
This project was financially supported by the Medical Research Council of Canada (MT-12889 and R-13272) and the Heart and Stroke Foundation of Québec. M. Yanagisawa is an investigator of the Howard Hughes Medical Institute, P. D’Orléans-Juste is a scholar of the Fonds de la recherche en santé du Québec, and N. Berthiaume is in receipt of a studentship of the Heart and Stroke Foundation of Canada. The authors gratefully acknowledge the secretarial assistance of Pascale Martel and Helen Morin and the efficient technical assistance of Shelley Dixon and Sahar Seyedkalal.
- Received November 29, 1999.
- Revision received January 3, 2000.
- Accepted June 6, 2000.
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