Julie Labonté

Function of the endothelinB receptor in cardiovascular physiology and pathophysiology

One of the two receptors by which the potent vasoactive effects of endothelin (ET)-1 are mediated is the ET(B) receptor (ET(BR)), which is found in several tissues, but, more importantly from a cardiovascular point of view, on the endothelial cell. The endothelial cell also has the unique capability of releasing ET-1, as well as other factors, such as the endothelial-derived relaxing factors and prostacyclin, which counteract the myotropic effects of the peptide. The secretory and contractile responses to ET-1 rely on G-protein-coupled ET(BR)s, as well as ET(A)-G-protein-coupled receptor-like proteins. The mitogenic properties of ET-1 via ET(A) receptors (ET(AR)s) coupled to mitogen-activated protein kinases and tyrosine kinases on the vascular smooth muscle may occur in conjunction with the anti-apoptotic characteristics of the endothelial ET(BR)s. Interestingly, most of the relevant antagonists and agonists for both ET(AR)s and ET(BR)s have been developed by the pharmaceutical industry. This highlights the therapeutical potential of compounds that act on ET receptors. In normal as well as in physiopathological conditions, the ET(BR) plays an important role in the control of vascular tone, and must be taken into account when using ET receptor antagonists for the treatment of cardiovascular diseases. For the management of congestive heart failure, renal failure and primary pulmonary hypertension, the most recent literature supports the use of selective ET(AR) antagonists rather than mixed antagonists of ET(AR)s and ET(BR)s. Nonetheless, validation of this view will have to await the first clinical trials comparing the actions of ET(A) to mixed ET(A)/ET(B) receptor antagonists.

Heterozygous Knock-Out of ETB Receptors Induces BQ-123–Sensitive Hypertension in the Mouse

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.

Tissue-engineered human vascular media with a functional endothelin system.

Background—Cardiovascular diseases remain a major cause of death and disability in the Western world. Among the various approaches adopted to counteract the morbidity associated with these diseases, surgical procedures and cardiac and vascular xenotransplantations or allotransplantations are routinely performed. The suitable vascular graft would be as close as possible to the native and healthy vessel composed exclusively of human components provided by the patient and would adapt to the donor’s hemodynamics. We have developed such a tissue-engineered human blood vessel reconstructed with human cells. Because endothelin is the most potent vasopressor known to date, we were interested in investigating the functionality of the endothelinergic system in our reconstructed human blood vessel. Methods and Results—Vasoconstriction studies were performed with nonselective and selective agonists and antagonists to demonstrate that ETA receptors were present and functional in tissue-engineered human vascular media constructed with the self-assembly method. Reverse-transcriptase polymerase chain reaction studies demonstrated that mRNA of the ETA but not the ETB receptor was present in these human tissue–engineered blood vessels. Furthermore, we demonstrated that the endothelin-converting enzyme, the main enzyme responsible for the formation of the biologically active endothelin peptides, was present and functional in these same bioengineered vascular media. Conclusions—Our results suggest that the media component of our tissue-engineered blood vessel has the potential of controlling vascular resistance via the presence of functional endothelin ETA receptors and endothelin-converting enzyme.

Endothelin-1 (1-31) Is an Intermediate in the Production of Endothelin-1 After Big Endothelin-1 Administration In Vivo

The precursor of endothelin-1, big endothelin-1, can be hydrolyzed by chymase to generate endothelin-1 (1-31) in vitro. In the present study, we explored the processes involved in the production of endothelin-1 (1-31) as well as its pharmacodynamic characteristics in the rabbit in vivo. Endothelin-1 (1-31) (1 nmol/kg, injected into the left cardiac ventricle) induced a monophasic increase of mean arterial blood pressure similarly to big endothelin-1 (1-38), whereas endothelin-1 induces a biphasic response. Phosphoramidon, a dual neutral endopeptidase and endothelin-converting enzyme inhibitor, blocked both pressor responses to endothelin-1 (1-31) and big endothelin-1 but not those afforded by endothelin-1. Thiorphan, a neutral endopeptidase inhibitor, markedly inhibited the response to endothelin-1 (1-31) but only weakly reduced that of big endothelin-1. In contrast, CGS 35066, an endothelin-converting enzyme inhibitor, was significantly more efficient against the pressor response to big endothelin-1 than to endothelin-1 (1-31). Furthermore, injection of big endothelin-1 concomitantly with phosphoramidon induced an increase in endothelin-1 (1-31) plasma levels. Finally, intracardiac-administered endothelin-1 (1-31) induced an increase of endothelin-1 plasma levels, which are markedly reduced by phosphoramidon and thiorphan but not by CGS 35066. Our results thus demonstrate that endothelin-1 (1-31) is an alternate intermediate in the production of endothelin-1 after big endothelin-1 administration in the rabbit in vivo.

Role of endothelin receptors in the hypertensive state of kinin B 2 knockout mice subjected to a high-salt diet

Mice with disruption of the kinin B(2) receptor (B(2)KO mice) are sensitive to salt-rich diets, which causes hypertension. The aim of the study was to assess the role of endothelin-1 (ET-1) and angiotensin-II in hypertensive B(2)KO mice on a salt-rich diet. We also wanted to verify if there is an upregulation of the mRNA expression of the precursors or receptors for these hormones. Two groups of B(2)KO mice (20-25 g) were investigated. The first group received an 8% NaCl diet with 1% NaCl in drinking water (HS) and the second was fed with normal food with tap water (NS). The antagonists tested were the ET(A) receptor antagonist BQ-123 (1 and 5 mg/kg), the ET(B) receptor antagonist BQ-788 (0.25 and 1 mg/kg), the angiotensin receptor type 1 antagonist losartan (10 mg/kg) and the angiotensin-converting enzyme inhibitor captopril (3 mg/kg). These were injected intraperitoneally 30 min prior to blood pressure measurement by the tail-cuff method. We also studied the level of expression of preproET-1, ET-1 receptors, angiotensinogen and angiotensin receptors by RNA extraction from the heart and kidneys of these mice followed by reverse transcriptase (RT)-PCR. B(2)KO mice (HS) were hypertensive after 8 weeks compared with B(2)KO mice on normal diet (HS, 93.4+/-1.5 mmHg, n=7; NS, 61.4+/-2.7 mmHg, n=7). In the HS group, the mean arterial blood pressure was significantly reduced by BQ-123 (5 mg/kg) to 61.9+/-1.8 mmHg (n=7), by BQ-788 (1 mg/kg) to 58.8+/-2.6 mmHg (n=6), by losartan (10 mg/kg) to 73.2+/-1.7 mmHg (n=8) and by captopril (3 mg/kg) to 86.0+/-2.3 mmHg (n=8). The expression studied by RT-PCR did not show any difference (either in precursors or receptors expression) between hypertensive and normal mice. The four antagonists used seemed to reverse the hypertension. These results suggest that ET-1 and angiotensin-II are probably involved in the mechanism that leads to hypertension since the effect of these hormones is probably not compensated by kinins in B(2)KO mice. Further studies are necessary to understand the implication of the cross-talk between these hormones in the hypertensive state.

Role of ETB and B2 receptors in the ex vivo platelet inhibitory properties of endothelin and bradykinin in the mouse.

We have developed a model to study the inhibitory properties of endogenous autacoids triggered by systemically‐administered vasoactive peptides, on platelet aggregation ex vivo in the mouse. Adenosine diphosphate (ADP) (0.5 – 10 μM) induces a concentration‐dependent aggregation of platelet‐rich plasma derived from C57BL/6 mice. Intravenously‐administered endothelin‐1 (0.01 – 1 nmol kg−1), the selective ETB agonist, IRL‐1620 (0.01 – 1 nmol kg−1) or bradykinin (1 – 100 nmol kg−1) significantly reduced in a dose‐dependent fashion the ADP‐induced platelet aggregation. The non‐selective cyclo‐oxygenase (COX) inhibitor, indomethacin, a selective COX‐2 inhibitor NS‐398 or the prostacyclin synthase inhibitor, tranylcypromine (10 mg kg−1), markedly reduced the inhibitory properties of endothelin‐1, whereas only a combination of both indomethacin, NS‐398 or tranylcypromine and L‐NAME (10 mg kg−1) were required to abolish the response to bradykinin. An ETB‐selective antagonist (BQ‐788) or knockout of the B2 receptor gene (in B2 knockout mice) abolishes the platelet inhibitory properties of endothelin‐1 and bradykinin, respectively. Our results suggest that intravenously‐administered endothelin‐1 and bradykinin, through ETB and B2 receptor activation, respectively, inhibit platelet aggregation ex vivo in the mouse. The inhibitory properties of endothelin‐1 require the activation of COX‐2 and the subsequent generation of prostacyclin. In addition to the two previously mentioned factors, nitric oxide is required for the anti‐aggregatory effects of bradykinin.

Cardiovascular diseases: new insights from knockout mice.

Knockout (KO) mice models have generated a wealth of new information on the developmental and physiopathological roles of several hormones and their receptors. In these mice, KO of a specific gene can be lethal at embryonic stages or during early adulthood. Furthermore, in conditions of non-lethality, KO mice may compensate for the repression of a particular protein expression. As a result of these two aspects, various phenotypic expressions occur in KO mice models for several peptides and their respective receptors, as well as for the enzymes involved in their processing.

Pivotal Role of Mouse Mast Cell Protease 4 in the Conversion and Pressor Properties of Big-Endothelin-1

The serine protease chymase has been reported to generate intracardiac angiotensin-II (Ang-II) from Ang-I as well as an intermediate precursor of endothelin-1 (ET-1), ET-1 (1–31) from Big-ET-1. Although humans possess only one chymase, several murine isoforms are documented, each with its own specific catalytic activity. Among these, mouse mast cell protease 4 (mMCP-4) is the isoform most similar to the human chymase for its activity. The aim of this study was to characterize the capacity of mMCP-4 to convert Big-ET-1 into its bioactive metabolite, ET-1, in vitro and in vivo in the mouse model. Basal mean arterial pressure did not differ between wild-type (WT) and mMCP-4(−/−) mice. Systemic administration of Big-ET-1 triggered pressor responses and increased blood levels of immunoreactive (IR) ET-1 (1–31) and ET-1 that were reduced by more than 50% in mMCP-4 knockout (−/−) mice compared with WT controls. Residual responses to Big-ET-1 in mMCP-4(−/−) mice were insensitive to the enkephalinase/neutral endopeptidase inhibitor thiorphan and the specific chymase inhibitor TY-51469 {2-[4-(5-fluoro-3-methylbenzo[b]thiophen-2-yl)sulfonamido-3-methanesulfonylphenyl]thiazole-4-carboxylic acid}. Soluble fractions from the lungs, left cardiac ventricle, aorta, and kidneys of WT but not mMCP-4(−/−) mice generated ET-1 (1–31) from exogenous Big-ET-1 in a TY-51469-sensitive fashion as detected by high-performance liquid chromatography/ matrix-assisted laser desorption/ionization-mass spectrometry. Finally, pulmonary endogenous levels of IR-ET-1 were reduced by more than 40% in tissues derived from mMCP-4(−/−) mice compared with WT mice. Our results show that mMCP-4 plays a pivotal role in the dynamic conversion of systemic Big-ET-1 to ET-1 in the mouse model.

Further pharmacological evaluation of a novel synthetic peptide bradykinin B2 receptor agonist

Abstract We recently identified a novel human B2 receptor (B2R) agonist [Hyp3,Thi5,NChg7,Thi8]-bradykinin (NG291) with greater in vitro and in vivo potency and duration of action than natural bradykinin (BK). Here, we further examined its stability and selectivity toward B2R. The hypotensive, antithrombotic, and profibrinolytic functions of NG291 relative to BK and its analogue ([Hyp3,Thi5,(4-Me)Tyr8(ΨCH2NH)Arg9]-BK) (RMP-7) were also tested. Contraction assays using isolated mouse stomachs (containing kinin B1R, B2R, and kininase I- and II-like activities) showed that NG291 is a more potent contractant than BK and is inhibited by HOE-140 (B2R antagonist) but unaffected by R954 (B1R antagonist), whereas both decreased the potency of BK. In stomach tissues from B2R knockout mice, BK maintained its activity via B1R, whereas NG291 had no contractile effect, indicating that it was selective for B2R. Unlike BK, NG291 was not degraded by rabbit lung ACE. Comparing intravenously administered BK and NG291 revealed that NG291 exhibited more potent and prolonged hypotensive action and greater antithrombotic and profibrinolytic activities. These effects were of comparable magnitude to RMP-7 and were absent in B2R knockout mice. We concluded that NG291 is a novel biostable B2R-selective agonist that may prove suitable for investigating the (pre)clinical cardioprotective efficacy of B2R activation.

Enhanced or repressed pressor responses to endothelin-1 or IRL-1620, respectively, in eNOS knockout mice.

Nitric oxide (NO) has been shown to be a physiological antagonist of the potent pressor agent endothelin-1, a transcriptional and post-transcriptional repressor of the production of this particular peptide as well as an endogenous modulator of vascular resistance in several animal species as well as in man. The aim of the present study was to assess the hemodynamic properties of endothelin-1 in endothelial NO synthase knockout mice (eNOS (-/-) KO). Ketamine- xylazine anesthetized eNOS (-/-) KO mice show a significantly higher mean arterial pressure (+25.7 ± 4.3 mmHg, n = 10) than their wild-type congeners (C57BL/6 background). eNOS (-/-) KO mice, when subjected to systemically administered endothelin-1 or the selective endothelin-B agonist IRL-1620 (1 nmol/kg, each), responded by markedly enhanced and reduced pressor responses (systolic, diastolic and mean arterial pressure), respectively, when compared with wild-type mice. Surprisingly, plasma levels of immunoreactive endothelin are not significantly different in endothelial nitric oxide synthase knockout mice and wild-type mice. Our results suggest that chronic repression of endothelial nitric oxide synthase affects in opposite fashion the functionality of endothelin-A and endothelin-B receptor-dependent responses. The increase in mean arterial pressure afforded in endothelial nitric oxide synthase knockout mice does not appear to involve an increase in production of endogenous endothelin in vivo.