ETB Receptor Blockade Potentiates the Pressor Response to Big Endothelin-1 But Not Big Endothelin-2 in the Anesthetized Rabbit
Abstract—The precursor of endothelin-1, big endothelin-1, is considered to be a more reliable marker of systemic production of vasoactive peptide. However, it is largely unclear whether ETB receptor–dependent clearance and endothelium-derived relaxing factors affect the precursor in a similar manner to mature ET-1. These ETB-dependent modulations of big ET-1 and big ET-2 pressor properties were therefore studied in the anesthetized rabbit. When injected into the left cardiac ventricle, ET-1 and ET-2 (0.01 to 1 nmol/kg) each induced biphasic responses (a depressor followed by a pressor response), whereas big ET-1 and big ET-2 (0.1 to 3 nmol/kg) caused only protracted pressor responses. The highest dose of big ET-1 caused significantly greater responses than ET-1, ET-2, or big ET-2. A selective ETA receptor antagonist, BQ-123 (1 mg/kg), markedly reduced pressor responses to all 4 peptides, whereas blockade of ETB receptors with BQ-788 (0.25 mg/kg) sharply potentiated the responses to ET-1, ET-2, and big ET-1, but not to big ET-2. Indomethacin (10 mg/kg) sharply potentiated the pressor response to ET-1 (1 nmol/kg), but not big ET-1, at all time points. In control animals, ET-1, but not big ET-1, also triggered an indomethacin-sensitive increase in circulating prostacyclin. Finally, systemically administered big ET-1, but not big ET-2, induced a phosphoramidon-sensitive increase in plasma IR-ET. Our results suggest a significant limiting role of ETB receptors on pressor responses to big ET-1. In contrast, the same receptor entities do not modulate the hemodynamic properties of the ET-2 precursor, given that, unlike big ET-1, it is poorly converted in the pulmonary or systemic circulation in anesthetized rabbits.
- endothelium-derived relaxing factor
- endothelin-converting enzyme
The potent vasoconstrictor peptide endothelin-1 (ET-1), and its isomers ET-2 and ET-3 are formed through proteolytic cleavage of their immediate precursors, big ET-1, big ET-2, and big ET-3, by phosphoramidon-sensitive endothelin-converting enzymes (ECEs).1 ECEs display pronounced activity in vivo, such that big ET-1, which shows very low affinity for endothelin ET receptors, is as potent as its active metabolite ET-1 as a hypertensive agent on an equimolar basis.2 As big ET-1 plasma levels in the human, rabbit, and rat are higher than those of its active metabolite, studies have suggested that the precursor might be the actual factor released from endothelial cells.3 Interestingly, big ET-1 plasma levels have been shown to correlate closely with severity of chronic heart failure in patients and to constitute a good prognostic marker of kidney function and survival.4
When administered intravascularly, big ET-1 induces only a monophasic pressor response, whereas its active metabolite, ET-1, induces a characteristic transient depressor effect, due to vasodilation, before onset of its pressor effect.1 2 This discrepancy in hemodynamic effects of exogenous ET-1 and big ET-1 has been ascribed to their differential abilities to stimulate vasoactive ET receptors; the precursor is virtually unable to activate vasodilator ETB receptors.5 However, we have recently shown that pretreatment with an ETB antagonist, BQ-788, potentiates the pressor response induced by big ET-1 in the anesthetized rabbit.6 This suggests that, at least in the rabbit, ETB receptors effectively modulate the pressor response to big ET-1 after its conversion to ET-1, possibly through the release of nitric oxide. Furthermore, big ET-1 has been shown to release prostacyclin (PGI2) from the rabbit lung and to inhibit indomethacin-sensitive platelet aggregation in the rabbit,2 6 the latter phenomenon being dependent on an increase in circulatory PGI2 levels after activation of ETB receptors.2 6 7
On the other hand, we have shown in the guinea pig in vivo that big ET-1 and big ET-2 have different vasopressor and bronchoconstrictor profiles of action, which possibly reflects the differential affinity of the latter peptide for ECE of the systemic vasculature compared to that of the pulmonary circulation.8 9 Furthermore, unlike big ET-1, big ET-2 has been shown to be inactive in generating eicosanoids from perfused lungs in several animal species.8 9 These results also suggest that tissue and cellular localizations of ECE are highly important determinants of the pressor properties of the big ETs.
Though plasma big ET levels are being increasingly recognized as important markers for cardiovascular diseases, the relation between big ET processing by the ECE in vivo and the population of ET receptors subsequently activated by its active metabolite has yet to be investigated. In the present study, we compared the differential capacities of big ET-1 and big ET-2 to activate vasodilator ETB receptors after their ECE-dependent conversion to either ET-1 or ET-2, respectively.
Experiments were performed on New Zealand White rabbits of either sex (1.3 to 1.6 kg) anesthetized with an intramuscular injection of a mixture of ketamine (48 mg/kg) and xylazine (7 mg/kg) and ventilated as previously reported.10 All experimental procedures conformed to the guiding principles for animal experimentation as enunciated by the Canadian Council on Animal Care and approved by the Ethical Committee on Animal Research of the Université de Sherbrooke Medical School.
Dose-response curves of the pressor effects of big ET-1, big ET-2, ET-1, and ET-2 (0.01 to 3 nmol/kg; 1 dose of a single agonist per animal) were first established in anesthetized animals. The antagonists BQ-123 (1 mg/kg), BQ-788 (0.25 mg/kg), and phosphoramidon or the neutral endopeptidase inhibitor CGS 2459211 were administered intra-arterially (5 mg/kg) 5 minutes before intra-arterial administration of ET-1 (0.25 and 1 nmol/kg), ET-2 (0.25 and 1 nmol/kg), big ET-1 (0.5, 1 and 3 nmol/kg), or big ET-2 (3 nmol/kg). Mean arterial pressure (MAP) was monitored for ≥30 minutes after a single administration of each agonist per animal.
Plasma Prostacyclin and Endothelin Radioimmunoassay
Blood samples (1 mL) taken before and at 1, 2.5, 5, 15, and 30 minutes after injection of the peptides were collected through a carotid cannula in trisodium citrate (0.35% final volume). Samples were centrifuged for 1 minute at 15 000g for plasma separation and stored at −80°C until assayed. Plasma prostacyclin levels were monitored with a radioimmunoassay for the stable metabolite of prostacyclin, 6-keto-PGF1α. The 6-keto-PGF1α antiserum (Sigma Chemical Co) has 100% cross-reactivity with 6-keto-PGF1α, 23% with PGE1, 4% with PGE2, 7% with PGF2α, and <1% with thromboxane B2.
Plasma immunoreactive endothelin levels from 400-μL samples were prepared and measured with a double-antibody assay (RPA-555; Amersham) as previously reported.10 Recoveries of ET-1 were 53% (n=4), and plasma immunoreactive endothelin (IR-ET) concentrations are shown uncorrected for the extraction recovery. Cross-reactivity of the endothelin antiserum for ET-1, ET-2, ET-3, big ET-1 (1-38), and big ET-1 (22-38) was 100%, 144%, 52%, 0.4%, and <0.0033%, respectively.
Plasma samples (600 μL) for high-performance liquid chromatography (HPLC) analysis were obtained at time points 0 and 2.5 minutes from 4 different rabbits and were prepared and analyzed as previously reported.10 For calibration, synthetic ET-1, ET-2, ET-3, and big ET-1 were run separately, and the elution positions of the standards were determined by ultraviolet absorbance (210 nm).
Drugs and Solutions
ET-1, ET-2, and big ET-1 (1-38) were purchased from American Peptide and BQ-788, big ET-2 (1-38), and phosphoramidon (a neutral endopeptidase and ECE inhibitor) from Peptide International. BQ-123 was synthesized by Dr Witold Neugebauer (Department of Pharmacology, Université de Sherbrooke). Heparin and PBS were purchased from Sigma. All drugs were dissolved in PBS (pH 7.4) except for BQ-788 and BQ-123, which were diluted in 20% DMSO (1 mg/mL stock solutions) and subsequently in PBS.
Results are expressed as mean±SEM. Statistical analyses were done by ANOVA followed by Dunnett’s test for multiple comparisons to evaluate the variation compared to basal values (time 0 minutes). Comparisons between groups were made by Student’s t test. Probability values <0.05 were considered significant.
Increase in Mean Arterial Pressure by Big ET-1 and Big ET-2 and Their Active Metabolites in the Anesthetized Rabbit
Basal MAP in ketamine/xylazine-anesthetized rabbits was 71.7±1.9 mm Hg, and mean heart rate was 162±4 bpm (n=113). Both ET-1 and ET-2 caused similar increases in MAP at the highest dose administered (1 nmol/kg; ΔMAP ET-1, 32.7±3.0 mm Hg, n=15; ΔMAP ET-2, 30.4±3.0 mm Hg, n=8). Higher doses of either peptide triggered significant cardiotoxic effects. Big ET-1 produced a significantly higher maximal increase in MAP at 1 nmol/kg (46.8±3.1 mm Hg, n=9) than ET-1 (P<0.05). Interestingly, big ET-2 induced a significantly smaller increase in MAP than big ET-1 at the highest dose tested (3 nmol/kg; Figure 1A⇓). As shown in Figure 1B⇓, the pressor effects of big ET-1 (0.5 and 1 nmol/kg) or big ET-2 (3 nmol/kg), in contrast to the depressor and pressor effects of either ET-1 or ET-2 (0.25 nmol/kg), were markedly reduced by phosphoramidon (5 mg/kg) (P<0.05, n=4 each). In contrast, CGS 24592 (5 mg/kg) did not affect the pressor effect induced by both big ET-1 (0.5 nmol/kg) and big ET-2 (3 nmol/kg) (ΔMAP big ET-1, 20.4±3.2 mm Hg, n=9; in presence of CGS, 22.2±1.0 mm Hg, n=3; ΔMAP big ET-2, 21.8±2.3 mm Hg, n=4; in presence of CGS, 21.6±3.8 mm Hg, n=3).
Effects of ETA and ETB Receptor Antagonists on Big ET-1 and Big ET-2 Pressor Responses in the Anesthetized Rabbit
Left intraventricular administration of ET-1 (0.25 nmol/kg) produced the characteristic biphasic pressor response to MAP (Figures 1B⇑ and 2A⇓). The pressor effect of ET-1 (0.25 nmol/kg) was significantly potentiated by pretreatment of the rabbit with BQ-788 (0.25 mg/kg; P<0.05, n=4). Furthermore, the initial depressor response to ET-1 (0.25 nmol/kg) was abolished after BQ-788 administration (P<0.05, n=4; Figure 2A⇓). A 5-minute pretreatment of the animal with BQ-123 (1 mg/kg), a selective ETA receptor antagonist, significantly reduced the hypertensive effect of ET-1 (0.25 nmol/kg) in terms of magnitude and duration (P<0.05, n=4) and prolonged the hypotensive response to the peptide (Figure 2A⇓). Intra-arterial administration of big ET-1 (0.5 nmol/kg), in contrast to that of ET-1, produced only a sustained pressor effect in the rabbit (ΔMAP, 20.4±3.2 mm Hg; n=9; Figure 2B⇓). BQ-123 (1 mg/kg) markedly reduced the response to big ET-1 (0.5 nmol/kg; ΔMAP in presence of BQ-123 [1 mg/kg], 4.5±1.1 mm Hg, P<0.05, n=7; Figure 2B⇓). Interestingly, the ETB receptor antagonist BQ-788 (0.25 mg/kg) significantly potentiated the pressor response to big ET-1 (0.5 nmol/kg; ΔMAP in presence of BQ-788 [0.25 nmol/kg], 32.0±4.9 mm Hg, P<0.05, n=7; Figure 2B⇓).
Similarly to ET-1, intra-arterial administration of ET-2 (0.25 nmol/kg) also produced a biphasic pressor response (Figures 1B⇑ and 2C⇑). As observed for ET-1, BQ-788 (0.25 mg/kg) potentiated the ET-2–induced pressor response and abolished the initial vasodepressor effect (P<0.05, n=4; Figure 2C⇑). BQ-123 (1 mg/kg) significantly reduced the hypertensive phase without affecting the maximal depressor response (Figure 2C⇑). Big ET-2 (3 nmol/kg) produced a sustained increase in blood pressure (ΔMAP, 21.8±2.3 mm Hg, n=6; Figure 2D⇑), which was roughly equivalent to that produced by big ET-1 (0.5 nmol/kg) and was likewise virtually abolished by pretreatment of the rabbit with BQ-123 (1 mg/kg) (ΔMAP, 2.9±1.0 mm Hg, P<0.05, n=7). However, in contrast to the big ET-1–induced pressor response, that caused by big ET-2 (3 nmol/kg) was not significantly altered by pretreatment with the ETB receptor antagonist BQ-788 (0.25 mg/kg) (ΔMAP, 25.1±3.0 mm Hg, n=7).
Plasma Immunoreactive Endothelin Levels After Big ET-1 or Big ET-2 Administration
Figure 3⇓ depicts variations in plasma levels of IR-ET (relative to basal levels) after big ET-1 or big ET-2 administration. Big ET-1 (0.5 nmol/kg) produced a significant elevation in IR-ET levels in the anesthetized rabbit. This elevation was maximal at time 2.5 minutes after injection and remained significantly elevated when compared with basal levels until the 5-minute time point (basal levels IR-ET, 9.7±2.5 fmol/mL; 2.5 minutes, 14.8±2.4 fmol/mL; P<0.05, n=7; Figure 3A⇓). IR-ET plasma levels, after big ET-1 administration at 3 nmol/kg, failed to produce any additional elevation in IR-ET plasma levels compared with the 0.5 nmol/kg dose, but levels remained significantly higher than baseline up to 15 minutes after administration. Pretreatment with phosphoramidon (5 mg/kg) fully prevented the increase in plasma IR-ET levels induced by big ET-1 (0.5 nmol/kg). Furthermore, variations in plasma IR-ET levels triggered by big ET-1 (0.5 nmol/kg) injection were unaffected by pretreatment with either BQ-788 (Figure 3A⇓) or BQ-123 (maximal variation in IR-ET, 4.0±1.3 fmol/mL; n=4).
The full cross-reactivity of the nonselective ET antibody for ET-1 and ET-2 also enabled us to monitor variations in IR-ET levels after big ET-2 (3 nmol/kg) administration in the present study. Big ET-2 (3 nmol/kg) administration in the anesthetized rabbit did not produce any detectable elevation of IR-ET basal levels (Figure 3B⇑). Moreover, ETB receptor blockade by BQ-788 did not unmask any increase of IR-ET after big ET-2 (3 nmol/kg) administration.
Furthermore, reversed-phase HPLC analyses coupled to the specific radioimmunoassay for endothelins was used to further identify the immunoreactive component of the plasma samples. The elution profile of the samples before big ET-1 administration revealed a major peak at 23 minutes of elution time, which coincided with the retention time of the authentic ET-1 standard (Figure 4⇓). A pronounced and selective increase in IR-ET in this major peak was detected in samples collected after big ET-1 (0.5 nmol/kg) administration.
Prostacyclin-Releasing Properties of ET-1 and ET-2 and Their Precursors
Figure 5A⇓ shows that pretreatment of the rabbit with indomethacin (10 mg/kg) significantly potentiated the pressor responses to ET-1 (1 nmol/kg) without affecting the magnitude of the initial depressor response. In sharp contrast, indomethacin treatment did not influence the pressor response to big ET-1 (1 nmol/kg; Figure 5B⇓). Concomitant analysis of plasma PGI2 levels revealed that ET-1 (1 nmol/kg) significantly increased plasma PGI2 levels, an effect that was abolished by indomethacin (10 mg/kg) pretreatment (P<0.05; n=4), whereas big ET-1 was ineffective for alteration of plasma PGI2 levels either at 1 nmol/kg (basal, 0.28±0.07 ng/mL; in presence of big ET-1, 0.34±0.09 ng/mL; Figure 5C⇓) or at 3 nmol/kg (results not shown).
Because of its different pharmacokinetic characteristics, big ET-1 has been suggested to be a more reliable marker of cardiovascular diseases than ET-1.4 Indeed, the latter peptide has a relatively short circulating half-life, and its plasma concentration is actively controlled by ETB clearance receptors on vascular endothelium.12 13 It is therefore of interest that, in the present study, we have shown that the selective ETB antagonist sharply potentiates the pressor response to big ET-1, as previously reported for mature ET-1.6
Our results would suggest that ECE is sufficiently active to transform big ET-1 into enough of its active metabolite to enable not only contraction of underlying vascular smooth muscle, but also some spillover of ET-1 back into the blood. The latter phenomena is controlled by ETB clearance receptors and is well correlated with the sharp rise in plasma IR-ET observed after administration of the precursor. The increase in IR-ET triggered by intracardiac big ET-1 administration was confirmed by HPLC as comprising mature ET-1 and, interestingly, was fully abrogated by prior injection of phosphoramidon, as also recently demonstrated in human forearm.14 Interestingly, big ET-1 at a dose of 3 nmol/kg did not promote a higher plasma level of IR-ET-1 than at the dose of 0.5 nmol/kg, albeit IR-ET-1 could be detected at further time points at the higher dose. The present study confirms earlier results that show that dynamic conversion of big ET-1 in the rabbit significantly raises plasma IR-ET levels.15 Furthermore, our results are in conflict with the suggestion made by Corder and Vane16 that the increase in plasma IR-ET after administration of big ET-1 may not be due to mature ET-1, but instead to the contaminant precursor itself, as detected by the same antiserum with a previously reported cross-reactivity of 0.4%.15
The pressor effects of both ET-1 and ET-2 are each potentiated to similar extents by the selective ETB antagonist BQ-788. This finding correlates well with the fact that the 3 natural endothelin isopeptides possess the same affinity for the ETB receptor.17 On the other hand, we have recently shown that the pulmonary circulations of different animal species8 9 are less efficient for conversion of big ET-2 versus big ET-1. First, in the present study, big ET-2 was much less potent than big ET-1 to induce phosphoramidon-sensitive increases in MAP. Second, we were unable to detect a significant increase in plasma immunoreactive levels of ET-2 after administration of big ET-2 in vivo. Third, unlike the effects caused by administration of exogenous ET-2, pressor responses to big ET-2 were entirely unaffected by treatment with the selective ETB antagonist. These 3 observations allow us to suggest that the precursor of ET-2 is only poorly converted in the pulmonary circulation or elsewhere in the systemic circulation in vivo.
The maximal pressor effect of ET-1 was significantly lower than that of big ET-1 in the anesthetized rabbit. In contrast, when treated with the cyclooxygenase inhibitor indomethacin, both the precursor and ET-1 induced identical maximal pressor responses. This suggests that ET-1 is more efficient than big ET-1 for triggering the release of vasomodulatory prostacyclin, as illustrated in the present study. Interestingly, only a higher dose of 3 nmol/kg of big ET-1 (but not 1 nmol/kg) was shown to inhibit platelet aggregation ex vivo in the anesthetized rabbit.2 Because big ET-1 (1 nmol/kg) pressor effects are not potentiated by indomethacin but are sharply enhanced by the ETB antagonist BQ-788, we can therefore conclude that nitric oxide but not vasodilatory prostaglandins released after ETB receptor activation is a significant modulator of big ET-1–induced pressor responses.
In summary, we show in the present study that the pressor effects of big ET-1, but not of big ET-2, are potentiated by the ETB antagonist BQ-788. The distinct susceptibilities of big ET-1 and big ET-2–induced pressor effects to potentiation by BQ-788 seem to correlate closely with the phosphoramidon-sensitive increase in plasma IR-ET levels after big ET-1 but not big ET-2 administration. Given that phosphoramidon equally abolished the pressor responses to either big ET-1 or big ET-2, our results suggest a more effective conversion of big ET-1 than big ET-2 by ECE, which leads to spillover of ET-1 (but not ET-2) back into the vascular compartment to evoke activation of ETB receptor–dependent release of nitric oxide. If one considers that big ET-2 (1-38) is poorly converted in the pulmonary circulation of several species,8 9 false ECE substrates derived from the structure of the ET-2 precursor may lead to moieties that interfere with the systemic production of ETs without affecting the delicate NO/ET-1 balance in the lungs.
This project was financially supported by the Medical Research Council of Canada (M.R.C.C.) (MA-16612 and GR-13915) and the Heart and Stroke Foundation of Québec (H.S.F.Q.). P.D.-J. is a scholar of the Fonds de la Recherche en Santé du Québec (F.R.S.Q.). J.P.G. has received fellowships from the H.S.F. of Canada and from Fonds pour la Recherche et l’Aide aux Chercheurs (F.C.A.R.). G.A.R. is a scholar of the Brazilian National Research Council (CNPq). We gratefully acknowledge Dr Arco Jeng (Novartis, Summit, NJ) for the generous supply of CGS 24592 and Helen Morin for secretarial assistance.
- Received June 6, 1999.
- Revision received July 12, 1999.
- Accepted November 12, 1999.
D’Orléans-Juste P, Lidbury PS, Télémaque S, Warner TD, Vane JR. Human big endothelin releases prostacyclin in vivo and in vitro through a phosphoramidon-sensitive conversion to endothelin-1. J Cardiovasc Pharmacol. 1991;17:S251–S255.
Kos T, Pacher R., Wimmer A., Bojic A, Hulsmann M, Frey B, Mayer G, Yilmaz N, Skvarilova L, Spinar J, Vitovec J, Toman J, Woloszcuk W, Stanek B. Relationship between kidney function, hemodynamic variables and circulating big endothelin levels in patients with severe refractory heart failure. Wien Klin Wochenschrift. 1998;110:89–95.
Haleen SJ, Davis LS, LaDouceur DM, Keiser JA. Why big endothelin-1 lacks a vasodilator response. J Cardiovasc Pharmacol. 1993;22:S271—S273.
Gratton JP, Cournoyer G, D’Orléans-Juste P. Endothelin-B receptor-dependent modulation of the pressor and prostacyclin-releasing properties of dynamically converted big-endothelin-1 in the anesthetized rabbit. J Cardiovasc Pharmacol. 1998;31:S161—S163.
D’Orléans-Juste P, Gratton JP, Bkaily G, Giaid A. Activity and distribution of endothelin-converting enzyme in the lung. In: Goldie RG, Hay DWP, eds. Pulmonary Actions of the Endothelins. Basel, Switzerland: Birkhauser-Verlag, AG; 1999: chapter 4; 53–73.
Gratton JP, Cournoyer G, Löffler BM, Sirois P, D’Orléans-Juste P. ETB receptor and nitric oxide synthase blockade induce BQ-123-sensitive pressor effects in the rabbit. Hypertension. 1997;30:1204–1209.
Dupuis J, Goresky CA, Fournier A. Pulmonary clearance of circulating endothelin-1 in dogs in vivo: exclusive role of ETB receptors. J Appl Physiol. 1996;81:1510–1515.