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Departments of 1Pharmacology and 2Anatomy and Cell Biology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Québec, Canada
Submitted 11 April 2005 ; accepted in final form 29 April 2005
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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clearance; telemetry
The role of ET-1 in cardiovascular disorders, demonstrated by the beneficial properties of ET receptor antagonists, has been well documented in preclinical studies (24). However, the preferential use of selective ETA over nonselective ETA/ETB receptor antagonists remains debatable. In this regard, two main hypotheses are proposed. First, the contractile component following ETB receptor activation is dominant; hence, this receptor should be blocked concomitantly with the ETA receptor. Second, the physiological antagonism displayed by the ETB receptor on the endothelium is crucial, inasmuch as it opposes vasodilator factors released from that cell layer to the vasoconstrictor properties of ET-1. With regard to this physiological antagonism, a selective ETA receptor blockade should be favored. Thus, to address these two hypotheses, the precise role of the ETB receptor, depending on its vascular location and function (i.e., dilatation or constriction), must be determined.
Selective ET receptor antagonists have been developed and are useful tools to test these hypotheses. Atrasentan (ABT-627), a nonpeptidic ETA receptor antagonist, is under clinical evaluation in hormone-refractory prostate cancer patients (21). A-192621, a structurally related compound, is highly selective and efficient in blocking ETB receptors in vitro and in vivo (27, 29). Using these two antagonists injected intravenously in the Syrian Golden hamster, we previously reported that, combined with an acute treatment with atrasentan, a complete blockade of ETB receptors (i.e., located on endothelial cells and VSMCs) was required to prevent the acute increase in blood pressure triggered by exogenous ET-1 (15). Conversely, hypotensive responses afforded by selective blockade of the ETA receptor with atrasentan, after exogenous ET-1 administration, were abolished when endothelial ETB receptors were concomitantly blocked by A-192621 (15).
On the other hand, the efficacy of selective ETA receptor antagonism has been demonstrated in heart failure conditions in the cardiomyopathic hamster (30). However, the contributions of endogenous ET-1 and both ET receptors in controlling blood pressure in noncardiomyopathic hamsters have not been explored. Furthermore, there has been no attempt, to the best of our knowledge, to distinguish physiological roles of endothelial and VSMC ETB receptors in a chronic setting of ET receptor blockade.
In the present study, we have thus first explored the above mentioned aspects in the Syrian Golden hamster. Hemodynamic variables were measured after ET receptor blockade in anesthetized animals as well as in conscious hamsters by radio- telemetry for the first time in this particular animal model. Second, experiments were conducted in anesthetized animals after chronic treatments to assess pressor responses to exogenous ET-1 to characterize the impact of ETB receptor blockade on ETA-mediated pressor effects.
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MATERIALS AND METHODS |
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Dose-Dependent Pressor Response Curves in Anesthetized Hamsters
Male Syrian Golden hamsters (Charles River, Montréal, PQ, Canada; 145–185 g body wt) were anesthetized with a mixture of ketamine and xylazine (87 and 13 mg/kg im, respectively) supplemented as required throughout the experiment. Polyethylene catheters (PE-10, Becton Dickinson, Franklin Lakes, NJ) were inserted in the left jugular vein for drug administration and in the right carotid artery for recording of hemodynamic variables by means of a blood pressure analyzer (model 400, Micromed, Louisville, KY), as previously described (15). After 20 min of equilibration, basal hemodynamic variables were recorded for 15 min, and pressor responses to intravenously injected ET-1 or IRL-1620, a selective ETB receptor agonist (0.01–2.5 nmol/kg for both agonists), were recorded over 30 min. Data were analyzed by determination of the maximal increases in mean arterial blood pressure (MAP) after agonist injection. Because of the tachyphylactic properties of ET pressor responses, each hamster received only a single dose of one agonist.
Effects of Chronic ET Receptor Antagonists on Hemodynamic Variables of Anesthetized Hamsters
In another set of experiments, 45 animals were separated into 5 groups with distinct treatments: 1) 0.9% saline containing 2 mol equivalents of sodium hydroxide solution (vehicle), 2) atrasentan (6 mg·kg–1·day–1), 3) A-192621 (0.5 mg·kg–1·day–1), 4) A-192621 (30 mg·kg–1·day–1), and 5) a mixture of atrasentan and A-192621 (6 and 30 mg·kg–1·day–1, respectively). Antagonists (atrasentan and A-192621) were administered by gavage twice a day, at 8 AM and 6 PM, over 16 days.
On day 14, at around 3 PM, hamsters in each group were lightly anesthetized with 2% halothane (MTC Pharmaceuticals, Cambridge, ON, Canada). A blood sample (1.5 ml) was harvested by orbital sinus puncture, centrifuged for 1 min at 19,900 g for plasma separation, and stored at –80°C until determination of plasma immunoreactive ET (irET) levels. Each hamster was observed for 15 min during reversal of anesthesia, verified for any physical aftereffects, and subsequently returned to a cage until the day of the experiment.
On day 16, 2–4 h after the morning gavage, all hamsters were anesthetized with a mixture of ketamine and xylazine (87 and 13 mg/kg im, respectively), and basal hemodynamic variables were recorded as specified above. Experiments using intravenously injected IRL-1620 (0.25 nmol/kg) or ET-1 (0.25 nmol/kg) were also conducted after baseline recordings. For this last series of experiments, data were analyzed by evaluation of the maximal variation of MAP starting from baseline (i.e., 2 s before injection of the agonist) and by calculation of the area under the curve using GraphPad Prism software (San Diego, CA).
Effects of a Chronic ETB Antagonist on Hemodynamic Variables in Conscious Hamsters
Radio-telemetry. In another group of hamsters, a telemetry probe was implanted on the basis of a previously reported method in the murine model (6). Briefly, hamsters were anesthetized with a mixture of ketamine and xylazine (87 and 13 mg/kg im, respectively) and treated with an analgesic (buprenorphine hydrochlorate, 0.5 mg/kg sc). Under sterile conditions, a 25- to 30-mm skin incision was made along the ventral neck area. After isolation of the left common carotid artery, vessel cannulation forceps were used to insert the tip of the telemetry probe (model TA11PA-C20, Data Science International, St. Paul, MN) into the lumen of the artery. The transmitter, placed subcutaneously along the left flank of the thoracic region, was then secured with Vetbond, and the incision was sutured with 4-0 silk. A 10-day recovery period was allowed for each animal before initiation of experiments. Subsequently, hemodynamic variables [systolic arterial blood pressure (SBP), MAP, and diastolic arterial blood pressure (DBP), pulse pressure, and heart rate (HR)] were monitored.
Chronic treatment with high dose of A-192621. Experiments in instrumented-conscious hamsters started with 2 days of baseline variable recording. Gavages were then initiated with 7 days of vehicle administration (0.9% saline containing 2 mol equivalents of sodium hydroxide) followed by 14 days of gavage with A-192621 (30 mg·kg–1·day–1 at 8 AM and 6 PM) and 3 days of washout, without any drug or vehicle administration. Averaged hemodynamic data were monitored daily for 15 s/min, from 3:00 to 4:30 PM.
After completion of the study and euthanasia of animals by CO2 inhalation, the exact location of the catheter tip into the aortic arch was confirmed.
Measurement of Plasma irET Levels
Plasma irET levels were measured by radioimmunoassay (model RPA-555, Amersham Biosciences, Baie d’Urfé, PQ, Canada), as previously described (14). ET recovery was determined at 65% by addition of exogenous ET to the samples before extraction. Plasma irET concentrations are shown uncorrected for extraction recovery.
Drugs and Solutions
Atrasentan and A-192621, which have been reported to be effective in vitro (29) and in vivo (27), were obtained from Abbott Laboratories (Abbott Park, IL). ET-1 and IRL-1620 were purchased from American Peptide (Sunnyvale, CA) and dissolved in phosphate-buffered saline.
Statistical Analyses
Values are means ± SE. Statistical analyses were performed using GraphPad Instat software (San Diego, CA) by ANOVA, followed by Tukey’s post hoc test for multiple comparisons, unless otherwise noted (see Fig. 4). Comparisons between only two groups were made using Student’s t-test. P < 0.05 was considered significant.
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RESULTS |
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As previously reported (16) and illustrated in Fig. 1A in the anesthetized hamster, IRL-1620, a selective ETB receptor agonist, induces a characteristic transient hypotensive phase followed by a sustained pressor phase. Conversely, ET-1 only triggers a sustained increase in MAP (Fig. 1B), as previously shown in the same animal model (16).
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Chronic blockade of the ETA receptor with atrasentan induced a significant decrease of basal MAP as well as SBP and DBP (Fig. 3). A-192621 (0.5 mg·kg–1·day–1) did not alter basal MAP, in contrast to the higher dose (30 mg·kg–1·day–1) of the same antagonist (Fig. 3). Interestingly, on simultaneous administration of atrasentan and A-192621 (30 mg·kg–1·day–1), no variation of basal arterial blood pressures was noted compared with the vehicle-treated group.
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Effects of Chronic ETB Receptor Blockade on Blood Pressure Variables in Conscious Hamsters
Basal SBP, MAP, and DBP were not significantly different in conscious and ketamine-xylazine-anesthetized hamsters (Table 1). However, HR was markedly lower in anesthetized hamsters than in conscious animals.
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Pulse pressure (Fig. 4B) and HR (Fig. 4C) were stable during the 26-day experiment, although A-192621 had a tendency to reduce HR in conscious hamsters, as previously reported in the rat model (22).
Effects of Chronic Treatments With Atrasentan and/or A-192621 on Plasma irET Levels
Plasma irET levels of vehicle-treated hamsters averaged 3.2 ± 0.2 fmol/ml (Fig. 5; n = 13). Neither atrasentan nor A-192621 at 0.5 mg·kg–1·day–1 altered these levels. However, A-192621 at 30 mg·kg–1·day–1 significantly increased irET levels by 115% compared with the control group. In addition, a combined blockade of ETA and ETB receptors very markedly enhanced plasma irET-1 by >1,000% (Fig. 5).
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Variations of MAP after intravenous injection of IRL-1620 in vehicle- and A-192621-treated hamsters are summarized in Table 2. A-192621 at 0.5 mg·kg–1·day–1 markedly reduced the transient hypotensive, but not the sustained pressor, response induced by IRL-1620. A-192621 at 30 mg·kg–1·day–1 also reduced the hypotensive phase, but, in addition, efficiently inhibited the increase in MAP induced by IRL-1620 (Table 2).
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A-192621 (0.5 mg·kg–1·day–1) enhanced the maximal increase in MAP (Fig. 6A) as well as the long-lasting pressor effects of intravenously injected ET-1, as determined by area under the curve (Fig. 6B).
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Simultaneous blockade of ETA and ETB receptors with atrasentan and A-192621 (at 6 and 30 mg·kg–1·day–1, respectively) markedly potentiated the ET-1-induced increase of maximal MAP variation and the sustained pressor response in a fashion similar to that observed with A-192621 treatment alone (Fig. 6).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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With regard to basal physiological variables, MAP and plasma irET were not modified after 2 wk of treatment with the lower dose of A-192621. Efficacy of this low dose of antagonist on endothelial ETB receptors was, however, confirmed, because the antagonist markedly reduced the transient depressor but not the sustained pressor phase induced by IRL-1620.
In contrast, the higher dose of A-192621 significantly reduced both hemodynamic responses. Furthermore, this high dose of antagonist induced a hypertensive state in anesthetized animals that was associated with an enhanced plasma irET level. These observations are in accordance with the data reported by Williams et al. (28) and Reinhart et al. (23) in rats and nonhuman primates, respectively. In addition, Goddard et al. (13) recently reported that acute treatment with a peptidoic ETB receptor antagonist (BQ-788) also increased plasma ET-1 levels 120 min after a 15-min intravenous infusion at 300 nmol/min in human subjects. A notable difference between the present results in hamsters and those reported by Goddard et al. (13) in humans is that, in the latter study, administration of BQ-788 with an ETA receptor antagonist, BQ-123, did not trigger a further increase of irET-1 compared with administration of the ETB receptor antagonist alone. We suggest that the difference is due to the acute experimental setting adopted by Goddard et al. (13) in contrast to the chronic 2-wk protocol used in the present study. Furthermore, one cannot exclude interspecies variations.
On the other hand, radio-telemetric results in the present study indicate that all baseline pressure-related variables were not affected by ketamine-xylazine, with the notable exception of HR, which was decreased by the anesthetics. Furthermore, in our experimental setting, treatment with A-192621 tended to reduce HR, albeit not significantly. Interestingly, MAP was markedly increased in conscious hamsters chronically treated with the ETB receptor antagonist.
Thus blockade of endothelial and VSMC ETB-dependent hemodynamic responses with the higher dose of A-192621 triggers a marked hypertension in nonanesthetized and anesthetized hamsters.
In this condition of endothelial and VSMC ETB receptor blockade, addition of the ETA receptor antagonist atrasentan prevented the hypertensive state induced by A-192621 in anesthetized animals. This dual antagonism also induced a further increase of circulating irET levels. This phenomenon may rely on the impeded binding of ET-1 to ETA receptors combined with the full blockade of ETB receptors after concomitant ETA and ETB receptor antagonism.
It is known that lungs, kidneys, and liver are the primary organs involved in ET-1 plasma clearance, through receptor- or non-receptor-mediated phenomena (5, 12). Although the former mechanism deserves to be fully explored and the latter mechanism may be important when ET receptors are blocked, our results suggest that non-receptor-mediated clearance is insufficient to avoid the development of hypertension in the hamster model.
Hence, notwithstanding their contribution in the hypotensive response triggered by IRL-1620, our results demonstrate that, in the hamster, ETB receptors expressed on endothelial cells are only remotely involved in ET clearance. Conversely, VSMC ETB receptors appear to be significantly involved in removing excess of circulating ET-1.
In contrast to the concept summarized above, the current wisdom suggests that endothelial ETB receptors are responsible for clearance of the endogeneous peptide (11). In the present study, the contribution of endothelial and VSMC ETB receptors to ET clearance was not directly investigated at the cellular level, inasmuch as such a study would be extremely difficult to perform in a dynamic in vivo system. Nonetheless, our results suggest that ETB receptors involved in the hypotensive properties of the 21-amino acid peptide may be restricted to a physiological modulation, rather than an important role, in clearance of the endogenous ET-1.
Alternatively, endothelial ETB receptors may be involved in the clearance of ET-1 only when the VSMC ETB receptor population is unavailable for reducing the circulating levels of the peptide. When endothelial and VSMC ETB receptors are blocked, the spillover of circulating ET-1 levels is indeed exacerbated, as shown in the present study.
It has been suggested that ETB receptors expressed in the kidney are involved in the control of MAP in rats fed a high-salt diet (22). It has also been proposed that intrarenal ET-1 modulates sodium excretion via an ETB receptor-dependent mechanism (17). Recently developed transgenic animal models with systemic or cell-specific gene alteration further support this view (1, 18). Consequently, in vivo investigations assessing functions of renal cell-specific ETB receptors may be pivotal in our understanding of the role of ETB receptors in salt-dependent and salt-independent hypertension.
In the present report, the ETB receptor population involved in the depressor response to IRL-1620 significantly modulates the increase of MAP triggered by exogenously administered ET-1. Studies addressing such regulation have been performed in several animal models (14, 19). However, the present pharmacological approach highlights the prominent role possibly displayed by the vascular endothelium through the release of vasodilatory substances such as nitric oxide and prostacyclin (9). Furthermore, the potent and selective ETA receptor antagonist atrasentan reduces the long-lasting pressor effects of ET-1 only when endothelial and VSMC ETB receptors are pharmacologically unaltered. This particular result confirms the physiological antagonism afforded by the mixture of ETA and ETB antagonists.
ETB receptor blockade has already been suggested to oppose the in vivo effects of another selective ETA receptor antagonist (BQ-123) (2, 16). In the hamster, on the other hand, acute administration of atrasentan with A-192621 (6 and 30 mg/kg iv, respectively) abolished the pressor responses to ET-1 (15). Surprisingly, we report here that the same combination of antagonists, in a chronic setting, potentiates the pressor response to ET-1. Indeed, the low dose of A-192621 only reduced the hypotensive response induced by IRL-1620, whereas the higher dose of the same antagonist efficiently reduced the pressor phase of the selective ETB receptor agonist. In Fig. 6, the low dose of A-192621 and the mixture of atrasentan and A-192621 (at 30 mg·kg–1·day–1) similarly potentiated the pressor response to ET-1, demonstrating a loss of antagonism of the ETA receptor blocker when it was administered concomitantly with the ETB receptor antagonist.
Furthermore, it is noteworthy that the dose of A-192621 used to reduce the pressor response to IRL-1620 was much higher than that required to virtually abolish the hypotensive phase induced by the same ETB receptor-selective agonist. This result supports the notion that ETB receptors on the endothelium are far more sensitive than those on the VSMCs (26).
Importantly, the efficacy of atrasentan is likely reduced as a result of the marked increase in endogenous ET plasma levels (>1,000%) with simultaneous ETA and ETB blockade.
Thus our results obtained in healthy Syrian Golden hamsters suggest that selective ETA receptor antagonists would be preferable to nonselective ETA/ETB receptor blockers with higher affinity for the ETA than the ETB receptor, such as bosentan (7), to avoid in chronic settings the vasopressor effects associated with the selective interference of ETB receptors.
In conclusion, our data demonstrate the key role of ETB receptors in the physiological modulation of MAP and exogenously ET-1-induced pressor responses in the hamster. To prevent the hypertensive state induced by increased circulating ET-1 levels, VSMC ETB receptors may be involved in processes whereby ET-1 is cleared from the plasma. In addition, vasodilator substances released by the endothelial ETB receptor are pivotal in counteraction of ET-1-induced pressor responses.
Because efficacy of a selective ETA receptor antagonism has been demonstrated in the cardiomyopathic hamster (30), characteristics of ETB receptors highlighted in the present study may need to be revisited in this pathophysiological animal model.
Altogether, the present results may contribute to a better understanding of the roles of endothelial and VSMC ETB receptors in the rational design of ET receptor antagonists in diseases such as congestive heart failure (25), hypertension (20), and coronary artery diseases (3).
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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) and IRL-1620 (
) in anesthetized hamsters. Average basal MAP was 99.0 ± 2.1 mmHg (n = 59). Values are means ± SE of number of animals in parentheses. **P < 0.01 vs. IRL-1620 at the same dose.
