INTRODUCTION

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ENDOTHELIN (ET) peptides are a family of structurally related 21-amino acid peptides capable of exerting an array of physiological effects, many with the potential to alter arterial pressure and circulatory function. Of the three isopeptides, ET-1 is recognized as the most physiologically relevant isoform and is synthesized as a precursor molecule by endothelial cells and other cell types (14, 21, 33). ET-1 is cleaved from a larger, inactive precursor polypeptide through a sequence of highly specific proteolytic steps. It potently constricts the peripheral vasculature, alters renal hemodynamic and tubular function, stimulates mitogenesis in vitro, and interacts with an array of cell and tissue types through activation of cell surface, ligand-specific receptors (14, 21, 33).

The physiological effects of ET-1 are mediated by two distinct, differentially expressed receptor subtypes designated ET_A and ET_B, each coded by a specific gene and possessing some overlap in their tissue distribution (1, 30). In the peripheral vasculature, ET_A receptors are expressed primarily on the surface membrane of vascular smooth muscle cells where they mediate, in a large part, the potent and characteristically sustained vasoconstrictor response associated with administration of exogenous ET peptides. ET_B receptors are also expressed by vascular smooth muscle cells albeit less densely than ET_A receptors and may mediate a portion of ET-induced vasoconstriction in many vascular beds (14, 19, 33). Also, and in contrast to ET_A receptors, ET_B receptors are highly expressed on vascular endothelial cells, where they mediate the rapid, transient vasodilation produced by exogenous ET through the enhanced generation of nitric oxide and prostaglandin-related substances (14, 15, 21, 33). Relative to ET_A receptors, the role of ET_B receptors in vascular homeostasis has been difficult to define, because ET_B receptors are known to mediate constriction in some vascular beds (3, 19, 27, 32) but also mediate transient hypotension when challenged appropriately (14, 21, 33). Acute blockade of ET_B receptors produces prompt increases in plasma concentrations of immunoreactive ET-1 in both the dog and rat, suggesting that the receptor functions as a clearance receptor and eliminates ET-1 from the plasma compartment (8, 9). The clearance function of the ET_B receptor is aided by equal affinity for all three ET isoforms, whereas the ET_A receptor selectively binds only ET-1 at physiological concentrations of the peptide (14).

Genetic disruption of ET_B receptor expression has been reported to increase arterial pressure in both mice and rats in association with increased circulating ET-1 levels (10, 22). Also, chronic administration of the ET_B-selective antagonist A-192621 produces or exacerbates experimental hypertension in rats (2, 20, 28, 35). Given the potential for ET_B activation to alter both vascular and renal function, the mechanism by which ET_B suppression induces hypertension is of considerable interest. In conscious mice in which ET_B expression is reduced to approximately one-eighth of normal (complete ET_B knockout is a lethal construct), arterial pressure is elevated relative to wild-type controls; acute ET_A blockade reduces but does not normalize blood pressure compared with control animals (22). In rats, sustained pharmacological blockade of ET_B receptors produces hypertension (28, 35), a response that is abolished by subsequent ET_A + ET_B blockade (28). Thus ET_A receptors may at least partly mediate the hypertension produced by suppression or blockade of ET_B receptors in rats.

The objective of the present study was to characterize the effects of sustained systemic ET_B receptor blockade on arterial pressure homeostasis and plasma ET-1 levels in conscious cynomolgus primates by using radiotelemetry techniques. In addition, we tested the hypothesis that the increase in arterial pressure produced by ET_B receptor blockade is indirectly mediated by activation of the ET_A receptor.

	METHODS

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Animal preparation. Seven male cynomolgus primates weighing 5.3-8.2 kg were used in this study. All procedures were approved by Abbott Laboratories' Institutional Animal Care and Use Committee and carried out in American Association for Accreditation of Laboratory Animal Care accredited facilities. For surgical implantation of the telemetry transmitter, anesthesia was induced with ketamine (10 mg/kg im) followed by isofluorane (1-1.5%) as described previously (29). With the use of aseptic techniques, a ventral midline incision was made, and a telemetry transmitter (model TL10M2-D70-PC or D70-PCT, Data Sciences International) was anchored in the abdominal cavity with nonabsorbable suture. A gel-filled catheter connected to the transmitter was tunneled to the femoral area. Via a small femoral incision, the catheter was inserted into a femoral artery, advanced into the abdominal aorta, and secured, and the femoral incision was closed. The abdominal incision was closed in layers. In addition, a venous vascular access port (Access Technologies) was implanted subcutaneously and contralateral to the telemetry transmitter; the connecting catheter was secured in the femoral vein in a manner similar to the arterial catheter. Preoperatively, the animals were treated with K⁺ penicillin (20,000 U/kg iv) and gentamicin (2 mg/kg iv). Postoperatively, buprenorphine (0.01 mg/kg im bid) was given to provide analgesia. Amoxicillin (5 mg/kg) and trimethoprim and sulfadiazine (Tribrissen, 30 mg/kg) were given postoperatively for a period of 5-7 days. The vascular access ports were flushed with sterile saline biweekly or after each use and filled with a sterile solution of saline containing heparin (1,000 U/ml) and gentamicin (3.2 mg/ml).

Experimental protocol. A 4-day control period was carried out to establish baseline values for MAP, heart rate, hematocrit, and plasma concentrations of ET-1 and electrolytes before treatment. Throughout the entire 24-day protocol, the animals were fed banana (or banana containing the active drug) twice daily at 12-h intervals (7:30 AM and 7:30 PM). On completion of the 4-day control period, the ET_B antagonist A-192621 (35) was placed in cored bananas and administered twice daily. Dose-dependent systemic ET_B receptor blockade was accomplished by administering A-192621 at 0.1, 1.0, and 10 mg/kg (bid) in ascending order; each dose was administered for a period of 4 days. After completion of the fourth day of high-dose (10 mg/kg) ET_B blockade, the animals were coadministered A-192621 (high dose) and the ET_A selective antagonist atrasentan (5 mg/kg bid) (24) for another 4 days. Subsequently, the animals were given plain banana during a final, 4-day posttreatment period. Atrasentan, also known as ABT-627 or A-147627, is a pharmacologically active enantiomer of A-127722, a chiral ET_A antagonist that has been described previously (23).

Analytic methods. Plasma concentrations of sodium and potassium were measured in fresh heparinized plasma by using a Synchron El-Ise Electrolyte System (Beckman). Hematocrit was determined by using a micromethod. Plasma concentrations of ET-1 were determined by ELISA (QuantiGlo, R&D Systems; Minneapolis, MN).

Statistical analysis. Results are expressed as the group means ± SE. Experimental and recovery data were compared with control using ANOVA with Dunnett's t-test for repeated measures (7).

	RESULTS

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Daily values for 24-h MAP and heart rate and responses to increasing levels of systemic ET_B blockade followed by simultaneous blockade of ET_B + ET_A receptors are summarized in Fig. 1. Administration of the ET_B antagonist A-192621 at the low dose (0.1 mg/kg bid) had no effect on MAP or heart rate. MAP tended to increase in response to 1.0 mg/kg A-192621, but these changes were not statistically significant. In contrast, MAP increased from a control value of 79 ± 3 to 87 ± 3 mmHg (P < 0.05) during the first day of the high-dose ET_B blockade and to 89 ± 3 mmHg (P < 0.05) on the fourth day. Subsequently, when ET_A blockade was superimposed on ET_B blockade by coadministration of A-192621 + atrasentan, MAP fell from the hypertensive value of 89 ± 3 to 75 ± 2 mmHg (P < 0.05) during the first day. MAP decreased even further on the second day of dual ET_B + ET_A blockade to 70 ± 3 or 9 mmHg below control; MAP was 71 ± 4 mmHg (P < 0.05) on the fourth day of ET_B + ET_A blockade. MAP remained suppressed at or near these hypotensive levels throughout the 4-day posttreatment period. Thus sustained hypertension was produced by the high dose of A-192621 and reversed to below baseline levels by dual ET_B + ET_A blockade, suggesting ET_A receptors mediate the hypertensive effects of ET_B blockade in nonhuman primates.

View larger version (44K): [in this window] [in a new window]   Fig. 1.   Effects of three levels of systemic endothelin-B (ETB) receptor blockade and effects of coblockade of ETA and ETB receptors on 24-h values for mean arterial pressure and heart rate in conscious primates. Values are means ± SE; n = 7 primates. *P < 0.05 vs. pretreatment baseline values.

Heart rate was not significantly different from control values on any day during administration of the ET_B receptor antagonist A-192621 (Fig. 1). However, coblockade of ET_B + ET_A receptors subsequent to ET_B blockade-induced hypertension elicited a pronounced and sustained tachycardia in association with marked hypotension; heart rate increased from a control value of 113 ± 5 to 132 ± 5 beats/min (P < 0.05) during the first day of dual ET_B + ET_A blockade and increased to 133 ± 7 beats/min on the fourth day of the treatment period. Subsequently, heart rate remained elevated during the first day of the posttreatment period. Heart rate declined toward control values during the second day and was not significantly different from control values thereafter.

The hematocrit (control = 47 ± 1%) was not significantly affected by the low dose of A-191621 but did decrease modestly, but significantly, on subsequent days; hematocrit decreased to 44 ± 1% (P < 0.05) on the first day of the 1 mg/kg dose of A-192621 and was 45 ± 1% (P < 0.05) 8 days later, following the first coadministration of A-192621 + atrasentan. After 4 days of dual ET_B + ET_A blockade, hematocrit fell to 41 ± 1% (P < 0.05) and declined to 40 ± 1% (P < 0.05) 4 days after treatment was terminated.

Plasma sodium (control = 145.3 ± 1.6 meq/l; n = 5) and plasma potassium (control = 3.8 ± 0.2 meq/l; n = 5) concentrations were not significantly different from control at any time during the experimental protocol.

The effects of dose-dependent ET_B blockade and subsequent coblockade of ET_B + ET_A receptors on the plasma concentration of ET-1 in conscious primates are summarized in Fig. 2. Plasma ET-1 concentration was not significantly affected by the low and middle dose of the ET_B antagonist A-192621. In contrast, plasma ET-1 concentration increased from a control value of 2.1 ± 0.3 to 7.24 ± 1.0 pg/ml (P < 0.05) 3 h after administration of the first high dose (10 mg/kg) of A-192621; on the fourth day of ET_B blockade, plasma ET-1 concentration increased to 11.03 ± 2.37 pg/ml (P < 0.05). Subsequent coadministration of A-192621 + atrasentan caused plasma ET-1 concentration to increase further to 26.64 ± 3.72 and 28.65 ± 2.89 pg/ml (P < 0.05) on the first and fourth day of the dual treatment period, respectively. On the first day when administration of A-192621 + atrasentan was terminated, plasma ET-1 concentration remained elevated at 25.54 ± 4.23 pg/ml (P < 0.05); at 4 days posttreatment, plasma ET-1 concentration had declined and was not different from pretreatment values.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Effects of systemic ETB and ETB + ETA blockade on plasma concentration of ET-1 in conscious primates. Values are means ± SE; n = 7 primates. *P < 0.05 vs. pretreatment baseline values.

	DISCUSSION

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In a recent study from our laboratory, we demonstrated that ET_A receptor blockade in normal conscious cynomolgus primates produces a sustained hypotension and a modest elevation in plasma ET-1 concentration (29). In the present study, also in conscious primates, we demonstrate a dose-dependent hypertension in response to sustained, systemic ET_B receptor blockade; this hypertension is completely abolished by subsequent dual blockade of ET_B + ET_A receptors. Thus selective, systemic blockade of ET_A or ET_B receptors produces markedly different effects on arterial pressure and ET-1 levels in the conscious primate, whereas the hypertension produced by ET_B blockade is mediated by activation of the ET_A receptor.

Acute systemic blockade of ET_B receptors increases arterial pressure or systemic vascular resistance in experimental animals and healthy human volunteers (34), respectively. Genetic suppression of ET_B receptor expression has been shown to produce hypertension in mice (22) and salt-dependent hypertension in rats (10, 20). In normal rats on a normal salt intake, prolonged administration of the selective ET_B antagonist A-192621 produces hypertension (27, 33) and exacerbates the hypertension produced by a high-salt diet (28). Chronic administration of A-192621 also exacerbates angiotensin II-induced hypertension in rats (2) but has been reported to either increase (26) or have no effect (20) on systolic pressure in deoxycorticosterone acetate-salt rats. Finally, blockade of ET_B receptors with A-192621 has been shown to markedly increase plasma levels of ET-1 in the rat, whereas the effects of ET_A blockade on plasma ET-1 concentration are modest to negligible in rats and primates (24, 29).

Whereas the hypertensive effects of ET_B suppression have been relatively consistent between studies, the mechanism by which ET_B deficiency produces hypertension in experimental animals has been unclear. Because ET_B activation can lead to transient hypotension and dilation of some vascular beds, a loss of ET_B-dependent vasodilator tone (leading to increased vasoconstriction) has been suggested as a possible mediator of this hypertensive response. Second, elevations in plasma ET-1 levels and increased ET_A activation as a result of reduced clearance of ET-1 is another potential mechanism and one supported by the present study. Finally, given the dominant role of the kidney in regulating arterial pressure (6, 12), the renal effects of altered ET_B activity must also be considered. Because low-level exogenous ET-1 (and ET_B receptor activation) elicits acute diuretic and natriuretic responses in vivo (31, 33), it has been hypothesized that intrarenal blockade of ET_B receptors may increase sodium and fluid reabsorption at the level of the renal tubule, thereby producing antinatriuretic effects that, if maintained, would lead to retention of fluid and electrolytes and possible increases in arterial pressure (17).

In the present study, subacute, oral administration of the ET_B-selective antagonist A-192621 produced a sustained increase in MAP and concomitant increases in plasma ET-1 concentration. To our knowledge, this is the first demonstration of hypertension produced by ET_B receptor blockade in a nonrodent species. At the highest level of ET_B blockade tested, 24-h MAP values remained elevated throughout the 4-day treatment period, achieving values 10 mmHg above control while plasma ET-1 concentration increased fivefold.

Once ET_B blockade-induced hypertension was established for 4 days, we quantified the contribution of ET_A receptors in mediating this response by coadministering the orally active, ET_A-selective antagonist atrasentan while continuing the hypertensive dose of A-192621. As a result, MAP fell promptly from hypertensive levels to values significantly below control on the first day of dual ET_B + ET_A blockade. Moreover, MAP continued to decrease, and, by the second day of dual blockade, MAP fell another 5 mmHg. This hypotensive response was accompanied by a marked and sustained tachycardia that likely reflects a compensatory response to the sustained fall in MAP. Finally, compared with baseline values, the hypotension produced by ET_A blockade in monkeys made hypertensive by ET_B antagonism was similar to that of monkeys possessing a fully intact ET system (29). Viewed collectively, these data suggest that the hypotension produced by ET_A blockade in the present study was not appreciably altered by, or dependent on, pretreatment with the ET_B antagonist A-191621. Thus these data indicate that hypertension produced by sustained ET_B receptor blockade in the conscious nonhuman primate is mediated indirectly, but completely, by activation of ET_A receptors, presumably through increased circulating concentrations of ET-1.

A similar ET_A dependency of ET_B-induced hypertension has been reported recently in several studies in rats. In telemetry-instrumented rats fed a low-salt diet and studied under a paradigm similar to that used in the present study, chronic administration of A-192621 produced hypertension that was abolished by subsequent coadministration of A-192621 + atrasentan (28). In the same study, rats fed a high-salt diet displayed increased arterial pressure, a response that was exacerbated by ET_B blockade with A-192621. Subsequent coadministration of A-192621 + atrasentan did not ameliorate the salt-dependent hypertension but did negate the additional increase in arterial pressure produced by A-192621.

In hypertensive ET_B receptor-deficient mice (22) or ET_B-deficient rats (10), acute administration of an ET_A receptor antagonist reduced but failed to completely normalize arterial pressure within several hours of treatment; the effects of long-term treatment were not assessed. It is our view that the partial amelioration of hypertension in ET_B-deficient rodents by acute ET_A blockade is consistent with the present study in which the hypotensive effects of ET_A antagonism were not fully apparent until the second 24-h period.

In addition to causing hypertension in conscious nonhuman primates, systemic blockade of ET_B receptors by A-192621 produced a dose-dependent increase in the plasma ET-1 concentration. At hypertensive levels of ET_B blockade, the plasma ET-1 concentration increased to more than threefold above control values within 3 h of treatment. This relatively rapid increase suggests that reduced clearance of ET-1 is the primary contributor to enhanced plasma ET-1 levels produced by acute ET_B blockade, because the temporal response is not consistent with increased protein synthesis.

When ET_A blockade was superimposed on preexisting ET_B blockade, the plasma ET-1 concentration increased again, also within 3 h of treatment, achieving levels ~2.5-fold above those produced by ET_B blockade alone or ~14-fold above control. This novel observation is in contrast to the effects of ET_A-selective blockade where plasma ET-1 levels remained unchanged during the first day and nearly doubled after 4 days (29). These data are consistent with acute studies in rats (18) and data generated in rats studied after 3 days of combined or selective ET_A or ET_B receptor blockade (24). In the present study, the acute, additive effects of ET_A blockade on ET-1 levels during preexisting ET_B blockade suggest a modest, net clearance function for the ET_A receptor, an observation that is consistent with reports of cellular internalization of ET_A-ET-1 receptor-ligand complexes (4). The effects of sustained ET_B blockade and elevated ET-1 levels on ET_A receptor expression and turnover patterns are unknown.

Because the ET system is largely viewed to function as a paracrine or autocrine system, it is important to consider the relevance of plasma ET-1 to changes in circulatory and end-organ function. Numerous studies have reported increased plasma levels of ET-1 in association with hypertension and other pathologies in patients and experimental animals (21, 36). In the present study, a threefold increase in plasma ET-1 concentration occurred in association with marked hypertension during ET_B blockade in conscious primates. These results are similar to those reported by Wilkins et al. (37) in which conscious chronically instrumented dogs were infused with ET-1 to produce sustained increases in plasma ET-1 levels two- to threefold above baseline, causing chronic hypertension. Thus, in both conscious dogs and nonhuman primates, modest increases in plasma ET-1 concentration produced through independent means (intravenous ET-1 infusion or systemic ET_B receptor blockade) caused a sustained hypertension. Therefore, excess plasma ET-1, whether derived directly from intravenous infusion or through spillover caused by reduced clearance of ET-1 during ET_B receptor blockade, produces sustained hypertension, suggesting the circulating peptide is freely accessible to functional sites in the kidney and other tissues involved in the long-term control of arterial pressure.

Studies in the anesthetized rat (13, 16), anesthetized dog (5), and conscious dog (31) demonstrate that low-level ET-1 or ET_B receptor activation produces natriuretic and diuretic effects. Studies performed in isolated rat nephron segments are consistent with these in vivo observations (11, 25). Thus it has been hypothesized that at normal levels of endogenous activation, ET_B receptors exert a natriuretic tone such that loss of this tone through ET_B suppression or ET_B blockade may produce antinatriuetic effects and therefore cause hypertension (17). However, this hypothesis is not supported by the present study in the conscious nonhuman primate or other studies in rats (28), because the hypertension produced by ET_B blockade was completely abolished by adding ET_A receptor blockade to the treatment regimen in both species.

The present data provide important insight regarding the function of ET_B and ET_A receptors and their influence on circulatory function. The ET_B receptor appears to play an important, overall clearance function that allows the receptor to modulate arterial pressure homeostasis indirectly, by minimizing ET_A tone through the clearance of ET-1 from the plasma. Because the ET_B receptor displays equal affinities for the other ET isoforms, the receptor is well suited to perform a clearance function. In addition, these data and those from our previous study point to the importance of the ET_A receptor in the long-term maintenance of normal arterial blood pressure in the nonhuman primate and an important interdependence of ET_A and ET_B receptors in determining the influence of endogenous ET-1 on circulatory homeostasis.

In summary, the present study demonstrates that continuous, systemic blockade of ET_B receptors in conscious primates produces a sustained hypertension and concomitant marked elevations in plasma ET-1 concentration. Moreover, the hypertensive effects of systemic ET_B blockade were completely abolished by subsequent ET_A blockade, with arterial pressure falling to below baseline values despite further increases in plasma ET-1 concentration. These results demonstrate that in the nonhuman primate, hypertension induced by ET_B blockade is wholly mediated through the indirect activation of ET_A receptors, presumably via increased circulating ET-1. These data suggest a critical clearance function for ET_B receptors, one that, by virtue of the potent ability of ET_A receptors to alter long-term blood pressure control, is especially relevant to normal circulatory homeostasis.