\E Role for Endothelin-1 in Angiotensin II– Mediated Hypertension
Abstract Experiments in cultured vascular smooth muscle cells have shown that angiotensin II (Ang II) stimulates expression of endothelin-1. We sought to examine role of endothelin-1 in the effects of Ang II in vivo. Ang II infusion in rats (0.7 mg/kg per day for 5 days) was associated with marked increases in vascular smooth muscle endothelin-1 levels, as assessed by immunostaining. Administration of the selective endothelin type A (ETA) receptor antagonist PD 155080 (50 mg/kg per day) abrogated the hypertensive response to a 5-day infusion of Ang II (0.7 mg/kg per day), as did losartan (25 mg/kg per day). ETA receptor blockade during Ang II–mediated hypertension was associated with marked elevations of plasma endothelin-1 levels. Ang II–mediated hypertension was associated with heightened vascular responsiveness to a variety of vasoconstrictor agents except endothelin-1. Blockade of ETA receptor invariably corrected this vasoconstrictor hyperresponsiveness. We conclude that some of the vascular effects of Ang II thought to be unique to this hormone are likely mediated by endothelin-1.
Traditionally, it has been assumed that the vasoconstriction and hypertension caused by Ang II is related to a direct action on the AT1 receptor and subsequent activation of second messenger pathways immediately linked to the receptor. These signaling events include increases in intracellular calcium, stimulation of protein kinase C, and activation of other phosphorylation pathways.1 During the past 5 years, it has been shown that Ang II can stimulate expression of preproendothelin mRNA and protein in both cultured vascular smooth muscle2 and endothelial3 4 cells. This has been shown to occur via activation of the AT1 receptor2 3 5 6 and presumably involves activation of transcription via activator protein-1/protein kinase C–mediated mechanisms. Furthermore, in rat cultured cardiomyocytes, endogenously produced ET-1 contributes to the hypertrophic response to Ang II.5
These observations in tissue culture raise the possibility that ET-1 might in part be responsible for alterations of vascular tone encountered in conditions in which Ang II is chronically elevated. This hypothesis is attractive because ET-1 is an extremely potent vasoconstrictor and may have other effects on BP regulation, such as stimulation of aldosterone synthesis7 and of conversion of Ang I to Ang II. Despite these considerations, the evidence that endothelin has any role in hypertension is inconclusive. Although plasma levels of ET-1 are elevated in patients with essential hypertension, there is a poor correlation between these levels and the degree of hypertension.8 Furthermore, mice deficient in the ET-1 gene have paradoxically elevated BP.9
Recently, selective antagonists of ET-1 receptors have become available that permit the study of the role of this peptide in various pathophysiological conditions. In the present study, we examined the hypothesis that locally generated ET-1 might contribute to alterations of vascular tone and hypertension caused by chronic elevations of angiotensin by examining the effects of a selective ETA receptor antagonist on Ang II–mediated hypertension.
Male Sprague-Dawley rats (250 to 300 g) were housed under constant temperature and humidity with a 12-hour light/dark cycle. Before and during the experimental period, all rats had free access to a standard rat chow and water. The rats were anesthetized with intraperitoneal ketamine (80 mg/kg) and xylazine (10 mg/kg). With the use of sterile techniques, a catheter (medical-grade Tygon) was implanted through the left carotid artery and advanced so that its tip was in the ascending aorta. The catheter was then externalized between the scapulae and secured by a polyester felt disk placed subcutaneously. The catheter was filled with a solution of 50% glucose and 500 IU/mL heparin and plugged with a nylon pin. After catheter implantation, the rats were housed individually until they had regained their preoperative weights and appeared healthy (6 to 8 days after catheter implantation).
At the end of the recovery period, the rats were reanesthetized, a skin incision was made in the abdominal region, and osmotic minipumps (Alzet model 2001, Alza Corp) were implanted subcutaneously (day 0). The minipumps were loaded with either Ang II (0.7 mg/kg per day, n=13) or vehicle (saline, n=13). In six of the animals in each of these groups, the ETA receptor antagonist PD 155080 (50 mg/kg per day) was administered twice daily by gavage feeding. To establish that Ang II effects were mediated by the AT1 receptor, an additional group of animals was treated with losartan (25 mg/kg per day, n=6) added to the drinking water.
Arterial Pressure Measurements
The animals were handled daily and exposed to the environment eventually used for BP measurement. On day 5 of osmotic minipump implantation, mean arterial BP was measured in conscious animals. After BP recording, the animals were given a lethal injection of sodium pentobarbital. After injection but before death, heparin (2500 U) was given via intracardiac injection. The aortas were then removed and used in subsequent studies.
Isolated Vascular Ring Experiments
Five-millimeter ring segments of the thoracic aorta were suspended in individual organ chambers for measurement of isometric tension and were studied using methods previously described.10 Responses of various vasoconstrictors, including serotonin, phenylephrine, ET-1 (all 1 nmol/L to 100 μmol/L), and KCl (5 to 80 mmol/L) were examined by cumulative addition of the various agents to the organ chamber.
Measurements of ET-1 Plasma Concentrations
Blood samples were obtained at the time of death and transferred to a chilled EDTA (2 mg/mL) tube. The chilled samples were centrifuged at 3000g for 15 minutes at 4°C, and plasma was stored at −20°C until assayed. ET-1 was extracted from 1 mL of plasma with 1.5 mL of extraction solvent composed of acetone/HCl (1 mol/L)/water (40:1:5). The mixture was centrifuged for 20 minutes at 3000 rpm and 4°C. The supernatant was dried down with a centrifugal evaporator, and the pellet was reconstituted in sample diluent and assayed using a solid-phase enzyme-linked immunosorbent assay kit (Parameter, R&D Systems). Optical density readings of unknown samples were plotted against a standard curve of synthetic ET-1–spiked rat plasma samples over a range of 1 to 113 pg/mL.
Two polyclonal antisera against human ET-1 were used as described recently11 12 —one against the C-terminal of ET-1 and the other against the C-terminal fragment of big ET-1 (big ET22-38). A commercial antiserum against human ET-1 (Peninsula Laboratories) was also used. In addition, antiserum to von Willebrand factor (factor VIII–related antigen) (Dako) was used as an endothelial cell marker. The avidin-biotin-peroxidase complex method was used as previously described. Negative controls were prepared with the specific antiserum absorbed with the cross-reactive endothelins or with nonimmune serum instead of primary antiserum, or by omitting steps in the avidin-biotin-peroxidase procedure. For each antisera, three sections were stained.
Data are expressed as mean±SEM. Comparisons between groups of animals or treatments were made with one-way ANOVA. When significance was indicated, a Student-Newman-Keuls post hoc analysis was used. To examine interactions between Ang II or sham treatment and treatment with PD 155080, two-way ANOVA was used, in which treatment with Ang II was assigned as one independent variable and treatment with PD 155080 as the other independent variable. Significance was considered at a value of P<.05.
In control animals, the mean arterial pressure was 98±5 mm Hg. In rats receiving only Ang II infusion for 5 days, the mean arterial pressure was 185±5 mm Hg. Administration of the ETA receptor antagonist PD 155080 markedly attenuated the pressor response to Ang II (128±5 mm Hg) and had a minimal effect on BP in rats not receiving Ang II (Fig 1⇓). As expected, losartan also prevented the increase in BP caused by Ang II.
Plasma ET-1 Concentrations
Plasma ET-1 levels averaged 1.62±0.36 pg/mL in control rats. At the end of 5 days of Ang II infusion, ET-1 levels were slightly but not significantly increased to 1.89±0.17 pg/mL (Fig 2⇓). Treatment with PD 155080 increased circulating ET-1 levels in animals receiving infusions of either vehicle or Ang II. The greatest increase in circulating ET-1 concentration was, however, in animals receiving both Ang II and PD 155080 (3.94±0.47 pg/mL). Losartan lowered ET-1 levels in Ang II–treated animals to values below those observed in control animals (Fig 2⇓).
Isometric Tension Studies
Vessels from rats treated with Ang II were more sensitive to KCl than control vessels, as evidenced by an EC50 of 18±2 versus 26±1 mmol/L (P<.05). Treatment with the ETA receptor antagonist normalized this increased sensitivity in vessels from Ang II–treated animals, while having no effect in controls (Fig 3⇓ and Table 1⇓). Likewise, treatment with losartan prevented the increased sensitivity to KCl in vessels from rats treated with Ang II (Table 1⇓).
Sensitivity to both serotonin and phenylephrine (reflected by EC50 values) was markedly increased in vessels from Ang II–treated animals (Fig 3⇑ and Table 1⇑). These values were normalized by treatment with the endothelin receptor antagonist (Table 1⇑ and Fig 4⇓). Peak responses to phenylephrine and serotonin were also increased in vessels from animals receiving Ang II infusion (Fig 3⇑ and Table 2⇓). Peak responses to phenylephrine, but not to serotonin, were normalized by treatment with the ETA antagonist (Fig 4⇓ and Table 2⇓). Losartan likewise prevented the increase in sensitivity and peak responses to serotonin and phenylephrine (Tables 1⇑ and 2⇓).
In contrast to the generalized increase in responses to vasoconstrictors such as phenylephrine, serotonin, and KCl, constrictions in response to ET-1 of vessels from rats treated with Ang II were suppressed compared with controls (Fig 3⇑ and Table 2⇑). Losartan prevented this effect of Ang II (Table 2⇑).
Immunohistochemical Analysis of ET-1 Expression
In control rat aorta, only faint staining for either ET-1 or big endothelin was visible (Fig 5A⇓). In contrast, in Ang II–treated animals, staining for ET-1 was readily apparent in the media (Fig 5B⇓). Staining for big endothelin appeared slightly increased in the aortas of Ang II–treated rats (Fig 5C⇓ and 5D⇓). No staining was apparent when the primary antibody was omitted (Fig 5E⇓).
Previous studies of cultured endothelial cells, vascular smooth muscle cells, and cardiomyocytes have shown that Ang II potently increases preproendothelin mRNA and ET-1 protein.2 3 4 5 These studies of cultured cells raise the possibility that some of the vascular effects of Ang II in vivo might be mediated by endogenously expressed endothelin. The present studies suggest that this hypothesis is correct. A substantial portion of the hypertension caused by Ang II, and the enhanced vasoconstrictor responsiveness in Ang II–mediated hypertension, was prevented by concomitant administration of the selective ETA receptor antagonist PD 155080. This enhanced vascular responsiveness was accompanied by an increase in synthesis of ET-1 in the vessel wall, as demonstrated by immunohistochemistry. It is conceivable that a higher dose of this agent would have been more effective in lowering BP. The dose used in this study has been found to completely prevent ET-1–induced constriction of the hindlimb in rabbits. Furthermore, after oral gavage, a dose approximately one third of this produces plasma levels substantially in excess of the IC50 values in Wistar-Kyoto rats.13
To our knowledge, this is the first report to show that endothelin might participate in the alteration of either vascular reactivity or BP in Ang II–induced hypertension. Reports on the involvement of endothelin in various experimental models of hypertension have conflicted.14 15 Our present findings suggest that in the setting of hypertension caused by elevations of Ang II, endothelin-receptor antagonists may be effective BP-lowering agents.
An interesting finding in the present study is the effect of the ETA receptor antagonist PD 155080 on plasma levels of ET-1. PD 155080 produced a modest increase in plasma ET-1 in control animals and a marked increase in plasma ET-1 in Ang II–treated animals. The mechanisms underlying this increase remain unclear. The ETA receptor (blocked by PD 155080) is not thought to be involved in the clearance of ET-1, and it is therefore unlikely that a change in clearance participated in this phenomenon.16 ET-1 is tightly bound by its receptors, and it is possible that the antagonist simply displaced the peptide from vascular receptors, resulting in spillover into the plasma. Notwithstanding the mechanisms responsible for this increase in plasma ET-1, the data are compatible with an increase in ET-1 synthesis caused by Ang II.
Related to the possible activation of endogenous endothelin production, it is of interest that constrictions in response to ET-1 of vessels from Ang II–treated rats were paradoxically reduced compared with those from control animals. Although other explanations are possible, this finding is compatible with the possibility that vascular endothelin receptors were occupied by endogenous endothelin, thus preventing the additional constrictor effect of exogenously added ET-1. This conclusion is in keeping with the observation that ET-1 immunostaining is increased in the aortas of Ang II–treated rats. A similar situation has been observed in the case of prolonged nitroglycerin treatment, in which an increase in vascular ET-1 immunoreactivity is associated with increased responses to several vasoconstrictor substances and paradoxically decreased constrictions to ET-1.
It is now well accepted that even low concentrations of ET-1, which alone produce either no or minimal vasoconstriction, can substantially enhance vasoconstrictions to numerous other vasoconstrictor agents.12 17 18 This process seems to involve activation of protein kinase C, in that it can be prevented by several chemically unrelated protein kinase C antagonists. Of note, the enhanced vasoconstrictor responses to phenylephrine, KCl, and serotonin found in vessels from animals treated with Ang II mirror responses that we have observed in vessels incubated with low concentrations of ET-1.12
In addition to direct vascular actions of ET-1, it is also likely that enhanced ET-1 production could contribute to hypertension via other mechanisms. It has been reported that endothelin can increase aldosterone synthesis, which could augment sodium and water retention and predispose to a volume-dependent form of hypertension.7 Furthermore, ET-1 has been shown to enhance conversion of Ang I to Ang II. This might result in a positive feedback–like situation in which Ang II could stimulate ET-1 production, which could in turn increase Ang II production. Finally, ET-1 is a mitogen for vascular smooth muscle,19 20 and it is conceivable that over the long term, increased levels of ET-1 might promote vascular hypertrophy and narrowing of the vascular lumen, resulting in elevated peripheral vascular resistance.
The mechanisms whereby ET-1 protein synthesis and preproendothelin mRNA are increased in response to Ang II remain poorly defined. It has been postulated that this is due to Ang II activation of protein kinase C and consequent activation of c-Fos and c-Jun binding to activator protein-1 sites in the endothelin promoter. In vivo, this process may be even more complex. It is known that nitric oxide can inhibit ET-1 expression. Recently, we showed that chronic elevations of Ang II increase vascular superoxide production via activation of NADH/NADPH-dependent oxidases.10 This increase in vascular superoxide results in a loss of the bioactivity of endothelium-derived nitric oxide, probably via a radical-radical reaction between superoxide and nitric oxide. It is therefore conceivable that loss of the effect of nitric oxide via Ang II–induced oxidase activation might contribute to an increase in ET-1 expression. It is also possible that changes in redox state caused by Ang II might stimulate transcription of the preproendothelin gene in a manner similar to that observed recently for other genes. Although controversial, there is evidence that c-Fos and c-Jun activation is stimulated by oxidant stress. In particular, reactive oxygen intermediates may be important in c-Fos and c-Jun heterodimer binding to activator protein-1 in response to Ang II in myoblasts.21
One potential explanation for these findings is that PD 155080 might nonspecifically inhibit Ang II binding to the AT1 receptor. We believe that this is unlikely. In additional experiments, we found that very high concentrations of PD 155080 (1 mmol/L) had no effect on the constriction of rat aortas in response to Ang II.
In summary, we have shown that Ang II–mediated hypertension is associated with enhanced production of ET-1 in vivo. The obligatory role of endothelin in mediating some of the effects of Ang II was further substantiated by the effects of selective ETA receptor blockade. Although several previous reports have shown that Ang II can stimulate ET-1 expression in tissue culture, this is, to our knowledge, the first demonstration that increased endogenous synthesis of ET-1 might contribute to Ang II–mediated hypertension in vivo. The interactions between these two hormones may be of importance in other conditions in which both have been shown to be elevated, such as myocardial infarction22 23 and congestive heart failure.24 25 Some of the beneficial effects of angiotensin-converting enzyme inhibition in congestive heart failure26 27 28 thus may stem from favorable modulation of endothelin levels.
Selected Abbreviations and Acronyms
|Ang I, II||=||angiotensin I, II|
|AT1||=||angiotensin II type 1 (receptor)|
- Received November 7, 1996.
- Revision received November 26, 1996.
- Accepted January 2, 1997.
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