Effect of an Endothelin Antagonist on Hemodynamic Responses to Angiotensin II
We determined changes in blood pressure, cardiac output, and total peripheral conductance evoked by intravenous infusions of angiotensin II (Ang II) in conscious, unrestrained normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) before and after pretreatment with bosentan, a nonselective endothelin antagonist. Blood pressure was recorded by radiotelemetry and cardiac output by ultrasonic transit-time flow probes. Bosentan per se failed to affect basal blood pressure and evoked only small changes in cardiac output and total peripheral conductance in both strains. The pressor effects of Ang II were exaggerated in SHR compared with WKY. Strikingly, bosentan pretreatment blunted the increases in blood pressure, the fall in cardiac output, and the decreases in conductance evoked by lower doses of Ang II but not higher doses of the peptide. This effect was observed in both rat strains but was more pronounced in SHR. These data suggest that endothelin contributes to the hemodynamic effects of Ang II in both SHR and WKY and that endothelin may contribute to the exaggerated pressor responsiveness of SHR to Ang II.
Angiotensin II (Ang II) has been reported to increase ET-1 production from cultured endothelial1 and mesangial2 cells and to induce endothelin release from the perfused mesenteric vascular bed of SHR.3 Ang II also evokes a large increase in preproendothelin mRNA.1 Recently, we reported that Ang II–evoked tension responses in the isolated rat mesenteric and tail arteries were endothelin dependent because responses to Ang II were attenuated in the presence of either the endothelin-converting enzyme inhibitor phosphoramidon4 or the endothelin subtype A receptor (ETA)–selective peptide antagonist BQ-123.5 Thus, considerable in vitro evidence suggests that endothelin may contribute to the cardiovascular actions of Ang II, but the issue has not been addressed directly at the hemodynamic level. Accordingly, we recorded BP by radiotelemetry and CO with ultrasonic transit-time flow probes in conscious rats. We monitored the changes in these variables in response to Ang II in both the presence and absence of the endothelin antagonist bosentan. Another objective was to determine whether any endothelin component in the hemodynamic effects of Ang II was different in hypertensive and normotensive strains.
All surgical procedures and protocols were conducted under the guidelines published by the Canadian Council on Animal Care and approved by the animal care committee at the University of Saskatchewan.
Ultrasonic flow probes (2.5 SB series, Transonic Systems) were implanted on the ascending aorta of 18- to 22-week-old anesthetized (50 mg/kg sodium pentobarbital) male SHR and WKY (Taconic Farms, Germantown, NY). A catheter connected to a radiotelemetry capsule (TA11PA-C40, Data Sciences) was implanted in the femoral artery of each rat for BP recording. The fluid-filled sensor catheter was inserted into the femoral artery and pushed so that the catheter tip reached the abdominal aorta above the iliac bifurcation. A second catheter was implanted into a femoral vein for administration of Ang II, ET-1, and bosentan.
Ten days later, BP and CO were recorded in conscious and unrestrained rats. Pressure data were collected with a computer-driven data-acquisition system (Dataquest IV, Data Sciences). The pressure waveform was sampled every 30 seconds with a 5-second sample duration. The validity of the telemetric system has been established in our laboratory previously. CO was recorded by feeding the signal from the flowmeter (Transonic Systems) to a pen recorder (Grass Instrument Co). The probes were calibrated in situ at the end of each experiment. TPC was calculated as the quotient of CO (milliliters per minute) and BP (millimeters of mercury).
After a 2-hour control period, a single dose of Ang II was infused intravenously at a rate of 3, 9, 30, or 90 ng/kg per minute for 6 minutes (Fig 1⇓). Responses to Ang II returned to control values within 10 minutes after the infusion was stopped. The selection of the Ang II dose was randomized to control for any time- or sequence-related variations. After a 2-hour recovery period, 30 mg/kg bosentan was injected intravenously as a bolus. This dose was based on previous studies6 and on results shown in Fig 2⇓. Thirty minutes after bosentan, the Ang II infusion was repeated. Finally, 24 hours later, Ang II was infused a third time to serve as a postrecovery control. The 2-hour interval between the initial control response to Ang II and the response in the presence of bosentan was chosen because in preliminary experiments, this interval yielded reproducible results to repeated Ang II administration. The 30-minute period after bosentan was chosen for the second Ang II infusion because the response to bosentan had reached a plateau by this time, and it was well before recovery from the effects of bosentan began (see below).
To ascertain that the bosentan used in our studies was active and a competitive antagonist to endothelin, we studied the relationship between the endothelin dose and the hemodynamic response in both SHR and WKY. Bolus doses of ET-1 at 0.01, 0.03, 0.06, 0.1, and 0.3 nmol/kg were infused in a cumulative fashion every 10 minutes before and after bosentan injection (30 mg/kg). The initial depressor response and prolonged pressor component were plotted. In preliminary experiments, we had determined the onset of recovery from the effects of bosentan by measuring the extent of inhibition of the response to ET-1 (0.1 nmol/kg) at different times. Recovery from the effects of bosentan began 2 hours after its administration, and recovery was complete within 24 hours.
To confirm that bosentan was selective for endothelin receptors and did not inhibit Ang II receptors, endothelium-denuded ring preparations of the thoracic segments of the aorta (length, 5 mm) were isolated from WKY as described in detail elsewhere.5 Each tissue was exposed to a single cumulative concentration-response determination for Ang II (0.5 nmol/L to 10 μmol/L) or ET-1 (10 pmol/L to 10 μmol/L). Concentration-response curves to Ang II or ET-1 were also determined in aortic rings in the presence of bosentan (100 nmol/L) or the Ang II type 1 (AT1) receptor–selective antagonist losartan (100 nmol/L). These antagonists were maintained in the organ bath for 20 minutes before the addition of agonists. At the end of the experiment, aortas were blotted dry and weighed, and cross-sectional areas were calculated to express the data of tension developed at each concentration of the agonist per unit area for each blood vessel.
Ang II octapeptide and ET-1 were obtained from American Peptide Co. Peptides were dissolved in saline containing 1% bovine serum albumin (Sigma Chemical Co). Bosentan (sodium salt), kindly provided by Dr M. Clozel (F Hoffman–La Roche Ltd), was dissolved in sterile water. The AT1 receptor–selective antagonist losartan was obtained from DuPont-Merck Pharmaceutical Co. Phenylephrine, acetylcholine chloride, and Triton X-100 used in the tension experiments were purchased from Sigma.
All values are expressed as mean±SE. Responses in the presence or absence of bosentan were compared by ANOVA for repeated measures. Simultaneous multiple comparisons were performed with modified t tests. For the tension studies, each cumulative concentration-response curve was analyzed individually for estimation of the concentration required to produce 50% of the maximal response (EC50) and the maximal increase in tension (Emax). Pooled values are shown in Table 2⇓.
Values for BP, CO, and TPC before Ang II infusion and after bosentan treatment in nine SHR and seven WKY (four determinations in each rat) are shown in Table 1⇓. Before bosentan, BP was significantly higher in SHR than in WKY, and the elevated pressure appeared to be due to the elevated resistance, ie, reduced TPC. Indeed, CO was significantly lower in SHR although the decrease was small (≈15%). Bosentan failed to change arterial pressure in either SHR or WKY. Bosentan evoked small increases in TPC and CO, but these changes were not significant. Overall, the changes evoked by bosentan were unremarkable.
Responses to Ang II
Ang II induced dose-related decreases in TPC and CO, and the vasoconstriction was associated with an increase in BP (Fig 1⇑). The pressor effects of Ang II were exaggerated (P<.001) in SHR (y=25.5 logx+6.7) compared with WKY (y=16.6 logx+5.0). Pretreatment with bosentan significantly blunted the increases in BP (P<.01), the fall in CO (P<.01), and the decreases in TPC (P<.001) evoked by the lower doses of Ang II but not the higher doses of the peptide. This effect was observed in both rat strains but was more pronounced in SHR. Indeed, bosentan abolished the exaggerated pressor and TPC responsiveness of SHR to 3 ng/kg per minute Ang II.
Responses to ET-1
ET-1 evoked a transient depressor response followed by a prolonged pressor response. These responses were due to changes in the resistance function of the circulation, as TPC and CO varied inversely to the changes in BP. The depressor component was observed only at higher doses of the peptide (Fig 2⇑). Bosentan pretreatment attenuated the pressor responses to ET-1 and abolished the depressor response seen at higher doses.
In the presence of losartan, the concentration-response curve to Ang II was shifted to the right, with significant increases (P<.01) in the EC50 value (Table 2⇓). In contrast to losartan, the concentration-response curve to Ang II in the presence of bosentan did not differ significantly from control: EC50 values did not change significantly (P>.9). Emax in the presence of either antagonist remained unaltered. The concentration-response curve to ET-1 was shifted to the right in the presence of bosentan but not of losartan (Table 2⇓).
The contribution of the endothelin system to BP regulation in the SHR is controversial, but the present work appears to be the first in which BP was measured by radiotelemetry. We found that bosentan per se failed to induce any change in BP in both rat strains. The bosentan was active because it antagonized the effects of exogenous administration of ET-1 in conscious rats (Fig 2⇑) and the tension responses to ET-1 in ring preparations of rat aorta (Table 2⇑) in a competitive fashion. These results parallel those of other researchers.6 7 In contrast to our findings with bosentan, both BQ-123, an ETA antagonist, and SB 209670, a nonselective ETA/ETB antagonist, have been reported to reduce BP in SHR8 9 and transgenic rats10 when infused over prolonged periods. On the other hand, other researchers have reported no change of BP with BQ-123 and bosentan in SHR.11 12 Pressure in these previous studies was monitored with exteriorized catheters or by the tail-cuff method. Control BP values are higher and responses to BP-lowering agents appear to be exaggerated in SHR when BP is measured by either of these methods,13 suggesting a stress-related component to these methods that may be a confounding factor leading to different responses. Telemetry avoids this confounding variable. The failure of bosentan to lower BP in SHR does not preclude a role for the peptide because blockade of one control system typically activates other compensatory systems.14 Indeed, it has been shown recently that simultaneous administration of an Ang II antagonist and an endothelin antagonist evoked a much greater fall in pressure than either antagonist alone.10
Although bosentan alone failed to change control BP in our study, it markedly altered the hemodynamic responses evoked by Ang II. The blunted responsiveness to the lower doses of Ang II in the presence of bosentan is the major finding reported in this article, suggesting a role for an endothelin-dependent component that contributes to the pressor activity of Ang II at the hemodynamic level. Moreover, the tension study confirms that the hemodynamic data could not be due to partial inhibition of Ang II receptors by bosentan because the antagonist failed to inhibit tension responses evoked by Ang II in aortic rings, which lack an Ang II–evoked endothelin component.5
This contribution of endothelin to the pressor activity of Ang II was exerted at the level of the resistance vessels, as TPC responses to Ang II were enhanced when the endothelin system remained fully functional, whereas decreases in CO tended to oppose the BP-elevating effect resulting from the vasoconstrictor activity of the peptide. These results are consistent with our in vitro data, which showed that Ang II–evoked tension responses in the rat mesenteric and rat tail arteries were attenuated by pretreatment with either an endothelin-converting enzyme inhibitor4 or an ETA antagonist.5
The exaggerated pressor responsiveness of SHR to Ang II compared with WKY was evident over the entire dose range in rats with a functional endothelin system. Importantly, bosentan abolished the exaggerated pressor responsiveness of SHR to the lowest dose of Ang II (3 ng/kg per minute). This observation suggests that endothelin contributes to the exaggerated pressor responsiveness of SHR to low doses of Ang II. The mechanism of this enhanced responsiveness is unknown. Although endothelin binding sites have been reported to be similar or reduced in SHR compared with WKY,15 16 we have shown that ETB receptor–mediated calcium signaling in vascular smooth muscle is enhanced in SHR.17 Thus, an increased activation of the events that accompany stimulation of ETB receptors by Ang II could contribute to the exaggerated pressor response observed in this strain. An increase in Ang II binding sites in SHR could also contribute to the enhanced responsiveness of SHR.18 19
In summary, the results suggest that endothelin contributes to the pressor activity of Ang II at least at the lower dose range in both SHR and WKY and that this endothelin component contributes to the exaggerated responsiveness of SHR to Ang II. Finally, the contribution of endothelin to the pressor activity of Ang II is mediated through an increase in total peripheral resistance.
Selected Abbreviations and Acronyms
|Ang II||=||angiotensin II|
|SHR||=||spontaneously hypertensive rat(s)|
|TPC||=||total peripheral conductance|
This work was supported by a grant-in-aid from the Heart and Stroke Foundation of Saskatchewan and the Medical Research Council of Canada. We thank Dr Martine Clozel (F Hoffman–La Roche Ltd, Basel, Switzerland) for a generous supply of bosentan.
Reprint requests to Dr J. Robert McNeill, Department of Pharmacology and Cardiovascular Risk Factor Reduction Unit (CRFRU), College of Medicine, University of Saskatchewan, Saskatoon, SK, S7N 5E5, Canada.
- Received February 9, 1996.
- Revision received March 11, 1996.
- Revision received July 2, 1996.
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