(Hypertension. 2000;35:130.)
© 2000 American Heart Association, Inc.
Scientific Contributions |
Angiotensin III Depressor Action in the Conscious Rabbit Is Blocked by Losartan but not PD 123319
From the Department of Physiology, East Tennessee State University College of Medicine, Johnson City.
Correspondence to Brian P. Rowe, PhD, Department of Physiology, Box 70,576, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614-0576. E-mail rowe{at}etsu.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract—Vasodilator and vasodepressor properties of angiotensins have been reported, and mediation by prostaglandins or nitric oxide has been proposed. Other studies indicate that angiotensin AT2 receptors might mediate a depressor action, and the present study was designed to delineate and explore this possibility in a conscious rabbit model. Large intravenous boluses of angiotensin III (15 nmol/kg) produced a predictable pressor peak (82±4 mm Hg) followed by a depressor phase (20±3 mm Hg), whereas equipressor doses of angiotensin II were less effective at producing depressor responses. Angiotensin-(1–7) did not exert a depressor action, and the reduced potency of angiotensin IV (relative to angiotensin III) was similar for both the pressor and depressor phases (
100-fold). It is clear that specific
angiotensin IV or angiotensin-(1–7) receptors
do not mediate depressor effects in this model. The AT1
antagonist losartan (1 mg/kg) blocked both the
pressor and depressor components of the angiotensin III
response, whereas the AT2 antagonist PD 123319
(35 mg/kg) had no effect on either element of the response. The data
obtained with the angiotensin receptor subtype–selective
compounds, losartan and PD 123319, suggest that the depressor
action is an AT1-mediated effect and give no indication
that AT2 receptors could be involved. Paradoxically, the
greater potency of angiotensin III as a vasodepressor
belies the conclusion that the response is AT1-mediated,
because AT1 receptors have a greater affinity for
angiotensin II versus angiotensin III.
Key Words: angiotensin III • receptors, angiotensin • blood pressure • rabbits • losartan
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Angiotensin II is well known as a potent vasoconstrictor, but many reports indicate that angiotensins exert vasodilator properties. Early in vitro work showed vasorelaxation by angiotensins in some vascular beds,1 2 3 and Campbell et al4 reported a vasodepressor phase after the characteristic pressor response to angiotensin II in conscious rabbits. We established that vasodilator prostaglandins contribute to the depressor response in conscious rabbits.5 Depressor or dilator responses to angiotensins are not duplicated by other vasopressors, tend to be manifested at high angiotensin concentrations, and might be prominent in specific vascular beds. For example, the cerebral and renal circulations appear susceptible to angiotensinogenic dilatation.2 6 7
New pharmacological tools became available in 19898 9 for the investigation of receptor mechanisms for depressor responses. Scheuer and Perrone10 reported depressor responses in barbiturate-anesthetized rats that were accentuated by the AT1 antagonist losartan and blocked by the putative AT2 antagonist PD 123319. Angiotensin III was a more potent depressor than was angiotensin II, and this is consistent with an AT2-mediated response because angiotensin III binds less effectively than angiotensin II at AT1 receptors but has a comparable affinity at AT2 receptors.11 An AT2-mediated dilator action is further supported by a study of Li et al,12 who reported that angiotensin III contractile responses in rat aorta were accentuated by PD 123177 but that angiotensin II and IV responses were unaffected. Other interesting differences between angiotensin II and III contractile responses in rat aorta were described by Sim and Yuan13 : des-Asp angiotensin I attenuated the contractile responses to angiotensin III but potentiated the response to angiotensin II, and the authors state that the 2 peptides exert their effects at 2 different receptors. Vasodilatation by angiotensins in the cerebral circulation might occur by different mechanisms, in view of the fact that it appears to be blocked by both AT 1 and AT2 antagonists and seems to be similar to arginine-induced vasodilatation.6 14
The various reports prompted us to reevaluate the angiotensin depressor response with the use of the conscious rabbit model described previously.5 Our experiments were designed to test the hypothesis that the depressor response is mediated by AT2 receptors and that depressor responses to angiotensin III will be more prominent than those produced by angiotensin II. Our experiments also address the hypothesis that angiotensinogenic depressor responses could be mediated by non-AT1/AT2 receptors, because of the experiments by Haberl6 described above and the observation that angiotensin IV and angiotensin-(1–7) have dilator or depressor activity.15 16
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Animal Preparation
Male New Zealand White rabbits (2.7 to 3.1 kg) were allowed food (Harlan Teklad 8630[W]) and water ad libitum and were monitored for steady weight gain before experimentation. All procedures were approved by the institutional committee governing animal care. A conscious rabbit was placed in a standard restraining box, and blood vessels in the ear were cannulated after application of the local anesthetic lidocaine (2% Xylocaine, Astra Pharmaceuticals). The central ear artery was cannulated with Clay-Adams PE-50 tubing (Beckton Dickinson & Co) after cutdown. The cannula contained heparinized saline (40 U/mL) and was connected to a Statham pressure transducer (model P23Db). Arterial blood pressure was monitored with a Grass polygraph (model 7D), and mean arterial pressure was obtained as the electronically damped pulse pressure. A PE-50 cannula was placed in the marginal ear vein via percutaneous puncture and was used for the administration of drugs. All drugs were infused in a volume of
0.1 mL and were flushed with 1 mL of
saline. Angiotensin peptides were given as 4 different doses in the initial experiments. Each dose was given at 20-minute intervals, and a 30-minute period was allowed between consecutive dose ranges. There is potential for a large dose of angiotensin to affect the response to a subsequent angiotensin bolus (tachyphylaxis).17 Randomization of dose sequence is one option that could be used to overcome sensitivity changes, but we elected not to use this approach because a large number of observations would be required for a valid analysis. A symmetric dose administration schedule was used instead; angiotensin peptides were administered in ascending and descending doses in alternate animals, and data obtained in this fashion were reproducible. Some experiments were conducted with a single repeating dose of angiotensin III (33 nmol/kg) in place of multiple dose sequences. These doses were given at 30-minute intervals.
Statistics
The data were evaluated as changes in mean arterial
pressure and expressed as mean±SEM. The initial experiments were
analyzed by ANOVA with repeated measures. In some experiments,
responses before and after drug treatment were compared with identical
protocols before and after vehicle administration. These data were
processed by a 2-way ANOVA with repeated measures for the dose-sequence
element (Abstat, Anderson-Bell Corp).
Materials
Angiotensin analogues were obtained from Bachem, and
PD 123319 was from Research Biochemicals. Losartan was a gift
from Merck & Co, Rahway, NJ.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Comparison of Angiotensin Peptides
A biphasic response to angiotensin III is illustrated in Figure 1. There is animal-to-animal variation in the magnitude of the depressor response, and it was <8 mm Hg in a small number of animals; these were excluded from further study. Figure 2 details the blood pressure responses to a dose range of the heptapeptide. Three identical dose ranges were obtained at 90-minute intervals consecutively. Pressor-response tachyphylaxis can occur with large doses of angiotensin in the rabbit but is not evident here because the pressor responses to angiotensin III are well maintained for each consecutive dose-range administration. The depressor response to angiotensin III is less well maintained. There was a significant decline in the response to doses of 15 nmol/kg (separated by 90 minutes): 20±3, 11±2, and 11±3 mm Hg (P<0.05).
|
|
The depressor response to angiotensin III is considerably more prominent than the depressor response to angiotensin II, but the reverse is true for the pressor responses (Figure 1, bottom trace). Table 1 shows the blood pressure responses to the first dose range of angiotensin III depicted in Figure 2 and a dose range of angiotensin II at which the pressor potency is comparable to that of angiotensin III. Mean arterial blood pressure rises by 93±7 mm Hg after 5 nmol/kg angiotensin II, but the subsequent depressor phase (5±2 mm Hg) is small in relation to comparable doses of angiotensin III and is quite variable. Differences between angiotensin II and III are apparent also when the duration of the response is examined (Figure 1). The duration of the pressor response to 15 nmol/kg angiotensin III was 2.1±0.1 minutes (pressor response 82±4 mm Hg; see Table 1), whereas the duration of the response to angiotensin II at a comparable pressor dose (1.5 nmol/kg, pressor response 77±9 mm Hg) was significantly longer at 2.8±0.2 minutes (P<0.01, unpaired t test).
|
The differences in responses to angiotensins II and III
suggest the involvement of multiple receptor subtypes. The
angiotensin-(1–7) heptapeptide and the
angiotensin-(3–8) hexapeptide (angiotensin IV)
were evaluated because they can function through
non-AT1/AT2
receptors.18 19 Both of these peptides have low potency at
AT1 receptors and are very weak vasoconstrictors,
but we explored the possibility that they might be relatively more
potent as vasodepressors. Table 1 shows the blood pressure
responses to both peptides and compares the responses with
angiotensins II and III. Angiotensin IV is
100-fold less potent than angiotensin III as a pressor
agent and comparably less potent as a depressor, providing no evidence
for depressor actions via different receptors. The
angiotensin-(1–7) heptapeptide had little demonstrable
effect on either pressor or depressor mechanisms, even in milligram
quantities.
AT1 and AT2 Subtype–Selective
Antagonists
The blood pressure responses to angiotensin III were
evaluated before and after blockade of AT1
receptors with losartan (Table 2). Losartan markedly attenuated
the pressor response to angiotensin III, confirming
blockade of AT1 receptors. Surprisingly, the
depressor responses were completely abolished also. The
losartan data suggest that both pressor and depressor responses
are mediated via AT1 receptors, whereas the
paradoxically higher potency of angiotensin III as a
depressor suggests that the depressor response is mediated by a
non-AT1 receptor.
|
The paradox was evaluated further by examining the effect of PD 123319, a selective AT2 antagonist, on the depressor response. This experiment was conducted differently: 3 single large doses of angiotensin III (33 nmol/kg) were given 30 minutes apart, and PD 123319 (35 mg/kg, n=4) was given 2 minutes before the second dose (Figure 3). A parallel protocol in which vehicle was substituted for PD 123319 is shown also (n=8). Pressor responses to angiotensin III were unaffected in any treatment group. A 2-way ANOVA with repeated measures revealed a significant decline in the depressor response with time (P=0.008), but there was no significant difference between the PD 123319 group and the control group. There is no effective method to test AT2 blockade, but the amount of PD 123319 given (35 mg/kg) was larger than that used by many investigators.10 Angiotensin III responses were tested at 2 and 30 minutes after PD 123319 administration to maximize the likelihood of AT2 receptor blockade. The number of observations in the PD 123319 group (n=4) was limited by the extreme cost of the compound.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The results confirm an angiotensin-induced depressor response in the conscious rabbit model as described previously.5 The response is evident at high concentrations of angiotensin (>0.5 nmol/kg), and we confirm the observation of Scheuer and Perrone10 that angiotensin III is a more potent depressor than is angiotensin II. The depressor phase follows the well-known pressor response after an intravenous bolus of the peptide. The depressor phase was reproducible with repeated administration of angiotensin III at the smaller doses, but the magnitude of the response to the larger dose (15 nmol/kg) fell significantly from an initial value of 20±3 mm Hg to 11±2 and 11±3 mm Hg. The accompanying pressor components were well maintained, which suggests that the mechanisms for the pressor and depressor responses are different. A qualitative inspection of the responses in Figure 1 indicates that a large depressor component of the angiotensin III response is associated with a truncated pressor phase. It could be argued that in these circumstances an abbreviated pressor phase accentuates the expression of a depressor component, and this notion might be supported if angiotensin III had a shorter half-life than angiotensin II. There is no information on the biological half-lives of the peptides in the rabbit, but they are comparable in the rat (14 versus 16 seconds; angiotensin III versus II, respectively).20 Moreover, when the depressor phase is blocked by cyclooxygenase inhibitors, the pressor component is prolonged.5 It seems likely then that the depressor component has a slow onset and a longer duration but can dominate and mask the late pressor phase. Studies with low-dose infusions of angiotensin II indicate that an elevation in mean arterial pressure of only 8 mm Hg incorporates a depressor element of 3 to 6 mm Hg, which can be revealed by prostaglandin synthesis inhibition.21 In humans, angiotensin pressor responses of only 20 mm Hg generate a significant depressor component, which intensifies during pregnancy.22 This provides evidence that physiological concentrations of angiotensin could exert meaningful depressor activity, although it is not evident with low-dose bolus administration.
The desensitization of the depressor action relative to the pressor response and the higher depressor potency of angiotensin III provide compelling evidence that the 2 responses are mediated by different receptor mechanisms. The hypothesis is further consolidated by the findings of Scheuer and Perrone10 showing that the depressor phase in rats is mediated by AT2 receptors and the studies of Endo et al7 implicating AT2 receptors in renal vasodilatation. Additionally, the AT2-selective compound PD 123319 enhances the contractile response to angiotensin III, but not angiotensin II, in rat aorta,12 and des-Asp1 angiotensin I attenuates the contractile response to angiotensin III, but not angiotensin II, in the same tissue.13 In view of this overwhelming evidence, it was astonishing that in our experiments losartan abolished the depressor component of the angiotensin III response, whereas PD 123319 had no effect. The dose of losartan was less than that used by most investigators; it is highly improbable that this dose influenced AT2 receptors because residual pressor responses indicate incomplete blockade even at AT1 receptors. It is difficult to evaluate the in vivo effect of PD 123319 on AT2 receptors in the absence of a good biological response to test antagonism. Moreover, PD 123319 has a short biological half-life (22 minutes; J. Keiser, personal communication, 1998). These problems were addressed by giving PD 123319 at 3.5 times the amount given in the experiments reported by Scheuer and Perrone, and the responses to a single dose of angiotensin III were examined 2 to 30 minutes after PD 123319 administration to maximize the window for blockade of AT2 receptors.
The argument that the depressor response is losartan sensitive but mechanistically distinct from AT1 actions might be resolved in several ways. One possibility is that the depressor response could be mediated by a non-AT1/AT2 receptor that happens to be losartan sensitive and PD 123319 insensitive. The most likely candidates would be receptors that use angiotensin-(1–7) or angiotensin IV as primary agonists. Biological actions of angiotensin-(1–7) might be mediated by unique non-AT1/AT2 receptors,18 and a depressor response for angiotensin-(1–7) has been reported.16 23 Angiotensin IV also functions via non-AT1/AT2 receptors19 and can cause renal vasodilatation.15 Angiotensin IV was 30 to 50 times less potent than angiotensin III as a pressor agent and was equally less potent as a depressor in our hands. This argues against a depressor action mediated via receptors with a high affinity for angiotensin IV and actually strengthens the case for a depressor action linked to AT1 receptors. Angiotensin-(1–7) produced weak pressor responses even in milligram quantities, and there was no evidence of a depressor response. Thus, the depressor response in the rabbit model is not mediated by receptors with affinity for angiotensin-(1–7).
It is difficult to define the mechanism of the depressor response reported in the present study. It is not a reflex phenomenon because it does not occur with other pressors and is relatively less prominent with angiotensin II, despite its superlative pressor action. We discounted depressor actions mediated by the putative non-AT1/AT2 receptors responsible for responses to angiotensin-(1–7) and angiotensin IV, but we cannot exclude the possibility of further angiotensin receptors that are, as yet, uncharacterized. Despite evidence to the contrary, we cannot positively exclude an AT2-mediated depressor response. For example, acute elevations in blood pressure might facilitate the transfer of angiotensin peptides across the blood-brain barrier, where angiotensin III could initiate AT2-mediated vasodilatation. If PD 123319 does not adequately access central nervous system tissue, the AT2-mediated depressor action would be unresponsive to circulating PD 123319 but blocked by the AT1 antagonist by virtue of its antihypertensive properties. Another tenuous explanation is that the depressor response is mediated by AT1 receptors but that the response does not exhibit a classic structure-activity profile. This could occur if internalized AT1/angiotensin III initiates atypical postreceptor events or angiotensin III activates unidentified receptors that synergize with AT1.
When the findings of depressor/dilator actions of angiotensins reported by various laboratories are taken into consideration, the evidence supports and refutes roles for prostaglandins, nitric oxide, AT1, AT2, and non-AT1/AT2 receptors. It appears likely that multiple mechanisms are operative in various species and various locations.
|
Acknowledgments |
|---|
This study was supported by a grant from the American Heart Association, Southeast Affiliate. Losartan was a gift from Merck & Co, Inc. The assistance of Lili Xin is acknowledged.
Received February 10, 1999; first decision March 9, 1999; accepted August 25, 1999.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Blumberg AL, Denny SE, Marshall GR, Needleman P. Blood vessel-hormone interactions: angiotensin, bradykinin, and prostaglandins. Am J Physiol. 1977;232:H305–H310.
2. Toda N, Miyazaki M. Angiotensin-induced relaxation in isolated dog renal and cerebral arteries. Am J Physiol. 1981;240:H247–H254.
3.
Fleming JT, Joshua IG. Mechanism of biphasic
arteriolar response to angiotensin II. Am J
Physiol. 1984;247:H88–H94.
4. Campbell WG, Donohue JA, Duket LH. Alteration in responses to bradykinin, angiotensin I, and angiotensin II during the induction phase of one kidney, one wrapped hypertension and associated arterial disease in rabbits. Am J Pathol. 1980;98:457–484.[Medline] [Order article via Infotrieve]
5. Rowe BP, Nasjletti A. Biphasic blood pressure response to angiotensin II in the conscious rabbit: relation to prostaglandins. J Pharmacol Exp Ther. 1983;255:559–563.
6. Haberl RL Role of angiotensin receptor subtypes in the response of rabbit brain arterioles to angiotensin. Stroke. 1994;25:1476–1480.[Abstract]
7. Endo Y, Arima S, Yaoita H, Omata K, Tsunoda K, Takeuchi K, Abe K, Ito S. Function of angiotensin II type 2 receptor in the postglomerular efferent arteriole. Kidney Int. 1997;52(suppl 63):S205–S207.
8. Chiu AT, Herblin WF, McCall DE, Ardecky RJ, Carini DJ, Duncia JV, Pease LJ, Wong PC, Wexler RR, Johnson AL, Timmermans PBMWM. Identification of angiotensin II receptor subtypes. Biochem Biophys Res Commun. 1989;165:196–203.[Medline] [Order article via Infotrieve]
9. Whitebread S, Mele M, Kamber B, de Gasparo M. Preliminary biochemical characterization of two angiotensin II receptor subtypes. Biochem Biophys Res Commun. 1989;163:284–291.[Medline] [Order article via Infotrieve]
10.
Scheuer DA, Perrone MH. Angiotensin type 2
receptors mediate depressor phase of biphasic pressure response to
angiotensin. Am J Physiol. 1993;264:R917–R923.
11. Chang RS, Lotti VJ, Chen TB, Faust KA. Two angiotensin II binding sites in rat brain revealed using [125I] Sar1 Ile8 angiotensin II and selective non peptide antagonists: identification of angiotensin II receptor subtypes. Biochem Biophys Res Commun. 1990;171:813–817.[Medline] [Order article via Infotrieve]
12. Li Q, Zhang J, Pfaffendorf M, van Zwieten PA. Comparative effects of angiotensin II and its degradation products angiotensin III and angiotensin IV in rat aorta. Br J Pharmacol. 1995;116:2963–2970.[Medline] [Order article via Infotrieve]
13. Sim MK, Yuan HT. Effects of des-Asp-angiotensin I on the contractile action of angiotensin II and angiotensin III. Eur J Pharmacol. 1995;278:175–178.[Medline] [Order article via Infotrieve]
14.
Haberl RL, Decker PJ, Einhäupl KM.
Angiotensin degradation products mediate
endothelium-dependent dilation of rabbit brain
arterioles. Circ Res. 1991;68:1621–1627.
15. Handa RK, Krebs LT, Harding JW, Handa SE. Angiotensin IV AT4-receptor system in the rat kidney. Am J Physiol. 1998;274(pt 2):F290–F299.
16.
Iyer SN, Chappell MC, Averill DB, Diz DI, Ferrario CM.
Vasodepressor actions of angiotensin-(1–7) unmasked during
combined treatment with lisinopril and losartan.
Hypertension. 1998;31:699–705.
17. Rowe BP, Noble AR, Munday KA. Cross-tachyphylaxis of the pressor response to angiotensins in conscious rabbits. Pflugers Arch. 1979;380:13–17.[Medline] [Order article via Infotrieve]
18. Santos RAS, Campagnole-Santos MJ, Baracho NCV, Fontes MAP, Silva LCS, Neves LAA, Oliveira DR, Caligiorne SM, Rodrigues ARV, Gropen C, Carvalho WS, Simoes AC, Khosla MC. Characterization of a new angiotensin antagonist selective for angiotensin-(1–7): evidence that the actions of angiotensin-(1–7) are mediated by specific angiotensin receptors. Brain Res Bull. 1994;35:293–298.[Medline] [Order article via Infotrieve]
19.
Zhang JH, Stobb JW, Hanesworth JM, Sardinia MF, Harding
JW. Characterization and purification of the bovine adrenal
angiotensin IV receptor (AT4) using
[125I]benzoylphenylalanine-angiotensin
IV as a specific photolabel. J Pharmacol Exp Ther.. 1998;287:416–424.
20.
Al-Merani SA, Brooks DP, Chapman BJ, Munday KA. The
half-lives of angiotensin II, angiotensin
II-amide, angiotensin III,
Sar1-Ala8-angiotensin
II and renin in the circulatory system of the rat. J Physiol
(Lond). 1978;278:471–490.
21. Rowe BP. The effect of prostaglandin and kinin synthesis inhibition on blood pressure during infusion of angiotensin II in the conscious rabbit. Clin Exp Hypertens. 1984;A6:1281–1295.
22.
Pipkin FB, Baker PN. Angiotensin II has
depressor effects in pregnant and non-pregnant women.
Hypertension. 1997;30:1247–1252.
23. Brosnihan KB, Li P, Ganten D, Ferrario CM. Estrogen protects transgenic hypertensive rats by shifting the vasoconstrictor-vasodilator balance of RAS. Am J Physiol. 1997;273:R1908–R1915.
This article has been cited by other articles:
 |
|
 |
|
  N. Toda, K. Ayajiki, and T. Okamura Interaction of Endothelial Nitric Oxide and Angiotensin in the Circulation Pharmacol. Rev., March 1, 2007; 59(1): 54 - 87. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||