Angiotensin-(1eC7) Acts as a Vasodepressor Agent Via Angiotensin II Type 2 Receptors in Conscious Rats
the Department of Pharmacology, Monash University, Melbourne, Australia.
Abstract
Given that angiotensin-(1eC7) (Ang-[1eC7]) has been frequently reported to exert direct in vitro vascular effects but less often in vivo, we investigated whether a vasodepressor effect of Ang-(1eC7) could be unmasked acutely in conscious spontaneously hypertensive rats (SHR) against a background of angiotensin II type 1 (AT1) receptor blockade. Mean arterial pressure (MAP) and heart rate were measured over a 5-day protocol in various groups of rats randomized to receive the following drug combinations: saline, AT1 receptor (AT1R) antagonist candesartan (0.01 or 0.1 mg/kg IV) alone, Ang-(1eC7) (5 pmol/min) alone, candesartan plus Ang-(1eC7), and candesartan plus Ang-(1eC7) and angiotensin II type 2 (AT2) receptor (AT2R) antagonist PD123319 (50 e蘥/kg per minute). In Wistar-Kyoto (WKY) rats, saline, Ang-(1eC7), or candesartan alone caused no significant alteration in MAP, whereas Ang-(1eC7) coadministered with candesartan caused a marked, sustained reduction in MAP. A similar unmasking of a vasodepressor response to Ang-(1eC7) during AT1R blockade was observed in SHR. Moreover, the AT2R antagonist PD123319 markedly attenuated the enhanced depressor response evoked by the Ang-(1eC7)/candesartan combination in SHR and WKY rats, whereas in other experiments, the putative Ang-(1eC7) antagonist A-779 (5 and 50 pmol/min) did not attenuate this vasodepressor effect. In separate experiments, the bradykinin type 2 receptor antagonist HOE 140 (100 e蘥/kg IV) or the NO synthase inhibitor N-nitro-L-arginine methyl ester (1 mg/kg IV) abolished the depressor effect of Ang-(1eC7) in the presence of candesartan. Collectively, these results suggest that Ang-(1eC7) evoked a depressor response during AT1R blockade via activation of AT2R, which involves the bradykinineCNO cascade.
Key Words: receptors, angiotensin rats, spontaneously hypertensive
Introduction
It is widely accepted that the angiotensin II (Ang II) type 1 receptor (AT1R) accounts for the majority of cardiovascular effects evoked by Ang II, such as contraction/pressor activity and growth-promoting effects leading to cardiac and vascular hypertrophy.1 In contrast, the Ang II type 2 receptor (AT2R) is thought to exert opposing effects, including vasodepressor and antigrowth effects.2,3
In addition, the N-heptapeptide fragment angiotensin-(1eC7) (Ang-(1eC7)) evokes a range of central and peripheral effects, including vasodilatation, that are not always sensitive to AT1R or AT2R blockade.4 For example, Ang-(1eC7) has been reported previously to directly cause relaxation in preparations such as coronary and mesenteric arteries5eC7 as well as to potentiate the vasodepressor effects of bradykinin in conscious rats.7eC9 Although we could not confirm this bradykinin potentiation, we confirmed the report10 that Ang-(1eC7) caused a slow-onset depressor response with chronic infusion in conscious spontaneously hypertensive rats (SHR).11
Given that Ang-(1eC7) has been reported frequently to exert direct in vitro vascular effects but less often in vivo, we hypothesized that like the AT2R agonist CGP4211212 a vasodepressor effect of Ang-(1eC7) would be unmasked acutely in conscious SHR against a background of AT1R blockade. Our reasoning was based on the likelihood that any potential vasoconstrictor effect of Ang-(1eC7) would be negated by AT1R blockade, thus allowing an AT2R component of the peptide to be evaluated. In addition, the D-ala analogue of Ang-(1eC7), A-779, is reported to be an antagonist of Ang-(1eC7).13 Therefore, the second aim of the present study was to determine whether any Ang-(1eC7)eCevoked depressor effect was mediated by AT2R or via a non-AT1/AT2R, A-779eCsensitive mechanism. Finally, we examined whether blockade of NO and bradykinin, both of which are linked to AT2R signaling and Ang-(1eC7) receptor signaling, altered cardiovascular effects of Ang-(1eC7).4,14
Methods
A total of 66 male 16- to 18-week-old SHR and Wistar-Kyoto (WKY) rats, weighing 300 to 350 g were used. These animals were obtained from Biological Research Laboratories (Austin Repatriation Medical Centre, Australia) and were maintained on a 12-hour light/dark cycle with free access to food and water.
General Procedure
On the day before experimentation, rats were anesthetized (ketamine and xylazine; 75 mg/kg and 10 mg/kg IP, respectively; supplemented as required) and catheters inserted into the right carotid artery and right jugular vein for direct blood pressure measurement and drug administration, respectively. Approximately 24 hours after surgery, the carotid artery was connected to a pressure transducer (Gould Inc.) attached to a MacLab-8 data acquisition system (ADInstruments). Mean arterial pressure (MAP) and heart rate (HR) were derived from the phasic blood pressure signal.
Experimental Protocol
Eight groups of rats underwent experimental protocols during which basal MAP and HR were recorded using a similar within-animal protocol tested over 4 to 5 days, as reported previously.12 Groups 1 (WKY rats) and 2 (SHR) were randomly assigned to receive 1 of the following IV treatments on each of the experimental days: (1) saline infusion (0.35 mL/h for 4 hours); (2) a bolus injection of candesartan (0.1 mg/kg in WKY rats or 0.01 mg/kg in SHR) plus a 4-hour infusion of saline; (3) Ang-(1eC7) infusion (5 pmol/min for 4 hours); (4) a 4-hour Ang-(1eC7) infusion commenced at the same time as a bolus injection of candesartan; and (5) a 4-hour Ang-(1eC7) infusion in the presence of candesartan and PD123319 (50 e蘥/kg per minute infusion). PD123319 was only given for 2 hours to determine any washout effect of AT2R blockade. The dose of Ang-(1eC7) is based on previous studies,11,15 whereas the different doses of the AT1R antagonist used in the 2 strains provided a low level of AT1R blockade that was used previously to unmask a vasodilator effect of CGP4211212 In addition, Ang II was administered (10 ng IV) before and 2 hours after commencement of the daily treatment protocol to assess level of AT1R blockade.
Additional WKY rats (group 3) and SHR (group 4) received candesartan alone or in combination with Ang-(1eC7), the putative Ang-(1eC7) antagonist A-779 (5 pmol/min for 2 hours) alone, or in combination with Ang-(1eC7) and candesartan. This initial dose of A-779 was derived from a study by Lima et al in which equimolar doses of Ang-(1eC7) and the Ang-(1eC7) receptor antagonist A-779 were used.9 A 10-fold higher dose of A-779 (50 pmol/min) was also investigated in SHR using the same protocol (group 5).
Analogous experiments were performed with either the bradykinin type 2 receptor antagonist HOE 140 (100 e蘥/kg IV; group 6) or the NO synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME; 1 mg/kg IV; group 7) instead of A-779. In preliminary dose-ranging studies with these inhibitors, it was found that they attenuated vasodepressor responses to the Ang-(1eC7)/candesartan combination for 24 hours. Therefore, in these series of experiments, a washout day was left after either HOE 140 (group 6) or L-NAME (group 7) before another treatment was performed. Finally, a 10-fold higher dose of candesartan (0.1 mg/kg IV) was also tested in SHR in combination with Ang-(1eC7) (group 8).
Candesartan and HOE 140 (also known as JE 049) were kind gifts from AstraZenica, Sweden, and Aventis Pharma Deutschland GmbH, respectively. PD123319 was synthesized by CSIRO (Clayton, Australia),16 whereas Ang-(1eC7) (Auspep), L-NAME (Sigma), and A-779 (Bachem) were purchased from commercial sources. Doses of candesartan, Ang-(1eC7), PD123319, A-779, and L-NAME were based on previous studies,9,11,12,15 whereas HOE 140 was shown in preliminary studies to block the depressor effects evoked by bradykinin (data not shown).
Statistical Analysis
All data in Figures 1 through 5 are presented as mean±SEM. Differences in MAP and HR from baseline measurements for groups 1 through 8 were analyzed using a 1-way ANOVA with repeated measures (GraphPad PRISM). Differences in MAP between treatments and treatment/time interactions were analyzed using a 2-way ANOVA with repeated measures. Differences in MAP for Ang II pressor doses before and 2 hours after the commencement of treatment were analyzed using a paired t test. Statistical significance was accepted at P<0.05.
Results
The baseline MAP and HR for each experimental day in each group are listed in supplemental Tables I and II (available online at http://hyper.ahajournals.org). There was negligible difference in basal values between different days, indicating that individual daily treatments had no persistent effect on MAP and HR on subsequent days. Generally, the effects of various treatments had minimal effect on HR except for modest tachycardia in response to candesartan, as reported previously.12
In both rat strains, infusion of saline or Ang-(1eC7) (5 pmol/min) alone had no effect on MAP (Figure 1A and 1B); therefore, these treatments were not always performed in subsequent groups. In WKY rats, candesartan (0.1 mg/kg IV) had no effect on MAP (<10 mm Hg reductions), but when infused with Ang-(1eC7) (5 pmol/min), this combination caused a sustained decrease in MAP that was attenuated by the additional infusion of the AT2R antagonist PD123319 (50 e蘥/kg per minute), an inhibitory effect that waned when the PD123319 infusion was stopped (Figure 1A). In SHR, candesartan (0.01 mg/kg IV) caused a small decrease in MAP (<10 mm Hg reductions), and like in WKY rats, a greater depressor effect was unmasked when combined with Ang-(1eC7) (5 pmol/min); this effect was also blocked by administration of PD123319 (Figure 1B).
Infusion of the putative Ang-(1eC7) antagonist A-779 (5 pmol/min) alone exerted minimal effect on MAP in WKY rats and SHR (Figure 2A and 2B). Moreover, the ability of Ang-(1eC7) to unmask a depressor response in the presence of candesartan was not affected when A-779 was infused with the Ang (1eC7)/candesartan combination in WKY rats and SHR (Figure 2A and 2B). Furthermore, a 10-fold higher dose of A-779 (50 pmol/min) failed to ablate the Ang-(1eC7)/candesartan combined vasodepressor response (Figure 3). It should be noted that when PD123319 was infused alone into SHR, there was no significant alteration of MAP (Figure 3).
The coadministration of HOE 140 (100 e蘥/kg IV) or L-NAME (1 mg/kg IV) with Ang-(1eC7)/candesartan was also examined in SHR. In both cases, these inhibitors abolished the enhanced depressor effect evoked by Ang-(1eC7) and candesartan (Figure 4A and 4B).
Pressor responses evoked by Ang II (10 ng IV) were also tested in the aforementioned groups, whereby Ang II was administered at least 30 minutes before the commencement of a daily treatment and then 2 hours later when the treatments were well established (Table 1). As expected, when given at doses of 0.1 mg/kg (WKY rats) and 0.01 mg/kg (SHR), candesartan significantly attenuated but did not completely abolish the Ang IIeCmediated pressor responses. The AT1R-mediated inhibition was not increased further when Ang-(1eC7) was infused in combination with candesartan; nor was it markedly affected by any of the other combinations (Table 1).
Pressor Responses ( mm Hg) Evoked by Ang II (10 ng IV) Before and After Treatments, as Indicated, in WKY and SHR
In separate experiments, candesartan was administered at a 10-fold higher dose in SHR, in which it caused a modest antihypertensive effect (20 mm Hg). The addition of Ang-(1eC7) during AT1R blockade caused a vasodepressor response that was greater than that seen with candesartan alone (Figure 5).
Discussion
The novel findings of the present study were that Ang-(1eC7) evoked a vasodepressor effect in conscious WKY rats and SHR in the presence of partial AT1R blockade, an effect that was mediated via AT2R and did not appear to involve a non-AT1/AT2R, A-779eCsensitive site.
Previous studies using Ang-(1eC7) have reported vasoconstrictor17,18 and vasodilator7,17,19 actions of this peptide. In addition, the Ang II receptor subtype involved in its cardiovascular actions is under dispute because the peptide has been reported to act at either AT1R or AT2R, as well as via non-AT1/AT2R sites.4,20,21
In the present study, Ang-(1eC7) given acutely evoked only minimal effects on MAP and HR. Conceivably, Ang-(1eC7) could cause AT1R-mediated vasoconstriction and AT2R-mediated vasodilatation, which would then cancel each other out under basal conditions. Indeed, it was only when Ang-(1eC7) and candesartan were coadministered, that a marked decrease in MAP in SHR and WKY rats was revealed. This vasodepressor effect was greater than that seen with either agent alone. Generally, candesartan caused only relatively small reductions in MAP when infused alone; thus, these doses were chosen because we have shown previously that this protocol optimized the potential unmasking of AT2R-mediated depressor responses.12 Such partial AT1R blockade not only caused submaximal reductions in MAP, which would enable further reductions to be detected, it also allowed one to determine whether the combination of Ang-(1eC7) and candesartan caused a greater reduction in the Ang IIeCmediated pressor responses, which may suggest that Ang-(1eC7) caused additional AT1R blockade. Strikingly, the AT2R antagonist PD123319 markedly attenuated the enhanced depressor response caused by Ang-(1eC7)/candesartan, suggesting that Ang-(1eC7) acts as a vasodepressor agent via the AT2R. It is likely that this effect is via peripheral vasodilation because we have shown recently in analogous studies that CGP42112caused AT2R-mediated depressor effects together with regional vasodilation.22 Moreover, Ang-(1eC7) still evoked a depressor response in the presence of a 10-fold greater dose of candesartan.
In the present study, when Ang-(1eC7) and A-779 were used at equimolar doses as in previous studies,8,9 the Ang-(1eC7) antagonist failed to block the Ang-(1eC7)eCinduced depressor response against a background a AT1R blockade. The fact that a 10-fold greater dose of A-779 was also ineffective adds further weight to our results suggesting that Ang-(1eC7) may act via the AT2R. Other researchers have suggested that Ang-(1eC7) may act via a "PD-sensitive" site because the peptide has low AT2R affinity in rat brain and in cultured cells, although the AT2R antagonist blocked a number of in vitro effects of Ang-(1eC7).4 However, these studies were performed before A-779 became available,4 which does not allow a direct comparison between antagonists to be made. Moreover, a unique Ang-(1eC7) binding site was postulated in cultured endothelial cells that did not express either AT1R or AT2R,23 which highlights the need to examine in vivo mechanisms.
Thus, we would argue that Ang-(1eC7) was acting via conventional AT2R because this peptide was PD123319 sensitive and A-779 insensitive in an in vivo assay we established using the AT2R agonist CGP4211212,22 which was confirmed by Carey et al.24 PD123319 was infused for a shorter period (2 hours) than Ang-(1eC7) to determine whether vasodepressor responses would return after stopping the AT2R antagonist infusion. Indeed, this was the case in WKY rats but not SHR. Interestingly, the Ang-(1eC7)eCmediated depressor effects, in the presence of partial AT1R blockade, in both strains persisted after the termination of the Ang-(1eC7) infusion. Given that Ang-(1eC7) is metabolized rapidly, it is possible that the prolonged depressor effects are a result of the activation of downstream signaling mechanisms. The depressor effects of Ang-(1eC7), although generally greater in SHR, occurred in both strains, which differs from our previous results using CGP42112 in which the AT2R agonist was ineffective in WKY rats.12,22 Whether or not this reflects subtle differences between strains with respect to AT2R distribution or interactions with different ligands remains to be determined. At this stage, there are no other analogous studies available for strain comparisons.
The fact that Ang-(1eC7), like CGP4211212,22 exerted no effect on MAP unless there was some AT1R blockade suggests a complex interaction between AT2R and AT1R. In a previous study, CGP42112did not cause a greater depressor response in the presence of a 10-fold higher concentration (0.1 mg/kg) of candesartan,12 whereas in the present study, Ang-(1eC7) still evoked a depressor response in combination with candesartan (0.1 mg/kg). This finding, together with the ability of Ang-(1eC7) to evoke depressor effects in both strains, may suggest a greater AT2R selectivity of Ang-(1eC7) in this experimental model. Interestingly, Ang-(1eC7) was shown recently to exhibit greater AT2R selectivity than Ang II in basolateral membranes of proximal tubules. Whereas PD123319 abolished the Ang-(1eC7)eC and Ang IIeCinduced inhibition of Na+-ATPase activity, the AT2R antagonist inhibited the effects of Ang-(1eC7) at lower concentrations than required for Ang II.20 Others have reported that Ang II receptor subtypes may exist as heterodimers in transfected cells expressing AT1R and AT2R, whereby AT2R negatively regulates AT1R signaling but via a mechanism independent of either agonist or PD123319.25 Although the results of the current in vivo study were very different from the molecular study,25 both help to illustrate the complex interplay that can occur between Ang II receptor subtypes. As already mentioned, it is possible that Ang-(1eC7) may exhibit opposing vascular actions that cancel one another. Alternatively, AT1/AT2R cross-talk may play a role,3 whereby the removal of (predominant) tonic AT1R-mediated vasoconstriction, caused by endogenous Ang II, may be necessary to unmask any AT2R-mediated vasodilation.
In addition, other studies also support an AT2R action of Ang-(1eC7).26eC28 Ang-(1eC7) was reported recently to mediate NO and superoxide release, which was markedly attenuated by PD123319 (90%) and to a lesser extent by A-779 (50%) and AT1R blockade (60%),29 although the relatively high concentration of Ang-(1eC7) used raises the issue of selectivity of response. Moreover, Ang-(1eC7)eCmediated relaxation in rat isolated aorta was unmasked in the presence of an AT1R antagonist;30 however, the effect of PD123319 on this response was not tested.
On the other hand, the putative Ang-(1eC7) antagonist A-779 has been reported to attenuate many effects of Ang-(1eC7), including potentiation of bradykinin-induced hypotension5,8,9 and relaxation,19,31 as well as vasodepressor effects in salt-sensitive hypertensive rats.32 In our previous study, in which Ang-(1eC7) caused a slow-onset depressor response over several days in SHR,11 there was equivocal evidence for an inhibitory effect of A-779. On the one hand, there was no significant reduction in basal MAP when this compound was coinfused with Ang-(1eC7); however, at the same time, this combination was not significantly different from the antihypertensive effect when Ang-(1eC7) was infused alone.11 In a different in vivo model, it was reported recently that Ang-(1eC7) reduced MAP acutely in Dahl salt-sensitive rats, and this effect was partly blocked by A-779, although the effect of AT2R blockade against Ang-(1eC7) was not tested.32 However, Nakamura et al reported that in salt-depleted SHR, A-779 caused a greater reversal of the antihypertensive effect of losartan than did PD123319, although these were relatively small effects.33
In this study, we also ruled out the possibility that Ang-(1eC7) acted as an AT1R antagonist to cause an additive AT1R block when combined with candesartan. Ang-(1eC7) is reported to act as an AT1R antagonist, but this effect occurred at doses 10 to 100x greater than those used in the present study.21 Moreover, Ang-(1eC7) itself did not lower basal MAP or inhibit the pressor effect evoked by Ang II, nor did it increase the inhibitory effect of candesartan on Ang IIeCmediated pressor responses when given in combination with the AT1R antagonist such that one would expect to see whether Ang-(1eC7) was acting as an AT1 antagonist.
Given the likely involvement of AT2R, we investigated the potential signaling pathways involving bradykinin and NO because these mediators have well-established roles in AT2R signal transduction.3,14,34 The vasodepressor effect of Ang-(1eC7) was abolished by HOE 140 or L-NAME. Interestingly, Ang-(1eC7) was reported to cause vasodilatation of canine and porcine isolated coronary arteries via a bradykinin/NO pathway,5eC8 and this was blocked predominantly by AT2R antagonism.6 Ang-(1eC7) was also reported to cause stimulation of endothelium-dependent NO release in cultured bovine aortic endothelial cells, and this was mediated via a bradykinin/NO pathway, which was attenuated by up to 90% using an AT2R antagonist.29 Many previous in vitro35,36 and in vivo34 studies have identified a link between AT2R activation and bradykinin and NO production. Therefore, on the basis of the present and previous11,12,24 findings, we suggest the AT2R may be activated by endogenous peptides such as Ang II and Ang-(1eC7), as well as by exogenous CGP42112 initiating a process involving bradykinin type 2 receptors and NO production leading to vasodilatation.35,36
Indeed, our results would indicate that Ang-(1eC7) may contribute to some of the counter-regulatory AT2R effects that oppose the AT1R-mediated effects.3 The potential importance of Ang-(1eC7) is underscored by the emergence of newly discovered peptide processing pathways involving the enzyme angiotensin-converting enzyme 2 (ACE2).37 For example, it is known that ACE2 can use Ang II as a substrate in the formation of Ang-(1eC7), and ACE2 is expressed predominantly in vasculature, heart, and kidneys.37 Therefore, it is possible that physiologically relevant levels of Ang-(1eC7) may be produced, particularly because it is known that ACE inhibitors increase levels of the heptapeptide by 5- to 50-fold.38
Perspectives
Increasingly, shortened peptide fragments of Ang II are being reported to exert physiological effects. In the present study, Ang-(1eC7) appears to have a role as a vasodepressor agent via activation of the AT2R, in the presence of partial AT1R blockade, and this vasodepressor mechanism involves the bradykinineCNO cascade. In addition, new pathways for processing Ang-(1eC7) and other peptides, such as ACE2, have been elucidated recently, thus emphasizing the potential importance of these peptides, particularly because Ang-(1eC7) levels are increased during ACE inhibition and AT1 blockade. Indeed, the possibility that Ang-(1eC7) may contribute to the therapeutic effect of AT1R antagonists, in much the same way as Ang II itself acting on uninhibited AT2Rs, requires further investigation. Collectively, the present results indicate that one should not only consider Ang II itself as an endogenous activator of AT2R but possibly other angiotensin peptides, including Ang-(1eC7).
Acknowledgments
This work was supported in part by grants from the National Health and Medical Research Council of Australia. The assistance provided by Dr Emma Jones in the initial phase of this work is gratefully acknowledged.
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Campbell DJ, Kladis A, Duncan AM. Effects of converting enzyme inhibitors on angiotensin and bradykinin peptides. Hypertension. 1994; 23: 439eC449., http://www.100md.com(Pia E. Walters; Tracey A.)
Abstract
Given that angiotensin-(1eC7) (Ang-[1eC7]) has been frequently reported to exert direct in vitro vascular effects but less often in vivo, we investigated whether a vasodepressor effect of Ang-(1eC7) could be unmasked acutely in conscious spontaneously hypertensive rats (SHR) against a background of angiotensin II type 1 (AT1) receptor blockade. Mean arterial pressure (MAP) and heart rate were measured over a 5-day protocol in various groups of rats randomized to receive the following drug combinations: saline, AT1 receptor (AT1R) antagonist candesartan (0.01 or 0.1 mg/kg IV) alone, Ang-(1eC7) (5 pmol/min) alone, candesartan plus Ang-(1eC7), and candesartan plus Ang-(1eC7) and angiotensin II type 2 (AT2) receptor (AT2R) antagonist PD123319 (50 e蘥/kg per minute). In Wistar-Kyoto (WKY) rats, saline, Ang-(1eC7), or candesartan alone caused no significant alteration in MAP, whereas Ang-(1eC7) coadministered with candesartan caused a marked, sustained reduction in MAP. A similar unmasking of a vasodepressor response to Ang-(1eC7) during AT1R blockade was observed in SHR. Moreover, the AT2R antagonist PD123319 markedly attenuated the enhanced depressor response evoked by the Ang-(1eC7)/candesartan combination in SHR and WKY rats, whereas in other experiments, the putative Ang-(1eC7) antagonist A-779 (5 and 50 pmol/min) did not attenuate this vasodepressor effect. In separate experiments, the bradykinin type 2 receptor antagonist HOE 140 (100 e蘥/kg IV) or the NO synthase inhibitor N-nitro-L-arginine methyl ester (1 mg/kg IV) abolished the depressor effect of Ang-(1eC7) in the presence of candesartan. Collectively, these results suggest that Ang-(1eC7) evoked a depressor response during AT1R blockade via activation of AT2R, which involves the bradykinineCNO cascade.
Key Words: receptors, angiotensin rats, spontaneously hypertensive
Introduction
It is widely accepted that the angiotensin II (Ang II) type 1 receptor (AT1R) accounts for the majority of cardiovascular effects evoked by Ang II, such as contraction/pressor activity and growth-promoting effects leading to cardiac and vascular hypertrophy.1 In contrast, the Ang II type 2 receptor (AT2R) is thought to exert opposing effects, including vasodepressor and antigrowth effects.2,3
In addition, the N-heptapeptide fragment angiotensin-(1eC7) (Ang-(1eC7)) evokes a range of central and peripheral effects, including vasodilatation, that are not always sensitive to AT1R or AT2R blockade.4 For example, Ang-(1eC7) has been reported previously to directly cause relaxation in preparations such as coronary and mesenteric arteries5eC7 as well as to potentiate the vasodepressor effects of bradykinin in conscious rats.7eC9 Although we could not confirm this bradykinin potentiation, we confirmed the report10 that Ang-(1eC7) caused a slow-onset depressor response with chronic infusion in conscious spontaneously hypertensive rats (SHR).11
Given that Ang-(1eC7) has been reported frequently to exert direct in vitro vascular effects but less often in vivo, we hypothesized that like the AT2R agonist CGP4211212 a vasodepressor effect of Ang-(1eC7) would be unmasked acutely in conscious SHR against a background of AT1R blockade. Our reasoning was based on the likelihood that any potential vasoconstrictor effect of Ang-(1eC7) would be negated by AT1R blockade, thus allowing an AT2R component of the peptide to be evaluated. In addition, the D-ala analogue of Ang-(1eC7), A-779, is reported to be an antagonist of Ang-(1eC7).13 Therefore, the second aim of the present study was to determine whether any Ang-(1eC7)eCevoked depressor effect was mediated by AT2R or via a non-AT1/AT2R, A-779eCsensitive mechanism. Finally, we examined whether blockade of NO and bradykinin, both of which are linked to AT2R signaling and Ang-(1eC7) receptor signaling, altered cardiovascular effects of Ang-(1eC7).4,14
Methods
A total of 66 male 16- to 18-week-old SHR and Wistar-Kyoto (WKY) rats, weighing 300 to 350 g were used. These animals were obtained from Biological Research Laboratories (Austin Repatriation Medical Centre, Australia) and were maintained on a 12-hour light/dark cycle with free access to food and water.
General Procedure
On the day before experimentation, rats were anesthetized (ketamine and xylazine; 75 mg/kg and 10 mg/kg IP, respectively; supplemented as required) and catheters inserted into the right carotid artery and right jugular vein for direct blood pressure measurement and drug administration, respectively. Approximately 24 hours after surgery, the carotid artery was connected to a pressure transducer (Gould Inc.) attached to a MacLab-8 data acquisition system (ADInstruments). Mean arterial pressure (MAP) and heart rate (HR) were derived from the phasic blood pressure signal.
Experimental Protocol
Eight groups of rats underwent experimental protocols during which basal MAP and HR were recorded using a similar within-animal protocol tested over 4 to 5 days, as reported previously.12 Groups 1 (WKY rats) and 2 (SHR) were randomly assigned to receive 1 of the following IV treatments on each of the experimental days: (1) saline infusion (0.35 mL/h for 4 hours); (2) a bolus injection of candesartan (0.1 mg/kg in WKY rats or 0.01 mg/kg in SHR) plus a 4-hour infusion of saline; (3) Ang-(1eC7) infusion (5 pmol/min for 4 hours); (4) a 4-hour Ang-(1eC7) infusion commenced at the same time as a bolus injection of candesartan; and (5) a 4-hour Ang-(1eC7) infusion in the presence of candesartan and PD123319 (50 e蘥/kg per minute infusion). PD123319 was only given for 2 hours to determine any washout effect of AT2R blockade. The dose of Ang-(1eC7) is based on previous studies,11,15 whereas the different doses of the AT1R antagonist used in the 2 strains provided a low level of AT1R blockade that was used previously to unmask a vasodilator effect of CGP4211212 In addition, Ang II was administered (10 ng IV) before and 2 hours after commencement of the daily treatment protocol to assess level of AT1R blockade.
Additional WKY rats (group 3) and SHR (group 4) received candesartan alone or in combination with Ang-(1eC7), the putative Ang-(1eC7) antagonist A-779 (5 pmol/min for 2 hours) alone, or in combination with Ang-(1eC7) and candesartan. This initial dose of A-779 was derived from a study by Lima et al in which equimolar doses of Ang-(1eC7) and the Ang-(1eC7) receptor antagonist A-779 were used.9 A 10-fold higher dose of A-779 (50 pmol/min) was also investigated in SHR using the same protocol (group 5).
Analogous experiments were performed with either the bradykinin type 2 receptor antagonist HOE 140 (100 e蘥/kg IV; group 6) or the NO synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME; 1 mg/kg IV; group 7) instead of A-779. In preliminary dose-ranging studies with these inhibitors, it was found that they attenuated vasodepressor responses to the Ang-(1eC7)/candesartan combination for 24 hours. Therefore, in these series of experiments, a washout day was left after either HOE 140 (group 6) or L-NAME (group 7) before another treatment was performed. Finally, a 10-fold higher dose of candesartan (0.1 mg/kg IV) was also tested in SHR in combination with Ang-(1eC7) (group 8).
Candesartan and HOE 140 (also known as JE 049) were kind gifts from AstraZenica, Sweden, and Aventis Pharma Deutschland GmbH, respectively. PD123319 was synthesized by CSIRO (Clayton, Australia),16 whereas Ang-(1eC7) (Auspep), L-NAME (Sigma), and A-779 (Bachem) were purchased from commercial sources. Doses of candesartan, Ang-(1eC7), PD123319, A-779, and L-NAME were based on previous studies,9,11,12,15 whereas HOE 140 was shown in preliminary studies to block the depressor effects evoked by bradykinin (data not shown).
Statistical Analysis
All data in Figures 1 through 5 are presented as mean±SEM. Differences in MAP and HR from baseline measurements for groups 1 through 8 were analyzed using a 1-way ANOVA with repeated measures (GraphPad PRISM). Differences in MAP between treatments and treatment/time interactions were analyzed using a 2-way ANOVA with repeated measures. Differences in MAP for Ang II pressor doses before and 2 hours after the commencement of treatment were analyzed using a paired t test. Statistical significance was accepted at P<0.05.
Results
The baseline MAP and HR for each experimental day in each group are listed in supplemental Tables I and II (available online at http://hyper.ahajournals.org). There was negligible difference in basal values between different days, indicating that individual daily treatments had no persistent effect on MAP and HR on subsequent days. Generally, the effects of various treatments had minimal effect on HR except for modest tachycardia in response to candesartan, as reported previously.12
In both rat strains, infusion of saline or Ang-(1eC7) (5 pmol/min) alone had no effect on MAP (Figure 1A and 1B); therefore, these treatments were not always performed in subsequent groups. In WKY rats, candesartan (0.1 mg/kg IV) had no effect on MAP (<10 mm Hg reductions), but when infused with Ang-(1eC7) (5 pmol/min), this combination caused a sustained decrease in MAP that was attenuated by the additional infusion of the AT2R antagonist PD123319 (50 e蘥/kg per minute), an inhibitory effect that waned when the PD123319 infusion was stopped (Figure 1A). In SHR, candesartan (0.01 mg/kg IV) caused a small decrease in MAP (<10 mm Hg reductions), and like in WKY rats, a greater depressor effect was unmasked when combined with Ang-(1eC7) (5 pmol/min); this effect was also blocked by administration of PD123319 (Figure 1B).
Infusion of the putative Ang-(1eC7) antagonist A-779 (5 pmol/min) alone exerted minimal effect on MAP in WKY rats and SHR (Figure 2A and 2B). Moreover, the ability of Ang-(1eC7) to unmask a depressor response in the presence of candesartan was not affected when A-779 was infused with the Ang (1eC7)/candesartan combination in WKY rats and SHR (Figure 2A and 2B). Furthermore, a 10-fold higher dose of A-779 (50 pmol/min) failed to ablate the Ang-(1eC7)/candesartan combined vasodepressor response (Figure 3). It should be noted that when PD123319 was infused alone into SHR, there was no significant alteration of MAP (Figure 3).
The coadministration of HOE 140 (100 e蘥/kg IV) or L-NAME (1 mg/kg IV) with Ang-(1eC7)/candesartan was also examined in SHR. In both cases, these inhibitors abolished the enhanced depressor effect evoked by Ang-(1eC7) and candesartan (Figure 4A and 4B).
Pressor responses evoked by Ang II (10 ng IV) were also tested in the aforementioned groups, whereby Ang II was administered at least 30 minutes before the commencement of a daily treatment and then 2 hours later when the treatments were well established (Table 1). As expected, when given at doses of 0.1 mg/kg (WKY rats) and 0.01 mg/kg (SHR), candesartan significantly attenuated but did not completely abolish the Ang IIeCmediated pressor responses. The AT1R-mediated inhibition was not increased further when Ang-(1eC7) was infused in combination with candesartan; nor was it markedly affected by any of the other combinations (Table 1).
Pressor Responses ( mm Hg) Evoked by Ang II (10 ng IV) Before and After Treatments, as Indicated, in WKY and SHR
In separate experiments, candesartan was administered at a 10-fold higher dose in SHR, in which it caused a modest antihypertensive effect (20 mm Hg). The addition of Ang-(1eC7) during AT1R blockade caused a vasodepressor response that was greater than that seen with candesartan alone (Figure 5).
Discussion
The novel findings of the present study were that Ang-(1eC7) evoked a vasodepressor effect in conscious WKY rats and SHR in the presence of partial AT1R blockade, an effect that was mediated via AT2R and did not appear to involve a non-AT1/AT2R, A-779eCsensitive site.
Previous studies using Ang-(1eC7) have reported vasoconstrictor17,18 and vasodilator7,17,19 actions of this peptide. In addition, the Ang II receptor subtype involved in its cardiovascular actions is under dispute because the peptide has been reported to act at either AT1R or AT2R, as well as via non-AT1/AT2R sites.4,20,21
In the present study, Ang-(1eC7) given acutely evoked only minimal effects on MAP and HR. Conceivably, Ang-(1eC7) could cause AT1R-mediated vasoconstriction and AT2R-mediated vasodilatation, which would then cancel each other out under basal conditions. Indeed, it was only when Ang-(1eC7) and candesartan were coadministered, that a marked decrease in MAP in SHR and WKY rats was revealed. This vasodepressor effect was greater than that seen with either agent alone. Generally, candesartan caused only relatively small reductions in MAP when infused alone; thus, these doses were chosen because we have shown previously that this protocol optimized the potential unmasking of AT2R-mediated depressor responses.12 Such partial AT1R blockade not only caused submaximal reductions in MAP, which would enable further reductions to be detected, it also allowed one to determine whether the combination of Ang-(1eC7) and candesartan caused a greater reduction in the Ang IIeCmediated pressor responses, which may suggest that Ang-(1eC7) caused additional AT1R blockade. Strikingly, the AT2R antagonist PD123319 markedly attenuated the enhanced depressor response caused by Ang-(1eC7)/candesartan, suggesting that Ang-(1eC7) acts as a vasodepressor agent via the AT2R. It is likely that this effect is via peripheral vasodilation because we have shown recently in analogous studies that CGP42112caused AT2R-mediated depressor effects together with regional vasodilation.22 Moreover, Ang-(1eC7) still evoked a depressor response in the presence of a 10-fold greater dose of candesartan.
In the present study, when Ang-(1eC7) and A-779 were used at equimolar doses as in previous studies,8,9 the Ang-(1eC7) antagonist failed to block the Ang-(1eC7)eCinduced depressor response against a background a AT1R blockade. The fact that a 10-fold greater dose of A-779 was also ineffective adds further weight to our results suggesting that Ang-(1eC7) may act via the AT2R. Other researchers have suggested that Ang-(1eC7) may act via a "PD-sensitive" site because the peptide has low AT2R affinity in rat brain and in cultured cells, although the AT2R antagonist blocked a number of in vitro effects of Ang-(1eC7).4 However, these studies were performed before A-779 became available,4 which does not allow a direct comparison between antagonists to be made. Moreover, a unique Ang-(1eC7) binding site was postulated in cultured endothelial cells that did not express either AT1R or AT2R,23 which highlights the need to examine in vivo mechanisms.
Thus, we would argue that Ang-(1eC7) was acting via conventional AT2R because this peptide was PD123319 sensitive and A-779 insensitive in an in vivo assay we established using the AT2R agonist CGP4211212,22 which was confirmed by Carey et al.24 PD123319 was infused for a shorter period (2 hours) than Ang-(1eC7) to determine whether vasodepressor responses would return after stopping the AT2R antagonist infusion. Indeed, this was the case in WKY rats but not SHR. Interestingly, the Ang-(1eC7)eCmediated depressor effects, in the presence of partial AT1R blockade, in both strains persisted after the termination of the Ang-(1eC7) infusion. Given that Ang-(1eC7) is metabolized rapidly, it is possible that the prolonged depressor effects are a result of the activation of downstream signaling mechanisms. The depressor effects of Ang-(1eC7), although generally greater in SHR, occurred in both strains, which differs from our previous results using CGP42112 in which the AT2R agonist was ineffective in WKY rats.12,22 Whether or not this reflects subtle differences between strains with respect to AT2R distribution or interactions with different ligands remains to be determined. At this stage, there are no other analogous studies available for strain comparisons.
The fact that Ang-(1eC7), like CGP4211212,22 exerted no effect on MAP unless there was some AT1R blockade suggests a complex interaction between AT2R and AT1R. In a previous study, CGP42112did not cause a greater depressor response in the presence of a 10-fold higher concentration (0.1 mg/kg) of candesartan,12 whereas in the present study, Ang-(1eC7) still evoked a depressor response in combination with candesartan (0.1 mg/kg). This finding, together with the ability of Ang-(1eC7) to evoke depressor effects in both strains, may suggest a greater AT2R selectivity of Ang-(1eC7) in this experimental model. Interestingly, Ang-(1eC7) was shown recently to exhibit greater AT2R selectivity than Ang II in basolateral membranes of proximal tubules. Whereas PD123319 abolished the Ang-(1eC7)eC and Ang IIeCinduced inhibition of Na+-ATPase activity, the AT2R antagonist inhibited the effects of Ang-(1eC7) at lower concentrations than required for Ang II.20 Others have reported that Ang II receptor subtypes may exist as heterodimers in transfected cells expressing AT1R and AT2R, whereby AT2R negatively regulates AT1R signaling but via a mechanism independent of either agonist or PD123319.25 Although the results of the current in vivo study were very different from the molecular study,25 both help to illustrate the complex interplay that can occur between Ang II receptor subtypes. As already mentioned, it is possible that Ang-(1eC7) may exhibit opposing vascular actions that cancel one another. Alternatively, AT1/AT2R cross-talk may play a role,3 whereby the removal of (predominant) tonic AT1R-mediated vasoconstriction, caused by endogenous Ang II, may be necessary to unmask any AT2R-mediated vasodilation.
In addition, other studies also support an AT2R action of Ang-(1eC7).26eC28 Ang-(1eC7) was reported recently to mediate NO and superoxide release, which was markedly attenuated by PD123319 (90%) and to a lesser extent by A-779 (50%) and AT1R blockade (60%),29 although the relatively high concentration of Ang-(1eC7) used raises the issue of selectivity of response. Moreover, Ang-(1eC7)eCmediated relaxation in rat isolated aorta was unmasked in the presence of an AT1R antagonist;30 however, the effect of PD123319 on this response was not tested.
On the other hand, the putative Ang-(1eC7) antagonist A-779 has been reported to attenuate many effects of Ang-(1eC7), including potentiation of bradykinin-induced hypotension5,8,9 and relaxation,19,31 as well as vasodepressor effects in salt-sensitive hypertensive rats.32 In our previous study, in which Ang-(1eC7) caused a slow-onset depressor response over several days in SHR,11 there was equivocal evidence for an inhibitory effect of A-779. On the one hand, there was no significant reduction in basal MAP when this compound was coinfused with Ang-(1eC7); however, at the same time, this combination was not significantly different from the antihypertensive effect when Ang-(1eC7) was infused alone.11 In a different in vivo model, it was reported recently that Ang-(1eC7) reduced MAP acutely in Dahl salt-sensitive rats, and this effect was partly blocked by A-779, although the effect of AT2R blockade against Ang-(1eC7) was not tested.32 However, Nakamura et al reported that in salt-depleted SHR, A-779 caused a greater reversal of the antihypertensive effect of losartan than did PD123319, although these were relatively small effects.33
In this study, we also ruled out the possibility that Ang-(1eC7) acted as an AT1R antagonist to cause an additive AT1R block when combined with candesartan. Ang-(1eC7) is reported to act as an AT1R antagonist, but this effect occurred at doses 10 to 100x greater than those used in the present study.21 Moreover, Ang-(1eC7) itself did not lower basal MAP or inhibit the pressor effect evoked by Ang II, nor did it increase the inhibitory effect of candesartan on Ang IIeCmediated pressor responses when given in combination with the AT1R antagonist such that one would expect to see whether Ang-(1eC7) was acting as an AT1 antagonist.
Given the likely involvement of AT2R, we investigated the potential signaling pathways involving bradykinin and NO because these mediators have well-established roles in AT2R signal transduction.3,14,34 The vasodepressor effect of Ang-(1eC7) was abolished by HOE 140 or L-NAME. Interestingly, Ang-(1eC7) was reported to cause vasodilatation of canine and porcine isolated coronary arteries via a bradykinin/NO pathway,5eC8 and this was blocked predominantly by AT2R antagonism.6 Ang-(1eC7) was also reported to cause stimulation of endothelium-dependent NO release in cultured bovine aortic endothelial cells, and this was mediated via a bradykinin/NO pathway, which was attenuated by up to 90% using an AT2R antagonist.29 Many previous in vitro35,36 and in vivo34 studies have identified a link between AT2R activation and bradykinin and NO production. Therefore, on the basis of the present and previous11,12,24 findings, we suggest the AT2R may be activated by endogenous peptides such as Ang II and Ang-(1eC7), as well as by exogenous CGP42112 initiating a process involving bradykinin type 2 receptors and NO production leading to vasodilatation.35,36
Indeed, our results would indicate that Ang-(1eC7) may contribute to some of the counter-regulatory AT2R effects that oppose the AT1R-mediated effects.3 The potential importance of Ang-(1eC7) is underscored by the emergence of newly discovered peptide processing pathways involving the enzyme angiotensin-converting enzyme 2 (ACE2).37 For example, it is known that ACE2 can use Ang II as a substrate in the formation of Ang-(1eC7), and ACE2 is expressed predominantly in vasculature, heart, and kidneys.37 Therefore, it is possible that physiologically relevant levels of Ang-(1eC7) may be produced, particularly because it is known that ACE inhibitors increase levels of the heptapeptide by 5- to 50-fold.38
Perspectives
Increasingly, shortened peptide fragments of Ang II are being reported to exert physiological effects. In the present study, Ang-(1eC7) appears to have a role as a vasodepressor agent via activation of the AT2R, in the presence of partial AT1R blockade, and this vasodepressor mechanism involves the bradykinineCNO cascade. In addition, new pathways for processing Ang-(1eC7) and other peptides, such as ACE2, have been elucidated recently, thus emphasizing the potential importance of these peptides, particularly because Ang-(1eC7) levels are increased during ACE inhibition and AT1 blockade. Indeed, the possibility that Ang-(1eC7) may contribute to the therapeutic effect of AT1R antagonists, in much the same way as Ang II itself acting on uninhibited AT2Rs, requires further investigation. Collectively, the present results indicate that one should not only consider Ang II itself as an endogenous activator of AT2R but possibly other angiotensin peptides, including Ang-(1eC7).
Acknowledgments
This work was supported in part by grants from the National Health and Medical Research Council of Australia. The assistance provided by Dr Emma Jones in the initial phase of this work is gratefully acknowledged.
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