Angiotensin-(1-7) Induces Bradykinin-Mediated Hypotensive Responses in Anesthetized Rats
Abstract Angiotensin-(1-7) [Ang-(1-7)] reportedly potentiates hypotensive responses to bradykinin. We studied whether increases in circulating bradykinin would alter responses to Ang-(1-7). In rats anesthetized with thiobutabarbital, bradykinin infusion (5 μg/kg per minute IA) resulted in a rapid decrease in mean arterial pressure (MAP) of about 20 mm Hg (P<.01, n=9), although MAP slowly increased by 10 mm Hg after 15 minutes. When Ang-(1-7) (20, 80, and 380 nmol per rat IA) was given during bradykinin infusion, it elicited hypotension at 80 and 380 nmol (ΔMAP: −15±2.7 and −21±3.3 mm Hg, respectively; P<.001); this hypotension was not affected by the angiotensin type 1 antagonist L-158,809 (200 μg/kg IA), the angiotensin type 2 antagonist PD 123319 (10 mg/kg IA), saralasin, or sarthran (10 μg/kg per minute). The bradykinin type 2 receptor antagonist icatibant (30 μg per rat) eliminated the hypotensive responses to Ang-(1-7), which now increased MAP at all doses tested (P<.005). Thus in the presence of bradykinin, Ang-(1-7) induces hypotensive responses that are blocked by icatibant and unaffected by angiotensin receptor antagonists. Ang-(1-7) given to saline-infused rats elicited hypertensive responses at all doses (ΔMAP: 6.4±1.5, 12±1.6, and 16.3±2.7 mm Hg, respectively; P<.01); these responses were abolished by L-158,809 and sarthran. In rats pretreated with saralasin, Ang-(1-7) induced hypotension at 80 and 380 nmol (ΔMAP: −7.7±2.3 and −9.5±2.7, respectively; P<.05), whereas icatibant abolished this response. Thus in the rat, Ang-(1-7) can decrease blood pressure by a mechanism involving the bradykinin type 2 receptor and participates with bradykinin in a vasodepressor pathway that may serve a counterregulatory role, modulating the vasoconstrictor effects of Ang II.
Angiotensin-(1-7) is formed from Ang I and Ang II by peptidases other than ACE.1 2 Ang-(1-7) has biological actions that can often be distinguished from those of Ang II, originally demonstrated by Kohara et al2 and Benter et al.3 4 Ang-(1-7) induced long-lasting hypotension in the pithed rat3 and dilated piglet pial arteries.5 In isolated perfused cat mesenteric and hindquarters preparations, Ang-(1-7) induced both vasoconstrictor and vasodilator responses depending on the dose.6 The vasoconstrictor responses are likely due to activation of AT1 receptors, since they were blocked by losartan. The vasodilator responses were partially blocked by an NO synthase inhibitor, indicating that Ang-(1-7) can release NO.6
There are data suggesting an interaction between Ang-(1-7) and bradykinin and/or bradykinin receptors. In canine coronary arteries, Ang-(1-7) released NO, and this release was blunted by icatibant (Hoe 140), a bradykinin type 2 (B2) receptor antagonist.7 Pörsti et al8 reported that Ang-(1-7) induced relaxation in isolated perfused and precontracted porcine coronary arteries and that this effect was lessened by icatibant. Paula et al9 reported that Ang-(1-7) potentiated the hypotensive response to bradykinin, whereas Brosnihan et al,10 working with canine coronary arteries, found that Ang-(1-7) directly relaxed precontracted rings by a mechanism involving NO and bradykinin receptors; they postulated that it acts via a non-AT1/AT2 receptor. Somewhat different results were found by Gorelik and Scicli11 using isolated precontracted porcine coronary artery rings. They reported that Ang-(1-7) by itself at 10−9 to 10−5 mol/L was unable to induce relaxation; however, when given after bradykinin, Ang-(1-7) consistently relaxed the rings by a mechanism involving B2 receptors. Thus we hypothesize that Ang-(1-7) induces hypotension in the presence of bradykinin.
To test this hypothesis, we studied whether Ang-(1-7) can decrease blood pressure in rats when given alone or in the presence of vasodepressor amounts of bradykinin. We also evaluated the contribution of bradykinin and angiotensin receptors to the blood pressure responses induced by Ang-(1-7).
Bradykinin was purchased from Bachem and Ang-(1-7) from Bachem Bioscience. Ang II, saralasin ([Sar1,Val5,Ala8]Ang II), and sarthran ([Sar1,Thr8]Ang II) were obtained from Sigma Chemical Co. Icatibant (Hoe 140), L-158,809 (AT1 receptor antagonist), and PD 123319 were generously supplied by Hoechst-Marian Roussel Pharmaceuticals, DuPont-Merck, and Parke-Davis Pharmaceuticals, respectively. Thiobutabarbital (Inactin) was obtained from RBI.
Angiotensin and bradykinin peptides were freshly dissolved in 0.9% saline from concentrated aliquots kept in Eppendorf vials at −70°C until used. Once thawed, the aliquot was discarded.
All protocols were approved by the Institutional Experimental Animal Care Committee. Male Sprague-Dawley rats (375 to 400 g) bred at Charles River Laboratories (Wilmington, Mass) were used. Water was given ad libitum, but food was withheld on the night preceding the experiments. Rats were anesthetized with thiobutabarbital (125 mg/kg body wt IP). A tracheostomy was performed to facilitate spontaneous breathing. A heparinized indwelling catheter (PE-10 fused to PE-50) was inserted into the abdominal aorta via the left femoral artery for direct blood pressure measurements. Two catheters (PE-10 fused to PE-50) were placed close to the root of the aorta via the right carotid artery, one for continuous infusion and the other for bolus injections. All drugs were dissolved in 0.9% NaCl and given as a bolus (100 μL, followed by 100 μL of 0.9% NaCl), except for bradykinin, saralasin, and sarthran, which were given as intra-arterial infusions.
Arterial Blood Pressure Measurements
MAP was monitored with a solid-state strain-gauge transducer (Gould) connected to an ink recorder (model 2200S, Gould). Changes in blood pressure were estimated from the records by measuring maximal changes from baseline, which was defined as the average MAP observed during the 5 minutes preceding the bolus injection.
Protocol 1: Blood Pressure Response to Ang-(1-7) in Rats Infused With Bradykinin
After a 60-minute stabilization period, an intra-arterial bolus injection of Ang-(1-7) at different doses (20, 80, and 380 nmol per rat) was given to either saline-infused rats or rats receiving an intra-arterial infusion of bradykinin (5 μg/kg per minute at 50 μL/min). In the rats receiving bradykinin, responses to Ang-(1-7) were studied beginning 15 minutes after the bradykinin infusion was begun. A minimum of 5 minutes was allowed between bolus injections to allow reproducible responses to Ang-(1-7). Bolus saline injections were administered intra-arterially as controls to determine nonspecific changes in MAP.
Protocol 2: Effects of Angiotensin and Bradykinin Receptor Antagonists on the MAP Response to Ang-(1-7) in Bradykinin-Treated Rats
In this protocol, we studied the influence of angiotensin receptor antagonists and a B2 receptor antagonist, icatibant (Hoe 140), on the hypotensive response to bolus injections of Ang-(1-7) in rats receiving a bradykinin infusion. The following antagonists were studied: (1) the AT1 receptor antagonist L-158,809 (125 μg/kg IA); (2) the AT2 receptor antagonist PD 123319 (10 mg/kg IA) (these two antagonists were given just before the bradykinin [or vehicle] infusion was started); (3) the AT1/AT2 receptor antagonist saralasin (10 μg/kg per minute IA); (4) the AT1/AT2 receptor antagonist sarthran (10 μg/kg per minute IA) (these two antagonists were given simultaneously with bradykinin); and (5) the B2 receptor antagonist icatibant (30 μg per rat IA), which was given 15 minutes after the bradykinin infusion was started.
Protocol 3: Effects of Angiotensin Receptor Antagonists on the MAP Responses to Ang-(1-7) in Saline-Infused Rats
In this protocol, we studied the effects of angiotensin receptor antagonists on the blood pressure response to bolus intra-arterial injections of Ang-(1-7) in control rats infused with saline alone, using the same antagonists and doses as in protocol 2.
Protocol 4: Effects of a B2 Receptor Antagonist on the Blood Pressure Response to Ang-(1-7) in Saralasin-Treated Rats
In protocol 3, we observed that Ang-(1-7) induced hypotension in saralasin-treated rats. In protocol 4, we studied whether this hypotensive response would be affected by the B2 receptor antagonist icatibant (30 μg per rat IA), which was administered 15 minutes after the saralasin infusion was started.
The doses of the AT1 and AT1/AT2 receptor antagonists used abolished the responses to an intra-arterial Ang II bolus of 5 ng per rat, a dose that increased MAP by more than 20 mm Hg.
Unless otherwise indicated, data are mean±SEM. Data were analyzed by two-sample t test. When variances were found to be unequal, we used Wilcoxon’s rank sum test. This test is analogous to the two-sample t test but is based on the ranking of the data and does not require the equal variance and normality assumptions of the t test. An adjusted α level of .01 was used to determine statistical significance, since multiple comparisons were done.
MAP Response to Ang-(1-7) in Rats Infused With Bradykinin
Acute injections of Ang-(1-7) induced hypotensive responses. Ang-(1-7) was given intra-arterially together with bradykinin (5 μg/kg per minute) (Fig 1⇓, Table⇓). In the rats infused with bradykinin, MAP rapidly fell at the start of the infusion, going from 99.6±3.5 to 76.7±4.1 mm Hg within 1 minute (P<.01, n=9). Just before the challenges with the angiotensin peptide were initiated (15 minutes after the infusion was started), MAP was 87.6±2.7 mm Hg (P=.047 versus control).
Effects of Angiotensin and Bradykinin Receptor Antagonists on the MAP Response to Ang-(1-7) in Bradykinin-Treated Rats
Responses to 80 nmol Ang-(1-7) per rat, either alone or in the presence of angiotensin receptor antagonists, are shown in Fig 2⇓. The concentrations of L-158,809, saralasin, and sarthran we used abolished the response to 5 ng Ang II IA per rat.
No statistical differences were observed between responses to Ang-(1-7) alone or in the presence of angiotensin receptor antagonists. Although a trend toward decreased responses was observed in the L-158,809–treated rats, it did not reach statistical significance. In the bradykinin-infused rats pretreated with the bradykinin antagonist icatibant, Ang-(1-7) induced only a hypertensive response, as shown in Fig 2⇑.
To determine whether Ang II–mediated hypotensive responses could be unmasked in the presence of bradykinin and an AT1 antagonist, we tested whether Ang II (5 ng per rat IA) would also decrease MAP in rats pretreated with L-158,809 and bradykinin infusion. In saline-infused rats, Ang II increased MAP by 23.8±5.1 mm Hg. In rats treated with L-158,809 alone or in the presence of bradykinin, Ang II failed to alter blood pressure.
Effects of Angiotensin Antagonists and a Bradykinin Receptor Antagonist on the Blood Pressure Responses to Ang-(1-7)
Intra-arterial administration of Ang-(1-7) (20, 80, and 380 nmol per rat) in saline-infused rats resulted in a hypertensive response (Fig 3⇓, Table⇑). This response was not affected by the AT2 antagonist PD 123319 but was completely blocked by the AT1 antagonists L-158,809 and sarthran. Ang-(1-7) induced a mild hypotensive response in rats treated with saralasin. The data corresponding to responses to 80 nmol Ang-(1-7) per rat are shown in Fig 4⇓. In rats pretreated with saralasin, icatibant abolished the hypotensive response to Ang-(1-7) (Fig 5⇓).
We found that in anesthetized rats, intra-arterial Ang-(1-7) given alone as a bolus injection at concentrations of 20 to 380 nmol increased blood pressure, whereas in rats receiving a vasodepressor dose of bradykinin, Ang-(1-7) consistently induced hypotension. In both cases, there was no clear dose response, because there were no statistical differences between 80 and 380 nmol per rat (P=.15). This hypotensive response was not due to stimulation of known Ang II receptors, as it was not significantly blocked by an AT1 antagonist, an AT2 antagonist, or two related but different AT1/AT2 receptor antagonists, sarthran and saralasin. The AT1 antagonist L-158,809 tended to decrease the hypotensive response to Ang-(1-7) (although not to a significant degree), suggesting that the AT1 receptor may somehow be involved in the responses to Ang-(1-7); however, if that were true, saralasin and sarthran would show the same inhibitory tendency, which was not the case. The inability of these antagonists to alter the hypotensive response to Ang-(1-7) in bradykinin-infused rats suggests that the response is probably not mediated by AT1 receptors.
Treating bradykinin-infused rats with a B2 receptor antagonist eliminated the vasodepressor effects of Ang-(1-7) and converted the response induced by Ang-(1-7) from hypotension to hypertension. Thus Ang-(1-7)–induced hypotension requires the presence of vasodepressor bradykinin activity.
To ascertain whether the responses we observed were specific for Ang-(1-7), in a few experiments we tested whether hypotension was also observed with Ang II, reasoning that if the hypotensive response was not selective for Ang-(1-7), we should also see it with Ang II. Since the potent vasoconstrictor effects of Ang II may conceal concomitant vasodepressor activity, we performed these studies in the presence of the AT1 antagonist L-158,809, which was not able to suppress Ang- (1-7)–induced hypotension. Ang II failed to show any hypotensive response. These data suggest that Ang-(1-7) selectively induces hypotension in the presence of bradykinin.
The precise mechanisms activated by Ang-(1-7) are not clear. The fact that angiotensin receptor antagonists had no effect suggests that hypotension is not mediated by any of the known angiotensin receptors. It has been suggested that Ang-(1-7) acts via a receptor pharmacologically distinguishable from AT1 or AT2 receptor subtypes. For example, sarthran antagonized the response to Ang-(1-7) in arreflexic rats, whereas losartan and saralasin did not.3 Sarthran also blocked the direct vasorelaxant effect of Ang-(1-7) in isolated canine coronary rings, whereas an AT1 or AT2 blocker was ineffective.10 Sarthran exhibited no such ability to block the effects of Ang-(1-7) in bradykinin-treated rats in the present experiments. Mediation via nonreceptor mechanisms is possible, but it is not clear what they might be. It has been reported that Ang-(1-7) is an AT1 receptor blocker12 ; but since Ang-(1-7) induced hypotension in rats already treated with blocking doses of an AT1 antagonist, AT1 blockade cannot be involved.
It is noteworthy that sarthran ([Sar1,Thr8]-Ang II), which differs from saralasin ([Sar1,Val5,Ala8]-Ang II) with regard to both the amino acid in position 5 (Ile versus Val) and its carboxyl-terminal amino acid (Thr versus Ala), was unable to reveal a hypotensive response to Ang-(1-7). Both are considered effective AT1/AT2 antagonists. At this time we do not have an explanation for the differential effects of saralasin and sarthran; however, as mentioned above, they have been reported to affect Ang-(1-7) responses differently.3
It has been proposed that Ang-(1-7) may release bradykinin from endothelial cells,13 14 thus increasing local bradykinin concentrations. It is difficult to envisage such a mechanism as explaining the effects of Ang-(1-7) shown in this part of the study, since the rats were already receiving conspicuous amounts of bradykinin. Although we did not measure circulating bradykinin, the dose we used (5 μg/kg per minute IA) initially resulted in marked hypotension, suggesting that circulating bradykinin levels were very high. It is highly unlikely that any site could release enough bradykinin to add significantly to the already excessive kinin concentrations in the circulation.
Other studies have linked some of the pharmacological actions of angiotensin peptides to bradykinin. Seyedi et al7 reported that both Ang II and to a lesser extent Ang-(1-7) were able to release NO from canine coronary microvessels. In the study of Seyedi et al, the effects of the angiotensin peptides were also blocked by icatibant. These and other studies8 9 10 as well as the present data show a link between angiotensin peptides [particularly Ang-(1-7)] and bradykinin- and/or bradykinin receptor–mediated responses in vascular tissue, with aspects that appear to depend, among other things, on the experimental model and species involved.
When Ang-(1-7) was given intra-arterially to saline-infused rats, it induced hypertension. This increase in MAP was mediated by AT1 receptors, since it was opposed by an AT1 receptor antagonist and by sarthran, a nonselective angiotensin receptor blocker, but not by an AT2 antagonist. Other researchers have reported that Ang-(1-7) is a weak activator of AT1 receptors15 16 ; the present study confirms this, since hundreds of micrograms of Ang-(1-7) were needed to increase MAP by 20 mm Hg as opposed to only nanogram amounts of Ang II.
The results obtained by injection of Ang-(1-7) into saralasin-treated rats were puzzling, because we anticipated blockade of the hypertensive response; instead, Ang-(1-7) induced a small but consistent hypotensive response, which was obliterated by the B2 receptor blocker. Thus in the presence of saralasin, Ang-(1-7) elicited a mild hypotensive response, which was mediated by activation of B2 receptors and did not require exogenous bradykinin. One explanation for these results might be that Ang-(1-7) potentiated endogenous bradykinin, which would imply that activation of the renin-angiotensin system is accompanied by activation of bradykinin-dependent responses. Thus the Ang-(1-7)–bradykinin/bradykinin receptor tandem would potentially act as a counterregulatory system opposing the vasoconstrictor and progrowth activities of Ang II. For this to occur, endogenous Ang-(1-7) would need to reach concentrations enabling it to exert these effects, a distinct possibility according to reported data.2 In any case, the data suggest that Ang-(1-7) can alter or propel bradykinin- and/or bradykinin receptor–mediated vasodepressor responses from subthreshold to above-threshold levels. It is not clear whether these responses are due to pathways similar to those involved in the response to Ang-(1-7) in the presence of excess bradykinin; however, one possibility is that excess bradykinin further potentiated responses to Ang-(1-7).
Conceptually, these data imply that when activated, the renin-angiotensin system can produce an angiotensin peptide, Ang-(1-7), that can exert a negative functional feedback on the vasoconstrictor activity of Ang II by increasing the responses to kinins (which are potent vasodilators). In addition, treatment with ACE inhibitors increases Ang-(1-7) in tissue as well as plasma17 and endothelial cells.1 Bradykinin appears to mediate a number of the cardiovascular effects of ACE inhibitors.18 19 20 Perhaps the newly found link between bradykinin and Ang-(1-7) participates in the mechanism or mechanisms whereby ACE inhibitors exert their cardiovascular effects.
The doses of Ang-(1-7) we used, as well as those used in some other studies,3 4 9 are not physiological. Responses to acute administration of a drug do not mimic the effects of prolonged and chronic changes in an endogenous compound or the effects of local (tissue) increases in concentration; however, they do demonstrate a potentially important biological effect. Further efforts should be made to elucidate its relevance.
In summary, we have shown that in thiobutabarbital-anesthetized rats, Ang-(1-7) induces hypotension when given in the presence of vasodepressor bradykinin concentrations. This hypotensive response is not significantly inhibited by an AT1, AT2, or nonselective AT1/AT2 antagonist but is completely reversed (converted back into hypertension) by icatibant, a B2 antagonist. In saline-infused rats, Ang-(1-7) induces a hypertensive response that is not affected by the AT2 antagonist PD 123319 and is blocked by an AT1 antagonist as well as the AT1/AT2 antagonist sarthran. In contrast, Ang-(1-7) induces a mild hypotensive response in saralasin-treated rats, which in turn is abolished by icatibant. These data suggest that in the rat, Ang-(1-7) participates together with bradykinin in a vasodepressor pathway. We conclude that the renin-angiotensin and kallikrein-kinin systems may be connected by an interaction between Ang-(1-7) and bradykinin- and/or bradykinin receptor–mediated responses.
Selected Abbreviations and Acronyms
|AT1, AT2||=||angiotensin II type 1, type 2 (receptors)|
|MAP||=||mean arterial pressure|
This research was supported in part by grant 15-PO1-HL-28982 from the National Heart, Lung, and Blood Institute of the National Institutes of Health. We are grateful to Hoechst-Roussel for icatibant, DuPont-Merck for L-158,809, and Parke-Davis Pharmaceuticals (a subsidiary of Warner-Lambert) for PD 123319.
- Received September 18, 1996.
- Revision received October 28, 1996.
- Accepted January 6, 1997.
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