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Synergistic effect of angiotensin-(1–7) on bradykinin arteriolar dilation in vivo
Peptides 20 (1999) 1195–1201
Synergistic effect of angiotensin-(1–7) on bradykinin arteriolar dilation in vivo Maria Aparecida Oliveiraa, Zuleica Bruno Fortesa, Robson A.S. Santosb, Malesh C. Koslac, Maria Helena C. De Carvalhoa,* a
Department of Pharmacology, Institute of Biomedical Sciences, University of Sa˜o Paulo, Av. Prof. Lineu Prestes, 1524, Cidade Universita´ria, 05508-900 CEP Sa˜o Paulo, Brazil b Department of Physiology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil c Cleveland Clinic Foundation, Cleveland, OH, USA Received 3 March 1999; accepted 8 June 1999
Abstract The interaction between angiotensin [Ang-(1–7)] and bradykinin (BK) was determined in the mesentery of anesthetized Wistar rats using intravital microscopy. Topical application of BK and Ang-(1–7) induced vasodilation that was abolished by the BK B2 receptor antagonist HOE-140 and the Ang-(1–7) antagonist A-779, respectively. BK (1 pmol)-induced vasodilation, but not SNP and ACh responses, was potentiated by Ang-(1–7) 10 pmol and 100 pmols. The effect of 100 pmol of Ang-(1–7) on BK-induced vasodilation was abolished by A-779, indomethacin, and L-nitroarginine methyl esther, whereas losartan was without effect. Enalaprilat treatment enhanced the BK- and Ang-(1–7)-induced vasodilation and the potentiating effect of Ang-(1–7) on BK vasodilation. The potentiation of BK-induced vasodilation by Ang-(1–7) is a receptor-mediated phenomenon dependent on cyclooxygenase-related products and NO release. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Bradykinin; Angiotensin-(1–7); Microcirculation; Prostaglandins; Nitric oxide; Angiotensin-converting enzyme; Renin-angiotensin system; Angiotensin-(1–7) antagonist
1. Introduction Angiotensin-(1–7) [Ang-(1–7)], one of the bioactive components of the renin-angiotensin system, has two major characteristics: its formation through an angiotensin converting enzyme (ACE)-independent pathway [28,29] and the selectivity of its biologic actions [29]. It has been shown recently that Ang-(1–7) can interact with bradykinin (BK), enhancing its biologic effect [23–24]. In vivo Ang-(1–7) has been shown to potentiate the hypotensive effect of BK in normotensive [24] and hypertensive [18] rats. In vitro Ang(1–7) potentiates the vasodilatory effect of BK in isolated dog coronary arteries [3,17] and the vasoconstrictor action in dog femoral veins [10]. An amplification by Ang-(1–7) of the release of arachidonic acid induced by an ACE-resistant BK analog in Chinese hamster ovary cells cotransfected
* Corresponding author. E-mail address: [email protected] (M.H.C. De Carvalho)
with cDNAs of both human BK B2 receptor and ACE also was described recently [6]. The observations that Ang-(1–7) potentiates BK may be particularly important especially considering that Ang-(1–7) can increase in plasma after chronic blockade of AT1 receptors [7] or ACE inhibition [4,15]. Therefore, it is possible that the interaction of Ang-(1–7) with BK contributes to the beneficial cardiovascular effects obtained with these treatments. A physiological role for Ang-(1–7), in contributing to the antihypertensive actions of lisinopril treatment, was demonstrated in SHR [12]. In addition to the interaction with BK, a direct vasodilatory action of Ang-(1–7) was described [3,17,26]. The heptapeptide has been reported as a vasodilator in vitro in pig [26] and dog [3,17] coronary arteries and in feline isolated mesenteric and hindquarter vascular beds [22]. One important question that arises from these observations is whether the direct vasodilation and/or BK potentiation of Ang-(1–7) could occur at the level of resistance vessels, the most functionally important site for determining peripheral
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vascular resistance [31]. In this study, we addressed this issue by evaluating the direct effect of Ang-(1–7) and its interaction with BK at the level of secondary (A2) resistance vessels by using an in vivo in situ mesenteric blood vessel preparation.
2. Materials and methods 2.1. Intravital microscopy The rats were anesthetized with chloral hydrate (400 – 450 mg/kg, subcutaneously (s.c.)), and the mesentery was arranged for microscopic observation in situ [8,32]. In brief, the animals were kept on a special board, heated at 37°C, which included a transparent plate on which the tissue to be transilluminated was placed. The mesentery was kept moist and warm by irrigating the tissue with warmed (37°C) Ringer–Locke’s solution (pH 7.2–7.4) containing 1% gelatin. In a series of experiments, a 500-line television camera was combined with a tri-ocular microscope to facilitate observation of the enlarged image (3400⫻) on the video screen. An image-splitting micrometer was adjusted to the phototube of the microscope. The image splitter sheared the optical image into two separate images and displaced one with respect to the other. By rotating the image splitter in the phototube, the shearing is maintained in a direction at right angles to the axis of the vessel. The displacement of one image from the other allowed measurement of the vessel diameter [2]. Blood vessels were classified according to their branching order beginning at the capillary level and reaching up to the arteriolar side [9]. The smallest, precapillary arterioles were classified as A4, fed by the terminal arterioles (A3) branching from large arterioles (A2). A2 arterioles (15–25 m) were selected for study, and any changes in vessel diameter were estimated following the topical application of vasoactive drugs or the diluent Ringer-Locke solution. The drugs, dissolved in Ringer–Locke’s solution, were added to the preparation in a standard volume of 0.01 ml and were removed by washing with warmed Ringer–Locke’s solution. 2.2. Experimental protocols 2.2.1. Effect of BK and Ang-(1–7) alone and in combination To choose the doses to be used in the potentiation studies, the effects of BK (in doses of 1, 10, and 30 pmol, n ⫽ 7–16 for each dose) and Ang-(1–7) (in doses of 1, 10, 100, and 1000 pmol, n ⫽ 11–15 for each dose) were tested. For the potentiation experiments, Ang-(1–7) was added to the preparation 30 s before the application of BK. To verify if BK would potentiate the vasodilation induced by Ang-(1– 7), the nonapeptide was applied 30 s before Ang-(1–7).
2.2.2. Specificity of the Ang-(1–7) potentiating activity To verify the specificity of the potentiating effect of Ang-(1–7) on BK-induced vasodilation, doses of sodium nitroprusside (38 pmol, n ⫽ 5) or acetylcholine (1.6 nmol, n ⫽ 6) equipotent to BK were tested. For these experiments, the dose of Ang-(1–7) was added to the preparations 30 s before the application of each of these agents. 2.2.3. Participation of nitric oxide (NO) and prostanoids on the Ang-(1–7) potentiating activity To investigate whether NO is involved in the interaction between BK and Ang-(1–7), L-nitroarginine methyl esther (L-NAME, 10 nmol topically applied), a NO synthase inhibitor, was used. The dose and the time delay necessary for the effect of this agent were chosen in preliminary experiments. L-NAME was added to the preparations 3 min prior to test BK alone or combined with Ang-(1–7) (n ⫽ 9). In another series of experiments, the animals were treated with indomethacin (5 mg/kg, intramuscularly (i.m.), 30 min before, n ⫽ 8), to inhibit the cyclooxygenase pathway. In the treated animals, BK and BK and Ang-(1–7) were tested with a 3-min interval between them. The dose and the time delay were selected because they did not change basal diameter and/or blood pressure. 2.2.4. Effect of receptor antagonists on the Ang-(1–7) potentiating activity A-799, a specific Ang-(1–7) receptor antagonist [1,30], HOE 140, a specific BK B2 receptor antagonist, and losartan (15 mg/kg i.v. 40 min before the experiment, n ⫽ 6), to block AT1 receptors, were used. In the treated animals, BK and BK and Ang-(1–7) were tested with 3-min intervals between them. The dose and the time delay necessary for the specific effects of these agents were chosen in preliminary experiments. A-779 (100 pmol, n ⫽ 10) or HOE 140 (100 pmol, n ⫽ 10) were added to the preparations 15 s and 1 min, respectively, prior to test BK, Ang-(1–7), or the combination of both. These treatments did not change basal diameters and/or blood pressure. 2.2.5. Effect of ACE inhibition To test the interference of ACE on the BK, Ang-(1–7), and BK ⫹ Ang-(1–7) effects, enalaprilat (10 mg/kg i.v. 30 min before the experiment, n ⫽ 6) was used. 2.2.6. Statistical analysis The data were analysed with standard statistical analyses, i.e. analysis of variance with Tukey-Kramer (post hoc) test and Student’s paired or unpaired t-test where appropriate. All values are reported as mean ⫾ SEM. Statistical significance was set at P ⬍ 0.05. 2.2.7. Drugs Ang-(1–7) was purchased from Bachem. Acetylcholine, bradykinin, indomethacin, and sodium nitroprusside were purchased from Sigma (St. Louis, MO, USA). The drugs
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Fig. 1. Bar graphs show the increase (%) observed in rat mesenteric arteriolar diameter induced by topical application of bradykinin (BK) in A and angiotensin-(1–7) [Ang-(1–7)] in B. Results are mean ⫾ SEM. Numbers inside bars indicate number of preparations tested. In A, * P ⬍ 0.001 and † P ⬍ 0.001 vs. BK 1 pmol and BK 10 pmol, respectively. In B, * P ⬍ 0.05 and † P ⬍ 0.001 vs. Ang-(1–7) 1 pmol; ‡ P ⬍ 0.001 vs. Ang-(1–7) 10 pmol and § P ⬍ 0.001 vs. Ang-(1–7) 100 pmol.
were dissolved in Ringer–Locke gelatin solution, except indomethacin, which was dissolved in Tris buffer, pH 8.1. Enalaprilat (MK 422) was from Merck, Sharp & Dohme. 7 D-Ala -Ang-(1–7) was synthetized by M.C.K. at Cleveland Clinic Foundation (Cleveland, OH, USA).
3.2. Specificity of the BK potentiation by Ang-(1–7) To study the specificity of the potentiation of BK (1 pmol) by Ang-(1–7) (100 pmol), acetylcholine (1.6 nmol), and sodium nitroprusside (38 pmol) were tested. The vasodilation induced by these agents was not significantly altered by Ang-(1–7) (Table 1).
3. Results 3.1. Vascular effects of BK and Ang-(1–7) alone and in combination BK in doses of 1, 10, and 30 pmol and Ang-(1–7) in doses of 1, 10, 100, and 1000 pmol topically applied induced a vasodilation (expressed as percentage increase in initial diameters) on rat mesenteric arterioles studied in vivo (Fig. 1). The vasodilation elicited by BK (1 pmol) was potentiated significantly by Ang-(1–7) 10 pmol and 100 pmol. On the other hand, the lower dose (1 pmol) or the higher dose (1000 pmol) of Ang-(1–7) did not modify the BK vasodilation (Fig. 2). Based on these results, the doses of 1 pmol of BK and 100 pmol of Ang-(1–7) were chosen for the additional experiments—first, because they consistently elicited a small, but statistically significant, increase in arteriolar diameter (3–5%) when tested alone (Fig. 1) and second, because those doses used in conjunction elicited the highest potentiating effect of Ang-(1–7) on BK-induced vasodilaton (Fig. 2). It is important to mention that the vasodilation elicited by Ang-(1–7) (100 pmol) (4.9 ⫾ 0.4% increase, n ⫽ 4) was not increased by prior addition of BK (1 pmol) (5.8 ⫾ 1.0% increase, n ⫽ 4).
Fig. 2. Bar graphs show the increase (%) observed in rat mesenteric arteriolar diameter induced by topical application of BK and BK after Ang-(1–7). Results are mean ⫾ SEM. Numbers inside bars indicate number of preparations tested. * P ⬍ 0.05 and † P ⬍ 0.001 vs. BK 1 pmol.
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Table 1 Lack of potentiation of Ang-(1–7) on acetylcholine and sodium nitroprusside Agent\Ang-(1–7) 100 pmol
Absent
Present
n
BK 1 pmol Ach 1.6 nmol SNP 36 pmol
4.3 ⫾ 0.5 6.5 ⫾ 0.3 4.4 ⫾ 0.3
11.1 ⫾ 1.2* 5.0 ⫾ 0.5 3.7 ⫾ 0.7
7 6 5
Values are mean ⫾ SEM. n, number of animals used. * P ⬍ 0.001 vs. values in the absence of Ang-(1–7).
3.3. Contribution of NO and prostanoids to the BK and Ang-(1–7) effects alone or in combination To evaluate prostanoid contribution to the Ang-(1–7) (100 pmol) vasodilation and potentiating effect on the responsiveness of the mesenteric arterioles to BK (1 pmol), the cyclooxygenase inhibitor indomethacin (5 mg/kg i.m.), was used. After 30 min of treatment with indomethacin, the vasodilation induced by Ang-(1–7) was slightly reduced (P ⬍ 0.05, Table 2), whereas that induced by BK was unaltered (Fig. 3). On the other hand, the potentiation of BK-induced vasodilation elicited by Ang-(1–7) was abolished (Fig. 3). To test whether the direct or the potentiating effect of Ang-(1–7) on BK vasodilation was mediated by NO, the effect of the NO synthase inhibitor (L-NAME) on the rat mesenteric arterioles was studied. As seen in Fig. 4, LNAME (10 nmol topically applied) abolished the potentiating effect of Ang-(1–7) (100 pmol) on BK (1 pmol) vasodilation without interfering with the vasodilation induced by BK. The vasodilation induced by Ang-(1–7) was slightly reduced (P ⬍ 0.05) by L-NAME treatment (Table 2).
Fig. 3. Bar graphs show the increase (%) observed in rat mesenteric arteriolar diameter induced by topical application of BK and BK after Ang-(1–7), in untreated and indomethacin (5 mg/kg i.m.)-treated animals. Results are mean ⫾ SEM. Numbers inside bars indicate number of animals tested. * P ⬍ 0.001 vs. BK in untreated animals; † P ⬍ 0.001 vs. Ang-(1–7)⫹ BK in untreated animals
before and 0.5 ⫾ 0.5% increase, n ⫽ 10 after HOE 140 treatment, P ⬍ 0.0001). As a consequence of the BK B2 blokade by HOE 140, the potentiation of BK by Ang-(1–7) was not observed [Ang-(1–7)⫹ BK before 12.0 ⫾ 0.4% increase, n ⫽ 10 and 1.1 ⫾ 0.7% increase, n ⫽ 10, P ⬍ 0.0001 after HOE 140 treatment]. In contrast, the Ang-(1–7) vasodilation was not affected (Table 2). Pretreatment with the specific Ang-(1–7) receptor antagonist A-799 (100 pmol) did not interfere with BK (1 pmol)induced vasodilation whereas the potentiating effect of
3.4. Effects of blockade of BK, angiotensin II, and Ang-(1–7) receptors on the vascular effect of BK and Ang-(1–7) alone or in combination Administration of the specific BK B2 receptor antagonist HOE 140, at a dose of 100 pmol, nearly abolished the vasodilation produced by BK (4.1 ⫾ 0.3% increase, n ⫽ 10 Table 2 Effect of antagonists and enzyme inhibitors on Ang-(1–7) vasodilation Group
Untreated
Treated
n
Indomethacin 5 mg/kg i.m. L-NAME 10 nmol t.a. HOE 100 pmol t.a. Losartan 15 mg/kg i.v. Enalaprilat 10 mg/kg i.v. A-779 100 pmol t.a.
2.7 ⫾ 0.4 4.7 ⫾ 0.5 4.7 ⫾ 0.5 3.8 ⫾ 0.4 4.4 ⫾ 0.5 4.4 ⫾ 0.5
1.3 ⫾ 0.5* 3.2 ⫾ 0.6* 4.6 ⫾ 0.3 3.4 ⫾ 0.2 8.1 ⫾ 0.4† ⫺0.5 ⫾ 0.5‡
8 9 10 6 6 10
Values are mean ⫾ SEM of the percentage increase in arteriolar diameter induced by Ang-(1–7) topically applied. *P ⬍ 0.05; † P ⬍ 0.001; ‡ P ⬍ 0.0001 vs. untreated values. n, number of animals; t.a., topically applied.
Fig. 4. Bar graphs show the increase (%) observed in rat mesenteric arteriolar diameter induced by bradykinin (BK) and BK after Ang-(1–7) topically applied, in untreated and L-NAME (10 nmol topical)-treated preparations. Results are mean ⫾ SEM. Numbers inside bars indicate number of preparations tested. * P ⬍ 0.05 vs. BK in untreated preparations; † P ⬍ 0.001 vs. Ang-(1–7)⫹ BK in untreated preparations.
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Table 3 Effect of receptor antagonists and enzyme inhibitors on basal arteriolar diameters in micrometers Group
Untreated
Treated
n
Indomethacin 5 mg/kg, IM L-NAME 10 nmol t.a. HOE 100 pmol t.a. Losartan 15 mg/kg i.v. Enalaprilat 10 mg/kg i.v. A-779 100 pmol t.a.
17.9 ⫾ 0.8 16.0 ⫾ 1.1 23.2 ⫾ 2.2 20.1 ⫾ 0.8 18.8 ⫾ 0.8 16.7 ⫾ 1.0
18.7 ⫾ 0.8 15.8 ⫾ 1.0 23.2 ⫾ 2.2 19.9 ⫾ 0.6 18.9 ⫾ 1.3 16.5 ⫾ 0.9
14 10 9 9 11 9
Values are mean ⫾ SEM. n, number of animals used; t.a., topically applied.
Fig. 5. Bar graphs show the increase (%) observed in rat mesenteric arteriolar diameter induced by BK and BK after Ang-(1–7) topically applied, in untreated and A-779 (100 pmol, topical)-treated preparations. Results are mean ⫾ SEM. Numbers inside bars indicate number of preparations tested. * P ⬍ 0.001 vs. BK in untreated preparations; † P ⬍ 0.001 vs. Ang-(1–7) ⫹ BK in untreated preparations.
Ang-(1–7) (100 pmol) on BK (1 pmol) response was reduced markedly (Fig. 5). The direct vasodilating effect of Ang-(1–7) was abolished completely by A-779 (Table 2). On the other hand, the angiotensin II AT1 receptor antagonist losartan at a dose of 15 mg/kg i.v. (30 min before the experiment) had no effect on Ang-(1–7) (Table 2), BK vasodilation (Fig. 6), or on the BK potentiation by Ang(1–7) (Fig. 6). 3.5. Effect of ACE inhibition on BK potentiation by Ang-(1–7)
pressure levels of 39.8 ⫾ 3.1 mmHg (n ⫽ 6, P ⬍ 0.002), no differences were observed in arteriolar diameters (Table 3). As expected, mesenteric arteriolar vasodilation produced by BK (1 pmol) was increased approximately 2-fold by the treatment with the ACE inhibitor enalaprilat (MK 422 10 mg/kg). ACE inhibition also substantially increased the direct vasodilation of Ang-(1–7) 100 pmol (Table 2) and the potentiating effect of Ang-(1–7) on BK response (Fig. 7). 3.6. Effect of the treatments on basal mesenteric arteriolar diameters As shown in Table 3, none of the treatments modified the basal mesenteric arteriolar diameters. 4. Discussion In this study we showed that Ang-(1–7) produces vasodilation of resistance mesenteric blood vessel in vivo-in situ
Although enalaprilat treatment (10 mg/kg i.v. 30 min before the experiment) caused a decrease in basal blood
Fig. 6. Bar graphs show the increase (%) observed in rat mesenteric arteriolar diameter induced by BK and BK after Ang-(1–7) topically applied, in untreated and losartan (15 mg/kg i.v.)-treated animals. Results are mean ⫾ SEM. Numbers inside bars indicate number of animals used. * P ⬍ 0.001 vs. BK in untreated animals.
Fig. 7. Bar graphs show the increase (%) observed in rat mesenteric arteriolar diameter induced by BK and BK after Ang-(1–7) topically applied, in untreated and enalaprilat (10 mg/kg i.v.)-treated animals. Results are mean ⫾ SEM. Numbers inside bars indicate number of animals used. * P ⬍ 0.001 vs. BK in untreated animals; † P ⬍ 0.001 vs. Ang-(1– 7)⫹BK in untreated animals.
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and potentiates the vasodilation produced by BK. Furthermore, evidence was obtained for the participation of cyclooxygenase products and nitric oxide release in the mechanism of BK potentiation by Ang-(1–7). Both the direct vasodilator effect and the BK-potentiating activity of Ang(1–7) were blocked by the selective Ang-(1–7) antagonist A-779. This is the first demonstration indicating that Ang(1–7) and BK can interact not only at the level of large arteries [3,17] but also in resistance vessels modulating vascular tone by a receptor-mediated mechanism. Several studies have described the vascular effects of Ang-(1–7). A vasodilatatory effect of Ang-(1–7) has been described in dog [3,17] and pig [26] coronary arteries, in the cat hindlimbs, [22] and in piglet cerebral microvessels [19]. Although Ang-(1–7) is a vasodilator in most regions, a vasoconstrictor activity was observed in isolated heart preparation of rats [21] and hamsters [16]. Taken together, these observations suggest that the vascular actions of Ang-(1–7) can vary according to the vascular bed and/or with different species. It is particularly important, however, that, in resistance vessels of the mesenteric territory, the heptapeptide produced vasodilation with topical application of doses as low as 10 pmol. The vasodilation produced by Ang-(1–7) was reduced but not abolished by pretreatment with L-NAME or indomethacin (Table 2). These observations are important particularly when comparing these findings with the effect of these treatments on the BK-potentiating activity of Ang-(1– 7). Each treatment by itself blocked the BK-potentiating activity of Ang-(1–7) suggesting that each intracellular transduction system can evoke the potentiating response. However, if one is blocked, the other system cannot be activated. It appears that the systems are essentially in series with the participation of both being required for the activation of the BK-potentiating response by Ang-(1–7). The fact that Ang-(1–7) did not alter the vasodilation produced by acetylcholine or sodium nitroprusside suggests that the BKpotentiating activity of Ang-(1–7) cannot be evoked nonspecifically by agents that also release NO. This agrees with previous findings [17,23]. We did not determine the site with which Ang-(1–7) is interacting to increase BK vasodilation. Several possibilities can be raised including interaction of both peptides at the level of the smooth muscle layer and facilitation of the endothelium-mediated vasodilation produced by BK through an action of Ang-(1–7) on endothelial [13] or smooth muscle cells [14]. Indeed, both peptides have been shown to act on endothelial and smooth muscle cells, releasing prostaglandins E2 and I2 and NO from endothelial [11,13] and smooth muscle cells [14,25]. It has been reported that in vascular smooth muscle cells, Ang-(1–7) triggers the release of arachidonic acid through a receptor-mediated mechanism involving stimulation of CaM kininase II, with subsequent enhancement of cytoplasmatic phospholipase A2 activity, via mitogen-activating protein kinase activation [20]. Whether a similar mecha-
nism is involved in the vascular actions of Ang-(1–7) in mesenteric vessels remains to be determined. The vasodilation produced by BK combined with Ang(1–7) followed a bell-shaped dose–response curve. Maximal potentiation was attained with 100 pmol of Ang-(1–7), whereas when increasing the dose of the heptapeptide to 1 mol the BK-potentiating activity disappeared (Fig. 2). On the other hand, the direct vasodilation obtained with the 1-mol dose of Ang-(1–7) was significantly greater than other doses of Ang-(1–7) (Fig. 1). Of interest, a bell-shaped dose–response curve was obtained for the release of arachidonic acid or 6-keto-prostaglandin F1␣ by Ang-(1–7) in cultured vascular smooth muscle cells by Muthalif et al. [20]. These observations may be related to the activation of enzymes, such as lipoxygenase, that could be involved in the modulation of NO and prostanoid production by the combination of Ang-(1–7) [20] and BK [27]. However, our data do not allow evaluation of this possibility. As described previously in freely moving rats, treatment with the ACE inhibitor enalaprilat did not prevent the interaction of Ang-(1–7) with BK [18,24]. This result, together with the observation that pretreatment with indomethacin or L-NAME essentially abolished the BK-potentiating activity of Ang-(1–7), further indicates that the mechanism of the facilitatory effect of Ang-(1–7) on the vasodilation produced by BK is not dependent on an interference with the ACE catalytic activity [5,6] or on facilitation of the crosstalk between ACE and the BK B2 receptors [6]. According to our results and previous findings [6,17, 23,24], the interaction of Ang-(1–7) with BK is complex, involving, in addition to the mechanism listed above, a receptor-mediated mechanism linked to phospholipase A2/ cyclooxygenase activation and a prostanoid or nonprostanoid pathway dependent on modulation by NO. It has been suggested recently [6] that some actions of Ang-(1–7) could be mediated by potentiation of endogenous BK through facilitation of a ‘cross-talk’ mechanism between ACE and the BK B2 receptors. According to this report, binding of Ang-(1–7) to ACE would facilitate the cross-talk of ACE and the B2 receptors leading to potentiation of endogenous BK, independently of an interference with BK hydrolysis. This appears not to be the mechanism involved in the vasodilation produced by Ang-(1–7) in the mesenteric vascular bed: the vasodilation produced by Ang(1–7) was not blocked by pretreatment with the B2 receptor antagonist HOE 140 in a dose effective to block an equipotent BK effect. Furthermore, the Ang-(1–7) effect was completely blocked by its selective antagonist A-779, which did not alter the BK vasodilatatory effect and was rather increased by treatment with the ACE inhibitor enalaprilat, a condition that would be expected to reduce or abolish the Ang-(1–7) effect according to the mechanism proposed by Deddish et al. [6]. Additionally, the BK-potentiating activity of Ang-(1–7) is apparently not dependent on interaction with AT1 receptors subtypes because losartan did not inter-
M.A. Oliveira et al. / Peptides 20 (1999) 1195–1201
fere with it. Similar findings were obtained previously by Li et al. [17] in dog coronary arteries. In summary, we have found that, in mesenteric resistance vessels, besides producing vasodilation, Ang-(1–7) facilitates the vasodilation of BK. The BK-potentiating activity is an Ang-(1–7) receptor-mediated phenomenon dependent on cyclooxygenase-related products and NO release, but not on interference with the catalytic activity of ACE or facilitation of cross-talk between ACE and the BK B2 receptor. The observation that the interaction of Ang-(1–7) with BK is rather facilitated in ACE inhibitor-treated rats suggests that this interaction contributes to the pharmacological effects of this class of drugs.
Acknowledgments Support was provided by Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo, Conselho Nacional de Pesquisa, Fundac¸ao de Amparo a` Pesquisa do Estado de Minas Gerais, and Financiadora de Estudos e Projetos-PRONEX.
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[13] Jaiswal N, Diz DI, Chappell MC, Khosla MC, Ferrario CM. Stimulation of endothelial cell prostaglandin production by angiotensin peptides: characterization of receptors. Hypertension 1992;19(suppl. II):55. [14] Jaiswal N, Jaiswal RK, Tallant EA, Diz DI, Ferrario CM. Alterations in prostaglandin production in spontaneously hypertensive rat smooth muscle cells. Hypertension 1993;21:900 –5. [15] Kohara K, Brosnihan KB, Ferrario CM. Angiotensin (1–7) in the spontaneously hypertensive rat. Peptides 1993;14:883–91. [16] Kumagai H, Khosla MC, Ferrario C, Fouad–Tarazi FM. Biological activity of angiotensin-(1–7) heptapeptide in the hamster heart. Hypertension 1990;15:33. [17] Li P, Chappell M. C, Ferrario CM, Brosnihan KB. Angiotensin-(1–7) augments bradykinin-induced vasodilation by competing with ACE and releasing nitric oxide. Hypertension 1997;29:394 – 400. [18] Lima CV, Paula RD, Resende FL, Khosla MC, Santos RAS. Potentiation of the hypotensive effect of bradykinin by short-term infusion of angiotensin-(1–7) in normotensive and hypertensive rats. Hypertension 1997;30:542– 8. [19] Meng W, Busija DW. Comparative effects of angiotensin-(1–7) and angiotensin II on piglet pial arterioles. Stroke 1993;24:2041–5. [20] Muthalif MM, Benter IF, Uddin MR, Harper HL, Malik KU. Signal transduction mechanisms involved in angiotensin-(1–7)-stimulated arachidonic acid release and prostanoid synthesis in rabbit aortic smooth muscle cells. J Pharmacol Exp Ther 1998;284:388 –98. [21] Neves LAA, Almeida AP, Khosla MC, Santos RAS. Metabolism of angiotensin I in isolated rat hearts. Effect of angiotensin converting enzyme inhibitors. Biochem Pharmacol 1995;50:1451–9. [22] Osei SY, Ahima RS, Minkes RK, Weaver JP, Khosla MC, Kadowitz PJ. Differential response to ang-(1–7) in the feline mesenteric and hidquarter vascular beds. Eur J Pharmacol 1993;234:35– 42. [23] Paula RD, Lima CV, Khosla MC, Santos RAS. Angiotensin-(1–7) potentiates the hypotensive effect of bradykinin in conscious rats. Hypertension 1995;26:1154 –9. [24] Paula RD, Lima CV, Khosla MC, Santos RAS. Papel do o´xido nı´trico na potenciac¸a˜o do efeito hipotensor da bradicinina pela angiotensina(1–7). X Reunia˜o Annual da Federac¸a˜o de Sociedade de Biologia Experimental 1995;1:131. [25] Peredo HA, Feleder EC, Adler Graschinsky E. Differential effects of acetylcholine and bradykinin on prostanoid release from the rat mesenteric bed: role of endothelium and of nitric oxide. Prostaglandins Leukot Essent Fatty Acids 1997;56:253– 8. [26] Po¨rsti I, Bara AT, Busse R, Hecker M. Release of nitric oxide by angiotensin-(1–7) from porcine coronary endothelium: implications for a novel angiotensin receptor. Br J Pharmacol 1994;111:652– 4. [27] Saban MR, Saban R, Bjorling DE. Kinetics of peptide-induced release of inflammatory mediators by the urinary bladder. Br J Urol 1997;80:742–7. [28] Santos RAS, Brosnihan KB, Chappell MC, Pesquero JL, Chernicky CL, Greene LJ, Ferrario CM. Converting enzyme activity and angiotensin metabolism in the dog brainstem. Hypertension 1988;11:153–7. [29] Santos RAS, Campagnole–Santos MJ. Central and peripheral actions of angiotensin-(1–7). Braz J Med Biol Res 1994;27:1033– 47. [30] Santos RAS, Campagnole–Santos MJ, Baracho NCV, Fontes MAP, Silva LCS, Neves LAA, Oliveira DR, Caligioni SM, Rodrigues ANV, Gropen C Jr, Carvalho WS, Simo˜es e Silva 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– 8. [31] Vanhoutte PM, Lu¨scher TF. Peripheral mechanism in cardiovascular regulation: transmitters receptors and endothelium. In: Tarazi RC, Zanchetti A, editors. Handbook of Hypertension: Physiology and Pathophysiology of Hypertension, Vol. 8. Regulatory Mechanisms. Amsterdam: Elservier Science; 1996. pp. 96 –123. [32] Zweifach BW. Indirect methods for regional blood flow. I. Microscopic observation of circulation in rat mesoappendix and dog omentum. Use in study of vasotropic substances. Methods Med Res 1948; 1:131– 8.
Synergistic effect of angiotensin-(1–7) on bradykinin arteriolar dilation in vivo Maria Aparecida Oliveiraa, Zuleica Bruno Fortesa, Robson A.S. Santosb, Malesh C. Koslac, Maria Helena C. De Carvalhoa,* a
Department of Pharmacology, Institute of Biomedical Sciences, University of Sa˜o Paulo, Av. Prof. Lineu Prestes, 1524, Cidade Universita´ria, 05508-900 CEP Sa˜o Paulo, Brazil b Department of Physiology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil c Cleveland Clinic Foundation, Cleveland, OH, USA Received 3 March 1999; accepted 8 June 1999
Abstract The interaction between angiotensin [Ang-(1–7)] and bradykinin (BK) was determined in the mesentery of anesthetized Wistar rats using intravital microscopy. Topical application of BK and Ang-(1–7) induced vasodilation that was abolished by the BK B2 receptor antagonist HOE-140 and the Ang-(1–7) antagonist A-779, respectively. BK (1 pmol)-induced vasodilation, but not SNP and ACh responses, was potentiated by Ang-(1–7) 10 pmol and 100 pmols. The effect of 100 pmol of Ang-(1–7) on BK-induced vasodilation was abolished by A-779, indomethacin, and L-nitroarginine methyl esther, whereas losartan was without effect. Enalaprilat treatment enhanced the BK- and Ang-(1–7)-induced vasodilation and the potentiating effect of Ang-(1–7) on BK vasodilation. The potentiation of BK-induced vasodilation by Ang-(1–7) is a receptor-mediated phenomenon dependent on cyclooxygenase-related products and NO release. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Bradykinin; Angiotensin-(1–7); Microcirculation; Prostaglandins; Nitric oxide; Angiotensin-converting enzyme; Renin-angiotensin system; Angiotensin-(1–7) antagonist
1. Introduction Angiotensin-(1–7) [Ang-(1–7)], one of the bioactive components of the renin-angiotensin system, has two major characteristics: its formation through an angiotensin converting enzyme (ACE)-independent pathway [28,29] and the selectivity of its biologic actions [29]. It has been shown recently that Ang-(1–7) can interact with bradykinin (BK), enhancing its biologic effect [23–24]. In vivo Ang-(1–7) has been shown to potentiate the hypotensive effect of BK in normotensive [24] and hypertensive [18] rats. In vitro Ang(1–7) potentiates the vasodilatory effect of BK in isolated dog coronary arteries [3,17] and the vasoconstrictor action in dog femoral veins [10]. An amplification by Ang-(1–7) of the release of arachidonic acid induced by an ACE-resistant BK analog in Chinese hamster ovary cells cotransfected
* Corresponding author. E-mail address: [email protected] (M.H.C. De Carvalho)
with cDNAs of both human BK B2 receptor and ACE also was described recently [6]. The observations that Ang-(1–7) potentiates BK may be particularly important especially considering that Ang-(1–7) can increase in plasma after chronic blockade of AT1 receptors [7] or ACE inhibition [4,15]. Therefore, it is possible that the interaction of Ang-(1–7) with BK contributes to the beneficial cardiovascular effects obtained with these treatments. A physiological role for Ang-(1–7), in contributing to the antihypertensive actions of lisinopril treatment, was demonstrated in SHR [12]. In addition to the interaction with BK, a direct vasodilatory action of Ang-(1–7) was described [3,17,26]. The heptapeptide has been reported as a vasodilator in vitro in pig [26] and dog [3,17] coronary arteries and in feline isolated mesenteric and hindquarter vascular beds [22]. One important question that arises from these observations is whether the direct vasodilation and/or BK potentiation of Ang-(1–7) could occur at the level of resistance vessels, the most functionally important site for determining peripheral
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vascular resistance [31]. In this study, we addressed this issue by evaluating the direct effect of Ang-(1–7) and its interaction with BK at the level of secondary (A2) resistance vessels by using an in vivo in situ mesenteric blood vessel preparation.
2. Materials and methods 2.1. Intravital microscopy The rats were anesthetized with chloral hydrate (400 – 450 mg/kg, subcutaneously (s.c.)), and the mesentery was arranged for microscopic observation in situ [8,32]. In brief, the animals were kept on a special board, heated at 37°C, which included a transparent plate on which the tissue to be transilluminated was placed. The mesentery was kept moist and warm by irrigating the tissue with warmed (37°C) Ringer–Locke’s solution (pH 7.2–7.4) containing 1% gelatin. In a series of experiments, a 500-line television camera was combined with a tri-ocular microscope to facilitate observation of the enlarged image (3400⫻) on the video screen. An image-splitting micrometer was adjusted to the phototube of the microscope. The image splitter sheared the optical image into two separate images and displaced one with respect to the other. By rotating the image splitter in the phototube, the shearing is maintained in a direction at right angles to the axis of the vessel. The displacement of one image from the other allowed measurement of the vessel diameter [2]. Blood vessels were classified according to their branching order beginning at the capillary level and reaching up to the arteriolar side [9]. The smallest, precapillary arterioles were classified as A4, fed by the terminal arterioles (A3) branching from large arterioles (A2). A2 arterioles (15–25 m) were selected for study, and any changes in vessel diameter were estimated following the topical application of vasoactive drugs or the diluent Ringer-Locke solution. The drugs, dissolved in Ringer–Locke’s solution, were added to the preparation in a standard volume of 0.01 ml and were removed by washing with warmed Ringer–Locke’s solution. 2.2. Experimental protocols 2.2.1. Effect of BK and Ang-(1–7) alone and in combination To choose the doses to be used in the potentiation studies, the effects of BK (in doses of 1, 10, and 30 pmol, n ⫽ 7–16 for each dose) and Ang-(1–7) (in doses of 1, 10, 100, and 1000 pmol, n ⫽ 11–15 for each dose) were tested. For the potentiation experiments, Ang-(1–7) was added to the preparation 30 s before the application of BK. To verify if BK would potentiate the vasodilation induced by Ang-(1– 7), the nonapeptide was applied 30 s before Ang-(1–7).
2.2.2. Specificity of the Ang-(1–7) potentiating activity To verify the specificity of the potentiating effect of Ang-(1–7) on BK-induced vasodilation, doses of sodium nitroprusside (38 pmol, n ⫽ 5) or acetylcholine (1.6 nmol, n ⫽ 6) equipotent to BK were tested. For these experiments, the dose of Ang-(1–7) was added to the preparations 30 s before the application of each of these agents. 2.2.3. Participation of nitric oxide (NO) and prostanoids on the Ang-(1–7) potentiating activity To investigate whether NO is involved in the interaction between BK and Ang-(1–7), L-nitroarginine methyl esther (L-NAME, 10 nmol topically applied), a NO synthase inhibitor, was used. The dose and the time delay necessary for the effect of this agent were chosen in preliminary experiments. L-NAME was added to the preparations 3 min prior to test BK alone or combined with Ang-(1–7) (n ⫽ 9). In another series of experiments, the animals were treated with indomethacin (5 mg/kg, intramuscularly (i.m.), 30 min before, n ⫽ 8), to inhibit the cyclooxygenase pathway. In the treated animals, BK and BK and Ang-(1–7) were tested with a 3-min interval between them. The dose and the time delay were selected because they did not change basal diameter and/or blood pressure. 2.2.4. Effect of receptor antagonists on the Ang-(1–7) potentiating activity A-799, a specific Ang-(1–7) receptor antagonist [1,30], HOE 140, a specific BK B2 receptor antagonist, and losartan (15 mg/kg i.v. 40 min before the experiment, n ⫽ 6), to block AT1 receptors, were used. In the treated animals, BK and BK and Ang-(1–7) were tested with 3-min intervals between them. The dose and the time delay necessary for the specific effects of these agents were chosen in preliminary experiments. A-779 (100 pmol, n ⫽ 10) or HOE 140 (100 pmol, n ⫽ 10) were added to the preparations 15 s and 1 min, respectively, prior to test BK, Ang-(1–7), or the combination of both. These treatments did not change basal diameters and/or blood pressure. 2.2.5. Effect of ACE inhibition To test the interference of ACE on the BK, Ang-(1–7), and BK ⫹ Ang-(1–7) effects, enalaprilat (10 mg/kg i.v. 30 min before the experiment, n ⫽ 6) was used. 2.2.6. Statistical analysis The data were analysed with standard statistical analyses, i.e. analysis of variance with Tukey-Kramer (post hoc) test and Student’s paired or unpaired t-test where appropriate. All values are reported as mean ⫾ SEM. Statistical significance was set at P ⬍ 0.05. 2.2.7. Drugs Ang-(1–7) was purchased from Bachem. Acetylcholine, bradykinin, indomethacin, and sodium nitroprusside were purchased from Sigma (St. Louis, MO, USA). The drugs
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Fig. 1. Bar graphs show the increase (%) observed in rat mesenteric arteriolar diameter induced by topical application of bradykinin (BK) in A and angiotensin-(1–7) [Ang-(1–7)] in B. Results are mean ⫾ SEM. Numbers inside bars indicate number of preparations tested. In A, * P ⬍ 0.001 and † P ⬍ 0.001 vs. BK 1 pmol and BK 10 pmol, respectively. In B, * P ⬍ 0.05 and † P ⬍ 0.001 vs. Ang-(1–7) 1 pmol; ‡ P ⬍ 0.001 vs. Ang-(1–7) 10 pmol and § P ⬍ 0.001 vs. Ang-(1–7) 100 pmol.
were dissolved in Ringer–Locke gelatin solution, except indomethacin, which was dissolved in Tris buffer, pH 8.1. Enalaprilat (MK 422) was from Merck, Sharp & Dohme. 7 D-Ala -Ang-(1–7) was synthetized by M.C.K. at Cleveland Clinic Foundation (Cleveland, OH, USA).
3.2. Specificity of the BK potentiation by Ang-(1–7) To study the specificity of the potentiation of BK (1 pmol) by Ang-(1–7) (100 pmol), acetylcholine (1.6 nmol), and sodium nitroprusside (38 pmol) were tested. The vasodilation induced by these agents was not significantly altered by Ang-(1–7) (Table 1).
3. Results 3.1. Vascular effects of BK and Ang-(1–7) alone and in combination BK in doses of 1, 10, and 30 pmol and Ang-(1–7) in doses of 1, 10, 100, and 1000 pmol topically applied induced a vasodilation (expressed as percentage increase in initial diameters) on rat mesenteric arterioles studied in vivo (Fig. 1). The vasodilation elicited by BK (1 pmol) was potentiated significantly by Ang-(1–7) 10 pmol and 100 pmol. On the other hand, the lower dose (1 pmol) or the higher dose (1000 pmol) of Ang-(1–7) did not modify the BK vasodilation (Fig. 2). Based on these results, the doses of 1 pmol of BK and 100 pmol of Ang-(1–7) were chosen for the additional experiments—first, because they consistently elicited a small, but statistically significant, increase in arteriolar diameter (3–5%) when tested alone (Fig. 1) and second, because those doses used in conjunction elicited the highest potentiating effect of Ang-(1–7) on BK-induced vasodilaton (Fig. 2). It is important to mention that the vasodilation elicited by Ang-(1–7) (100 pmol) (4.9 ⫾ 0.4% increase, n ⫽ 4) was not increased by prior addition of BK (1 pmol) (5.8 ⫾ 1.0% increase, n ⫽ 4).
Fig. 2. Bar graphs show the increase (%) observed in rat mesenteric arteriolar diameter induced by topical application of BK and BK after Ang-(1–7). Results are mean ⫾ SEM. Numbers inside bars indicate number of preparations tested. * P ⬍ 0.05 and † P ⬍ 0.001 vs. BK 1 pmol.
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Table 1 Lack of potentiation of Ang-(1–7) on acetylcholine and sodium nitroprusside Agent\Ang-(1–7) 100 pmol
Absent
Present
n
BK 1 pmol Ach 1.6 nmol SNP 36 pmol
4.3 ⫾ 0.5 6.5 ⫾ 0.3 4.4 ⫾ 0.3
11.1 ⫾ 1.2* 5.0 ⫾ 0.5 3.7 ⫾ 0.7
7 6 5
Values are mean ⫾ SEM. n, number of animals used. * P ⬍ 0.001 vs. values in the absence of Ang-(1–7).
3.3. Contribution of NO and prostanoids to the BK and Ang-(1–7) effects alone or in combination To evaluate prostanoid contribution to the Ang-(1–7) (100 pmol) vasodilation and potentiating effect on the responsiveness of the mesenteric arterioles to BK (1 pmol), the cyclooxygenase inhibitor indomethacin (5 mg/kg i.m.), was used. After 30 min of treatment with indomethacin, the vasodilation induced by Ang-(1–7) was slightly reduced (P ⬍ 0.05, Table 2), whereas that induced by BK was unaltered (Fig. 3). On the other hand, the potentiation of BK-induced vasodilation elicited by Ang-(1–7) was abolished (Fig. 3). To test whether the direct or the potentiating effect of Ang-(1–7) on BK vasodilation was mediated by NO, the effect of the NO synthase inhibitor (L-NAME) on the rat mesenteric arterioles was studied. As seen in Fig. 4, LNAME (10 nmol topically applied) abolished the potentiating effect of Ang-(1–7) (100 pmol) on BK (1 pmol) vasodilation without interfering with the vasodilation induced by BK. The vasodilation induced by Ang-(1–7) was slightly reduced (P ⬍ 0.05) by L-NAME treatment (Table 2).
Fig. 3. Bar graphs show the increase (%) observed in rat mesenteric arteriolar diameter induced by topical application of BK and BK after Ang-(1–7), in untreated and indomethacin (5 mg/kg i.m.)-treated animals. Results are mean ⫾ SEM. Numbers inside bars indicate number of animals tested. * P ⬍ 0.001 vs. BK in untreated animals; † P ⬍ 0.001 vs. Ang-(1–7)⫹ BK in untreated animals
before and 0.5 ⫾ 0.5% increase, n ⫽ 10 after HOE 140 treatment, P ⬍ 0.0001). As a consequence of the BK B2 blokade by HOE 140, the potentiation of BK by Ang-(1–7) was not observed [Ang-(1–7)⫹ BK before 12.0 ⫾ 0.4% increase, n ⫽ 10 and 1.1 ⫾ 0.7% increase, n ⫽ 10, P ⬍ 0.0001 after HOE 140 treatment]. In contrast, the Ang-(1–7) vasodilation was not affected (Table 2). Pretreatment with the specific Ang-(1–7) receptor antagonist A-799 (100 pmol) did not interfere with BK (1 pmol)induced vasodilation whereas the potentiating effect of
3.4. Effects of blockade of BK, angiotensin II, and Ang-(1–7) receptors on the vascular effect of BK and Ang-(1–7) alone or in combination Administration of the specific BK B2 receptor antagonist HOE 140, at a dose of 100 pmol, nearly abolished the vasodilation produced by BK (4.1 ⫾ 0.3% increase, n ⫽ 10 Table 2 Effect of antagonists and enzyme inhibitors on Ang-(1–7) vasodilation Group
Untreated
Treated
n
Indomethacin 5 mg/kg i.m. L-NAME 10 nmol t.a. HOE 100 pmol t.a. Losartan 15 mg/kg i.v. Enalaprilat 10 mg/kg i.v. A-779 100 pmol t.a.
2.7 ⫾ 0.4 4.7 ⫾ 0.5 4.7 ⫾ 0.5 3.8 ⫾ 0.4 4.4 ⫾ 0.5 4.4 ⫾ 0.5
1.3 ⫾ 0.5* 3.2 ⫾ 0.6* 4.6 ⫾ 0.3 3.4 ⫾ 0.2 8.1 ⫾ 0.4† ⫺0.5 ⫾ 0.5‡
8 9 10 6 6 10
Values are mean ⫾ SEM of the percentage increase in arteriolar diameter induced by Ang-(1–7) topically applied. *P ⬍ 0.05; † P ⬍ 0.001; ‡ P ⬍ 0.0001 vs. untreated values. n, number of animals; t.a., topically applied.
Fig. 4. Bar graphs show the increase (%) observed in rat mesenteric arteriolar diameter induced by bradykinin (BK) and BK after Ang-(1–7) topically applied, in untreated and L-NAME (10 nmol topical)-treated preparations. Results are mean ⫾ SEM. Numbers inside bars indicate number of preparations tested. * P ⬍ 0.05 vs. BK in untreated preparations; † P ⬍ 0.001 vs. Ang-(1–7)⫹ BK in untreated preparations.
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Table 3 Effect of receptor antagonists and enzyme inhibitors on basal arteriolar diameters in micrometers Group
Untreated
Treated
n
Indomethacin 5 mg/kg, IM L-NAME 10 nmol t.a. HOE 100 pmol t.a. Losartan 15 mg/kg i.v. Enalaprilat 10 mg/kg i.v. A-779 100 pmol t.a.
17.9 ⫾ 0.8 16.0 ⫾ 1.1 23.2 ⫾ 2.2 20.1 ⫾ 0.8 18.8 ⫾ 0.8 16.7 ⫾ 1.0
18.7 ⫾ 0.8 15.8 ⫾ 1.0 23.2 ⫾ 2.2 19.9 ⫾ 0.6 18.9 ⫾ 1.3 16.5 ⫾ 0.9
14 10 9 9 11 9
Values are mean ⫾ SEM. n, number of animals used; t.a., topically applied.
Fig. 5. Bar graphs show the increase (%) observed in rat mesenteric arteriolar diameter induced by BK and BK after Ang-(1–7) topically applied, in untreated and A-779 (100 pmol, topical)-treated preparations. Results are mean ⫾ SEM. Numbers inside bars indicate number of preparations tested. * P ⬍ 0.001 vs. BK in untreated preparations; † P ⬍ 0.001 vs. Ang-(1–7) ⫹ BK in untreated preparations.
Ang-(1–7) (100 pmol) on BK (1 pmol) response was reduced markedly (Fig. 5). The direct vasodilating effect of Ang-(1–7) was abolished completely by A-779 (Table 2). On the other hand, the angiotensin II AT1 receptor antagonist losartan at a dose of 15 mg/kg i.v. (30 min before the experiment) had no effect on Ang-(1–7) (Table 2), BK vasodilation (Fig. 6), or on the BK potentiation by Ang(1–7) (Fig. 6). 3.5. Effect of ACE inhibition on BK potentiation by Ang-(1–7)
pressure levels of 39.8 ⫾ 3.1 mmHg (n ⫽ 6, P ⬍ 0.002), no differences were observed in arteriolar diameters (Table 3). As expected, mesenteric arteriolar vasodilation produced by BK (1 pmol) was increased approximately 2-fold by the treatment with the ACE inhibitor enalaprilat (MK 422 10 mg/kg). ACE inhibition also substantially increased the direct vasodilation of Ang-(1–7) 100 pmol (Table 2) and the potentiating effect of Ang-(1–7) on BK response (Fig. 7). 3.6. Effect of the treatments on basal mesenteric arteriolar diameters As shown in Table 3, none of the treatments modified the basal mesenteric arteriolar diameters. 4. Discussion In this study we showed that Ang-(1–7) produces vasodilation of resistance mesenteric blood vessel in vivo-in situ
Although enalaprilat treatment (10 mg/kg i.v. 30 min before the experiment) caused a decrease in basal blood
Fig. 6. Bar graphs show the increase (%) observed in rat mesenteric arteriolar diameter induced by BK and BK after Ang-(1–7) topically applied, in untreated and losartan (15 mg/kg i.v.)-treated animals. Results are mean ⫾ SEM. Numbers inside bars indicate number of animals used. * P ⬍ 0.001 vs. BK in untreated animals.
Fig. 7. Bar graphs show the increase (%) observed in rat mesenteric arteriolar diameter induced by BK and BK after Ang-(1–7) topically applied, in untreated and enalaprilat (10 mg/kg i.v.)-treated animals. Results are mean ⫾ SEM. Numbers inside bars indicate number of animals used. * P ⬍ 0.001 vs. BK in untreated animals; † P ⬍ 0.001 vs. Ang-(1– 7)⫹BK in untreated animals.
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and potentiates the vasodilation produced by BK. Furthermore, evidence was obtained for the participation of cyclooxygenase products and nitric oxide release in the mechanism of BK potentiation by Ang-(1–7). Both the direct vasodilator effect and the BK-potentiating activity of Ang(1–7) were blocked by the selective Ang-(1–7) antagonist A-779. This is the first demonstration indicating that Ang(1–7) and BK can interact not only at the level of large arteries [3,17] but also in resistance vessels modulating vascular tone by a receptor-mediated mechanism. Several studies have described the vascular effects of Ang-(1–7). A vasodilatatory effect of Ang-(1–7) has been described in dog [3,17] and pig [26] coronary arteries, in the cat hindlimbs, [22] and in piglet cerebral microvessels [19]. Although Ang-(1–7) is a vasodilator in most regions, a vasoconstrictor activity was observed in isolated heart preparation of rats [21] and hamsters [16]. Taken together, these observations suggest that the vascular actions of Ang-(1–7) can vary according to the vascular bed and/or with different species. It is particularly important, however, that, in resistance vessels of the mesenteric territory, the heptapeptide produced vasodilation with topical application of doses as low as 10 pmol. The vasodilation produced by Ang-(1–7) was reduced but not abolished by pretreatment with L-NAME or indomethacin (Table 2). These observations are important particularly when comparing these findings with the effect of these treatments on the BK-potentiating activity of Ang-(1– 7). Each treatment by itself blocked the BK-potentiating activity of Ang-(1–7) suggesting that each intracellular transduction system can evoke the potentiating response. However, if one is blocked, the other system cannot be activated. It appears that the systems are essentially in series with the participation of both being required for the activation of the BK-potentiating response by Ang-(1–7). The fact that Ang-(1–7) did not alter the vasodilation produced by acetylcholine or sodium nitroprusside suggests that the BKpotentiating activity of Ang-(1–7) cannot be evoked nonspecifically by agents that also release NO. This agrees with previous findings [17,23]. We did not determine the site with which Ang-(1–7) is interacting to increase BK vasodilation. Several possibilities can be raised including interaction of both peptides at the level of the smooth muscle layer and facilitation of the endothelium-mediated vasodilation produced by BK through an action of Ang-(1–7) on endothelial [13] or smooth muscle cells [14]. Indeed, both peptides have been shown to act on endothelial and smooth muscle cells, releasing prostaglandins E2 and I2 and NO from endothelial [11,13] and smooth muscle cells [14,25]. It has been reported that in vascular smooth muscle cells, Ang-(1–7) triggers the release of arachidonic acid through a receptor-mediated mechanism involving stimulation of CaM kininase II, with subsequent enhancement of cytoplasmatic phospholipase A2 activity, via mitogen-activating protein kinase activation [20]. Whether a similar mecha-
nism is involved in the vascular actions of Ang-(1–7) in mesenteric vessels remains to be determined. The vasodilation produced by BK combined with Ang(1–7) followed a bell-shaped dose–response curve. Maximal potentiation was attained with 100 pmol of Ang-(1–7), whereas when increasing the dose of the heptapeptide to 1 mol the BK-potentiating activity disappeared (Fig. 2). On the other hand, the direct vasodilation obtained with the 1-mol dose of Ang-(1–7) was significantly greater than other doses of Ang-(1–7) (Fig. 1). Of interest, a bell-shaped dose–response curve was obtained for the release of arachidonic acid or 6-keto-prostaglandin F1␣ by Ang-(1–7) in cultured vascular smooth muscle cells by Muthalif et al. [20]. These observations may be related to the activation of enzymes, such as lipoxygenase, that could be involved in the modulation of NO and prostanoid production by the combination of Ang-(1–7) [20] and BK [27]. However, our data do not allow evaluation of this possibility. As described previously in freely moving rats, treatment with the ACE inhibitor enalaprilat did not prevent the interaction of Ang-(1–7) with BK [18,24]. This result, together with the observation that pretreatment with indomethacin or L-NAME essentially abolished the BK-potentiating activity of Ang-(1–7), further indicates that the mechanism of the facilitatory effect of Ang-(1–7) on the vasodilation produced by BK is not dependent on an interference with the ACE catalytic activity [5,6] or on facilitation of the crosstalk between ACE and the BK B2 receptors [6]. According to our results and previous findings [6,17, 23,24], the interaction of Ang-(1–7) with BK is complex, involving, in addition to the mechanism listed above, a receptor-mediated mechanism linked to phospholipase A2/ cyclooxygenase activation and a prostanoid or nonprostanoid pathway dependent on modulation by NO. It has been suggested recently [6] that some actions of Ang-(1–7) could be mediated by potentiation of endogenous BK through facilitation of a ‘cross-talk’ mechanism between ACE and the BK B2 receptors. According to this report, binding of Ang-(1–7) to ACE would facilitate the cross-talk of ACE and the B2 receptors leading to potentiation of endogenous BK, independently of an interference with BK hydrolysis. This appears not to be the mechanism involved in the vasodilation produced by Ang-(1–7) in the mesenteric vascular bed: the vasodilation produced by Ang(1–7) was not blocked by pretreatment with the B2 receptor antagonist HOE 140 in a dose effective to block an equipotent BK effect. Furthermore, the Ang-(1–7) effect was completely blocked by its selective antagonist A-779, which did not alter the BK vasodilatatory effect and was rather increased by treatment with the ACE inhibitor enalaprilat, a condition that would be expected to reduce or abolish the Ang-(1–7) effect according to the mechanism proposed by Deddish et al. [6]. Additionally, the BK-potentiating activity of Ang-(1–7) is apparently not dependent on interaction with AT1 receptors subtypes because losartan did not inter-
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fere with it. Similar findings were obtained previously by Li et al. [17] in dog coronary arteries. In summary, we have found that, in mesenteric resistance vessels, besides producing vasodilation, Ang-(1–7) facilitates the vasodilation of BK. The BK-potentiating activity is an Ang-(1–7) receptor-mediated phenomenon dependent on cyclooxygenase-related products and NO release, but not on interference with the catalytic activity of ACE or facilitation of cross-talk between ACE and the BK B2 receptor. The observation that the interaction of Ang-(1–7) with BK is rather facilitated in ACE inhibitor-treated rats suggests that this interaction contributes to the pharmacological effects of this class of drugs.
Acknowledgments Support was provided by Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo, Conselho Nacional de Pesquisa, Fundac¸ao de Amparo a` Pesquisa do Estado de Minas Gerais, and Financiadora de Estudos e Projetos-PRONEX.
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