Introduction

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Several recent studies have suggested that angiotensin fragments may have biological activity. Ang 1-7 is a heptapeptide that results from removal of the carboxy-terminal phenylalanine from Ang II. Ang 1-7 apparently induces biological responses which in some cases resemble Ang II and in other cases differ. Both weak vasoconstrictor and dilator activity have been reported in hamster coronary arteries, piglet pial arterioles and the mesenteric and hindquarters vascular beds of the cat (Kumagai et al., 1990; Meng and Busija, 1993; Osei et al., 1993). Hypotensive responses to Ang 1-7 have been reported in pithed rats, spontaneously hypertensive rats and dogs with renovascular hypertension (Benter et al., 1993, 1995; Nakamoto et al., 1995). Both prostanoids and NO have been implicated as mediating the vasodilator activity of Ang 1-7 (Brosnihan et al., 1996; Meng and Busija, 1993; Nakamoto et al., 1995; Osei et al., 1993; Paula et al., 1995).

Recently some reports linked part of the vascular effect of Ang 1-7 to bradykinin. Ang 1-7 induced relaxation which was attenuated by icatibant (Hoe 140), a bradykinin B₂ receptor antagonist, and potentiated by an ACEi in precontracted isolated perfused porcine coronary arteries (Pörsti et al., 1994). In rats, Ang 1-7 (5 nmol) had no effect on blood pressure by itself, but potentiated the hypotensive response to bradykinin. This effect was increased further by treatment with an ACEi (Paula et al., 1995). Brosnihan et al. (1996) reported that Ang 1-7-induced relaxation was greatly attenuated (75%) by a bradykinin receptor antagonist.

Abbas et al. (1997) studied whether Ang 1-7 can decrease blood pressure in anesthetized normotensive rats when given either alone or in the presence of vasodepressor amounts of bradykinin. They found that whereas Ang 1-7 by itself did not decrease blood pressure, it did induce hypotension in the presence of bradykinin. Thus we hypothesized that kinins must be present to reveal the relaxant effect of Ang 1-7, and that these kinins mediate at least part of the vasodilator response to Ang 1-7. To test this hypothesis, we measured responses to Ang 1-7 in precontracted porcine coronary rings, either alone or in the presence of bradykinin. We found that in this in vitro preparation Ang 1-7 induced relaxation only in the presence of bradykinin. Because some angiotensin receptor antagonists have been reported to inhibit responses to Ang 1-7 (Ferrario et al., 1997), we then tested whether the relaxation induced by Ang 1-7 in bradykinin-stimulated rings was affected by angiotensin and/or bradykinin receptor antagonists and whether NO was involved in mediating these responses.

	Methods

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Materials

Angiotensin 1-7 and substance P were purchased from Bachem Bioscience (Bubendorg, Switzerland) and bradykinin from Bachem (Torrance, CA). The bradykinin receptor antagonist icatibant (Hoe 140) and the ACEi ramiprilat were generously supplied by Hoechst-Marian Roussel Pharmaceuticals (Somerville, NJ), the AT₁ receptor antagonist losartan by Dupont Merck (Wilmington, DE) and the AT₂ receptor antagonist PD123319 by Parke-Davis Pharmaceuticals (Ann Arbor, MI). The thromboxane receptor agonist U-46619 used to contract the rings was provided by Upjohn-Pharmacia (Kalamazoo, MI). The Ang 1-7 analog 7-D-Ala-Ang 1-7 was generously provided by M. Chappell (Dept. of Hypertension, Bowman Gray School of Medicine, Winston Salem, NC) and M. Khosla (Cleveland Clinic, Cleveland, OH). Other angiotensin peptides, the cyclooxygenase inhibitor meclofenamate and chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). All peptide solutions were prepared on the day of the experiment. Unless otherwise indicated, they were dissolved in Krebs-Henseleit solution, pH 7.4.

Experimental Protocol

Hearts from freshly sacrificed pigs were obtained from a local slaughterhouse and transported to the laboratory in ice-cold Krebs-Henseleit buffer. With a Petri dish placed on crushed ice, the circumflex coronary arteries were dissected, cleaned of adipose and connective tissue and cut into rings 3 to 5 mm wide. The rings were mounted on stainless steel hooks and suspended in an 8.0-ml tissue bath containing Krebs-Henseleit solution (mM: 120.0, NaCl; 4.7, KCl; 1.2, MgSO₄; 1.2, KH₂PO₄; 11.1, glucose; 2.5, CaCl_2; 25.0, NaHCO₃) gassed with 5% CO₂ in O₂ and maintained at pH 7.4 and 37°C. Changes in contractile tension were recorded with a Grass force displacement transducer (model 7D polygraph, Grass Instrument Co., Quincy, MA) coupled to an ink-writing oscillograph. The rings were stretched to a passive force of 5 g, a force previously determined to be optimal. This was followed by a 60-min equilibration period, washing the tissues every 15 min, with two to four complete renewals of the bath each time. Thereafter the rings were exposed to 60 mM KCl and the levels of contraction for each individual ring noted. Developed force was 4 ± 1 g. Tension was returned to base line by repeated washings (at least eight times) and the rings precontracted with the thromboxane mimetic U46619 to 60 to 80% of the maximal tension induced by 60 mM KCl, which was achieved with 10 to 50 nmol/l U46619. After vascular tone stabilized, the rings were challenged first with 10⁹ M bradykinin and then with increasing successive doses until maximal relaxation was obtained (10⁸-10⁷ M). Despite the continuous presence of bradykinin (rings were not rinsed out), vascular tone was restored spontaneously in about 15 min. After tone stabilized, Ang 1-7 (10⁸-10⁵ M, n = 5) was added. Responses to Ang 1-7 were expressed as per cent relaxation from this stable value; we used 5 × 10⁶ M in all subsequent experiments, because this concentration gave 40 to 50% relaxation. Only rings that relaxed in response to bradykinin were used. When other angiotensin peptides were studied, they were added instead of Ang 1-7 at the same concentration, 5 × 10⁶ M.

All experiments were performed with a paired design. After the initial response to Ang 1-7 was determined (control, taken as 100%), rings were rinsed repeatedly until basal tone returned, then precontracted again with U-46619 to determine the effect of inhibitors or antagonists on the relaxant effect of a second addition of Ang 1-7. Unless otherwise indicated, when the effect of inhibitors or receptor antagonists was studied, they were added after dose-dependent bradykinin-induced relaxation and 15 min before Ang 1-7. The second response to Ang 1-7 was 100 to 120% of the first (n.s; n = 4). The general protocol is given in figure 1.

View larger version (7K): [in this window] [in a new window]   Fig. 1.   Representative tracing showing the general protocol. After the pig coronary rings were precontracted with the thromboxane receptor agonist U-46619, the rings were relaxed with bradykinin. Although bradykinin was not rinsed out, the rings contracted again, returning to the initial tone. The dots show when the different compounds were added to the incubation bath. Ant, antagonist; Inh, inhibitor.

To determine whether relaxation induced by Ang 1-7 depended on bradykinin activation of bradykinin receptors, we used a) carboxypeptidase B, a potent kininase, and b) icatibant (Hoe 140), a potent and specific bradykinin B₂ receptor antagonist. To study whether cyclooxygenase or NO synthase was involved, the rings were preincubated with the cyclooxygenase inhibitor meclofenamate and the NOS inhibitor L-NAME. In some rings N^G-nitro-L-arginine was used instead of L-NAME; but because results were identical with both drugs, we only used L-NAME in further experiments. To determine whether relaxation involved guanylate cyclase products, we used methylene blue, a guanylyl cyclase inhibitor. To study whether ACE inhibition was involved in the effects of Ang 1-7, we first measured the effect of Ang 1-7 on purified ACE and then determined whether the ACEi ramiprilat induced relaxation when given alone and also altered the effects of Ang 1-7. To determine whether blocking AT₁ and/or AT₂ angiotensin receptors affected responses to Ang 1-7, we used the nonpeptidic compounds losartan, a selective AT₁ antagonist, and PD123319, a selective AT₂ antagonist. To study whether the effects of Ang 1-7 involved activation of Ang receptors, we tested the effects of the Ang II analogs sarthran (Sar¹-Thr⁸-Ang II) and saralasin (Sar¹-Val⁵-Ala⁸-Ang II) and the Ang 1-7 analog 7-D-Ala-Ang 1-7. In some experiments we tested whether another peptide known to induce endothelium-dependent relaxation could replace bradykinin. For this we used the tachykinin substance P.

In some experiments (n = 4), we measured bradykinin in the tissue bath by radioimmunoassay. Bradykinin was measured a) at the time of maximal relaxation (taken as 100%); b) when the rings had reached the precontracted tone, immediately before Ang 1-7; and c) during the maximal relaxation induced by Ang 1-7.

To study the participation of the endothelium in relaxation, it was removed by gentle mechanical rubbing with a cotton-tipped applicator. Absence of the endothelium was confirmed by lack of relaxation in response to 10⁷ M bradykinin.

ACE activity. To determine the effect of Ang 1-7 on ACE activity, porcine ACE (ACE control-E kit, Procedure 305-UV, Sigma Diagnostics, St. Louis, MO) and the substrate N-[3-(2-furyl)acryloyl]-L-phenylalanylglycylglycine (0.5 mM) were used. Ang 1-7 and the ACEi ramiprilat were dissolved in 300 mM NaCl. Solutions containing Ang 1-7 or ramiprilat were incubated for 15 min at 37°C with the ACE standard dissolved in Tris-buffered saline, pH 8.2 (72 U/l), for a total volume of 0.2 ml. Changes in absorbance (340 nm) on addition of the sample to the substrate were determined with an autoanalyzer (Hitachi 717) as described by the manufacturer. The rate of decrease in absorbance is directly proportional to ACE activity, which was calculated as units per liter of sample. Changes in ACE activity induced by Ang 1-7 or ramiprilat are expressed as per cent activity of the ACE standard incubated with vehicle.

Biostatistics. All concentrations reported in this work indicate the final levels in the organ chambers. Relaxation was calculated as per cent U-46619-induced tone and is expressed as mean ± S.E.M. of n hearts, with values from each individual heart taken as the average from two to four coronary rings. All experiments were performed in pairs, with the first relaxation response to Ang 1-7 as a control for the second response which was obtained in the presence of the inhibitor. This approach is valid because in time-control experiments (n = 4) the second response to Ang 1-7 did not decrease, ranging from 100 to 120% of the first response (n.s.). For the purposes of statistical analysis, we compared initial tension (T_i) and final tension (T_f) for each ring. Multiple measurements of T_f and T_i were evaluated for each sample. A two-sided paired comparison was used to evaluate differences in mean T_i and T_f values in each treatment group separately. To account for the multiple measurements, a ratio estimation approach derived from sampling theory was used (Cochran, 1977), which approximates the dependence of the data within a cluster. This approach also was used to compare per cent relaxation between various treatments. To assess differences between groups, T_f was adjusted for T_i by analysis of covariance. Because the data were gathered in clusters, we used the generalized estimating equations approach (Liang and Zeger, 1986). We started with the assumption that the slopes relating the dependent variable to the covariate in the two groups were equal; if not, the two regression equations were fit and compared. P < .05 was considered significant.

	Results

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Effects of Ang 1-7 either alone or after treatment with bradykinin. Exogenous Ang 1-7 (10⁸-10⁵ M) alone (in the absence of bradykinin) had no effect on precontracted coronary artery rings (n = 17) (fig. 2). In addition we used PGF₂ as a contractile agent instead of U-46619 (n = 5). Still Ang 1-7 alone did not induce relaxation. In contrast, addition of cumulative doses of bradykinin induced dose-dependent relaxation. Despite the continuous presence of bradykinin, this relaxation was transient, with vascular tone returning to the precontracted level. When bradykinin was measured in the bath at the time the precontracted tone was reached, degradation was only 15.7 ± 5.3% (n = 4). The half-life of bradykinin in the bath was 30.5 ± 2.9 min.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Representative tracings showing the effect of Ang 1-7 on tension in precontracted pig circumflex coronary artery rings in the absence (upper panel) and presence (lower panel) of bradykinin (BK). Ang 1-7-induced relaxation was observed only in rings pretreated with BK. Final molar concentrations in the bath are shown. Dots show when Ang 1-7 or BK was added to the bath.

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Effects of a kinin-degrading peptidase and a bradykinin B₂ receptor antagonist. To determine whether intact bradykinin needs to be present for Ang 1-7 to induce relaxation, carboxypeptidase B (2 U/ml), which inactivates bradykinin, was added to the bath 15 min before Ang 1-7. In rings containing carboxypeptidase B, Ang 1-7 failed to produce relaxation (fig. 3). To determine whether Ang 1-7 induces relaxation by a mechanism involving bradykinin B₂ receptors, the rings were preincubated with the bradykinin B₂ receptor antagonist icatibant (10⁶ M), whereupon Ang 1-7-induced relaxation was abolished (fig. 3).

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Effect of a bradykinin (BK) B2 receptor antagonist and carboxypeptidase B, a kininase, on Ang 1-7-induced relaxation in BK-pretreated rings. Icatibant (10 M) or carboxypeptidase B (2 U/ml) was added after BK-induced relaxation subsided and tone returned to the precontracted level, and 15 min before adding Ang 1-7. Responses are expressed as per cent relaxation with respect to Ang 1-7-induced relaxation during the control period in the same ring, which was taken as 100%. Data are expressed as mean ± S.E.M. of three hearts (two to four rings per heart).

Effects of Ang II and other Ang peptides. We tested whether other Ang peptides also would relax the rings in the presence of bradykinin. Unlike Ang 1-7, neither Ang I, Ang II nor Ang 3-8 (Ang IV) (5 × 10⁶ M) elicited relaxation (2.2 ± 1.3%, 6.1 ± 2.3% and 2.2 ± 1.1%, respectively; final vs. initial tone = n.s.) (fig. 4).

View larger version (11K): [in this window] [in a new window]   Fig. 4.   Comparative responses to Ang 1-7 (n = 22), Ang I (n = 5), Ang II (n = 12), and Ang 3-8 (n = 4) in pig coronary arteries pretreated with BK. Each Ang peptide (5 × 10 M) was added to the bath 15 min after tone returned to the precontracted level. BK was not rinsed out of the bath. Data are expressed as mean ± S.E.M. of the number of hearts indicated above (two to four rings per heart).

Effects of Ang 1-7 after treatment with substance P. To determine whether responses to Ang 1-7 also would be observed after treatment with a peptide other than bradykinin and known to induce endothelium-dependent relaxation, we tested a tachykinin, substance P (10⁸ M). At this concentration substance P induced maximal relaxation which was also transient, returning to the precontracted tone just as with bradykinin; however, Ang 1-7 did not induce any response in the presence of substance P (% relaxation = 0; n = 3).

Endothelial participation in the relaxant effect of Ang 1-7. Removal of the endothelium completely abolished the vasodilator effect of bradykinin as well as that of post-bradykinin Ang 1-7 (n = 2; not shown).

Effects of inhibitors of cyclooxygenases and NO synthases. To determine whether prostanoids were involved in the relaxant response to Ang 1-7, the rings were preincubated with the cyclooxygenase inhibitor meclofenamate (10⁶ M). However, the relaxation induced by Ang 1-7 was not altered; in the presence of meclofenamate it was 89.9 ± 20.6% of the control response to Ang 1-7 (n = 7; n.s.). To determine whether the endothelial NO synthase pathway is involved in the relaxant effect of Ang 1-7, the rings were incubated with the NO synthase inhibitor (L-NAME, 10⁶ M), which prevented relaxant responses to Ang 1-7. Incubation with methylene blue (10⁵ M), an inhibitor of guanylate cyclase, also abolished the relaxant effect of Ang 1-7. These data are shown in figure 5.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Effect of Meclofenamate, L-NAME and methylene blue on Ang 1-7-induced relaxation in BK-pretreated rings. Meclofenamate (10 M), L-NAME (10 M) or methylene blue (10 M) was added to coronary artery rings and Ang 1-7 administered 15 min later. Responses are expressed as per cent relaxation with respect to Ang 1-7-induced relaxation during the control period in the same ring, which was taken as 100%. Data are expressed as mean ± S.E.M. of five to seven hearts.

Ang 1-7 and ACE activity. To see whether Ang 1-7 could affect ACE, we measured ACE activity in the presence of different concentrations of Ang 1-7. At 10⁶ M, Ang 1-7 inhibited ACE activity by 30%, whereas the ACEi ramiprilat at 10⁷ M inhibited nearly 100% (table 1).

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View larger version (12K): [in this window] [in a new window]   Fig. 6.   Effect of the ACEi ramiprilat on Ang 1-7-induced relaxation in BK-pretreated rings. Ramiprilat (10 M) was added to the bath after BK-induced relaxation subsided and tone returned to the precontracted level; Ang 1-7 was added 15 min later. Addition of ramiprilat caused no change in the tone of the rings. Responses are expressed as per cent relaxation with respect to Ang 1-7-induced relaxation during the control period in the same ring, which was taken as 100%. Data are expressed as mean ± S.E.M. of six hearts.

Effects of AT₁ and AT₂ nonpeptidic antagonists on relaxant responses to Ang 1-7. To study whether the relaxant effect of Ang 1-7 could be mediated by AT₁ and/or AT₂ receptors, experiments were carried out in the presence of losartan (10⁶ M), an AT₁ receptor blocker, and PD123319 (10⁶ M), an AT₂ receptor antagonist. PD123319 did not alter the relaxing response to bradykinin (n = 2). When rings previously stimulated by bradykinin were then treated with PD123319, Ang 1-7-induced vasodilation was reduced significantly by 69.9 ± 6.2%. Higher doses of PD 123319 (10⁵ M; n = 2) did not increase inhibition. Preincubation of the rings with losartan resulted in a smaller but still significant 29.3 ± 7.3% reduction in Ang 1-7-induced relaxation. Further, the inhibitory activity of both angiotensin receptor blockers was not additive, because in the presence of both losartan and PD123319, Ang 1-7-induced relaxation was still reduced by 70% of control, similar to that obtained with PD123319 alone (fig. 7).

View larger version (27K): [in this window] [in a new window]   Fig. 7.   Effects of the selective AT1 antagonist losartan and the AT2 antagonist PD123319 (both 10 M) on Ang 1-7-induced relaxation in BK-pretreated rings. Each antagonist was added to the bath after BK-induced relaxation subsided and tone returned to the precontracted level, 15 min before adding Ang 1-7. Both lessened the response to Ang 1-7, but PD123319 was more effective. There was no difference in the inhibition observed with PD123319 alone or combined with losartan. Responses are expressed as per cent relaxation with respect to Ang 1-7-induced relaxation during the control period in the same ring, which was taken as 100%. Data are expressed as mean ± S.E.M. of seven hearts.

Effects of a nonselective Ang receptor antagonist and an Ang 1-7 analog. Neither of the two nonselectiveAT₁/AT₂ receptor antagonists we tested, Sar¹-Thr⁸-Ang II (sarthran) and Sar¹-Val⁵-Ala⁸-Ang II (saralasin) (10⁶-10⁵ M), inhibited Ang 1-7-induced relaxation of bradykinin-treated rings (fig. 8). When we tested a synthetic analog of Ang 1-7, 7-D-Ala-Ang 1-7 (10⁵ M), the response to Ang 1-7 was 119.7 ± 18.4% of control (n = 3; n.s.).

View larger version (28K): [in this window] [in a new window]   Fig. 8.   Effect of the nonselective AT1/AT2 antagonists sarthran and saralasin on the relaxant response to Ang 1-7 in coronary artery rings relaxed with bradykinin. Each antagonist was added to the bath after BK-induced relaxation subsided and tone returned to the precontracted level, 15 min before adding Ang 1-7. Neither altered tone by itself or decreased the response to Ang 1-7; in fact, saralasin significantly increased relaxation (P < .05 vs. Ang 1-7). Responses are expressed as per cent relaxation with respect to Ang 1-7-induced relaxation during the control period in the same ring, which was taken as 100%. Data are expressed as mean ± S.E.M. of five hearts.

	Discussion

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We found that in isolated precontracted porcine coronary artery rings, Ang 1-7 alone was not able to induce relaxation at concentrations up to 10⁵ M, whereas under the same conditions bradykinin induced almost complete relaxation at 10⁸ to 10⁷ M. The relaxation induced by bradykinin was not maintained; although bradykinin remained in the chamber, vascular tone returned to the basal contracted state within about 10 to 20 min after the last addition of bradykinin. When Ang 1-7 was added at this time, it induced clear dose-dependent relaxation. Thus, under these conditions, Ang 1-7-induced relaxation required the presence of bradykinin. This contrasts with the results reported by Pörsti et al (1994) in porcine coronary artery rings and Brosnihan et al (1996) using canine coronary artery rings. They reported that Ang 1-7 at micromolar concentrations induced relaxation which was affected to varying degrees by the kinin analog icatibant (Hoe 140), a highly specific bradykinin B₂ receptor antagonist, which suggests that at least part of the response to Ang 1-7 is kinin-mediated and therefore kinins must be present. We are not aware of any obvious reasons why we were unable to observe a direct relaxant effect with Ang 1-7. It was not the result of differences in the nature of the contractile agent used, because relaxation to Ang 1-7 alone was not observed in rings precontracted with PGF₂ instead of U46619. Perhaps differences in species and experimental conditions (superfused vessels vs rings, differences in endogenous kinin production, or varying levels of plasma kallikrein and/or high-molecular-weight kininogen in the vascular wall) might explain these discrepancies.

The relaxation evoked by Ang 1-7 resembles the effect of ACE inhibitors described in canine and bovine coronary arteries (Mombouli et al., 1991; Auch-Schwelk et al., 1993). In these studies, different ACEi elicited vasorelaxation in the presence of subthreshold concentrations of bradykinin even after kinins were rinsed out. The mechanism involved is not clear. Besides inhibition of kinin degradation, it has been proposed that ACEi increase vasodilator responses to bradykinin by some other mechanism (Mombouli et al., 1995). Angiotensin peptides (Salgado and Krieger, 1983; Textor et al., 1981) and particularly Ang 1-7 reportedly can inhibit ACE activity (Li et al., 1997). We also found that ACE can be inhibited by Ang 1-7. Thus the concentration of Ang 1-7 we used (5 × 10⁶ M) could have partially inhibited endothelial ACE in the rings and thereby induced or magnified kinin-mediated relaxation. If so, then any ACEi might be expected to induce kinin-mediated relaxation when added instead of Ang 1-7. To address this possibility, we used a potent ACEi, ramiprilat, at concentrations 10 times higher than the concentration needed to fully inhibit ACE in vitro. At such doses ramiprilat markedly potentiated the relaxant effect of kinins in coronary rings, which indicates that it blocks ACE. However, ramiprilat did not induce relaxation; moreover, responses to Ang 1-7 were not affected by pretreatment with ramiprilat. In addition, when bradykinin (10⁸ M) was added instead of Ang 1-7, there was no relaxant response. Together these data indicate that Ang 1-7-induced relaxation after bradykinin is not caused by either partial ACE inhibition or magnified kinin-mediated relaxation. On the other hand, we cannot discard the possibility that ACE inhibition is one component of the mechanism behind the Ang 1-7 potentiation of kinin-induced hypotension reported by Paula et al. (1995) in rats and the kinin-induced relaxation reported by Brosnihan et al. (1996) in canine coronary artery rings. It is conceivable that relaxation to Ang 1-7 given after a prior relaxant response to bradykinin has waned (present work) and potentiation of kinins by Ang 1-7 are caused by different mechanisms.

It is not clear why the rings returned to the precontracted state after bradykinin-induced relaxation. This was not caused by bradykinin degradation. The half-life of bradykinin in the bath we found is quite similar to that reported previously (Auch-Schwelk et al., 1993); 30 min after the initial dose, the concentration of bradykinin left in the bath would have been sufficient to induce substantial relaxation in precontracted rings, as shown by the dose response to bradykinin (figs. 1 and 2). During these studies we observed that Ang 1-7 did not alter the rate of kinin disappearance from the bath or increase kinin concentrations in the bath, which suggests that it does not act by significantly inhibiting kininases or releasing bound kinins. The response to bradykinin is endothelium-dependent; de-endothelialized rings did not relax in response to Ang 1-7, which indicates that they also depend on the endothelium. No response to Ang 1-7 was observed if the rings were relaxed with the tachykinin substance P, which indicates that Ang 1-7 relaxation is observed selectively with bradykinin. In addition, the relaxant response to Ang 1-7 depended on activation of the bradykinin B₂ receptor by the bradykinin present in the bath, because it was not observed if bradykinin was degraded with carboxypeptidase B and also was abolished by a bradykinin B₂ receptor antagonist, icatibant. This suggests some type of interaction between Ang 1-7 and the bradykinin receptor. Based on similar transient responses to bradykinin (falling perfusion pressure) and restoration of vasodilation by an ACEi, it has been postulated that ACEi might interact with the bradykinin receptor (Hecker et al., 1994), and recently it has been demonstrated that ACEi altered the rate of internalization and affinity state of the bradykinin B₂ receptor (Minshall et al., 1997). Likewise, restoration of the relaxant response to bradykinin by Ang 1-7 may involve regulation of the bradykinin receptor after it has been activated by the agonist. The fact that the relaxant response to bradykinin was transient and tone returned to the precontracted level despite the relaxant concentration of bradykinin, coupled with the lack of relaxation in response to bradykinin added de novo, suggests receptor desensitization. The nature of the process(es) leading to desensitization of the bradykinin receptor is still unresolved (Freedman and Lefkowitz, 1996; Blaukat et al., 1996; Olmos et al., 1995; Munoz and Leeb-Lundberg, 1992), although it may be similar to that of other G-protein-coupled receptors (Leeb-Lundberg et al., 1987). Perhaps Ang 1-7 transiently affects the process(es) which lead to resensitization of the bradykinin receptor, although at this time this is just speculation.

Both methylene blue and L-NAME obliterated the vasodilator response to Ang 1-7, whereas the cyclooxygenase inhibitor meclofenamate had no inhibitory effect. These data indicate that Ang 1-7 relaxed precontracted rings primarily by releasing NO and activating guanylate cyclase. Endothelium-dependent vasodilation by bradykinin is mediated by NO, prostacyclins and EDHF, a compound similar to epoxyeicosatrienoic acid, which is derived from metabolism of arachidonic acid by cytochrome P450 (Campbell et al., 1996). Although bradykinin-induced relaxation in the presence of inhibitors of NOS and cyclooxygenase is EDHF-dependent, in porcine epicardial arteries NO is almost completely responsible for endothelium-mediated relaxation in the absence of agents that inhibit cyclooxygenase or NO synthase, consistent with the data which indicate that Ang 1-7 acts via stimulation of NO. A NOS enzyme was purified recently from rat cerebellum. This NOS uses bradykinin as a substrate to generate NO, and this reaction can be inhibited by both typical NOS inhibitors such as L-NAME and also by bradykinin receptor antagonists (Chen and Rosazza, 1996). At this time no other information is available about this NOS, but its discovery implies that bradykinin may be linked to NO generation by mechanisms other than activation of its receptor.

In contrast to Ang 1-7, no relaxation was seen with Ang I, Ang II or Ang IV; thus responses to Ang 1-7 are not caused by interaction with the putative Ang IV receptor (Wright et al., 1995). The differing responses to Ang 1-7 and Ang II suggest that Ang 1-7 stimulates a mechanism other than activation of the AT₁ or AT₂ receptor. They also suggest that Ang 1-7 is unique among angiotensin peptides in its ability to stimulate or enhance endothelium-derived responses after desensitization of the bradykinin B₂ receptor secondary to exposure to bradykinin. A receptor for Ang 1-7 which is distinct from the AT₁ or AT₂ receptor has been inferred from functional studies (Tallant et al., 1997; Ferrario et al., 1991); and this hypothesis is strengthened by the discovery that the synthetic Ang 1-7 analog 7-D-Ala-Ang 1-7 (A-779) blocks some of the biological effects of Ang 1-7 (Santos et al., 1994, 1996) as well as the recent report of a specific binding site for this peptide in endothelial cells which is displaced selectively by A-779 (Tallant et al., 1997). Although there is no direct evidence of the existence of a discrete gene coding for this putative non-AT₁/AT₂ receptor, the ability of Ang 1-7 to stimulate the AT₁ receptor at high concentrations (affinity 1 µM) complicates a pharmacological approach to the question of Ang 1-7-elicited responses. Reports involving inhibition of responses to Ang 1-7 by AT₁ and/or AT₂ antagonists are not consistent, because there are reports describing a) attenuation of responses by AT₂ antagonists (Jaiswal et al., 1993), b) attenuation by AT₁ antagonists (Handa et al., 1996; Seyedi et al., 1995; Garcia and Garvin, 1994), c) inhibition by both AT₁ and AT₂ antagonists (Gironacci et al., 1994), d) lack of inhibition by either AT₁ or AT₂ antagonists (Pörsti et al., 1994; Freeman et al., 1996; Brosnihan et al., 1996) and e) competition between losartan and Ang 1-7 for the same binding sites (Mahon et al., 1994). Inhibition of responses to Ang 1-7 by angiotensin analogs such as sarthran, sarile or saralasin is also variable (Brosnihan et al., 1996; Freeman et al., 1996; Jaiswal et al., 1992; Seyedi et al., 1995).

We wanted to find out whether angiotensin antagonists affect the relaxation response to Ang 1-7 after bradykinin. Ang 1-7-induced relaxation was decreased in all samples tested with the AT₂ antagonist PD123319, with a mean blockade of 70%. Preincubation with losartan, an AT₁ antagonist, had a smaller and more variable effect, with a mean blockade of 30%. In the presence of both antagonists, the response remained inhibited by 70%, which indicates that inhibition is not additive. These results could be interpreted as suggesting that the response to Ang 1-7 is mediated primarily by the AT₂ receptor, with only minor involvement of the AT₁ receptor; however, this is inconsistent with Ang II's lack of effect. Responses to stimulation of AT₁ and AT₂ receptors may oppose each other (Scheuer and Perrone, 1993; Siragy and Carey, 1996; Nakajima et al., 1995; Stoll et al., 1995). Thus the effects of Ang II on vascular tone mediated by AT₁ and AT₂ receptors may cancel each other out. We reasoned that if stimulation of AT₂ receptors was responsible for the relaxation, then treating the rings with Ang II when AT₁ receptors are blocked would result in relaxation. Thus we challenged the rings with Ang II in the presence of the AT₁ antagonist losartan, always after bradykinin-induced relaxation. No relaxation was observed, which suggests that Ang II stimulation of the AT₂ receptor does not mimic responses to Ang 1-7. Furthermore, Ang 1-7-induced relaxation was not inhibited by either sarthran or saralasin, both nonselective AT₁/AT₂ receptor antagonists; in fact, saralasin tended to potentiate relaxation. This finding coupled with the fact that Ang II had no effect suggests that responses to Ang 1-7 are not caused by stimulation of known Ang II receptors or by putative non-AT₁ or AT₂ receptors. As mentioned earlier, A-779 selectively displaces Ang 1-7 from endothelial binding sites and is a specific inhibitor of the central effects of Ang 1-7 as well as its antidiuretic effects (Santos et al., 1994,1996; Tallant et al., 1997; Ambühl et al., 1994); however, in our study it was unable to block the response to Ang 1-7 when given at 10⁵ M. Taken together, these results are perplexing, because they indicate that responses to Ang 1-7 are not mediated via either AT₁ or AT₂ and suggests they are not mediated by non-angiotensin AT₁/AT₂ receptors either. Yet they are still partially sensitive to PD123319, and to a lesser degree losartan. The ability of losartan and PD123319 to discriminate between AT₁ and AT₂ receptors exceeds 3 orders of magnitude; however, the fact that they are Ang II-selective does not mean they do not bind to or interact with other non-Ang receptors or proteins and thereby modify the response to angiotensin peptides (Handa et al., 1996; Li et al., 1996; Bertolino et al., 1994). It is also possible that some of the effects of losartan (and by extension other nonpeptidic antagonists) are the result of actions at sites other than the AT₁ receptor; data supporting this notion have been summarized recently (Speth et al., 1995). Thus studies with selective Ang II receptor ligands must consider the possibility that some of the actions of these agents may be independent of their effect on the Ang II receptor. Losartan and PD 123319 share a common benzamidazole structure (Chiu et al., 1990). Our findings suggest that these compounds may recognize the site(s) affected by Ang 1-7 after bradykinin. The lack of additive effects between PD123319 and losartan suggests that they act on a similar site(s) already maximally inhibited by PD123319.

The present data point to an unusual interaction between Ang 1-7, a byproduct of the metabolism of angiotensin, and bradykinin, a potent vasodilator. In the presence of bradykinin, Ang 1-7 induced kinin/NO-mediated relaxation through a novel pathway. Recently we reported similar findings in anesthetized rats (Abbas et al., 1997). Blood pressure of rats receiving a hypotensive infusion of bradykinin first decreased and then slowly rose. Bolus injections of large doses of Ang 1-7 induced kinin-mediated hypotensive responses. Ang 1-7 given alone (in the absence of bradykinin) induced AT₁-mediated hypertensive responses. Injections of Ang 1-7 into saralasin-treated rats induced a mild hypotensive response which was obliterated by the bradykinin B₂ receptor blocker, which suggests that it was mediated by activation of bradykinin B₂ receptors and endogenous bradykinin. Together the present data and those reported by others (Ferrario et al., 1997) suggest that the Ang 1-7-bradykinin/bradykinin receptor/NO tandem potentially would act as a counter-regulatory system, opposing the vasoconstrictor and pro-growth activities of Ang II.

Conceptually these data imply that when the renin-angiotensin system is activated, it can produce an angiotensin peptide, Ang 1-7, which may exert a negative functional feedback on the vasoconstrictor activity of Ang II by increasing responses to kinins (which are potent vasodilators). In addition, treatment with ACEi induces increases in Ang 1-7 (Kohara et al., 1993; Campbell et al., 1991, 1994). Bradykinin apparently mediates many of the cardiovascular effects of ACEi (Scicli and Carretero, 1996; Carretero and Scicli, 1995). Perhaps the newly found link between bradykinin and Ang 1-7 participates in the mechanism(s) whereby ACEi exert their kinin-mediated cardiovascular effects.

The doses of Ang 1-7 needed to have any effect, as well as those used in most other studies, are supraphysiological (Brosnihan et al., 1996; Trachte et al., 1990; Benter et al., 1995; Nakamoto et al., 1995; Paula et al., 1995). These in vitro experiments cannot mimic the effects of prolonged and chronic changes in tissue concentrations of Ang 1-7 and/or bradykinin. However, they do demonstrate an unusual and potentially important biological effect. These data suggest that Ang 1-7 participates with bradykinin in a vasodepressor pathway and that the renin-angiotensin and kallikrein-kinin systems may be connected by an interaction between Ang 1-7 and bradykinin- and/or bradykinin B₂ receptor-mediated responses. The mechanism(s) involved in this interaction as well as its physiological relevance remain to be clarified.

In summary, Ang 1-7, a metabolite of Ang I, failed to induce relaxation of precontracted porcine coronary rings when given alone at concentrations up to 10⁵ M. Bradykinin (10¹⁰-10⁷ M) induced potent relaxation which was not sustained, returning to precontracted levels within 15 to 20 min. In these bradykinin-stimulated rings, Ang 1-7 (5 × 10⁶ M) relaxed the rings by approximately 50%. This response was not mimicked by Ang I, Ang II, Ang IV or Ang II in the presence of losartan. Icatibant and the kininase carboxypeptidase B completely abolished Ang 1-7-induced relaxation, as did the NOS inhibitor L-NAME and the guanyl cyclase inhibitor methylene blue. Ang 1-7 inhibited ACE by 30% at 10⁶ M, but relaxation was not observed when the rings were treated with the potent ACE inhibitor ramiprilat instead of Ang 1-7. Ang 1-7-induced relaxation was decreased by 30% by the AT₁ antagonist losartan (10⁶-10⁵ M) and blunted by 70% by the AT₂ antagonist PD-123319. However, relaxant responses to Ang 1-7 were not decreased by the nonselective Ang antagonists saralasin or sarthran. In the presence of bradykinin, Ang 1-7 acts by a PD-123319-sensitive pathway, releasing NO from endothelial cells via the bradykinin B₂ receptor.