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CARDIOVASCULAR

Do the Cardiovascular Effects of Angiotensin-Converting Enzyme (ACE) I Involve ACE-Independent Mechanisms? New Insights from Proline-Rich Peptides of Bothrops jararaca

Center for Applied Toxinology/Centro de Pesquisa, Inovac çãoe Difusão, Laboratório Especial de Toxinologia Aplicada (D.I., A.C.M.d.C.) and Laboratório de Farmacologia, Departamento de Farmacologia (B.C.P.), Instituto Butantan, Sao Paulo, Brazil; Laboratório de Hipertensão, Departamento de Fisiologia e Biofísica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gevais, Belo Horizonte, Brazil (R.A.S.S., G.M.E., C.H.X., J.d.A.S., E.P.M., L.T.M.); and CEA, iBiTecS, Service d'Ingénierie Moléculaire des Protéines (SIMOPRO), Gif sur Yvette, France (V.D.)

Received February 1, 2007; accepted May 1, 2007.

	Abstract

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Angiotensin-converting enzyme (ACE) inhibitors were developed based on proline-rich oligopeptides found in the venom of Bothrops jararaca (Bj) previously known as bradykinin-potentiating peptides (BPPs). However, the exact mechanism of action of BPPs remains unclear. The role of the ACE in the cardiovascular effects of two of naturally proline-rich oligopeptides (Bj-BPP-7a and Bj-BPP-10c) was evaluated in vitro and in vivo. Bj-BPP-7a does not potentiate the cardiovascular response to bradykinin and is a weak inhibitor of ACE C and N sites (K_i = 40,000 and 70,000 nM, respectively), whereas Bj-BPP-10c is a strong bradykinin potentiator and inhibitor of the ACE C site (K_i = 0.5 versus 200 nM for N site). Strikingly, both peptides, in doses ranging from 0.47 to 71 nmol/kg, produced long-lasting reduction (>6 h) in the mean arterial pressure of conscious spontaneously hypertensive rats (maximal change, 45 ± 6 and 53 ± 6 mm Hg for Bj-BPP-7a and Bj-BPP-10c, respectively). The fall in blood pressure was accompanied by variable degrees of bradycardia. In keeping with the absence of relationship between ACE-inhibitory and antihypertensive activities, no changes in the pressor effect of angiotensin I or in the hypotensive effect of bradykinin were observed at the peak of the cardiovascular effects of both peptides. Our results indicate that the antihypertensive effect of two Bj-BPPs containing the motif Ile-Pro-Pro is unrelated to their ability for inhibiting ACE or potentiating bradykinin (BK), indicating as a major component ACE and BK-independent mechanisms. These results are in line with previous observations suggesting ACE inhibition-independent mechanisms for angiotensin I-converting enzyme inhibitor.

Subsequently, a pentapeptide and a nonapeptide were isolated from the Bj venom and fully characterized (Ferreira et al., 1970; Ondetti et al., 1971; Stewart et al., 1971). The corresponding synthetic peptides displayed transient potentiation of BK ex vivo and in vivo. In addition, one of these peptides, SQ 20.881 (Bj-BPP-9a), produced an antihypertensive effect, as shown in animal models and human subjects (Bianchi et al., 1973; Gavras et al., 1975). Vane's suggestion implied that the antihypertensive activity and the potentiation of BK by this Bj-BPP were a consequence of somatic angiotensin I-converting enzyme (sACE) inhibition since the in vivo experiments showed that the Bj-BPPs blocked bradykinin degradation and inhibited the conversion of Ang I to Ang II (Stewart et al., 1971). The Bj venom peptides raised enormous interest, and only the lack of oral activity prevented their general therapeutic use (Ondetti and Cushman, 1981; Hayashi et al., 2003). Thus, the development of a specific nonpeptidic sACE inhibitor, effective by oral route, became mandatory for pharmaceutical application.

The structure-activity studies by Cushman and Ondetti, from The Squibb Institute, suggested that the C-terminal proline of the Bj-BPPs specifically interacted with putative subsites, or pockets, at the active site of the sACE. Extensive studies on the binding to the Zn²⁺ of the active site led to the generation of nonpeptide compounds that combined proline with a sulfhydryl group, increasing the inhibitory potency. The best compound was named captopril, a very potent ACE inhibitor, the first truly useful antihypertensive drug designed to bind to the active site of ACE (Ondetti and Cushman, 1981). Captopril was shown to reproduce all known pharmacological and enzymatic features of the Bj-BPPs, consequently strongly reducing the interest in the Bj-peptides themselves (Case et al., 1978; Antonaccio et al., 1979).

However, recent results revived the interest in these peptides. Three reasons explain the renewed interest: a number of the Bj-BPPs can distinguish between the N and C active sites of sACE (Cotton et al., 2002). In fact, sACE has two homologous and functionally distinct active sites (Wei et al., 1991), one of which, the active site at the C domain (C site), is slightly more effective in hydrolyzing some vasoactive peptides, like bradykinin and angiotensin I (Perich et al., 1992; Jaspard et al., 1993); 2) the isolation and identification of novel Bj-BPPs in the crude venom (Ianzer et al., 2004); and 3) the presence of several Bj-BPPs in the C-type natriuretic peptide precursor of the snake brain may reveal novel neuropeptides (Murayama et al., 1997; Hayashi et al., 2003).

We have previously described the isolation of a cDNA coding for a Bj-BPP precursor protein from the venom gland and found in the neuroendocrine areas of the Bj-brain, containing seven tandemly arranged Bj-BPP sequences at the N terminus and one C-type natriuretic peptide sequence at the C terminus (C site) (Murayama et al., 1997; Hayashi et al., 2003). Among the endogenous, BPP-9a and BPP-10c turned out to be the most efficient inhibitors of the sACE, displaying high selectivity toward the C-active site (Cotton et al., 2002; Hayashi et al., 2003).

It has been suggested that Bj-BPPs may act by an ACE-independent mechanism (Camargo and Ferreira, 1971; Greene et al., 1972; Mueller et al., 2005, 2006). A similar possibility has been raised for conventional ACEI (Marks et al., 1980; Mittra and Singh, 1998; Houben et al., 2000; Ignjatovic et al., 2002). In the present study, we tested whether this possibility is true by selecting two peptides, a heptapeptide and a decapeptide, among 19 Bj-BPP sequences according to their totally distinct properties concerning BK potentiation in isolated guinea pig ileum (Ianzer et al., 2004). We observed a sustained antihypertensive activity of both Bj-BPPs in spontaneously hypertensive rats (SHRs). Strikingly, this activity was not related to the inhibition of sACE in vitro or to the bradykinin potentiation or blockade of the pressor effect of angiotensin I in vivo.

	Materials and Methods

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Drugs
Human wild-type somatic ACE was obtained through stable expression in Chinese hamster ovary cells transfected with appropriate ACE cDNA. The expression and purification of ACE were performed as previously described (Wei et al., 1991) and was kindly provided by Pierre Corvol (Institut National de la Santé et de la Recherche Médicale, Paris, France). Mca-Ala-Ser-Asp-Lys-N3(2,4-dinitrophenyl)L-2,3-diaminopropionyl (Mca-Ala) and Mca-Ser-Asp-Lys-N3(2,4-dinitrophenyl)L-2,3-diaminopropionyl (Mca-Ser) were prepared following the procedure previously described (Dive et al., 1999). BPP-7a (PyroGlu-Asp-Gly-Pro-Ile-Pro-Pro-OH), BPP-10c (PyroGlu-Asn-Trp-Pro-His-Pro-Gln-Ile-Pro-Pro-OH), bradykinin, captopril, angiotensin I, and angiotensin II were purchased from Bachem Bioscience (King of Prussia, PA). The peptides were dissolved in isotonic sterile saline (0.9% NaCl) (Asterflex, Belo Horizonte, Minas Gevais, Brazil) just before use in vivo.

In Vitro Assay: Enzyme Assays for Selective Inhibition of the ACE Active Sites
The inhibitions of somatic ACE determined for Bj-BPP-7a were performed as previously described (Cotton et al., 2002). In summary, the synthetic BPP was dissolved in buffer containing either the Mca-Ala or Mca-Ser substrate. The reactions were initiated by addition of the recombinant human ACE. The assays were carried out at 25°C in 50 mM HEPES, pH 6.8, 200 mM NaCl, and 10 µM ZnCl₂. Continuous assays were performed by recording the fluorescence increase at 390 nm ( _ex = 340 nm) induced by the cleavage of Mca-Ala and Mca-Ser substrates by ACE, monitored using a Biolumin 960 photon counter spectrophotometer (Molecular Dynamics, Sunnyvale, CA) equipped with a device control and a plate shaker. The substrate and enzyme concentrations for the experiments were chosen so as to remain well below 10% of substrate utilization and observe initial rates. Reported inhibition percentages corresponded to the average of three independent experiments.

Cell Culture
Commercially available cell lines Chinese hamster ovary, obtained from the American Type Culture Collection (Manassas, VA), were used for cell culture experiments. Cells were cultured 3 days or more as a monolayer (94/16-mm Petri dish) in culture medium recommended for each cell type. Cells stably transfected with AT₁ receptors cDNA driven by a cytomegalovirus promoter and selected by neomycin (Pesquero et al., 1994) were obtained from Dr. J. E. Krieger.

Binding Studies in Cell Cultures: Competition Experiments
AT₁ receptor-transfected cells were incubated with ¹²⁵I-Ang II (0.4 nM) in 24-well plates for 60 min at 4°C in 300 µl of serum-free medium (Dulbecco's modified Eagle's medium) supplemented with 0.2% BSA, 0.005% bacitracin, 100 µM phenylmethylsulfonyl fluoride, and 500 µM o-phenanthroline. Competition experiments were performed by preincubating the cells with Ang II (1 µM) (0.01 to 1 µM) or Bj-BPP-7a and Bj-BPP-10c (1 pM to 10 µM). After two washes with ice-cold serum-free Dulbecco's modified Eagle's medium, cells were disrupted with 0.1% Triton X-100 in water at 22 to 24°C. Bound radioactivity in the cell lysate was measured in a gamma counter (1275 MINIGAMMA; GE Energy, Little Chalfont, Buckinghamshire, UK). Experiments were made in duplicate (n = 3–6 for each peptide). Ang II was labeled with ¹²⁵I by the chloramine T method and purified by high-performance liquid chromatography, as described (Pinheiro et al., 2004).

Ex Vivo Assay: BK Potentiation on Isolated Guinea Pig Ileum
Animals. Experiments were carried out in five female guinea pigs (160–200 g) bred at the Instituto Butantan (Sao Paulo, Brazil). The animals had free access to food and water and were submitted to a light/dark cycle (12 h each) before the preparation for the experiments. All experimental protocols were performed in accordance to the guidelines for the human use of laboratory animals of our institute and approved by local authorities.

Experimental Protocol. The BK-potentiation assays on isolated guinea pig ileum were performed as previously described (Ianzer et al., 2006). After a 20-h fasting period, approximately 15 cm of the distal ileum of the female guinea pigs was removed immediately after death and washed thoroughly with Tyrode's solution (137 mM NaCl, 2.7 mM KCl, 1.36 mM CaCl₂, 0.49 mM MgCl₂, 0.36 mM NaH₂PO₄, 11.9 mM NaHCO₃, and 5.04 mM D-glucose). Segments (2.5 cm) of the ileum were isotonically mounted under a 1-g load in a 10.5-ml muscle bath containing Tyrode's solution at 37°C and bubbled with air. Muscular contraction was recorded on a Gould 2600 polygraph (Gould Instrument Systems Inc., Valley View, OH). One unit of BK potentiation is defined as the amount of potentiator (nanomoles) necessary to transform the effects of the single dose of BK into that produced by the double dose.

In Vivo Assays: Blood Pressure Recording in Rats
Animals. Experiments were carried out in 50 male SHRs (280–350 g) and 33 Wistar rats (250–320 g) bred at the animal facility of the Biological Science Institute (CEBIO, Universidade Federal de Minas Gerais, Minas Gerais, Brazil). The animals had free access to food and water and were submitted to a light/dark cycle (12 h each) before the preparation for the experiments. All experimental protocols were performed in accordance to the guidelines for the human use of laboratory animals of our institute and approved by local authorities.

Arterial Pressure Measurements. The cardiovascular parameters, pulsatile arterial pressure, mean arterial pressure (MAP), and heart rate (HR) were monitored by a solid-state strain gauge transducer connected to a computer through a data acquisition system (MP 100; BIOPAC Systems, Inc., Goleta, CA). The pulse arterial pressure, MAP, and HR were monitored simultaneously during experiments in different monitor channels and recorded in the computer hard disk for late analysis.

Experimental Protocols
Protocol 1: Inhibition of the Pressor Effect of Angiotensin I on Blood Pressure in Anesthetized Wistar Rats. The surgery was performed under anesthesia with 12% urethane i.p. (1.0 ml/100 g body weight). A polyethylene catheter (PE-10 connected to PE-50) was introduced into abdominal aorta through the left femoral artery for measurements of cardiovascular parameters and into right femoral vein for i.v. injection. During the experimental procedure, the temperature of the animal was maintained at 36 to 37°C (rectal temperature) through an electrical heating blanket.

Before drug administration, the cardiovascular parameters of the rat were monitored for 10 min. After this period, i.v. injections of Ang I (20 and 40 ng) were made. After administration of Bj-BPP (45 nmol), Ang I (40 ng) was injected in regular intervals (5, 10, 20, 30, 40, 50, 60, 70, and 90) (n = 4 for each Bj-BPP). ACE inhibition was estimated by the time (minutes) needed for 50% recovery of the response to 40 ng of Ang I before the administration of Bj-BPPs.

Protocol 2: Potentiation of the Hypotensive Effect of Bradykinin by Bj-BPP in Anesthetized Wistar Rats. This assay was performed according to (Ianzer et al., 2006) as follows. After surgery (performed according to protocol 1), the cardiovascular parameters were monitored for 10 min. After this period, i.v. injections of BK (0.5 and 1.0 µg) were made. After administration of Bj-BPP, BK (0.5 µg) was injected at regular intervals (5, 10, 15, 20, 25, 30, 40, 50, 60, 75, and 90 min). Different doses of Bj-BPP were injected to achieve the dose corresponding to the potentiating unit: dose necessary to transform the effects of the single dose of BK (0.5 µg) into that produced by the double dose (Bj-BPP-7a, n = 7; Bj-BPP-10c, n = 5).

Protocol 3: Effect of Bj-BPP on Blood Pressure of Conscious SHRs and Wistar Rats. Twenty hours before the experiment, under anesthesia with 2.5% tribromoethanol (1.0 ml/100 g of body weight) i.p., a polyethylene catheter (PE-10 connected to PE-50) was introduced into abdominal aorta through a femoral artery for measurements of cardiovascular parameters and into a femoral vein for i.v. injection. After recovery from anesthesia, the rats were kept in individual cages with free access to water and chow until the end of the experiments.

Before drug administration, the cardiovascular parameters were monitored for 1 h (baseline period). After this period, i.v. bolus injection of the Bj-BPPs or vehicle (NaCl 0.9%) in a total volume of 0.5 ml was made. Bj-BPPs doses of 71, 14.2, 2.37, and 0.47 nmol/kg and a captopril dose of 71 nmol/kg were used in SHRs (n = 5–8 for each dose), and Bj-BPPs doses of 71 and 0.47 nmol/kg were used in Wistar rats (n = 5–6 for each dose). The cardiovascular parameters were monitored continuously for 6 h after drug administration. MAP and HR values were computed at 2-min intervals for the entire recording period. Each animal received two randomly selected treatments [drugs (Bj-BPPs or captopril) or vehicle]. On the 2nd experimental day, all animals presented MAP and HR levels similar to the basal period of the 1st day.

Protocol 4: Potentiation of Bradykinin Hypotensive Effects and Inhibition of Angiotensin I Pressor Effect during Cardiovascular Effects of the Bj-BPPs in Conscious SHRs. The surgery was performed according to protocol 3. Before drug administration, the cardiovascular parameters were monitored for 40 min (baseline period). After this period, i.v. bolus injection of BK (0.5 and 1.0 µg) and Ang I (20 and 40 ng) were made to obtain the control responses. Bj-BPP-5a and captopril (71 nmol/kg), Bj-BPP-10c (2.37 and 71 nmol/kg), or saline (0.9%) in a total volume of 0.5 ml were administrated in SHRs (n = 4–5 for each BPP). Injections of BK (0.5 µg) were made at 10 and 210 min after Bj-BPP, and injections of Ang I (40 ng) were made at 20 and 220 min after Bj-BPP. MAP and HR values were computed at 2-min intervals for the entire recording period.

View larger version (41K): [in this window] [in a new window]   Fig. 1. Time course of the effect of i.v. administration of Bj-BPPs on the MAP and HR of SHRs. A and B, Bj-BPP-7a; C and D, Bj-BPP-10c. The i.v. injections of BPPs (0.47 nmol/kg) and vehicle were made at the time 0, indicated by the solid line.

	Results

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In Vitro and in Vivo Inhibition of sACE and ex Vivo and in Vivo Potentiation of BK. In vitro, Bj-BPP-7a presented a weak inhibition of sACE and showed no clear preference for either active site of ACE. Its inhibition constants for either site (micromolar range) was 4 orders of magnitude higher than that of Bj-BPP-10c (nanomolar range). The potency of sACE inhibition by the two Bj-BPPs, both in vitro and in vivo, was proportional to the degree of BK potentiation in both bioassays. Indeed, in vivo, Bj-BPP-7a neither inhibited ACE nor potentiated BK. On the other hand, in vivo, the ACE inhibition by Bj-BPP-10c presented a half-life of approximately 30 min (Table 1).

View larger version (24K): [in this window] [in a new window]   Fig. 2. Maximal changes in MAP and HR produced by Bj-BPPs in SHRs. A and B, Bj-BPP-7a; C and D, Bj-BPP-10c. Bj-BPPs were administered in doses ranging from 0.47 to 71 nmol/kg. The data show the mean ± S.E.M. of maximal (peak) changes observed in each rat with each dose. *, p < 0.05; **, p < 0.01; and *** p < 0.001 compared with the change observed with vehicle.

Figure 2, A and C, shows the peak changes in MAP produced by Bj-BPP-7a and Bj-BPP-10c in SHRs. Figure 3, A and C, shows the mean changes in MAP and HR in the 6 h following administration of both peptides. In sharp contrast with its weak potency as ACE inhibitor or bradykinin potentiator, Bj-BPP-7a produced a marked and sustained fall in the MAP of SHRs even at doses as low as 0.47 nmol/kg (peak change, –33 ± 5 versus –11 ± 3 mm Hg in vehicle-treated SHRs, p < 0.05; mean change over a 6-h period, –14 ± 3 versus –1 ± 3 mm Hg in vehicle-treated SHRs, p < 0.01). The maximal change in MAP was obtained with the dose of 14.2 nmol/kg (peak change, –45 ± 6 mm Hg, p < 0.01; mean change, –22 ± 4 mm Hg, p < 0.001). However, this effect was not statistically different from that obtained with the lowest dose tested (0.47 nmol/kg) (Figs. 2A and 3A). The changes in MAP were associated with significant reductions in HR (peak change, –78 ± 15 bpm for 0.47 nmol/kg and –89 ± 18 bpm for 14.2 nmol/kg, p < 0.05) (Figs. 2B and 3B).

View larger version (22K): [in this window] [in a new window]   Fig. 3. Average of the changes in MAP and HR over 6 h following administration of Bj-BPPs in SHRs. A and B, Bj-BPP-7a; C and D, Bj-BPP-10c. Bj-BPPs were administered in doses ranging from 0.47 to 71 nmol/kg. The data are presented as mean ± S.E.M. of averaged changes of the period of observation (6 h) *, p < 0.05; **, p < 0.01; and ***, p < 0.001 compared with the change with vehicle.

The maximal changes in MAP and HR usually occurred after 160 min of the Bj-BPP injection (Table 2). However, no clear relationship could be established between the changes in MAP and HR.

In Wistar rats, no significant changes in MAP and HR were observed after i.v. administration of Bj-BPPs with the dose of 0.47 nmol/kg, considering either the peak change or mean change (Figs. 4 and 5). At the dose of 71 nmol/kg, both Bj-BPPs caused a significant decrease in MAP when the peak changes were considered (–28 ± 4 mm Hg for Bj-BPP-7a and –20 ± 1 mm Hg for Bj-BPP-10c, p < 0.05 and p < 0.01, respectively; Fig. 4, A and C). The maximal changes in HR were –50 ± 9 bpm for Bj-BPP-7a (71 nmol/kg) and –69 ± 8 bpm for Bj-BPP-10c (0.47 nmol/kg); however, these changes were not statistically significant when compared with vehicle (–40 ± 17 bpm). On the other hand, considering the average of the changes in the entire period of observation (6 h), no noticeable effect on MAP or HR was observed (Fig. 5). Similar to SHRs, the maximal changes in cardiovascular parameters started after 160 min of the Bj-BPP injection (Table 2).

View larger version (20K): [in this window] [in a new window]   Fig. 4. Maximal changes in MAP and HR of Bj-BPPs in normotensive Wistar rats. A and B, Bj-BPP-7a; C and D, Bj-BPP-10c. Bj-BPPs were administered in two different doses: 0.47 and 71 nmol/kg. The data are presented as mean ± S.E.M. of maximal (peak) changes observed in each rat with each dose. *, p < 0.05; and **, p < 0.01 compared with the change observed with vehicle.

View larger version (18K): [in this window] [in a new window]   Fig. 5. Average of the changes in MAP and HR over 6 h following administration of Bj-BPPs in normotensive Wistar rats. A and B, Bj-BPP-7a; C and D, Bj-BPP-10c. Bj-BPPs were administered in two different doses: 0.47 and 71 nmol/kg. The data are presented as mean ± S.E.M. of averaged changes of period of observation (6 h).

Effect of BK and Ang I in the Arterial Blood Pressure of Conscious SHRs before and after Bj-BPP Administration. The results presented in Fig. 6A show that the extent of hypotension following the injection of BK was not affected by the administration of Bj-BPP-7a (71 nmol/kg), either after 10 or 210 min of administration. Accordingly, the extent of the increase in blood pressure following administration of Ang I was not affected by the injection of Bj-BPP-7a (71 nmol/kg). On the other hand, Bj-BPP-10c at 71 nmol/kg produced a modest, but statistically significant, potentiation of BK without interfering with the Ang I pressor effect, at both time points (Fig. 6C). Both Bj-BPPs produced a significant decrease in MAP (Fig. 6, B and D). A similar but more intense BK potentiation was observed with captopril at the same molar dose (71 nmol/kg or 15.4 µg/kg). As observed with both Bj-BPPs, no significant changes in the pressor effect of Ang I were observed (Fig. 6E). In sharp contrast with the Bj-BPPs, despite changing the BK hypotensive effect, captopril did not affect blood pressure levels (Fig. 6F). With a lower dose of Bj-BPP-10c (2.37 nmol/kg), no effect was observed in the BK response at both time points (10 and 210 min after administration), whereas a significant reduction in MAP was observed for up to 6 h (Fig. 7, A and B).

View larger version (21K): [in this window] [in a new window]   Fig. 6. Effect of i.v. administration of Bj-BPPs or captopril (71 nmol/kg) on the mean arterial pressure and on the blood pressure change produced by i.v. injection of BK and Ang I in SHRs. A, C, E, and G, influence of Bj-BPPs, captopril, and vehicle on the depressor effect of BK and the pressor effect of Ang I. Gray bars, hypotensive effect of bradykinin (0.5 µg); black bars, pressor response of angiotensin I (40 ng). B, D, F, and H, time course of the effect of Bj-BPPs, captopril, and vehicle on the MAP of SHRs (white bars). The data are presented as mean ± S.E.M. (n = 4–6); *, p < 0.05; **, p < 0.01; and ***, p < 0.001.

View larger version (14K): [in this window] [in a new window]   Fig. 7. Effect of i.v. administration of Bj-BPP-10c (2.37 nmol/kg) on the mean arterial pressure and on the blood pressure change produced by i.v. injection of BK and Ang I in SHRs. A, influence of Bj-BPP-10c on the depressor effect of BK and the pressor effect of Ang I. Gray bars, hypotensive effect of bradykinin (0.5 µg); black bars, pressor response of angiotensin I (40 ng). B, time course of the effect of Bj-BPP-10c on the MAP of SHRs (white bars). The data are presented as mean ± S.E.M. (n = 5); *, p < 0.05; **, p < 0.01; and ***, p < 0.001.

Binding Studies. To test whether the dissociation of the ACE inhibitory activity of Bj-BPPs from their antihypertensive effect could be due to blockade of AT₁ receptors, we determined the effect of both peptides on the binding of ¹²⁵I-Ang II to AT₁ receptor-transfected cells. No significant displacement was observed with both peptides in concentrations ranging from 10^–12 to 10^–5 M (Fig. 8).

View larger version (21K): [in this window] [in a new window]   Fig. 8. Competition for 125I-Ang II binding to AT1-transfected Chinese hamster ovary cells by Ang II and Bj-BPPs. Competition curves were generated by adding increasing concentrations of Ang II (10–11 to 10–6 M) or Bj-BPP-7a or Bj-BPP-10c (10–12 to 10–5 M) to the incubation buffer containing 0.4 nM of 125I-Ang II. Data are presented as mean ± S.E.M. of three to six independent experiments.

	Discussion

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However, the mechanisms of the bradykinin potentiation as well as the mechanism of the antihypertensive effect of ACEI appears to be more complex than previously suspected (Mittra and Singh, 1998; Marcic et al., 1999; Houben et al., 2000). Bradykinin potentiation by ACEI might involve beside ACE inhibition, induction of a cross-talk mechanism between ACE and B₂ receptors (Mittra and Singh, 1998; Marcic et al., 1999; Houben et al., 2000; Mueller et al., 2006). Indeed, more than 30 years ago, an ACE-independent mechanism for BK potentiation by ACEI of B. jararaca venom was suggested (Camargo and Ferreira, 1971; Greene et al., 1972).

Likewise, there are scarce but consistent reports showing that ACEI can produce vasodilation by mechanism that cannot be ascribed to ACE inhibition (Marks et al., 1980; Antonaccio et al., 1979; Mittra and Singh, 1998; Houben et al., 2000; Mueller et al., 2006). For example, Houben et al. (2000) have found that quinaprilat but not enaprilat produced significant vasodilation with 15 min of administration. At this time, both drugs completely blocked the Ang I pressor effect. Nakamura et al. (1992) have observed that although enalaprilat was without effect on basal forearm blood flow or vascular resistance, it significantly augmented the increase in blood flow and reduction in forearm resistance induced by acetylcholine. Captopril lowers blood pressure of animals with different models hypertension including situations where the renin-angiotensin system is not responsible for blood pressure maintenance (Marks et al., 1980). In addition, captopril has been shown to produce vasodilation in vitro in conditions that lisinopril was without effect (Mittra and Singh, 1998). Our data are in line with these previous observations. Taken all together, the current available data show that an ACEI can produce: 1) BK potentiation without blood pressure-lowering effect (captopril in our study), 2) antihypertensive effect without changing the hypotensive action of BK or the hypertensive effect of Ang I or Ang II (Bj-BPPs 7a and 10c in our study), 3) blockade of the pressor effect of Ang I without producing vasodilation (Houben et al., 2000), 4) vasorelaxation that can be dissociated from ACE inhibition (captopril) (Mittra and Singh, 1998), and 5) facilitation of NO release induced by acetylcholine without producing vasodilation directly (Nakamura et al., 1992). Furthermore, the antihypertensive effect of ACE inhibitors does not correlate with Ang II plasma levels (Marks et al., 1980; Duncan et al., 1999), and a similar dissociation has been described for bradykinin (Stanziola et al., 1999). In addition, it has been suggested that ACEI can act as an antioxidant (de Cavanagh et al., 1997) or can also act as B1 receptor agonist (Ignjatovic et al., 2002). It should be also pointed out that proline-rich peptides, which were the basis for the development of ACEI (Ondetti et al., 1977) having the same C-terminal motif (Ile-Pro-Pro), produced similar and potent antihypertensive effects, despite the fact that their ACE inhibitory potency differ about 80,000-fold (Bj-BPP-7a as compared with Bj-BPP-10c) (Table 1; this study). Therefore, although ACE inhibition is apparently the main mechanism of the antihypertensive effects of ACEI, other mechanisms unrelated to interference with the hydrolytic activity of ACE are obviously involved. More important, our study and the available data strongly support the hypothesis that ACEIs are not a single (uniform) class of antihypertensive drugs. Actually, they may present completely distinct mechanisms of action. In this regard, the contribution of other factors such as the increase of the vasodilator Ang-(1–7) should be considered in future studies (Duncan et al., 1999; Stanziola et al., 1999).

As mentioned above, the present study clearly demonstrates that, at least in SHRs, ACE inhibition cannot be applied to describe the antihypertensive effect of the proline-rich oligopeptides since the peptide concentrations used were far below those required to inhibit ACE in vivo. In fact, the hypotensive effect of the Bj-BPPs was also independent of BK potentiation, whether or not this effect is a consequence of the ACE inhibition. Therefore, in contrast to results that have been suggested for currently used ACEI, our results strongly suggest that the reduced formation of Ang II and BK potentiation are not essential mechanisms involved in the antihypertensive action of ACEI. We have also shown that the antihypertensive effect of Bj-BPPs cannot be ascribed to direct effects on AT₁ receptors.

Interestingly, Bj-BPPs caused a significant reduction instead of an increase of HR, as would be expected by the unloading of baroreceptors due to hypotension or if it was mediated by BK (Buñag et al., 1975). Whether this reflects a consequence of the tendency to a decrease in the locomotion activity following administration of the two Bj-BPPs (data not shown) or a direct effect of these peptides on the heart, such as interference in the autonomic activity modulating the heart pacemaker and/or changes in the baroreflex control of heart rate, remains to be clarified. It should be pointed out that our current data do not allow discarding a contribution of heart rate (cardiac output) changes to the blood pressure effects of Bj-BPPs, although a clear relationship between changes in BP and HR could not be established.

Herein we present one interesting result; the hypotensive effect of Bj-BPPs, especially in low doses, was observed in SHRs but not in normotensive animals. This observation suggests that the antihypertensive effect produced in SHRs is not due to a nonspecific effect of both peptides on blood vessels or in the heart. In conclusion, synthetic compounds, displaying properties similar to these Bj-BPPs, which produced antihypertensive effect without affecting the complex physiological role of ACE, could represent an attractive alternative for the treatment of human hypertension and other cardiovascular diseases.

	Acknowledgements

 
We acknowledge José Roberto Silva for excellent technical assistance and Neusa Lima for secretarial assistance. We thank Jöel Cotton (CEA, Saclay) for technical assistance in the enzymatic measurements of ACE and Beatriz L. Fernandes for critical reading.

	Footnotes

 
This study was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (Center for Applied Toxinology/Centro de Pesquisa, Inovac çãoe Difusão-FAPESP), by COINFAR Pesquisa e Desenvolvimento Ltda, and by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

D.I. and R.A.S.S. contributed equally to this work.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.120873.

ABBREVIATIONS: ACE, angiotensin I-converting enzyme; Bj, Bothrops jararaca; BK, bradykinin; BPP, bradykinin-potentiating peptide; sACE, somatic angiotensin-converting enzyme; Ang, angiotensin; ACEI, angiotensin I-converting enzyme inhibitor; SHR, spontaneously hypertensive rat; Mca-Ala, Mca-Ala-Ser-Asp-Lys-N3(2,4-dinitrophenyl)L-2,3-diaminopropionyl; Mca-Ser, Mca-Ser-Asp-Lys-N3(2,4-dinitrophenyl)L-2,3-diaminopropionyl; AT, angiotensin receptor; MAP, mean arterial pressure; HR, heart rate.

Address correspondence to: Dr. Antonio C.M. Camargo, Center for Applied Toxinology-CAT/CEPID, Instituto Butantan, Av. Vital Brasil, 1500, São Paulo, SP 05530-900, Brazil. E-mail: camur{at}macbbs.com.br

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