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A new structurally atypical bradykinin-potentiating peptide isolated from Crotalus durissus cascavella venom (South American rattlesnake)
Toxicon 90 (2014) 36e44
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A new structurally atypical bradykinin-potentiating peptide isolated from Crotalus durissus cascavella venom (South American rattlesnake) Denise M. Lopes a, Norberto E.G. Junior a, Paula P.C. Costa a, Patrícia L. Martins a, udia F. Santos a, Ellaine D.F. Carvalho b, c, Maria D.F. Carvalho b, c, d, Cla s C. Fonteles a, Daniel C. Pimenta e, Bruno A. Cardi d, Manasse a, 1 Nilberto R.F. Nascimento , Krishnamurti M. Carvalho b, d, *, 1 , rio de Farmacologia Cardiovascular e Renal, Instituto Superior de Ci^ Laborato encias Biom edicas, Universidade Estadual do Ceara , Brazil Fortaleza, Ceara b , Brazil GENPHARMA LTDA, Fortaleza, Ceara c , Brazil Faculdade de Medicina Christus, Fortaleza, Ceara d , rio de Toxinologia e Farmacologia Molecular, Instituto Superior de Ci^ Laborato encias Biom edicas, Universidade Estadual do Ceara , Brazil Fortaleza, Ceara e ~o Paulo, Brazil Instituto Butantan, Sa a
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 April 2014 Received in revised form 7 July 2014 Accepted 23 July 2014 Available online 1 August 2014
Venom glands of some snakes synthesize bradykinin-potentiating peptides (BPP's) which increase bradykinin-induced hypotensive effect and decrease angiotensin I vasopressor effect by angiotensin-converting enzyme (ACE) inhibition. The present study shows a new BPP (BPP-Cdc) isolated from Crotalus durissus cascavella venom: Pro-Asn-Leu-Pro-Asn-TyrLeu-Gly-Ile-Pro-Pro. Although BPP-Cdc presents the classical sequence IPP in the C-terminus, it has a completely atypical N-terminal sequence, which shows very low homology with all other BPPs isolated to date. The pharmacological effects of BPP-Cdc were compared to BBP9a from Bothrops jararaca and captopril. BPP-Cdc (1 mM) significantly increased BK-induced contractions (BK; 1 mM) on the guinea pig ileum by 267.8% and decreased angiotensin I-induced contractions (AngI; 10 nM) by 62.4% and these effects were not significantly different from those of BPP9a (1 mM) or captopril (200 nM). Experiments with 4-week hypertensive 2K-1C rats show that the vasopressor effect of AngI (10 ng) was decreased by 50 mg BPP-Cdc (69.7%), and this result was similar to that obtained with 50 mg BPP9a (69.8%). However, the action duration of BPP-Cdc (60 min) was 2 times greater than that of BPP-9a (30 min). On the other hand, the hypotensive effect of BK (250 ng) was significantly increased by 176.6% after BPP-Cdc (50 mg) administration, value 2.5 times greater than that obtained with BPP9a administered at the same doses (71.4%). In addition, the duration of the action of BPP-Cdc (120 min) was also at least 4 times greater than that of BPP-9a (30 min). Taken together, these results suggest that BPP-Cdc presents
Keywords: Bradykinin-potentiating peptide Angiotensin-converting enzyme Hypertension Crotalus durissus cascavella venom
Abbreviations: ACE, angiotensin-converting enzyme; AngI, angiotensin I; AngII, angiotensin II; BK, bradykinin; BPPs, bradykinin-potentiating peptides; BPP-Cdc, bradykinin-potentiating peptides from Crotalus durissus cascavella. rio de Toxinologia e Farmacologia Molecular, Instituto Superior de Cie ^ncias Biome dicas, Universidade Estadual do Ceara , * Corresponding author: Laborato , Brazil. Tel./fax: þ55 85 3486 6221. Av. Paranjana 1700, Campus do Itaperí, CEP 60.740-000, Fortaleza, Ceara E-mail address: [email protected] (K.M. Carvalho). 1 These authors contributed equally to the paper. http://dx.doi.org/10.1016/j.toxicon.2014.07.011 0041-0101/© 2014 Published by Elsevier Ltd.
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more selective action on arterial blood system than BPP9a. Besides the inhibition of ACE, it may present other mechanisms of action yet to be elucidated. © 2014 Published by Elsevier Ltd.
1. Introduction The Bothrops jararaca venom contains enzymes that induce the release of kinins from plasma kininogen (Rocha e Silva et al., 1949). This venom was later found to contain a fraction (bradykinin-potentiating factor), which was able to potentiate the bradykinin actions on various isolated organs and hypotensive effect in the cat (Ferreira and Rocha e Silva, 1965). Thereafter, nine biologically active peptides (bradykinin-potentiating peptides, BPPs) were isolated from this venom and shown to potentiate bradykinininduced contraction in the isolated guinea pig ileum and to increase the hypotensive effect of bradykinin, whose actions are due to inhibition of bradykinin degradation (Ferreira et al., 1970). The inhibition of bradykinin degradation by these BPBs was also associated with the inhibition of the conversion of angiotensin I to its active metabolite angiotensin II (Stewart et al., 1971). These crucial findings paved the way for the later development of angiotensin converting enzyme (ACE) inhibitors, such as captopril the prototype compound of this group, for the treatment of hypertension and heart failure (Cushman and Ondetti, 1991; Camargo et al., 2012). Nevertheless, recent discoveries have pointed that some BPPs can distinguish between the N or C-terminal catalytic domains of ACE and these discoveries have renewed the interest in the search for more selective peptides (Perich et al., 1992; Jaspard et al., 1993; Cotton et al., 2002; Ianzer et al., 2007). Usually, the potentiation of bradykinin and the inhibition of ACE activity are related to the antihypertensive activity of these compounds, but exceptions to this rule suggest that these effects can occur independently, i.e., BPPs may act by an ACE-independent mechanism (Camargo and Ferreira, 1971; Greene et al., 1972; Mueller et al., 2005; Ianzer et al., 2007). For instance, Ianzer and coworkers (2004) demonstrated that a BPP isolated from B. jararaca venom inhibited both ECA and neutral endopeptidase (NEP) and strongly potentiated the hypotensive activity of bradykinin. Although BPPs have been initially isolated from genus Bothrops (Ferreira et al., 1970; Ondetti et al., 1971; Cintra et al., 1990; Ferreira et al., 1992; Murayama et al., 1997; Hayashi et al., 2003; Ianzer et al., 2004; BPPs deposited in NCBI bank (AAP 83422 and AAL 09426), several others were purified from other genera of snakes, such as Crotalus (Politi et al., 1985; Wermelinger et al., 2005; Higuchi et al., 2006; Gomes et al., 2007; Coutinho-Neto et al., 2013), Lachesis (Soares et al., 2005), Agkistrodon (Kato and Suzuki, 1971; Kato et al., 1973; Ferreira et al., 1995; Yanoshita et al., 1999; Murayama et al., 2000), Trimeresurus (Higuchi et al., 1999) and Vipera (Komori and Sugihara, 1990). In the present study, a new atypical BPP (BPP-Cdc) was isolated for the first time from the Crotalus durissus cascavella (C. d. cascavella) venom, its amino acid sequence
determined, pharmacodynamics performed on the guinea pig ileum and its effects on blood pressure of Goldblatt 2R1C hypertensive rats was examined. 2. Material and methods 2.1. The venom of C. d. cascavella The venom of C. d. cascavella was a gift from the Labo rio de Animais Peçonhentos do Instituto de Cie ^ncias rato dicas da Universidade Estadual do Ceara . Biome 2.2. Reagents All chemicals, salts and standards were purchased from SigmaeAldrich (St Louis, MO, USA), unless stated otherwise. 2.3. Purification of the BPP-Cdc A 20 mg/mL solution of crude venom of C. d. cascavella in 0.05% trifluoroacetic acid (TFA) (1:4; w/v) was prepared and centrifuged at 17,000g for 60 min. The supernatant was fractionated by reverse-phase high performance liquid chromatography (RP-HPLC-Shimadzu Co.) using a C18 column (Shim-pack PREP 25 250 mm). Peptides were eluted with a 0e60% gradient of acetonitrile containing 0.05% TFA, over a period of 60 min at a flow rate of 5 mL/min, detected by UV absorbance at 214 nm (Fig. 1A), lyophilized and stored at 25 C. Aliquots of these fractions were dissolved in saline and probed in the guinea pig ileum in order to check whether they could either potentiate BK- or antagonize angiotensin I-induced contractions. In order to confirm the purity of the fraction containing the BPP-Cdc activity, an aliquot was submitted for re-chromatography, under the same conditions described above (Fig. 1B). 2.4. Animals Albino male Guinea pigs (300e400 g) and Wistar rats gio (250e300 g) were obtained from the vivarium of Cole Christus. The animals were maintained under controlled conditions (temperature: 23 ± 1 C; relative humidity: 55 ± 1%; and light: 8:00 AM to 8:00 PM) and received a normal diet (Purina Chow®) and tap water ad libitum. All the protocols were approved by the Ceara State University Animal Ethics Committee under the protocol #084393384-0. 2.5. Mass spectrometry Electrospray ionization mass spectrometry (ESI-MS) was performed on a Perkin Elmer-Sciex API-300 triple quadrupole mass spectrometer operated in positive ion mode. The samples were injected using in 50% acetonitrile
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2.7. Pharmacological assays 2.7.1. Guinea pig ileum This model was used in order to compare the efficacy of BPP-Cdc, BPP-9a and captopril in potentiating BK-induced contractions and to antagonize angiotensin I-induced contractions as described by Rubin et al. (1978). We also tested whether BPP-Cdc could nonspecifically increase the contractions to acetylcholine, histamine or whether it could antagonize angiotensin II-induced contractions. Briefly, albino guinea pigs (250e300 g) were sacrificed by cervical displacement and the ileum was isolated and excised discarding 10 cm of the ileum next to the ileocecal valve. Strips (2 cm) were mounted vertically under 1 g resting tension in organ baths for isotonic recording of contractions. The tissues were attached to isotonic lever for frontal inscriptions in a smoked drum kymograph. The tissues were kept in Tyrode solution (pH 7.4) containing (in mM): NaCl 137; KCl 2.7; CaCl2 1.36; NaH2PO4 0.36; MgCl2 0.5; NaHCO3 11.9 and glucose 5.5 and gassed with room air by means of an aquarium pump. In the first set of protocols, a single submaximal BKinduced contraction, chosen from a previous curve (BK 15, 30 or 60 nM), was probed in the absence or presence of 1 mM of BBP-Cdc, 1 mM BBP-9a or 20 nM captopril in separate protocols (n ¼ 6/each). In other groups, submaximal contractions induced by a single concentration, chosen from a curve of angiotensin I (AgI) or II (AgII) (10e30 nM), acetylcholine (ACh) (0.3e1 mM) or histamine (Hist) (1e10 mM) were probed in the absence or presence of 1 mM BPP-Cdc. The contraction evoked by angiotensin I (AgI) was also probed in the absence or presence of 1 mM of BBP-Cdc, 1 mM BBP-9a or 200 nM captopril. In another set of experiments, a concentrationeresponse curve to BK (1, 3, 10, 30, 100 and 500 nM) was performed in the absence or presence of 1 mM BPP-Cdc.
Fig. 1. Reversed-phase HPLC purification and mass spectrometry of BPP-Cdc. (A) A 20 mg/mL solution of crude venom of C. d. cascavella in 0.05% trifluoroacetic acid (TFA) (1:4; w/v) was prepared and centrifuged at 17,000g for 60 min. The supernatant was injected into a Shim-pack CLC-ODS C18 column (25 250 mm) eluted with a 0e60% gradient of acetonitrile containing 0.05% TFA over a period of 60 min at a flow rate of 5 mL/min. Fractions containing BPP-Cdc were eluted at 17 min (17% of acetonitrile) (arrow and horizontal bar), lyophilized and stored at 25 C. (B) An aliquot of 10 mg of this fraction was submitted to re-chromatography under the same conditions. (C) A 10 mL aliquot of BPP-Cdc at 5 pmol/mL in 50% acetonitrile (v/v) in 5% acetic acid (v/v) was analyzed by electrospray mass spectrometry. The abbreviation ufs stands for unit full scale.
(v/v) in 5% acetic acid (v/v) at a flow rate of 20 mL/h. The concentration of the samples was typically 5 pmol/mL (Fig. 1C). 2.6. Amino acid sequencing For the amino acid sequence, the peptide was subjected to Edman degradation (1950) using a Shimadzu PPSQ-21 automated protein sequencer, following the manufacturer's standard instructions.
2.7.2. Blood pressure effects in 2K-1C Goldblatt hypertensive rats Male Wistar rats were anesthetized with xylazine (10 mg/ Kg; i.p) and ketamine (90 mg/kg; i.p). Thereafter, the left renal artery was partially obstructed with a 0.2 mm wide silver clip. The control animals were subjected to the same surgical procedure but without partial renal artery occlusion. All experiments were performed 4 weeks after the renal surgery. After 4 weeks the rats were anesthetized with xylazine (10 mg/Kg; i.p) and ketamine (90 mg/kg; i.p) and the carotid artery cannulated with a PE-50 cannula for direct recordings of blood pressure using a Statham pressure transducer (P23, Gould, Oxnard, CA, USA) registered in a 4channel Desk model physiograph (Narco Biosystems, Houston, TX, USA) and the right femoral vein was cannulated with a PE-10 cannula for drug injection. The animals were randomly assigned to one of three groups to study the effects of BPP-Cdc (50 mg bolus injection), BPP-9a (50 mg bolus injection) or captopril (50 mg bolus injection), respectively, in the responses to bolus intravenous injection of Ang I (10 ng) or BK (250 ng). The animals were first injected with BK or AngI twice, with 10 min interval between each injection, to test reproducibility. Thereafter, BPP-Cdc, BPP-9a or captopril were
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injected and after stabilization of the response, usually 2 min after injection, the injections of BK or AngI were repeated. Each agonist was analyzed four-times with 15 min interval between subsequent injections. 2.8. Statistical analysis The data were presented as mean ± SEM of at least 6 animals per group. Statistical significance was determined by analysis of variance (ANOVA) followed by the multiple comparison test of TukeyeKramer or Dunnett with p value set at 0.05. When applicable, paired or unpaired Student's t test was also used with significant values of p < 0.05. 3. Results 3.1. Purification and primary structure determination of BPP-Cdc BPP-Cdc purification was achieved by one RP-HPLC step and the peak corresponding to its activity was eluted at 17 min (17% of acetonitrile) (Fig. 1A, arrow and horizontal bar). Approximately 300 mg of purified peptide was obtained from 40 mg of the crude venom. To confirm the purity of BPP-Cdc, an aliquot of this fraction (10 mg) was submitted to re-chromatography, under the same conditions, yielding a single active peak (Fig.1B). The BPP-Cdc molecular mass was 1194.49 ± 1.0 Da as determined by electrospray ionization mass spectrometry (ESI-MS) (Fig. 1C). The BPP-Cdc was fully sequenced by the classical Edman degradation (1950) using a Shimadzu PPSQ-21 automated protein sequencer and its chemical structure was determined as: Pro-Asn-Leu-Pro-Asn-Tyr-Leu-Gly-Ile-Pro-Pro (PNLPNYLGIPP). 3.2. Pharmacological assays 3.2.1. Guinea pig ileum BPP-Cdc increased BK-induced contractions in the guinea-pig ileum but did not change acetylcholine,
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histamine or angiotensin II evoked contractions (Fig. 2A). The contractions elicited by BK were increased by 267.8% (p < 0.05) (Fig. 2A) by 1 mM BPP-Cdc, and this result was not significantly different from those obtained by 1 mM BPP-9a or by 200 nM captopril (data not shown). Furthermore, BPP-Cdc, BPP-9a and captopril, used at the same concentrations utilized in the BK-potentiating test, were able to inhibit 40e60% AngI-induced contractions (p < 0.05) with no statistically significant difference among the percent inhibitions produced by them (Fig. 2B). The cumulative concentrationeresponse curve to BK (1e500 nM) was displaced to the left and upwards after BPP-Cdc incubation (1 mM). The calculated pD2 value for BK in the absence of BPP-Cdc was 7.7 [7.8e7.5] and 7.9 [8.0e7.7] in the presence of this peptide. BPP-Cdc increased the maximal contractile response to the last cumulative concentration of BK in 112.9 ± 21.5% (Fig. 3) and similar results were obtained with BPP-9a or captopril (data not shown). 3.2.2. Blood pressure effects in 2K-1C Goldblatt hypertensive rats The peak hypotensive response to intravenous bolus injection of BK was increased by both BPP-Cdc and BPP-9a in 4-week hypertensive 2K-1C rats (Fig. 4A, B). The control hypotensive effect of BK was 13.7 ± 4.1 mmHg before the administration of BPP-Cdc (50 mg) and 37.9 ± 9.5 mmHg 2 min after its administration (176.6% increase) (t1 ¼ 15 min). This response kept increasing not only until the last experimental period (t4 ¼ 60 min) with e 46.4 ± 3.5 mmHg response to BK (238.7% increase from control) (Fig. 4A), but at least for more 60 min without significant alterations (data not shown). However, BPP9a (50 mg) only promoted an increased BK-induced hypotension from control at 15 min (t1) (71.4%) and 30 min (t2) (54.3%), but not at 45 min (t3) and 60 min (t4) (Fig. 4B). The vasopressor effect of AngI was inhibited by the intravenous injection of BPP-Cdc or BPP-9a administration in 2K-1C hypertensive rats (Fig. 5A, B). The control
Fig. 2. (A) Effect of BPP-Cdc (1 mM) in the phasic contractions elicited by bradykinin (BK), angiotensin II (Ang II), histamine or acetylcholine. The data are expressed as mean ± 1 SE (n ¼ 6), paired Student t test(p > 0.05). (B) Effects of vehicle (v), 1 mM BPP-Cdc, 1 mM BPP-9a or 200 nM captopril in the phasic contractions elicited by angiotensin I (Ang I). The data are expressed as mean ± 1 SE (n ¼ 6), p > 0.05 vs. Ang I þ V (vehicle: saline), ANOVA followed by TukeyeKramer.
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Fig. 3. Cumulative concentrationeresponse curve to bradykinin (BK; 1e500 nM) in the absence or presence of BPP-Cdc (1 mM; n ¼ 6). The data are expressed as mean ± 1 SE. *p < 0.05 vs. control, ANOVA followed by TukeyeKramer.
vasopressor effect of AngI was 33.0 ± 4.4 mmHg before the administration of BPP-Cdc (50 mg) and 10.0 ± 4.3 mmHg 2 min after its administration (69.7% inhibition) (t1 ¼ 15min). This response was inhibited until the last experimental period (t4 ¼ 60 min) with e 19.2 ± 2.29 mmHg response to BK (42% inhibition) (Fig. 5A). However, BPP-9a only promoted an inhibition on vasopressor effect of Ang I at 15 min (t1) (71.4% inhibition) and 30 min (t2) (54.3% inhibition), but not at 45 min (t3) or 60 min (t4) (Fig. 5B).
4. Discussion BPPs were first described in the B. jararaca venom as pyroglutamyl proline-rich oligopeptides containing 5e13 amino acid residues, and are recognized by an invariable proline residue at the C-terminus (Camargo et al., 1971). These peptides, which have been the prototype for development of modern anti-hypertensive drugs (Cushman and Ondetti, 1991), have been also identified in the last three decades in venom of several other species of snakes from different genera, such as Crotalus, Lachesis, Agkistrodon, Trimeresurus and Vipera (Fig. 6).
As of yet, BPP-Cdc is the only BPP isolated from C. d. cascavella. The molecular mass of 1194.49 Da as determined by mass spectrometry is in agreement with amino acid sequence calculated mass of 1195.39: Pro-Nle-Leu-Pro-NleTyr-Leu-Gly-Ile-Pro-Pro. Direct and full sequencing by Edman degradation was possible with BPP-Cdc, due to the absence of pyroglutamic acid (
Fig. 4. Effects of BPP-Cdc and BPP-9a on the hypotensive response to BK. (A) and (B): potentiating effects of BPP-Cdc and BPP-9a in the hypotensive response to BK, respectively; drugs were injected as a bolus in the femoral vein of 4-week hypertensive 2K-1C rats. BK ¼ bradykinin (250 ng); BBP-Cdc (50 mg), BBP- 9a (50 mg). BK was injected four-times with a 15 min interval between subsequent injections. t ¼ time in minutes after administration of BPP's. The data are expressed as mean ± 1 SE (n ¼ 6), ANOVA followed by Dunnett, *p < 0.05 vs. control (BK).
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Fig. 5. Effects of BPP-Cdc and BPP-9a on the vasopressor effect to Ang I. (A) and (B): inhibition of the vasopressor effect of Ang I by BPP-Cdc and BPP-9a, respectively; drugs were injected as a bolus in the femoral vein of 4-week hypertensive 2K-1C rats. Ang I ¼ 10 ng; BBP-Cdc ¼ 50 mg, BBP- 9a ¼ 50 mg. Ang I was injected four-times with a 15 min interval between subsequent injections. t ¼ time in minutes after administration of BPP's. The data are expressed as mean ± 1 SE (n ¼ 6), ANOVA followed by Dunnett, *p < 0.05 vs. control (Ang I).
sensitization. The rapid increased contractile responses to BK in the guinea pig ileum after 2 min incubation period to BPP-Cdc, as occurred with BPP-9a or captopril, may be more related to sensitization of the bradykinin B2 receptor than to ACE inhibition, as shown previously in the guinea pig ileum and atria (Minshall et al., 1997; Rabito et al., 1998; Minshall et al., 2000). By using CHO cells this group has also shown that agents that react with either one of the active centers of ACE can enhance the action of agonists on the B2 receptor (Marcic et al., 1999). Furthermore, other results disprove the ACE inhibition as the only single mechanism and also the direct interaction of potentiating peptides with the bradykinin receptors in transfected COS-7 cells as molecular mechanism of potentiation, showing a stimulation of inositol phosphates (IPn) formation independently from the B2 receptor (Mueller et al., 2005). Thus, taken together, these results give evidence that the potentiation of the bradykinin action can occur by different mechanisms, depending on the system and on the applied potentiating factor. On the other hand, BPP-Cdc, like BPP-9a and captopril, after 30-min incubation decreased the amplitude of angiotensin I-induced contractions in the guinea pig ileum with the same magnitude (Fig. 3B), suggesting an inhibition of the catalytic activity of ACE and consequently a decreased production of Ang II. To reinforce this hypothesis, we have also shown that in vivo BPP-Cdc also decreased the acute pressor effect of Ang I in 4-week hypertensive 2K-1C rats. However, although the effect of BPP-Cdc on acute pressor effect of Ang I, at the same doses, was similar to that BPP-9a, the duration of its action (60 min) was at least two times higher than that of BPP-9a (30 min). Despite the high degree of sequence identity between the N- and C-domains of human somatic ACE, they differ in substrate/inhibitor specificity (Cotton et al., 2002; Hayashi et al., 2003; Hayashi and Camargo, 2005). The N-domain is specific for the degradation of the AcSDKP tetrapeptide that controls hematopoietic stem cell proliferation and differentiation (Rousseau et al., 1995), whereas the C-domain is primarily involved in blood pressure regulation through the degradation of Ang I
(Jaspard et al., 1993) and the inactivation of vasodilator peptide bradykinin (Villard and Soubrier, 1996). Interesting, while it was shown that the decapeptide BPP-10c (
Fig. 6. Comparison of the BPP-Cdc amino acid sequence with those of other BPPs from snake venoms. Identical residues are boxed. aGomes et al., 2007; bHiguchi et al., 2006; cPoliti et al., 1985; dCoutinho-Neto et al., 2013; eWermelinger et al., 2005; fFerreira et al., 1970; gMurayama et al., 1997; hHayashi et al., 2003; iIanzer et al., 2004; jOndetti et al., 1971; kFerreira et al., 1992; lBPPs deposited in NCBI bank (AAP 83422 and AAL 09426); mCintra et al., 1990; nSoares et al., 2005; oKato et al., 1973; pKato and Suzuki, 1971; qMurayama et al., 2000; rYanoshita et al., 1999; sFerreira et al., 1995; tHiguchi et al., 1999; uKomori and Sugihara, 1990.
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production, a natural precursor molecule for NO synthesis, and consequently reducing the arterial blood pressure (Guerreiro et al., 2009; Camargo et al., 2012). Therefore, further studies will be necessary to investigate whether BPP-Cdc may be involved in arginine metabolism and thus to activate endothelial nitric oxide. Finally, as occurs in the guinea ileum pig, another possible mechanism of action of BPP-Cdc on vascular system is the augmented BK effects on B2 receptors. ACE inhibitors can augment BK effects on B2 receptor indirectly only when enzyme and receptor molecules are sterically close, possibly forming a heterodimer (Marcic et al., 1999). Thus, ACE inhibitors and other agents with affinity for the active center likely alter the heterodimer interaction to promote a conformation of the B2 receptor that can more efficiently induce signal transduction (Marcic et al., 2000). In conclusion, the pharmacological effects of BPP-Cdc in vitro decreasing the amplitude of angiotensin I-induced contractions in the guinea pig ileum and in vivo reducing the acute pressor effect of Ang I in 4 week hypertensive 2K-1C rats, were similar to those BPP-9a, suggesting that both peptides act inhibiting the ACE activity. Furthermore, the long duration of action of BPP-Cdc in these experiments may also suggest that it has more affinity for ACE than BPP-9a. On the other hand, the bradykinin-potentiation effects in vivo of BPPCdc on the hypotensive effect to BK in 4 week hypertensive 2K-1C rats were higher than those of BPP-9a in amplitude (60%) and duration (100%) and these results may also suggest the involvement of other ACE-independent mechanisms to be elucidated. Furthermore, further studies will be required to understand the relationship between the atypical chemical structure of BPP-Cdc and its pharmacological activity, as well as to fully elucidate its mechanism of action. Acknowledgments This work was supported by FUNCAP, CNPq, CAPES and GENPHARMA. We thank Rosa Germana da Silva for the rio for the writing technical assistance and Francisco Robe assistance of the paper. Conflict of interest The authors declare no conflict of interest regarding the content of this manuscript. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.toxicon.2014.07. 011. References Camargo, A., Ferreira, S.H., 1970. Action of a bradykinin potentiating factor (BPF) and dimercaprol (BAL) on the responses to bradykinin of isolated preparations of rat intestines. Br. J. Pharmacol. 42, 305e307. Camargo, A., Ferreira, S.H., 1971. Action of bradykinin potentiating factor (BPF) and dimercaprol (BAL) on the responses to bradykinin of isolated preparations of rat intestines. Br. J. Pharmacol. 42 (2), 305e307. Camargo, A.C.M., Ianzer, D., Guerreiro, J.R., Serrano, S.M.T., 2012. Bradykinin-potentiating peptides: beyond captopril. Toxicon 59, 516e523.
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cardiovascular effects of angiotensin-converting enzyme (ACE) I involve ACE-independent mechanisms? new insights from prolinerich peptides of Bothrops jararaca. J. Pharmacol. Exp. Ther. 322 (2), 795e805. Jaspard, E., Wei, L., Alhenc-Gelas, F., 1993. Differences in the properties and enzymatic specificities of the two active sites of angiotensin Iconverting enzyme (kininase II): studies with bradykinin and other natural peptides. J. Biol. Chem. 268, 9496e9503. Johnston, C.I., Fabris, B., Yamada, H., Mendelsohn, F.A., Cubela, R., Sivell, D., Jackson, B., 1989. Comparative studies of tissue inhibition by angiotensin converting enzyme inhibitors. J. Hypertens. Suppl. 7 (5), 11e16. Review. Kato, H., Suzuki, T., 1971. Bradykinin-potentiating peptides from the venom of Agkistrodon halys blomhoffi. Isolation of five bradykinin potentiators and the amino acid sequences of two of them potentiators B and C. Biochemistry 10 (6), 972e980. Kato, H., Suzuki, T., Okada, K., Kimura, T., Sakakibara, S., 1973. Structure of potentiator A, one of the five bradykinin-potentiating peptides from the venom of Agkistrodon halys blomhoffii. Experientia 29, 574e575. Komori, Y., Sugihara, H., 1990. Characterization of a new inhibitor for angiotensin converting enzyme from the venom of Vipera aspis aspis. Int. J. Biochem 22, 767e771. €s, E.G., 1999. Enhancement of Marcic, B., Deddish, P.A., Jackman, H.L., Erdo bradykinin and resensitization of its B2 receptor. Hypertension 33 (3), 835e843. € s, E.G., Minshall, R.D., Tan, F., Marcic, B., Deddish, P.A., Skidgel, R.A., Erdo 2000. Replacement of the transmembrane anchor in angiotensin Iconverting enzyme (ACE) with a glycosylphosphatidylinositol tail affects activation of the B2 bradykinin receptor by ACE inhibitors. J. Biol. Chem. 275, 16110e16118. Minshall, R.D., Tan, F., Nakamura, F., Rabito, S.F., Becker, R.P., Marcic, B., € s, E.G., 1997. Potentiation of the actions of bradykinin by angioErdo tensin I-converting enzyme inhibitors. The role of expressed human bradykinin B2 receptors and angiotensin I-converting enzyme in CHO cells. Circ. Res. 81 (5), 848e856. €s, E.G., Rabito, S.F., 2000. Minshall, R.D., Nedumgottil, S.J., Igi c, R., Erdo Potentiation of the effects of bradykinin on its receptor in the isolated guinea pig ileum. Peptides 21 (8), 1257e1264. Mueller, S., Gothe, R., Siems, W.D., Vietinghoff, G., Paegelow, I., Reissmann, S., 2005. Potentiation of bradykinin actions by analogues of the bradykinin potentiating nonapeptide BPP9a. Peptides 26 (7), 1235e1247. Murayama, N., Hayashi, M.A., Ohi, H., Ferreira, L.A., Fernandes, B.L., Yamane, T., Camargo, A.C.M., 1997. Cloning and sequence analysis of a Bothrops jararaca cDNA encoding a precursor of seven bradykininpotentiating peptides and a Ctype natriuretic peptide. Proc. Natl. Acad. Sci. U. S. A. 94 (4), 1189e1193. Murayama, N., Michel, G.H., Yanoshita, R., Samejima, Y., Saguchi, K., Ohi, H., Fujita, Y., Higuchi, S., 2000. cDNA cloning of bradykinin-
potentiating peptides-C-type natriuretic peptide precursor, and characterization of the novel peptide Leu3-blomhotin from the venom of Agkistrodon blomhoffi. Eur. J. Biochem 267, 4075e4080. Ondetti, M.A., Williams, N.J., Sabo, E.F., Pluscec, J., Weaver, E.R., Kocy, O., 1971. Angiotensin-converting enzyme inhibitors from the venom of Bothrops jararaca. Isolation, elucidation of structure, and synthesis. Biochemistry 1971 (10), 4033e4039. Perich, R.B., Jackson, B., Johnston, C.I., 1992. Variation in angiotensinconverting enzyme (ACE) inhibitor affinity at two binding sites on rat pulmonary ACE: influence on bradykyinin hydrolysis. Clin. Exp. Pharmacol. Physiol. 19, 353e357. Politi, V., De Luca, G., Di Stazio, G., Schinina, E., Bossa, F., 1985. A new peptide from Crotalus atrox snake venom. Pept. Suppl. 3, 343e346. €s, E.G., Rabito, S.F., Minshall, R.D., Swaminathan, B., Tan, M.N., Igíc, R., Erdo 1998. Mode of potentiation of the effects of bradykinin by angiotensin I converting enzyme inhibitors on the guinea pig ileum. Anesthesiology 89 (3A), 635 (abstr.). Rocha e Silva, M., Beraldo, W.T., Rosenfeld, G., 1949. Bradykinin, a hypotensive and smooth muscle stimulating factor released from plasma globulin by snake venoms and by trypsin. Am. J. Physiol. 156 (2), 261e273. Rousseau, A., Michaud, A., Chauvet, M.-T., Lenfant, M., Corvol, P., 1995. The hemoregulatory peptide N-acetyl-Ser-Asp-Lys-Pro is a natural and specific substrate of the N-terminal active site of human angiotensinconverting enzyme. J. Biol. Chem. 270, 3656e3661. Rubin, B., Antonaccio, M.J., Horovitz, Z.P., 1978. Captopril (SQ 14,225) (D3-mercapto-2-methylpropranoyl-L-proline): a novel orally active inhibitor of angiotensin-converting enzyme and antihypertensive agent. Prog. Cardiovasc. Dis. 21 (3), 183e194. Review. Soares, M.R., Oliveira-Carvalho, A.L., Wermelinger, L.S., Zingali, R.B., Ho, P.L., et al., 2005. Identification of novel bradykininpotentiating peptides and C-type natriuretic peptide from Lachesis muta venom. Toxicon 46, 31e38. Stewart, J.M., Ferreira, S.H., Greene, L.J., 1971. Bradykinin-potentiating peptide PCA-Lys-Trp-Ala-Pro. An inhibitor of the pulmonary inactivation of bradykinin and conversion of angiotensin I to II. Biochem. Pharmacol. 20, 1557e1567. Villard, E., Soubrier, F., 1996. Molecular biology and genetics of the angiotensin-I-converting enzyme: potential implications in cardiovascular diseases. Cardiovasc. Res. 32 (6), 999e1007. Wermelinger, L.S., Dutra, D.L.S., Oliveira-Carvalho, A.L., Soares, M.R., Bloch Jr., C., Zingali, R.B., 2005. Fast analysis of low molecular mass compounds present in snake venom: identification of ten new pyroglutamate-containing peptides. Rapid Commun. Mass Spectrom. 19, 1703e1708. Yanoshita, R., Kasuga, A., Inoue, S., Ikeda, K., Samejima, Y., 1999. Blomhotin: a novel peptide with smooth muscle contractile activity identified in the venom of Agkistrodon halys blomhoffii. Toxicon 37, 1761e1770.
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Toxicon journal homepage: www.elsevier.com/locate/toxicon
A new structurally atypical bradykinin-potentiating peptide isolated from Crotalus durissus cascavella venom (South American rattlesnake) Denise M. Lopes a, Norberto E.G. Junior a, Paula P.C. Costa a, Patrícia L. Martins a, udia F. Santos a, Ellaine D.F. Carvalho b, c, Maria D.F. Carvalho b, c, d, Cla s C. Fonteles a, Daniel C. Pimenta e, Bruno A. Cardi d, Manasse a, 1 Nilberto R.F. Nascimento , Krishnamurti M. Carvalho b, d, *, 1 , rio de Farmacologia Cardiovascular e Renal, Instituto Superior de Ci^ Laborato encias Biom edicas, Universidade Estadual do Ceara , Brazil Fortaleza, Ceara b , Brazil GENPHARMA LTDA, Fortaleza, Ceara c , Brazil Faculdade de Medicina Christus, Fortaleza, Ceara d , rio de Toxinologia e Farmacologia Molecular, Instituto Superior de Ci^ Laborato encias Biom edicas, Universidade Estadual do Ceara , Brazil Fortaleza, Ceara e ~o Paulo, Brazil Instituto Butantan, Sa a
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 April 2014 Received in revised form 7 July 2014 Accepted 23 July 2014 Available online 1 August 2014
Venom glands of some snakes synthesize bradykinin-potentiating peptides (BPP's) which increase bradykinin-induced hypotensive effect and decrease angiotensin I vasopressor effect by angiotensin-converting enzyme (ACE) inhibition. The present study shows a new BPP (BPP-Cdc) isolated from Crotalus durissus cascavella venom: Pro-Asn-Leu-Pro-Asn-TyrLeu-Gly-Ile-Pro-Pro. Although BPP-Cdc presents the classical sequence IPP in the C-terminus, it has a completely atypical N-terminal sequence, which shows very low homology with all other BPPs isolated to date. The pharmacological effects of BPP-Cdc were compared to BBP9a from Bothrops jararaca and captopril. BPP-Cdc (1 mM) significantly increased BK-induced contractions (BK; 1 mM) on the guinea pig ileum by 267.8% and decreased angiotensin I-induced contractions (AngI; 10 nM) by 62.4% and these effects were not significantly different from those of BPP9a (1 mM) or captopril (200 nM). Experiments with 4-week hypertensive 2K-1C rats show that the vasopressor effect of AngI (10 ng) was decreased by 50 mg BPP-Cdc (69.7%), and this result was similar to that obtained with 50 mg BPP9a (69.8%). However, the action duration of BPP-Cdc (60 min) was 2 times greater than that of BPP-9a (30 min). On the other hand, the hypotensive effect of BK (250 ng) was significantly increased by 176.6% after BPP-Cdc (50 mg) administration, value 2.5 times greater than that obtained with BPP9a administered at the same doses (71.4%). In addition, the duration of the action of BPP-Cdc (120 min) was also at least 4 times greater than that of BPP-9a (30 min). Taken together, these results suggest that BPP-Cdc presents
Keywords: Bradykinin-potentiating peptide Angiotensin-converting enzyme Hypertension Crotalus durissus cascavella venom
Abbreviations: ACE, angiotensin-converting enzyme; AngI, angiotensin I; AngII, angiotensin II; BK, bradykinin; BPPs, bradykinin-potentiating peptides; BPP-Cdc, bradykinin-potentiating peptides from Crotalus durissus cascavella. rio de Toxinologia e Farmacologia Molecular, Instituto Superior de Cie ^ncias Biome dicas, Universidade Estadual do Ceara , * Corresponding author: Laborato , Brazil. Tel./fax: þ55 85 3486 6221. Av. Paranjana 1700, Campus do Itaperí, CEP 60.740-000, Fortaleza, Ceara E-mail address: [email protected] (K.M. Carvalho). 1 These authors contributed equally to the paper. http://dx.doi.org/10.1016/j.toxicon.2014.07.011 0041-0101/© 2014 Published by Elsevier Ltd.
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more selective action on arterial blood system than BPP9a. Besides the inhibition of ACE, it may present other mechanisms of action yet to be elucidated. © 2014 Published by Elsevier Ltd.
1. Introduction The Bothrops jararaca venom contains enzymes that induce the release of kinins from plasma kininogen (Rocha e Silva et al., 1949). This venom was later found to contain a fraction (bradykinin-potentiating factor), which was able to potentiate the bradykinin actions on various isolated organs and hypotensive effect in the cat (Ferreira and Rocha e Silva, 1965). Thereafter, nine biologically active peptides (bradykinin-potentiating peptides, BPPs) were isolated from this venom and shown to potentiate bradykinininduced contraction in the isolated guinea pig ileum and to increase the hypotensive effect of bradykinin, whose actions are due to inhibition of bradykinin degradation (Ferreira et al., 1970). The inhibition of bradykinin degradation by these BPBs was also associated with the inhibition of the conversion of angiotensin I to its active metabolite angiotensin II (Stewart et al., 1971). These crucial findings paved the way for the later development of angiotensin converting enzyme (ACE) inhibitors, such as captopril the prototype compound of this group, for the treatment of hypertension and heart failure (Cushman and Ondetti, 1991; Camargo et al., 2012). Nevertheless, recent discoveries have pointed that some BPPs can distinguish between the N or C-terminal catalytic domains of ACE and these discoveries have renewed the interest in the search for more selective peptides (Perich et al., 1992; Jaspard et al., 1993; Cotton et al., 2002; Ianzer et al., 2007). Usually, the potentiation of bradykinin and the inhibition of ACE activity are related to the antihypertensive activity of these compounds, but exceptions to this rule suggest that these effects can occur independently, i.e., BPPs may act by an ACE-independent mechanism (Camargo and Ferreira, 1971; Greene et al., 1972; Mueller et al., 2005; Ianzer et al., 2007). For instance, Ianzer and coworkers (2004) demonstrated that a BPP isolated from B. jararaca venom inhibited both ECA and neutral endopeptidase (NEP) and strongly potentiated the hypotensive activity of bradykinin. Although BPPs have been initially isolated from genus Bothrops (Ferreira et al., 1970; Ondetti et al., 1971; Cintra et al., 1990; Ferreira et al., 1992; Murayama et al., 1997; Hayashi et al., 2003; Ianzer et al., 2004; BPPs deposited in NCBI bank (AAP 83422 and AAL 09426), several others were purified from other genera of snakes, such as Crotalus (Politi et al., 1985; Wermelinger et al., 2005; Higuchi et al., 2006; Gomes et al., 2007; Coutinho-Neto et al., 2013), Lachesis (Soares et al., 2005), Agkistrodon (Kato and Suzuki, 1971; Kato et al., 1973; Ferreira et al., 1995; Yanoshita et al., 1999; Murayama et al., 2000), Trimeresurus (Higuchi et al., 1999) and Vipera (Komori and Sugihara, 1990). In the present study, a new atypical BPP (BPP-Cdc) was isolated for the first time from the Crotalus durissus cascavella (C. d. cascavella) venom, its amino acid sequence
determined, pharmacodynamics performed on the guinea pig ileum and its effects on blood pressure of Goldblatt 2R1C hypertensive rats was examined. 2. Material and methods 2.1. The venom of C. d. cascavella The venom of C. d. cascavella was a gift from the Labo rio de Animais Peçonhentos do Instituto de Cie ^ncias rato dicas da Universidade Estadual do Ceara . Biome 2.2. Reagents All chemicals, salts and standards were purchased from SigmaeAldrich (St Louis, MO, USA), unless stated otherwise. 2.3. Purification of the BPP-Cdc A 20 mg/mL solution of crude venom of C. d. cascavella in 0.05% trifluoroacetic acid (TFA) (1:4; w/v) was prepared and centrifuged at 17,000g for 60 min. The supernatant was fractionated by reverse-phase high performance liquid chromatography (RP-HPLC-Shimadzu Co.) using a C18 column (Shim-pack PREP 25 250 mm). Peptides were eluted with a 0e60% gradient of acetonitrile containing 0.05% TFA, over a period of 60 min at a flow rate of 5 mL/min, detected by UV absorbance at 214 nm (Fig. 1A), lyophilized and stored at 25 C. Aliquots of these fractions were dissolved in saline and probed in the guinea pig ileum in order to check whether they could either potentiate BK- or antagonize angiotensin I-induced contractions. In order to confirm the purity of the fraction containing the BPP-Cdc activity, an aliquot was submitted for re-chromatography, under the same conditions described above (Fig. 1B). 2.4. Animals Albino male Guinea pigs (300e400 g) and Wistar rats gio (250e300 g) were obtained from the vivarium of Cole Christus. The animals were maintained under controlled conditions (temperature: 23 ± 1 C; relative humidity: 55 ± 1%; and light: 8:00 AM to 8:00 PM) and received a normal diet (Purina Chow®) and tap water ad libitum. All the protocols were approved by the Ceara State University Animal Ethics Committee under the protocol #084393384-0. 2.5. Mass spectrometry Electrospray ionization mass spectrometry (ESI-MS) was performed on a Perkin Elmer-Sciex API-300 triple quadrupole mass spectrometer operated in positive ion mode. The samples were injected using in 50% acetonitrile
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2.7. Pharmacological assays 2.7.1. Guinea pig ileum This model was used in order to compare the efficacy of BPP-Cdc, BPP-9a and captopril in potentiating BK-induced contractions and to antagonize angiotensin I-induced contractions as described by Rubin et al. (1978). We also tested whether BPP-Cdc could nonspecifically increase the contractions to acetylcholine, histamine or whether it could antagonize angiotensin II-induced contractions. Briefly, albino guinea pigs (250e300 g) were sacrificed by cervical displacement and the ileum was isolated and excised discarding 10 cm of the ileum next to the ileocecal valve. Strips (2 cm) were mounted vertically under 1 g resting tension in organ baths for isotonic recording of contractions. The tissues were attached to isotonic lever for frontal inscriptions in a smoked drum kymograph. The tissues were kept in Tyrode solution (pH 7.4) containing (in mM): NaCl 137; KCl 2.7; CaCl2 1.36; NaH2PO4 0.36; MgCl2 0.5; NaHCO3 11.9 and glucose 5.5 and gassed with room air by means of an aquarium pump. In the first set of protocols, a single submaximal BKinduced contraction, chosen from a previous curve (BK 15, 30 or 60 nM), was probed in the absence or presence of 1 mM of BBP-Cdc, 1 mM BBP-9a or 20 nM captopril in separate protocols (n ¼ 6/each). In other groups, submaximal contractions induced by a single concentration, chosen from a curve of angiotensin I (AgI) or II (AgII) (10e30 nM), acetylcholine (ACh) (0.3e1 mM) or histamine (Hist) (1e10 mM) were probed in the absence or presence of 1 mM BPP-Cdc. The contraction evoked by angiotensin I (AgI) was also probed in the absence or presence of 1 mM of BBP-Cdc, 1 mM BBP-9a or 200 nM captopril. In another set of experiments, a concentrationeresponse curve to BK (1, 3, 10, 30, 100 and 500 nM) was performed in the absence or presence of 1 mM BPP-Cdc.
Fig. 1. Reversed-phase HPLC purification and mass spectrometry of BPP-Cdc. (A) A 20 mg/mL solution of crude venom of C. d. cascavella in 0.05% trifluoroacetic acid (TFA) (1:4; w/v) was prepared and centrifuged at 17,000g for 60 min. The supernatant was injected into a Shim-pack CLC-ODS C18 column (25 250 mm) eluted with a 0e60% gradient of acetonitrile containing 0.05% TFA over a period of 60 min at a flow rate of 5 mL/min. Fractions containing BPP-Cdc were eluted at 17 min (17% of acetonitrile) (arrow and horizontal bar), lyophilized and stored at 25 C. (B) An aliquot of 10 mg of this fraction was submitted to re-chromatography under the same conditions. (C) A 10 mL aliquot of BPP-Cdc at 5 pmol/mL in 50% acetonitrile (v/v) in 5% acetic acid (v/v) was analyzed by electrospray mass spectrometry. The abbreviation ufs stands for unit full scale.
(v/v) in 5% acetic acid (v/v) at a flow rate of 20 mL/h. The concentration of the samples was typically 5 pmol/mL (Fig. 1C). 2.6. Amino acid sequencing For the amino acid sequence, the peptide was subjected to Edman degradation (1950) using a Shimadzu PPSQ-21 automated protein sequencer, following the manufacturer's standard instructions.
2.7.2. Blood pressure effects in 2K-1C Goldblatt hypertensive rats Male Wistar rats were anesthetized with xylazine (10 mg/ Kg; i.p) and ketamine (90 mg/kg; i.p). Thereafter, the left renal artery was partially obstructed with a 0.2 mm wide silver clip. The control animals were subjected to the same surgical procedure but without partial renal artery occlusion. All experiments were performed 4 weeks after the renal surgery. After 4 weeks the rats were anesthetized with xylazine (10 mg/Kg; i.p) and ketamine (90 mg/kg; i.p) and the carotid artery cannulated with a PE-50 cannula for direct recordings of blood pressure using a Statham pressure transducer (P23, Gould, Oxnard, CA, USA) registered in a 4channel Desk model physiograph (Narco Biosystems, Houston, TX, USA) and the right femoral vein was cannulated with a PE-10 cannula for drug injection. The animals were randomly assigned to one of three groups to study the effects of BPP-Cdc (50 mg bolus injection), BPP-9a (50 mg bolus injection) or captopril (50 mg bolus injection), respectively, in the responses to bolus intravenous injection of Ang I (10 ng) or BK (250 ng). The animals were first injected with BK or AngI twice, with 10 min interval between each injection, to test reproducibility. Thereafter, BPP-Cdc, BPP-9a or captopril were
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injected and after stabilization of the response, usually 2 min after injection, the injections of BK or AngI were repeated. Each agonist was analyzed four-times with 15 min interval between subsequent injections. 2.8. Statistical analysis The data were presented as mean ± SEM of at least 6 animals per group. Statistical significance was determined by analysis of variance (ANOVA) followed by the multiple comparison test of TukeyeKramer or Dunnett with p value set at 0.05. When applicable, paired or unpaired Student's t test was also used with significant values of p < 0.05. 3. Results 3.1. Purification and primary structure determination of BPP-Cdc BPP-Cdc purification was achieved by one RP-HPLC step and the peak corresponding to its activity was eluted at 17 min (17% of acetonitrile) (Fig. 1A, arrow and horizontal bar). Approximately 300 mg of purified peptide was obtained from 40 mg of the crude venom. To confirm the purity of BPP-Cdc, an aliquot of this fraction (10 mg) was submitted to re-chromatography, under the same conditions, yielding a single active peak (Fig.1B). The BPP-Cdc molecular mass was 1194.49 ± 1.0 Da as determined by electrospray ionization mass spectrometry (ESI-MS) (Fig. 1C). The BPP-Cdc was fully sequenced by the classical Edman degradation (1950) using a Shimadzu PPSQ-21 automated protein sequencer and its chemical structure was determined as: Pro-Asn-Leu-Pro-Asn-Tyr-Leu-Gly-Ile-Pro-Pro (PNLPNYLGIPP). 3.2. Pharmacological assays 3.2.1. Guinea pig ileum BPP-Cdc increased BK-induced contractions in the guinea-pig ileum but did not change acetylcholine,
39
histamine or angiotensin II evoked contractions (Fig. 2A). The contractions elicited by BK were increased by 267.8% (p < 0.05) (Fig. 2A) by 1 mM BPP-Cdc, and this result was not significantly different from those obtained by 1 mM BPP-9a or by 200 nM captopril (data not shown). Furthermore, BPP-Cdc, BPP-9a and captopril, used at the same concentrations utilized in the BK-potentiating test, were able to inhibit 40e60% AngI-induced contractions (p < 0.05) with no statistically significant difference among the percent inhibitions produced by them (Fig. 2B). The cumulative concentrationeresponse curve to BK (1e500 nM) was displaced to the left and upwards after BPP-Cdc incubation (1 mM). The calculated pD2 value for BK in the absence of BPP-Cdc was 7.7 [7.8e7.5] and 7.9 [8.0e7.7] in the presence of this peptide. BPP-Cdc increased the maximal contractile response to the last cumulative concentration of BK in 112.9 ± 21.5% (Fig. 3) and similar results were obtained with BPP-9a or captopril (data not shown). 3.2.2. Blood pressure effects in 2K-1C Goldblatt hypertensive rats The peak hypotensive response to intravenous bolus injection of BK was increased by both BPP-Cdc and BPP-9a in 4-week hypertensive 2K-1C rats (Fig. 4A, B). The control hypotensive effect of BK was 13.7 ± 4.1 mmHg before the administration of BPP-Cdc (50 mg) and 37.9 ± 9.5 mmHg 2 min after its administration (176.6% increase) (t1 ¼ 15 min). This response kept increasing not only until the last experimental period (t4 ¼ 60 min) with e 46.4 ± 3.5 mmHg response to BK (238.7% increase from control) (Fig. 4A), but at least for more 60 min without significant alterations (data not shown). However, BPP9a (50 mg) only promoted an increased BK-induced hypotension from control at 15 min (t1) (71.4%) and 30 min (t2) (54.3%), but not at 45 min (t3) and 60 min (t4) (Fig. 4B). The vasopressor effect of AngI was inhibited by the intravenous injection of BPP-Cdc or BPP-9a administration in 2K-1C hypertensive rats (Fig. 5A, B). The control
Fig. 2. (A) Effect of BPP-Cdc (1 mM) in the phasic contractions elicited by bradykinin (BK), angiotensin II (Ang II), histamine or acetylcholine. The data are expressed as mean ± 1 SE (n ¼ 6), paired Student t test(p > 0.05). (B) Effects of vehicle (v), 1 mM BPP-Cdc, 1 mM BPP-9a or 200 nM captopril in the phasic contractions elicited by angiotensin I (Ang I). The data are expressed as mean ± 1 SE (n ¼ 6), p > 0.05 vs. Ang I þ V (vehicle: saline), ANOVA followed by TukeyeKramer.
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D.M. Lopes et al. / Toxicon 90 (2014) 36e44
Fig. 3. Cumulative concentrationeresponse curve to bradykinin (BK; 1e500 nM) in the absence or presence of BPP-Cdc (1 mM; n ¼ 6). The data are expressed as mean ± 1 SE. *p < 0.05 vs. control, ANOVA followed by TukeyeKramer.
vasopressor effect of AngI was 33.0 ± 4.4 mmHg before the administration of BPP-Cdc (50 mg) and 10.0 ± 4.3 mmHg 2 min after its administration (69.7% inhibition) (t1 ¼ 15min). This response was inhibited until the last experimental period (t4 ¼ 60 min) with e 19.2 ± 2.29 mmHg response to BK (42% inhibition) (Fig. 5A). However, BPP-9a only promoted an inhibition on vasopressor effect of Ang I at 15 min (t1) (71.4% inhibition) and 30 min (t2) (54.3% inhibition), but not at 45 min (t3) or 60 min (t4) (Fig. 5B).
4. Discussion BPPs were first described in the B. jararaca venom as pyroglutamyl proline-rich oligopeptides containing 5e13 amino acid residues, and are recognized by an invariable proline residue at the C-terminus (Camargo et al., 1971). These peptides, which have been the prototype for development of modern anti-hypertensive drugs (Cushman and Ondetti, 1991), have been also identified in the last three decades in venom of several other species of snakes from different genera, such as Crotalus, Lachesis, Agkistrodon, Trimeresurus and Vipera (Fig. 6).
As of yet, BPP-Cdc is the only BPP isolated from C. d. cascavella. The molecular mass of 1194.49 Da as determined by mass spectrometry is in agreement with amino acid sequence calculated mass of 1195.39: Pro-Nle-Leu-Pro-NleTyr-Leu-Gly-Ile-Pro-Pro. Direct and full sequencing by Edman degradation was possible with BPP-Cdc, due to the absence of pyroglutamic acid (
Fig. 4. Effects of BPP-Cdc and BPP-9a on the hypotensive response to BK. (A) and (B): potentiating effects of BPP-Cdc and BPP-9a in the hypotensive response to BK, respectively; drugs were injected as a bolus in the femoral vein of 4-week hypertensive 2K-1C rats. BK ¼ bradykinin (250 ng); BBP-Cdc (50 mg), BBP- 9a (50 mg). BK was injected four-times with a 15 min interval between subsequent injections. t ¼ time in minutes after administration of BPP's. The data are expressed as mean ± 1 SE (n ¼ 6), ANOVA followed by Dunnett, *p < 0.05 vs. control (BK).
D.M. Lopes et al. / Toxicon 90 (2014) 36e44
41
Fig. 5. Effects of BPP-Cdc and BPP-9a on the vasopressor effect to Ang I. (A) and (B): inhibition of the vasopressor effect of Ang I by BPP-Cdc and BPP-9a, respectively; drugs were injected as a bolus in the femoral vein of 4-week hypertensive 2K-1C rats. Ang I ¼ 10 ng; BBP-Cdc ¼ 50 mg, BBP- 9a ¼ 50 mg. Ang I was injected four-times with a 15 min interval between subsequent injections. t ¼ time in minutes after administration of BPP's. The data are expressed as mean ± 1 SE (n ¼ 6), ANOVA followed by Dunnett, *p < 0.05 vs. control (Ang I).
sensitization. The rapid increased contractile responses to BK in the guinea pig ileum after 2 min incubation period to BPP-Cdc, as occurred with BPP-9a or captopril, may be more related to sensitization of the bradykinin B2 receptor than to ACE inhibition, as shown previously in the guinea pig ileum and atria (Minshall et al., 1997; Rabito et al., 1998; Minshall et al., 2000). By using CHO cells this group has also shown that agents that react with either one of the active centers of ACE can enhance the action of agonists on the B2 receptor (Marcic et al., 1999). Furthermore, other results disprove the ACE inhibition as the only single mechanism and also the direct interaction of potentiating peptides with the bradykinin receptors in transfected COS-7 cells as molecular mechanism of potentiation, showing a stimulation of inositol phosphates (IPn) formation independently from the B2 receptor (Mueller et al., 2005). Thus, taken together, these results give evidence that the potentiation of the bradykinin action can occur by different mechanisms, depending on the system and on the applied potentiating factor. On the other hand, BPP-Cdc, like BPP-9a and captopril, after 30-min incubation decreased the amplitude of angiotensin I-induced contractions in the guinea pig ileum with the same magnitude (Fig. 3B), suggesting an inhibition of the catalytic activity of ACE and consequently a decreased production of Ang II. To reinforce this hypothesis, we have also shown that in vivo BPP-Cdc also decreased the acute pressor effect of Ang I in 4-week hypertensive 2K-1C rats. However, although the effect of BPP-Cdc on acute pressor effect of Ang I, at the same doses, was similar to that BPP-9a, the duration of its action (60 min) was at least two times higher than that of BPP-9a (30 min). Despite the high degree of sequence identity between the N- and C-domains of human somatic ACE, they differ in substrate/inhibitor specificity (Cotton et al., 2002; Hayashi et al., 2003; Hayashi and Camargo, 2005). The N-domain is specific for the degradation of the AcSDKP tetrapeptide that controls hematopoietic stem cell proliferation and differentiation (Rousseau et al., 1995), whereas the C-domain is primarily involved in blood pressure regulation through the degradation of Ang I
(Jaspard et al., 1993) and the inactivation of vasodilator peptide bradykinin (Villard and Soubrier, 1996). Interesting, while it was shown that the decapeptide BPP-10c (
Fig. 6. Comparison of the BPP-Cdc amino acid sequence with those of other BPPs from snake venoms. Identical residues are boxed. aGomes et al., 2007; bHiguchi et al., 2006; cPoliti et al., 1985; dCoutinho-Neto et al., 2013; eWermelinger et al., 2005; fFerreira et al., 1970; gMurayama et al., 1997; hHayashi et al., 2003; iIanzer et al., 2004; jOndetti et al., 1971; kFerreira et al., 1992; lBPPs deposited in NCBI bank (AAP 83422 and AAL 09426); mCintra et al., 1990; nSoares et al., 2005; oKato et al., 1973; pKato and Suzuki, 1971; qMurayama et al., 2000; rYanoshita et al., 1999; sFerreira et al., 1995; tHiguchi et al., 1999; uKomori and Sugihara, 1990.
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production, a natural precursor molecule for NO synthesis, and consequently reducing the arterial blood pressure (Guerreiro et al., 2009; Camargo et al., 2012). Therefore, further studies will be necessary to investigate whether BPP-Cdc may be involved in arginine metabolism and thus to activate endothelial nitric oxide. Finally, as occurs in the guinea ileum pig, another possible mechanism of action of BPP-Cdc on vascular system is the augmented BK effects on B2 receptors. ACE inhibitors can augment BK effects on B2 receptor indirectly only when enzyme and receptor molecules are sterically close, possibly forming a heterodimer (Marcic et al., 1999). Thus, ACE inhibitors and other agents with affinity for the active center likely alter the heterodimer interaction to promote a conformation of the B2 receptor that can more efficiently induce signal transduction (Marcic et al., 2000). In conclusion, the pharmacological effects of BPP-Cdc in vitro decreasing the amplitude of angiotensin I-induced contractions in the guinea pig ileum and in vivo reducing the acute pressor effect of Ang I in 4 week hypertensive 2K-1C rats, were similar to those BPP-9a, suggesting that both peptides act inhibiting the ACE activity. Furthermore, the long duration of action of BPP-Cdc in these experiments may also suggest that it has more affinity for ACE than BPP-9a. On the other hand, the bradykinin-potentiation effects in vivo of BPPCdc on the hypotensive effect to BK in 4 week hypertensive 2K-1C rats were higher than those of BPP-9a in amplitude (60%) and duration (100%) and these results may also suggest the involvement of other ACE-independent mechanisms to be elucidated. Furthermore, further studies will be required to understand the relationship between the atypical chemical structure of BPP-Cdc and its pharmacological activity, as well as to fully elucidate its mechanism of action. Acknowledgments This work was supported by FUNCAP, CNPq, CAPES and GENPHARMA. We thank Rosa Germana da Silva for the rio for the writing technical assistance and Francisco Robe assistance of the paper. Conflict of interest The authors declare no conflict of interest regarding the content of this manuscript. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.toxicon.2014.07. 011. References Camargo, A., Ferreira, S.H., 1970. Action of a bradykinin potentiating factor (BPF) and dimercaprol (BAL) on the responses to bradykinin of isolated preparations of rat intestines. Br. J. Pharmacol. 42, 305e307. Camargo, A., Ferreira, S.H., 1971. Action of bradykinin potentiating factor (BPF) and dimercaprol (BAL) on the responses to bradykinin of isolated preparations of rat intestines. Br. J. Pharmacol. 42 (2), 305e307. Camargo, A.C.M., Ianzer, D., Guerreiro, J.R., Serrano, S.M.T., 2012. Bradykinin-potentiating peptides: beyond captopril. Toxicon 59, 516e523.
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