NO production and potassium channels activation induced by Crotalus durissus cascavella underlie mesenteric artery relaxation

Toxicon 133 (2017) 10e17

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NO production and potassium channels activation induced by Crotalus durissus cascavella underlie mesenteric artery relaxation ~ es a, W.P. Vasconcelos b, I.A. Medeiros b, R.C. Veras b, S.S. Santos a, R.L.C. Jesus a, L.O. Simo L.L. Casais-E-Silva a, D.F. Silva Profa. Dra a, * a b

Department of Bioregulation, Federal University of Bahia, Salvador, BA, 40110-902, Brazil ~o Pessoa, Paraíba, 58059-900, Brazil Department of Pharmaceutical Sciences, Federal University of Paraiba, Joa

a r t i c l e i n f o

a b s t r a c t

Article history: Received 15 February 2017 Received in revised form 12 April 2017 Accepted 16 April 2017 Available online 18 April 2017

Animal toxins are natural resources for pharmacological studies. The venom of Crotalus durissus cascavella (C.d. cascavella) may be a source in the bio-prospecting of new anti-hypertensive agents. The aim of this study was to investigate vascular effects of the venom of C.d. cascavella in normotensive rats. Studies were performed using isolated mesenteric artery segments and aortic endothelial cells. The cumulative administration of the venom of C.d. cascavella (0.001e30 mg/mL) on phenylephrine (Phe; 10 mM) precontracted rings induced a concentration-dependent vasorelaxation in the presence of vascular endothelium (Emax ¼ 47.9 ± 5.0% n ¼ 8), and its effect was almost abolished in the absence of endothelium (Emax ¼ 5.8± 2.4% n ¼ 5 (***p < 0.001)). Tissue viability was maintained as there was no difference in the contractile capacity of rings before and after the administration of venom. The vasorelaxant effect of the venom was also abolished when arteries were pre-contracted with potassium chloride (KCl; 80 mM) (Emax ¼ 6.4± 0.9% n ¼ 5, ***p < 0.001). When assessing the participation of endothelium-derived relaxing factors, it was noted that non-selective COX inhibition with indomethacin (10 mM) caused a significant reduction in the vasorelaxant effect of C.d. cascavella (*p < 0.05). When investigating the participation of NO released by endothelium, there was a significant reduction of the vasorelaxant effect of venom in rings treated with L-NAME (100 mM; Emax ¼ 17.5± 2.2% n ¼ 6; **p < 0.01). Similar results were noted in the presence of ODQ (10 mM), an inhibitor of soluble guanylyl cyclase (Emax ¼ 11.2± 3.5%, n ¼ 6) and PTIO (100 mM), a stable radical scavenger for nitric oxide (Emax ¼ 10.77± 3.6%, n ¼ 6). Moreover, the venom induced the release of NO by isolated aortic endothelial cells through amperometric studies. When assessing the participation of Kþ channels on the vasodilatory response of the venom, tyrode solution with 20 mM of KCl caused a significant reduction in the relaxation response (p < 0.001) (Emax ¼ 21.3 ± 8%, n ¼ 7), as did inhibitor of delayed rectifier Kþ channels (4-amynopiridine 1 mM; Emax ¼ 9.5 ± 1.3, %, n ¼ 5, ***p < 0.001), and vasorelaxation was almost abolished in the presence of Iberiotoxin (IbTx 100 nM). Therefore, these results suggest that the venom of C.d. cascavella induces vasorelaxation in superior mesenteric artery rings of normotensive rats in an endothelium-dependent manner. Specifically, the venom stimulates the generation of endothelium-derived relaxing factors, especially NO, and activates vascular smooth muscle hyperpolarization through Kþ channels. These data illustrate that C.d. cascavella is a source of bioactive molecules and therefore has therapeutic potential in the treatment of cardiovascular diseases such as hypertension. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Crotalus durissus cascavella Mesenteric artery Endothelium-dependent Nitric oxide Soluble guanyly cyclase Potassium channels

1. Introduction Natural products derived from plants and animals are an * Corresponding author. Department of Bioregulation, Federal University of Bahia, Av. Reitor Miguel Calmon, S/N, Vale do Canela, Salvador, BA, 40110-902, Brazil. E-mail address: [email protected] (D.F. Silva). http://dx.doi.org/10.1016/j.toxicon.2017.04.010 0041-0101/© 2017 Elsevier Ltd. All rights reserved.

excellent source for the development of new therapeutic agents. Prodrugs of natural origin have played an important role over the years in the emergence of synthetic drugs, and represent sources of low cost and easy accessibility (Kumar et al., 2014). Among the natural products, animal venoms constitute complex mixtures of various bioactive compounds involved on human envenomation, which also can provide therapeutically useful

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molecules. They present a wide variety of biological activities and different targets such as the cardiovascular system (Horta et al., 2016; Hodgson and Isbister, 2009; Joseph et al., 2004). Cardiovascular envenomation symptoms are manifested clinically by hypotension, vasodilation, vasoconstriction, arrhythmia, violent contractions of the heart, collapse and possible cardiac arrest (Aguilar et al., 2007; Hodgson and Isbister, 2009; Accary et al., 2016). Hypotensive effects of snake venoms could be due to the direct effects of venom components or by indirect effects due to ischemia or obstruction of vasculature (Tibballs, 1998). In relation to the direct effects of venom, specific hypotensive toxins, which differ widely in their structure, mechanism, and site of action, have been isolated from snake venoms causing different effects on cardiovascular system (Joseph et al., 2004). Based on this, different compounds were obtained from venoms, such as bradykinin potentiating peptide (BPP), detected for the first time in the venom of Bothrops jararaca 1965 (Ferreira, 1965; Cushman et al., 1982; Ferreira and Silva, 1965 cited by Camargo et al., 2012), natriuretic peptides, blockers of L-type calcium channel, vascular endothelial growth factors (VEGFs), sarafotoxin, and antagonists of a-adrenergic receptors (Joseph et al., 2004; Koh and Kini, 2012; Accary et al., 2016; Horta et al., 2016). Crotalus genus is represented in Brazil by a single species, Crotalus durissus, and five subspecies: C. d. terrificus; C. d. collilineatus; C. d. cascavella; C. d. ruruima; C. d. marajoensis, distributed on a huge geographic area. This genre is popularly known as rattlesnake (Boldrini-Franca et al., 2010). In Brazil, envenomation caused by Crotalus snake produces serious health complications such as neurotoxicity, respiratory paralysis, hypotension, coagulation disorders, myotoxicity, acute renal failure, and shock (Barraviera et al., 1995). The subspecies of interest in this study, C.d. cascavella is found mainly in the Northeast region of Brazil (Boldrini-Franca et al., 2010). Studies with venom subspecies have shown that there are a range of therapeutic possibilities from secondary metabolites of this complex chemical compound. The crotapotin isolated from C.d. cascavella showed bactericidal effect on gram-negative bacteria (Oliveira et al., 2003), confirmed by further studies The C.d. cascavella venom also possesses a an antiparasitic, specifically antileishmaniose effect,, which is dependent on hydrogen peroxide dis-Baptista et al., 2006). In vivo production (Toyama et al., 2006 . Ra assays conducted in mice comparing three subspecies of Crotalus durrisus identified that products with antileshimaniose effects are present in higher quantities in the C.d. cascavella subspecies, and that these could serve as potential agents against cutaneous leishmaniasis (Passero et al., 2007). Evangelista et al. (2008) isolated a natriuretic peptide from C.d. cascavella venom that produces renal and vascular effects characterized by a dose-dependent decrease of mean arterial pressure and increase of nitrite production. This effect was also confirmed for the crude venom, which induced decreased heart rate and respiratory frequency (Evangelista et al., 2011). Despite the well documented hypotensive effect of C.d cascavella venom (Evangelista et al., 2011), there has been limited characterization of the cardiovascular mechanisms of these venoms. Therefore, in the current study, we aimed to investigate the mechanism of the vasodilation induced by C.d. cascavella venom in normotensive rats. 2. Material and methods

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Ofiology Regional Center and Bahia Venomous Animals in the Federal University of Bahia (NOAP-UFBA, BA, Brazil) and stored at 20  C. Fresh stock solutions were prepared daily in distilled water and stored on ice until used. 2.2. Drugs and solutions L-Phenylephrine (Phe), acetylcholine chloride, 4-Aminopyridine, prostaglandin F2a, ODQ (1H-[1,2,4] Oxadiazolo-[4,3-a] Quinoxalin 1 e One), indomethacin, barium chloride (BaCl2), iberiotoxin, PTIO (2Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide) and L-NAME (Nu-Nitro-L-arginine methyl ester hydrochloride) were obtained from Sigma Chemical Co. All drugs were dissolved in distilled water, with the exception of indomethacin, PTIO and ODQ which were dissolved in 5% sodium bicarbonate, ethanol and dimethyl sulfoxide (DMSO), respectively, and diluted in water Milli Q. Kþ-depolarizing solutions (KCl 20 and 80 mM) were prepared by replacing 20 or 80 mM of KCl in Tyrode's solution with an equimolar concentration of NaCl, respectively. 2.3. Animals Male Wistar rats (12 weeks, 250e300 g) were obtained from the Neurosciences Laboratory of the Health Sciences Institute of the Federal University of Bahia (ICS/UFBA). The animals were housed under controlled temperature (21 ± 1 C) and lighting (lights on: 06:00e18:00 h), with free access to food and water. The study was carried out in accordance with the Guide for the Care and Use of Laboratory Animals, adopted by the US National Institutes of Health, and with the general guidelines of the Brazilian Society of Laboratory Animal Science (SBCAL). The study protocol was approved by the Ethics Committee on Animal Use the Health Sciences Institute - ICS from Federal University of Bahia (CEUA-ICS/ UFBA, protocol No. 072/2014). 2.4. Isolation of superior mesenteric arteries Wistar normotensive rats were euthanized in carbon dioxide chamber, and the superior mesenteric arteries were cleaned of connective tissue (Silva et al., 2011). Briefly, the superior mesenteric artery was removed and cleaned from connective tissue and fat. Rings (1e2 mm) were obtained and placed in physiological Tyrode's solution (composition in mM: NaCl 158.3; KCl 4.0; CaCl2$2H2O 2.0; MgCl2$6H2O 1.05; NaHCO3 10.0; NaH2PO4$H2O 0.42; glucose 5.6), maintained at 37  C, and gassed with a carbogenic mixture (95% O2 and 5% CO2). Rings were stabilized with an optimal resting tension of 0.75 g, which had been determined previously by lengthetension relationship experiments and studies using the optimal contraction to 10 mM Phe against passive tension. The tissues were then allowed to equilibrate for 60 min while resting tension was readjusted to 0.75 g when necessary. The isometric contraction was recorded by a force transducer (FORT-10; WPI, Sarasota, USA) coupled to an amplifiererecorder (Miobath-4, WPI) and to a personal computer equipped with an analog-todigital converter board. Endothelial removal was qualitatively assessed by the failure to relax to acetylcholine (10 mM) after contractile tone was induced by Phe (10 mM). Rings that relaxed less than 10% were considered endothelium denuded, and relaxation more than 90% was considered endothelium intact. After the washout, the rings were pre-contracted again with Phe (10 mM) to produce a similar level of pre-contraction.

2.1. The venom of C.d.cascavella 2.5. Effect of venom of C.d. cascavella on mesenteric rings Lyophilized C.d. cascavella venom obtained from adult snakes from Ibiquera, BA, Brazil. Snakes belonged to the Serpentarium

After equilibration, endothelium-intact and -denuded superior

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mesenteric artery rings were contracted with Phe (105 M), then challenged with cumulative concentrations of C.d. cascavella (0.001, 0.01, 0.1, 0.3, 1, 3, 10 and 30 mg/mL). The ability of C.d. cascavella venom to attenuate the 80 mM KCl and PGF2a (10 mM)-induced contraction was also examined for endothelium-intact arteries. To investigate the mechanism involved in C.d. cascavella venominduced relaxation, the preparations with functional endothelium were pre-incubated with different pharmacological agents: ODQ (10 mM), a soluble guanylyl cyclase (sGC) inhibitor; L-NAME (100 mM), non-selective inhibitor of nitric oxide synthase NOS; indomethacin (10 mM),non-selective inhibitor of the enzyme cyclooxygenase (COX); PTIO (100 mM), a stable radical scavenger for nitric oxide (Dantas et al., 2014); Iberiotoxin (100 nM) a selective blocker of BKCa channels (Smith et al., 1986; Reis et al., 2013) and 4AP (1 mM), a voltage-operated Kþ channel (KV) blocker (Ghisdal and Morel, 2001). Furthermore, Tyrode's solution with elevated Kþ (20 mM equimolar replacement of NaCl with KCl) to attenuate Kþ efflux was used to investigate the involvement of Kþ channels (Silva et al., 2011). To examine the reversibility of the venom-induced responses, in some experiments, the preparations were washed three times after venom addition and a second contraction with phenylephrine (105 M) was obtained. In other experiments, the venom was heated (20 min, 100  C) before the test. 2.6. Nitric oxide measurements 2.6.1. Obtaining and cell viability The thoracic aorta was removed and immediately immersed in Tyrode solution, which was freed from surrounding connective and adipose tissue. It was then opened longitudinally, and endothelial cells were collected by mechanical scraping. The pellet was washed twice and re-suspended (106 cells/ml) in HEPES medium supplemented with 0.5% fetal bovine serum (FBS) (Baudin et al., 2007; van Beijnum et al., 2008). Initially, the cells were pre-incubated with 1 mg/ml antibody “anti-CD 31-rat endothelium” (OX43 - PE, sc-53109, Santa Cruz Biotechnology, USA) for 45 min at 4  C, in the absence light (Hewett and Murray, 1993). The cells were then washed twice with HEPES medium supplemented with 0.5% FBS and centrifuged 400 g for 5 min) and analyzed with 10,000 cells per sample by flow cytometry using the FACSCalibur (Becton Dickinson, San Jose, CA). Cytofluorographic analysis was performed using a BectoneDickinson FACScan with an argon ion laser tuned to 488 nm at 15 mW output. For each sample, 10,000 cells were analyzed, possible non-cellular particles were removed from the analysis by conducting gates. Cells with fluorescence of positive PE channel (with Lowpass of 556 nm, lem: 485/42 nm) were considered CD-31 positive, confirming the endothelial origin (van Beijnum et al., 2008). Cell viability was determined using the 7-amino actinomycin D (7AAD), a waterproof membrane probe used for marking non-viable cells. Cells washed and aliquoted in 106 cells/100 mL were treated with 5uL of 7-AAD and incubated for 30 min at 4  C in the dark. Viable cells were obtained from dot-plot graph scatter versus side-7-AAD (fluorescence in the PerCP channel). Unlabelled cells were used as negative control. 2.6.2. Determination of nitric oxide release using NO microsensors Nitric oxide was measured according to the manufacturer's instructions. Briefly, microsensors (ISSO-NOP3005, WPI, USA) were connected to an acquisition system (TBR 4100 e Free Radical Analyser, WPI, FL, USA) and kept immersed for 2 h in CuCl2 (0.1 M) to polarize and to balance the amperage within a range of acceptability between 150 and 3500 pA. The microsensors were calibrated by decomposition of SNAP (S-nitroso-N-acetyl-d,l-penicillamine)

by using a solution of CuCl2 (0.1 M) as active catalyst. The standard curve was constructed by plotting amperage versus concentration of NO released by SNAP. Then, the previously obtained cell suspension was placed in the measuring chamber node (NOCHM-4, WPI, FL, USA) coupled to microsensor used for calibration, and the concentration of NO captured before and after the addition of increasing concentrations venom C.d. cascavella (1; 3; 10; 30; 90 mg/ mL). 2.7. Statistical analysis Values were expressed as mean ± S.E.M. When appropriate, statistical significance was determined by Student's t-test or oneway ANOVA following Bonferroni's post-test, using GraphPad Prism© software, version 5.0 (GraphPad Software Inc., La Jolla, CA, USA). Two-sided p < 0.05 was considered statistically significant. 3. Results 3.1. C.d. cascavella induces endothelium-dependent relaxation of mesenteric artery Fig. 1 shows that C.d. cascavella venom relaxed the Phe-induced contraction in artery segments with intact endothelium in a concentration-dependent manner (Emax ¼ 47.9 ± 5.0% n ¼ 8). In endothelium-denuded vessels, the vasorelaxant effects induced by venom was almost abolished (Emax ¼ 5.8± 2.4%, n ¼ 5, ***p < 0.001). Heating the venom (20 min, 100  C) significantly decreased the dilation induced by C.d. cascavella (**p < 0.01) when compared to control (Emax ¼ 20.5± 4.0%, n ¼ 5). 3.2. C.d. cascavella venom contracts arteries under passive tone In isolated rat superior mesenteric artery rings, in the presence or absence of the vascular endothelium, cumulative venom addition (0.001e30 mg/mL) without pre-contraction to Phe (10 mM) resulted in a significant small contraction only with 30 mg/mL (*p < 0.05) compared to the basal tension before venom addition

Fig. 1. Vasorelaxant effect of the C.d. cascavella venom on superior mesenteric artery rings. Logarithmic concentration-response curves to C. d. cascavella in mesenteric artery rings pre-contracted with phenylephrine (10 mM), in the presence (C), absence (-) of functional endothelium or after heating the venom (heated venom - ◊) n ¼ 5]. Results are means ± S.E.M. of n ¼ 5e8. The data were examined using one-way ANOVA followed by the Bonferroni post-test. *p < 0.05, **p < 0.01, ***p < 0.001 vs endothelium intact.

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(Fig. 2). To assess the tissue viability after concentration response curves to C.d. cascavella venom (from 0.001 to 30 mg/ml), a subsequent Phe-induced contraction (10 mM) was performed. There was no significant difference in the contractile ability of vascular rings after administration of cumulative concentrations of venom (Emax ¼ 65.3 ± 6.4, n ¼ 6; Emax ¼ 68.5 ± 8.0; n ¼ 6, contraction induced by Phe before and after venom, respectively), indicating that the venom (at least the concentrations investigated) did not induce toxic effects or directly interfere with the arterial contractile machinery. 3.3. Effects of C.d. cascavella venom on contraction induced by depolarization with high Kþ concentration or PGF2a To better explore the mechanism of venom-induced vasorelaxation, the arteries were contracted with 80 mM KCl or PGF2a. Fig. 3 shows that C.d. cascavella-mediated relaxation was almost abolished (vessels pre-contracted by KCl 80 mM; Emax ¼ 6.4± 0.9%, n ¼ 5) when compared to the control (vessels pre-contracted by Phe; Emax ¼ 47.9± 5.0%, n ¼ 8). Furthermore, in vessels contracted by PGF2a, the vasodilation induced by venom was significantly decreased (Emax ¼ 18.7± 1.3%, n ¼ 6; ***p < 0.001 vs Phe). 3.4. Participation of endothelium-derived relaxing factors on the vasorelaxation induced by venom of C.d. cascavella NO and prostacyclin are known to be endothelium-derived relaxing factors. In endothelium-intact vessels, rings were preincubated for 30 min with indomethacin (10 mM), L-NAME (100 mM), PTIO (100 mM) and ODQ (10 mM). In the presence of indomethacin, the relaxant effect of C.d. cascavella was significantly reduced (Fig. 4A, Phe, Emax ¼ 47.9± 5.0%, n ¼ 8; Phe þ indomethacin, Emax ¼ 29.1± 7.1%, n ¼ 6). In the presence of LNAME, the concentration-response curve to C.d. cascavella was shifted to the right, with significant changes in the vascular effects of venom (Emax ¼ 17.5± 2.2%, n ¼ 6, **p < 0.01) as shown in Fig. 5B. Similarly, in the presence of PTIO, extracellular NO scavenger, vasorelaxant effect caused by the venom of C.d. cascavella was attenuated significantly (Emax ¼ 10.77± 3.6%, n ¼ 6, ***p < 0.001 vs Phe).

Fig. 3. The pharmacological efficacy of C.d. cascavella is higher in vessels stimulated by a1-adrenergic agonist than by KCl or PGF2a. Logarithmic concentrationeresponse curves to C.d. cascavella in mesenteric artery rings pre-contracted by phenylephrine (C, 105 M), KCl (-, 80 mM) or PGF2a (:, 10 mM). Results are means ± S.E.M. of n ¼ 5 to 8 experiments. The data were examined using one-way ANOVA followed by the Bonferroni post-test **p < 0.01, ***p < 0.001 vs. phenylephrine.

Finally, the presence of ODQ significantly inhibited the vasorelaxant effect induced by venom (Emax 11.2± 3.5%, n ¼ 6, ***p < 0.001) (Fig. 4B). 3.5. C.d. cascavella venom increases the NO production in isolated endothelial cells Nitric oxide release by C.d. cascavella was also assessed in isolated aortic endothelial cells by using amperometric technique. Previously, it was confirmed that the cells had over 75% incorporation of endothelial cells and that they were viable for experimentation as shown in Fig. 5 (A, B and C). The results show that the C.d. cascavella venom significantly increases NO (*p < 0.05) after cumulative addition of venom, in a concentration-dependent manner (Fig. 5D). 3.6. Vasodilatation induced by C.d. cascavella venom involves Kþ channels activation To evaluate the participation of Kþ channels in the vasorelaxant response induced by the venom of C.d. cascavella, experiments were performed using vascular rings with a functional endothelium and contracted with Phe (10 mM) in the presence of a Tyrode solution containing KCl 20 mM. The partial blocking of the Kþ efflux by increasing the extracellular Kþ concentration ([Kþ]e) to 20 mM induced a significant reduction (***p < 0.001) of the vasorelaxant effect after cumulative administration of C. d. cascavella (0.001e30 mg/mL) with significant changes in the values of maximum effect (Emax ¼ 21.3± 8%, n ¼ 7) (Fig. 6). Interestingly, the venom-induced vasodilation was almost abolished in the presence of both Iberiotoxin (IbTx 100 nM) and 4-AP (1 mM). These findings indicate that Kþ channel activation, mainly Kv and BKCa, plays an important role in the relaxant effect of C.d. cascavella venom in mesenteric arteries. 4. Discussion

Fig. 2. Effect of the C.d. cascavella venom on intrinsic tone. Logarithmic concentration-response curves to C.d. cascavella in mesenteric artery rings at basal tone (not pre-contracted), in the presence (C) or absence (-) of functional endothelium. The data were examined using unpaired Student's t-tests *p < 0.05, **p < 0.01 vs. endothelium intact.

Snake venoms are bioactive compounds, consisting of a complex biochemical mixture, formed largely of proteins. In addition, researches with these compounds to discovery of the mechanisms of

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Fig. 4. Evaluation of nitric oxide synthesis, bioavailability, and activity on the relaxation induced by of C.d. cascavella. (A) Logarithmic concentrationeresponse curves to C. d. cascavella in endothelium intact rat mesenteric artery, pre-contracted by phenylephrine (-, control, n ¼ 8) in the presence of indomethacin (:, 10 mM, n ¼ 6) and (B) in the presence of L - NAME (:, 100 mM, n ¼ 6), ODQ (;, 10 mM, n ¼ 6) or PTIO (A,100 mM, n ¼ 6). Values are expressed as means ± S.E.M. The data were examined using one-way ANOVA followed by the Bonferroni post-test *p < 0.05, **p < 0.01, ***p < 0.001 vs. control.

Fig. 5. NO measured by amperometry in CD31 positive cells. Dot plot showing (A) collected cells gating in P1 for further analysis; (B) Gating P2 were used to separate live and dead cells based on 7-AAD uptake and (C) gating to quantitate/separate CD-31 positive cells based on difference in fluorescence intensity between autofluorescence; (D) NO concentration measured by amperometry. *p < 0.05 vs. 0 mg/mL; FSC eForward scatter, SSC e Side scatter.

action and their targets can be important to obtained new pharmacological and biotechnological tools with various biological activities (Calvete, 2009, 2013; Warrell, 2010; De Sousa, 2011). The effect of C.d. cascavella venom in isolated rat mesenteric artery rings has not been reported in the literature. Although previous studies demonstrated that the venom from this snake induced vasodilation and hypotension (Evangelista et al., 2011), the precise molecular

mechanism(s) of action involved in these effects had not been fully elucidated until now. The present work clearly shows that the vasorelaxant mechanism of the C.d. cascavella venom involves endothelium-derived relaxing factors, in particular, NO, with strong evidence for stimulation of sGC/cGMP pathway, followed by BKca, Kir and Kv channel activation and hyperpolarization of the vascular smooth muscle. In

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Fig. 6. Activation of Kþ channels are involved of the relaxation induced by C.d. cascavella. (A) Logarithmic concentrationeresponse curves to C. d. cascavella in mesenteric artery rings pre-contracted by phenylephrine (-, control) and in the presence of KCl (, 20 mM), 4-AP (:,1 mM) and IbTx (A, 100 nM). Values are expressed as means ± S.E.M. of n ¼ 5e8. The data were examined using unpaired Student's t-tests *p < 0.05, **p < 0.01, ***p < 0.001 vs. control.

addition, our study demonstrated that the vasodilation induced by C.d. cascavella venom involves thermosensitive molecules within venom. In vivo studies described a hypotensive effect of C.d. cascavella venom, reduced mean arterial pressure, heart rate and increased production of nitrite, a stable metabolite of NO (Evangelista et al., 2011). Others studies described renal effects with increased perfusion pressure, urinary flow and glomerular filtration rate (Evangelista et al., 2008). However, the vascular signaling involved was not previously identified. Recently, Lopes et al. have isolated a new bradykinin potentiating peptide (BPP) from venom of C.d. cascavella. BPP was found to inhibit the pressor effect of angiotensin II in hypertensive rats and to reduce the contraction induced by angiotensin II on guinea pig ileum. These data suggest a decreased production of angiotensin II with very similar results to that shown by captopril (Lopes et al., 2014). The vascular endothelium plays a strategic control role in vascular hemodynamics, The modulation of the contractile state of the vessels by endothelial cells is done by releasing different contracting and relaxing factors. The relaxation factors are collectively called endothelium-derived relaxing factors (EDRFs), represented mainly by NO, prostacyclin (PGI2), and the endothelium-derived hyperpolarizing factor (Lüescher and Barton, 1997; Galley and Webster, 2004; Kang, 2014). In order to investigate the role of endothelium in the vasorelaxant effect induced by C. d. cascavella, experimental protocols were performed in the presence and absence of functional endothelium. The results showed that the vasorelaxant response was significantly reduced by the absence of the endothelium, indicating that EDRFs are essential for the vasodilator effect of the venom. Previous studies in rat thoracic aorta with natriuretic peptide isolated from the same snake venom, also showed an endotheliumdependent vasorelaxant effect (Evangelista et al., 2008). To evaluate if the effect of the C.d. cascavella venom in mesenteric artery rings depended on a specific contraction pathway, experiments was performed with high Kþ-solution (KCl; 80 mM) and PGF2a, as well as Phe. The Tyrode solution with KCl 80 mM promotes a depolarization of the plasma membrane with subsequent activation voltage-sensitive calcium channels (Vergara et al., 1998;

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guinou et al., 2014). On the other hand, PGF2a is an agonist of FP Gue receptor (Takayama et al., 1996; Pastel et al., 2015). In our experimental conditions, the venom was not able to relax the contractions induced by high concentrations of K. Similar results were observed in the thoracic aorta with a natriuretic peptide of the same subspecies (Evangelista et al., 2008), or other species such as Crotalus oreganus abyssus (Da Silva et al., 2012). Furthermore, subspecies Crotalus durissus cumanensis were not able to induce relaxation in solutions with lower Kþ concentrations (40 mM) (Pereira et al., 2011). Additionaly, our results demonstrated a significant attenuation of the vasorelaxant effect of the venom on contractions induced by PGF2a, compared to contractions induced by Phe. These results suggest a probable preference of the venom in promoting relaxation against contractions induced by drug-receptor coupling, when compared to contraction induced by high-Kþ solution. However, the vasodilation induced by venom was more effective in contraction induced by Phe compared with PGF2a, both of which induce drug-receptor activation. This predilection for rings contracted by Phe (compared to PGF2a) may be due to the fact that a1-adrenergic receptors are capable of promoting phosphorylation of eNOS (NOS3) at the stimulatory residue Ser1177 and resulting in a possible increase in activity of this enzyme (Looft-Wilson et al., 2013). However, further studies are needed to evaluate this hypothesis. In analyzing the possible mechanisms responsible for the relaxation induced by the venom of C.d. cascavella, the results suggest the involvement of NO in this response, since vasorelaxation was attenuated after non selective inhibition of NOS enzyme, using L-NAME. The sGC present in vascular smooth muscle cells is the main cellular target of NO to induce vascular relaxation. The NO binds to the heme of sGC site and positively modulates its activity. The activation of the sGC leads to an increase in cGMP production. Through various signaling pathways, cGMP leads to a reduction of cytosolic Ca2þ concentration ([Ca2þ]c) in vascular smooth muscle cells and subsequent vascular relaxation (Sandoo et al., 2010 . Kang, 2014). In our study, it was shown that NO/sGC is critical to the relaxation induced by the venom, as the selective inhibitor of sGC (ODQ 10 mM) prevented relaxation. These results further reinforce that the relaxation stimulated by the venom of C.d. cascavella must be due to the release of endothelial-derived NO and consequent activation of sGC. Nitric oxide possesses different sites of production, release and action (Costa et al., 2016). To confirm that the observed vascular effect was due to the release of NO from endothelial cells, cell culture experiments were conducted and NO was measured directly with an amperometric technique. The result of the release of NO from isolated aortic cells corroborate the probable mechanism of action of venom in isolated arteries. In tests conducted with indomethacin, a non-selective COX inhibitor, the vascular response from the venom of C.d. cascavella was significantly reduced, at least in part, suggesting that vasorelaxing cyclooxygenase derivatives participated in the vasorelaxation induced by the venom of C.d. cascavella. It is worth mentioning that the tissue viability was maintained after cumulative addition of venom, since the contractions induced by Phe did not change when compared to contractions before incubation with venom. This suggests that the venom does not induce toxic activity or irreversible binding to its biological targets in vascular tissue with the concentrations used in this study. Additionally, venoms are bioactive compounds with a variety of biological activities, consisting mainly of proteins. In the present study, it was shown that the components of the venom C.d. cascavella that participates in the vasorelaxant effect are sensitive to

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high temperatures, since the effect was significantly attenuated after heating. This result indicates that the relaxing components of the venom of C.d. cascavella probably have peptide nature. Different studies have confirmed the hypothesis formulated concerning the chemical nature of the venoms (Jiang et al., 2008; Rodrigues et al., 2014). Potassium channels play an important role in the regulation of vascular function through vascular smooth muscle hyperpolarization. Moreover, they are direct and indirect targets of the actions of NO in vascular tissues. NO can induce activation of channels for Kþ ATP-sensitive (KATP) channels and to Kþ activated by calcium (K2þ ca ) (Costa and Assreuy, 2005; Zhang et al., 2014). The participation of these channels in the vasorelaxant response induced by C.d. cascavella was evaluated using arteries incubated in 20 mM KCl, Under this concentration, Kþ efflux is partially blocked, thereby minimizing relaxation mediated by opening channels for Kþ (Silva et al., 2011; Adeagbo and Triggle, 1993). The elevation of extracellular Kþ (4e20 mM) significantly attenuates the concentrationdependent vasorelaxation induced by the venom suggesting that the relaxing response elicited by the venom of C.d. cascavella involves Kþ channels and vascular smooth muscle hyperpolarization. Experiments were also performed to obtain concentrationresponse curves C.d. cascavella venom in the presence of some Kþ channels blockers. When tested in the presence of IBTX (100 nM) the selective blocker of BKCa (Smith et al., 1986) or 4-AP, a selective blocker of Kv channels (Ghisdal and Morel, 2001), the vasorelaxant effect induced by the venom of C.d. cascavella was significantly attenuated, strongly suggesting the involvement of these channels. It is important to note that NO also has an important role among vasoactive factors influencing the membrane potential and varying the activity of the Kþ channel, through cGMP-dependent protein kinase (PKG). The KCa channels can be activated by cAMPdependent protein kinases or cGMP and/or directly by G protein (Costa and Assreuy, 2005). Some studies suggest that NO also modulates the activation of KATP via sGC/cGMP/PKG (Zhang et al., 2014). 5. Conclusions Taken together, these results demonstrate that the venom of C.d. cascavella induces vasodilatation in rat isolated mesenteric artery in an endothelium-dependent manner, and particularly involving NO. Furthermore, Kþ channels, most likely through Kv and BKCa channels, in vascular smooth muscle are involved in the relaxation induced by the venom of C.d. cascavella. These results show promise in the identification of possible molecules for therapeutic purposes in preventing and treating cardiovascular diseases. Conflict of interest We confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. Acknowledgments This work was funded by the Conselho Nacional de Desenvolgico (CNPq) and Fundaça ~o de Amparo a  vimento Científico e Tecnolo Pesquisa do Estado da Bahia (FAPESB - grant number RED 0033/ 2014). Appendix A. Supplementary data Supplementary data related to this article can be found at http://

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