Cardioprotective effects of the proline-rich oligopeptide Bj-PRO-7a in spontaneously hypertensive rats.

The proline-rich oligopeptide from Bothrops jararaca snake venom, Bj-PRO-7a, promotes acute effects in blood pressure in hypertensive animals. However, the cardiac effects of this heptapeptide are completely unknown. Thus, we sought to evaluate whether the Bj-PRO-7a could protect against cardiac remodelling in spontaneously hypertensive rats (SHR). SHR were treated with Bj-PRO-7a (71 nmol/kg/day, s.c.) or saline for 28 days. Wistar rats were used as control. Systolic blood pressure (SBP) and heart rate (HR) were measured by tail-cuff plethysmography. Cardiomyocyte diameter and interstitial and perivascular fibrosis of the left ventricle (LV) were evaluated using Picrosirius staining. Immunofluorescence was used to detect collagen I and III. Fibroblast proliferation was assessed by immunohistochemistry to detect proliferating cell nuclear antigen (PCNA). Protein expression was assessed by western blot. The superoxide dismutase and catalase activities and the concentration of lipid peroxidation products were evaluated in the LV. The SBP and HR were not different between treated and non-treated SHR at the end of the treatment. However, Bj-PRO-7a attenuated the cardiomyocyte hypertrophy, deposition of interstitial and perivascular fibrosis and collagen I, and positive PCNA-labelled fibroblasts. This peptide also reduced the increased levels of TBARS, expression and activity of catalase, and activity of SOD in LV from SHR. Also, the Bj-PRO-7a increased the expression of metalloproteinases-2 in SHR hearts. These findings demonstrate that the Bj-PRO-7a reduced the pathological cardiac remodelling in a pressure-independent manner in hypertensive rats through mechanisms mediated by oxidative stress regulation.

Stating asymmetry in neural pathways: methodological trends in autonomic neuroscience.

Aim: Many particularities concerning interhemispheric differences still need to be explored and unveiled. Functional and anatomical differential features found between left and right brain sides are best known as asymmetries and are consequence of the unilateral neuronal recruitment or predominance that is set to organize some function. The outflow from different neural pathways involved in the autonomic control of the cardiovascular system may route through asymmetrically relayed efferences (ipsilateral/lateralized and/or contralateral). In spite of this, the literature reporting on the role of central nuclei involved in the autonomic control is not always dedicated on these interhemispheric comparisons. Considering the recent reports demonstrating that asymmetries may set differential functional responses, it is worth checking differences between right and left sides of central regions. This review aims to inspire neuroscientists with the idea that studying the interhemispheric differences may deepen the understanding on several centrally controlled responses, with special regard to the autonomic functions underlying the cardiovascular regulation. Conclusions: Thus, an avenue of knowledge may unfold from a field of research that requires further exploration.

Insights into cardiovascular effects of proline-rich oligopeptide (Bj-PRO-10c) revealed by structure-activity analyses: dissociation of antihypertensive and bradycardic effects.

We have previously reported that the proline-rich decapeptide from Bothrops jararaca (Bj-PRO-10c) causes potent and sustained antihypertensive and bradycardic effects in SHR. These activities are independent of ACE inhibition. In the present study, we used the Ala-scan approach to evaluate the importance of each amino acid within the sequence of Bj-PRO-10c (Pyr(1)-Asn(2)-Trp(3)-Pro(4)-His(5)-Pro(6)-Gln(7)-Ile(8)-Pro(9)-Pro(10)). The antihypertensive and bradycardic effects of the analogues Bj-PRO-10c Ala(3), Bj-PRO-10c Ala(7), Bj-PRO-10c Ala(8) were similar to those of Bj-PRO-10c, whereas the analogues Bj-PRO-10c Ala(2), Bj-PRO-10c Ala(4), Bj-PRO-10c Ala(5), Bj-PRO-10c Ala(9), and Bj-PRO-10c Ala(10) kept the antihypertensive activity and lost bradycardic activity considerably. In contrast, Bj-PRO-10c Ala(1) and Bj-PRO-10c Ala(6) were unable to provoke any cardiovascular activity. In summary, we demonstrated that (1) the Pyr(1) and Pro(6) residues are essential for both, the antihypertensive and bradycardic effects of Bj-PRO-10c; (2) Ala-scan approach allowed dissociating blood pressure reduction and bradycardic effects. Conformational properties of the peptides were examined by means of circular dichroism (CD) spectroscopy. The different Ala-scan analogues caused either an increase or decrease in the type II polyproline helix content compared to Bj-PRO-10c. The complete loss of activity of the Pro(6) â†’ Ala(6) mutant is probably due to the fact that in the parent peptide the His(5)-Pro(6) bond can exist in the cis configuration, which could correspond to the conformation of this bond in the bound state. Current data support the Bj-PRO-10c as a promising leader prototype to develop new agents to treat cardiovascular diseases and its co-morbidities.

Peptidomics of three Bothrops snake venoms: insights into the molecular diversification of proteomes and peptidomes.

Snake venom proteomes/peptidomes are highly complex and maintenance of their integrity within the gland lumen is crucial for the expression of toxin activities. There has been considerable progress in the field of venom proteomics, however, peptidomics does not progress as fast, because of the lack of comprehensive venom sequence databases for analysis of MS data. Therefore, in many cases venom peptides have to be sequenced manually by MS/MS analysis or Edman degradation. This is critical for rare snake species, as is the case of Bothrops cotiara (BC) and B. fonsecai (BF), which are regarded as near threatened with extinction. In this study we conducted a comprehensive analysis of the venom peptidomes of BC, BF, and B. jararaca (BJ) using a combination of solid-phase extraction and reversed-phase HPLC to fractionate the peptides, followed by nano-liquid chromatography-tandem MS (LC-MS/MS) or direct infusion electrospray ionization-(ESI)-MS/MS or MALDI-MS/MS analyses. We detected marked differences in the venom peptidomes and identified peptides ranging from 7 to 39 residues in length by de novo sequencing. Forty-four unique sequences were manually identified, out of which 30 are new peptides, including 17 bradykinin-potentiating peptides, three poly-histidine-poly-glycine peptides and interestingly, 10 L-amino acid oxidase fragments. Some of the new bradykinin-potentiating peptides display significant bradykinin potentiating activity. Automated database search revealed fragments from several toxins in the peptidomes, mainly from l-amino acid oxidase, and allowed the determination of the peptide bond specificity of proteinases and amino acid occurrences for the P4-P4' sites. We also demonstrate that the venom lyophilization/resolubilization process greatly increases the complexity of the peptidome because of the imbalance caused to the venom proteome and the consequent activity of proteinases on venom components. The use of proteinase inhibitors clearly showed different outcomes in the peptidome characterization and suggested that degradomic-peptidomic analysis of snake venoms is highly sensitive to the conditions of sampling procedures.

Median preoptic nucleus mediates the cardiovascular recovery induced by hypertonic saline in hemorrhagic shock.

Changes in plasma osmolarity, through central and peripheral osmoreceptors, activate the median preoptic nucleus (MnPO) that modulates autonomic and neuroendocrine adjustments. The present study sought to determine the participation of MnPO in the cardiovascular recovery induced by hypertonic saline infusion (HSI) in rats submitted to hemorrhagic shock. The recordings of mean arterial pressure (MAP) and renal vascular conductance (RVC) were carried out on male Wistar rats (250-300 g). Hemorrhagic shock was induced by blood withdrawal over 20 min until the MAP values of approximately 60 mmHg were attained. The nanoinjection (100 nL) of GABAA agonist (Muscimol 4 mM; experimental group (EXP)) or isotonic saline (NaCl 150 mM; control (CONT)) into MnPO was performed 2 min prior to intravenous overload of sodium through HSI (3 M NaCl, 1.8 mL/kg, b.wt.). Hemorrhagic shock reduced the MAP in control (62 ± 1.1 mmHg) and EXP (61 ± 0.4 mmHg) equipotently. The inhibition of MnPO impaired MAP (CONT: 104 ± 4.2 versus EXP: 60 ± 6.2 mmHg) and RVC (CONT: 6.4 ± 11.4 versus EXP: -53.5 ± 10.0) recovery 10 min after HSI. The overall results in this study demonstrated, for the first time, that the MnPO plays an essential role in the HSI induced resuscitation during hypovolemic hemorrhagic shock.

Interaction of Angiotensin-(1-7) with kinins in the kidney circulation: Role of B1 receptors.

Changes in renal hemodynamics impact renal function during physiological and pathological conditions. In this context, renal vascular resistance (RVR) is regulated by components of the Renin-Angiotensin System (RAS) and the Kallikrein-Kinin System (KKS). However, the interaction between these vasoactive peptides on RVR is still poorly understood. Here, we studied the crosstalk between angiotensin-(1-7) and kinins on RVR. The right kidneys of Wistar rats were isolated and perfused in a closed-circuit system. The perfusion pressure and renal perfusate flow were continuously monitored. Ang-(1-7) (1.0-25.0 nM) caused a sustained, dose-dependent reduction of relative RVR (rRVR). This phenomenon was sensitive to 10 nM A-779, a specific Mas receptor (MasR) antagonist. Bradykinin (BK) promoted a sustained and transient reduction in rRVR at 1.25 nM and 125 nM, respectively. The transient effect was abolished by 4 µM des-Arg9-Leu8-bradykinin (DALBK), a specific kinin B1 receptor (B1R) antagonist. Accordingly, des-Arg9-bradykinin (DABK) 1 µM (a B1R agonist) increased rRVR. Interestingly, pre-perfusion of Ang-(1-7) changed the sustained reduction of rRVR triggered by 1.25 nM BK into a transient effect. On the other hand, pre-perfusion of Ang-(1-7) primed and potentiated the DABK response, this mechanism being sensitive to A-779 and DALBK. Binding studies performed with CHO cells stably transfected with MasR, B1R, and kinin B2 receptor (B2R) showed no direct interaction between Ang-(1-7) with B1R or B2R. In conclusion, our findings suggest that Ang-(1-7) differentially modulates kinin's effect on RVR in isolated rat kidneys. These results help to expand the current knowledge regarding the crosstalk between the RAS and KKS complex network in RVR.

Asymmetry in the control of cardiac performance by dorsomedial hypothalamus.

Dorsomedial hypothalamus (DMH) plays a key role in integrating cardiovascular responses to stress. We have recently reported greater heart rate responses following disinhibition of the right side of the DMH (R-DMH) in anesthetized rats and greater suppression of stress-induced tachycardia following inhibition of the R-DMH in conscious rats [both compared with similar intervention in the left DMH (L-DMH)], suggesting existence of right/left side asymmetry in controlling cardiac chronotropic responses by the DMH. The aim of the present study was to determine whether similar asymmetry is present for controlling cardiac contractility. In anesthetized rats, microinjections of the GABAA antagonist bicuculline methiodide (BMI; 40 pmol/100 nl) into the DMH-evoked increases in heart rate (HR), left ventricular pressure (LVP), myocardial contractility (LVdP/dt), arterial pressure, and respiratory rate. DMH disinhibition also precipitated multiple ventricular and supraventricular ectopic beats. DMH-induced increases in HR, LVP, LVdP/dt, and in the number of ectopic beats dependent on the side of stimulation, with R-DMH provoking larger responses. In contrast, pressor and respiratory responses did not depend on the side of stimulation. Newly described DMH-induced inotropic responses were rate-, preload- and (largely) afterload-independent; they were mediated by sympathetic cardiac pathway, as revealed by their sensitivity to ß-adrenergic blockade. We conclude that recruitment of DMH neurons causes sympathetically mediated positive chronotropic and inotropic effects, and that there is an asymmetry, at the level of the DMH, in the potency to elicit these effects, with R-DMH > L-DMH.

Behavioral effects evoked by the beta globin-derived nonapeptide LVV-H6.

LVV-hemorphin-6 (LVV-h6) is bioactive peptide and is a product of the degradation of hemoglobin. Since LVV-h6 effects are possibly mediated by opioid or AT4/IRAP receptors, we hypothesized that LVV-h6 would modify behavior. We evaluated whether LVV-h6 affects: i) anxiety-like behavior and locomotion; ii) depression-like behavior; iii) cardiovascular and neuroendocrine reactivity to emotional stress. Male Wistar rats ( ± 300 g) received LVV-h6 (153 nmol/kg i.p.) or vehicle (NaCl 0.9% i.p.). We used: i) open field (OF) test for locomotion; ii) elevated plus maze (EPM) for anxiety-like behavior; iii) forced swimming test (FST) for depression-like behavior and iv) air jet for cardiovascular and neuroendocrine reactivity to stress. Diazepam (2 mg/kg i.p.) and imipramine (15 mg/kg i.p.) were used as positive control for EPM and FST, respectively. To evaluate the LVV-h6 mechanisms, we used: the antagonist of oxytocin (OT) receptors (atosiban - ATS 1 and 0.1 mg/kg i.p.); the inhibitor of tyrosine hydroxylase (Alpha-methyl-p-tyrosine - AMPT 200 mg/kg i.p.) to investigate the involvement of catecholaminergic paths; and the antagonist of opioid receptors (naltrexone - NTX 0.3 mg/kg s.c.). We found that LVV-h6: i) evoked anxiolytic-like effect; ii) evoked antidepressant-like effect in the FST; and iii) did not change the locomotion, neuroendocrine and cardiovascular responses to stress. The LVV-h6 anxiolytic-like effect was not reverted by ATS and AMPT. However, the antidepressant effects were reverted only by NTX. Hence, our findings demonstrate that LVV-h6 modulates anxiety-like behavior by routes that are not oxytocinergic, catecholaminergic or opioid. The antidepressant-like effects of LVV-h6 rely on opioid pathways.

Characterization of urinary metabolites from four synthetic bradykinin potentiating peptides (BPPs) in mice.

BPPs have been identified in the venom of the Bothrops jararaca snake, or deduced from precursor proteins expressed either in the venom gland or in the brain of the snake. Their potentiating activity on bradykinin (Bk) is assumed to occur through a somatic angiotensin-converting enzyme (sACE) inhibitory mechanism. We have demonstrated that synthetic BPPs show remarkable functional differences, despite their high amino acid sequence similarities. Recently, we demonstrated that BPP-10c, after i.p. administration, was found in its intact form and in the form of a unique metabolite (des-Pro(10) BPP-10c) in mouse urine. Given this finding, we selected a number of BPPs with different structure-activities - BPP-5a (

Tissue distribution in mice of BPP 10c, a potent proline-rich anti-hypertensive peptide of Bothrops jararaca.

The snake venom proline-rich peptide BPP 10c is an active somatic angiotensin-converting enzyme (sACE) inhibitors. Recently we demonstrated that the anti-hypertensive effect of BPP 10c is not related to the inhibition of sACE alone, thus suggesting that this enzyme is not its only target for blood pressure reduction. In the present work, a biodistribution study in Swiss mice of [(125)I]-BPP 10c in the absence or in the presence of a saturating concentration of captopril, a selective active-site inhibitor of sACE, demonstrated that: (1) [(125)I]-BPP 10c was present in several organs and the renal absorption was significantly high; (2) [(125)I]-BPP 10c showed a clear preference for the kidney, maintaining a high concentration in this organ in the presence of captopril for at least 3h; (3) The residual amount of [(125)I]-BPP 10c in the kidney of animals simultaneously treated with captopril suggest that the peptide can interact with other targets different from sACE in this organ. We also showed that Cy3-labeled BPP 10c was internalized by human embryonic kidney cells (HEK-293T). Taken together, these results suggest that sACE inhibition by captopril affects the tissue distribution of [(125)I]-BPP 10c and that the anti-hypertensive effects of BPP 10c are not only dependent on sACE inhibition.

Involvement of GABAergic and Adrenergic Neurotransmissions on Paraventricular Nucleus of Hypothalamus in the Control of Cardiac Function.

Sympathetic premotor neurons of the paraventricular hypothalamus (PVN) play a role in hemodynamics adjustments during changes in body fluid homeostasis. However, PVN contribution to the tonic control of cardiac function remains to be systematically studied. In this study, we assessed whether GABAergic and adrenergic synapses, known for being active in the PVN, are involved in the control of cardiac function. Adult male Wistar rats (250-350 g; n = 27) were anesthetized with urethane (1.2-1.4 g/kg i.p.) and underwent catheterization of femoral artery to record blood pressure and heart rate. The femoral vein was used to inject the vasoactive agents phenylephrine (10 µg/kg) and sodium nitroprusside (10 µg/kg) and to supplement anesthesia. The cardiac left ventricle was catheterized to record left ventricular pressure and its derivative. Craniotomy allowed for injections (100 nL) into the PVN of: muscimol (20 mM), bicuculline methiodide (0.4 mM), propranolol (10 mM), isoproterenol (100 µM), phentolamine (13 mM), phenylephrine (30 nM). We found that: (i) inhibition of PVN by muscimol, reduced arterial pressure, cardiac chronotropy and inotropy; (ii) disinhibition of PVN neurons by bicuculline evoked positive chronotropy and inotropy, and increase blood pressure; (iii) PVN alpha adrenergic receptors control cardiac chronotropy and inotropy; (iv) beta adrenergic receptors of the PVN do not influence cardiac function; (v) afterload does not contribute to the PVN-evoked inotropy. Our results indicate that the modulation of the activity of PVN neurons exerted by GABAergic and adrenergic mechanisms contribute to the control of cardiac function.

Ghrelin potentiates cardiac reactivity to stress by modulating sympathetic control and beta-adrenergic response.

Prior evidence indicates that ghrelin is involved in the integration of cardiovascular functions and behavioral responses. Ghrelin actions are mediated by the growth hormone secretagogue receptor subtype 1a (GHS-R1a), which is expressed in peripheral tissues and central areas involved in the control of cardiovascular responses to stress. AIMS: In the present study, we assessed the role of ghrelin - GHS-R1a axis in the cardiovascular reactivity to acute emotional stress in rats. MAIN METHODS AND KEY FINDINGS: Ghrelin potentiated the tachycardia evoked by restraint and air jet stresses, which was reverted by GHS-R1a blockade. Evaluation of the autonomic balance revealed that the sympathetic branch modulates the ghrelin-evoked positive chronotropy. In isolated hearts, the perfusion with ghrelin potentiated the contractile responses caused by stimulation of the beta-adrenergic receptor, without altering the amplitude of the responses evoked by acetylcholine. Experiments in isolated cardiomyocytes revealed that ghrelin amplified the increases in calcium transient changes evoked by isoproterenol. SIGNIFICANCE: Taken together, our results indicate that the Ghrelin-GHS-R1a axis potentiates the magnitude of stress-evoked tachycardia by modulating the autonomic nervous system and peripheral mechanisms, strongly relying on the activation of cardiac calcium transient and beta-adrenergic receptors.

Identification of novel bradykinin-potentiating peptides (BPPs) in the venom gland of a rattlesnake allowed the evaluation of the structure-function relationship of BPPs.

Aiming to extend the knowledge about the diversity of bradykinin-potentiating peptides (BPPs) and their precursor proteins, a venom gland cDNA library from the South American rattlesnake (Crotalus dursissus terrificus, Cdt) was screened. Two novel homologous cDNAs encoding the BPPs precursor protein were cloned. Their sequence contain only one single longer BPP sequence with the typical IPP-tripeptide, and two short potential BPP-like molecules, revealing a unique structural organization. Several peptide sequences structurally similar to the BPPs identified in the precursor protein from Cdt and also from others snakes, were chemically synthesized and were bioassayed both in vitro and in vivo, by means of isolated smooth muscle preparations and by measurements of blood pressure in anaesthetized rats, respectively. We demonstrate here that a pyroglutamyl residue at the N-terminus with a high content of proline residues, even with the presence of a IPP moiety characteristic of typical BPPs, are not enough to determine a bradykinin-potentiating activity to these peptides. Taken together, our results indicate that the characterization of the BPPs precursor proteins and identification of characteristic glutamine residues followed by proline-rich peptide sequences are not enough to predict if these peptides, even with a pyroglutamyl residue at the N-terminus, will present the typical pharmacological activities described for the BPPs.

Novel pharmaceutical composition of bradykinin potentiating penta peptide with beta-cyclodextrin: physical-chemical characterization and anti-hypertensive evaluation.

This work describes chemical properties and anti-hypertensive activity of an oral pharmaceutical formulation obtained from the complexation of beta-cyclodextrin (beta-CD) with bradykinin potentiating penta peptide (BPP-5a) founded in the Bothrops jararaca poison. Physical chemistry characterizations were recorded in order to investigate the intermolecular interactions between species in complex. Circular dichroism data indicated conformational changes of BPP-5a upon complexation with beta-CD. ROESY and theoretical calculations showed a selective approximation of triptophan moiety into cavity of beta-CD. Isothermal titration calorimetry data indicated an exothermic formation of the complex, which is accomplished by reduction of entropy. The anti-hypertensive activity of the BPP-5a/beta-CD complex has been evaluated in spontaneous hypertensive rats, showing better results than pure BPP-5a.

The hemoglobin derived peptide LVV-hemorphin-7 evokes behavioral effects mediated by oxytocin receptors.

LVV-hemorphin-7 (LVV-h7) is bioactive peptide resulting from degradation of hemoglobin ß-globin chain. LVV-h7 is a specific agonist of angiotensin IV receptor. This receptor belongs to the class of insulin-regulated aminopeptidases (IRAP), which displays oxytocinase activity. Herein, our aims were to assess whether: i) LVV-h7 modifies centrally organized behavior and cardiovascular responses to stress and ii) mechanisms underlying LVV-h7 effects involve activation of oxytocin (OT) receptors, probably as result of reduction of IRAP proteolytic activity upon OT. Adult male Wistar rats (270-370g) received (i.p.) injections of LVV-h7 (153nmol/kg), or vehicle (0.1ml). Different protocols were used: i) open field (OP) test for locomotor/exploratory activities; ii) Elevated Plus Maze (EPM) for anxiety-like behavior; iii) forced swimming test (FST) test for depression-like behavior and iv) air jet for cardiovascular reactivity to acute stress exposure. Diazepam (2mg/kg) and imipramine (15mg/kg) were used as positive control for EPM and FST, respectively. The antagonist of OT receptors (OTr), atosiban (1 and 0,1mg/kg), was used to determine the involvement of oxytocinergic paths. We found that LVV-h7: i) increased the number of entries and the time spent in open arms of the maze, an indicative of anxiolysis; ii) provoked antidepressant effect in the FS test; and iii) increased the exploration and locomotion; iv) did not change the cardiovascular reactivity and neuroendocrine responses to acute stress. Also, increases in locomotion and the antidepressant effects evoked by LVV-h7 were reverted by OTr antagonist. We conclude that LVV-h7 modulates behavior, displays antidepressant and anxiolytic effects that are mediated in part by oxytocin receptors.

Hemorphin and hemorphin-like peptides isolated from dog pancreas and sheep brain are able to potentiate bradykinin activity in vivo.

Hemorphins are biologically active peptides, derived from hemoglobin, which presents a number of physiological activities. Proteolytic generation of these peptides is not fully understood; however, among their roles, is to provoke reduction on blood pressure. In this work, this particular biological effect was chosen as the monitor for the selection of mammalian vasoactive peptides. By combining high-performance liquid chromatography and mass spectrometry, including 'de novo' sequencing, several hemorphin-like peptides were identified presenting bradykinin potentiating activity. Moreover, taking LVV-hemorphin-7 as model compound, we evaluated its biological effect on blood pressure of anaesthetized rats. By summarizing all the results, it is possible to present the hemorphins as a family of proteolytically generated peptides that are able to potentiate bradykinin activity in vivo.

Polystyrene-type resin used for peptide synthesis: application for anion-exchange and affinity chromatography.

This paper deals with an unusual application for a copolymer of styrene-1% divinylbenzene bearing high amount of aminomethyl groups for anion-exchange and affinity chromatography. The so-called aminomethyl resin (AMR), to date only employed for peptide synthesis, swelled appreciably in water and was used successfully to purify negatively charged peptides. By correlating swelling degree of beads with pH of the media, it was possible to estimate that the AMR amino group pK(a) is approximately 5.5. In addition, the synthesized acetyl-(NANP)3-AMR succeeded in the affinity interaction with large antibody molecules related to malaria transmission and raised previously against this dodecapeptide sequence.

Identification of snake bradykinin-potentiating peptides (BPPs)-simile sequences in rat brain--Potential BPP-like precursor protein?

Bradykinin-potentiating peptides (BPPs) from the South American pit viper snake venom were the first natural inhibitors of the human angiotensin I-converting enzyme (ACE) described. The pioneer characterization of the BPPs precursor from the snake venom glands by our group showed for the first time the presence of the C-type natriuretic peptide (CNP) in this same viper precursor protein. The confirmation of the BPP/CNP expression in snake brain regions correlated with neuroendocrine functions stimulated us to pursue the physiological correlates of these vasoactive peptides in mammals. Notably, several snake toxins were shown to have endogenous physiological correlates in mammals. In the present work, we expressed in bacteria the BPPs domain of the snake venom gland precursor protein, and this purified recombinant protein was used to raise specific polyclonal anti-BPPs antibodies. The correspondent single protein band immune-recognized in adult rat brain cytosol was isolated by 2D-SDS/PAGE and/or HPLC, before characterization by MS fingerprint analysis, which identified this protein as superoxide dismutase (SOD, EC 1.15.1.1), a classically known enzyme with antioxidant activity and important roles in the blood pressure modulation. In silico analysis showed the exposition of the BPP-like peptide sequences on the surface of the 3D structure of rat SOD. These peptides were chemically synthesized to show the BPP-like biological activities in ex vivo and in vivo pharmacological bioassays. Taken together, our data suggest that SOD protein have the potential to be a source for putative BPP-like bioactive peptides, which once released may contribute to the blood pressure control in mammals.

Identification of five new bradykinin potentiating peptides (BPPs) from Bothrops jararaca crude venom by using electrospray ionization tandem mass spectrometry after a two-step liquid chromatography.

Bradykinin potentiating peptides (BPPs) from Bothrops jararaca venom were described in the middle of 1960s and were the first natural inhibitors of the angiotensin-converting enzyme displaying strong anti-hypertensive effects in human subjects. The BPPs can be recognized by their typical pyroglutamyl proline-rich oligopeptide sequences presenting invariably a proline residue at the C-terminus. In the present study, we identified 18 BPPs, most of them already described for the B. jararaca venom. We isolated and sequenced new peptides ranging from 5 to 14 amino acid residues exhibiting similar amino acid sequence features. The applied methodology consisted of a strait two-step liquid chromatography, followed by mass spectrometry analysis. Besides the amino acid sequence homology, the corresponding synthetic peptides were able to potentiate bradykinin on the isolated guinea-pig ileum.

Excitatory amino acid receptors mediate asymmetry and lateralization in the descending cardiovascular pathways from the dorsomedial hypothalamus.

The dorsomedial hypothalamus (DMH) and lateral/dorsolateral periaqueductal gray (PAG) are anatomically and functionally connected. Both the DMH and PAG depend on glutamatergic inputs for activation. We recently reported that removal of GABA-ergic tone in the unilateral DMH produces: asymmetry, that is, a right- (R-) sided predominance in cardiac chronotropism, and lateralization, that is, a greater increase in ipsilateral renal sympathetic activity (RSNA). In the current study, we investigated whether excitatory amino acid (EAA) receptors in the DMH-PAG pathway contribute to the functional interhemispheric difference. In urethane (1.2 to 1.4 g/kg, i.p.) anesthetized rats, we observed that: (i) nanoinjections of N-methyl D-aspartate (NMDA 100 pmol/100 nl) into the unilateral DMH produced the same right-sided predominance in the control of cardiac chronotropy, (ii) nanoinjections of NMDA into the ipsilateral DMH or PAG evoked lateralized RSNA responses, and (iii) blockade of EAA receptors in the unilateral DMH attenuated the cardiovascular responses evoked by injection of NMDA into either the R- or left- (L-) PAG. In awake rats, nanoinjection of kynurenic acid (1 nmol/100 nL) into the L-DMH or R- or L-PAG attenuated the tachycardia evoked by air stress. However, the magnitude of stress-evoked tachycardia was smallest when the EAA receptors of the R-DMH were blocked. We conclude that EAA receptors contribute to the right-sided predominance in cardiac chronotropism. This interhemispheric difference that involves EAA receptors was observed in the DMH but not in the PAG.