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Background and aims Metformin has been known to promote cardiovascular benefits in humans and animal models, even in non-diabetic subjects. However, its chronic effects on hypertension-related autonomic dysfunction remain poorly understood. Therefore, we evaluate the cardiac autonomic effects of chronic metformin in hypertensive rats. Methods and results Twelve-week-old male SHR and Wistar rats were separated into 3 groups: WN (Wistar normotensive); SC (SHR hypertensive control); and SM (SHR: Metformin 300 mg/kg/day for 30 days). Spontaneous and induced (by phenylephrine and sodium nitroprusside) baroreflexes were analysed in catheterised rats. Next, cardiac autonomic tone was evaluated through heart rate shift by atropine (parasympathetic) or atenolol (sympathetic). Plasma TNFα was assessed by ELISA. Western blot analyses of inflammatory, oxidant and antioxidant proteins were performed. Cardiac parasympathetic tone and baroreflex function were lower in SC than in WN, whereas cardiac sympathetic tone was higher. Metformin treatment in non-diabetic hypertensive rats reduced the resting heart rate, attenuated the cardiac sympathetic tone and improved baroreflex (especially in the offsetting of rising BP), while blood pressure and glycaemia remained unchanged. Cardiac sympathetic tone correlated negatively with spontaneous baroreflex. Metformin reduced plasma TNFα levels and decreased tissue expression of COX2 and NOX2 (which were positively correlated), without affecting SOD1 and SOD2. Conclusion Chronic metformin presented anti-inflammatory and antioxidant effects and, independently of alterations in glycaemia, it improved cardiac autonomic parameters that are impaired in hypertension, being related to end-organ damage and mortality. These findings open up perspectives for future innovative uses of metformin in cardiovascular diseases, especially in hypertension.

The biological activities observed upon envenomation by Scorpaena plumieri could be linked to both the venom and the skin mucus. Through a proteomic/functional approach we analyzed protein composition and biological activities of the venom and skin mucus. We identified 885 proteins: 722 in the Venomous Apparatus extracts (Sp-VAe) and 391 in the Skin Mucus extract (Sp-SMe), with 494 found exclusively in Sp-VAe, being named S. plumieri Venom Proteins (Sp-VP), while 228 were found in both extracts. The majority of the many proteins identified were not directly related to the biological activities reported here. Nevertheless, some were classified as toxins/potentially interesting molecules: lectins, proteases and protease inhibitors were detected in both extracts, while the pore-forming toxin and hyaluronidase were associated with Sp-VP. Proteolytic and anti-microbial activities were linked to both extracts, while the main toxic activities – cardiovascular, inflammatory, hemolytic and nociceptive – were elicited only by Sp-VAe. Our study provided a clear picture on the composition of the skin mucus and the venom. We also show that the classic effects observed upon envenomation are produced by molecules from the venomous gland. Our results add to the growing catalogue of scorpaeniform fish venoms and their skin mucus proteins. Significance In this study a large number of proteins – including classical and non-classical toxins – were identified in the venomous apparatus and the skin mucus extracts of the Scorpaena plumieri fish through shotgun proteomic approach. It was shown that the toxic effects observed upon envenomation are elicited by molecules originated from the venomous gland. These results add to the growing catalogue of scorpaeniform fish venoms and their skin mucus proteins – so scarcely explored when compared to the venoms and bioactive components of terrestrial animals. Data are available via ProteomeXchange with identifier PXD009983

Fish venom cytolysins are multifunctional proteins that in addition to their cytolytic/hemolytic effects display neurotoxic, cardiotoxic and inflammatory activities, being described as "protein lethal factors". A pore-forming cytolysin called Sp-CTx (Scorpaena plumieriCytolytic Toxin) has been recently purified from the venom of the scorpionfish Scorpaena plumieri. It is a glycoprotein with dimeric constitution, comprising subunits of approximately 65 kDa. Previous studies have revealed that this toxin has a vasorelaxant activity that appears to involve the L-arginine-nitric oxide synthase pathway; however its cardiovascular effects have not been fully comprehended. The present study examined the cardiovascular effects of Sp-CTx in vivo and in vitro. In anesthetized rats Sp-CTx (70 μg/kg i.v) produced a biphasic response which consisted of an initial systolic and diastolic pressure increase followed by a sustained decrease of these parameters and the heart rate. In isolated rats hearts Sp-CTx (10(-9) to 5 × 10(-6) M) produced concentration-dependent and transient ventricular positive inotropic effect and vasoconstriction response on coronary bed. In papillary muscle, Sp-CTx (10(-7) M) also produced an increase in contractile isometric force, which was attenuated by the catecholamine releasing agent tyramine (100 μM) and the β-adrenergic antagonist propranolol (10 μM). On isolated ventricular cardiomyocytes Sp-CTx (1 nM) increased the L-type Ca(2+) current density. The results show that Sp-CTx induces disorders in the cardiovascular system through increase of sarcolemmal calcium influx, which in turn is partially caused by the release of endogenous noradrenaline.

Background High plasma cholesterol levels are able to trigger several pathophysiological events, including inflammation, cell damage and especially oxidative stress. Previously, studies have shown that sildenafil exhibited antioxidant effects in several experimental models. Here we evaluate the role of sildenafil in liver redox equilibrium of apolipoprotein E knockout (apoE-KO) mice. Methods ApoE-KO mice were divided in two groups: one group received the PDE5 inhibitor sildenafil (40 mg/kg/day) for 3 weeks (apoE-KO + Sil) and was compared to a second group of apoE-KO mice, which received only the vehicle (water) for 3 weeks (apoE-KO). Control group (C57 mice) received only a standard chow diet. At the age of 18 weeks, mice livers were collected for the measurement of intracellular ROS levels and apoptotic cells by flow cytometry analysis, and mitochondria isolation for proteomic analysis. Results Compared to the control group, liver cells from apoE-KO presented some typical redox imbalance features: higher levels of intracellular ROS (global oxidative stress ˜60%, superoxide anion ˜82%, and peroxynitrite/hydroxyl radical ˜53%), higher amounts of apoptotic cells (up to ˜19%) and higher mitochondrial intensity of catalase (+339%) and transferrin spots (+914%). After treatment with sildenafil, apoE-KO presented ROS levels and the number of apoptotic cells similar to those observed in C57. In addition, when compared to apoE-KO, apoE-KO + Sil showed lower spots volumes of catalase (-23%) and transferrin (-71%) and up-regulation of urate oxidase (+94%). Conclusion The treatment with sildenafil is able to induce beneficial changes in liver mitochondrial protein dynamics, which restores the redox homeostasis contributing to a potential hepatoprotection.

In the present study, we successfully demonstrated for the first time the existence of cardiac proteomic differences between non-selectively bred rats with distinct intrinsic exercise capacities. A proteomic approach based on two-dimensional gel electrophoresis coupled to mass spectrometry was used to study the left ventricle (LV) tissue proteome of rats with distinct intrinsic exercise capacity. Low running performance (LRP) and high running performance (HRP) rats were categorized by a treadmill exercise test, according to distance run to exhaustion. The running capacity of HRPs was 3.5-fold greater than LRPs. Protein profiling revealed 29 differences between HRP and LRP rats (15 proteins were identified). We detected alterations in components involved in metabolism, antioxidant and stress response, microfibrillar and cytoskeletal proteins. Contractile proteins were upregulated in the LVs of HRP rats (α-myosin heavy chain-6, myosin light chain-1 and creatine kinase), whereas the LVs of LRP rats exhibited upregulation in proteins associated with stress response (aldehyde dehydrogenase 2, α-crystallin B chain and HSPβ-2). In addition, the cytoskeletal proteins desmin and α-actin were upregulated in LRPs. Taken together, our results suggest that the increased contractile protein levels in HRP rats partly accounted for their improved exercise capacity, and that proteins considered risk factors to the development of cardiovascular disease were expressed in higher amounts in LRP animals.

Spatially distinct mitochondrial subpopulation may mediate myocardial pathology through permeability transition pore opening (MPTP). The goal of this study was to assess sex differences on the two spatially distinct mitochondrial subpopulations: subsarcolemmal mitochondria (SSM) and intermyofibrillar mitochondria (IFM) based on morphology, membrane potential, mitochondrial function, oxidative phosphorylation, and MPTP. Aged matched Wistar rats were used to study SSM and IFM. Mitochondrial size was larger in SSM than in IFM in both genders. However, SSM internal complexity, yield, and membrane potential were higher in male than in female. The maximal rate of mitochondrial respiration, states 3 and 4, using glutamate + malate as substrate, were higher in IFM and SSM in the male group compared to female. The respiratory control ratio (RCR-state3/state 4), was not different in both SSM and IFM with glutamate + malate. The ADP:O ratio was found higher in IFM and SSM from female compared to males. When pyruvate was used, state 3 was found unchanged in both IFM and SSM, state 4 was also greater in male IFM compared to female. The RCR increased in the SSM while IFM remained the same. State 4 was higher in male SSM while in the IFM remained the same. The IFM presented a higher Ca(2+) retention capacity compared with SSM, however, there was a greater sensitivity to Ca(2+)-induced MPTP in SSM and IFM in the male group compared to female. In conclusion, our data show that spatially distinct mitochondrial subpopulations have sex-based differences in oxidative phosphorylation, morphology, and calcium retention capacity.

PnTx4(6-1), henceforth renamed δ-Ctenitoxin-Pn1a (δ-CNTX-Pn1a), a peptide from Phoneutria nigriventer spider venom, initially described as an insect toxin, binds to site 3 of sodium channels in nerve cord synaptosomes and slows down sodium current inactivation in isolated axons in cockroaches (Periplaneta americana). δ-CNTX-Pn1a does not cause any apparent toxicity to mice, when intracerebroventricularly injected (30 μg). In this study, we evaluated the antinociceptive effect of δ-CNTX-Pn1a in three animal pain models and investigated its mechanism of action in acute pain. In the inflammatory pain model, induced by carrageenan, δ-CNTX-Pn1a restored the nociceptive threshold of rats, when intraplantarly injected, 2 h and 30 min after carrageenan administration. Concerning the neuropathic pain model, δ-CNTX-Pn1a, when intrathecally administered, reversed the hyperalgesia evoked by sciatic nerve constriction. In the acute pain model, induced by prostaglandin E2, intrathecal administration of δ-CNTX-Pn1a caused a dose-dependent antinociceptive effect. Using antagonists of the receptors, we showed that the antinociceptive effect of δ-CNTX-Pn1a involves both the cannabinoid system, through CB1 receptors, and the opioid system, through μ and δ receptors. Our data show, for the first time, that δ-Ctenitoxin-Pn1a is able to induce antinociception in inflammatory, neuropathic and acute pain models.

Poisoning by organophosphorus insecticides is often accompanied by cardiac complications which may be serious and even fatal. However, the effects of these compounds on the cardiovascular mechanisms involved in blood pressure regulation are not known. The aim of this study was to evaluate the effects of a sublethal dose (8 mg/kg, i.p.) of the organophosphorus methamidophos on chemoreceptor (CR) and Bezold-Jarisch (BJR) cardiovascular reflexes. Male Wistar rats were treated with single intraperitoneal injections of methamidophos in saline (n=23) or saline (0.9 percent, n=20) and underwent catheterization of femoral artery and vein one day after the injections. Cardiovascular recordings were performed 24h after the catheterization procedure. Plasma cholinesterase (ChE) activity was measured 24h after similar treatments in separate groups (n=10/group). The bradycardic component of CR and BJR was significantly attenuated in animals treated with methamidophos. The ChE activity was 80 percent reduced in the methamidophos-treated animals. Methamidophos impairment of the bradycardic component of two important cardiovascular reflexes may contribute to the cardiovascular toxicity associated with acute organophosphorus insecticides exposure.

The aim of the present study was to investigate the cardiovascular activity of Scorpaena plumieri venom in both in vivo and in vitro models. In anesthetized rats, doses of the venom (14-216 microg protein/kg) induced a transient increase in the mean arterial pressure. However at higher dose (338 microg protein/kg) this effect was followed by a sudden hypotension and the animal evolved to death. The heart rate was temporarily increased and followed by bradycardia using dosesor =108 microg/kg. In isolated rat hearts the crude venom (5-80 microg protein) produced dose-dependent positive ventricular chronotropic, inotropic, lusitropic and coronary vasoconstriction responses. Partial purification of an active fraction (CF, cardiovascular fraction) which reproduced the cardiovascular effects induced by crude venom on isolated hearts was achieved by conventional gel filtration chromatography. Adrenergic blockades, prazosin and propranolol, significantly attenuated these responses. The coronary vasoconstriction response to CF was also attenuated by chemical endothelium denudation. In conclusion, the data showed that S. plumieri fish venom induces disorders in the cardiovascular system. It also suggests that alpha(1) and beta-adrenergic receptors, and the vascular endothelium, are involved at least partially, in these cardiac effects.

Background Several studies show that the consumption of vegetable oils, such as soybean oil, rich in polyunsaturated fatty acids (PUFAs) has beneficial health effects by preventing or reducing the risk factors of cardiovascular diseases. While the demonstration of beneficial effects of the consumption of unsaturated fatty acids on the cardiovascular system has been proven in a macroscopic level, the molecular/cellular mechanisms responsible for this phenomenon are poorly understood. Methods In this work, a comparative proteomic approach, two-dimensional gel electrophoresis (2-DE) coupled to mass spectrometry (MALDI-TOF/TOF), was applied to investigate proteome differences in the left ventricle (LV) of rats that received 0.1 mL of soybean oil intramuscularly for 15 days (treated group - TR) and rats that had not (control group - CT). Results Soybean oil treatment improved left ventricular function, TR animals presented lower value of LVEDP and significantly changed LV proteome. The protein profile of VE revealed differences in the expression of 60 protein spots (p

A potent insecticidal toxin, β/δ-PrIT1, molecular mass of 5598.86 [M+H](+), was characterized from Phoneutria reidyi spider venom. Its partial amino acid sequence showed high similarity with insecticidal spider toxins from the genus Phoneutria. β/δ-PrIT1 was very toxic (LD50 = 4 nmol/g) to flies (Musca domestica), but not to mice (Mus musculus). Kinetic studies showed that (125)I-β/δ-PrIT1 binds to two distinct sites in insect sodium channels, with close affinity (Kd1 = 34.7 pM and Kd2 = 35.1 pM). Its association is rather fast (t1/2(1) = 1.4 min, t1/2(2) = 8.5 min) and its dissociation is a slower process (t1/2(1) = 5.4 min, t1/2(2) = 32.8 min). On rat brain synaptosomes β/δ-PrIT1 partially competed (∼30%) with the beta-toxin (125)I-CssIV, but did not compete with the alpha-toxin of reference (125)I-AaII, nor with the beta-toxin (125)I-TsVII. On cockroach nerve cord synaptosomes, β/δ-PrIT1 did not compete with the anti-insect toxin (125)I-LqqIT1, but it competed (IC50 = 80 pM) with the "alpha-like" toxin (125)I-BomIV. In cockroach neurons, β/δ-PrIT1 inhibited the inactivation of Nav-channels and it shifted the sodium channel activation to hyperpolarizing potentials. These results indicate two different binding sites for β/δ-PrIT1, leading to two different pharmacological responses. β/δ-PrIT1 is one of the most toxic spider toxins to insects without apparent toxicity to mammals, and provide new model for the development of insecticides.

In this work we describe some biological properties and a partial biochemical characterization of the Scorpanea plumieri crude venom. The fresh venom induced a decrease in blood pressure, cardiac and respiratory frequency, and exhibited hemorrhagic, hemolytic and proteolytic activities. The LD(50) (i.v. mouse) was 0.28 mg/kg. The pharmacological activities were found to be very unstable and this fact could be associated with proteolytic activity. Enzymes which hydrolyze casein and gelatin were found in this venom. A gelatinolytic protease (Sp-GP) was purified to homogeneity from S. plumieri venom through a combination of three chromatographic steps: gel filtration on Sephacryl S-200; ion exchange on DEAE-cellulose and reverse-phase/HPLC on a Vydac C4 column. The purified protease was approximately 2% of the whole protein in the soluble crude venom. The molecular mass of the Sp-GP scorpionfish gelatinase estimated by SDS-PAGE was around 80,000 Da under reducing conditions and 72,000 Da under non-reducing conditions. Attempts to determine the N-terminal sequence by automatic Edman degradation were unsuccessful, probably due to blockage of the N-terminal group. Gelatinolytic activity was optimal at pH 7-8. This is the first report of the isolation and characterization of a scorpionfish venom protease.

Several pools of neurotoxic peptides obtained from fractionated Phoneutria nigriventer venom induce different toxicological effects. One of them, PhTx4, is highly toxic towards insects and displays only a slight toxicity when injected in mice. Also, this fraction contains a class of peptides that are able to inhibit glutamate uptake in preparations of mammalian central nervous systems (CNS). In this work a new toxin called PnTx4-3 was isolated from the PhTx4 fraction by reverse phase and anion exchange steps using high performance liquid chromatography (HPLC). Edman sequencing of PnTx4-3 revealed that it was a polypeptide of 48 amino acid residues, containing 10 cysteines cross-linked by five disulfide bridges. The molecular mass measured by ES-Q-TOF mass spectrometry was 5199.49±0.64 Da, which is very close to the calculated mass from amino acid sequence (5199.99 Da). This toxin induces immediate excitatory effects when injected intrathoracically in house flies and cockroaches. Intracerebroventricular injections of 30 μg of PnTx4-3 in mice resulted in no apparent signs of intoxication. In order to make an orthologous comparison, pharmacological characterisation were carried out in rat brain synaptosomes by using [ 3 H]- l -glutamate, showed that the whole PhTx4 fraction as well as the pure toxins PnTx4-3, Tx4(6-1) and Tx4(5-5) obtained of this fraction, were able to inhibit the glutamate uptake in the micromolar concentration range. PnTx4-3 inhibits the glutamate uptake in a dose dependent manner, with an IC 50 of approximately 1 μM. PnTx4-3 is highly homologous to the Tx4(6-1) and Tx4(5-5) toxins previously described from the same fraction.

The mechanism of miltefosine-resistance in Leishmania spp. has been partially determined in experimental resistant lines; however, studies using clinical isolates with different miltefosine susceptibilities are still needed. In our study, we used a proteomic 2D-DIGE/MS approach to study different protein abundances in miltefosine-sensitive and -resistant Leishmania infantum chagasi isolates from visceral leishmaniasis patients with different miltefosine treatment outcomes. The high-resolution proteome obtained from these isolates showed 823 matched spots and 46 spots exhibited different abundances between the isolates. Out of these differentially expressed spots, 26 (56.5%) showed greater and 20 (43.5%) showed lower expression of the resistant isolate compared to the sensitive isolate. MALDI/TOF–TOF mass spectrometry allowed the identification of 32 spots with unique protein identification correspondent to 22 non-redundant proteins. Most of the proteins up-regulated in the proteome miltefosine-resistant isolates were associated with redox homeostasis, stress response, protection to apoptosis, and drug translocation. These differentially expressed proteins are likely involved in miltefosine natural resistance and suggest that the miltefosine-resistance mechanism in Leishmania is multifactorial. Biological significance Visceral leishmaniasis (VL) is a serious disease with a challenging treatment plan requiring the prolonged and painful applications of poorly tolerated toxic drugs. Therefore, the identification of miltefosine, an effective and safe oral drug, was considered a significant advancement in leishmaniasis therapy. However, different sensitivities to miltefosine in Leishmania have been observed in clinically relevant species, and the biological mechanism by which clinical isolates of Leishmania acquire drug resistance is poorly understood. Our work aims to elucidate the mechanism of natural resistance to miltefosine in Leishmania by studying the isolates from VL patients who displayed different miltefosine treatment outcomes.

The Scorpaena plumieri fish venom induces a severe pain and edema, observed both clinically and experimentally. In order to understand more about the envenomation syndrome, the present study characterized experimentally the local acute inflammatory response induced by S. plumieri venom (SpV) in a mouse model of tissue injury. Our results demonstrated that the local inflammatory response provoked after 2 h of SpV injection in footpad of mice is characterized by release of pivotal pro-inflammatory mediators (TNF, IL-6 and MCP-1). These mediators could be associated with histopathological changes observed into paw tissue, characterized by cellular infiltration, mainly neutrophils. Additionally, an investigation of edema formation pathways involved in inflammatory response was performed. SpV-induced edema was reduced significantly by previous administration of aprotinin or icatibant (HOE-140). However, the pre-treatment with diclofenac sodium and promethazine had less effect on this response. These results demonstrate that the kallikrein-kinin system (KKS) plays a major role in the edema formation. Despite the whole venom hydrolyzed the kallikrein synthetic substrate S-2302 (Pro-Phe-Arg-pNA), its main pro-inflammatory fraction was devoid of kininogenase activity. Our results demonstrate that SpV evokes a complex inflammatory reaction stimulating a secretion of TNF, IL-6, MCP-1 and leukocytes recruitment at the site of venom injection. In addition provide clear evidence of the involvement of the KKS in inflammatory response induced by S. plumieri venom.

Venomous fish are often involved in human accidents and symptoms of envenomation include local (intense pain and swelling) and systemic effects (cardiovascular and neurological disorders). However the only commercially available antivenom is against the Indo-Pacific stonefish Synanceja trachynis S tone f ish A nti v enom (SFAV). The aim of the present study was to evaluate the potential of SFAV in neutralising the in vivo effects of some toxic activities of scorpionfish Scorpaena plumieri venom (SpV), and the in vitro immuno cross-reactivity. The SpV (7.5–100 μg/animal) caused nociceptive and dose-dependent edematogenic responses in the mice footpad. In rats SpV (300 μg/kg, i.v.) produced immediate and transient increase in arterial blood pressure and decrease in heart rate. Prior incubation of SpV with SFAV (1 μg SpV/1 U SFAV) abolished the inflammatory response, and significantly attenuated the cardiovascular effects induced by SPV. Western blotting analysis on two-dimensional SDS-PAGE of S plumieri venom proteins using SFAV proved that the epitopes recognized by SFAV are shared with the ∼98 kDa proteins. This is the first report of venom similarities between Indo-Pacific and Atlantic venomous fish, suggesting that the SpV compound responsible for inflammatory and cardiovascular effects possesses similar biochemical and antigenic properties to those found in stonefish venom.

The proteomes of the venoms of the Brazilian wandering “armed” spiders Phoneutria nigriventer , Phoneutria reidyi , and Phoneutria keyserlingi , were compared using two-dimensional gel electrophoresis. The venom components were also fractionated using a combination of preparative reverse phase HPLC on Vydac C4, analytical RP-HPLC on Vydac C8 and C18 and cation exchange FPLC on Resource S at pH 6.1 and 4.7, or anion exchange HPLC on Synchropak AX-300 at pH 8.6. The amino acid sequences of the native and S-pyridyl-ethylated proteins and peptides derived from them by enzymatic digestion and chemical cleavages were determined using a Shimadzu PPSQ-21 A automated protein sequencer, and by MS/MS collision induced dissociations. To date nearly 400 peptides and proteins (1.2– 27 kDa) have been isolated in a pure state and, of these, more than 100 have had their complete or partial amino acid sequences determined. These sequences demonstrate, as might be expected, that the venoms of P. reidyi and P. keyserlingi (Family: Ctenidae) both contain a similar range of isoforms of the neurotoxins as those previously isolated from P. nigriventer which are active on neuronal ion (Ca 2+ , Na + and K + ) channels and NMDA-type glutamate receptors. In addition two new families of small (3–4 kDa) toxins, some larger protein (> 10 kDa) components, and two serine proteinases of the venom of P. nigriventer are described. These enzymes may be responsible for some of the post-translational modification observed in some of the venom components.