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Cardiovascular effects induced by snake venoms, in spite of having a crucial role in the outcome of the envenomation, have been less studied than other toxic activities displayed by these venoms. In this study we evaluated acute cardiovascular responses to Bothrops leucurus venom - Bl-V - both in vivo, in anesthetized rats, and in vitro, in isolated rat mesenteric resistance arteries. Bl-V (10–100 μg protein/kg) caused dose-dependent hypotension, followed by gradual recovery (2–20 min) to basal levels, and induced dose-dependent (1–20 μg/mL) vasodilation in pre-contracted arteries, what was more pronounced when the endothelium remained intact. These effects were partially counteracted by pre-treatment with indomethacin (cyclooxygenase inhibitor). Prior incubation of Bl-V with commercial pentavalent Bothrops antivenom also attenuated the cardiovascular effects induced by the venom, in spite of it not being among the venoms used for the development of the bothropic antivenom. Through an approach based on two chromatographic steps and mass spectrometry (MALDI-ToF and MALDI-ISD), a component with acute cardiovascular effects was isolated and identified as the basic phospholipase blD-PLA2, previously purified from the venom of B. leucurus. Taken together, our results show that, at low doses, the venom of B. leucurus induces transient, acute hypotension in anesthetized rats following systemic vasodilation in a dose-dependent way. In addition, we provide clear evidence of the involvement of the enzymatic activity of blD-PLA2 in this cardiovascular response, acting via the production of vasodilating prostanoids.

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

Proteins that account for the hemolytic activity found in scorpaeniform fish venoms are responsible for the majority of the effects observed upon envenomation, for instance, neurotoxic, cardiotoxic and inflammatory effects. These multifunctional toxins, described as protein lethal factors and referred to as cytolysins, are known to be extremely labile molecules. In the present work, we endeavored to overcome this constraint by determining optimal storage conditions for Sp-CTx, the major bioactive component from the scorpionfish Scorpaena plumieri venom. This cardiotoxic hemolytic cytolysin is a large dimeric glycoprotein (subunits of ≈65 kDa) with pore-forming ability. We were able to establish storage conditions that allowed us to keep the toxin partially active for up to 60 days. Stability was achieved by storing Sp-CTx at −80 and −196 °C in the presence of glycerol 10% in a pH 7.4 solution. It was demonstrated that the hemolytic activity of Sp-CTx is calcium dependent, being abolished by EDTA and zinc ions. Furthermore, the toxin exhibited its maximal hemolytic activity at pH between 8 and 9, displaying typical N- and O- linked glycoconjugated residues (galactose (1–4) N-acetylglucosamine and sialic acid (2–3) galactose in N- and/or O-glycan complexes). The hemolytic activity of Sp-CTx was inhibited by phosphatidylglycerol and phosphatidylethanolamine, suggesting a direct electrostatic interaction lipid - toxin in the pore-formation mechanism of action of this toxin. In addition, we observed that the hemolytic activity was inhibited by increasing doses of cholesterol. Finally, we were able to show, for first time, that Sp-CTx is at least partially responsible for the pain and inflammation observed upon envenomation. However, while the edema induced by Sp-CTx was reduced by pre-treatment with aprotinin and HOE-140, pointing to the involvement of the kallikrein-kinin system in this response, these drugs had no significant effect in the toxin-induced nociception. Taken together, our results could suggest that, as has been already reported for other fish cytolysins, Sp-CTx acts mostly through lipid-dependent pore formation not only in erythrocytes but also in other cell types, which could account for the pain observed upon envenomation. We believe that the present work paves the way towards the complete characterization of fish cytolysins.

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.

The most poisonous fish species found along the Brazilian coast is the spotted scorpionfish Scorpaena plumieri. Though hardly ever life-threatening to humans, envenomation by S. plumieri can be quite hazardous, provoking extreme pain and imposing significant socioeconomic costs, as the victims may require days to weeks to recover from their injuries. In this review we will walk the reader through the biological features that distinguish this species as well as the current epidemiological knowledge related to the envenomation and its consequences. But above all, we will discuss the challenges involved in the biochemical characterization of the S. plumieri venom and its compounds, focusing then on the successful isolation and pharmacological analysis of some of the bioactive molecules responsible for the effects observed upon envenomation as well as on experimental models. Despite the achievement of considerable progress, much remains to be done, particularly in relation to the non-proteinaceous components of the venom. Therefore, further studies are necessary in order to provide a more complete picture of the venom’s chemical composition and physiological effects. Given that fish venoms remain considerably less studied when compared to terrestrial venoms, the exploration of their full potential opens a myriad of possibilities for the development of new drug leads and tools for elucidating the complex physiological processes.