Methanolic extract of Rhinella schneideri (Anura: Bufonidae) poison causes ultrastructural changes in nerve terminals of phrenic nerve-diaphragm preparations in mice / Extracto metanólico de Rhinella schneideri (Anura: Bufonidae) causa cambios ultraestructurales en las terminaciones nerviosas de preparaciones de nervio frénico de ratón

Abstract Rhinella schneideri (or Bufo paracnemis), popularly known in Brazil as cururu toad, is also found in other countries in South America. The cardiovascular effects of this poison are largely known and recently was shown that it is capable to affect the neuromuscular junction on avian and mice isolated preparation. In this work, we used transmission electron microscopy to investigate the ultrastructure of the motor nerve terminal and postsynaptic junctional folds of phrenic nerve-hemidiaphragm preparations incubated for either 5 or 60 min with the methanolic extract of R. schneideri (50 µg/mL). In addition, the status of the acetylcholine receptors (AChR) was examined by TRITC-α-bungarotoxin immunofluorescence location at the endplate membrane. The results show that 5 min of incubation with the gland secretion extract significantly decreased (32 %) the number of synaptic vesicles into the motor nerve terminal, but did not decrease the electron density on the top of the junctional folds where nicotinic receptors are concentrated; however, 60 min of incubation led to significant nerve terminal reloading in synaptic vesicles whereas the AChR immunoreactivity was not as marked as in control and after 5 min incubation. Muscle fibers were well-preserved but intramuscular motor axons were not. The findings corroborated pharmacological data since the decrease in the number of synaptic vesicles (5 min) followed by recovery (60 min) is in accordance with the transient increase of MEPPs frequency meaning increased neurotransmitter release. These data support the predominant presynaptic mode of action of the R. schneideri, but do not exclude the possibility of a secondary postsynaptic action depending on the time the preparation is exposed to poison. Rev. Biol. Trop. 66(3): 1290-1297. Epub 2018 September 01.

Resumen Rhinella schneideri (o Bufo paracnemis), conocido popularmente en Brasil como sapo cururu, también se encuentra en otros países de América del Sur. Los efectos cardiovasculares de este veneno son ampliamente conocidos y recientemente se demostró que es capaz de afectar la unión neuromuscular en la preparación aislada de aves y ratones. En este trabajo, utilizamos microscopía electrónica de transmisión para investigar la ultraestructura de la terminación nerviosa motora y pliegues de unión postsináptica de preparaciones de nervio frénico-hemidiafragma incubadas durante 5 o 60 min con el extracto metanólico de R. schneideri (50 μg/mL). Además, se examinó el estado de los receptores de acetilcolina (AChR) mediante la ubicación de inmunofluorescencia de TRITC-α-bungarotoxina en la membrana de la placa terminal. Los resultados muestran que 5 min de incubación con el extracto de secreción de glándula disminuyeron significativamente (32 %) el número de vesículas sinápticas en el terminal del nervio motor, pero no disminuyeron la densidad electrónica en la parte superior de los pliegues de unión donde se concentran los receptores nicotínicos. Sin embargo, 60 min de incubación condujeron a una recarga significativa de los terminales nerviosos en las vesículas sinápticas, mientras que la inmunorreactividad del AChR no fue tan marcada como en el control y después de 5 min de incubación. Las fibras musculares estaban bien conservadas, pero los axones motores intramusculares no. Los hallazgos corroboraron los datos farmacológicos ya que la disminución en el número de vesículas sinápticas (5 min) seguida de recuperación (60 min) está de acuerdo con el aumento transitorio de la frecuencia de MEPPs, lo que significa una mayor liberación de neurotransmisores. Estos datos apoyan el modo de acción presináptico predominante de R. schneideri, pero no excluyen la posibilidad de una acción postsináptica secundaria dependiendo del tiempo en que la preparación esté expuesta al veneno.

Development of a double sandwich fluorescent ELISA to detect rattlesnake venom in biological samples from horses with a clinical diagnosis of rattlesnake bite.

Rattlesnake bites in horses are not uncommon and the clinical outcomes are widely variable. Treatment of horses with anti-venom is often cost prohibitive and could have negative consequences; therefore, the development of a quantitative test to determine if anti-venom therapy is indicated would be valuable. The objective of this study was to develop an ELISA to detect rattlesnake venom in biological samples from clinically bitten horses. Nineteen horses were enrolled in the study. Urine was available from 19 horses and bite site samples were available from 9 horses. A double sandwich fluorescent ELISA was developed and venom was detected in 5 of 9 bite site samples and 12 of 19 urine samples. In order to determine if this assay is useful as a guide for treatment, a correlation between venom concentration and clinical outcome needs to be established. For this, first peak venom concentration needs to be determined. More frequent, consistent sample collection will be required to define a venom elimination pattern in horses and determine the ideal sample collection time to best estimate the maximum venom dose. This report describes development of an assay with the ability to detect rattlesnake venom in the urine and at the bite site of horses with a clinical diagnosis of rattlesnake bite.

Antibody responses to natural rattlesnake envenomation and a rattlesnake toxoid vaccine in horses.

Antivenom antibody titers following administration of rattlesnake venom for antivenom production in horses are well documented; however, antivenom antibody titers following natural rattlesnake envenomation in horses are not. Antibody titers produced in response to the commercially available rattlesnake venom vaccine are also not published. Our study objectives were to measure antivenom antibody titers in rattlesnake-bitten horses and compare them to titers in horses vaccinated with the rattlesnake venom vaccine. Additionally, titers were compared in pregnant versus nonpregnant horses to assess the affect of pregnancy on vaccine response and were measured pre- and postsuckle in foals of vaccinated mares to detect passive transfer of vaccine immunoglobulins. Blood samples were collected from 16 rattlesnake-bitten horses. Thirty-six horses (11 pregnant mares, 12 nonpregnant mares, 13 geldings) were vaccinated using a Crotalus atrox venom toxoid vaccine. Blood was collected before administering each vaccination and 30 days following the third vaccination. Blood was collected from foals of vaccinated mares pre- and postsuckle. All serum was assayed for anti-Crotalus atrox venom antibodies using an enzyme-linked immunosorbent assay (ELISA). Rattlesnake-bitten horses had higher (P = 0.001) titers than vaccinated horses. There was no significant difference between titers in vaccinated pregnant versus nonpregnant horses. One mare had a positive titer at foaling, and the foals had positive postsuckle titers. Antivenom antibody titer development was variable following natural envenomation and vaccination, and vaccine-induced titers were lower than natural envenomation titers. Further studies are required to determine if natural or vaccine antivenom antibody titers reduce the effects of envenomation.

Increase of the cytotoxic effect of Bothrops jararacussu venom on mouse extensor digitorum longus and soleus by potassium channel blockers and by Na(+)/K(+)-ATPase inhibition.

We investigated the myotoxicity of Bothrops jararacussu crude venom and other cytolytic agents on mouse isolated extensor digitorum longus (EDL) and soleus (SOL) muscles, which present distinct properties: EDL is a fast-twitch, white muscle with predominantly glycolytic fibers, while SOL is slow-twitch, red muscle with predominantly oxidative fibers. Muscles were exposed to B. jararacussu crude venom (25 microg/ml) and other crotaline venoms (Agkistrodon contortrix laticinctus; Crotalus viridis viridis; Crotalus durissus terrificus) at the same concentration. Basal creatine kinase (CK) release to bathing solution was 0.43+/-0.06 for EDL and 0.29+/-0.06 for SOL (U g(-)(1) h(-)(1), n=36 for each muscle). Sixty minutes after exposure to B. jararacussu venom, EDL presented higher increase in the rate of CK release than SOL, respectively, 13.2+/-1.5 and 2.9+/-0.7 U g(-)(1)h(-)(1), n=10-12. Muscle denervation, despite decreasing CK content, did not affect sensitivities to B. jararacussu venom. Ouabain and potassium channel blockers (TEA; clotrimazole; glibenclamide) increased the rate of CK release by B. jararacussu in EDL and SOL muscles, decreasing and almost abolishing the different sensitivity. When we exposed EDL or SOL muscles to Naja naja, Apis mellifera venoms (25 microg/ml), or Triton X-100 (0.01%), they showed similar rate of CK release. Our present data suggest that a mechanism involving intracellular calcium regulation or potassium channels may participate in the different sensitivity of EDL and SOL to B. jararacussu venom.

A molecular mechanism for Lys49-phospholipase A2 activity based on ligand-induced conformational change.

Agkistrodon contortrix laticinctus myotoxin is a Lys(49)-phospholipase A(2) (EC 3.1.1.4) isolated from the venom of the serpent A. contortrix laticinctus (broad-banded copperhead). We present here three monomeric crystal structures of the myotoxin, obtained under different crystallization conditions. The three forms present notable structural differences and reveal that the presence of a ligand in the active site (naturally presumed to be a fatty acid) induces the exposure of a hydrophobic surface (the hydrophobic knuckle) toward the C terminus. The knuckle in A. contortrix laticinctus myotoxin involves the side chains of Phe(121) and Phe(124) and is a consequence of the formation of a canonical structure for the main chain within the region of residues 118-125. Comparison with other Lys(49)-phospholipase A(2) myotoxins shows that although the knuckle is a generic structural motif common to all members of the family, it is not readily recognizable by simple sequence analyses. An activation mechanism is proposed that relates fatty acid retention at the active site to conformational changes within the C-terminal region, a part of the molecule that has long been associated with Ca(2+)-independent membrane damaging activity and myotoxicity. This provides, for the first time, a direct structural connection between the phospholipase "active site" and the C-terminal "myotoxic site," justifying the otherwise enigmatic conservation of the residues of the former in supposedly catalytically inactive molecules.

Membrane depolarization is the initial action of crotoxin on isolated murine skeletal muscle.

Although much is known about the pathogenesis of crotoxin-induced muscle damage, the initial site and action of the toxin is still not clear. In this study we used an electrochromic fluorescent dye, Di-4-ANEPPS, to measure the changes in membrane potential of isolated murine omohyoid muscle to determine if depolarization could be one of the initial effects of crotoxin. Omohyoid isolates were pre-loaded with 1 microM Di-4-ANEPPS, exposed to various crotoxin treatments, and the change in fluorescence was recorded using either a dual-wavelength spectrofluorometer or digital imaging. Spectrofluorometry indicated that crotoxin depolarized isolated omohyoid muscles within 4 min as indicated by an increase in fluorescence to 122% of control values. Crotoxin also induced depolarization of extensor digitorum longus and soleus muscles as indicated by an increase in fluorescence of 140 and 110% of the control, respectively. Fluorescent images obtained from omohyoid muscle preparations exposed to crotoxin and Di-4-ANEPPS revealed localized areas of increased fluorescence, muscle contractions, derangement of myofibrils, and differing sensitivity to crotoxin of different muscle cells. Light microscopy results confirmed this variable disruption of muscle cell integrity and differing sensitivity to crotoxin. An increase in creatine kinase release rates confirmed damage to the plasma membrane. We conclude that plasma membrane depolarization is most likely the earliest indicator of cell damage from crotoxin and is quickly followed by hypercontraction of myofilaments, disruption of the plasma membrane, release of creatine kinase and necrosis.

Preclinical assessment of the ability of polyvalent (Crotalinae) and anticoral (Elapidae) antivenoms produced in Costa Rica to neutralize the venoms of North American snakes.

Polyvalent (Crotalinae) and anticoral (Elapidae) antivenoms produced by Instituto Clodomiro Picado, Costa Rica, were assessed for their ability to neutralize various toxic activities of the venoms of North American snakes of the genera Crotalus, Agkistrodon and Micrurus, in assays involving preincubation of venom and antivenom. When the intraperitoneal route of injection was utilized, polyvalent (Crotalinae) antivenom was effective in the neutralization of the venoms of Crotalus atrox, Crotalus adamanteus, Crotalus viridis viridis, Crotalus horridus atricaudatus, Agkistrodon contortrix contortrix and Agkistrodon piscivorus piscivorus, whereas the venom of Crotalus scutulatus was not neutralized. When the intravenous route was used, results differed depending on the "challenge dose" of venom employed. Polyvalent antivenom neutralized all venoms when mice were challenged with 2 LD(50)s of venom. When 5 LD(50)s were used, antivenom neutralized the venoms of C. atrox, C. adamanteus, C. v. viridis and C. h. atricaudatus, being ineffective in the neutralization of C. scutulatus, A. c. contortrix and A. p. piscivorus. Polyvalent antivenom was effective in the neutralization of hemorrhagic and myotoxic activities of all venoms studied. It also neutralized coagulant activity of C. adamanteus venom, whereas most of the venoms were devoid of clotting activity on plasma in vitro. Moreover, it neutralized defibrinating activity of the only three venoms that induced this effect (i.e. C. adamanteus, A. c. contortrix and A. p. piscivorus). Anticoral (Elapidae) antivenom neutralized lethality induced by the venom of Micrurus fulvius, using either the intravenous or the intraperitoneal routes of injection. Moreover, it neutralized myotoxic effect of this venom as well. It is concluded that polyvalent antivenom neutralizes lethality and other activities of most of the crotaline venoms tested. However, since it is ineffective in neutralizing the lethal effect of C. scutulatus venom, it is suggested that a venom containing presynaptically-active neurotoxic phospholipases A(2) related to "mojave toxin" needs to be introduced in the immunizing mixture in order to increase the neutralizing scope of this product in North America. Anticoral antivenom is highly effective in the neutralization of the venom of M. fulvius.

Skeletal muscle degeneration induced by venom phospholipases A2: insights into the mechanisms of local and systemic myotoxicity.

Local and systemic skeletal muscle degeneration is a common consequence of envenomations due to snakebites and mass bee attacks. Phospholipases A2 (PLA2) are important myotoxic components in these venoms, inducing a similar pattern of degenerative events in muscle cells. Myotoxic PLA2s bind to acceptors in the plasma membrane, which might be lipids or proteins and which may differ in their affinity for the PLA2s. Upon binding, myotoxic PLA2s disrupt the integrity of the plasma membrane by catalytically dependent or independent mechanisms, provoking a pronounced Ca2+ influx which, in turn, initiates a complex series of degenerative events associated with hypercontraction, activation of calpains and cytosolic Ca(2+)-dependent PLA2s, and mitochondrial Ca2+ overload. Cell culture models of cytotoxicity indicate that some myotoxic PLA2s affect differentiated myotubes in a rather selective fashion, whereas others display a broad cytolytic effect. A model is presented to explain the difference between PLA2s that induce predominantly local myonecrosis and those inducing both local and systemic myotoxicity. The former bind not only to muscle cells, but also to other cell types, thereby precluding a systemic distribution of these PLA2s and their action on distant muscles. In contrast, PLA2s that bind muscle cells in a more selective way are not sequestered by non-specific interactions with other cells and, consequently, are systemically distributed and reach muscle cells in other locations.

Characterization of aquaporin-4 in muscle and muscular dystrophy.

Aquaporins are a growing family of transmembrane proteins that transport water and, in some cases, glycerol and urea across cellular membranes. Aquaporin-4 (AQP4) is enriched at the sarcolemma of skeletal muscle and may play a role in accommodating the rapid changes in cell volume and hydrostatic forces that occur during contraction in order to prevent damage to the sarcolemma. Recent evidence has shown that AQP4 is absent in dystrophin-deficient mdx mice, suggesting that AQP4 associates with dystrophin and has a role in the dystrophic process. To examine the relationship between aquaporins and muscle disease, and between aquaporins and dystrophin, we have investigated aquaporin expression in various mouse models of muscular dystrophy and cardiomyopathy before and after the onset of pathology. We find that AQP4 is expressed in prenecrotic mdx muscle despite the absence of dystrophin and that AQP4 is lost after the onset of muscle degeneration. Analysis of various dystrophin transgenic mice reveals that AQP4 is lost even when the dystrophin-glycoprotein complex is present, suggesting that loss of AQP4 is not directly resulting from loss of the DGC. AQP4 was also lost in muscular dystrophies caused by primary mutations in the sarcoglycan genes. Taken together, our data demonstrate that AQP4 loss in skeletal muscle correlates with muscular dystrophy and is a common feature of pathogenesis.