Phoneutria Nigriventer Spider Venom Research Articles

Lethality and histopathological alterations caused by Phoneutria nigriventer spider venom from Argentina: Neutralization of lethality by experimental and therapeutic antivenoms

Although the spiders of the genus Phoneutria cause envenomation and their presence has been described in several provinces of the north of Argentina, they are not as common as other spiders of sanitary importance. In the present work, we studied the toxicity of samples of venom of Phoneutria spiders from the provinces of Misiones (where severe envenomation and deaths by Phoneutria have been recorded) and Jujuy (where no deaths have been recorded and severe envenomations are not frequent). To this end, we assessed the lethal potency in mice and guinea pigs and the histopathological alterations caused by both venoms, as well as the neutralization by the commonly used therapeutic antivenom produced by the Butantan Institute in Brazil and by an experimental antivenom developed with venom of P. nigriventer from Misiones. There were no differences in the lethality of the venoms of spiders from both regions. Post mortem examination showed that the heart and lungs were the most affected organs, while important pulmonary edema was seen macroscopically. Histological analysis showed edema, atelectasis, emphysema and cardiac lesion in both experimental models. The antivenoms assayed showed good neutralization of the venoms in the two experimental models. Despite the different geographic origins, the venoms showed similar toxicity and both the experimental antivenom and therapeutic antivenmos were able to neutralize the venoms of Argentinean P. nigriventer.

δ-Ctenitoxin-Pn1a, a Peptide from Phoneutria nigriventer Spider Venom, Shows Antinociceptive Effect Involving Opioid and Cannabinoid Systems, in Rats.

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.

Are Synchronized Changes in Connexin-43 and Caveolin-3 a Bystander Effect in a Phoneutria nigriventer Venom Model of Blood-Brain Barrier Breakdown?

Upregulation of caveolin-3 (Cav-3) or connexin-43 (Cx43) in astrocytes has been associated with important brain pathologies. We used Phoneutria nigriventer spider venom (PNV), which induces blood-brain barrier breakdown in rats, in order to investigate Cav-3 and Cx43 expression in the cerebellum over critical periods of rat envenomation. By immunofluorescence, western blotting (WB), and transmission electron microscopy (TEM), we assessed changes at 1, 2, 5, 24, and 72h post-venom. WB showed immediate increases in Cav-3 and Cx43 at 1h (interval of greatest manifestations of envenomation) that persisted at 5h (when there were signs of recovery) and peaked at 24h when no signs of envenomation were detectable. At 2 and 72h, Cav-3 was downregulated and Cx43 had returned to baseline. PNV markedly intensified Cx43 in molecular, Purkinje and granular layers and Cav-3 in astrocytes whose colocalization to increased GFAP suggests interaction between reactive astrogliosis and Cav-3 upregulation. TEM showed swollen perivascular astrocytic end-feet and synaptic contact alterations that had generally resolved by 72h. It is uncertain whether such PNV-induced synchronized changes are an interactive effect between Cav-3 and Cx43, or a bystander effect. Evidences indicate that Cav-3 downregulation coupled to Cx43 return to baseline at 72h when no signs of envenomation were visible, suggesting homeostasis reestablishment. This experimental model is relevant to studying mechanisms involved in neurological disorders associated with Cav-3 overexpression.

Caveolae as a target for Phoneutria nigriventer spider venom

An important transcellular transport mechanism in the blood-brain barrier (BBB) involves caveolae, which are specialized delta-shaped domains of the endothelial plasma membrane that are rich in cholesterol, glycosphingolipids and the scaffolding protein Caveolina-1 (Cav-1). In this work, we investigated whether the increase in endocytosis and transendothelial vesicular trafficking in rat cerebellum after blood-brain barrier breakdown (BBBb) induced by Phoneutria nigriventer spider venom (PNV) was mediated by caveolae. The expression of Cav-1, phosphorylated Cav-1 (pCav-1), dynamin-2 (Dyn2), Src kinase family (SKF) and matrix-metalloproteinase-9 (MMP9), proteins involved in caveolar dynamics and BBB opening, was investigated. Immunofluorescence, western blotting (WB) and transmission electron microscopy were used to assess changes at 1, 2, 5, 24 and 72h post-venom. WB showed upregulation of Cav-1, Dyn2 and MMP9 at 1, 5 and 72h (corresponding, respectively, to intervals when intoxication was most evident, when signs of recovery were present, and when no intoxication was detectable). In contrast, pCav-1 and SKF, which are essential for internalization and transport, decreased when Cav-1 and Dyn2, proteins essential for caveolar formation, were increased. Overall, these changes indicated that vesicular trafficking across the endothelium (high pCav/SKF levels) coincided with lower numbers of caveolae (Cav-1/Dyn2 downregulation) and lower expression of MMP9. Thus, the internalization (disassembly) of caveolae alternates with caveolar neoformation (assembly), resulting in changes in caveolar density in the endothelium membrane. These caveolar dynamics imply tensional mechanical stress that is important in triggering key signaling mechanisms. We conclude that PNV-induced breakdown of transcellular transport in the BBB is caused by an increase in caveolae-mediated endocytosis; this effect was correlated with the progression of temporal signs of envenoming. Caveolar dynamics are probably involved in shear stress and BBBb regulatory mechanisms in this experimental model.

Triggering of protection mechanism against Phoneutria nigriventer spider venom in the brain.

Severe accidents caused by the “armed” spider Phoneutria nigriventer cause neurotoxic manifestations in victims. In experiments with rats, P. nigriventer venom (PNV) temporarily disrupts the properties of the BBB by affecting both the transcellular and the paracellular route. However, it is unclear how cells and/or proteins participate in the transient opening of the BBB. The present study demonstrates that PNV is a substrate for the multidrug resistance protein-1 (MRP1) in cultured astrocyte and endothelial cells (HUVEC) and increases mrp1 and cx43 and down-regulates glut1 mRNA transcripts in cultured astrocytes. The inhibition of nNOS by 7-nitroindazole suggests that NO derived from nNOS mediates some of these effects by either accentuating or opposing the effects of PNV. In vivo, MRP1, GLUT1 and Cx43 protein expression is increased differentially in the hippocampus and cerebellum, indicating region-related modulation of effects. PNV contains a plethora of Ca2+, K+ and Na+ channel-acting neurotoxins that interfere with glutamate handling. It is suggested that the findings of the present study are the result of a complex interaction of signaling pathways, one of which is the NO, which regulates BBB-associated proteins in response to PNV interference on ions physiology. The present study provides additional insight into PNV-induced BBB dysfunction and shows that a protective mechanism is activated against the venom. The data shows that PNV has qualities for potential use in drug permeability studies across the BBB.

Evidences of endocytosis via caveolae following blood–brain barrier breakdown by Phoneutria nigriventer spider venom

Spider venoms contain neurotoxic peptides aimed at paralyzing prey or for defense against predators; that is why they represent valuable tools for studies in neuroscience field. The present study aimed at identifying the process of internalization that occurs during the increased trafficking of vesicles caused by Phoneutria nigriventer spider venom (PNV)-induced blood–brain barrier (BBB) breakdown. Herein, we found that caveolin-1α is up-regulated in the cerebellar capillaries and Purkinje neurons of PNV-administered P14 (neonate) and 8- to 10-week-old (adult) rats. The white matter and granular layers were regions where caveolin-1α showed major upregulation. The variable age played a role in this effect. Caveolin-1 is the central protein that controls caveolae formation. Caveolar-specialized cholesterol- and sphingolipid-rich membrane sub-domains are involved in endocytosis, transcytosis, mechano-sensing, synapse formation and stabilization, signal transduction, intercellular communication, apoptosis, and various signaling events, including those related to calcium handling. PNV is extremely rich in neurotoxic peptides that affect glutamate handling and interferes with ion channels physiology. We suggest that the PNV-induced BBB opening is associated with a high expression of caveolae frame-forming caveolin-1α, and therefore in the process of internalization and enhanced transcytosis. Caveolin-1α up-regulation in Purkinje neurons could be related to a way of neurons to preserve, restore, and enhance function following PNV-induced excitotoxicity. The findings disclose interesting perspectives for further molecular studies of the interaction between PNV and caveolar specialized membrane domains. It proves PNV to be excellent tool for studies of transcytosis, the most common form of BBB-enhanced permeability.

Vascular endothelial growth factor increases during blood-brain barrier-enhanced permeability caused by Phoneutria nigriventer spider venom.

Phoneutria nigriventer spider accidental envenomation provokes neurotoxic manifestations, which when critical, results in epileptic-like episodes. In rats, P. nigriventer venom (PNV) causes blood-brain barrier breakdown (BBBb). The PNV-induced excitotoxicity results from disturbances on Na+, K+ and Ca2+ channels and glutamate handling. The vascular endothelial growth factor (VEGF), beyond its angiogenic effect, also, interferes on synaptic physiology by affecting the same ion channels and protects neurons from excitotoxicity. However, it is unknown whether VEGF expression is altered following PNV envenomation. We found that adult and neonates rats injected with PNV showed immediate neurotoxic manifestations which paralleled with endothelial occludin, β-catenin, and laminin downregulation indicative of BBBb. In neonate rats, VEGF, VEGF mRNA, and Flt-1 receptors, glutamate decarboxylase, and calbindin-D28k increased in Purkinje neurons, while, in adult rats, the BBBb paralleled with VEGF mRNA, Flk-1, and calbindin-D28k increases and Flt-1 decreases. Statistically, the variable age had a role in such differences, which might be due to age-related unequal maturation of blood-brain barrier (BBB) and thus differential cross-signaling among components of the glial neurovascular unit. The concurrent increases in the VEGF/Flt-1/Flk-1 system in the cerebellar neuron cells and the BBBb following PNV exposure might imply a cytokine modulation of neuronal excitability consequent to homeostatic perturbations induced by ion channels-acting PNV neuropeptides. Whether such modulation represents neuroprotection needs further investigation.

Expression of VEGF and Flk-1 and Flt-1 Receptors during Blood-Brain Barrier (BBB) Impairment Following Phoneutria nigriventer Spider Venom Exposure

Apart from its angiogenic and vascular permeation activity, the vascular endothelial growth factor (VEGF) has been also reported as a potent neuronal protector. Newborn rats with low VEGF levels develop neuron degeneration, while high levels induce protective mechanisms in several neuropathological conditions. Phoneutria nigriventer spider venom (PNV) disrupts the blood-brain barrier (BBB) and causes neuroinflammation in central neurons along with excitotoxic signals in rats and humans. All these changes are transient. Herein, we examined the expression of VEGF and its receptors, Flt-1 and Flk-1 in the hippocampal neurons following envenomation by PNV. Adult and neonatal rats were evaluated at time limits of 2, 5 and 24 h. Additionally, BBB integrity was assessed by measuring the expression of occludin, β-catenin and laminin and neuron viability was evaluated by NeuN expression. VEGF, Flt-1 and Flk-1 levels increased in PNV-administered rats, concurrently with respective mRNAs. Flt-1 and Flk-1 immunolabeling was nuclear in neurons of hippocampal regions, instead of the VEGF membrane-bound typical location. These changes occurred simultaneously with the transient decreases in BBB-associated proteins and NeuN positivity. Adult rats showed more prominent expressional increases of the VEGF/Flt-1/Flk-1 system and earlier recovery of BBB-related proteins than neonates. We conclude that the reactive expressional changes seen here suggest that VEGF and receptors could have a role in the excitotoxic mechanism of PNV and that such role would be less efficient in neonate rats.

Temporal relationship between aquaporin-4 and glial fibrillary acidic protein in cerebellum of neonate and adult rats administered a BBB disrupting spider venom

Two astrocyte markers, the glial water channel aquaporin-4 (AQP4) and the glial fibrillary acidic protein (GFAP), have been implicated in several physiological and pathological conditions in the central nervous system (CNS) as well as in blood–brain barrier breakdown (BBBb). By color segmentation the immunoreactivity of both proteins, we demonstrate that the expression of AQP4 and GFAP was increased in the cerebellum of neonate (14-day-old, P14) and adult (8-week-old) rats administered Phoneutria nigriventer spider venom (PNV) known to cause perivascular edema, BBBb and convulsion. In the cerebellum's gray matter, PNV produced a major response, especially in the granular layer. Parallel increases in AQP4 and GFAP expression occurred 24 h after envenomation in the white matter of P14 and in the molecular layer of adults, as well as in the granular layer 2 h after envenomation. In the Purkinje layer there was a tendency of increased AQP4, for both, neonates (5 h), and adults (2 and 24 h). Moreover, PNV also provoked nonparallel upregulation of both markers with prevalence of upregulation of AQP 4 for P14 rats, and GFAP for adults. The major expression of both proteins was in the gray matter. The data indicates a venom effect in water/electrolyte balance in the cerebellum and the participation of AQP4 in these effects. Age-related and time-related regional differences probably reflect specificity in AQP4 distribution in different astrocytic membrane domains as well as its participation in K+ buffering and neural activity. This study is the first to associate astrocytic AQP4 expression and reactive gliosis in a model of BBB permeability promoted by P. nigriventer venom. Our data provide compelling evidence that AQP4 expression was increased in the cerebellum of rats administered PNV.

Erectile Function Is Improved in Aged Rats by PnTx2‐6, a Toxin from Phoneutria nigriventer Spider Venom

Age-associated erectile dysfunction (ED) involves a decrease in nitric oxide (NO) availability and impaired relaxation. PnTx2-6, a toxin from the Phoneutria nigriventer spider, has been demonstrated to improve erectile function via NO/cyclic guanosine monophosphate (cGMP) pathway. This spider's venom is characterized by several symptoms, including erection. PnTx2-6 has been implicated in this phenomenon. Animal venoms have been postulated as potential drugs to treat ED. PnTx2-6 toxin improves erectile function in aged rats via NO/cGMP. We investigated the effect of PnTx2-6 in the erectile function of aged rats. ED was evaluated through changes in intracavernosal pressure/mean arterial pressure ratio during electrical field stimulation (EFS) of the pelvic ganglion of aged and adult rats (70 vs. 14 weeks). In functional studies, EFS-induced relaxation of corpus cavernosum (CC) strips were performed with or without PnTx2-6 (10-8M). The decrease in erectile function associated with age was partially restored 15-20 minutes after injection of PnTx2-6 and further improved by sildenafil. PnTx2-6 enhanced EFS-induced relaxation, as well as cGMP levels in CC, from young and aged rats. Relaxation due to PnTx2-6 was further increased after 30 minutes incubation with Y-27632, a Rho-kinase inhibitor (10-6 M), in aging CC. Nitric oxide synthase (NOS) activity in aged and young cavernosal tissue was increased by incubation with PnTx2-6 (10 minutes). However, this toxin did not modify NOS expression. PnTx2-6 improves penile relaxation in aged rats, via increased NOS activity and NO release, resulting in enhanced cGMP levels.

Effect of Phoneutria nigriventer Venom on the Expression of Junctional Protein and P-gp Efflux Pump Function in the Blood–Brain Barrier

Phoneutria nigriventer spider venom (PNV) contains Ca(2+), K(+) and Na(+) channel-acting peptides that affect neurotransmitter release and causes excitotoxicity in PNS and CNS. It has been demonstrated that PNV causes blood-brain barrier (BBB) breakdown of hippocampal microvessels time-dependently through enhanced microtubule-mediated vesicular transport. Herein, it is hypothesized that PNV can cause BBB breakdown in the hippocampus and cerebellum time-dependently through other molecular mechanisms. The BBB integrity was assessed through the analysis of expression of Poly-glycoprotein (P-gp) efflux transporter protein, laminin from basement membrane and endothelial tight junctional and adhesion junctional (TJ/AJ) proteins. Phosphatase and tensin homolog (PTEN) and protein phosphatase 2A (PP2A) expression, which are known to have a role in the phosphorylation of junctional proteins and BBB opening, were also investigated. Astrocytes P-gp activity was determined by flow cytometry. The study demonstrated temporary decreased expression of laminin, TJ and AJ proteins (ZO1//occludin//claudin-5//beta-catenin) and P-gp (more prominently in hippocampus), which was completely or partially resolved between 2 and 5h (and more quickly for cerebellum). PNV inhibited P-gp activity in astrocytes. PP2A phosphorylation, which inhibits the enzyme activity, was increased in both regions (15-45min); however the phosphorylation level returned to baseline after 2h. In conclusion, PNV disrupts paracellular transport in the BBB and possesses substrates for the active P-gp efflux transporter located in the BBB complex. Further studies into cellular mechanisms of astrocyte/endothelial interactions, using PNV as tool, may identify how astrocytes regulate the BBB, a characteristic that may be useful for the temporary opening of the BBB.

Increased cavernosal relaxation by Phoneutria nigriventer toxin, PnTx2-6, via activation at NO/cGMP signaling

Erectile dysfunction mechanisms in diabetic patients are multifactorial and often lead to resistance to current therapy. Animal toxins have been used as pharmacological tools to study penile erection. Human accidents involving the venom of Phoneutria nigriventer spider are characterized by priapism. We hypothesize that PnTx2-6 potentiates cavernosal relaxation in diabetic mice by increasing cGMP. This effect is nNOS dependent. Cavernosal strips were contracted with phenylephrine (10−5 M) and relaxed by electrical field stimulation (EFS, 20V, 1–32 Hz) in the presence or absence of PnTx2-6 (10−8 M).Cavernosal strips from nNOS and eNOS knocaut (KO) mice, besides nNOS inhibitor (10−5M), were used to evaluate the role of this enzyme in the potentiation effect evoked by PnTx2-6. Tissue cGMP levels were determined after stimulation with PnTx2-6 in presence or absence of L-NAME (10−4M) and ω-conotoxin GVIA (10−6M), an N-type calcium channel inhibitor. Results showed PnTx2-6 enhanced cavernosal relaxation in diabetic mice (65%) and eNOS KO mice, but not in nNOS KO mice. The toxin effect in the cavernosal relaxation was abolished by nNOS inhibitor. cGMP levels are increased by PnTx2-6, however L-NAME abolished this enhancement as well as ω-conotoxin GVIA. We conclude PnTx2-6 facilitates penile relaxation in diabetic mice through a mechanism dependent on nNOS, probably via increasing NO/cGMP production.

Determination of sphingomyelinase-D activity of Loxosceles venoms in sphingomyelin/cholesterol liposomes containing horseradish peroxidase

Based on degradation of sphingomyelin/cholesterol liposomes containing entrapped horseradish peroxidase, we evaluated the Sphingomyelinase-D (SMase-D) activity of scorpion, spider and snake venoms by monitoring spectrophotometrically the product of oxidation of HRP released. The results indicate that Loxosceles crude venoms (Loxosceles intermedia, Loxosceles laeta, Loxosceles gaucho and Loxosceles similis) displayed SMase-D activity in a concentration-dependent manner. Furthermore, this activity was blocked by the anti-loxoscelic antivenom. However, Tityus serrulatus scorpion venom, Phoneutria nigriventer spider venom and Bothrops jararaca, Crotalus durissus, Lachesis muta and Micrurus frontalis snake venoms did not show measurable SMase-D activity.

Functional expression of a recombinant toxin – rPnTx2-6 – active in erectile function in rat

In the current study, the putative cDNA for PnTx2-6 toxin of the Phoneutria nigriventer spider venom was cloned and expressed as tioredoxin fusion protein in the cytoplasm of Escherichia coli. The fusion protein was purified from the bacterial extracts by combination of immobilized Ni–ion affinity and gel filtration chromatographies. Then, it was cleaved by enterokinase and the generated recombinant PnTx2-6 (rPnTx2-6) was further purified by reverse-phase HPLC. Likewise the native toxin purified from the spider venom, rPnTx2-6 potentiates the erectile function when injected in rats. This result indicates that the production of functional recombinant PnTx2-6 might be an alternative to provide this basic and valuable tool for study, as well as for further understanding such complex physiological system, including its correlation with the central nervous system and local tissue factors.

Improvement of relaxation in Type II diabetic mice corpus cavernosum by PhTx2‐6 toxin from Phoneutria nigriventer spider

Type II diabetes mellitus (T2DM) is a major risk factor for development of erectile dysfunction (ED). Mechanisms causing ED in T2DM are multifactorial and often lead to resistance to current therapy. Animal toxins have been used as pharmacological tools to study penile erection. Human accidents involving the venom of Phoneutria nigriventer spider are characterized by priapism. The toxin PhTx2‐6 purified from this venom inhibits sodium (Na+) channel inactivation in neuronal‐type Na+ channels. We hypothesize that PhTx2‐6 potentiates cavernosal relaxation in T2DM mice. Cavernosal strips were pre‐contracted with phenylephrine (10−5 M) and relaxed by electrical field stimulation (EFS) in the presence or absence of PnTx2‐6 (10−8 M). cGMP levels from mice tissue were determined after stimulation with PhTx2‐6 in presence or absence of L‐NAME (10−4M). Results showed that PhTx2‐6 enhanced cavernosal relaxation in diabetic mice (± 65%) and the toxin was not able to reverse the inhibition by L‐NAME. cGMP levels are increased by PhTx2‐6 (± 1.57 vs. basal ±0.86), however L‐NAME abolished this enhancement. We conclude that PhTx2‐6 facilitates penile relaxation in diabetic mice probably via increasing NO release and consequently cGMP production. This toxin may be an important tool for development of new therapeutic approaches to treat ED in T2DM.

Neuroinflammation and astrocytic reaction in the course of Phoneutria nigriventer (armed-spider) blood–brain barrier (BBB) opening

Phoneutria nigriventer spider venom (PNV) causes uneven BBB permeability throughout different cerebral regions. Little is known about cellular and molecular responses which course with the PNV-induced BBB opening. We investigate by immunohistochemistry (IHC) and Western blotting (WB), the GFAP, S100, IFN-γ and TNF-α proteins expression in hippocampus and cerebellum after different time-points from venom or saline intravenous injection. All proteins variably altered its expression temporally and regionally. WB showed increased GFAP content at 15–45 min followed by a shift below the control level which was less pronounced in hippocampus. IHC showed reactive gliosis during all the trial period. In cerebellum, GFAP was mostly immunodetected in astrocytes of the molecular layer (Bergmann glia), as was S100 protein. The maximum S100 immunolabeling was achieved at 5 h. IFN-γ and TNF-α, expressed mostly by hippocampal neurons, increased along the trial period, suggesting a role in BBB permeability. In envenomed animals, closer contacts astrocyte–astrocyte, granule cells–granule cells and astrocytes-Purkinje cells were observed in cerebellum. Closer contacts between neurons–neurons–astrocytes–astrocytes were also seen in hippocampus. PNV contains serotonin, histamine, Ca 2+ channels-blocking toxins, some of which affect glutamate release. The hypothesis that such substances plus the cytokines generated, could have a role in BBB permeability, and that calcium homeostasis loss and disturbance of glutamate release are associated with the marked GFAP/S100 reaction in Bergmann glia is discussed. The existence of a CNS mechanism of defense modulated differentially for fast synthesis and turnover of GFAP, S100, IFN-γ and TNF-α proteins was evident. A clear explanation for this differential modulation is unclear, but likely result from regional differences in astrocytic/neuronal populations, BBB tightness, and/or extent/distribution of microvasculature and/or ion channels density/distribution. Such differences would respond for transient characteristics of BBB disruption. This in vivo model is useful for studies on drug delivery throughout the CNS and experimental manipulation of the BBB.

NNOS mediates relaxation in corpus cavernosum mice strips improved by Tx2‐6 toxin from Phoneutria nigriventer spider via cGMP increase

A toxin from the venom of Phoneutria nigriventer spider, Tx2‐6, causes priapism in mice and relaxation of rat cavernosal tissue. The aim of this study is to investigate the involvement of NOS isoforms and cGMP in the action of the toxin Tx2‐6 on erectile tissue. We isolated cavernosum strips from eNOS and nNOS knock out (KO) mice. The strips were incubated with bretylium tonsilate (3x10−5M) to block adrenergic neurotransmission. Following contraction induced by phenylephrine (10−5 M), the strips were made to relax in response to electrical field stimulation (EFS). Cumulative concentration‐response curves to acetylcholine (10−9 to 10−5M) were performed in absence and presence of Tx2‐6. cGMP levels were analyzed using kit (Cayman) in presence of Tx2‐6 and this toxin plus L‐NAME (10−4M). The action of the N‐type calcium channels antagonist, ω‐conotoxin GVIA (10−6M) on EFS was evaluated. Results showed relaxation increased in the presence Tx2‐6 toxin (10nM) in wild type and eNOS KO mice but not in nNOS KO mice. Acetylcholine seems not be involve in the relaxation improved by Tx2‐6. cGMP levels significantly increased in the presence of Tx2‐6. This increase was abolished by L‐NAME and ω‐conotoxin GVIA. This study indicates that Tx2‐6 improves the erectile response, probably via activation of calcium channels by slowing the inactivation of Na+ channels in nitrergic nerves facilitating NO production from nNOS and consequently increased cyclic GMP.

Characterization of the mechanisms underlying the inflammatory response to Polistes lanio lanio (paper wasp) venom in mouse dorsal skin

Stings by Polistes wasps can cause life-threatening allergic reactions, pain and inflammation. We examined the changes in microvascular permeability and neutrophil influx caused by the venom of Polistes lanio a paper wasp found in southeastern Brazil. The intradermal injection of wasp venom caused long-lasting paw oedema and dose-dependently increased microvascular permeability in mouse dorsal skin. SR140333, an NK 1 receptor antagonist, markedly inhibited the response, but the NK 2 receptor antagonist SR48968 was ineffective. The oedema was reduced in capsaicin-treated rats, indicating a direct activation of sensory fibres. Dialysis of the venom partially reduced the oedema and the remaining response was further inhibited by SR140333. Mass spectrometric analysis of the venom revealed two peptides (QPPTPPEHRFPGLM and ASEPTALGLPRIFPGLM) with sequence similarities to the C-terminal region of tachykinin-like peptides found in Phoneutria nigriventer spider venom and vertebrates. Wasp venom failed to release histamine from mast cells in vitro and spectrofluorometric assay of the venom revealed a negligible content of histamine in the usual dose of P. l. lanio venom (1 nmol of histamine/7 μg of venom) that was removed by dialysis. The histamine H 1 receptor antagonist pyrilamine, but not bradykinin B 1 or B 2 receptor antagonists, inhibited venom-induced oedema. In conclusion, P. l. lanio venom induces potent oedema and increases vascular permeability in mice, primarily through activation of tachykinin NK 1 receptors by substance P released from sensory C fibres, which in turn releases histamine from dermal mast cells. This is the first description of a neurovascular mechanism for P. l. lanio venom-mediated inflammation. The extent to which the two tachykinin-like peptides identified here contribute to this neurogenic inflammatory response remains to be elucidated.

C-FOS and n-NOS reactive neurons in response to circulating Phoneutria nigriventer spider venom

Drugs and neurotoxins activate specific neural circuits by increasing or decreasing the formation and release of neurotransmitters, such as nitric oxide (NO), and by inducing immediate early genes, such as FOS. We have previously shown that Phoneutria nigriventer spider venom (PNV) impairs the microtubule-dependent transcellular barrier of the blood–brain interface and causes structural alterations in perivascular astrocytic end-feet without producing morphological changes in central neuronal cells. In the present study, we used FOS and neuronal nitric oxide synthase (n-NOS) immunolabeling to investigate the ability of PNV to activate the central nervous system. Three groups of rats were used: the first group received a sublethal dose of PNV (850 μg/kg, via a tail vein), the second received an equal volume of 0.9% saline (sham group) and the third group received no injection. Envenomed rats showed salivation, lachrymation, tremors and flaccidity followed by spastic paralysis of the hind limbs and convulsions. Cryosections (30 μm thick) were serially collected at 600 μm intervals for free-floating immunohistochemical analysis. FOS-like positive neurons predominated in motor-related areas such as dorsolateral (dlPAG) and ventral periaqueductal gray matter (vPAG), frontal (FCM) and parietal motor cortex (PCM), and periventricular thalamic nucleus (PTN) and in acute stress-related areas (rhinal cortex and lateral septal nuclei). The greatest relative increases in FOS-like positive neurons occurred in the vPAG, PCM and PTN motor-related areas. n-NOS-positive neurons predominated in the periventricular thalamic nuclei, followed by the dorsolateral periaqueductal gray matter and parietal cortex motor area. The marked activation of motor areas and, to a lesser extent, of acute stress-related areas suggested the involvement of neuronal pathways in these regions in the response to envenoming by PNV. In addition, the occurrence of n-NOS immunolabeling in some anatomical regions with FOS-like positive neurons suggests that NO may modulate the response to PNV in these regions.

Acute blood–brain barrier permeabilization in rats after systemic Phoneutria nigriventer venom

A highly controlled transport of substances at the interface between blood and brain characterizes the blood–brain barrier (BBB), fundamental for maintenance of the homeostasis of the cerebral milieu. In this study, we investigated the time course (15 min, 1, 2, and 5 h) of BBB opening induced by intravenous (i.v.) injection of Phoneutria nigriventer spider venom (PNV) using quantitative and morphological approaches on cerebellum and hippocampus vessels for assessment of BBB permeability. The results showed vasogenic edema and tracer extravasation faster and severalfold higher in hippocampus than in cerebellum. Reactive astrocytes with swollen perivascular end-feet processes were found only in cerebellum. An immediate and total degradation of laminin in capillaries occurred resulting in the disappearance of the basement membrane. In medium-sized vessels, this effect was less prominent. The changes were transient, with cerebellum in general presenting a faster recovery. However, at 5 h laminin was overexpressed, principally in hippocampus. The rapid and abrupt shift of laminin expression in capillaries (at 15 min) coincided with the immediate and severe signs of intoxication shown by the animals, but not with the peak of leakage of vessels and vasogenic edema, which occurred later (1–2 h). The findings suggest a complex regulatory mechanism, since the extension of BBB impairment caused by PNV depends on the region of the SNC, and on the vessels types. It is suggested that the components of the BBB (gliovascular unit) have a critical role in these differences. P. nigriventer venom can be a useful tool to explore the mechanisms of BBB.