Phoneutria Nigriventer Venom Research Articles

Recombinant Production, NMR Solution Structure, and Membrane Interaction of the Phα1β Toxin, a TRPA1 Modulator from the Brazilian Armed Spider Phoneutria nigriventer.

Phα1β (PnTx3-6) is a neurotoxin from the spider Phoneutria nigriventer venom, originally identified as an antagonist of two ion channels involved in nociception: N-type voltage-gated calcium channel (CaV2.2) and TRPA1. In animal models, Phα1β administration reduces both acute and chronic pain. Here, we report the efficient bacterial expression system for the recombinant production of Phα1β and its 15N-labeled analogue. Spatial structure and dynamics of Phα1β were determined via NMR spectroscopy. The N-terminal domain (Ala1-Ala40) contains the inhibitor cystine knot (ICK or knottin) motif, which is common to spider neurotoxins. The C-terminal α-helix (Asn41-Cys52) stapled to ICK by two disulfides exhibits the µs-ms time-scale fluctuations. The Phα1β structure with the disulfide bond patterns Cys1-5, Cys2-7, Cys3-12, Cys4-10, Cys6-11, Cys8-9 is the first spider knottin with six disulfide bridges in one ICK domain, and is a good reference to other toxins from the ctenitoxin family. Phα1β has a large hydrophobic region on its surface and demonstrates a moderate affinity for partially anionic lipid vesicles at low salt conditions. Surprisingly, 10 µM Phα1β significantly increases the amplitude of diclofenac-evoked currents and does not affect the allyl isothiocyanate (AITC)-evoked currents through the rat TRPA1 channel expressed in Xenopus oocytes. Targeting several unrelated ion channels, membrane binding, and the modulation of TRPA1 channel activity allow for considering Phα1β as a gating modifier toxin, probably interacting with S1-S4 gating domains from a membrane-bound state.

Comparison between a Phoneutria nigriventer toxin treatment and galantamine treatment in a memory deficit mouse model

Acetylcholine modulates circuits related to cognitive functions (attention, cognitive flexibility, memory, and social interaction). Consequences of low acetylcholine levels in cholinergic synaptic clefts include memory and sociability impairments. VAChT KDHET mice exhibit diminished vesicular acetylcholine transporter protein production, which could evoke low acetylcholine quantal release and memory and sociability issues. It has been demonstrated that the PhKv toxin, isolated from the Phoneutria nigriventer (Brazilian wandering spider) venom, inhibits the enzyme responsible for acetylcholine hydrolysis, acetylcholinesterase. This could result in acetylcholine remaining longer in the synaptic cleft, as well as alleviating cognitive problems caused by low levels of this neurotransmitter. Hence, here we intended to investigate VAChT KDHET mice cognitive deficits in a behavioral assay - the novel object recognition task - and examine the potential effect of the PhKv toxin in memory improvement of this mouse model. First, we noted that VAChT KDHET mice showed impaired object recognition memory. We also observed that these deficits, especially those related to short-term memory in the behavioral test, are sex-related. Subsequently, to assess the effects of PhKv toxin in object recognition memory, we injected PhKv or galantamine in VAChT KDHET mice and compared their performance in the novel object recognition task. We noted that mice treated with PhKv performed similarly to mice treated with galantamine or both vehicles in this behavioral assay. Finally, we observed that mice treated with both vehicles displayed memory improvements compared to non-treated (naive) mutant mice. Together, our results imply that PhKv may have an effect on memory improvement, which might be further explored to elucidate its mechanisms of action.

Isobolographic analysis reveals antinociceptive synergism between Phα1β recombinant toxin and morphine in a model of cancer pain in C57BL/6J mice.

Background:Phoneutria nigriventer venom contains Phα1β. This toxin and its recombinant form have a remarkable analgesic potential that is associated with blockage of voltage-gated calcium channels and TRPA1 receptors. Although morphine is a mainstay drug to treat moderate and severe pain related to cancer, it has serious and dose-limiting side effects. Combining recombinant Phα1β and morphine to treat pain is an interesting approach that has been gaining attention. Therefore, a quantitative and reliable method to establish the strength of the antinociceptive interaction between these two substances is necessary. The present study was designed to investigate the nature of the functional antinociceptive (analgesic) interaction between Phα1β recombinant toxin and morphine in a model of cancer pain.Methods:Melanoma was produced by intraplantar inoculation of B16-F10 cells into the right paw of C57BL/6J mice. Von Frey filaments measured the paw-withdrawal threshold after intrathecal administration of morphine, recombinant Phα1β, and their combination. Thermal hyperalgesia was assessed using Hargreaves apparatus. The degree of interaction was evaluated using isobolographic analysis. Spontaneous and forced motor performance was assessed with the open-field and rotarod tests, respectively.Results:Co-administration of recombinant Phα1β and morphine synergistically reverses the melanoma-induced mechanical hyperalgesia. The potency of the mixture, measured as the effective dose to reach 50% of maximum possible effect (MPE) in ameliorating mechanical hyperalgesia, was about twice fold higher than expected if the interaction between morphine and recombinant Phα1β was merely additive. Treatment with the combination at doses necessary to reach 50% of MPE caused no spontaneous nor forced motor alterations.Conclusion:The combinatorial use of recombinant Phα1β and morphine allows significant and effective dose reduction of both agents, which has translational potential for opioid-sparing approaches in pain management related to cancer.

The peptide Phα1β, from spider venom, acts as a TRPA1 channel antagonist with antinociceptive effects in mice.

Peptides from venomous animals have long been important for understanding pain mechanisms and for the discovery of pain treatments. Here, we hypothesized that Phα1β, a peptide from the venom of the armed spider Phoneutria nigriventer, produces analgesia by blocking the TRPA1 channel. Cultured rat dorsal root ganglion (DRG) neurons, human fetal lung fibroblasts (IMR90) or HEK293 cells expressing the human TRPA1 (hTRPA1-HEK293), human TRPV1 (hTRPV1-HEK293) or human TRPV4 channels (hTRPV4-HEK293), were used for calcium imaging and electrophysiology. Nociceptive responses induced by TRPA1, TRPV1 or TRPV4 agonists or by bortezomib were investigated in mice. Phα1β selectively inhibited calcium responses and currents evoked by the TRPA1 agonist, allyl isothiocyanate (AITC), on hTRPA1-HEK293, IMR90 fibroblasts and DRG neurons. Phα1β did not affect calcium responses evoked by selective TRPV1 (capsaicin) or TRPV4 (GSK 1016790A) agonists on the various cell types. Intrathecal (i.t.) and intraplantar (i.pl.) administration of low doses of Phα1β (up to 300pmol per paw) attenuated acute nociception and mechanical and cold hyperalgesia evoked by AITC (i.t. or i.pl.), without affecting responses produced by capsaicin or hypotonic solution. Notably, Phα1β abated the TRPA1-dependent neuropathic pain-like responses induced by bortezomib. In vitro and in vivo inhibition of TRPA1 by Phα1β was reproduced by a recombinant form of the peptide, CTK 01512-2. Phα1β and CTK 01512-2 selectively target TRPA1, but not other TRP channels. This specific action underlines the potential of Phα1β and CTK 01512-2 for pain treatment.

Age-Related Modulations of AQP4 and Caveolin-1 in the Hippocampus Predispose the Toxic Effect of Phoneutria nigriventer Spider Venom.

We have previously demonstrated that Phoneutria nigriventer venom (PNV) causes blood–brain barrier (BBB) breakdown, swelling of astrocytes end-feet and fluid permeation into brain interstitium in rats. Caveolae and water channels respond to BBB alterations by co-participation in shear stress response and edema formation/resolution. Herein, we showed post-natal developmental-related changes of two BBB-associated transporter proteins: the endothelial caveolin-1 (Cav-1), the major scaffolding protein from caveolae frame, and the astroglial aquaporin-4 (AQP4), the main water channel protein expressed in astrocytic peri-vascular end-feet processes, in the hippocampus of rats intraperitoneally-administered PNV. Western blotting protein levels; immunohistochemistry (IHC) protein distribution in CA1, CA2, and CA3 subfields; and gene expression by Real Time-Polymerase Chain Reaction (qPCR) were assessed in post-natal Day 14 (P14) and 8–10-week-old rats over critical periods of envenomation. The intensity and duration of the toxic manifestations indicate P14 neonate rats more vulnerable to PNV than adults. Histologically, the capillaries of P14 and 8–10-week-old rats treated with PNV showed perivascular edema, while controls did not. The intensity of the toxic manifestations in P14 decreases temporally (2 > 5 > 24 h), while inversely the expression of AQP4 and Cav-1 peaked at 24 h when clinically PNV-treated animals do not differ from saline controls. IHC of AQP4 revealed that hippocampal CA1 showed the least expression at 2 h when toxic manifestation was maximal. Subfield IHC quantification revealed that in P14 rats Cav-1 peaked at 24 h when toxic manifestations were absent, whereas in 8–10-week-old rats Cav-1 peaked at 2 h when toxic signs were highest, and progressively attenuated such increases until 24 h, remaining though significantly above baseline. Considering astrocyte-endothelial physical and functional interactions, we hypothesize that age-related modulations of AQP4 and Cav-1 might be linked both to changes in functional properties of astrocytes during post-natal development and in the BBB breakdown induced by the venom of P. nigriventer.

Neuropharmacological effects of Phoneutria nigriventer venom on astrocytes

Bites from genus Phoneutria (Ctenidae, Araneomorpha) are the second most frequent source of spider accidents in Southeast Brazil. Severe envenoming from Phoneutria nigriventer produces vision disturbance, tremor and convulsion, suggesting that the CNS is involved; however, the mechanisms by which P. nigriventer venom (PNV) affects the CNS remain poorly understood. The present study aimed to investigate whether PNV directly impairs astrocytes. Cultured astrocytes were exposed to PNV, and intracellular Ca2+ release and signaling were measured (Fura-2/AM), Na+/K+-ATPase and Toll-like receptor 4 (TLR4) involvement were investigated, actin filaments were stained (Alexa™ 488-conjugated phalloidin probe), the G-actin/F-actin ratio was determined, and the expression level of connexin 43 (Cx43) was assessed. Incubation in Ca2+-free buffer did not change the Ca2+ responses. However, pre-incubation in thapsigargin/caffeine completely abolished these responses, suggesting that PNV-evoked Ca2+ transients were from intracellular Ca2+ stores. Pretreatment with a Na+/K+-ATPase antagonist (ouabain) or a TLR4 antagonist (LPS-RS) decreased or increased the Ca2+-evoked transients, respectively. Astrocytes showed altered actin filament structure after PNV exposure. PNV treatment increased the expression levels of Na+/K+-ATPase and Cx43 but decreased those of TLR4. The present results suggest that PNV directly affects astrocytes. Na+/K+-ATPase may thus represent a more specific drug target for controlling the neurotoxicity of PNV.

Characterization of the antinociceptive effect of PhTx3-4, a toxin from Phoneutria nigriventer, in models of thermal, chemical and incisional pain in mice

Venom-derived peptides constitute a unique source of drug prototypes for the pain management. Many of them can modulate voltage-gated calcium channels that are central in the processing of pain sensation. PhTx3-4 is a peptide isolated from Phoneutria nigriventer venom, which blocks high voltage-activated calcium channels with low specificity, thereby leading to neuroprotection in models of ischemia in vitro. The aim of the present work was evaluating the potential of intrathecal PhTx3-4 in the reversal of different nociceptive states in mice, furthermore assessing the potential of PhTx3-4 in triggering motor side effects. We found that bellow 100 pmol/site, PhTx3-4 did not cause major motor side effects. By comparison, ω-conotoxin MVIIA and ω-conotoxin MVIIC triggered motor side effects at the doses of 10 and 100 pmol/site, respectively. Also, PhTx3-4 (30 pmol/site) caused no significant alterations in the forced locomotor activity test (rotarod) and in the exploratory activity test (versamax). In a model of inflammatory persistent pain (formalin test), PhTx3-4 reversed nociceptive behavior both pre or post-administered, although this effect was observed only at the inflammatory phase of the test and not at the neurogenic phase. Comparatively, ω-conotoxin MVIIC was effective only when post-administered in the formalin test. Nonetheless, PhTx3-4 treatment was devoid of action in acute nociceptive thermal model (hotplate test), whereas morphine showed efficacy in this test. Efficacy of PhTx3-4 in the formalin test was associated with inhibition of formalin-induced glutamate release in the cerebrospinal fluid. PhTx3-4, but not ω-conotoxin MVIIC, reversed NMDA-induced nociceptive behavior indicating a putative role of PhTx3-4 at ionotropic glutamate receptors. Finally, we observed efficacy of PhTx3-4 in ameliorating mechanical hypersensitivity induced by paw incision, a post-operative and more clinically relevant pain model. Taken together, our data show that PhTx3-4 possesses antinociceptive effect in different models of pain in mice, suggesting that this toxin may serve as drug prototype for pain control.

MP43-09 THE EFFECT OF PNTX2-6 PROTEIN FROM PHONEUTRIA NIGRIVENTER SPIDER TOXIN ON IMPROVEMENT OF ERECTILE DYSFUNCTION IN A RAT MODEL OF CAVERNOUS NERVE INJURY.

You have accessJournal of UrologySexual Function/Dysfunction/Andrology: Basic Research I1 Apr 2014MP43-09 THE EFFECT OF PNTX2-6 PROTEIN FROM PHONEUTRIA NIGRIVENTER SPIDER TOXIN ON IMPROVEMENT OF ERECTILE DYSFUNCTION IN A RAT MODEL OF CAVERNOUS NERVE INJURY. Yong Sun Choi, Ae Ryang Jung, Sung-Hoo Hong, Sae Woong Kim, Tae-Kon Hwang, and Ji Youl Lee Yong Sun ChoiYong Sun Choi More articles by this author , Ae Ryang JungAe Ryang Jung More articles by this author , Sung-Hoo HongSung-Hoo Hong More articles by this author , Sae Woong KimSae Woong Kim More articles by this author , Tae-Kon HwangTae-Kon Hwang More articles by this author , and Ji Youl LeeJi Youl Lee More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.1166AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail Introduction and Objectives Patients bitten by Phoneutria nigriventer show diverse symptoms involving priapism. This spider venom has been found to augment erectile function by increasing nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) levels. PnTx2-6 of Phoneutria nigriventer venom may be utilized for pharmacological therapy for penile erection.This study was conducted to explore the therapeutic efficacy of subcutaneous PnTx2-6 injection in a rat model of bilateral cavernous nerve crush injury (BCNI) through increased nitric oxide and cyclic guanosine monophosphate (cGMP) levels. Methods Rats were randomly assigned into six groups: Normal group, BCNI group, PBS group (BCNI group with PBS injection), N/C group (BCNI group with Negative control injection), W/T group (BCNI group with wild type recombinant Baculovirus injection), and SEAN group (BCNI group with recombinant Baculovirus containing PnTx2-6 gene injection). Each rat was injected with respective group-assigned agents into the subcutaneous corpus cavernosum once every three days. Four weeks after the operation, we demonstrated ratios of intracavernous pressure (ICP) to mean arterial pressure (MAP). The contents of smooth muscle and collagen were evaluated by Masson¡¯s trichrome staining. The expression of neuronal nitric oxide synthase (nNOS), alpha smooth muscle (¥á-SMA), and endothelial nitric oxide synthase (eNOS) were evaluated by immunohistochemistry. The cyclic guanosine monophosphate (cGMP) level in the corpus cavernosum was determined by cGMP assay. Results The ratio of ICP/MAP was significantly higher in the SEAN group compared to the other experimental groups. The SEAN group also had increased smooth muscle/collagen ratio. The expression of nNOS, ¥á-SMA in SEAN groups was significantly increased, compared with others groups. However, eNOS levels were not significantly different across the groups. The cGMP level was increased in the SEAN group. Conclusions PnTx2-6 improved erectile function and prevented muscle atrophy in a rat model of BCNI via increased NO/cGMP. Therefore, PnTx2-6 may be utilized as a pharmacological therapy for post-prostatectomy ED. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e480 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Yong Sun Choi More articles by this author Ae Ryang Jung More articles by this author Sung-Hoo Hong More articles by this author Sae Woong Kim More articles by this author Tae-Kon Hwang More articles by this author Ji Youl Lee More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

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.

Upregulation of the vascular endothelial growth factor, Flt-1, in rat hippocampal neurons after envenoming by Phoneutria nigriventer; age-related modulation

This study characterizes the distribution and quantifies the expression of the tyrosine kinase receptor for the vascular endothelial growth factor (VEGF), Flt-1, in the rat hippocampus following intra-peritoneal injection of Phoneutria nigriventer venom (PNV). Post-natal day 14 (P14) and 8–10 weeks (adult) old rats were used and analyses were done at 1, 2, 5 and 24 h after venom exposure and compared with saline-injected counterparts. PNV-injected animals showed hippocampal venules with perivascular edema indicating blood–brain barrier (BBB) dysfunction. This was accompanied by significant overexpression of Flt-1 which though was not the same for CA1, CA2, CA3 and dentate gyrus (DG) hippocampal regions, neither for P14 and adult rats. Regional analysis using GIMP methodology showed that Flt-1 was constitutively distributed more densely in neurons of DG, followed by CA1/CA2 and CA3 of both control P14 and adult animals, without variation over time, but significantly more expressed in P14 than in adults. A time-course analysis showed that Flt-1 upregulation was progressive and that neurons VEGFR1/Flt-1+ of PNV-exposed animals are timely and regionally modulated depending on the hippocampal region, being CA2 the least responsive region regardless animal's age, whilst DG was the most susceptible with adult animals having higher upregulation than neonates. Since VEGF has been reported to confer protection in several pathological processes we suggest that VEGF may be involved in hippocampal neurons response via Flt-1 mediation following PNV envenoming; its higher upregulation in adult envenomed rats may be an indication that Flt-1 neuroprotective mediation is more efficient with age. The Flt-1 upregulation and the incidence of perivascular edema in young animals may indicate a pro-inflammatory role of the receptor.

Antinociceptive effect of Brazilian armed spider venom toxin Tx3-3 in animal models of neuropathic pain

Venoms peptides have produced exceptional sources for drug development to treat pain. In this study we examined the antinociceptive and side effects of Tx3-3, a peptide toxin isolated from Phoneutria nigriventer venom, which inhibits high-voltage-dependent calcium channels (VDCC), preferentially P/Q and R-type VDCC. We tested the effects of Tx3-3 in animal models of nociceptive (tail-flick test), neuropathic (partial sciatic nerve ligation and streptozotocin-induced diabetic neuropathy), and inflammatory (intraplantar complete Freund’s adjuvant) pain. In the tail-flick test, both intrathecal (i.t.) and intracerebroventricular (i.c.v.) injection of Tx3-3 in mice caused a short-lasting effect (ED50 and 95% confidence intervals of 8.8 [4.1–18.8] and 3.7 [1.6–8.4]pmol/site for i.t. and i.c.v. injection, respectively), without impairing motor functions, at least at doses 10–30 times higher than the effective dose. By comparison, ω-conotoxin MVIIC, a P/Q and N-type VDCC blocker derived from Conus magus venom, caused significant motor impairment at doses close to efficacious dose in tail flick test. Tx3-3 showed a long-lasting antinociceptive effect in neuropathic pain models. Intrathecal injection of Tx3-3 (30pmol/site) decreased both mechanical allodynia produced by sciatic nerve injury in mice and streptozotocin-induced allodynia in mice and rats. On the other hand, i.t. injection of Tx3-3 did not alter inflammatory pain. Taken together, our data show that Tx3-3 shows prevalent antinociceptive effects in the neuropathic pain models and does not cause adverse motor effects at antinociceptive efficacious doses, suggesting that this peptide toxin holds promise as a novel therapeutic agent for the control of neuropathic pain.The Brazilian armed spider Tx3-3, a new P/Q and R-type calcium channel blocker, effectively alleviates allodynia in animal neuropathic pain models.

Antiarrhythmogenic effects of a neurotoxin from the spider Phoneutria nigriventer

In this study, we evaluated the effects of PhKv, a 4584 Da peptide isolated from the spider Phoneutria nigriventer venom, in the isolated rat heart and in isolated ventricular myocytes. Ventricular arrhythmias were induced by occlusion of the left anterior descending coronary artery for 15 min followed by 30 min of reperfusion. Administration of native PhKv (240 nM) 1 min before or after reperfusion markedly reduced the duration of arrhythmias. This effect was blocked by atropine, thereby indicating the participation of muscarinic receptors in the antiarrhythmogenic effect of PhKv. Notably, recombinant PhKv (240 nM) was also efficient to attenuate the arrhythmias (3.8 ± 0.9 vs. 8.0 ± 1.2 arbitrary units in control group). Furthermore, PhKv induced a significant reduction in heart rate. This bradycardia was partially blunted by atropine and potentiated by pyridostigmine. To further evaluate the participation of acetylcholine on the PhKv effects, we examined the release of this neurotransmitter from neuromuscular junctions. It was found that Phkv (200 nM) significantly increased the release of acetylcholine in this preparation. Moreover, PhKv (250 nM) did not cause any significant change in action potential or Ca2+ transient parameters in isolated cardiomyocytes. Altogether, these findings show an important acetylcholine-mediated antiarrhythmogenic effect of the spider PhKv toxin in isolated hearts.

Erection induced by Tx2-6 toxin of Phoneutria nigriventer spider: Expression profile of genes in the nitric oxide pathway of penile tissue of mice

The peptides Tx2-5 and Tx2-6, isolated from the whole venom of “armed-spider” Phoneutria nigriventer venom, are directly linked with the induction of persistent and painful erection in the penis of mammals. The erection induced by Tx2-6 has been associated with the activation of nitric oxide synthases. There is a scarcity of studies focusing on the outcome of Tx2-6 at the molecular level, by this reason we evaluated the gene profile activity of this toxin at the nitric oxide (NO) pathway. After microarray analyses on cavernous tissue of mice inoculated with Tx2-6 we found that only 10.4% (10/96) of these genes were differentially expressed, showing a limited effect of the toxin on the NO pathway. We found the genes sparc, ednrb, junb, cdkn1a, bcl2, ccl5, abcc1 over-expressed and the genes sod1, s100a10 and fth1 under-expressed after inoculation of Tx2-6. The differential expressions of sparc and ednrb genes were further confirmed using real-time PCR. Interestingly, ednrb activates the l-arginine/NO/cGMP pathway that is involved in the relaxation of the cavernous body. Therefore the priapism induced by Tx2-6 is a consequence of a highly specific interference of this neurotoxin with the NO pathway.

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.

Tx1, from Phoneutria nigriventer spidervenom, interacts with dihydropyridine sensitive-calcium channels in GH3 cells

The aim of this work was to use the binding assay of tritiated-dihydropyridine and radioiodinated Tx1, isolated from the Phoneutria nigriventer venom, in order to show the presence of Cav1 calcium channels on pituitary tumour cell (GH3). We showed that GH3 cells have specific sites for 125I-Tx1, which are sensitive to nifedipine (~20%). Reverse competition assay with 3H-PN200-110 (40% inhibition) and electrophysiological data (50% inhibition) suggest that Cav1 calcium channels are target sites for this toxin. To summarize, Tx1 binds to specific sites on GH3 cells and this interaction results in Cav1 calcium channel blockade. 3H-PN200-110 and 125I-Tx1 binding assays proved to be useful tools to show the presence of calcium channels on GH3 cells.

How important are NK 1 receptors for influencing microvascular inflammation and itch in the skin? Studies using Phoneutria nigriventer venom

Pain and itch sensations are induced by depolarization of C-fibre nerves and possibly other types of fibres. We have evidence from several species, including mice, that skin plasma extravasation induced by the Phoneutria nigriventer spider venom (PNV) is dependent on tachykinin NK 1 receptors. We have now investigated the itching measured as bouts of scratching in response to intradermal (i.d.) PNV in wildtype (NK 1 +/+) and NK 1 receptor knockout (NK 1 −/−) mice. Mice, either NK 1 +/+ or NK 1 −/−, were given a single i.d. injection (0.05 ml) of test agent or vehicle into the shaved dorsal skin, in the intercostal region, in a randomized way. The bouts of scratching were recorded in a blinded manner for 60 min. Oedema formation was concomitantly assessed by the extravascular accumulation of i.v. injected 125I-albumin. The i.d. injection of either substance P (at a high dose of 100 nmol/site), or PNV (0.3–10 μg/site) induced oedema formation in NK 1 +/+ but substantially less was observed in NK 1 −/− mice, as previously reported. PNV also induced scratching, but significantly less scratching was observed in NK 1 −/− compared with NK 1 +/+ mice. In contrast, SP did not induce significant scratching at amounts up to 100 nmol in NK 1 +/+ mice. Experiments with an NK 1 receptor antagonist SR140333 (at doses that blocked PNV-induced oedema) revealed that whilst a local co-injection i.d. (1 nmol) in NK 1 +/+ mice had no effect on PNV (3 μg/site)-induced scratching (18.5 ± 3.7 vs. 14.4 ± 3.5 bouts, mean ± S.E.M., n = 5–7), systemic treatment with SR140333 (120 nmol/kg, i.v.) significantly inhibited scratching (14 ± 3.5 vs. 3.1 ± 1.2 bouts, n = 4–6; P < 0.05). These results indicate that NK 1 receptors are involved in mediating PNV-induced scratching and that the location of the receptors is unlikely to be skin. Thus, a distinct separation between endogenous microvascular and PNV nociceptive NK 1-dependent effects is suggested.

Neuroprotective effect on brain injury by neurotoxins from the spider Phoneutria nigriventer

The role of calcium channels blockers in ischemic condition has been well documented. The PhTx3 neurotoxic fraction of the spider Phoneutria nigriventer venom is a broad-spectrum calcium channel blocker that inhibits glutamate release, calcium uptake and also glutamate uptake in synaptosomes. In the present study we describe the effect of PhTx3 (1.0 μg/mL), ω-conotoxin GVIA (1.0 μmol/L) and ω-conotoxin MVIIC (100 nmol/L) on neuroprotection of hippocampal slices and SN56 cells subjected to ischemia by oxygen deprivation and low glucose insult (ODLG). After the insult, cell viability in the slices and SN56 cells was assessed by confocal microscopy and epifluorescence, using live/dead kit containing calcein-AM and ethidium homodimer. Confocal images of CA1 region of the rat hippocampal slices subjected to ischemia insult and treated with ω-conotoxin GVIA, ω-conotoxin MVIIC and PhTx3 showed a percentage of dead cells of 68%, 54% and 18%, respectively. The SN56 cells subjected to ischemia were almost completely protected from damage by PhTx3 while with ω-conotoxin GVIA or ω-conotoxin MVIIC the cell protection was only partial. Thus, PhTx3 provided robust ischemic neuroprotection showing potential as a novel class of agents that targets multiple components and exerts neuroprotection in in vitro model of brain ischemia.

Involvement of spinal neurokinins, excitatory amino acids, proinflammatory cytokines, nitric oxide and prostanoids in pain facilitation induced by Phoneutria nigriventer spider venom

The major local symptom of Phoneutria nigriventer envenomation is an intense pain, which can be controlled by infiltration with local anesthetics or by systemic treatment with opioid analgesics. Previous work showed that intraplantar (i.pl) injection of Phoneutria nigriventer venom in rats induces hyperalgesia, mediated peripherally by tachykinin and glutamate receptors. The present study examined the spinal mechanisms involved in pain-enhancing effect of this venom. Intraplantar injection of venom into rat hind paw induced hyperalgesia. This phenomenon was inhibited by intrathecal (i.t.) injection of tachykinin NK 1 (GR 82334) or NK 2 (GR 94800) receptor antagonists, a calcitonin gene-related peptide (CGRP) receptor antagonist (CGRP 8–37) and N-methyl- d-aspartate (NMDA; MK 801 and AP-5), non-NMDA ionotropic (CNQX), or metabotropic (AIDA and MPEP) glutamate receptor antagonists, suggesting the involvement of spinal neurokinins and excitatory amino acids. The role of proinflammatory cytokines, nitric oxide (NO), and prostanoids in spinally mediated pain facilitation was also investigated. Pharmacological blockade of tumour necrosis factor-α (TNFα) or interleukin-1β (IL-1β) reduced the hyperalgesic response to venom. Intrathecal injection of L-N 6-(1-iminoethyl)lysine (L-NIL), but not of 7-nitroindazole (7-NI), inhibited hyperalgesia induced by the venom, indicating that NO, generated by the activity of the inducible form of nitric oxide synthase, also mediates this phenomenon. Furthermore, indomethacin, an inhibitor of cyclooxigenases (COX), or celecoxib, a selective inhibitor of COX-2, abolished venom-induced hyperalgesia, suggesting the involvement of spinal prostanoids in this effect. These data indicate that the spinal mechanisms of pain facilitation induced by Phoneutria nigriventer venom involves a plethora of mediators that may cooperate in the genesis of venom-induced central sensitization.

Role of Ca 2+ in vascular smooth muscle contractions induced by Phoneutria nigriventer spider venom

Phoneutria nigriventer venom (PNV) contracts vascular tissues and increases arterial blood pressure. This study aimed to investigate the mechanisms involved on PNV-induced contractions of rabbit mesenteric and celiac arteries. Strips of mesenteric and celiac arteries were suspended in a cascade system and superfused with warmed and oxygenated Krebs solution. PNV was dialyzed in order to exclude the participation of biogenic amines in the contractions elicited by the venom. Noradrenaline (NA, 30–300 pmol), PNV (1–10 μg), Bay K-8644 (0.3–3 nmol) and KCl (10–100 μmol) dose-dependently contracted the preparations. Ca 2+-free solution reduced by 38 and 83% the PNV-induced contractions of mesenteric and celiac arteries, respectively. Subsequent infusion of EGTA (0.2 mM) suppressed the residual contractions. Nifedipine (1 μM) and verapamil (10 μM) abolished PNV- and Bay K-8644-evoked contractions, whereas those induced by NA were reduced to a lesser extent. Lanthanum chloride (0.2 mM) inhibited by 75–90% the mesenteric and celiac contractions mediated by PNV. Caffeine (2 mM) fully blocked contractions induced by NA (95% mean inhibition), but only partly reduced those induced by PNV (35% mean inhibition). Ryanodine (10 μM) inhibited by 50% the contractions evoked by NA, but had no effect on the PNV-induced contractions in both tissues. Our findings indicate that PNV contracts vascular smooth muscle mainly due to increased influx of Ca 2+ from extracellular sources.

PnTx4-3, a new insect toxin from Phoneutria nigriventer venom elicits the glutamate uptake inhibition exhibited by PhTx4 toxic fraction

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 [ 3H]- 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.