Phoneutria Nigriventer Research Articles

Cloning, cDNA sequence analysis and patch clamp studies of a toxin from the venom of the armed spider ( Phoneutria nigriventer)

The cDNAs ( Tx3-2 and Pn3A) encoding precursor of toxin Tx3-2 and an isoform called Pn3A have been isolated from a library constructed from stimulated venom glands of the spider Phoneutria nigriventer. The cDNA of Tx3-2 reveals the presence of a signal peptide of 21 amino acids and of an intervening propeptide (with 16 amino acids) preceding the toxin sequence, which was followed by additional amino acid residues at the C-terminus (C-terminal peptide), implying post-translational modifications of the synthesised peptide. The deduced amino acid sequence for the mature toxin confirms the previous sequence published. In addition, by using the whole-cell patch clamp technique, we have determined that purified Tx3-2 decreases L-type currents present in GH3 cells. Finally, the presence of the cDNA Pn3A, with high sequence identity with Tx3-2, reveals the existence of a putative new toxin showing, at the cDNA level, 85.4% identity in its whole segment.

Cloning of cDNAs encoding neurotoxic peptides from the spider Phoneutria nigriventer

A cDNA library made from venom glands of the spider Phoneutria nigriventer was constructed and used to clone neurotoxic peptides. A cDNA of about 360 nucleotides encoding the precursor for the toxin Tx2-1 active on mammals has been isolated. The deduced amino acid sequence for the mature polypeptide confirms the polypeptide sequence previously published. In addition, two new putative toxins called Pn2-1A and Pn2-5A have been characterized and their complete amino acid sequence show 92% similarity to Tx2-1 and 94% similarity to Tx2-5 respectively. The cDNAs revealed that the precursors contain signal peptides characterized by a very hydrophobic core and a propeptide interposed between the signal sequence and the peptide toxin.

Activation by Phoneutria nigriventer spider venom of autonomic nerve fibers in the isolated rat heart

In the isolated rat heart, Phoneutria nigriventer spider venom (10–100 μg) produced a dose-dependent and reversible rise in left ventricular developed pressure. A low dose (10 μg) of venom induced a short-lasting, positive inotropic effect ( P<0.05) with no change in heart rate or coronary flow. At a dose of 50 μg, the venom caused significant positive inotropic and chronotropic responses associated with occasional ventricular arrhythmia, whereas coronary flow was not significantly affected within 10 min after venom administration. The highest dose of venom (100 μg) caused bradycardia, transient cardiac arrest, rhythm disturbances and an increase in end diastolic pressure followed by a reduction in coronary flow. Hearts treated with the non-selective β-adrenoceptor antagonist propranolol (3 μM) and the selective β 1-adrenoceptor antagonist CGP-20712A (10 μM) were protected against all the cardiac actions of the venom. The selective β 2-adrenoceptor antagonist butoxamine (10 μM) slightly reduced the cardiac response to 50 μg, but not to 100 μg of venom. Butoxamine also prevented the reduction in coronary flow induced by 100 μg of venom. Hearts from reserpine-treated rats (5 mg kg −1 day −1, i.p., for 2 days) showed a marked decrease in all venom (≤100 μg)-induced cardiac responses. The muscarinic receptor antagonist atropine (1 μM) slightly potentiated the response to 50 μg of venom but had little or no effect on the responses to 100 μg of venom. The cardiac responses to venom (50–100 μg) were unaltered in hearts from rats treated with 8-methyl N-vanillyl-6-nonenamide (capsaicin; 50 mg/kg, s.c.). These findings indicate that P. nigriventer venom releases norepinephrine from cardiac sympathetic nerve endings and this may explain the observed increase in contractile force and heart rate.

Pathological changes induced by PhTx1 from Phoneutria nigriventer spider venom in mouse skeletal muscle in vitro

The `armed' spider Phoneutria nigriventer is responsible for most human accidents involving spiders in Brazil. The effects of fraction Tx1 (PhTx1) from the venom of this spider were investigated by physiological and morphological methods using the mouse phrenic nerve-diaphragm preparation. PhTx1 (1 and 5 μg) did not affect the twitch tension of muscle fibers under indirect electrical stimulation. At this same concentration, PhTx1 also did not alter the miniature end-plate potential (mepp) frequency and amplitude, nor did it change the resting membrane potential 60 min after addition to the preparation. Light microscopy (LM) revealed that in muscles incubated with PhTx1 a number of fibers were morphologically altered, as evidenced by microvacuolization and myofibril hypercontraction and loss within 15 min after toxin administration. Transmission electron microscopy (TEM) showed sarcoplasmic reticulum swelling, disorganization of the sarcomeres and mitochondrial damage, and occasionally, sarcolemmal discontinuities with a persisting basal membrane. The intra-muscular fascicles of the phrenic nerve showed myelinated axons with vacuolated myelin sheaths as well as peri- and intra-axoplasmic vacuoles. The neuromuscular junction changes were variable, but were rarely severe. Thus, although PhTx1 did not depolarize or hyperpolarize the neuromuscular junction, it was nevertheless toxic to a restricted number of muscle fibers and nerve structures. The site of action of PhTx1 may involve the sarcolemma and axolemma as suggested by the morphological abnormalities which could reflect hydroelectrolytic disturbances.

Investigation of the effect of PhTx2, from the venom of the spider Phoneutria nigriventer, on the release of [ 3H]-acetylcholine from rat cerebrocortical synaptosomes

Venoms from spiders are an important source of toxins that can help on the dissection of mechanisms involved in neurotransmission. Among them is the venom of the spider Phoneutria nigriventer that contains several toxic fractions with different targets in mammals and/or insects. We here report that one of these fractions (PhTx2) is able to evoke acetylcholine release from rat cortical synaptosomes and that this effect is dependent on extracellular calcium and is inhibited by the sodium channel blocker tetrodotoxin.

ω-Phonetoxin-IIA: a calcium channel blocker from the spider Phoneutria nigriventer.

A peptide with neurotoxic effect on mammals, purified from the venom of the spider Phoneutria nigriventer, was studied regarding its primary structure and its effects on voltage-gated calcium channels. The peptide, named ω-phonetoxin-IIA, has 76 amino acids residues, with 14 Cys forming 7 disulphide bonds, and a molecular weight of 8362.7 Da. The neurotoxicity is a consequence of the peptide’s blocking effects on high-voltage-activated (HVA) calcium channels. N-type HVA calcium channels of rat dorsal root ganglion neurons are blocked with affinity in the sub-nanomolar concentration range. The toxin also blocks L-type channels of rat β pancreatic cells, with an affinity 40 times lower. Although not studied in detail, evidence indicates that the toxin also blocks other types of HVA calcium channels, such as P and Q. No effect was observed on low-voltage-activated, T-type calcium channels. The significant homologies between ω-phonetoxin-IIA and the peptides of the ω-agatoxin-III family, and the overlapping inhibitory effects on calcium channels are discussed in terms of the structure-activity relationship.

Phoneutria nigriventer toxins block tityustoxin-induced calcium influx in synaptosomes.

Neurotoxins can help the understanding of mechanisms involved in neurotransmission. We here report that two neurotoxin isoforms, Tx3-3 and Tx3-4 obtained from the venom of the spider Phoneutria nigriventer inhibited the 45Ca2+ influx in rat cortical synaptosomes induced by the scorpion venom tityustoxin. The IC50 for Tx3-3 and Tx3-4 were 0.32 and 7.9 nM, respectively. The neurotoxins Tx3-3 and Tx3-4 are very effective in inhibiting 45Ca2+ influx and they should be useful in studies involving Ca(2+)-dependent processes.

ELISA for the detection of venom antigens in experimental and clinical envenoming by Loxosceles intermedia spiders

Enzyme linked immunosorbent assays (ELISA) were developed to detect antigens from Loxosceles intermedia spider venom. Hyperimmune horse anti- Loxosceles intermedia IgGs were prepared by immunoaffinity chromatography and used to set up a sandwich-type ELISA. The specificity of the assay was demonstrated by its capacity to correctly discriminate the circulating antigens in mice that were experimentally inoculated with L. intermedia venom from those inoculated with L. gaucho, L. laeta, and Phoneutria nigriventer spider venoms, Tityus serrulatus scorpion venom and Bothrops jararaca, Crotalus durissus terrificus, Lachesis muta muta and Micrurus frontalis snake venoms. Measurable absorbance signals were obtained with 0.8 ng of venom per assay. The ELISA also detected antigens in the sera of patients envenomed by L. intermedia. Therefore, after standardization for clinical use this ELISA may be a valuable tool for clinicians and epidemiologists.

Effects of capsiacin cream on nociceptive hypersensitivity induced by carrageenan in rats

Capsaicin, the pungent component of hot peppers, and the venom of the spider Phoneutria nigriventer are able to activate sensory nerves resulting in cutaneous neurogenic plasma extravasation. This study was undertaken to compare the ability of these substances to evoke oedema in the rat hind-paw and mechanisms underlying this effect. Subplantar injection of either Phoneutria nigriventer venom (PNV; 1–100 μg/paw) or capsaicin (10–200 μg/paw) caused a significant paw oedema that was potentiated by CGRP (10 pmol/paw). In rats treated neonatally with capsaicin to deplete neuropeptides, the paw oedema induced by either PNV (100 μg/paw) or capsaicin (100 μg/paw) was partially reduced (P<0.05). The tachykinin NK1 receptor antagonist SR140333 (0.2 μmol/kg; i.v.) prevented the paw oedema induced by the tachykinin NK1 receptor agonist GR73632 (30 pmol/paw) and partially reduced paw oedema induced by PNV or capsaicin. Treatment of rats with compound 48/80 (5 mg/kg; s.c. 3 days) or with both H1 receptor antagonist (mepyramine; 1 nmol/paw) and 5-HT receptor antagonist (methysergide; 1 nmol/paw) significantly inhibited PNV- or capsaicin-induced paw oedema. The combined treatment with mepyramine and methysergide and SR140333 further reduced PNV- and capsaicin-induced paw oedema. The bradykinin B2 receptor antagonist Hoe 140 affected neither PNV- nor capsaicin-induced responses. Our results suggest that PNV and capsaicin each induce paw oedema that is partially mediated by activation of sensory fibers culminating in the release of substance P as well as by activation of mast cells which in turn release amines such as histamine and 5-HT.

Reduction of voltage-dependent Mg 2+ block of NMDA receptor response by neuronal injury

Phoneutria nigriventer venom causes stimulation of capsaicin-sensitive primary afferent neurons in the rat dorsal skin, leading to neurogenic plasma protein extravasation due to the release of tachykinin NK1 receptor agonist. In this study we further investigated the mechanisms involved in the venom-induced activation of capsaicin-sensitive primary afferent neurons. The plasma extravasation in response to venom intradermally injected was measured in Wistar rats as the local accumulation of i.v. injected 125I-labelled human serum albumin into skin sites. The tachykinin NK1 receptor agonist, d-Ala-[l-Pro9,Me-Leu8]substance P-(7–11) (GR73632; 10–100 pmol/site), induced a significant plasma leakage that was abolished by the selective tachykinin NK1 receptor antagonist, (S)-1-[2-[3-(3,4-dichlorphenyl)-1 (3-isopropoxyphenylacetyl) piperidin-3-yl] ethyl]-4-phenyl-1 azaniabicyclo [2.2.2]octane chloride (SR140333; 1 nmol/site), whereas the leakage after venom (1–10 μg/site) was significantly inhibited (but not abolished) by SR140333. The calcitonin gene-related peptide (CGRP) receptor antagonist, CGRP-(8–37), failed to further reduce the residual plasma extravasation induced by venom plus SR140333. The μ-opioid receptor agonist, [d-Ala2,Me-Phe4,Gly-ol5]enkephalin (DAMGO), and the local anaesthetic, lignocaine, had no effect on the venom-induced plasma extravasation. Similarly, the L-, N- and P/Q-type voltage-sensitive Ca2+ channel blockers (verapamil, ω-conotoxin MVIIA and MVIIC, respectively) as well as the Na+ channel blockers, tetrodotoxin and carbamazepine, had no effect on the venom-induced effect. Neither the systemic treatment nor the local injection of ruthenium red prevented the venom-induced plasma extravasation. However, the vanilloid receptor antagonist, N-[2-(4-chlorophenyl) ethyl]-1,3,4,5-tetrahydro-7,8-dihydroxy-2H-2-benzazepine-2-carbothioamide (capsazepine; 120 μmol/kg, i.v.), reduced by 48% (P<0.05) the venom (10 μg/site)-induced plasma extravasation. A significant inhibitory effect was also observed with the P2 purinoceptor agonists, adenosine 5′-triphosphate (ATP; 10 and 30 nmol/site) and adenosine 5′-diphosphate (ADP; 10 nmol/site). The involvement of histamine and/or 5-hydroxytryptamine (5-HT) in the venom-induced plasma extravasation was ruled out since neither histamine and 5-HT receptor antagonists nor depletion of mast cells by compound 48/80 affected the venom response. This was further supported by the failure of venom to degranulate in vitro peritoneal mast cells. In conclusion, only vanilloid receptors and P2 prejunctional purinoceptors had an inhibitory effect on the neurogenic plasma extravasation evoked by P. nigriventer venom in rat dorsal skin.

Calcium channel antibodies and Lambert-Eaton myasthenic syndrome

The present study investigated the effects of pharmacological spinal inhibition of voltage-gated calcium channels (VGCC) in mouse pruritus. The epidural administration of P/Q-type MVIIC or PhTx3.3, L-type verapamil, T-type NNC 55-0396 or R-type SNX-482 VGCC blockers failed to alter the scratching behavior caused by the proteinase-activated receptor 2 (PAR-2) activator trypsin, injected into the mouse nape skin. Otherwise, trypsin-elicited pruritus was markedly reduced by the spinal administration of preferential N-type VGCC inhibitors MVIIA and Phα1β. Time-course experiments revealed that Conus magus-derived toxin MVIIA displayed significant effects when dosed from 1 h to 4 h before trypsin, while the anti-pruritic effects of Phα1β from Phoneutria nigriventer remained significant for up to 12 h. In addition to reducing trypsin-evoked itching, MVIIA or Phα1β also prevented the itching elicited by intradermal (i.d.) injection of SLIGRL-NH2, compound 48/80 or chloroquine, although they did not affect H2O2-induced scratching behavior. Furthermore, the co-administration of MVIIA or Phα1β markedly inhibited the pruritus caused by the spinal injection of gastrin-releasing peptide (GRP), but not morphine. Notably, the epidural administration of MVIIA or Phα1β greatly prevented the chronic pruritus allied to dry skin model. However, either tested toxin failed to alter the edema formation or neutrophil influx caused by trypsin, whereas they significantly reduced the c-Fos activation in laminas I, II and III of the spinal cord. Our data bring novel evidence on itching transmission mechanisms, pointing out the therapeutic relevance of N-type VGCC inhibitors to control refractory pruritus.

Phoneutria nigriventer spider venom induces oedema in rat skin by activation of capsaicin sensitive sensory nerves

Phoneeutria nigriventer venom induces oedema formation when injected in the rat dorsal skin and such oedema is, in part, dependent on the stimulation of tachykinin NK 1 receptors. This study investigated whether Phoneutria nigriventer venom acts directly on tachykinin NK 1 receptors, or indirectly to activate sensory neurones which in turn release a tachykinin NK 1 receptor agonist. The plasma extravasation induced by Phoneutria nigriventer venom (1–10 μg/site) in neonatally capsaicin (8-methyl N-vanillyl-6-nonenamide)-pretreated rats was substantially attenuated ( P<0.05) but the response to either the tachykinin NK 1 receptor agonist GR73632 (( δAva[ l-Pro 9, N-MeLeu10] substance P-(7–11) 30 pmol/site) or bradykinin (0.3–3 nmol/site) was not affected. These results indicate that Phoneutria nigriventer venom stimulates sensory nerves indirectly. The lack of effect of capsaicin-pretreatment on the GR73632 and bradykinin responses indicated that the tachykinin NK 1 and bradykinin B 2 receptors remained functional. There was no evidence to suggest that Phoneutria nigriventer venom contains a tachykinin NK 1 receptor agonist.

A toxin from the spider Phoneutria nigriventer that blocks calcium channels coupled to exocytosis.

1. The aim of the present experiments was to investigate the pharmacological action of a toxin from the spider Phoneutria nigriventer, Tx3-3, on the function of calcium channels that control exocytosis of synaptic vesicles. 2. Tx3-3, in confirmation of previous work, diminished the intracellular calcium increase induced by membrane depolarization with KCl (25 mM) in rat cerebrocortical synaptosomes. The toxin was very potent (IC50 0.9 nM) at inhibiting calcium channels that regulate calcium entry in synaptosomes. In addition, Tx3-3 blocked the exocytosis of synaptic vesicles, as measured with the fluorescent dye FM1-43. 3. Using omega-toxins that interact selectively with distinct neuronal calcium channels, we investigated whether the target of Tx3-3 overlaps with known channels that mediate exocytosis. The results indicate that the main population of voltage-sensitive calcium channels altered by Tx3-3 can also be inhibited by omega-agatoxin IVA, an antagonist of P/Q calcium channels. Omega-conotoxin GVIA, which inhibits N type calcium channels did not decrease significantly the entry of calcium or exocytosis of synaptic vesicles in depolarized synaptosomes. 4. It is concluded that Tx3-3 potently inhibits omega-agatoxin IVA-sensitive calcium channels, which are involved in controlling exocytosis in rat brain cortical synaptosomes.

New Genes for Male Accessory Gland Proteins in Drosophila melanogaster

The accessory gland of male insects produces components of the seminal fluid that alter the behavior, physiology and life span of the mated female, and contribute to her efficient storage and utilization of sperm. As a step towards understanding how this occurs, we have isolated genes encoding 12 previously unreported accessory gland-specific mRNAs from the fruit fly Drosophila melanogaster. We report here the restriction maps of the new genes, the chromosome positions — which are all autosomal — of the 11 non-repetitive genes, their expression patterns, and the sequences of the accessory gland proteins (Acps) encoded by nine of the genes. Eight of the proteins predicted from these sequences begin with putative secretion signals. Following their signal sequences, three of the predicted molecules are peptides and the other five are larger polypeptides with characteristics of cleavable prohormones. The ninth molecule, which has an N-terminal hydrophobic region but no consensus signal peptide cleavage site, is predicted to be a 716 amino acid glycoprotein. Of the nine proteins, two have intriguing similarities to sequences in protein databases. Acp76A is a 388 amino acid pro-protein which contains a signature sequence for the serpin class of protease inhibitors. The 115 amino acid Acp62F has a 28 amino acid region of high sequence similarity to a neurotoxin of the Brazilian armed spider Phoneutria nigriventer. Models are discussed in which Acp76A plays a role in the observed regulation of Acp proteolysis and/or in the coagulation of seminal fluid to form a mating plug, and in which Acp62F contributes to the reported toxicity of Drosophila seminal fluid.

Novel polypeptide toxins with crab lethality from the sea anemone Anemonia erythraea

The sea anemone Anemonia erythraea was found to contain polypeptide toxins with crab lethality as well as hemolysins. Three polypeptide toxins (AETX I, II and III) were isolated by gel filtration on Sephadex G-50 and reverse-phase HPLC on TSKgel ODS-120T. A geographic variation in toxin composition was suggested. The LD 50 against crabs of AETX I, II and III were estimated to be 2.2, 0.53 and 0.28 μg/kg, respectively, but none of the toxins showed lethality in mice. The amino acid sequences of the three toxins were deduced from sequencings of the whole molecules and their enzymatic fragments. Amino acid analyses and molecular mass determinations supported the accuracy of the deduced sequences. AETX I, comprising 47 amino acid residues including 6 half-Cys residues, is an analog of sea anemone type 1 toxins. On the other hand, AETX II and III, which are highly homologous with each other, are quite distinct from the known sea anemone polypeptide toxins in that they are composed of 59 residues including 10 half-Cys residues. Interestingly, both toxins have sequence similarities with neurotoxins isolated from the Brazilian `armed' spider Phoneutria nigriventer.

Acetylcholine release from rat brain cortical slices evoked by the fraction P4 of the venom of the spider Phoneutria nigriventer has Ca 2+ and temperature independent components

Fractionation of the venom of the spider Phoneutria nigriventer revealed that it was a mixture of several neurotoxic peptides. The peptides so far characterized either inhibited or induced neurotransmitter release. These effects were mediated by Ca 2+ channels or increasing Na + permeability through voltage sensitive Na +-channels, respectively. The pooled toxic components (fraction P4) showed stimulatory effects on acetylcholine release from brain cortical slices. In addition, a component of the observed effects resembling that of α-latrotoxin was identified, which was characterized by the ability to provoke release of acetylcholine (ACh) at low temperature and in a manner independent of extracellular Ca 2+ and of voltage sensitive Na +-channels.

Isolation and partial characterization of a polypeptide from Phoneutria nigriventer spider venom that relaxes rabbit corpus cavernosum in vitro

The venom of the Brazilian spider Phoneutria nigriventer was fractionated using a C18 μBondapack reverse-phase high-performance liquid chromatography column. The resulting fractions were assayed in the rabbit perfused corpus cavernosum tissue to identify those fractions responsible for the corpus cavernosum relaxation. Two fractions (A and B) with retention times of 18.1 and 36.7 min, respectively, induced relaxation of corpus cavernosum strips. Fraction A was selected for further biochemical characterization. Repurification of this fraction revealed the presence of a polypeptide (named PNV4) which migrates in sodium dodecyl sulphate-polyacrylamide gel electrophoresis as a single band consistent with a mol. wt close to 16,600. The amino acid composition of PNV4 showed the presence of 147 residues, a high content of Cys and a calculated mol. wt of 17,213 + Trp. The N-terminal amino acid sequence of PNV4 determined for its first 48 residues was AELTSCFPVGHECDGD- ASNCNCCGDDVYCGCGWGRWNCKCKVADQSYA.

The effect of a tachykinin NK1 receptor antagonist, SR140333, on oedema formation induced in rat skin by venom from the Phoneutria nigriventer spider.

1. The possibility that tachykinin NK1 receptors are involved in the plasma extravasation evoked by intradermal (i.d.) injection of Phoneutria nigriventer venom (PNV) in rat dorsal skin in vivo has been investigated. 2. Local oedema formation induced by the i.d. injection of test agents was measured by the extravascular accumulation of intravenously (i.v.) injected 125I-labelled human serum albumin over a 30 min period. 3. The tachykinin NK1 agonist, GR73632 (30 pmol per site), induced local oedema formation which was potentiated by co-injection with the neuropeptide vasodilator, calcitonin gene-related peptide (CGRP, 10 pmol per site). The non-peptide tachykinin NK1 receptor antagonist, SR140333 (0.03-1 nmol per site co-injected, i.d.) significantly inhibited (0.3 nmol per site, P < 0.05; 1 nmol per site, P < 0.001) local oedema formation induced by GR73632 with CGRP but not that induced by histamine (10 nmol per site) with CGRP. 4. PNV (0.03-0.3 microgram per site) injected i.d. induced dose-dependent local oedema formation. SR140333 (1 nmol per site, co-injected i.d.) inhibited oedema formation; with complete inhibition observed at doses of 0.03 microgram (P < 0.05) and 0.1 microgram (P < 0.001); and partial inhibition (50%) observed with the highest dose of PNV, 0.3 microgram (P < 0.05). 5. Local oedema formation induced by PNV was not affected by systemic pretreatment with the bradykinin B2 receptor antagonist, Hoe 140 (80 nmol kg-1, i.v.), which was used at a dose which significantly inhibited oedema formation by bradykinin (1 nmol per site). 6. Local oedema formation induced by PNV was significantly inhibited (P < 0.01) by co-injection of the histamine H1 receptor antagonist, mepyramine (2.5 nmol per site), together with the 5-hydroxytryptamine (5-HT) antagonist, methysergide (2.8 nmol per site). 7. In the presence of all three antagonists (mepyramine 2.5 nmol per site; methysergide, 2.8 nmol per site and SR140333 1 nmol per site), the plasma extravasation induced by PNV was further significantly inhibited (P < 0.001, when compared with PNV injected i.d. alone; P < 0.05 when compared with PNV co-injected with mepyramine and methysergide and P < 0.01, when compared with PNV co-injected with SR140333). 8. These results suggest that oedema formation evoked by i.d. PNV in rat skin may be partially mediated via a mechanism involving tachykinin NK1 receptors and that this effect is independent of histamine and 5-HT.

The effect of Phoneutria nigriventer (armed spider) venom on arterial blood pressure of anaesthetised rats

The changes induced in the mean arterial blood pressure of anaesthetised rats following the administration of armed spider ( Phoneutria nigriventer) venom have been investigated. The intravenous injection of Phoneutria nigriventer venom (0.1 mg/kg) evoked a brief and reversible decrease in the mean arterial blood pressure whereas a higher dose of venom (0.3 mg/kg) caused a biphasic response characterized by a short-lasting hypotension followed by a sustained and prolonged hypertension (40–50 min). These changes were accompanied by tachycardia, salivation, fasciculations, defecation and respiratory disturbances. Pretreatment of the animals with atropine (10 mg/kg), propranolol (100 mg/kg), phenoxybenzamine (100 mg/kg) and indomethacin (4 mg/kg) did not significantly affect the mean arterial blood pressure changes induced by Phoneutria nigriventer venom. Similarly, the bradykinin B 2 receptor antagonist Hoe 140 ( d-Arg-[Hyp 3,Thi 5, dTic 7,Oic 8]-bradykinin) (0.6 mg/kg), the PAF receptor antagonist WEB 2086 (3-(4-(2-chlorophenyl)-9-methyl-6 H-thieno-(3,2 f) (1,2,4)-triazolo-(4,3- a) (1,4)-diazepine-2-yl)-(4-morpholinyl)-1-propanone) (20 mg/kg), the tachykinin NK 1 receptor antagonist SR 140333 (( S)1-{2-[3-(3,4-dichlorophenyl)-1-(3-iso-propoxyphenyl acetyl) piperidin-3-yl] ethyl}-4-phenyl-1-azoniabicyclo[2.2.2] octane, chloride) (0.5 mg/kg), the tachykinin NK 2 receptor antagonist SR 48968 ( (S)-N-methyl- N[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide) (0.5 mg/kg) and the nitric oxide synthase inhibitor N ω-nitro- l-arginine methyl ester (10 mg/kg) had no significant effect on the mean arterial blood pressure changes induced by Phoneutria nigriventer venom. The increase in the blood pressure induced by Phoneutria nigriventer venom was also not significantly affected by either the angiotensin II receptor antagonist losartan (10 mg/kg) or the endothelin ET A receptor antagonist FR 139317 (( R)2-[( R)-2-[[1-(hexahydro-1 H-azepinyl]carbonyl]amino-4-methyl-pentanoyl]amino-3-[3-(1-methyl-1 H-indoyl)]propionyl] amino-3-(2-propionyl] amino-3-(2-pyridyl) propionic acid) (30 mg/kg). The ATP-dependent K + channel antagonist glibenclamide (50 mg/kg) reduced by 40% the hypotension induced by Phoneutria nigriventer venom without affecting the hypertensive response. Pretreatment of the animals with L-type Ca 2+ channel antagonists such as verapamil (10–100 μg/kg/min), diltiazem (40–120 μg/kg/min) and nifedipine (0.3–10 mg/kg) markedly attenuated the hypertension induced by Phoneutria nigriventer venom. Verapamil (30 μg/kg/min) and diltiazem (120 μg/kg/min) also promptly reversed the established hypertension induced by Phoneutria nigriventer venom when infused 8 min after venom injection. Our results indicate that the brief decrease of blood pressure induced by Phoneutria nigriventer venom is partially due to ATP-dependent K + channel activation. The prolonged hypertension seems to result from direct Ca 2+ entry into vascular and/or cardiac muscles.

A novel tool for the investigation of glutamate release from rat cerebrocortical synaptosomes: the toxin Tx3-3 from the venom of the spider Phoneutria nigriventer

The present experiments investigated the effect of some of the toxic components present in the venom of the spider Phoneutria nigriventer on the release of neurotransmitter. The toxic fraction, Phoneutria nigriventer toxin-3 (PhTx3), abolished Ca2+-dependent glutamate release, but did not alter Ca2+-independent secretion of glutamate when rat brain cortical synaptosomes were depolarized with 33 mM KCl. This effect was most likely due to interference with the entry of calcium through voltage-gated calcium channels, because PhTx3 reduced by 50% the increase in intrasynaptosomal free calcium induced by membrane depolarization, and did not affect the release of glutamate evoked by a calcium ionophore (ionomycin). A polypeptide (Tx3-3) present in the PhTx3 fraction reproduced the effects of the PhTx3 fraction on transmitter release and intrasynaptosomal free calcium in the low nanomolar range. We compared the alterations produced by the Tx3-3 with the actions of toxins known to block calcium channels coupled to exocytosis: the results indicated that the Tx3-3 inhibition of glutamate release and intrasynaptosomal calcium resemble that observed with omega-conotoxin MVIIC. We suggest that the Tx3-3 is a calcium-channel antagonist that blocked glutamate exocytosis.