Marco Aurélio Romano Silva

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

&NA; 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 Freunds 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, &ohgr;‐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 (30 pmol/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.

Association of antipsychotic induced weight gain and body mass index with GNB3 gene: A meta-analysis

It has been reported that C825T variant in the gene encoding the G-protein subunit beta3 (GNB3) is associated with antipsychotic-induced weight gain and obesity. We investigated the association of the GNB3 and antipsychotic-induced weight gain as well as body mass index (BMI) using meta-analytical techniques. Our analysis of 402 schizophrenia subjects showed a trend (p=0.072) only under a fixed-model. As it was observed heterogeneity among the studies (p=0.007), we re-analyzed using a random-effects framework and no significance was found (p=0.339). No evidence for bias publication was reported (p=0.868). Our analysis of 18,903 subjects showed a trend (p=0.053) associating CC and lower BMI under a fixed model. Although no significant association was found, the same pattern (CC and lower antipsychotic-induced weight gain) was observed. Our meta-analysis indicates that firmly establishing the role of pharmacogenetics in clinical psychiatry requires much larger sample sizes that have been reported.

An Evaluation of the Antinociceptive Effects of Phα1β, a Neurotoxin from the Spider Phoneutria nigriventer, and ω-Conotoxin MVIIA, a Cone Snail Conus magus Toxin, in Rat Model of Inflammatory and Neuropathic Pain

Voltage-sensitive calcium channels (VSCCs) underlie cell excitability and are involved in the mechanisms that generate and maintain neuropathic and inflammatory pain. We evaluated in rats the effects of two VSCC blockers, ω-conotoxin MVIIA and Phα1β, in models of inflammatory and neuropathic pain induced with complete Freund’s adjuvant (CFA) and chronic constrictive injury (CCI), respectively. We also evaluated the effects of the toxins on capsaicin-induced Ca2+ influx in dorsal root ganglion (DRG) neurons obtained from rats exposed to both models of pain. A single intrathecal injection of Phα1β reversibly inhibits CFA and CCI-induced mechanical hyperalgesia longer than a single injection of ω-conotoxin MVIIA. Phα1β and MVIIA also inhibited capsaicin-induced Ca2+ influx in DRG neurons. The inhibitory effect of Phα1β on capsaicin-induced calcium transients in DRG neurons was greater in the CFA model of pain, while the inhibitory effect of ω-conotoxin MVIIA was greater in the CCI model. The management of chronic inflammatory and neuropathic pain is still a major challenge for clinicians. Phα1β, a reversible inhibitor of VSCCs with a preference for N-type Ca2+ channels, has potential as a novel therapeutic agent for inflammatory and neuropathic pain. Clinical studies are necessary to establish the role of Phα1β in the treatment of chronic pain.

Phoneutria spider toxins block ischemia-induced glutamate release and neuronal death of cell layers of the retina.

Purpose: To investigate the effect of calcium channel blockers, spider toxins, on cell viability and the glutamate content of ischemic retinal slices. Methods: Rat retinal slices were subjected to ischemia via exposure to oxygen-deprived low-glucose medium for 45 minutes. Slices were either treated or not treated with the toxins PhTx3, Tx3-3, and Tx3-4. After oxygen-deprived low-glucose insult, glutamate content and cell viability were assessed in the slices by confocal and optical microscopy. Results: In the retinal ischemic slices that were treated with PhTx3, Tx3-3, and Tx3-4, confocal imaging showed a decrease in cell death of 79.5 ± 3.1%, 75.5 ± 5.8%, and 61 ± 3.8%, respectively. Neuroprotective effects were also observed 15, 30, 60, and 90 minutes after the onset of the retinal ischemic injury. As a result of the ischemia, glutamate increased from 6.2 ± 1.0 nMol/mg protein to 13.2 ± 1.0 nMol/mg protein and was inhibited by PhTx3, Tx3-3, and Tx3-4 to 8.6 ± 0.7, 8.8 ± 0.9, and 7.4 ± 0.8 nMol/mg protein, respectively. Histologic analysis of the live cells in the outer, inner, and ganglion cell layers of the ischemic slices showed a considerable reduction in cell death by the toxin treatment. Conclusion: Spider toxins reduced glutamate content and cell death of retinal ischemic slices.

Effects of α-scorpion toxin, tityustoxin on the release of [3H] dopamine of rat brain prefrontal cortical slices

The effect of tityustoxin (TsTX) on the release of [3H] dopamine in rat brain prefrontal cortical slices was investigated. The stimulatory effect of TsTX was dependent on incubation time and TsTX concentration with an EC50 of 0.05 microM. The release of [3H] dopamine stimulated by TsTX is dependent of Na+ channels and thus, was completely, inhibited by tetrodotoxin. Tityustoxin-induced release of [3H] dopamine was not blocked by ethylene glycol-bis(beta-aminoethyl) ether (EGTA) and thus was independent of extracellular calcium. However, [3H] dopamine release induced by TsTX was inhibited by 52% by BAPTA, a calcium chelator. Moreover, dantrolene (100 microM) and tetracaine (500 microM) partially inhibited by 38 and 29%, respectively, the tityustoxin-induced release of [3H] dopamine from prefrontal cortical slices suggesting a role from intracellular calcium increase. In conclusion, part of the TsTX-induced release [3H] dopamine may be due to an effect of the toxin on the reversal of the dopamine transporter (DAT), but the majority of the toxin stimulated release of [3H] dopamine involves the mobilization of intracellular calcium stores.

Phα1β toxin prevents capsaicin-induced nociceptive behavior and mechanical hypersensitivity without acting on TRPV1 channels

Phα1β toxin is a peptide purified from the venom of the armed spider Phoneutria nigriventer, with markedly antinociceptive action in models of acute and persistent pain in rats. Similarly to ziconotide, its analgesic action is related to inhibition of high voltage activated calcium channels with more selectivity for N-type. In this study we evaluated the effect of Phα1β when injected peripherally or intrathecally in a rat model of spontaneous pain induced by capsaicin. We also investigated the effect of Phα1β on Ca²⁺ transients in cultured dorsal root ganglia (DRG) neurons and HEK293 cells expressing the TRPV1 receptor. Intraplantar or intrathecal administered Phα1β reduced both nocifensive behavior and mechanical hypersensitivity induced by capsaicin similarly to that observed with SB366791, a specific TRPV1 antagonist. Peripheral nifedipine and mibefradil did also decrease nociceptive behavior induced by intraplantar capsaicin. In contrast, ω-conotoxin MVIIA (a selective N-type Ca²⁺ channel blocker) was effective only when administered intrathecally. Phα1β, MVIIA and SB366791 inhibited, with similar potency, the capsaicin-induced Ca²⁺ transients in DRG neurons. The simultaneous administration of Phα1β and SB366791 inhibited the capsaicin-induced Ca²⁺ transients that were additive suggesting that they act through different targets. Moreover, Phα1β did not inhibit capsaicin-activated currents in patch-clamp recordings of HEK293 cells that expressed TRPV1 receptors. Our results show that Phα1β may be effective as a therapeutic strategy for pain and this effect is not related to the inhibition of TRPV1 receptors.

Choline oxidase chemiluminéscent assay, after removal of eserine from medium, of acetylcholine released in vitro from brain slices ☆

A chemiluminescent method has been used recently for the determination of acetylcholine with limitations such as the presence of a cholinesterase inhibitor in the incubation medium, which is indispensable for the study of acetylcholine release by various agents. A modified procedure is presented in which the cholinesterase inhibitor eserine (physostigmine) is extracted from the medium. The results showed complete recovery when labelled acetylcholine was used. This modified procedure was used to determine the release of acetylcholine evoked by tityustoxin and ouabain. The results were comparable to those obtained by bioassay using a strip of guinea pig ileum.

The Effect of Spider Toxin PhTx3-4, ω-Conotoxins MVIIA and MVIIC on Glutamate Uptake and on Capsaicin-Induced Glutamate Release and [Ca2+]i in Spinal cord Synaptosomes

In spinal cord synaptosomes, the spider toxin PhTx3-4 inhibited capsaicin-stimulated release of glutamate in both calcium-dependent and -independent manners. In contrast, the conus toxins, ω-conotoxin MVIIA and ω-conotoxin MVIIC, only inhibited calcium-dependent glutamate release. PhTx3-4, but not ω-conotoxin MVIIA or ω-conotoxin MVIIC, is able to inhibit the uptake of glutamate by synaptosomes, and this inhibition in turn leads to a decrease in the Ca2+-independent release of glutamate. No other polypeptide toxin so far described has this effect. PhTx3-4 and ω-conotoxins MVIIC and MVIIA are blockers of voltage-dependent calcium channels, and they significantly inhibited the capsaicin-induced rise of intracellular calcium [Ca2+]i in spinal cord synaptosomes, which likely reflects calcium entry through voltage-gated calcium channels. The inhibition of the calcium-independent glutamate release by PhTx3-4 suggests a potential use of the toxin to block abnormal glutamate release in pathological conditions such as pain.

PhTx3-4, a Spider Toxin Calcium Channel Blocker, Reduces NMDA-Induced Injury of the Retina

The in vivo neuroprotective effect of PhTx3-4, a spider toxin N-P/Q calcium channel blocker, was studied in a rat model of NMDA-induced injury of the retina. NMDA (N-Methyl-d-Aspartate)-induced retinal injury in rats reduced the b-wave amplitude by 62% ± 3.6%, indicating the severity of the insult. PhTx3-4 treatment increased the amplitude of the b-wave, which was almost equivalent to the control retinas that were not submitted to injury. The PhTx3-4 functional protection of the retinas recorded on the ERG also was observed in the neuroprotection of retinal cells. NMDA-induced injury reduced live cells in the retina layers and the highest reduction, 84%, was in the ganglion cell layer. Notably, PhTx3-4 treatment caused a remarkable reduction of dead cells in the retina layers, and the highest neuroprotective effect was in the ganglion cells layer. NMDA-induced cytotoxicity of the retina increased the release of glutamate, reactive oxygen species (ROS) production and oxidative stress. PhTx3-4 treatment reduced glutamate release, ROS production and oxidative stress measured by malondialdehyde. Thus, we presented for the first time evidence of in vivo neuroprotection from NMDA-induced retinal injury by PhTx3-4 (-ctenitoxin-Pn3a), a spider toxin that blocks N-P/Q calcium channels.

Effects of tityustoxin and ouabain on release of acetylcholine from slices of cortex from the rat brain and on the acetylcholine content of cytoplasmic and crude vesicular fractions

In this paper, the effects of tityustoxin and ouabain on the release and mobilization of ACh from cytoplasmic and vesicular stores of slices of cortex from the brain of the rat are described. Tityustoxin induced an increase of release of ACh which was time-dependent and this effect was accompanied by a concomitant decrease of the ACh, measured in cytoplasmic and crude vesicular fractions. Unlike tityustoxin, ouabain did not increase the release and synthesis of ACh during a 2 min period of stimulation but later, at 5, 10 and 15 min, ouabain increased the release and synthesis of ACh from incubated slices of cortex. After 10 min, ouabain caused a reduction of the content of ACh from cytoplasmic and crude vesicular fractions.