Edited by Negri Alberto and Migliore Matteo.
INTRODUCTION
Phoneutria nigriventer is a spider belonging to the family Ctenidae, commonly known as banana spider. The aggressiveness of the species belonging to the genus Phoneutria and the high toxicity of their venom pose these spiders at the top in terms of hazard to humans, despite cases of serious envenomation are rare (0.5%).
A clinico-epidemiological study of bites by spiders of the genus Phoneutria.2000
The spider is quite large, reaching 15 centimeters in length of the body including the legs; the species lives in the forests and wetlands and is quite common in various parts of Brazil, and has also been found further south in northern Argentina, Uruguay and Paraguay. He lives mostly in the Amazon and Atlantic forest, and its propensity to hide in the banana plantations favors the contact with the men and enhances the reputation of dangerous spider.
VENOM
The Phoneutria nigriventer venom contains a variety of toxins that have in common the ability to act on voltage-gated sodium channels, calcium and potassium. In addition, they also present activity on glutamate transporter.
These toxins act on neuronal sodium channels, inhibiting them; this can lead to depolarization of muscle fibers and nerve endings in the vicinity of the neuromuscular junctions and the activation of the autonomic nervous system, to cause the release of neurotransmitters such as acetylcholine and catecholamines. The peptides present in its poison can induce contractions of vascular muscle and increase vascular permeability by activating the system of the tissue kallikrein and stimulating the release of nitric oxide (NO). The neurotoxin PhTx3, at lethal concentrations causes loss of muscle control and breathing problems, resulting in paralysis and eventual asphyxiation. In addition, the venom causes intense pain and inflammation for an excitatory effect on the serotonin 5-HT4 receptors of sensory nerves; this stimulation causes a release of neuropeptides such as substance P which triggers inflammation and acute pain.
This helps to understand the local and systemic symptoms brought by his bite, among which we find priapism (persistent and abnormal erection, often painful, only carried out by the corpora cavernosa of the penis, not accompanied by the usual sexual desire) which can lead to impotence, tachycardia, vomiting, diarrhea, numbness, edema and anaphylactic shock.
Phoneutria nigriventer venom: a cocktail of toxins that affect ion channels.2002
The venom of the spider P. nigriventer appears to be a rich source of channel-blocking toxins. A number of neurotoxic peptides have been purified from the venom of this spider. Furthermore, sequences obtained from P. nigriventer venom gland cDNAs suggest the existence of at least 30 toxic peptides. Although some of these peptides have homology among each other, the Phoneutria toxins are mostly unrelated to other known neurotoxins.
Toxins whose role has been better characterized and with a possible clinical relevance:
PnTx1
The first venom fraction, named PnTx1 causes neurotoxic symptoms like tail elevation, excitation, salivation and spastic paralysis, when administered by intracerebro-ventricular injection on mice. That's because it reversibly inhibites sodium currents carried out by Na v 1.2, Na v 1.7, Na v 1.4 and Na v 1.3 channels (nine subtypes of mammalian voltage-dependent sodium channels have been identified : Na v 1.1 to Na v 1.9).
The kinetics of inhibition of peak sodium current varies with membrane potential, in fact is reported that the apparent affinity of PnTx1 for the channel increases as the membrane is depolarized. One possible mechanism could be the stabilization of the inactivated state.
Since there are a number of channelopathies involving these channels, the study of PnTx1 opens a new perspective for understanding specific characteristics of the channel and the development of new drugs.
Inhibitory effect of the recombinant Phoneutria nigriventer Tx1 toxin on voltage-gated sodium channels.2012
Phoneutria nigriventer toxin 1: a novel, state-dependent inhibitor of neuronal sodium channels that interacts with micro conotoxin binding sites.2006
PnTx2
Among the many different toxins described in this venom, PnTx2-6 (part of the second fraction) stands out for the toxicity and lethality. It has been demonstrated that causes erection in anesthetized rats and mice. Male mice injected with this toxin present a dramatic intoxication with two early signs: penile erection and hypersalivation, followed by death, with signs of severe respiratory distress. Severe human accidents involving this spider are characterized by lung edema, therefore, it seems plausible to assume that death is caused by lung edema.
Physiological penile erection involves non-adrenergic non-cholinergic transmission (NANC) and the mediation by nitric oxide (NO) have been demonstrated by studies focused on the role of NO in the signs and symptoms of intoxication by Tx2, the results showed that pretreatment with an inhibitor of n-NOS, 7-NI eliminated all the signs and symptoms of intoxication by the toxin, including the deadly lung edema.
It was recently named “eretina” because when this toxin was injected directly into the mouse penis, it induced an erection. Priapism is one of several symptoms observed in accidental bites by the spider Phoneutria nigriventer, potential complications include ischemia, clotting of the blood retained in the penis (thrombosis), and damage to the blood vessels of the penis which may result in an impaired erectile function or impotence. In serious cases, the ischemia may result in gangrene, which could necessitate penis removal.
Relaxation of corpora cavernosal smooth muscle is associated with penile erection, corpora cavernosal smooth muscle contraction with flaccidity.
The trabeculae make up the walls of the sinusoids (lacunar spaces). In the flaccid state, the smooth muscle of the trabeculae is held in a state of tonic contraction that allows free venous drainage of the lacunar spaces through the subclinical venues. Following dorsal nerve stimulation, the relaxation of the smooth muscle in the inflow increases arterial supply to the lacunar spaces. In conjunction with relaxation of the trabecular smooth muscle, the spaces expand compressing the venues against the tunica albuginea, reducing outflow.
Acetylcholine may modulate the adrenergic contractile stimulus to cavernosal smooth muscle, promoting tumescence and erection but its principal action is upon the endothelium of vascular and sinusoidal tissue to liberate NO.
NO acts by stimulation of the intracellular enzyme guanylate cyclase which binds GTP and converts it to cGMP which induces relaxation of smooth muscle by the activation of an enzyme cascade, mediated through intracellular calcium levels. cGMP-dependent protein kinases PKG causes a phosphorylation of a protein, phospholambin, which normally inhibits calcium pumps, within the cell wall and sarcoplasmic reticulum, thereby maintaining a high level of intracellular calcium and smooth muscle contraction. Phosphorylated phospholambin is inactive, calcium pumps, therefore, become active and intracellular calcium levels are reduced, allowing smooth muscle to relax.
It is known that the vasodilator mediator NO can be produced by the sinusoidal endothelium of the corpus cavernous as well as by the penile nerves.
The role of nitric oxide in penile erection.2001
PnTx2−6 molecular modeling. It’s a cysteine-rich peptide toxins containing eight cysteine residues whose disulfide bridges are highlighted with different colours.
PnTx2-6 is a voltage dependent sodium channel selective toxin, slows Na+ channels inactivation in nitrergic neurons, allowing Ca2+ influx to facilitate NO/cGMP signalling, which promotes increased NO production. In addition, this relaxation effect is independent of phosphodiesterase enzyme type 5 inhibition.
PnTx2-5 is a similar toxin extracted from the same fraction, and differs from PnTx2-6 by five amino acid residues. Mice injected intraperitonealy with PnTx2-5 induced a toxic syndrome similar to PnTx2-6, including penile erection.
Possible mechanism of action of PnTx2-6 toxin in the penis. The neuronal depolarization caused by PnTx2-6 (since this toxin delay the inactivation period of Na+ channels) lead to an increase in Ca2+ influx, probably via activation of N-type Ca2+ channels, which in turn activates nNOS inducing NO production and improving relaxation of vascular smooth muscle, causing penile erection. Red arrows mean inhibition.
Since it has been demonstrated a therapeutic effect of PnTx2-6 and correlation of this toxin to increased NO production and/or bioavailability, this naturally existing polypeptide has the potential to emerge as a new pharmacological tool in conditions where vascular and endothelial dysfunction persists, and where increasing NO bioavailability may be able to reverse the dysfunction. In particular that use of PnTx2-6 can reverse hypertension-induced Erectile Dysfunction.
Blockade of neuronal nitric oxide synthase abolishes the toxic effects of Tx2-5, a lethal Phoneutria nigriventer spider toxin.2004
Increased cavernosal relaxation by Phoneutria nigriventer toxin, PnTx2-6, via activation at NO/cGMP signaling.2012
Nitric oxide-induced vasorelaxation in response to PnTx2-6 toxin from Phoneutria nigriventer spider in rat cavernosal tissue.2010
Phoneutria nigriventer spider toxin Tx2-6 causes priapism and death: a histopathological investigation in mice.2012
PnTx3
Voltage-sensitive ion channels are of fundamental importance for controlling the membrane potential and the venom of the spider Phoneutria nigriventer can represent a tool to investigate the functions of ion channels at the molecular and cellular levels.
One of the four toxic fractions of Phoneutria venom, PhTx3 (containing peptide toxins Tx3-1 through Tx3-6), has received considerable attention in the last few years, because is composed by peptides acting as potent blockers of tigh voltage-dependent Ca2+ channels (classified as L-, N-, P-, Q-, R-, or T-type based on the biophysical and pharmacological properties of the currents they mediate).
-as neuroprotector:
Cell calcium plays a key role in mediating ischemic neuronal damage and its intracellular concentration is critically regulated. High voltage-dependent calcium channels (VDCC) mediate the presynaptic Ca2+ influx that generates glutamate release during brain ischemia.
The glutamate-Ca2+ neurotoxicity hypothesis states that excessive release of glutamate causes pathological elevation of intracellular Ca2+ concentration which activates calcium-dependent processes leading to cell death. One cause of excessive glutamate release in the brain is ischemia. Glutamate excitotoxicity consists in a massive release of glutamate during ischemia, that leads to the hyper stimulation of glutamate receptors and subsequent Ca2+-induced free radical injury; this plays a central role in triggering neuronal damage.
Ischemic cell death in brain neurons.1999
The hippocampus has one of the highest rates of oxygen consumption in the brain, which may make it particularly susceptible to the glucose and oxygen deprivation.
Tx3–3 and Tx3–4 are two toxins isolated from the third fraction of this venom, and they act as N-and P/Q-type Ca2+-channel blockers inhibiting the increase in glutamate release, neuronal death, and loss of neurotransmission in hippocampus CA1 that result from the in vitro ischemia. Moreover, Tx3–3 and Tx3–4 are also effective preventing the in vitro neuronal cell death when they are applied after the onset of the induced ischemia.
Pharmacological agents, such as these spider toxins, might be valuable as part of the therapeutic strategy to reduce damage provoked by cerebral ischemia.
Phoneutria spider toxins block ischemia-induced glutamate release, neuronal death, and loss of neurotransmission in hippocampus.2009
-as analgesic:
Tx3-3 is an isoform of the fraction 3 of Phoneutria nigriventer venom, and it has been revelated as a high-threshold VDCC blocker, preferentially towards P/Q and R-type.
The presence of voltage-dependent calcium channels (VDCC )in pain-modulation areas, such as the spinal cord, dorsal root ganglia, and brainstem, indicate the essential role of these channel types in the processing of painrelated information in the central nervous system.
A significant functional role of VDCCs in neuropathic pain mechanisms has been substantiated by several lines of evidence; the armed spider toxin Tx3-3 effectively alleviates neuropathic pain, without promoting the adverse effects usually developed by VDCC blockers.
Furthermore, VDCCs seem to be implicated in the central pain sensitization that occurs following nerve injury and during inflammatory states. VDCCs regulate calcium influx, initiating the release of various neurotransmitters at the spinal and supraspinal levels. Thus, the inhibition of calcium-dependent glutamate elease by Tx3-3 could explain, at least in part, the fast antinociceptive effect produced by both spinal and supraspinal injection of this toxin.
Therefore, is believed that Tx3-3 produces its effect by blocking supraspinal VDCCs implicated in descending facilitatory pain pathway.
Tx3-3 don't change mechanical sensitivity except when alterations of nociceptive processing secondary to persistent injury are present, as in mechanical allodynia produced by nerve injury.
These results hold a significant promise that Tx3-3 can represent a novel therapeutic agent to manage neuropathic pain.
Antinociceptive effect of Brazilian armed spider venom toxin Tx3-3 in animal models of neuropathic pain.2011
PnTx4
The fourth fractionated part from Phoneutria nigriventer venom is called PnTx4 and several studies revelated how it is highly toxic towards insects and displays only a slight toxicity when injected in mice (in which it's able to inhibit glutamate uptake in preparations of central nervous system).
Tx4 (6-1), one of the toxins isolated from this fraction, is a basic 48 amino acids polypeptide highly reticulated by five disulfide bridges; it's an α-like toxin, since it binds to insect Na+ channel at site 3, slowing down sodium current inactivation and prolonging the axonal action potential in the same way as do scorpion α-toxins and other toxins active on insect Na+ channels.
PnTx4-3 (the sequence of PnTx4-3 is very similar to Tx4 (6-1), is 98% identical) is able to inhibit the glutamate uptake inducing immediate excitatory effects when injected intrathoracically in house flies and cockroaches.
PhTx4 seems to act at the level of the peripheric nervous system in insects, stimulating glutamate release at cockroach neuromuscular junctions and in mammals inhibiting the glutamate uptake in rat brain synaptosomes. The role of PnTx4-3 seems to be similar to the Tx4 one, prolonging evoked axonal action potentials in insect nervous system, due a slowing down of sodium current inactivation that causes the inhibition of glutamate uptake.
In fact several isoforms of glutamate transporters, named EAAT1 (GLAST), EAAT2 (GLT-1), EAAT3 (EAAC1), EAAT4 and EAAT5, have been identified in the central and peripheral nervous systems of different species. Their function relies on the Na+ and other ions gradient across the plasma membrane; changes in the concentration of these ions can alter transport function up to the point of reversing its activity, resulting in net glutamate release.
The toxin Tx4 (6-1) from the spider Phoneutria nigriventer slows down Na+ current inactivation in insect CNS via binding to receptor site 3.2002
PnTx4-3, a new insect toxin from Phoneutria nigriventer venom elicits the glutamate uptake inhibition exhibited by PhTx4 toxic fraction.2003
