The venom of the spider P. nigriventer has a cocktail of toxins active on mammal cells, including several neurotoxins that exert diverse biological effects. Human response to this spider venom is characterized by various symptoms, one of which is priapism.
This is the first study to demonstrate the effect of the toxin Tx2-6 purified from the venom of the spider P. nigriventer on rat erectile function. In the present study, we examined the effect of Tx2-6 on rat erectile function using an in vivo model, the effect of this toxin on the release of NO in rat corpus cavernosum and the potentiation effect of Tx2-6 on penile erection of animals presenting severe ED such as the DOCA-salt hypertensive rats.
The potentiating effect of Tx2-6 on rat penile erection seems to be mediated by relaxation of the vasculature and smooth muscle in the corpus cavernosum induced by the release of NO. This important relaxant agent could be synthesized in nerve terminals and endothelial cells lining blood vessels and lacuna spaces in the corpus cavernosum (Burnett et al., 1992
). The crucial role of NO derived from non-andrenergic, non-cholinergic (NANC) nerves and possibly from the corpus cavernosum sinusoidal endothelium in penile erection is widely accepted (Toda et al., 2005
). The mechanisms involved in the regulation of either contraction or relaxation of the corpus cavernosum and penile vasculature have been intensely investigated during the past decade (Andersson and Wagner, 1995
). NO has been suggested as the main mediator of NANC nerve-induced relaxation of the corpora cavernosa in vitro
, as well as of increased intracavernous pressure in vivo
in a variety of mammals, including rats (Burnett et al., 1992
), rabbits (Teixeira et al., 1998
), dogs (Hayashida et al., 1996
), monkeys (Okamura et al., 1998
) and humans (Leone et al., 1994
venom causes activation of voltage-dependent sodium channels in muscle and nerve cell membranes (Cordeiro et al., 1995
; Gomez et al., 2002
). Teixeira et al. (2003)
showed that this spider venom is active on vascular smooth muscle mainly due to increased influx of calcium from extracellular sources. The PhTx2-6 fraction has been proposed to be the main toxic fraction of the P. nigriventer
spider venom, responsible for increasing permeability of sodium channels (Mattiello-Sverzuta and Cruz-Hofling, 2000
It has been demonstrated that both the fraction PhTx2-6 and the purified toxin (Tx2-6) act on sodium channels slowing down its inactivation (Araujo et al., 1993
; Matavel et al., 2002
), an effect similar to that observed for α
-toxins from arthropods (Cestele and Catterall, 2000
). This change in the inactivation time would maintain a depolarized state of the nerve fibers resulting in an increase in calcium influx and consequently an increase in neurotransmitters release, as observed for acetylcholine (Moura et al., 1998
). Action potential opens tetrodotoxin-sensitive sodium channels in nitrergic nerve terminals and promote the Ca2+
influx possibly through N-type Ca2+
channels in blood vessels (Toda et al., 2005
). This seems to be the case in the corpus cavernosum, because relaxations elicited by nerve stimulation of human and canine corpus cavernosum strips were sensitive to ω
-conotoxin, an N-type Ca2+
channel blocker (Leone et al., 1994
; Okamura et al., 2001
). Increase in cytosolic calcium participates in activation of nNOS in the presence of calmodulin (Bredt and Snyder, 1990
). Neuronal release or synthesis of NO is dependent on intracellular bioavailability of Ca2+
in the rabbit corpus cavernosum (Saito et al., 1996
As mentioned before, intra-peritoneal injections of Tx2-5, a toxin that differs of Tx2-6 by seven amino acid residues, induced a toxic syndrome that included priapism and death, these effects being partially prevented by L-NAME and abolished by the nNOS-selective inhibitor 7-NI (Yonamine et al., 2004
). Studies of structure–function are of fundamental importance to clarify the activities of these toxins, concerning toxicity and potentiating of erection. Furthermore, possible differences within species, i.e. mice and rats, would be evaluated. Considering these data one could speculate that the potentiated erection induced by Tx2-6 could also point out to an important role of nNOS on the increased NO release. Further experiments are needed to confirm this conclusion.
There has been evidence that penile erection is driven by two distinct hemodynamic stages. The first one is dependent on NO release from NANC nerves in the penis causing dilation of arterioles and expansion of sinusoids. This stage is followed by the release of endothelium-derived relaxing factor from endothelial cells surrounding corpus cavernosum trabecules in response to shear stress. It is generally accepted that NO derived from eNOS is the major vasodilator for this second stage of erection (Burnett et al., 1992
; Hurt et al., 2002
). From our results, it is not clear yet whether Tx2-6 acts to stimulate NO release from NANC nerve terminals, to facilitate the release of acetylcholine from pre- and post-synaptic parasympathetic nerves, or to sensitize the corpus cavernosum endothelial cells to the increase of shear stress. Additional studies are required to reveal the precise mechanism by which Tx2-6 induces NO release facilitates penile erection.
The erection induced by Tx2-6 could be observed about 15 min after subcutaneous injection. It can be the necessary time for the toxin to reach its target. Preliminary experiments using Tx2-6 labeled with technetium-99 (data not shown) revealed that after 10 min the distribution of labeled toxin is predominantly in kidney and bladder. It is worth mentioning that this toxin was also found in the penile tissue. This suggests a possible existence of specific receptors on corpus cavernosum for the toxin. Other experiments are being carried out to clarify this point.
Our results clearly demonstrated for the firs time, that Tx2-6, a toxin purified from the venom of the P. nigriventer spider can induce erection and facilitate the ganglionic-stimulated penile erection in control rats. We have also shown that this toxin is able to induce and facilitate the release of NO from cavernosum tissue of normotensive and hypertensive rats. Moreover, the severely depressed erectile function of DOCA-salt hypertensive rats was essentially normalized by Tx2-6 administration. These data suggest that Tx2-6 is a potential tool for the development of new therapeutics for the treatment of ED.