Selective release of ATP from sympathetic nerves of rat vas deferens by the toxin TsTX-I from Brazilian scorpion Tityus serrulatus

Abstract

1. The effects of the main component of the Tityus serrulatus scorpion venom, toxin TsTX-I, were studied on the contractility and release of neurotransmitters in the rat vas deferens. Since TsTX-I is known to act on sodium channels, we used veratridine, another sodium channel agent, for comparison. 2. Toxin TsTX-I induced concentration-dependent contractions with an EC(50) value of 47.8+/-0.1 nM and a maximum effect of 84.4+/-10.4% of that for BaCl(2). 3. Contractions by TsTX-I were abolished by denervation or tetrodotoxin (0.1 microM), showing that the toxin effects depend on the integrity of sympathetic nerve terminals. 4. To check for the presence of a noradrenergic component, experiments were conducted after removal of adrenergic stores in nerve terminals by reserpinization (10 mg kg(-1), 24 h prior to experiments) or blockade of alpha(1) adrenoceptors by prazosin (30 microM), showing that these procedures did not modify the response to TsTX-I, and therefore that adrenoceptors were not involved in contractions. 5. To check for the presence of a purinergic component, experiments were carried out after blockade of P(2X) receptors by suramin (0.1 mM) or desensitization by alpha,beta-methylene-ATP (30 microM). These agents greatly abolished the contractile response to TsTX-I (about 83% by desensitization and 96% by suramin), showing the involvement of purinergic receptors. 6. The release of noradrenaline and purinergic agents (ATP, ADP, AMP and adenosine) was detected by HPLC. Together, the total release of purines in the presence of TsTX-I was about 42 times higher than in the control group. In contrast, TsTX-I did not modify the overflow of noradrenaline, showing that the release was selective for purines. 7. The release of purinergic agents was reduced by the N-type calcium channel blocker omega-conotoxin GVIA (1 microM) and by the P/Q-type blocker omega-conotoxin MVIIC (1 microM), showing that the effects of TsTX-I are calcium-dependent. 8. The results show that TsTX-I produced a selective release of purines from postganglionic sympathetic nerves in the rat vas deferens.

Figure 1

Concentration–response and time–response curves for toxin TsTX-I or VTD in the rat vas deferens. Each point represents the mean±s.e.mean (n=7–8) of the contraction induced by TsTX-I or VTD. Concentration–response curves are shown in panel a and the temporal course of contractions for each dose (time–response curves) is represented in panels b (TsTX-I) and c (VTD). Data are percent values in relation to maximum contraction induced by BaCl₂ (10 mM) applied at the beginning of experiments.

Figure 2

Effects of denervation on contraction induced by toxin TsTX-I or VTD in the rat vas deferens. Typical original traces of contractions induced by TsTX-I (150 nM) or VTD (100 μM) in normal or denervated vas deferens. At the beginning of the experiments, a single dose of BaCl₂ (10 mM) was applied to verify the integrity of the organ. In denervated tissue, tyramine (0.1 mM) was added before BaCl₂ to ensure that the organs were denervated (not shown). Organs that responded to tyramine were discarded. Dotted arrows indicate that recording was stopped after achieving the maximum effect of barium, followed by washout, and that recording was restarted before adding TsTX-I or VTD. Six experiments were performed with similar results.

Figure 3

Effects of reserpinization or desensitization of purinergic receptors on the contraction induced by toxin TsTX-I or VTD in the rat vas deferens. Each bar represents the mean±s.e.mean (n=7) of the maximum contraction induced by TsTX-I (45 nM) or VTD (10 μM). The drugs were added to the organs of rats without (control), or with injection of RES (10 mg kg⁻¹, 24 h before experiments), or to organs whose purinergic receptors were desensitized with α,β-mATP (mATP, 30 μM, 15 min before). The mATP+RES group represents organs of reserpinized animals with purinergic desensitization. Data are percent values in relation to the maximum contraction induced by BaCl₂ (10 mM applied at the beginning of experiment). ^*P<0.01 in relation to control group, #P<0.001 in relation to RES group.

Figure 4

Effects of PZS or SUR on the contraction induced by toxin TsTX-I or VTD in the rat vas deferens. Each bar represents the mean±s.e.mean (n=7) of the maximum contraction induced by TsTX-I (45 nM) or VTD (10 μM). Effects were obtained in the absence (Control) or presence of PZS (30 μM), SUR (0.1 mM) or both (PZS+SUR), applied 30 min before. Data are percent values in relation to maximum contraction induced by 10 mM of BaCl₂, applied at the beginning of the experiment. ^*P<0.001 in relation to control group, ^#P<0.001 in relation to PZS group, ^§P<0.01 in relation to SUR group.

Figure 5

Effects of toxin TsTX-I and VTD on neurotransmitter overflow in the rat vas deferens. Each bar represents the mean±s.e.mean (n=3–8) of the release of noradrenaline or purines (ATP, ADP, AMP or adenosine) in the absence (control) or presence of TsTX-I (150 nM) or VTD (30 μM). Note that since ATP is rapidly degraded by metabolic enzymes into ADP, AMP and adenosine, the total sum of the released nucleotides can be used as an estimate of ATP released, instead of the actual value for ATP alone (Todorov et al., 1996). Data are presented as pmol mg tissue⁻¹. ^*P<0.05 in relation to control group.