Suramin inhibits the early effects of PLA(2) neurotoxins at mouse neuromuscular junctions: A twitch tension study.

Several phospholipase A(2) (PLA(2)) neurotoxins from snake venoms can affect acetylcholine release at the neuromuscular junction. In isolated nerve-muscle preparations three distinct phases have been described for this phenomenon: An initial transient decrease in twitch tension; a second facilitatory phase during which twitch height is greater than control twitch height; and the last phase which causes a reduction in twitch height that finally results in paralysis. Suramin has been reported to inhibit the toxic effects of β-bungarotoxin and another PLA(2) neurotoxin, crotoxin in vitro and in vivo. We have further examined the effects of suramin on the three phases of the effects of the presynaptic PLA(2) neurotoxins β-bungarotoxin, taipoxin and ammodytoxin on mouse phrenic nerve-hemidiaphragm preparations. When preparations were pre-treated with suramin (0.3mM), the early biphasic effects (depression followed by facilitation) were abolished, and the time taken for final blockade induced by β-bungarotoxin, taipoxin and ammodytoxin A was significantly prolonged. In contrast, suramin did not significantly affect the facilitation induced by the potassium channel blocking toxin dendrotoxin I when applied under the same conditions. In addition, application of 0.3mM suramin did not prevent the facilitatory actions of 3,4-diaminopyridine (3,4-DAP) and tetraethylammonium chloride (TEA). Overall, the mechanism whereby suramin reduces the effects of PLA(2) neurotoxins remains elusive. Since suramin reduces both enzyme-dependent and enzyme-independent effects of the toxins, suramin is not acting as a simple enzyme inhibitor. Furthermore, the observation that suramin does not affect actions of standard K(+) channel blockers suggests that suramin does not stabilise nerve terminals.

INTRODUCTION

Suramin (an anti-trypanosomiasis drug and antagonist of P2 purinoceptors; Hoyle et al, 1990) has been reported to reverse the blocking action of non-depolarizing relaxants such as tubocurarine and pancuronium on twitch tension of rat diaphragm. This effect has not been observed on the paralysis caused by a depolarizing relaxant agent such as suxamethonium (Henning et al, 1992). Also suramin has an inhibitory effect on nerve terminal Ca2+ currents recorded from mouse triangularis sterni preparations (Henning et al, 1996; Lin et al, 2000). In addition, it has been reported that suramin can prevent the inhibitory effects of neurotoxins which block P-type Ca2+ channels, ω-conotoxin MVIIC and ω-agatoxin IVA, but has no effect on the non-selective Ca2+ channel blocker, Cd2+, on nerve-evoked muscle contractions of mouse diaphragm preparations (Lin et al, 2000). Suramin has also been shown to interfere with the pharmacological effects of some snake venoms and toxins, for example; the PLA2 activity of Bothrops jararacussu snake (Sifuentes et al, 2008), the myotoxic and paralyzing effect of bothropstoxin-I (Oliveira et al, 2003), and the pharmacology of some crotalid venoms (Arruda et al, 2002).

Suramin has also been shown to inhibit the physiological activity β-bungarotoxin, both in vivo and in vitro (Lin-Shiau and Lin, 1999). Suramin significantly delayed the time to paralysis induced by β-bungarotoxin in mice when administered intravenously 30min before toxin. Also, suramin at 0.3mM effectively delayed the neuromuscular blocking effect of β-bungarotoxin and crotoxin in mouse phrenic nerve-muscle preparations when applied 20 to 30min before, or after application of toxins. In contrast, suramin had no significant effect on the blocking action of a postsynaptic neurotoxin, α-bungarotoxin (Lin-Shiau and Lin, 1999). Recently, suramin was shown to antagonise the haematoxin action of Echis carinatus (Iran) snake venom, and significantly delaying time to death of envenomed mice (Fathi et al, 2010). Suramin also has a neuroprotective effect against β-bungarotoxin-induced cytotoxicity on cultured cerebellar granule neurons (Tseng and Lin-Shiau, 2003). In addition, suramin abolished the increase of frequency and amplitude of miniature end plate potentials (m.e.p.ps) induced by β-bungarotoxin (Lin-Shiau and Lin, 1999) and prolonged the time course of block of end plate potentials (e.p.ps) by β-bungarotoxin.

The purpose of the present study was to investigate the effects of suramin on a panel of prejunctionally active toxins, particularly looking at the early phases of their effects which are believed to be independent of their enzymatic activity.

MATERIALS AND METHODS

Reagents and materials

β-Bungarotoxin (T-5644, Lots 124H40081, 33H40141 and 68H4003) was supplied by Sigma Chemical Co Ltd (Poole, Dorset, England) and Latoxan, 20 Rue Leon Blum, 2600 Valence-France. Taipoxin was a gift from Dr David Eaker (Biochemistry Department, Uppsala University, Sweden) and was also purchased from Latoxan. Dendrotoxin I (DpI) was purchased from Ventoxin (Frederick, MD, USA). Two PLA2 toxins from the long-nosed viper (Vipera ammodytes ammodytes), ammodytoxin A and C, were gifts from Dr Igor Krizaj (Department of Biochemistry and Molecular Biology, University of Ljubljana, Slovenia). Tetraethylammonium chloride (TEA) and 3,4-diaminopyridine (3,4-DAP) and materials required for making salt solutions were purchased from Sigma Chemical Co Ltd or Gibco BRL Life Technologies Ltd. Suramin (sodium salt) was obtained from Bayer (Leverkusen, Germany).

Twitch tension recording of mouse phrenic nerve-hemidiaphragm preparations

Mouse hemidiaphragms and their phrenic nerves were removed from male mice (Balb C strain, 20-25gm) killed by CO2, in compliance with UK Home Office guidelines, immediately before experiments and placed in a dish containing physiological salt solution. The preparations were cleaned under the microscope of any connective tissue and the diaphragm was divided into two triangular or wedge-shaped parts. Each preparation was attached along its origin at the rib margin to a special tissue holder. The preparations were mounted in 10ml organ baths, under a resting tension of approximately 1gm in a physiological salt solution (Krebs solution) of the following composition: NaCl 118.4mM; KCl 4.7mM; NaHCO3 25mM; KH2PO4 1.2mM; MgSO4.7H2O 1.4mM; CaCl2 2.5mM; glucose 11.1mM, pH 7.3-7.4 and bubbled with oxygen containing 5% carbon dioxide and maintained at 27°C or 37°C. Twitches were evoked by stimulating the phrenic nerves via platinum ring electrodes (0.2Hz with square wave pulses of 0.1ms and sufficient strength to elicit maximal contractions) and recorded isometrically using Grass Model 79 and Grass Model 7D polygraphs, and Grass Force-Displacement Transducers FT03. In order to reveal any facilitation of neuromuscular transmission in twitch tension experiments, preparations were partially paralysed (to 15-20% of control) by either the addition of 9-10mM MgCl2 applied directly into the organ bath or by using physiological salt solution containing low Ca2+ (0.27-0.45mM). In some experiments, to ensure that direct stimulation of the nerve-muscle preparation did not contribute to the overall tension recorded, indirectly evoked twitches were blocked by adding successively greater concentrations of Mg2+ or low Ca2+ (less than 0.2mM) to the tissue bath and then re-adjusted with required amount of Mg2+ or Ca2+ to stabilise the twitch at 20% of control.

RESULTS

Effect of suramin on phrenic nerve-hemidiaphragm preparations

In order to determine the effect of suramin on twitch tension in the absence of PLA2 toxins, we first tested its effect on mouse phrenic nerve-hemidiaphragm preparations. The preparations were indirectly stimulated and partly paralysed with low Ca2+ or high Mg2+ at 27°C or 37°C. Under these conditions, suramin (0.3mM) blocked the twitches. The blocking effect of suramin appeared without any delay and was temperature-dependent. This effect was accelerated significantly by increasing the temperature from 27°C to 37°C. The time to block was 35 ±6.6min at 27°C (n = 3) and 13 ±3min at 37°C (n = 4) (Figure 1).

Figure 1.

Effect of suramin (0.3mM) on twitch tension of mouse hemi-diaphragm at 27°C (A) in low Ca2+ and (B) at 37°C, in high Mg2+ (9-11mM) Krebs solution. Preparations were stimulated indirectly at 0.2Hz with pulses of 0.1ms duration and voltage greater than required to produce the maximum response.

Effect of suramin on the responses to PLA2 neurotoxins

To monitor any changes in the amplitude of twitch tension caused by the PLA2 neurotoxins, it was necessary to maintain the twitch height at a stable level of 20 to 30% of control twitch height in the presence of suramin. This was achieved by adding a few drops (<100μl) of normal Krebs solution to the organ bath (at 4-6min intervals) until twitch tension height stabilised at desired height. Under these conditions the interaction between suramin and toxins was examined.

Phospholipase A2 neurotoxins cause triphasic changes on twitch tension of phrenic nerve-hemidiaphragm preparations. In view of the reported reversal action of suramin on neuromuscular blockade induced by these toxins, the effect of suramin on the triphasic action of three presynaptic PLA2 neurotoxins, β-bungarotoxin (3μg/ml = 0.15μM), taipoxin (1μg/ml = 20nM) and ammodytoxin A (10μg/ml = 0.2μM) was investigated.

After partial paralysis of preparations by reducing the concentration of Ca2+ (0.27-0.45mM) or increasing the concentration of Mg2+ (9-11mM), the preparations were pre-treated with 0.3mM suramin for 15-20min before application of toxins. Suramin abolished the early biphasic effects, i.e., depression and facilitation, and significantly prolonged the time taken to achieve twitch block. Figures 2-4 show the effects of suramin on the triphasic actions of β-bungarotoxin (n = 3), taipoxin (n = 4) and ammodytoxin A (n = 3), respectively.

A. Effect of β-bungarotoxin (3μg/ml = 0.15μM) on twitch tension of mouse hemi-diaphragm partly paralysed by low Ca2+ Krebs solution. B. Effect of suramin (0.3mM) on triphasic action of β-bungarotoxin (3μg/ml = 0.15μM) when applied 20 min before application of toxin. Preparations were stimulated indirectly at 0.2Hz with pulses of 0.1ms duration and voltage greater than required to produce the maximum response.

A. Effect of taipoxin (1μg/ml = 20nM) on twitch tension of mouse hemi-diaphragm partly paralysed by low Ca2+ Krebs solution. B. Effect of suramin (0.3mM) on the triphasic action of taipoxin (1μg/ml = 20nM) when applied 20 min before application of toxin. Preparations were stimulated indirectly at 0.2Hz with pulses of 0.1ms duration and voltage greater than required to produce the maximum response.

A. Effect of ammodytoxin A (10μg/ml = 2μM) on twitch tension of mouse hemi-diaphragm partly paralysis by high Mg2+ (9-11mM) Krebs solution. B. Effect of suramin (0.3mM) on the triphasic action of ammodytoxin when applied 20 min before application of toxin. Preparations were stimulated indirectly at 0.2Hz with pulses of 0.1ms duration and voltage greater than required to produce the maximum response.

Lack of effect of suramin on the facilitatory action of dendrotoxin I (DpI)

The effect of suramin on the facilitatory action of dendrotoxin I (DpI) (1μg/ml = 0.14μM) was investigated. Under the same conditions used for β-bungarotoxin, taipoxin, and ammodytoxin, preparations were incubated with 0.3mM suramin for 15-20min before application of DpI. In the control experiments, the facilitatory action of this toxin appeared without delay and without initial depression, the amplitude of twitch height increasing to more than twice of that of the control twitch height (263 ±13%) (n = 3) (Figure 5). Similar effects of DpI were observed in the experiments in which suramin was applied 20min before DpI. In these experiments, suramin did not significantly affect the facilitation by dendrotoxin I; twitch height increased to 275 ±11% of control.

Figure 5.

A. Effect of dendrotoxin I (DpI) (1μg/ml = 0.14 μM) on twitch tension of mouse hemi-diaphragm partly paralysed by high Mg2+ (9-11mM) Krebs solution. B. Lack of effect of suramin (0.3mM) on the facilitatory action of dendrotoxin I (DpI) (1μg/ml = 0.14μM) when applied 20min before application of toxin. Preparations were stimulated indirectly at 0.2Hz with pulses of 0.1ms duration and voltage greater than required to produce the maximum response.

Interaction of suramin with 3, 4-diaminopyridine (3, 4-DAP) and tetraethylammonium (TEA)

Under similar experimental condition to the previous experiments with PLA2 neurotoxins and dendrotoxin I, application of 0.3mM suramin in low Ca2+ at 27°C did not prevent the facilitatory actions of 3,4-DAP (0.1mM) and TEA (1-2mM) (Figure 6).

Figure 6.

Upper. Effect of 3,4-diaminopyridine (3,4-DAP) (0.1mM) on twitch tension of mouse hemi-diaphragm partly paralysis by high Mg2+ (9-11mM) Krebs solution. Middle and Lower. Lack of effect of suramin (0.3mM) on the facilitatory action of 3,4-DAP (0.1mM) and TEA (2mM) when applied 20min before application of chemical agents. Preparations were stimulated indirectly at 0.2Hz with pulses of 0.1ms duration and voltage greater than required to produce the maximum response. Control twitch tension in normal Krebs solution (B) Part paralysis of twitch by high Mg2+ (9-11mM).

DISCUSSION

The present study used twitch tension experiments on mouse hemidiaphragm preparations and revealed that the early effects of PLA2 neurotoxins, depression (phase I) and facilitiation (phase II), were abolished by suramin. Under the same conditions, suramin had no detectable effect on the facilitatory actions of dendrotoxin I (DpI), tetraethylammonium (TEA) and 3, 4-diaminopyridine (3,4-DAP).

It is generally accepted that the initial reduction of twitch tension (phase I) is due to the binding of the PLA2 toxin to the nerve terminal (Chang, 1985). Suramin abolished this early effect of PLA2 neurotoxins but only delayed the onset of the blocking phase. This suggests that suramin may compete with a PLA2 toxin acceptor on the nerve terminal to delay the binding of the toxins to their binding site, as twitch block eventually takes place in the continued presence of suramin. The facilatatory phase of β-Bungarotoxin, crotoxin, taipoxin, notexin and ammodytoxin coincides with a block a fraction of the K+ current recorded as perineural waveforms from mouse triangularis sterni preparations (Rowan and Harvey, 1988; Krizaj et al, 1995; Lin-Shiau and Lin, 1999). As suramin did not affect the facilitatory action of K+ channel blockers, TEA, 3,4-DAP and dendrotoxin it is unlikely that the pharmacology of suramin is mediated through potassium channels. Suramin is a polysulfate anionic compound rich in negative charges that can interact with positive charges on peptides and proteins, thus it is possible that suramin could directly bind with the PLA2 toxin. This interaction would be predicted to cause a conformational change which may delay binding to acceptors on the nerve terminals. However, it is difficult to explain the lack of effect of suramin on the neuromuscular blocking effect of a postsynaptic neurotoxin α-bungarotoxin or of the facilitatory toxin DpI, both of which also have positively charged residues. The mechanism of action of suramin at the neuromuscular junction is still not clear, although it was suggested that suramin inhibited Ca2+ entry to the nerve terminal by binding weakly to presynaptic voltage-dependent Ca2+ channels and reducing the release of acetylcholine (Henning et al, 1996; Lin et al, 2000). Such a mode of action can explain the twitch blocking effect of suramin on mouse nerve-hemidiaphragms partially paralysed by low Ca2+ or high Mg2+ but cannot account for the antagonistic effect of suramin on the neuromuscular effects of β-bungarotoxin, as Cd2+, a Ca2+ channel blocker, does not alter the pharmacology of such toxins on neuromuscular transmission (Lin-Shiau and Lin, 1999). Overall, the mechanism whereby suramin reduces the effects of PLA2 neurotoxins remains elusive. Since suramin reduces both enzyme-dependent and enzyme-independent effects of the toxins, suramin is not acting as a simple enzyme inhibitor. As suramin does not affect the actions of standard K+ channel blockers, it is not stabilising nerve terminals. Perhaps there is a direct physical interaction between suramin and the toxins as has been suggested to account for the effect of suramin on the myotoxic activity of Lys49 homologues (Murakami et al, 2007).