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The effect of temperature on the effects of the phospholipase A2 neurotoxins β-bungarotoxin and taipoxin at the neuromuscular junction
Toxicon 70 (2013) 86–89
Contents lists available at SciVerse ScienceDirect
Toxicon journal homepage: www.elsevier.com/locate/toxicon
Short communication
The effect of temperature on the effects of the phospholipase A2 neurotoxins b-bungarotoxin and taipoxin at the neuromuscular junction Behrooz Fathi a, *, Alan L. Harvey b, Edward G. Rowan b a b
Department of Pharmacology, School of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 161 Cathedral Street, Glasgow G4 0RE, UK
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 September 2012 Received in revised form 4 April 2013 Accepted 16 April 2013 Available online 3 May 2013
Snake venom neurotoxins with phospholipase A2 affect the neuromuscular junction with three distinct phases. There is a transient decrease in twitch height, followed by a facilitatory phase and finally a progressive blockade. It has been suggested that the initial phase is a direct consequence of the binding of the toxins to nerve terminals. This study was designed to determine whether the initial phase is present under conditions that would reduce the enzyme activity of the toxins. At 27 C, b-bungarotoxin and taipoxin exhibited all three phases, i.e. 5–6 min after exposure to the preparation, twitch height was significantly reduced (P < 0.5) to 50 4% and 64 9% of control respectively. This was followed by facilitation and subsequent blockade. However, at 20 C, neither toxin exhibited the first phase while the second phase, although reduced, clearly occurred and the blocking activity of these toxins always appeared. The data clearly demonstrate that the initial fall is temperature dependent as reducing the temperature from 27 C to 20 C blocks the first phase. As the second phase still occurs the toxins must have bound to their target. Therefore, the first phase cannot simply be a toxin binding step. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Phospholipase A2 neurotoxins b-Bungarotoxin Taipoxin Neuromuscular junction Temperature
Some snake venom phospholipases A2 (PLA2) like bbungarotoxin and taipoxin are presynaptic neurotoxins that affect the neuromuscular junction and modify neurotransmitter release. These neurotoxins exhibit a triphasic modulation of acetylcholine (ACh) release on isolated mammalian nerve-muscle preparations, which is reflected in changes in twitch height (see Rossetto and Montecucco, 2008; Punger car and Kri zaj, 2007, for reviews). The first phase is a transient initial reduction in the amount of ACh released in response to an action potential, and has been suggested to be caused by the binding of the toxins to their presynaptic receptors, although direct evidence for this is lacking. This effect is regarded as being independent of * Corresponding author. Tel.: þ98 (0)511 8813907, þ98 9159765651 (mobile). E-mail address: [email protected] (B. Fathi). 0041-0101/$ – see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxicon.2013.04.016
PLA2 enzyme activity (Chang and Su, 1982). It is followed by the second phase, the facilitatory phase, when ACh release is augmented and twitch height is increased. In mammalian, but not in amphibian preparations, the facilitatory phase is independent of PLA2 activity, suggesting that the toxins may induce their effects with different mechanisms of action at the amphibian and mammalian neuromuscular junctions. These two early phases are thought to be phospholipase activity-independent as they are still observed in experiments with p-bromophenacyl bromide (p-BPB)treated toxins and in the presence of Sr2þ, which inhibited enzyme activity of the PLA2 (Chang and Su, 1982; Rowan et al., 1990). Finally, the third phase is a progressive decline, leading to complete block of transmitter release and muscle paralysis. This phase is dependent on the PLA2 enzyme activity of the toxins (Chang et al., 1973) after they enter into the cytosol (Rigoni et al., 2008; Logonder et al.,
B. Fathi et al. / Toxicon 70 (2013) 86–89
2009). Most studies on the PLA2 neurotoxins have focused on their final neuromuscular blocking action. The initial blocking phase of toxicity has not been particularly investigated. The purpose of this study was to determine whether the initial phase caused by b-bungarotoxin and taipoxin can be affected by temperature. Lowering the temperature was used as a means to reduce the enzymatic activity of the toxins. Experiments were performed on mouse phrenic nervehemidiaphragm preparations (male Balb C strain, 20–25 g) maintained in standard Krebs–Henseleit solution at 20, 27 or 37 C. Phrenic nerves were stimulated at frequencies of 0.2 Hz with rectangular pulses of 0.1 ms duration and voltages greater than that required to produce maximal twitches. In order to reveal any facilitation of neuromuscular transmission, preparations were partly paralysed by addition of 9–10 mM MgCl2 applied directly to the bath. The twitch height was reduced to 15–20% of control and then allowed to stabilise for 20–30 min before applying toxins. b-Bungarotoxin (T-5644, Lots 124H40081, and 68H4003) was supplied by Sigma Chemical Co. Ltd., Poole,
87
Dorset, England. Taipoxin was a gift from Dr. David Eaker (Biochemistry Department, Uppsala University, Sweden) and was also purchased from Latoxan Company, 20 Rue Leon Blum, 2600 Valence-France. At 27 C, b-bungarotoxin (150 nM) and taipoxin (20 nM) exhibited all the cardinal signs of phospholipase A2 neurotoxins on twitch tension (Figs. 1 and 2), i.e. approximately 6 min after exposure to the toxin, twitch height was significantly reduced (P < 0.05, Student’s t-test) to 50 4% and 64 9% of control (n ¼ 3–7) respectively. This was followed by an increase in twitch tension to 400% and more than 500% respectively and the subsequent block of twitches (Figs. 1 and 2). However, at 20 C neither toxin exhibited the first phase while the second phase, although reduced, clearly occurred. For b-bungarotoxin, the times to final block at 20 C, 27 C and 37 C were 291 22 min, 229 10 min and 98 11 min, respectively (Fig. 1) which were significantly different (P < 0.05). For taipoxin, the times to final block at 20 C and 27 C were 70 3 min and 63 6 min respectively, which were not significantly different (Fig. 2). Taipoxin at 37 C did not exhibit the two
Twitch (% of control)
600
20 ° C
500
27 ° C
400
37 ° C
300 200 100 0 0
50
100
150
200
250
300
Time (min) 500
27° C
Twitch (% of control)
BuTx 400
300
20° C
200
100
37° C
0 0
5
10
15
20
Time (min) Fig. 1. Top – Effect of b-bungarotoxin (3 mg/ml ¼ 150 nM) on twitch tension of mouse phrenic nerve-hemidiaphragm preparations at 20 C, 27 C and 37 C in high Mg2þ (9–11 mM) solution. Preparations were stimulated indirectly at 0.2 Hz with pulses of 0.1 ms duration and voltage greater than required to produce the maximum response. Bottom – First 20 min in b-bungarotoxin (150 nM) at 20 C, 27 C and 37 C. At 20 C, the first phase depression was not evident.
88
B. Fathi et al. / Toxicon 70 (2013) 86–89
600
20° C
Twitch (% of control)
500
27° C
400 300 200 100 0 0
10
20
30
40
50
60
70
80
Time (min) 600 taipoxin
Twitch (% of control)
500
27 C 400
300
200
20 C
100
0 0
5
10
15
20
Time (min) Fig. 2. Top – Effect of taipoxin (20 nM) on twitch tension of mouse phrenic nerve-hemidiaphragm preparations at 20 C and 27 C in high Mg2þ (9–11 mM) solution. Preparations were stimulated indirectly at 0.2 Hz with pulses of 0.1 ms duration and voltage greater than required to produce the maximum response. Bottom – First 20 min taipoxin (20 nM) at 20 C and 27 C. At 20 C, the first phase depression was not evident.
early phases and only blocked the twitches in less than 20 min which was significantly faster than the times at the lower temperatures (P < 0.05). As it is expected that most enzymatic reactions increase with increasing the temperature, phospholipid hydrolysis by phospholipase enzymes should be temperaturedependent and it has been shown that the blocking phase is dependent on the PLA2 enzyme activity of toxins (Chang et al., 1973; Rowan et al., 1990). The enzyme activity of b-bungarotoxin, taipoxin, and crotoxin is reduced to 4–10% of control when the temperature is lowered from 37 C to 27 C (Su and Chang, 1984). The time taken for complete
block of neuromuscular transmission in the mouse hemidiaphragm by these toxins was markedly prolonged (five-fold) at 27 C compared to at 37 C (Su and Chang, 1984). The present results reconfirm that by lowering the temperature, the neuromuscular blocking activity of these toxins is delayed. The results also clearly demonstrate that the initial fall in twitch tension is temperature-dependent because reducing the temperature from 27 C to 20 C eliminated the first phase. However, the facilitation (phase 2) was almost not affected by lowering the temperature, indicating that the toxins must have bound to their target on the nerve terminal. The
B. Fathi et al. / Toxicon 70 (2013) 86–89
initial phase of the toxins’ action thus appears to require the enzymatic action of the toxins in order for it to be evident as a reduction in twitch height. An additional possibility is that the presently unknown receptor or binding site for the toxins is sensitive to temperature. However, this target for the enzymes’ action is different from the intracellular targets that are suggested to be responsible for the slow and irreversible failure of ACh release and twitch blockade. Acknowledgements This work was funded by Ferdowsi University of Mashhad, Mashhad-Iran, and Strathclyde University, Scotland. We thank members of the Strathclyde Institute of Pharmacy and Biomedical Sciences for their help and support. Conflict of interest None.
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References Chang, C.C., Chen, T.F., Lee, C.Y., 1973. Studies of presynaptic effect of bbungarotoxin on neuromuscular transmission. J. Pharmacol. Exp. Ther. 184, 339–345. Chang, C.C., Su, M.J., 1982. Presynaptic toxicity of the histidine-modified, phospholipase A2-inactive, b-bungarotoxin, crotoxin and notexin. Toxicon 20, 895–905. Logonder, U., Jenko-Pra znikar, Z., Scott-Davey, T., Punger car, J., Kri zaj, I., Harris, J.B., 2009. Ultrastructural evidence for the uptake of a neurotoxic snake venom phospholipase A2 into mammalian motor nerve terminals. Exp. Neurol. 219, 591–594. Punger car, J., Kri zaj, I., 2007. Understanding the molecular mechanism underlying the presynaptic toxicity of secreted phospholipases A2. Toxicon 50, 871–892. Rigoni, M., Paoli, M., Milanesi, E., Caccin, P., Rasola, A., Bernardi, P., Montecucco, C., 2008. Snake phospholipase A2 neurotoxins enter neurons bind specifically to mitochondria, and open their transition pores. J. Biol. Chem. 283, 34013–34020. Rossetto, O., Montecucco, C., 2008. Presynaptic neurotoxins with enzymatic activities. Handb. Exp. Pharmacol. 184, 129–170. Rowan, E.G., Pemberton, K.E., Harvey, A.L., 1990. On the blockade of acetylcholine release at mouse motor nerve terminals by b-bungarotoxin and crotoxin. Br. J. Pharmacol. 100, 301–304. Su, M.J., Chang, C.C., 1984. Presynaptic effects of snake venom toxins which have phospholipase A2 activity (b-bungarotoxin, taipoxin, crotoxin). Toxicon 22, 631–640.
Contents lists available at SciVerse ScienceDirect
Toxicon journal homepage: www.elsevier.com/locate/toxicon
Short communication
The effect of temperature on the effects of the phospholipase A2 neurotoxins b-bungarotoxin and taipoxin at the neuromuscular junction Behrooz Fathi a, *, Alan L. Harvey b, Edward G. Rowan b a b
Department of Pharmacology, School of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 161 Cathedral Street, Glasgow G4 0RE, UK
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 September 2012 Received in revised form 4 April 2013 Accepted 16 April 2013 Available online 3 May 2013
Snake venom neurotoxins with phospholipase A2 affect the neuromuscular junction with three distinct phases. There is a transient decrease in twitch height, followed by a facilitatory phase and finally a progressive blockade. It has been suggested that the initial phase is a direct consequence of the binding of the toxins to nerve terminals. This study was designed to determine whether the initial phase is present under conditions that would reduce the enzyme activity of the toxins. At 27 C, b-bungarotoxin and taipoxin exhibited all three phases, i.e. 5–6 min after exposure to the preparation, twitch height was significantly reduced (P < 0.5) to 50 4% and 64 9% of control respectively. This was followed by facilitation and subsequent blockade. However, at 20 C, neither toxin exhibited the first phase while the second phase, although reduced, clearly occurred and the blocking activity of these toxins always appeared. The data clearly demonstrate that the initial fall is temperature dependent as reducing the temperature from 27 C to 20 C blocks the first phase. As the second phase still occurs the toxins must have bound to their target. Therefore, the first phase cannot simply be a toxin binding step. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Phospholipase A2 neurotoxins b-Bungarotoxin Taipoxin Neuromuscular junction Temperature
Some snake venom phospholipases A2 (PLA2) like bbungarotoxin and taipoxin are presynaptic neurotoxins that affect the neuromuscular junction and modify neurotransmitter release. These neurotoxins exhibit a triphasic modulation of acetylcholine (ACh) release on isolated mammalian nerve-muscle preparations, which is reflected in changes in twitch height (see Rossetto and Montecucco, 2008; Punger car and Kri zaj, 2007, for reviews). The first phase is a transient initial reduction in the amount of ACh released in response to an action potential, and has been suggested to be caused by the binding of the toxins to their presynaptic receptors, although direct evidence for this is lacking. This effect is regarded as being independent of * Corresponding author. Tel.: þ98 (0)511 8813907, þ98 9159765651 (mobile). E-mail address: [email protected] (B. Fathi). 0041-0101/$ – see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxicon.2013.04.016
PLA2 enzyme activity (Chang and Su, 1982). It is followed by the second phase, the facilitatory phase, when ACh release is augmented and twitch height is increased. In mammalian, but not in amphibian preparations, the facilitatory phase is independent of PLA2 activity, suggesting that the toxins may induce their effects with different mechanisms of action at the amphibian and mammalian neuromuscular junctions. These two early phases are thought to be phospholipase activity-independent as they are still observed in experiments with p-bromophenacyl bromide (p-BPB)treated toxins and in the presence of Sr2þ, which inhibited enzyme activity of the PLA2 (Chang and Su, 1982; Rowan et al., 1990). Finally, the third phase is a progressive decline, leading to complete block of transmitter release and muscle paralysis. This phase is dependent on the PLA2 enzyme activity of the toxins (Chang et al., 1973) after they enter into the cytosol (Rigoni et al., 2008; Logonder et al.,
B. Fathi et al. / Toxicon 70 (2013) 86–89
2009). Most studies on the PLA2 neurotoxins have focused on their final neuromuscular blocking action. The initial blocking phase of toxicity has not been particularly investigated. The purpose of this study was to determine whether the initial phase caused by b-bungarotoxin and taipoxin can be affected by temperature. Lowering the temperature was used as a means to reduce the enzymatic activity of the toxins. Experiments were performed on mouse phrenic nervehemidiaphragm preparations (male Balb C strain, 20–25 g) maintained in standard Krebs–Henseleit solution at 20, 27 or 37 C. Phrenic nerves were stimulated at frequencies of 0.2 Hz with rectangular pulses of 0.1 ms duration and voltages greater than that required to produce maximal twitches. In order to reveal any facilitation of neuromuscular transmission, preparations were partly paralysed by addition of 9–10 mM MgCl2 applied directly to the bath. The twitch height was reduced to 15–20% of control and then allowed to stabilise for 20–30 min before applying toxins. b-Bungarotoxin (T-5644, Lots 124H40081, and 68H4003) was supplied by Sigma Chemical Co. Ltd., Poole,
87
Dorset, England. Taipoxin was a gift from Dr. David Eaker (Biochemistry Department, Uppsala University, Sweden) and was also purchased from Latoxan Company, 20 Rue Leon Blum, 2600 Valence-France. At 27 C, b-bungarotoxin (150 nM) and taipoxin (20 nM) exhibited all the cardinal signs of phospholipase A2 neurotoxins on twitch tension (Figs. 1 and 2), i.e. approximately 6 min after exposure to the toxin, twitch height was significantly reduced (P < 0.05, Student’s t-test) to 50 4% and 64 9% of control (n ¼ 3–7) respectively. This was followed by an increase in twitch tension to 400% and more than 500% respectively and the subsequent block of twitches (Figs. 1 and 2). However, at 20 C neither toxin exhibited the first phase while the second phase, although reduced, clearly occurred. For b-bungarotoxin, the times to final block at 20 C, 27 C and 37 C were 291 22 min, 229 10 min and 98 11 min, respectively (Fig. 1) which were significantly different (P < 0.05). For taipoxin, the times to final block at 20 C and 27 C were 70 3 min and 63 6 min respectively, which were not significantly different (Fig. 2). Taipoxin at 37 C did not exhibit the two
Twitch (% of control)
600
20 ° C
500
27 ° C
400
37 ° C
300 200 100 0 0
50
100
150
200
250
300
Time (min) 500
27° C
Twitch (% of control)
BuTx 400
300
20° C
200
100
37° C
0 0
5
10
15
20
Time (min) Fig. 1. Top – Effect of b-bungarotoxin (3 mg/ml ¼ 150 nM) on twitch tension of mouse phrenic nerve-hemidiaphragm preparations at 20 C, 27 C and 37 C in high Mg2þ (9–11 mM) solution. Preparations were stimulated indirectly at 0.2 Hz with pulses of 0.1 ms duration and voltage greater than required to produce the maximum response. Bottom – First 20 min in b-bungarotoxin (150 nM) at 20 C, 27 C and 37 C. At 20 C, the first phase depression was not evident.
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B. Fathi et al. / Toxicon 70 (2013) 86–89
600
20° C
Twitch (% of control)
500
27° C
400 300 200 100 0 0
10
20
30
40
50
60
70
80
Time (min) 600 taipoxin
Twitch (% of control)
500
27 C 400
300
200
20 C
100
0 0
5
10
15
20
Time (min) Fig. 2. Top – Effect of taipoxin (20 nM) on twitch tension of mouse phrenic nerve-hemidiaphragm preparations at 20 C and 27 C in high Mg2þ (9–11 mM) solution. Preparations were stimulated indirectly at 0.2 Hz with pulses of 0.1 ms duration and voltage greater than required to produce the maximum response. Bottom – First 20 min taipoxin (20 nM) at 20 C and 27 C. At 20 C, the first phase depression was not evident.
early phases and only blocked the twitches in less than 20 min which was significantly faster than the times at the lower temperatures (P < 0.05). As it is expected that most enzymatic reactions increase with increasing the temperature, phospholipid hydrolysis by phospholipase enzymes should be temperaturedependent and it has been shown that the blocking phase is dependent on the PLA2 enzyme activity of toxins (Chang et al., 1973; Rowan et al., 1990). The enzyme activity of b-bungarotoxin, taipoxin, and crotoxin is reduced to 4–10% of control when the temperature is lowered from 37 C to 27 C (Su and Chang, 1984). The time taken for complete
block of neuromuscular transmission in the mouse hemidiaphragm by these toxins was markedly prolonged (five-fold) at 27 C compared to at 37 C (Su and Chang, 1984). The present results reconfirm that by lowering the temperature, the neuromuscular blocking activity of these toxins is delayed. The results also clearly demonstrate that the initial fall in twitch tension is temperature-dependent because reducing the temperature from 27 C to 20 C eliminated the first phase. However, the facilitation (phase 2) was almost not affected by lowering the temperature, indicating that the toxins must have bound to their target on the nerve terminal. The
B. Fathi et al. / Toxicon 70 (2013) 86–89
initial phase of the toxins’ action thus appears to require the enzymatic action of the toxins in order for it to be evident as a reduction in twitch height. An additional possibility is that the presently unknown receptor or binding site for the toxins is sensitive to temperature. However, this target for the enzymes’ action is different from the intracellular targets that are suggested to be responsible for the slow and irreversible failure of ACh release and twitch blockade. Acknowledgements This work was funded by Ferdowsi University of Mashhad, Mashhad-Iran, and Strathclyde University, Scotland. We thank members of the Strathclyde Institute of Pharmacy and Biomedical Sciences for their help and support. Conflict of interest None.
89
References Chang, C.C., Chen, T.F., Lee, C.Y., 1973. Studies of presynaptic effect of bbungarotoxin on neuromuscular transmission. J. Pharmacol. Exp. Ther. 184, 339–345. Chang, C.C., Su, M.J., 1982. Presynaptic toxicity of the histidine-modified, phospholipase A2-inactive, b-bungarotoxin, crotoxin and notexin. Toxicon 20, 895–905. Logonder, U., Jenko-Pra znikar, Z., Scott-Davey, T., Punger car, J., Kri zaj, I., Harris, J.B., 2009. Ultrastructural evidence for the uptake of a neurotoxic snake venom phospholipase A2 into mammalian motor nerve terminals. Exp. Neurol. 219, 591–594. Punger car, J., Kri zaj, I., 2007. Understanding the molecular mechanism underlying the presynaptic toxicity of secreted phospholipases A2. Toxicon 50, 871–892. Rigoni, M., Paoli, M., Milanesi, E., Caccin, P., Rasola, A., Bernardi, P., Montecucco, C., 2008. Snake phospholipase A2 neurotoxins enter neurons bind specifically to mitochondria, and open their transition pores. J. Biol. Chem. 283, 34013–34020. Rossetto, O., Montecucco, C., 2008. Presynaptic neurotoxins with enzymatic activities. Handb. Exp. Pharmacol. 184, 129–170. Rowan, E.G., Pemberton, K.E., Harvey, A.L., 1990. On the blockade of acetylcholine release at mouse motor nerve terminals by b-bungarotoxin and crotoxin. Br. J. Pharmacol. 100, 301–304. Su, M.J., Chang, C.C., 1984. Presynaptic effects of snake venom toxins which have phospholipase A2 activity (b-bungarotoxin, taipoxin, crotoxin). Toxicon 22, 631–640.