Comparative Biochemistry and Physiology Part C 135 (2003) 205–214
A toxic fraction from scolopendra venom increases the basal release
of neurotransmitters in the ventral ganglia of crustaceans
a,
´ del Carmen Gutierrez
´
Marıa
*, Carolina Abarcaa, Lourival D. Possanib
a
´ Centro de Investigacion
´ en Biotecnologıa,
´ Universidad Autonoma
´
Laboratory de Neurofarmacologıa,
del Estado de Morelos,
Av. Universidad 1001, Col. Chamilpa, Cuernavaca Morelos C.P. 62210, Mexico
b
´ Department of Molecular Recognition and Structural Biology,
Instituto de Biotecnologıa,
´
´
Universidad Nacional Autonoma
de Mexico,
Avenida Universidad, 2001, Cuernavaca 62210, Mexico
Received 24 October 2001; received in revised form 10 April 2003; accepted 1 May 2003
Abstract
A toxic fraction from centipede (Scolopendra sp.) venom was tested in neurotransmitter release experiments. The
venom was fractionated by DEAE-cellulose with a linear gradient from 20 mM to 1.0 M of ammonium acetate pH 4.7.
Lethality tests were performed by injections into the third abdominal dorsolateral segment of sweet water crayfishes of
the species Cambarellus cambarellus. Only fraction V (TF) was toxic. Analysis by SDS-PAGE showed that this fraction
contains at least seven proteins. It induces an increase of basal gamma-amino butyric acid (GABA) and glutamate
release from ventral abdominal ganglia of C. cambarellus. Assays conducted with this fraction in the presence of several
drugs that affect ion channel function suggested that TF modifies membrane permeability by increasing basal release of
neurotransmitters was very likely through sodium channels.
䊚 2003 Elsevier Science Inc. All rights reserved.
Keywords: Gamma amino butyric acid; Glutamic acid; Ion channels; Channel blockers; Neurotransmitter release; Scolopendra; Toxin;
Naq permeability; Arthropod; Centipede
1. Introduction
In recent years, arthropod venoms have been
recognized to contain many neurotoxins, some of
which have been utilized as probes in the field of
neuroscience (Tipton and Dajas, 1994; Martos et
al., 1998). Most of these studies have been focused
on mammal and insect-specific neurotoxins from
venom of ants (Piek et al., 1991), scorpions
(Zlotkin, 1983; Possani, 1984; Zlotkin et al., 1985)
and spiders (Branton et al., 1987; Stapleton et al.,
1990; Gomes Figueiredo et al., 1995). There are
*Corresponding author. Tel.: q777-3-29-70-57; fax: q7772-29-70-30.
E-mail address:
´
[email protected] (M.d.C. Gutierrez).
few reports on venom from centipedes, despite the
fact that they have venom capable of causing
paralysis and dead to their preys.
Centipedes prey on many species of arthropods,
earthworms, snails and other small animals. They
are long, flattened, segmented animals. Most of
the segments have a pair of legs. Appendages on
the first segment behind the head have strong,
piercing, terminal segment with orifices of ducts
that lead to venom glands. Many species of centipedes can inflict venomous wounds on man, but
¨
these rarely result in death (Bucherl,
1971).
Our knowledge of the composition and properties of centipede venom is scarce. A few studies
based on crude extracts of whole glands and fangs
have reported the presence of serotonin in Scolo-
1532-0456/03/$ - see front matter 䊚 2003 Elsevier Science Inc. All rights reserved.
doi:10.1016/S1532-0456Ž03.00108-X
206
´
M.d.C. Gutierrez
et al. / Comparative Biochemistry and Physiology Part C 135 (2003) 205–214
pendra viridicornis (Welsh and Batty, 1963), and
histamine in Scolopendra subspinipes (Gomes et
al., 1983). Venom from Scolopendra morsitans
(Mohamed et al., 1980) contains both serotonin
and histamine. Further, the presence of several
protease-activities has been demonstrated in S.
morsitans (Zaid, 1958; Mohamed et al., 1983).
Related to its properties, Gomes et al. (1983) have
isolated a cardiotoxic protein (named toxin S)
from the venom of S. subspinipes with a molecular
mass of 60 kDa. In other studies, Mohamed et al.
(1980) and Gomes et al. (1983) investigated the
pharmacodynamic action of the venom of S. morsitans and S. subspinipes, respectively. More
recently, Stankiewicz et al. (1999) reported that
the application on the cockroach giant axon of a
Scolopendra sp. venom fraction (SC1) induced an
increase in the leak current correlated with a
decrease in the membrane resistance suggesting
the presence in SC1 of several components opening non-specific pores in the axonal membrane.
The mechanism is unknown, but the presence of
muscarinic agonists in SC1 was demonstrated.
We have observed that scolopendra venom can
be lethal to arthropods such as sweet water crayfishes, insect larvae’s and grasshoppers; but it is
not toxic to mice. Thus, in the present report, we
have tested the effect of a toxic fraction from
scolopendra venom on neurotransmitters release
using the isolated ventral abdominal nerve cord of
Cambarellus cambarellus, as a model system. We
have measured the in vitro release of endogenous
GABA and glutamate from the isolated ventral
ganglia of C. cambarellus under the effect of the
toxic fraction (TF). Additionally, we report some
chemical properties of TF. Finally, the results
obtained in assays of neurotransmitter release in
the presence of specific drugs that affect ion
channels (Naq, Kq or Ca2q) were analysed.
2. Materials and methods
2.1. Reagents and chemicals
Reagents and chemicals were obtained from the
following sources: All reagents were analytical
grade and were purchased from Baker (Ecatepec,
Mexico). Electrophoresis reagents and low range
molecular weight standards were from Bio-Rad
Laboratories (Rockville Centre, NY, USA). Gamma amino butyric acid (GABA), glutamic acid,
aminooxyacetic acid (AOA), tetrodotoxin (TTX),
choline chloride, bovine serum albumin, verapamil, 4-aminopyridine (4-AP), cobalt chloride,
ethyleneglycol-bis
(aminoethylether)-N,N,N,Ntetraacetic acid (EGTA) and O-phthaldialdehyde
(OPA) were from Sigma Chemical Co. (St. Louis,
USA). Solvents were HPLC grade from Merck
(Darmstadt, Germany). Nitrate membrane filters
13 mm diameters, 0.45 mm pore size cellulose
were obtained from Whatman Int. Ltd (England).
DEAE-cellulose was from Pharmacia Fine Chemicals (Uppsala, Sweden). All solutions were prepared with deionized, glass-distilled water and only
analytical grade reagents were used.
Protein concentration was estimated by the
method of Lowry et al. (1951) using bovine serum
albumin as standard.
2.2. Preparation of TF from Scolopendra sp.
venom
Scolopendra sp. were collected in the region of
´
Cuernavaca, Morelos State, Mexico.
Venom was
obtained at 4 8C after an electrical stimulation on
the scolopendra head. Fresh venom was partially
purified by anionyexchange chromatography on
DEAE-cellulose at 4 8C in a 6=1.3 cm column,
and eluted with a linear gradient of ammonium
acetate buffer pH 4.7 (from 20 mM to 1 M) at a
flow rate of 250 mlymin; aliquots of 750 ml were
collected. Toxic fraction was routinely identified
by testing toxicity of each one. Toxic fraction was
lyophilized and stored at y20 8C for further
experiments.
Protein content was estimated in these semipurified fractions assuming an A280 of 1.0s1 mg
proteinyml.
2.3. Assay of toxicity
Toxicity was assayed by injecting aliquots of 20
ml per animal (1 mgyml by absorbance) into the
third dorsolateral abdominal segment of the crustacean C. cambarellus. A toxic fraction was
defined as that capable of causing paralysis or
dead after injection.
2.4. SDS-polyacrylamide gel electrophoresis of the
venom and toxic fraction
Total venom and toxic fractions were electrophoresis in 12% polyacrylamide slab gel in the
´
M.d.C. Gutierrez
et al. / Comparative Biochemistry and Physiology Part C 135 (2003) 205–214
presence of 1% sodium dodecyl sulfate (SDS)
according to the method of Laemmli (1970). Gel
was fixed and stained with Coomassie brilliant
blue G. Low range molecular weight standards
were used as markers.
207
2.6. Statistics
ANOVA and Student’s-t tests were used for
statistical evaluations.
3. Results
2.5. Release experiments of endogenous GABA
and glutamic acid
Neurotransmitter release experiments were carried on through a modified superfusion procedure
according to Tapia et al. (1985). In brief, segmental ventral abdominal ganglia were dissected from
the crustacean and placed at 4 8C in the basal
medium. Ganglia were minced and placed on
Millipore filters lying on perfusion chambers
aligned in parallel. Minced ganglia were perfused
through a peristaltic pump (flow rate of 1.0 mly
min) with basal medium at 37 8C during 10 min
period. Thereafter, perfusate fractions (1.0 ml)
were collected. After 5 min a test medium was
substituted for the initial control medium and other
5 perfusate fractions were collected. At the end of
the experiment, the content of GABA and glutamic
acid into each collected fraction was determined
by HPLC, previous derivatization with O-phtaldialdehyde (Jones and Gilligan, 1983). Protein content in the minced ganglia was determined after
its homogenization in 1 ml of water.
The composition of basal medium (a modified
Krebs–Ringer medium) used to perfuse the ganglia preparation was: 120 mM NaCl, 4.7 mM KCl,
1.8 mM CaCl2, 0.8 mM MgSO4, 1 mM Tris–HCl
buffer (pH 7.4), 10 mM glucose. Amino oxyacetic
acid (AOA), 10 mM was present to block GABA
metabolism. The incubation medium was oxygenated with a mixture of 95% O2 y5% CO2 throughout the experiment.
The GABA and glutamic acid-releasing effect
by TF was first studied with regard to its dependence on the presence of external Na and Ca ions.
Therefore, test medium also contained TF (1 mgy
ml). The effect of TF was also tested in the
presence of Naq, Ca2q or Kq channel blockers.
These experiments were conducted in a similar
manner as before, but compounds were presented
in the basal medium throughout all the experiment
at the following final concentrations: EGTA 25
mM, cobalt chloride 1.25 mM, verapamil 20 mM,
TTX 1.0 mM, choline chloride 118 mM and 4-AP
20 mM.
3.1. Purification of toxic fraction (TF) from Scolopendra sp. venom
In Fig. 1, the profile of chromatographic separation of Scolopendra sp. venom applied on
DEAE-cellulose is shown. Aliquots were pooled
in six different fractions indicated by the horizontal
bars (I–VI). All toxic activity was found in fraction V (toxic fraction, TF). TF was eluted at 250
mM of acetate ammonium pH 4.7 indicates that it
is made of acidic components. As measured by
absorbance 6.7% from the total venom protein
corresponds to this fraction.
SDS-PAGE of total venom and toxic fraction is
also shown in Fig. 1. The electrophoresis protein
pattern of Scolopendra sp. venom revealed at least
16 protein bands; TF showed 8 with a main band
of 34.4 kDa.
3.2. Effect of TF on basal release of GABA and
glutamic acid
Ganglia were perfused with basal medium and
then exposed to the same medium containing TF.
Addition of TF produced an increase of neurotransmitter, 51 and 43% for GABA and glutamic acid,
respectively (Fig. 2).
In order to test if the release enhancement
evoked by TF was done by membrane damage,
the presence of phospholipase activity in TF was
assayed. Neither crude venom nor TF contained
phospholipase activity (data not shown).
3.3. Dependence of the effect of TF on external
Ca2q and Ca2q permeability
Neurotransmitter release is generally thought to
be calcium dependent (Turner et al., 1993; Dunlap
et al., 1995). Accordingly, we examined the influence of calcium on the TF-induced release of
GABA and glutamic acid. In the absence of
calcium (25 mM EGTA; Fig. 3a and b), TF was
not able to induce release neither for glutamic acid
nor for GABA. The substitution of cobalt for
208
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M.d.C. Gutierrez
et al. / Comparative Biochemistry and Physiology Part C 135 (2003) 205–214
Fig. 1. Ion exchange separation of Scolopendra venom. A sample (0.52 units of absorbance at 280 nm) of crude fresh venom was
dissolved in 0.750 ml ammonium acetate buffer 0.020 M pH 4.7 and it was applied to a DEAE-cellulose column (6=1.3 cm) at 4 8C
equilibrated with the same buffer. After elution of the unbound protein, a linear gradient from 0.02 to 1.0 M ammonium acetate buffer
pH 4.7 was initiated as indicated (arrow). Flow rate, 250 mlymin; fraction volume, 0.80 ml. Fraction V (TF) was toxic to crustaceans.
Inset: SDS-Polyacrylamide gel electrophoresis of Scolopendra venom. Samples were mixed with SDS buffer and then run in a SDSPAGE system (12.5% acrylamide). Gel lanes are as follows: lane 1, molecular mass standards: a: 97.4 kDa; b: 66.2 kDa; c: 45.0 kDa;
d: 31.0 kDa; e: 21.5 kDa and f: 14.4 kDa; lane 2, 40 mg of fresh crude whole Scolopendra venom and lane 3, 100 mg of TF. Asterisk
indicates the main component in TF.
calcium in the perfusing medium also blocked the
release induced by TF (Fig. 3c and d). In contrast
to these results, the presence of a low concentration
in the medium of verapamil (a Ca2q channel
blocker) did not affect the TF-induced release for
both neurotransmitters (Fig. 3e and f). These
results also indicate that in our preparation the
glutamate basal release is Ca2q-independent (Fig.
3a, c and e), since the blockers did not affect it;
although with verapamil there was a slight
increase. In contrast to that the GABA basal
release was Ca2q-dependent, omission of calcium
in the medium decreased GABA release (Fig. 3b
and d), except with verapamil where an increase
was observed from 13.1"1.96 to 17.4"0.91
pmolymg protein. In this condition, TF still evoked
the GABA release to 28.7 pmolymg protein
(P-0.05) (Fig. 3f).
3.4. Effect of TF in the presence of TTX and in
the absence of external Naq
The aperture of Naq channels could depolarize
the membrane and releasing neurotransmitters.
Therefore, the TF-induced release was tested in
the absence of external Naq (by replacing it for
choline chloride) or in the presence of TTX (a
Naq channel blocker). TTX completely inhibits
not only TF-induced release, but also basal neurotransmitters release as well. Besides, choline
chloride decreases the basal release of both neurotransmitters and blocks the TF releasing effect
(Fig. 4).
3.5. Effect of TF in the presence of 4-AP
The presence of 4-AP (a Kq channel blocker)
in the perfusing medium decreased the basal
´
M.d.C. Gutierrez
et al. / Comparative Biochemistry and Physiology Part C 135 (2003) 205–214
209
Fig. 2. Release of neurotransmitters evoked by TF. Release of GABA (Fig. 2a) or glutamic acid (Fig. 2b) stimulated by TF. Ganglion
segments were incubated at 4 8C in 5 ml of a modified Krebs–Ringer buffer (basal medium) pH 7.4 during 5 min. Elapsed time,
segments were placed on 0.45 mM filters in a perfusion chamber. They were perfused with the basal medium through a peristaltic
pump (flow rate of 1 mlymin). After a previous washed of 10 min, 5 aliquots (1 mlyaliquot) were collected. At min 5, basal medium
was changed to either basal medium in the absence (X) or in the presence of TF (1 mgyml) (⽧). Additionally, 5 aliquots were collected
in this condition. The perfusion system was maintained at 37 8C during all the experiment. The arrow indicates the minute in which
basal medium was changed. GABA and glutamic acid were measured by HPLC (see Methods). Values are mean"S.E.M of 6 independent experiments.
release for both neurotransmitters, 10% for glutamate and 33% for GABA, however, TF was still
able to release both GABA and glutamate (Fig.
5).
4. Discussion
Arthropod venoms as natural insecticides could
represent a new approach in the development of
bioinsecticides. Recently, neurotoxic proteins from
arthropod venoms, some of which specifically
attack insect sodium channels have been engineered into baculoviruses or polyhedrovirus to act
as biopesticides (Treacy and Rensner, 2000; Zlotkin et al., 2000). Moreover, neurotoxins, which
affect the process of neurotransmitters release may
be useful probes for neurochemical studies aimed
at the dissection of the molecular mechanism of
210
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et al. / Comparative Biochemistry and Physiology Part C 135 (2003) 205–214
Fig. 3. Dependence of the release of neurotransmitters evoked by TF in the absence of external Ca2q or in the presence of Co2q or
verapamil. Ganglia were perfused with basal Ca2q -free medium containing 25 mM EGTA (a, b), with basal medium in which Ca2q
was replaced for Co2q (c, d) or with basal medium containing 20 mM verapamil (e, f). In all cases, TF (1 mgyml) was added at 5
min (arrow) (j). Results are expressed as pmolymg protein of glutamic acid (a, c, e) or GABA (b, d, f). Values are mean"S.E.M of
6 independent experiments.
´
M.d.C. Gutierrez
et al. / Comparative Biochemistry and Physiology Part C 135 (2003) 205–214
211
pH 4.7, TF is assumed of acidic proteins. It
remains to be seen which of them is the toxic one.
Most of the studies with neurotoxins have been
achieved at the electrophysiological level and, in
some of them, the results have been correlated to
neurotransmitter release experiments involving different mechanisms of action (Sitges et al., 1986;
Elrick and Charlton, 1999; Lee et al., 2000).
Because of our study, we have shown that the
mechanism of neurotransmitter release in the ventral nerve cord of crustacean was sensitive to TF.
Our results suggest that TF could affect nerve
endings by enhancing basal release of endogenous
neurotransmitters from ventral abdominal ganglia
regardless of the transmitter involved (Fig. 2),
since the GABA and glutamate release were both
affected. Because TF lacks of phospholipase activity and it was incapable to enhance neurotransmitter release in the presence of high potassium (data
Fig. 4. Inhibition of the releasing effect of TF in the presence
of TTX and in the absence of external sodium. Ganglia were
perfused with basal medium containing 1 mM TTX (j, ⽧)
or with a modified basal medium in which Naq was replaced
by choline (h, e). In all cases, TF (1 mgyml) was added at
5 min (arrow)(j, h). Results are expressed as pmoly mg protein of glutamic acid (a) or GABA (b). Values are
mean"S.E.M of 6 independent experiments.
the neurotransmission involving ionic channels
(Narahashi et al., 1998).
In reference to Scolopendra sp., its venom is
only toxic to arthropods when it is administered
systemically without apparent effect on mice (data
not shown). Since the effect observed with the
venom on crustaceans was initially paralysis before
dead, we considered that this paralysis could be
related to the release of excitatory andyor inhibitory neurotransmitters, and consequently TF could
have a neurotoxic activity.
For this study, Scolopendra sp. venom was
partially purified on DEAE-cellulose. From six
fractions tested only one (TF) was toxic to C.
cambarellus. It contains at least eight proteins.
Because the separation was on DEAE-cellulose
Fig. 5. Release of neurotransmitters evoked by TF in the presence of 4-aminopyridine. Ganglia were perfused with basal
medium containing 20 mM 4-AP. TF (1 mgyml) was added at
5 min (arrow) (j). Results are expressed as pmoly mg protein
of glutamic acid (a) or GABA (b). Values are mean"S.E.M.
of 6 independent experiments.
212
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M.d.C. Gutierrez
et al. / Comparative Biochemistry and Physiology Part C 135 (2003) 205–214
not shown), we concluded that TF does not have
an indiscriminate effect promoting the leakage of
cellular content.
Several toxins from different sources have been
shown to interact in different ways with voltagedependent Naq channels and releasing neurotransmitters by inhibiting the sodium current
inactivation, either by its persistent activation or
by shifting the voltage dependence of activation
(Sitges et al., 1987; Gordon, 1997). In fact, the
realising effects of many neurotoxins are mediated
by an increase of Naq permeability that is blocked
by TTX.
If the releasing effect of TF was mediated by
an increase in Naq permeability, the presence in
the perfusion medium of either TTX, which is a
sodium channel blocker or choline chloride instead
Naq (sodium free medium) would impede an
increase of the neurotransmitters released by TF.
The results indicate that TF in the presence of
TTX or in sodium free medium was incapable of
inducing the release of GABA or glutamic acid
(Fig. 3), suggesting that TF induces neurotransmitter release through the activation of TTXsensitive Naq channels. In addition, the releasing
action of TF seems to depend on the sodium
gradient and the sodium equilibrium potential,
which are reversed in Naq-free medium, since the
absence of external Naq reduced the effectiveness
of TF.
Currently, neurotransmitter release requires the
presence of extracellular calcium ions (Katz and
Miledi, 1970), therefore, the dependence of calcium on the TF-induced release was tested. Although
the evoked release of neurotransmitters is Ca2qdependent, it has been reported as Ca2q-independent basal release for glutamate in synaptosomes
from cortical neurons of rat and mice (Leenders
et al., 2002). Our results point out that in crustaceans the glutamate basal release is also Ca2qindependent. In contrast to that the GABA basal
release showed a dependency for Ca2q except
when the medium was in the presence of verapamil. With regards to TF this fraction affects neither
the GABA nor glutamate release in a calcium-free
media obtained by replacing the omitted calcium
ions with EGTA. As well as in the presence of
EGTA, TF was not able to induce release of
neurotransmitters by replacing Co2q for Ca2q.
Since inorganic compounds such as Co2q inhibit
all calcium channels (Tsien et al., 1987), our
results suggest that a Ca2q influx through cobalt-
sensitive Ca2q channel might be directly induced
by TF or otherwise required for the release process
triggered by TF. It is interesting to note that
verapamil had no effect on the TF-induced neurotransmitter release, but the mechanism of neurotransmitters basal release was affected. Since
verapamil blocks L-type Ca2q channel in mammals (Hille, 1992), our results point out that
verapamil-sensitive Ca2q channel apparently is not
involved in the release evoked by TF, at least in
crustaceans. Similar results were obtained by
´
Lopez
et al. (2001), they were found in cortical
neurons from fetal rat brain, that verapamil blocked
the release of aspartate and glycine, but it did not
affect the high potassium evoked release of glutamate or GABA. From these results, they conclude
that the release of a particular amino acid neurotransmitter not only depends on the opening of the
voltage-dependent Ca2q channel, but also on the
effector which produces the opening, and that the
amount of amino acid release evoked by the
different depolarizing agents is not correlated with
the elevation of intracellular Ca2q produced by
them. We could explain our results obtained with
verapamil according to this explanation.
Finally, it is unlikely that TF blocks Kq channels. The presence of 4-AP, a Kq channel blocker
did not affect the TF-induced release. 4-AP blocks
Kq channels and the nerve terminals increase the
basal neurotransmitter release (Tapia and Sitges,
1982). Although our results showed that 4-AP
decreased basal release of both glutamate and
GABA, TF was still able to enhance the release
of both neurotransmitters. Since ganglia were preincubated with 4-AP, the endogenous neurotransmitter pool was probably diminished, thereby,
basal and TF-induced release was also diminished.
From this study, it remains to be seen whether
the same component in TF, which induces neurotransmitters release is involved in its lethality to
C. cambarellus. Because released experiments
have been carried out with partially fractionated
venom preparation, it is not still clear if the same
component would be responsible for both toxicity
and neurotransmitter release.
The results of this study suggest that TF modify,
presumably, ionic permeability of the membranes,
increasing basal release of neurotransmitters, most
probably through an increase of Naq permeability.
This possibility is supported by the fact that in the
absence of external sodium TF does not affect the
release of neurotransmitters. At present, however,
´
M.d.C. Gutierrez
et al. / Comparative Biochemistry and Physiology Part C 135 (2003) 205–214
participation of Ca2q channels cannot be discarded. It remains to clarify if TF can initiate the
release process by a mechanism different from
direct activation of Ca2q channels; thus, the
Ca2q dependence of the releasing effect of TF
appears to be linked to the role of this ion in the
coupling of stimulus and secretion. Further studies
using other techniques and preparations may be
conclusive on this matter.
Acknowledgments
The research presented here was supported by a
´
grant to M.C. Gutierrez
and L.D. Possani from the
´
Consejo Nacional de Ciencia y Tecnologıa,
CONACyT Grant No. 3731-M and Z-005. The
technical assistance of Lucero Valladares was
greatly appreciated.
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