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Comparative Biochemistry and Physiol
Biological and enzymatic activities of Micrurus sp. (Coral) snake venoms
Alessandra L. Cecchinia,b, Silvana Marcussic,f, Lucas B. Silveirac, Caroline R. Borja-Oliveirad,
Lea Rodrigues-Simionid, Susan Amarab, Rodrigo G. Stabelie, Jose R. Gigliof,
Eliane C. Arantesa, Andreimar M. Soaresc,g,*
aDepartamento de Fısica e Quımica, FCFRP, USP, Ribeirao Preto-SP, BrazilbHoward Hughes Medical Institute-Reseach Laboratories. Oregon Health Sciences University-Vollum Institute, Sam Jackson Park Road Portland, USA
cUnidade de Biotecnologia, Universidade de Ribeirao Preto, UNAERP, Ribeirao Preto-SP, BrazildDepartamento de Farmacologia, UNICAMP, Campinas-SP, Brazil
eLaboratorio de Bioquımica e Biotecnologia, Instituto de Pesquisas em Patologias Tropicais (IPEPATRO), Porto Velho-RO, BrazilfDepartamento de Bioquımica e Imunologia, FMRP, USP, Ribeirao Preto-SP, Brazil
gDepartamento de Analises ClVnicas, Toxicologicas e Bromatologicas, FCFRP, USP, Ribeirao Preto-SP, Brazil
Received 28 May 2004; received in revised form 11 November 2004; accepted 15 November 2004
Abstract
The venoms of Micrurus lemniscatus carvalhoi, Micrurus frontalis frontalis, Micrurus surinamensis surinamensis and Micrurus
nigrocinctus nigrocinctus were assayed for biological activities. Although showing similar liposome disrupting and myotoxic activities, M.
frontalis frontalis and M. nigrocinctus nigrocinctus displayed higher anticoagulant and phospholipase A2 (PLA2) activities. The latter
induced a higher edema response within 30 min. Both venoms were the most toxic as well. In the isolated chick biventer cervicis preparation,
M. lemniscatus carvalhoi venom blocked the indirectly elicited twitch-tension response (85F0.6% inhibition after a 15 min incubation at 5
Ag of venom/mL) and the response to acetylcholine (ACh; 55 or 110 AM), without affecting the response to KCl (13.4 mM). In mouse
phrenic nerve-diaphragm preparation, the venom (5 Ag/mL) produced a complete inhibition of the indirectly elicited contractile response after
50 min incubation and did not affect the contractions elicited by direct stimulation. M. lemniscatus carvalhoi inhibited 3H-l-glutamate uptake
in brain synaptosomes in a Ca2+, but not time, dependent manner. The replacement of Ca2+ by Sr2+ and ethylene glycol-bis(h-aminoethyl
ether) (EGTA), or alkylation of the venom with p-bromophenacyl bromide (BPB), inhibited 3H-l-glutamate uptake. M. lemniscatus
carvalhoi venom cross-reacted with postsynaptic a-neurotoxins short-chain (antineurotoxin-II) and long-chain (antibungarotoxin) antibodies.
It also cross-reacted with antimyotoxic PLA2 antibodies from M. nigrocinctus nigrocinctus (antinigroxin). Our results point to the need of
catalytic activity for these venoms to exert their neurotoxic activity efficiently and to their components as attractive tools for the study of
molecular targets on cell membranes.
D 2004 Elsevier Inc. All rights reserved.
Keywords: Coral snake venoms; Micrurus sp.; Micrurus lemniscatus carvalhoi; Liposome-disrupting activity; Myotoxicity; Neurotoxicity; Phospholipase A2;
Synaptosome; l-glutamate uptake
1095-6433/$ - s
doi:10.1016/j.cb
Abbreviation
nigrocinctus nig
oxiana; BPB, p
modified eagle m
saline; PLA2, p
dependent Na+ c
* Correspon
E-mail addr
ogy, Part A 140 (2005) 125–134
ee front matter D 2004 Elsevier Inc. All rights reserved.
pb.2004.11.012
s: anti-BGTX, antibodies against bungarotoxin from Bungarus multicinctus; anti-NGX, antibodies against nigroxin from Micrurus
rocinctus; anti-NT-I, antibodies against neurotoxin I from Naja naja oxiana; anti-NT-II, antibodies against neurotoxin II from Naja naja
-bromophenacyl bromide; CK, creatine kinase; DIBAC4(3), bis[1,3-dibutylbarbituric acid-(5)] trimethin-eoxolnol; DMEM, Dulbecco’s
edium; EGTA, ethylene glycol-bis(h-aminoethyl ether); GABA, g-aminobutyric acid; i.c.v., intracerebroventricular; PBS, phosphate buffered
hospholipase A2; RCCS, rat cortico-cerebral synaptosomes; TTX, tetrodotoxin; VDCC, voltage-dependent Ca++ channel; VDSC, voltage-
hannel.
ding author. Tel.: +55 16 603 6892; fax: +55 16 603 7030.
ess: [email protected] (A.M. Soares).
A.L. Cecchini et al. / Comparative Biochemistry and Physiology, Part A 140 (2005) 125–134126
1. Introduction
Coral snakes comprise a group of almost 50 species
from the genus Micrurus found in the Southern United
States and South America. They are a taxonomic
assembly of more than 120 species and subspecies,
achieving their greatest diversity near the equator (Rose
and Bernal-Carlo, 1987). However, the mode of action
of the venom of only a few species has been
investigated.
The signs and symptoms of envenomation by Micrurus
sp. are the result of a progressive blockade at the neuro-
muscular endplate and, in severe cases, death results from
respiratory arrest. Besides supportive clinical care, serother-
apy with heterologous antivenoms is the only treatment for
coral snake bite envenomation (Russel, 1983; Bolanos,
1984). Experimental studies suggest the presence of a
considerable spectrum of pharmacological activities of
Micrurus venoms. They induce neurophysiological changes
similar to those induced by a-neurotoxins, and some of
them also show postsynaptic effects (Goularte et al., 1983;
Vital-Brazil, 1987).
Some Micrurus venoms have been characterized
according to their neurotoxic activities. Micrurus cor-
allinus venom was described as having both presynap-
tic and postsynaptic actions. M. corallinus venom
produces an irreversible neuromuscular blockade, reduc-
ing evoked acetylcholine (ACh) release and increasing
the spontaneous release of ACh. M. lemniscatus and
M. frontalis venoms demonstrated only a postsynaptic
action (Vital-Brazil, 1987).
Micrurus venoms also showed myotoxicity (Gutierrez
et al., 1980, 1983, 1986, 1992) and cardiotoxicity when
injected intravenously (Ramsey et al., 1972). Common
characteristics as well as variability in some biological
activities among venoms from different Micrurus species
have been demonstrated in comparative studies (Gutierrez
et al., 1983, 1992; Aird and Da Silva, 1991; Tan and
Ponnudurai, 1992; Alape-Giron et al., 1994).
Electrophoretic, immunochemical and chromatographic
studies of Micrurus venoms have shown the presence of
components with profile similar to several toxins from other
elapids (Jorge-Da-Silva et al., 1991; Alape-Giron et al.,
1994, 1996, 1999). Almost all Micrurus venoms have a
high enzymatic phospholipase A2 (PLA2) activity but
different profiles for other enzymes (Aird and Da Silva,
1991; Tan and Ponnudurai, 1992). For example, some of
these venoms have anticholinesterase and anticoagulant
activities in vitro (Kumar et al., 1973; Tan and Ponnudurai,
1992; Alape-Giron et al., 1996).
Since knowledge on the mechanism of action of this
venom may be helpful in establishing protocols for the
treatment of persons envenomed by this species, we have
investigated the enzymatic and pharmacological effects
evoked by the venom from four coral snakes, especially
focusing on the neurotoxicity and inhibition of 3H-l-
glutamate uptake induced byMicrurus lemniscatus carvalhoi
venom.
2. Materials and methods
2.1. Materials
The venoms from Micrurus sp. (M. lemniscatus
carvalhoi, Micrurus frontalis frontalis and Micrurus
surinamensis surinamensis) were kindly supplied by Luiz
H. Anzaloni-Pedrosa, FMRP, USP, Brazil. Micrurus
nigrocinctus nigrocinctus venom and antineurotoxins anti-
bodies were kindly provided by Dr. Alberto Alape-Giron
(Instituto Clodomiro Picado, Universidad de Costa Rica,
San Jose, Costa Rica). 3H-l-Glutamate was purchased
from Perkin-Elmer Life Science; ScintiVerse, OptiPhase
Supermix and the 1900 TR Liquid Scintillation Analyser
were from Fisher Scientific, Wallac and Packard, respec-
tively. BRANDEL system—Biomedical Research Devel-
opment Laboratories, Gaithersburg, MD USA. All other
reagents used were purchased from Sigma-Aldrich and
Mallinckrodt.
2.2. Enzymatic and anticoagulant activities
Micrurus venom PLA2 activity was evaluated using egg
yolk as substrate (de Haas et al., 1968). Anticoagulant
activity was assessed as described earlier (Alvarado and
Gutierrez, 1988).
2.3. Myotoxic activity
The assay of plasma creatine kinase (CK) activity
was carried out using the CK-UV kinetic kit from
Sigma. Venoms (5 Ag) were injected intramuscularly
in the gastrocnemius muscle of 18–22 g male Swiss
mice (50 AL, n=6). Animals used as negative controls
were injected with phosphate buffered saline (PBS).
After 3 h, a blood sample was collected from the tail
in heparinized capillary tubes and centrifuged for
plasma separation (Soares et al., 2000a,b). The
enzyme activity was expressed in U/L, one unit
producing 1 Amol of NADH/min under the conditions
of the assay.
2.4. Edema-inducing activity
Groups of five male Swiss mice (18–22 g) were injected
subcutaneously in the subplantar region with 50 AL of
venom (3.5 Ag). At different intervals, the thickness of the
paw was measured with a low-pressure spring caliper
(Mitutoyo, Japan) as an index of edema (Soares et al.,
2000a,b). Zero time values were subtracted from the
corresponding final values, and the differences were
expressed as percentage increment.
A.L. Cecchini et al. / Comparative Biochemistry and Physiology, Part A 140 (2005) 125–134 127
2.5. Isolated chick biventer cervicis nerve-muscle and
phrenic nerve-diaphragm preparations
The biventer cervicis was removed from chicks, as
described by Ginsborg and Warriner (15), and mounted
under a tension of 0.5 g in a 5-mL organ bath containing
aerated (95% O2, 5% CO2) Krebs solution (pH 7.5, 37 8C)of the following composition (mM): NaCl 118.6, KCl 4.69,
CaCl2 1.88, KH2PO4 1.17, MgSO4 1.17, NaHCO3 25.0 and
glucose 11.65. The phrenic nerve-diaphragm preparation
was obtained from mice anesthetized with chloral hydrate
(300 mg/kg) and sacrificed by exsanguination. Hemidiaph-
ragms were mounted under a tension of 5 g in a 5 mL organ
bath (Bulbring, 1946) containing Tyrode solution (pH 7.4,
37 8C) of the following composition (mM): NaCl 137, KCl
2.7, CaCl2 1.8, MgCl2 0.49, NaH2PO4 0.42, NaHCO3 11.9
and glucose 11.1, aerated with 95% O2 and 5% CO2.
Indirect (0.1 Hz, 0.2 ms, 6–7 V) and direct (0.1 Hz, 0.2 ms,
50 V) stimulations (Grass S4 stimulator, Grass Instruments,
Quincy, MA, USA) were used, and contractions and
contractures were recorded via a force displacement-trans-
ducer (BG 25 GM, Kulite Semiconductor Products, Leonia,
NJ, USA) coupled to a Gould RS 3400 recorder (Gould,
Cleveland, OH, USA). The preparations were allowed to
stabilize for at least 15 min before the addition of M.
lemniscatus carvalhoi venom (5 or 10 Ag/mL). For biventer
cervicis preparations, contractures to exogenously applied
submaximal concentrations of acetylcholine (55 or 110 AM)
and KCl (13.4 mM) were obtained in the absence of nerve
stimulation prior to the addition of venom and at the end of
the experiment in order to test for the presence of neurotoxic
and myotoxic activities (Harvey et al., 1994).
2.6. Lethal dose 100% (LD100)
Male albino Swiss mice (18–22 g, n=6) were injected
intraperitoneally (100 AL, i.p.) and intramuscularly (50 AL,i.m.) with different amounts of Micrurus venoms, and
deaths were registered within 24 h.
2.7. Liposome-disrupting activity
Negatively charged liposomes (phosphatidylserine, 63
Amol; dicethylphosphate, 18 Amol; cholesterol, 9 Amol)
were obtained from Sigma-Aldrich. The assay was per-
formed according to Dıaz et al. (1991) incubating 20 AL of
the liposome suspension with 20 AL of the venom solution
(in PBS) for 30 min at 37 or 4 8C.
2.8. Synaptosome preparation
Synaptosome assay was performed to evaluate the
Micrurus venom activity on neuronal preparations. Synap-
tosomes were obtained from the cerebral cortex of young
Sprague–Dawley rats (200–250 g; Charles River Labora-
tory) and prepared following the method of Gray and
Whittaker (1962). Briefly, the cortex was homogenized
three times in 0.32 M sucrose and centrifuged at 1700�g for
10 min at 4 8C, yielding pellet 1 (P1). The supernatant was
collected and centrifuged at 21,000�g for 20 min at 4 8C,producing P2. P2 was dispersed in 0.32 M sucrose and
centrifuged at 60,000�g under a sucrose gradient from 1.2
to 0.8 M for 60 min at 4 8C. Three distinct phases were
obtained, and the synaptosome (intermediate) phase was
collected, diluted with cold Milli-Q water to a concentration
of 0.4 M and centrifuged at 24,400�g for 20 min at 4 8C. P3was obtained and dissolved with an appropriate volume of
Tyrode buffer in (mM); 136 NaCl; 5 KCl; 2.5 KH2PO4; 1
MgSO4; 25 Tris–HCl; 2 CaCl2; 5 glucose, pH7.4. When the
assay was performed in the absence of Ca2+, 7.6 mM Sr2+
and 1 mM ethylene glycol-bis(h-aminoethyl ether) (EGTA)
were used. Protein was determined, of the according
manufacturer (Pierce), with a BCA protein assay kit. The
amount of protein concentration used in each assay well was
30–45 Ag.
2.9. L-glutamate uptake assay
Approximately 100 nmol of 3H-l-glutamate (specific
activity 22.50 Ci/mM) were used in each well together
with 0.35, 0.71, 1.7, 3.55, 5.33, 7.1 and 10.6 ng of M.
lemniscatus carvalhoi venom. Different incubation times
(5, 10, 15 and 20 min), at room temperature, of the venom
with the synaptosomes were assayed, although the time
chosen for the following assays was 20 min. To eliminate
background interference of l-glutamate, 6.3 mM l-trans-
pyrrollidine-2,4-dicarboxylic acid (PDC) was used. The
assay employed 96-well plates, and the incubation time of
the synaptosome with the radioactive material was 3 min
followed by suction and filtration of the medium using the
BRANDEL system. OptiPhase Supermix Scintillation
liquid was used, and radioactivity was counted in
WALLAC 1450 Microbeta scintillation counter. Data were
converted in to 3H-l-Glu uptake rates and expressed as
fmol mg�1 min�1.
To determine the role of PLA2 activity on 3H-l-
glutamate uptake, the phospholipase A2 inhibitor p-bromo-
phenacyl bromide (BPB) at 0.5 AM, 5.0 AM and 2 mM,
preincubated with the different concentrations of the venom
for 1 h before the 3H-l-glutamate uptake experiment started,
was assayed. To evaluate the effect of phospholipase
activity of the venom on synaptic membranes, a modified
Tyrode buffer was prepared using 7.6 mM Sr2+ and 1 mM
EGTA instead of Ca2+, and the uptake assay was performed
as previously described (Cecchini et al., 2004).
2.10. Enzyme-immunoassays
Microplate wells (Dynatech Lab.) were coated with M.
lemniscatus carvalhoi venom at 0.5 Ag/well by overnight
incubation in 0.1 M Tris, 0.15 M NaCl, pH 9.0 buffer
(Angulo et al., 2001). After five washings with solution A
Table 1
Enzymatic and biological activities of Micrurus sp. snake venoms
Samples Coagulation time (min)a PLA2 activity (U/mg)b Rupture of liposomes (%)c Myotoxicity (U/L)d Edema (%)e
Ca++/PBS 3.50F0.5 0.00 0.00 365.35F9.67 9.58F0.58
Tween 2% – – 100.00 – 0.00
M. lemniscatus N45 38.5F3.5 53.12F1.8 1630.08F101.93 57.8F1.03
M. frontalis N45 89.9F2.1 59.83F1.2 1858.72F100.56 73.2F1.12
M. surinamensis N45 28.6F3.4 37.23F0.8 1104.85F112.35 80.5F0.89
M. nigrocinctus N45 75.3F1.9 56.02F1.3 2259.10F139.78 93.7F1.25
a Anticoagulant activity upon platelet rich plasma. Doses of 1.0 Ag of M. frontalis and M. nigrocinctus venoms, and doses of 50 Ag of M. lemniscatus and
M. surinamensis were used.b PLA2 activity by potenciometric tritiation. Doses of 30 Ag were used for all venoms.c Liposome disruption at 37 8C with doses of 5 Ag.d Myotoxic activity with doses of 5 Ag.e Edema inducing activity 30 min after injection of 3.5 Ag of venom.
Table 2
Lethality induced by Micrurus snakes venoms
Micrurus venoms Lethality (%)
Doses (Ag/mouse) i.p. i.m.
M. lemniscatus carvalhoi 5 33.3 33.3
10 66.7 45.5
20 100.0 100.0
M. frontalis frontalis 5 36.7 40.0
10 80 77.6
20 100.0 100.0
M. surinamensis surinamensis 5 46.7 35.6
10 80.0 68.8
20 100.0 100.0
M. nigrocinctus nigrocinctus 5 42.5 39.8
10 65.8 50.8
20 100.0 100.0
PBS 50 AL 0.0 0.0
A.L. Cecchini et al. / Comparative Biochemistry and Physiology, Part A 140 (2005) 125–134128
(0.05 M Tris, 0.15 M NaCl, 20 AM ZnCl2, 1 mMMgCl2, pH
7.4, buffer), the plates were air-dried and stored at 4 8C.Purified rabbit antibodies to different neurotoxins (Naja
naja oxiana neurotoxins I and II; Bungarus multicinctus a-
and h-bungarotoxins; Micrurus nigrocinctus a-nigroxin)
were added to triplicate wells, diluted (1:600) in solution A
containing 2% bovine serum albumin (BSA). After five
washes with solution A, they were incubated at room
temperature for 2 h. Bound antibodies were detected with
antirabbit immunoglobulin conjugated to alkaline phospha-
tase (Sigma-Aldrich), diluted 1:2000 with solution A-BSA
and incubated for 90 min. After washing, colour was
developed with p-nitrophenylphosphate, and absorbances
were recorded on a microplate reader at 410 nm. Normal
rabbit serum was used as a negative control, and crotamine
was included as an unrelated antigen.
2.11. C6 glioma cell assay
C6 glioma cells were cultivated in 6-well plates,
suplemented with Dulbecco’s modified eagle medium
(DMEM) in the absence of fetal bovine serum but in
presence of penicillin (100 U/mL)/streptomycin (100 Ag/mL), kept at 37 8C and 5% (v/v) CO2 and used 2 days
later. In order to evaluate the membrane potential, the
fluorescent compound bis[1,3-dibutylbarbituric acid-(5)]
trimethin-eoxolnol (DIBAC4(3)) was used, showing
absorbance at 493 nm and emission at 516 nm. DIBAC4(3)
is a lipophilic anion which becomes fluorescent when
bound to a protein or cell membrane (Cecchini et al.,
2004). Hyperpolarization reduces fluorescence due to
changes in the fluorescent anion intracellular concentration.
Depolarization increases it through an increase in the
permeability of cell membrane and so increasing the
concentration of the probe inside the cell. Glass slides
containing C6 cells were placed in an appropriate device,
DIBAC4(3) was added and the images were collected by a
confocal microscope with a Bro-Rad MRC-100 krypton–
argon scanning laser and a Nikon Diaphot 200 inverted
microscope provided with a 40� Nikon PahnApo lens.
Projection of images was analysed with the Bio-Rad Laser
Sharp 1024 program. All controls were submitted to
identical parameters.
2.12. Statistical analysis
Results were expressed as the meanFstandard deviation
(S.D.) of values obtained with the indicated number of
animals. The statistical significance of differences between
groups was evaluated using Student’s unpaired t-test. A p
value b0.05 was considered to indicate significance.
3. Results
The venom of the four coral snakes, M. lemniscatus
carvalhoi, M. frontalis frontalis, M. surinamensis surina-
mensis and M. nigrocinctus nigrocinctus, were assayed for
several biological activities. M. frontalis frontalis and M.
nigrocinctus nigrocinctus showed higher anticoagulant and
phospholipase A2 activities (Table 1). Regarding liposome
disrupting and myotoxicity, all venoms showed statistically
similar behavior (Table 1) except for M. surinamensis
surinamensis venom. M. nigrocinctus nigrocinctus induced
a higher edema response within the first 30 min of
A.L. Cecchini et al. / Comparative Biochemistry and Physiology, Part A 140 (2005) 125–134 129
inoculation of the venom when compared with other
Micrurus species, although the progression of the activity
ran similarly along the remaining time (Table 1).
The most lethal venoms, by both assay routes, were those
from M. frontalis frontalis and M. nigrocinctus nigrocinc-
tus, followed by M. surinamensis surinamensis (Table 2).
Except for the dose of 6 Ag (i.p.), when the venom of M.
frontalis frontalis showed twofold higher lethality compared
with the venom of M. lemniscatus carvalhoi, all doses used
presented similar responses (Table 2).
M. lemniscatus carvalhoi venom had a time- and
temperature-dependent inducting effect on liposome dis-
ruption (Fig. 1A). Myotoxicity and edema-inducing activity
were dose- and time-dependent (Fig. 1B and C).
M. lemniscatus carvalhoi venom produced a fast and
progressive blockade of the indirectly elicited twitch-tension
Fig. 1. Pharmacological activities induced by M. lemniscatus carvalhoi snake ven
min with M. lemniscatus carvalhoi venom. (B) Myotoxic activity induced by M
muscle of mice. (C) Edema-inducing activity of M. lemniscatus carvalhoi (3.5 AmeansFS.D. (n=6).
response, reaching 85F0.6% (n=3) inhibition after a 15-min
incubation at 5 Ag/mL in chick muscle and a complete
inhibition after a 50 min inhibition in mouse neuromuscular
preparation (Fig. 2). Furthermore, it completely abolished
the response to acetylcholine (55 or 110 AM) in chick
neuromuscular preparations. The contracture induced by the
addition of KCl (13.4 mM) in chick muscle remained
unaltered (data not shown), as the contractile response to
direct stimulation of mouse nerve-muscle preparation.
To evaluate the ability of M. lemniscatus carvalhoi
venom to inhibit 3H-l-glutamate uptake by brain synapto-
somes, assays with different incubation times and different
concentrations of the venom were carried out (Fig. 3).
Venom doses from 0.71 ng up showed significant inhibitory
effect. The venom effect was not time-dependent, and the
chosen incubation time was 20 min. Replacing extracellular
om. (A) Peroxidase release from liposomes incubated at 37 and 4 8C for 30
. lemniscatus carvalhoi 1, 3 and 6 h after injection into the gastrocnemius
g/50 AL) 0.25, 0.5, 1, 2 and 4 h after injection. Results are expressed as
Fig. 2. Effect of M. lemniscatus carvalhoi venom on neuromuscular
preparations of mammals (phrenic nerve diaphragm) and birds (biventer
cervicis). The time course of blockade of the indirectly elicited muscle
twitch by M. lemniscatus carvalhoi (5 Ag/mL) is indicated. Results are
expressed as meansFS.D. (n=3).
A.L. Cecchini et al. / Comparative Biochemistry and Physiology, Part A 140 (2005) 125–134130
Ca2+ by Sr2+ plus EGTA (Fig. 4A), we could observe that
the absence of Ca2+ completely abolished the venom
inhibitory activity. In addition, inhibition of the venom
PLA2 activity by BPB also inhibited 3H-l-Glu uptake
(Fig. 4B).
Myotoxic and neurotoxic activities induced by M.
lemniscatus carvalhoi venom were also partially inhibited
after incubation with p-BPB (data not shown). Fig. 5 shows
that M. lemniscatus carvalhoi venom shows a high cross-
reactivity against short-chain a-neurotoxins antibodies (anti-
a-neurotoxin-II) and also towards long-chain antibodies
(anti-a-bungarotoxin). This venom also reacts but to a lesser
extent with antimyotoxic phospholipase A2 antibodies from
the venom of M. nigrocinctus nigrocinctus (antinigroxin).
Verification of the progressive increase of fluorescence
due to the penetration of the probe in the cell lipid bilayer,
evidenced depolarization evoked by M. lemniscatus carval-
hoi snake venom. Hyperpolarization of the C6 glioma cell
was not observed. This is additional evidence that the
venom does not disrupt the cell, despite its hydrolytic
action, but acts on the cell ,surface altering its membrane
potential with the resulting alteration of l-glutamate uptake.
Fig. 3. Dose-dependent 3H-l-glutamate uptake RCCS inhibition by M.
lemniscatus carvalhoi venom. RCCS and the venom were incubated at RT
for 20 min prior the uptake assay. Doses from 0.71 ng up were significant
(*pb0.01).
4. Discussion
The four species of Micrurus venoms studied displayed
enzymatic, anticoagulant, edema-inducing, myotoxic and
liposome disrupting activities. Coral snake venoms are
known to display neurotoxic, myotoxic, hemorrhagic and
cardiovascular effects (Aird and Da Silva, 1991; Jorge-Da-
Silva et al., 1991; Gutierrez et al., 1992; Francis et al.,
1997). However, toxins sequence data have been gathered
for only four phospholipases, which proved to be similar to
Type I PLA2 from Old World elapids and for several
postsynaptic neurotoxins (Rosso et al., 1996; Silveira-de-
Oliveira et al., 2000; de Oliveira et al., 2003).
Francis et al. (1993) demonstrated that polyclonal anti-
bodies raised against hemorrhagic toxin from the Australian
Notechis scutatus scutatus snake venom recognized the
most abundant proteins in the venom of M. frontalis. These
proteins proved to be hemorrhagic phospholipase A2, which
are structurally similar to pancreatic phospholipase A2
(Francis et al., 1997). Our results show that the venoms
assayed have significant edema-inducing and myotoxic
activities (Table 1).
Phospholipase A2 toxins are implicated directly and
indirectly in the formation of edema (Lomonte et al., 1993).
Sanchez et al. (1992) detected an edema-inducing activity in
M. frontallis venom comparable to that induced by Bothrops
alternatus and Crotalus durissus subspecies. The authors
also reported that procoagulant, fibrinolytic, hemorrhagic
and necrotic activities were absent from M. frontalis venom.
Increased coagulation time shown by the four venoms
assayed (Table 1) agrees with literature data. Two sets of
proteins have been purified from the venom of the Brazilian
coral snake M. frontalis frontalis. One set appears to
represent isoforms of postsynaptic toxins, while the other
set shows phospholipase A2 activity. The toxic members of
this set promote hemorrhage in mice in a manner closely
resembling that produced by a N. scutatus scutatus PLA2
isolated from the venom of the Australian tiger snake
(Francis et al., 1997).
Liposome disruption is an evidence of high PLA2
activity. The venom of M. lemniscatus carvalhoi induced
Fig. 4. 3H-l-Glutamate uptake by RCCS. Different M. lemniscatus carvalhoi venom concentrations were incubated at RT with RCCS for 20 min, followed by
the uptake assay. (A) Substitution of 2 mM Ca2+ for 7.6 mM Sr2++1 mM EGTA. (B) Addition of 0.5 and 5 AM of BPB and incubated at RT for 1 h with M.
lemniscatus carvalhoi venom prior the uptake assay (*pb0.01).
A.L. Cecchini et al. / Comparative Biochemistry and Physiology, Part A 140 (2005) 125–134 131
this effect in a dose- and temperature-dependent manner.
This fact suggests that the fluidity of the biological
membrane is a key factor for the enzyme to perform its
activity (Dıaz et al., 1991; Soares et al., 2000a,b, 2001).
Fig. 5. Enzyme immunoassay cross-reactivity of a and h-neurotoxinsantibodies againstM. lemniscatus carvalhoi snake venom. Microplate wells
were coated with antigen (0.5 Ag/well), and the binding of antibodies (anti-
aNT-I, anti-aNT-II, anti-aBGTX, anti-hBGTX and antibodies against
nigroxin from M. nigrocinctus nigrocinctus [anti-NGX]) was detected as
described in Materials and methods. Cross-reactivity was expressed as a
percentage of the absorbance signal resulting from the binding of antibodies
to the homologous antigen (a-neurotoxins I and II, NT, from N. naja
oxiana, a and h-bungarotoxins, BGTX, from Bungarus multicinctus and
nigroxin, NGX, from M. nigrocinctus nigrocinctus venom). Crotamine was
included as an unrelated, negative control antigen. Each bar represents the
meanFS.D. (n=3).
M. lemniscatus carvalhoi venom showed high myotoxic
activity in mice and neurotoxic activity in isolated neuro-
muscular preparations of birds and mammals. The venom
was able to produce neuromuscular blockade and inhibited
the response to colinoceptor agonist acetylcholine, without
affecting the responses to KCl, in chick muscle preparations.
These results suggest that the venom acts preferentially on
postsynaptic nicotinic receptors, interfering with the neuro-
muscular transmission without affecting adjacent muscular
membranes. Due to venom myotoxicity in mice, as observed
by determination of plasma CK concentration, an inhibition
of the contractile response to direct stimulation in mice and
of the response to KCl in chick muscle could be expected.
Nevertheless, an opposite effect occurred probably because
the myotoxicity produced by the venom was not enough to
manifest evident effects in isolated neuromuscular prepara-
tions at the venom concentrations used. Similarly, recent
findings indicated that Micrurus dumerilii carinicauda
venom inhibit the twitch-tension elicited by indirect
stimulation and the response to acetylcholine, without
producing blockade of the response to KCl in chicks, but
caused myonecrosis at the same concentrations. The authors
suggested that the morphological changes did not adversely
influence muscle contractility and therefore did not cause
the venom-induced neuromuscular blockade (Serafim et al.,
2002).
Neurotoxicity is a prominent factor, which leads to
lethality (Table 2). It is already well established that PLA2s
activity participates in the events resulting from neuron
damage (Kolko et al., 1996). The convulsant properties of
low molecular mass toxins (PLA2s of~14 kDa) by intra-
cerebroventricular (i.c.v.) route are known for many years
(Vital-Brazil, 1972).
In addition, it is very important to investigate the mode of
action of the venoms of each Micrurus species and
subspecies because although the predominant effect of most
of them is the inhibition of the cholinoceptor, the venom of
M. corallinus is mainly presynaptic. For most cases,
A.L. Cecchini et al. / Comparative Biochemistry and Physiology, Part A 140 (2005) 125–134132
neostigmine must be employed to avoid asphyxia and death.
Neostigmine restores neuromuscular transmission if the
venom-induced blockade results from a reversible interac-
tion of its neurotoxins with the end-plate receptors. Never-
theless, when presynaptic action predominates, such as in
the poisoning induced by Micrurus corallinus, neostigmine
is ineffective (Vital-Brazil, 1987; Vital-Brazil and Vieira,
1996).
Secretory PLA2s play a relevant role in the events
resulting from neuronal injuries (Bazan et al., 1995). High
levels of l-glutamate in the synaptic cleft have been
considered consequent from diseases whose symptoms are
convulsion and nervous disturbances (Miller et al., 1997).
However, little evidence shows the participation of intra- or
extracellular Ca2+ in neurotransmitter uptake in mammalian
cells. Zhu et al. (1999) postulated that absence of
extracellular Ca2+ decreases l-glutamate uptake in synapto-
somes since removal of external Ca2+ promotes depolariza-
tion of the synaptosomal membrane and consequent
activation of voltage-dependent Na+ channels (VDSCs)
which are sensitive to tetrodotoxin (TTX). However, it is
not expected that depletion of extracellular Ca2+ promotes
an increase of intracellulaer Ca2+ through activation of
VDCCs. In addition, internal Ca2+ reserves would be
depleted, while involvement of any intrasynaptosomal
Ca2+ would be unlikely. Substitution of Sr2++EGTA for
Ca2+ does not decrease l-glutamate uptake significantly.
Our experiments were carried out within the linear portion
of l-glutamate uptake curve.
Our results show that extracellular Ca2+ ions do not
significantly interfere with 3H-l-glutamate uptake. Yet the
venom did not exert its inhibitory effect on 3H-l-Glu uptake
by rat cortico-cerebral synaptosomes (RCCS) in the absence
of extracellular Ca2+ (Fig. 4A). Catalytically, active venom
PLA2s are dependent on millimolar Ca2+concentrations, and
residues involved in Ca2+ binding and catalysis are highly
conserved, leading to a threshold level of identity of about
30% among venom sPLA2s from the same group (Hawgood
and Bon, 1995; Valentin and Lambeau, 2000). Moreover,
the absence or decrease of extracellular Ca2+ abolishes toxin
Fig. 6. Confocal microscopy of C6 glioma cells. (A) Control; (B) 2 min after a
activity, hence PLA2s need Ca2+, although at micromolar
concentrations, to exert their enzymatic activity. Irreversible
inactivation of PLA2s by BPB alkylation is used to
completely suppress their catalytic activity, central toxicity
and consequently, epileptogenic activity (Dorandeu et al.,
1998).
Alkylation by BPB partially inhibited myotoxic (58%)
and neurotoxic (65%) activities induced by M. lemniscatus
carvalhoi venom. Other PLA2s, as that of Naja mossambica
mossambica, also loses its central activity when treated with
BPB (Clapp et al., 1995). As for the apparent l-Glu uptake
impairment, one evidence suggests that membrane disrup-
tion may not be the main explanation since lactate
dehydrogenase (LDH) levels did not change for venom-
treated synaptosomes (data not shown). Therefore, the
uptake inhibition is not an effect of hydrolytic action of
the venom upon the membrane but upon the electrogenic
equilibrium of the synaptic membrane since evidence of
depolarization of C6 glioma cell membranes in the presence
of venom was obtained by confocal microscopy using a
fluorescent probe (Fig. 6; data not shown). The precise
mechanism of action of many venom components is still
unknown, but the participation of membrane proteins has
already been studied (He and Curry, 1995). Several papers
have reported that toxins inhibit the uptake of many
neurotransmitters, including g-aminobutyric acid (GABA),
noradrenalin, serotonin and choline (Ng and Howard, 1978,
1981; Smith et al., 1980), while studies on the uptake of
glutamate are more limited.
Elapid venoms usually comprise several isoforms of a-
neurotoxins (Endo and Tamiya, 1987). The high cross-
reactivity against antibodies against neurotoxin II from N.
naja oxiana (anti-NT-II) and antibodies against bungaro-
toxin from Bungarus multicinctus (anti-BGTX) antibodies
suggests that M. lemniscatus carvalhoi venom contains two
kinds of a-neurotoxins (short and long chains). It has been
demonstrated that antibodies against M. nigrocinctus nigro-
cinctus inhibit the binding of the a-neurotoxins to acetyl-
choline receptors (AChR), suggesting the presence of
postsynaptic a-neurotoxins with short and long chains
ddition of M. lemniscatus carvalhoi venom at 7.1 ng/mL. Bar=250 Am.
A.L. Cecchini et al. / Comparative Biochemistry and Physiology, Part A 140 (2005) 125–134 133
(Alape-Giron et al., 1996), alike those found by cross-
reactivity for the venom of M. lemniscatus carvalhoi.
Recent studies have also revealed that the toxic effects of
venom PLA2s cannot be easily correlated with their catalytic
activity, suggesting that they may specifically bind to target
proteins (Valentin et al., 1999). In spite of that, our results
suggest the need of catalytic activity for the venom to exert
its neurotoxic activity efficiently. These interactions make
these venom components attractive tools for the develop-
ment of therapeutic agents in the study of molecular targets
on cell membranes.
Acknowledgements
Authors acknowledge the financial support from Funda-
cao de Amparo a Pesquisa do Estado de Sao Paulo
(FAPESP), Conselho Nacional de Desenvolvimento Cientı-
fico e Tecnologico (CNPq) and Universidade de Ribeirao
Preto (UNAERP), the skilfull technical assistance of
Eliandra G. Silva (FAPESP, TT-II), Gildo Bernardo Leite
(UNICAMP) and the kind collaboration of Prof. Dr. Alberto
Alape-Giron and Prof. Dr. Jose Maria Gutierrez (Instituto
Clodomiro Picado, Universidad de Costa Rica).
References
Aird, S.D., Da Silva, N.J., 1991. Comparative enzymatic composition of
Brazilian coral snake (Micrurus) venoms. Comp. Biochem. Physiol., B
99, 287–294.
Alape-Giron, A., Lomonte, B., Gustafsson, B., Da Silva, N.J., Thelestam,
M., 1994. Electrophoretic and immunochemical studies of Micrurus
snake venoms. Toxicon 32, 713–723.
Alape-Giron, A., Stiles, B., Schmidt, J., Giron-Cortes, M., Thelestam, M.,
Jornvall, H., Bergman, T., 1996. Characterization of multiple nicotinic
acetylcholine receptor-binding proteins and phospholipases A2 from the
venom of the coral snake Micrurus nigrocinctus. FEBS Lett. 380,
29–32.
Alape-Giron, A., Persson, B., Cederlund, E., Flores-Diaz, M., Gutierrez,
J.M., Thelestam, M., Bergman, T., Jornvall, H., 1999. Elapid venom
toxins: multiple recruitements of ancient scaffolds. Eur. J. Biochem.
259, 225–234.
Alvarado, J., Gutierrez, J.M., 1988. Anticoagulant effect of myotoxic
phospholipase A2 isolated from the venom of the snake Bothrops asper
(Viperidae). Rev. Biol. Trop. 36, 563–565.
Angulo, Y., Nunez, C.E., Lizano, S., Soares, A.M., Lomonte, B., 2001.
Immunochemical properties of the N-terminal helix of myotoxin II, a
lysine-49 phospholipase A2 from Bothrops asper snake venom.
Toxicon 39, 879–887.
Bazan, N.G., Rodriguez de Turco, E.B., Allan, G., 1995. Mediators of
injury in neurotrauma: intracellular signal transduction and gene
expression. J. Neurotrauma. 12, 791–814.
Bolanos, R., 1984. Serpientes, Venenos y Ofidismo en Centroamerica.
Editorial Universidad de Costa Rica, San Jose, Costa Rica.
Bulbring, E., 1946. Observations on the isolated phrenic nerve-diaphragm
preparation of the rat. Br. J. Pharmacol. 1, 38–61.
Cecchini, A.L., Soares, A.M., Giglio, J.R., Amara, S., Arantes, E.C., 2004.
Inhibition of l-glutamate and GABA synaptosome uptake by crotoxin,
the major neurotoxin from Crotalus durissus terrificus venom.
J. Venom Anim. Toxins icl. Trop. Dis. 10, 260–279.
Clapp, L.E., Klette, K.L., DeCoster, M.A., Bernton, E., Petras, J.M., Dave,
J.R., Laskosky, M.S., Smallrige, R.C., Tortella, F.C., 1995. Phospho-
lipase A2-induced neurotoxicity in vitro and in vivo in rats. Brain Res.
693, 101–111.
de Haas, G.H., Postema, N.M., Nieuwenhuizen, W., van Deenen, L.L.M.,
1968. Purification and properties of phospholipase A from porcine
pancreas. Biochim. Biophys. Acta 159, 103–110.
de Oliveira, U.C., Assui, A., da Silva, A.R., de Oliveira, J.S., Ho, P.L.,
2003. Cloning and characterization of a basic phospholipase A2
homologue from Micrurus corallinus (coral snake) venom gland.
Toxicon 42, 249–255.
Dıaz, C., Gutierrez, J.M., Lomonte, B., Gene, J.A., 1991. The effect of
myotoxins isolated from Bothrops snake venoms on multilamellar
liposomes: relationship to phospholipase A2, anticoagulant and myo-
toxic activities. Biochim. Biophys. Acta 1070, 455–460.
Dorandeu, F., Antier, D., Pernot-Marino, I., Lapeyre, P., Lallement, G.,
1998. Venom phospholipase A2-induced impairment of glutamate
uptake: an indirect and nonselective effect related to phospholipid
hydrolysis. J. Neurosci. Res. 51, 349–359.
Endo, T., Tamiya, N., 1987. Current view on the structure–function
relationship of postsynaptic neurotoxins from snake venoms. Pharma-
col. Ther. 34, 403–451.
Francis, B., Williams, E.S., Seebart, C., Kaiser, I.I., 1993. Proteins isolated
from the venom of the common tiger snake (Notechis scutatus scutatus)
promote hypotension and hemorrhage. Toxicon 31, 447–458.
Francis, B.R., da Silva Junior, N.J., Seebart, C., Casais e Silva, L.L.,
Schmidt, J.J., Kaiser, I.I., 1997. Toxins isolated from the venom of the
Brazilian coral snake (Micrurus frontalis frontalis) include hemorrhagic
type phospholipases A2 and postsynaptic neurotoxins. Toxicon 35,
1193–1203.
Goularte, F.C.L., Cogo, J.C., Gutierrez, J.M., Rodrigues-Simioni, L., 1983.
Effects of Micrurus nigrocinctus snake venom on mouse and chick
neuromuscular preparation. Toxicon 31, 135–136.
Gray, E.G., Whittaker, V.P., 1962. The isolation of nerve endings from
brain: an electron-microscopic study of cell fragments derived by
homogenization and centrifugation. J. Anat. 96, 79–87.
Gutierrez, J.M., Chaves, F., Rojas, E., Bolanos, R., 1980. Local effects
induced by Micrurus nigrocinctus venom in white mice. Toxicon 18,
633–639.
Gutierrez, J.M., Lomonte, B., Portilla, E., Cerdas, L., Rojas, E., 1983.
Local effects induced by coral snake venoms: evidence of myonecrosis
after experimental inoculations of venoms of five species. Toxicon 21,
777–783.
Gutierrez, J.M., Arroyo, O., Chaves, F., Lomonte, B., Cerdas, L., 1986.
Pathogenenis of myonecrosis induced by coral snake (Micrurus
nigrocinctus) venom in mice. Br. J. Exp. Pathol. 67, 1–12.
Gutierrez, J.M., Rojas, E., da Silva, N.J., Nunez, J., 1992. Experimental
myonecrosis induced by the venoms of South American Micrurus
(coral snakes). Toxicon 30, 1299–1302.
Harvey, A.L., Barfaraz, A., Thompson, E., Faiz, A., Preston, S., Harris,
J.B., 1994. Screening of snake venoms for neurotoxic and myotoxic
effects using simple in vitro preparations from rodents and chicks.
Toxicon 32, 257–265.
Hawgood, B., Bon, C., 1995. Snake venom presynaptic toxins. In:
A., Tu (Ed.), Handbook of Natural Toxins. Marcel Dekker, New York,
pp. 3–52.
He, P., Curry, F.E., 1995. Measurement of membrane potential of
endothelial cells in single perfused microvessels. Microvasc. Res. 50,
183–198.
Jorge-Da-Silva, N.J., Griffin, P.R., Aird, S.D., 1991. Comparative
chromatography of Brazilian coral snake (Micrurus) venoms. Comp.
Biochem. Physiol., B 100, 117–126.
Kolko, M., DeCoster, M.A., de Turco, E.B., Bazan, N.G., 1996. Synergy by
secretory phospholipase A2 and glutamate on inducing cell death and
sustained arachidonic acid metabolic changes in primary cortical
neuronal cultures. J. Biol. Chem. 271, 32722–32728.
A.L. Cecchini et al. / Comparative Biochemistry and Physiology, Part A 140 (2005) 125–134134
Kumar, V., Rejent, T., Elliot, W., 1973. Anticholinesterase activity of elapid
venoms. Toxicon 11, 131–138.
Lomonte, B., Tarkowski, A., Hanson, L.A., 1993. Host response to Bothrops
asper snake venom. Analysis of edema formation, inflammatory cells,
and cytokine release in a mouse model. Inflammation 17, 93–105.
Miller, H.P., Levey, A.I., Rothstein, J.D., Tzigounis, A.V., Conn, P.J., 1997.
Alterations in glutamate transporter protein levels in kindling-induced
epilepsy. J. Neurochem. 68, 1564–1570.
Ng, R.H., Howard, B.D., 1978. Deenergization of nerve terminals by alfa-
bungarotoxin. Biochemistry 17, 4978–4986.
Ng, R.H., Howard, B.D., 1981. Inhibition by neurotoxic phospholipases
A2 of synaptosomal uptake of aminobutyric acid. J. Neurochem. 36,
310–312.
Ramsey, H.W., Taylor, W.J., Boruchow, I.B., Nyder, G.K., 1972.
Mechanism of shock produced by an elapid snake (Micrurus f. fulvius)
venom in dogs. Am. J. Physiol. 222, 782–786.
Rose, J.A., Bernal-Carlo, A., 1987. Las serpientes corales venenosas del
genero Leptomicrurus (serpentes, Elapidae) da Sulamerica com
descripcion de una nueva subespecie. Boll.-Mus. Reg. Sci. Nat. Torino
5, 573–608.
Rosso, J.P., Vargas-Rosso, O., Gutierrez, J.M., Rochat, H., Bougis, P.E.,
1996. Characterization of alpha-neurotoxin and phospholipase A2
activities from Micrurus venoms. Determination of the amino acid
sequence and receptor-binding ability of themajor alpha-neurotoxin from
Micrurus nigrocinctus nigrocinctus. Eur. J. Biochem. 238, 231–239.
Russel, F.E., 1983. Snake Venom Poisoning. Scholium, New York.
Sanchez, E.F., Freitas, T.V., Ferreira-Alves, D.L., Velarde, D.T., Diniz,
M.R., Cordeiro, M.N., Agostini-Cotta, G., Diniz, C.R., 1992. Biological
activities of venoms from South American snakes. Toxicon 30, 95–103.
Serafim, F.G., Reali, M., Cruz-Hofling, M.A., Fontana, M.D., 2002. Action
of Micrurus dumerilii carinicauda coral snake venom on the
mammalian neuromuscular junction. Toxicon 40, 167–174.
Silveira-de-Oliveira, J., Rossan de Brandao Prieto da Silva, A., Soares,
M.B., Stephano, M.A., de Oliveira Dias, W., Raw, I., Ho, P.L., 2000.
Cloning and characterization of an alpha-neurotoxin-type protein
specific for the coral snake Micrurus corallinus. Biochem. Biophys.
Res. Commun. 267, 887–891.
Smith, C.C.T., Bradford, H.F., Thompson, E.J., McDerma, J., 1980. Actions
of a-bungarotoxin on aminoacid transmitter release. J. Neurochem. 34,
487–494.
Soares, A.M., Andriao-Escarso, S.H., Angulo, Y., Lomonte, B., Gutierrez,
J.M., Marangoni, S., Toyama, M.H., Arni, R.K., Giglio, J.R., 2000a.
Structural and functional characterization of myotoxin I, a Lys49
phospholipase A2 homologue from Bothrops moojeni (Caissaca) snake
venom. Arch. Biochem. Biophys. 373, 7–15.
Soares, A.M., Guerra-Sa, R., Borja-Oliveira, C.R., Rodrigues, V.M.,
Rodrigues-Simioni, L., Rodrigues, V., Fontes, M.R.M., Lomonte, B.,
Gutierrez, J.M., Giglio, J.R., 2000b. Structural and functional character-
ization of BnSP-7, a Lys49 myotoxic phospholipase A2 homologue
from Bothrops neuwiedi pauloensis venom. Arch. Biochem. Biophys.
378, 201–209.
Soares, A.M., Andriao-Escarso, S.H., Rodrigues-Simioni, L., Arni, R.K.,
Bortoleto, R.K., Ward, R.J., Gutierrez, J.M., Giglio, J.R., 2001.
Dissociation of enzymatic and pharmacological properties of piratox-
ins-I and -III, two myotoxic phospholipases A2 from Bothrops pirajai
snake venom. Arch. Biochem. Biophys. 387, 188–196.
Tan, N.-H., Ponnudurai, G., 1992. The biological properties of venoms of
some American coral snakes (genus Micrurus). Comp. Biochem.
Physiol., B 101, 471–474.
Valentin, E., Lambeau, G., 2000. What can venom phospholipase A2 tell us
about functional diversity of mammalian secreted phospholipase A2.
Biochimie 82, 815–831.
Valentin, E., Gomashchi, F., Gelb, M.H., Lazdunski, M., Lambeau, G.,
1999. On the diversity of secreted phospholipase A2. Cloning, tissue
distribution, and functional expression of two novel mouse group II
enzymes. J. Biol. Chem. 274, 31195–31202.
Vital-Brazil, O., 1972. Neurotoxins from SouthAmerican rattlesnake venom.
Taiwan I Hsueh Hui Tsa Chih. J. Formos. Med. Assoc. 1, 394–400.
Vital-Brazil, O., 1987. Coral snake venoms: mode of action and
pathophysiology of experimental envenomation. Rev. Inst. Med. Trop.
Sao Paulo 29, 119–126.
Vital-Brazil, O., Vieira, R.J., 1996. Neostigmine in the treatment of snake
accidents caused by Micrurus frontalis: report of two cases (1). Rev.
Inst. Med. Trop. Sao Paulo 38, 61–67.
Zhu, B.G., Chen, Y.Z., Xing, B.R., 1999. Effect of calcium on the uptake of
glutamate by synaptosomes: possible involvement of two different
mechanisms. J. Neural Transm. 106, 257–264.