Biological and enzymatic activities of Micrurus sp. (Coral) snake venoms

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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


(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


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).


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).


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,


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.


(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).

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Biological and enzymatic activities of Micrurus sp. (Coral) snake venoms