Navarrete et al 2011 revision

Int. J. Morphol.,
29(3):733-741, 2011.

Molecular Characterization, Electrophysiological and
Contraceptive Effect of Chilean Latrodectus Venom

Caracterización Molecular, Electrofisiológica y Efecto Anticonceptivo
del Veneno de Latrodectus Chilena

**,*****
Patricia Navarrete Gómez; *David Ormeño; **Antonio Miranda;
***,*****
Raúl Sánchez Gutiérrez; *,*****Fernando Romero Mejía & *Jorge Parodi Rivera

NAVARRETE, G. P.; ORMEÑO, D.; MIRANDA, A.; SÁNCHEZ, G. R.; ROMERO, M. F. & PARODI, R. J. Molecular
characterization, electrophysiological and contraceptive effect of Chilean Latrodectus venom. Int. J. Morphol., 29(3):733-741, 2011.

SUMMARY: Since the 1970s, There have been studies of the venom of Latrodectus sp. spiders, in particular the latrotoxin
(LTX) of Latrodectus mactans. Many of the studies were aimed at understanding the action of the venom on the muscular system. Now
accepted that LTX is able to generate a calcium-permeable membrane pore and modulate the release of synaptic vesicles that activate a
receptor and induce cellular changes. Interestingly, when work began with venom obtained from the Latrodectus sp present in Chile, it
generated clinical indications similar to the bite of this spider in another country, with some differences in intensity. The purpose of the
first studies was to understand the systemic mechanisms of this venom, and other active compounds were studied for biological interest.
It was found that these molecules are capable of causing systemic effects such as changes in muscle contraction; of generating vascular
relaxation and synaptic and cellular modulation; and of altering potassium conductance channels. Based on this evidence, we suggested
biotechnological applications to characterize low molecular-weight compounds obtained from the Chilean Latrodectus venom and exploring
the effects on the electrophysiology in oocytes and neurons, and the contraceptive effect on spermatozoa.

KEY WORDS: Latrodectus venom; Oocytes; Neurons; Spermatozoa; Contraceptive.

INTRODUCTION

The Chilean spider Latrodectus mactans (L. mactans) (Shimuta et al., 1982). Our studies in the deferent vessel of
belongs to the Latrodectus genus which has worldwide the rat revealed that the effect induced by the L. mactans
distribution (Garb et al., 2004). It is known in Chile as "Araña venom is partially dependent of adrenergic and cholinergic
del trigo" (wheat spider) or "black widow", and is present in mediators (patent pending).
various regions of the country. Its bite generates a systemic
effect known as "latrodectism" or "systemic arachnoidism" Moreover, we tested the effect of Chilean venom in
in humans, in some cases causing death in both adults and hippocampal neuron, and reported the changes in synaptic
children (Schenone & Correa, 1985). activity, for have synaptic effects under control and postulate,
mechanism over neuromuscular union (Varghese et al.,
Venom collected from Chilean L. mactans in the VIII 2006).
and IX Regions of Chile was shown to induce a sustained
tonic effect in cardiac and smooth muscle (Romero et al., In a previous report we described that the venom of
2003). In smooth muscle, the mechanism of contraction is the Chilean black widow spider L. mactans increased
related to the permeability of sodium (Na+) and calcium intracellular calcium (Ca++i) concentration of the
(Ca ++) ions which modulate the contractile response spermatozoa (Romero et al., 2007). Furthermore, this venom
(Nouailhetas et al., 1985) that has a fast, phasic component is known to block the tetraethylammonium (TEA)-sensitive
followed by a slower more sustained tonic component potassium (K+) currents in neurons (Parodi & Romero, 2008;

*Center of Neurosciences and Peptides Biology- (CEBIOR), BIOREN, Universidad de La Frontera, Temuco, Chile.
**Department of Biophysics University of Sao Paulo, Federal University of Sao Paulo, Sao Paulo, Brazil.
***Centre of Excellence in Biotechnology in Reproduction (CEBIOR), BIOREN, Universidad de La Frontera, Temuco, Chile.
****Department of Preclinics Sciences, University of La Frontera, Temuco, Chile.
*****Doctorate in Sciences Applied Cellular and Molecular Biology, Universidad de La Frontera, Temuco, Chile, CONICYT Scholarship.

733

NAVARRETE, G. P.; ORMEÑO, D.; MIRANDA, A.; SÁNCHEZ, G. R.; ROMERO, M. F. & PARODI, R. J. Molecular characterization, electrophysiological and contraceptive effect of Chilean
Latrodectus venom. Int. J. Morphol., 29(3):733-741, 2011.

Parodi et al., 2010) as well as endogenous K+ currents of The initial work carried out in Chile showed that the
Xenopus laevis oocytes (Parodi et al., 2008); this current is effect of a bite of the Chilean spider generated symptoms of
similar to that recently described in bovine spermatozoa latrodectism, similar to, but more moderate than that of their
(Marconi et al., 2008). Finally, we analyzed how either Eurasian relatives (Schenone, 1966). The work of Dr.
depolarization or L. mactans venom alters the properties of Romero’s group showed effects similar to those reported in
the sperm cells assessed by microscope imaging, muscle preparations (Rauber, 1983-1984). The Chilean
morphology, Ca++i dynamics by ﬂuorometry, acrosome spider is harvested in the Eighth and Ninth Regions. The
reaction, DNA fragmentation and reactive oxygen species bite induces a sustained tonic effect in heart muscle and
(ROS) production in bovine spermatozoa. smooth muscle tissue (Romero et al., 2003). However, the
purification and analysis of the venom by Brazilian groups
Molecular characterization of Chilean Latrodectus demonstrated the absence of molecules of high molecular
venum extract. The venom contains at least 86 unique weight (Fig. 1), which could indicate the absence of LTX or
proteins (Duan et al., 2006), including several LTX similar toxins. Vessel contractility studies done in rats have
homologues which play a role in its toxicity in insects and demonstrated that the effect induced by the venom of the
crustaceans (Grishin, 1998), with only one, LTX, specifically Chilean L. mactans spider is partially independent of
targeting vertebrates (Rosenthal & Meldolesi, 1989). LTX adrenergic and cholinergic mediators (Romero et al., 2003).
is usually isolated from spider venom by conventional These venoms were found to contain only a large group of
chromatography (Frontali et al., 1976; Tzeng & Siekevitz, small peptides, less than 10 kDa, which were considered to
1978), but to achieve homogeneity and remove contaminants be low molecular weight components (Parodi & Romero,
(Pescatori et al., 1995; Volkova et al., 1995) which may 2008). These peptides have been studied and the mixture
endow the preparation with uncharacteristic properties found to have interesting cellular impacts which may explain
(Umbach et al.,1998), preparative native electrophoresis the clinical effects found in patients bitten by this spider.
should ideally be used (Ashton et al., 2000). This characteristic of the venom of Latrodectus spp spiders

Fig. 1. Total venom. The figure shows typical HPLC-mass obtained by the purification of different peptides present in the
Chilean L. mactans. The profile indicated the absence of molecules of high molecular weight, like LTX, in the samples.
Moreover, shows the molecular simulation of venom and poliacrilamide gel of 10% submitted to electrophoresis in denaturing
conditions. The location of the zones of the signal lines of the molecular weight is indicated (94, 67, 43, 30 y 20.1 KDa.).

734


found in Chile has been exploited to search for peptides of Electrophysiological effect of Chilean Latrodectus venum
biological interest in which it is easy to purify a natural form extract. The venom from the Chilean Black Widow spider
of these components. contains several small polypeptides. We have recently
demonstrated cellular effects of these peptides at the synaptic
As previously reported, in our laboratory and work level using whole-cell patch clamp techniques. Puriﬁed
toward development of a patent (Underway in countries venom from the glands of L. mactans was studied in 12 DIV
PCT) was to provide information about the component of rat hippocampal neuronal cultures (Fig. 2A and B). Our data
the Chilean venom, L. mactans. We described a fractionation show that polypeptides present in the venom from Chilean
of the venom by HPLC–MS, and found the absence of the L. mactans spiders increase spontaneous synaptic activity
higher weight component, like a-latrotoxin. This toxin, is in hippocampal neurons and changes the passive properties
the principal component in Black Widow venom and explains of the membrane, cells lines. We suggested, K+ current, for
most of the systemic effects secondary to this venom. A few the importance of this current over membrane potential
reports describe the low weight component, present in the (Gutman et al., 2003) and the control in the synaptic activity
venom but not explored. The Chilean venom, does not (Dodson & Forsythe, 2004; Yuan & Chen, 2006). Work by
present higher component (minor to 10 kDa) (Romero et Grider & Makhlouf (1988) demonstrated that the tonic
al., 2000) but, the Chilean venom can induce similar effects response in smooth muscle is dependent on the inﬂux of
to other Black Widow venom in the absence of a-latrotoxin. Ca++ into the cell. Similarly, synaptic activity is dependent

Fig. 2. Effects of Chilean L. mactans venom extract on cellular electrophysiology in neurons and oocytes. (A) Shows synaptic
currents obtained in the absence and presence of venom or boiled venom in neurons. (B) Shows miniature synaptic current frequency
in the presence of the venom. The bars are mean S.E. from 19 different neurons. (C) Shows plot from sample traces control and a
venom current from (D) in oocytes. The bars represent means ± SD of 6 different experiments, * was significant p<0.05.

735


on Ca++ homeostasis in the presynaptic button (Cousin & we have no evidence of blocking or modulatory effects on
Robinson, 2000). Therefore, the neuronal response observed either voltage gated Ca++ channels or the Ca++ gated -
in the presence of total venom could be related to Ca++ influx chloride currents. In contrast, a previous report explained
as a result of the change in membrane potential, secondary the blocking of Ca++ channels with the L. mactans venom
effect over Ca+v, for regulation of K+ channels (Pan & (Romero et al., 2007). The discrepancy with the present
Stringer, 1997; Yuan & Chen). These results indicate that results may be due to differences in the molecular identities
venom from Chilean spider L. mactans is capable of of voltage-activated Ca++-channels of frog oocytes and
increasing cell membrane resistance, prolonging the action sperm cells (Romero et al., 2007). Unfortunately, thus far,
potential and generating an increase in synaptic activity the Ca++ channels present in the oocyte have not been
demonstrating an interesting pharmacological effect of these cloned. K+ currents of the oocyte were also investigated
low molecular weight fragments. (Weber, 1999). The genes coding for the channels
responsible for these currents have not been fully
In an attempt to explain the effects of the venom on characterized, but due to their functional properties and TEA
ion channels, endogenous conductances of the frog oocyte sensitivity, we assumed they may be part of the Kv family.
were investigated (Fig. 2C and D) (Lu et al., 1990; Miledi, The L. mactans venom efficiently and reversibly blocked
1982; Parker & Miledi, 1988b; Parker & Ivorra, 1990). The the transient-outward K+-current, whereas no apparent
voltage-operated Ca++-and chloride-currents were not effects were found in other K+ conductances. In order to
sensitive to the concentrations of venom tested. In addition, confirm the expression of a Kv channel in the oocytes, we
the Ca++-dependent chloride-currents, which are mainly searched for the mRNA coding for the xKv 1.1 (Gutman et
generated by the bestrophins (Hartzell et al., 2005) were al.) channel which generates currents similar to those of
not affected when applying up to 5 µg/mL of the venom. the endogenous K + channel of the oocyte. RT-PCR
Therefore, in the venom extract used for these experiments indirectly evidenced the expression of this channel.

Fig. 3. Effects of Chilean L.
mactans venom extract on
electrophysiology cellular in
spermatozoa. (A) I/V relation in
the absence or presence of 7.5 µg/
mL venom in spermatozoa. (B)
Shows the membrane conductance
is reduced in spermatozoa exposed
to the venom. (C) Shows
comparative effect of venom, high
K+ and increasing concentrations
of TEA (10 to 50 mM) on the pre-
pulse current. The bars represent
means ± SD of 6 different
experiments, * was significant
p<0.05.

736


All conductances are crucial to spermatozoa capacitation. profiles of Kv channels (Darszon et al., 2006) among
After several minutes of exposure of the spermatozoa to the species have been observed. Nevertheless the functional
venom (Fig. 3A-C), the change in the membrane conductance data reported here and elsewhere (Darszon et al., 2006;
after exposure to venom suggests depolarization and Ca+v Marconi et al.; Navarro et al., 2008) is consistent with
channel voltage dependent (CCVD) activation in the the view that Kv channels are integral to the spermatozoa.
spermatozoa cells. This may reflect the blockage of voltage-
dependent K+ channels leading to the entrance of Ca+v from the Contraceptive effect of Chilean Latrodectus venum
extracellular medium in accord with previous studies that extract. There is strong experimental evidence that
suggested that the venom inhibits several K+ conductances mature mammalian spermatozoa have several ionic-
(Parodi & Romero; Parodi et al., 2008). Like the oocyte Kv1.1 conductances, including those driven by voltage
s (Parodi et al., 2008), this channel is also present in the dependent K+ channels (Darszon et al., 1996; Labarca et
spermatozoa midpiece (Darszon et al., 1999). Since Kv1.1 is al., 1995; Nuccitelli & Ferguson, 1994). In addition,
integral to membrane potential regulation (Gutman et al.) we recent reports in which a whole cell patch-clamp was
compared the effect with high K+ added in the medium. In both used to study spermatozoa ion-conductance have
cases the concentration of free Ca++ exhibited a transient rise described the functional properties of Ca++ channels
and sharp fall as detected by spermatozoal midpiece (CATsper), which are key components in the capacitation
fluorescence. However, arise in Ca++ was observed throughout process (Darszon et al., 2005; Wennemuth et al., 2000).
the cell after long periods of time. This is consistent with the Recently other reports have shown a K+ channel sensitive
view that a CCVD was present (Darszon et al., 2005). The to TEA in bovine spermatozoa (Marconi et al.) which is
currents blocked by the spider venom appear similar to those important for the control of membrane potential (Gutman
found in frog oocytes and neurons in culture (Parodi & Rome- et al.). Mammalian sperm acquire their functional
ro; Parodi et al., 2008) although differences in the expression capacity to fertilize an egg during their migration through
the male and female genital tract (Boni et al., 2007). In
this process the plasma membrane potential is suddenly
hyperpolarized by the activation of pH- sensitive K+ -
channels, leading to an increase in Ca+v permeability
(Kumar et al., 2000; Linares-Hernandez et al., 1998; Shi
& Ma, 1998). Previously we measured the Ca++ influx
induced by depolarization of the plasma membrane and
by venom isolated from the Chilean black widow spider
(L. mactans), and functional changes in the presence of
either high K+ or total venom. Our results indicate that
the venom increased the Ca++ influx, with an EC50 of
6.1 µg/mL and triggering the acrosome reaction in
43.26% of the cells and decreased the viability in 40%
(Fig. 4A and B). The application of potassium (10 mM
K+) or total venom (10 µg/mL) did not affect the
morphology or DNA stability of the sperm nor the
production of reactive oxygen species (ROS) (Fig. 5A
and B). We observed a reduction in viability and suggest
that this alteration is a consequence of capacitation and
the acrosomal reaction (Medeiros et al., 2002). We
explain the reduction in viability as methodological
condition and cell death mediated by spermatozoa
activation and the acrosomal reaction. This notion is
supported by the observation of the DNA integrity
measured by DNA fragmentation, in the absence or
presence of venom. No DNA fragmentation was
observed under any condition. The controls with DNase
Fig. 4. Venom induced acrosome reaction and decrease of viability I showed typical fragmentation and support our
spermatic. (A) Shows plot of percent live spermatozoa with intact
suggestion that there is no toxic effect of the venom in
acrosome in control, high potassium (10 mM) and venom (10 µg/mL)
and percent live spermatozoa (B). The bar are means ± E.R. of 5 different our model (Navarrete et al., 2010). Previous studies have
experiments, * was significant p<0.05. provided information about the spermicidal properties
737


of the venom of the Chilean spider L. mactans. Other natural Concluding Remarks. In the future, we are looking
sources of spermicidal action have been reported, such as scorpion to find more applications and effects derived from other
toxins and plant extracts (Harat et al., 2008; Lopez-Gonzalez et peptides present in the total venom, or maybe to refine
al., 2003). All these studies suggest new lines of research to the present cellular effect, to be able to reduce doses
identify the active compound(s) that mediate the modulation of and make it the best choice for pharmacological
ionic conductances, and that could provide a resource for a new applications. In the end, our experience with venom
generation of contraceptives. Ca++ influx in response to membrane from Chilean L. mactans suggested a new line of
depolarization with K+ has been reported as one of the key early application for other peptides, and suggested that we
events leading to the process of sperm capacitation. Therefore, it should not look only at the principal component to
is possible that components in the venom from the black widow explain all its toxic effects. In the future we hope to
spider may influence membrane properties in sperm leading to see more biotechnological applications derived from
capacitation. In conclusion, we find that bovine spermatozoa the small peptides present in this venom and others.
increases the Ca++i when exposed to aracnotoxin from L. mactans
and that this phenomenon produces the subsequent acrosome ACKNOWLEDGMENTS. Fernando Romero, Raúl
reaction in the spermatozoa, most probably through the blocking Sánchez, Antonio Miranda and Jorge Parodi were
of voltage-dependent K+ channels Thus, molecules derived from supported by the FONDEF-CONICYT Chile N°
venom could be isolated for biotechnological applications, DO5I10416. David Ormeño has a “Universidad de la
including the design of new contraceptives. New data is presently Frontera” M. Sc. fellowship. Patricia Navarrete is
being gathered that demonstrates the effects of the purified protein supported by a CONICYT grant.
and peptides.

NAVARRETE, G. P.; ORMEÑO, D.; MIRANDA, A.;
SÁNCHEZ, G. R.; ROMERO, M. F. & PARODI, R. J.
Caracterización molecular, electrofisiológica y efecto anti-
conceptivo del veneno de Latrodectus Chilena. Int. J.
Morphol., 29(3):733-741, 2011.

RESUMEN: Desde los años 70`, se han realizado
estudios con el veneno de arañas Latrodectus sp, en particu-
lar la latrotoxina (LTX) de Latrodectus mactans. Muchos de
estos estudios estuvieron enfocados a entender la acción del
veneno sobre el sistema muscular. Hoy en día es aceptado
que la LTX es capaz de generar un poro de membrana
permeable a calcio y modular la liberación de vesículas
sinápticas que activan un receptor e inducen cambios celula-
res. Interesantemente, cuando comenzamos a trabajar con el
veneno obtenido de Latrodectus sp. presente en Chile, ésto
generó indicaciones clínicas similares a la picadura de esta
araña en otros países, con algunas diferencias en su intensi-
dad. El propósito de estos primeros estudios fue entender los
mecanismos sistémicos de este veneno y además otros com-
puestos activos fueron estudiados para interés biológico. Se
ha encontrado que estas moléculas son capaces de causar
efectos sistémicos así como cambios en la contracción mus-
cular; generar relajación vascular y modulación sináptica y
celular; y de alterar los canales de conductancia de potasio.
Basados en estas evidencias, nosotros sugerimos usar apli-
caciones biotecnológicas para caracterizar los compuestos
de bajo peso molecular obtenidos del veneno de Latrodectus
Chilena y explorar los efectos sobre la electrofisiología en
ovocitos y neuronas, y el efecto anticonceptivo sobre los
Fig. 5. Venom not induced DNA fragmentation or ROS production in espermatozoides.
spermatozoa. (A) Shows plot of percent TUNEL positive spermatozoa
in control, DNase I, high potassium (10 mM) and venom (10 µg/mL) PALABRAS CLAVE: Veneno de Latrodectus;
and percent of live spermatozoa producing ROS (B). The bar are means Ovocitos; Neuronas; Espermatozoides; Anticonceptivo.
± E.R. of 5 different experiments, * was significant p<0.05.

738


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