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G ModelNEUTOX-899; No of Pages 6 NeuroToxicology xxx (2008) xxx–xxx Contents lists available at ScienceDirect NeuroToxicologyBrief communicationSynaptic effects of low molecular weight components from Chilean BlackWidow spider venomJorge Parodi *, Fernando RomeroLaboratorio de Neurociencia-CEBIOR, Departamento de Ciencia Preclinicas, Facultad de Medicina, Universidad de la Frontera, Montevideo 0870, Temuco, ChileA R T I C L E I N F O A B S T R A C TArticle history: a-Latrotoxin is the principal component of the venom from the euroasiatic Black Widow spider and hasReceived 23 May 2008 been studied for its pharmacological use as a synaptic modulator. Interestingly, smaller molecular weightAccepted 27 August 2008 fractions have been found to be associated with this toxin, but their cellular actions have not been studiedAvailable online xxx in detail. The venom from the Chilean Black Widow spider (Latrodectus mactans) does not produce a- latrotoxin, however it does contain several small polypeptides. We have recently demonstrated cellularKeywords: effects of these peptides at the synaptic level using whole-cell patch clamp techniques. Puriﬁed venomSynapses from the glands of L. mactans was studied in 12 DIV rat hippocampal neuronal cultures. Venom at aVenom concentration of 10 nM was able to decrease neuronal conductance thereby increasing membraneSpiderToxic resistance. This effect on the passive properties of the neurons induced a change in action potentialBlack Widow kinetics simulating the action of classic potassium channel blockers. These changes produced an increase in spontaneous synaptic activity in rat hippocampal cultures in the presence of the venom in a concentration- and time-dependent manner. These results indicate that venom from Chilean spider L. mactans is capable of increasing cell membrane resistance, prolonging the action potential and generating an increase in synaptic activity demonstrating an interesting pharmacological effect of these low molecular weight fragments. ß 2008 Elsevier Inc. All rights reserved.1. Introduction cholinergic mediators (patent pending). These results raise ques- tions about the mechanism of action of the toxin on smooth muscle. The Chilean spider Latrodectus mactans pertains to the genus We postulated ionic mechanism, to explain same of these systemicLatrodectus which is found worldwide (Garb et al., 2004) and is effects, based in the idea of described effects of low-molecularknown as the ‘‘Wheat Spider’’ or ‘‘Black Widow’’ and inhabits component from Black Widow venom, over muscle and alterationvarious regions of Chile. The bite from this spider in humans the cellular physiology (Kiyatkin et al., 1992; Grishin et al., 1993). Itproduces a systemic effect called latrodectism or systemic has been postulated that potassium currents, for the importance oflatrodectism and in some cases results in death (Schenone and these currents in the regulation of muscle contraction and theCorrea, 1985). participation over calcium homeostatic (Sanborn, 2000). A classic Venom collected from Chilean L. mactans in the VIII and IX regions synaptic model was affects for a-latrotoxin and high-molecularof Chile was shown to induce a sustained tonic effect in cardiac and weight peptides (Auger and Marty, 2000) and dependent ofsmooth muscle (Romero et al., 2003). In smooth muscle, the potassium channels for normal regulation of synaptic activitymechanism of contraction is related to the permeability of Na+ and (Pan and Stringer, 1997). We tested the effect of Chilean venom inCa2+ ions which modulate the contractile response (Nouailhetas hippocampal neuron, and reported the changes in synaptic activity,et al., 1985) that has a fast, phasic component followed by a slower for have synaptic effects under control and postulate, mechanismmore sustained tonic component (Shimuta et al., 1982). Our studies over neuromuscular union. For another way, we choice a basicin the deferent vessel of the rat revealed that the effect induced model of cell lines, for observation of passive electric properties ofby the L. mactans venom is partially dependent of adrenergic and membrane and not showed receptor or increased number of channels (Varghese et al., 2006). For these reason used HEK 293 cells for the observation of changes in membranes conductance. In the present study was to analyze the possible participation of potassium * Corresponding author at: Laboratorio de Neurociencia-CEBIOR, Facultad deMedicina, Universidad de la Frontera, Montevideo 0870, Temuco, Chile. conductance on the systemic effects observed with the venom of L.Tel.: +56 45 734041. mactans, recording cellular effects associated a changes in potassium E-mail address: firstname.lastname@example.org (J. Parodi). conductance in neurons and cells lines.0161-813X/$ – see front matter ß 2008 Elsevier Inc. All rights reserved.doi:10.1016/j.neuro.2008.08.006 Please cite this article in press as: Parodi J, Romero F. Synaptic effects of low molecular weight components from Chilean Black Widow spider venom, Neurotoxicology (2008), doi:10.1016/j.neuro.2008.08.006
G ModelNEUTOX-899; No of Pages 62 J. Parodi, F. Romero / NeuroToxicology xxx (2008) xxx–xxx2. Materials and methods scope (Nikon, Eclipse, TE200-u, Japan). The membrane potential was adjusted to À60 mV, the current recorded at 50 ms intervals2.1. Spider retrieval and ﬁltered at 2 kHz using commercially available software (Axon Instruments, Inc.). Tetrodotoxin (TTX, 100 nM), a Na+ voltage- Female adult L. mactans spiders from Chile were captured dependent channel blocker, was added to the external solution toduring the summer months (December 2005 and January 2006) block excessive synaptic activity evoked by action potentialsfrom the area of Alto Bio in the VIII region (728160 5100 W, 78450 2400 S) thereby allowing the visualization of miniature synaptic currentsas previously described, taking care not to damage breeding zones (mIPSCs and mEPSCs). The patch electrodes were made of(Romero et al., 2003). The specimens were maintained separate in borosilicate (WPI, Sarasota, USA) and prepared using a horizontalindividual jars for 30 days without food and given only water in pipette puller (Sutter Instruments, USA). The resistance of theorder to stimulate the production and concentration of venom in electrodes was less than 4 MV when ﬁlled with normal internalthe glands. solution. Stock solutions for assays containing pharmacological com-2.2. Venom retrieval pounds, drugs or different ligands were prepared weekly in deionized distilled water and kept at 4 8C. For fast application of The spiders were immersed in liquid nitrogen and after 1 min different compounds, a system of external tubes (200 mm internaltransferred to a phosphate saline buffer solution (PBS: NaH2PO4 diameter) situated 50 mm from the cells was used. Electrophysio-0.1 M, Na2HPO4 0.01 M, NaCl 1.35 M, pH 7.4) at 4 8C. The glands logical recordings were done in the presence and absence of differentwere removed and the membrane that binds them to the base was drugs, pharmacological compounds and ions. The solutions contain-sectioned. Each glands with its poison secreting, was placed into a ing the different compounds ﬂowed continuously from the inside oftube containing PBS (25 pairs of glands for 100 ml of PBS) and the tubes by gravity and locally bathed the cell under study.homogenized. The homogenate was immediately centrifuged at Neuronal recordings were done in the presence or absence of TTX1000 Â g for 15 min and the supernatant was subsequently (100 nM) depending on whether the desired information was foraliquoted, labeled total venom (TV) and frozen at À20 8C. The miniature currents or potential action recording, respectively.protein content of the TV was measured using the Bradfordtechnique, with small modiﬁcations (BioRad Protein Assay). 2.6. Data analyse2.3. Neuronal cultures The recordings were obtained in pClamp 9 from axon instrument. The graphic was made using Origin 6 or prism 4, for The animals were treated and handled according to NIH statistic analyze used Origin 6. The plotted are means Æ S.E. Forguidelines (National Institute of Health, USA). Timed (18 days) p < 0.05 the means are considered signiﬁcant.pregnant Sprague–Dawley rats were anaesthetized with ether andeuthanized by cervical dislocation. The embryos were quickly 3. Resultsremoved, decapitated and the brains placed in cold Hank’ssolution. Subsequently, the hippocampi were dissected; mechani- 3.1. Total venom alters cell membrane conductancecally dissociated using trypsin, collagenase and DNase III and theneurons were then plated at a density of 350,000 cells/ml onto Previous reports have shown that total venom from the Chilean35 mm culture plates containing a monolayer of glia cells. Neurons L. mactans spider is capable of altering normal processes in isolatedwere maintained in culture for 12 days in vitro (DIV) at which point organs (Romero et al., 2003; Romero et al., 2007). These effectsthey were used for experiments. The culture media consisted of have been reported as secondary to the action of the venom on80% MEM, 2 mM glutamine, and 10% fetal bovine serum. The media ionic channels and proton pumps such as the sodium/potassiumwas replaced with fresh culture media every 3 days. All drugs, exchanger (Robello, 1989; Chanturiya and Nikoloshina, 1994). It ispharmacological compounds and ions used in the different noteworthy that the Chilean species, although having similar toxicexperimental protocols were diluted in PBS buffer. effects, lacks the a-latrotoxin characteristic of the venom. We decided to investigate if there was a mechanism which would2.4. Cell cultures explain the changes at the isolated organ level that was related to some of the ionic alterations previously described. Therefore, we HEK Cells 293 (human embryonic kidney cells) are cultivated in studied the effect of extracellular application of TV from L. mactansD-MEM (Dulbecco’s-modiﬁed Eagle medium, Life Technologies, in HEK 293 cells. Voltage–current curves for membrane con-Inc.) supplemented by 10% of fetal bovine serum (Life Technologies ductance were performed, in voltage clamp mode, used aInc.) and streptomicina–penicillin (200 units each, Life Technol- depolarization protocol from À60 to 160 mV on HEK cells in theogies Inc). Growth is carried out fewer than 5% CO2 to 37 8C. The presence and absence of TV. Fig. 1 shows that HEK cells incubatedmedium is changed every 3 days. 30 min with TV present a decrease in the slope, for a conductance graph (Voltage vs. current plot) indicating an increase in2.5. Whole-cell patch clamp membrane resistance (the inverses of conductance for this graph) as compared with control. This ﬁnding suggests the possibility of The culture media in the plate was replaced with an external ionic channel closure producing an increase in membranesolution containing (in mM): 150 NaCl, 5.4 KCl, 2.0 CaCl2, 1.0 resistance or reduced the conductance (slope of the plot). To ﬁndMgCl2, 10 glucose and 10 HEPES (pH 7.4). The internal solution out if this observation was reproducible with other knowncontained (in mM): 120 KCl, 2.0 MgCl2, 2 ATP-Na2, 10 BAPTA, 0.5 molecules, we used the same protocol in cells cultured with TEAGTP, 10 HEPES (pH 7.4). The cells were stabilized at 22 8C for (a potassium channel blocker) or with gramicidin, an agent known30 min before starting the experiments. Current changes in the to perforate membranes (i.e. increase in conductance). Fig. 1Bneurons were detected using the whole-cell patch clamp demonstrates that both compounds vary the slope of the curves astechnique (Hamill et al., 1981) with an Axopatch-200B ampliﬁer compared to control. Interestingly, the channel blocker TEA had a(Axon Instruments, Inc., Burlingame, CA) and an inverted micro- similar effect as that observed with TV. The graph in Fig. 1C Please cite this article in press as: Parodi J, Romero F. Synaptic effects of low molecular weight components from Chilean Black Widow spider venom, Neurotoxicology (2008), doi:10.1016/j.neuro.2008.08.006
G ModelNEUTOX-899; No of Pages 6 J. Parodi, F. Romero / NeuroToxicology xxx (2008) xxx–xxx 3Fig. 1. Effects of Chilean Latrodectus mactans venom extract on membrane conductance. The data were obtained from HEK 293 cells. The cultures were exposed to chronictreatment with Chilean Latrodectus mactans venom extract (7.5 mg/ml, 30 min). (A) Shows an I–y relationship in absence or presence of venom. (B) Shows I–y curves thatcompare the effects of gramicidin (10 mg/ml) and TEA (100 nM) with the venom. (C) Shows values for membrane resistance in the absence and presence of venom. The barsare mean Æ S.E. from 16 different cells (*p < 0.05).demonstrates the membrane resistance in the absence and 3.3. TV increases synaptic transmission in cultured hippocampalpresence of the venom. Cells exposed in acute manner with TV neuronsshowed a signiﬁcant increase in membrane resistance. As previously mentioned, a-latrotoxin is capable of altering3.2. Latrodectus venom alters membrane and action potentials in synaptic activity, however smaller peptides appear to have ahippocampal neurons complementary effect. Our results suggest that in the absence of a- latrotoxin, such as in our Chilean L. mactans model, the smaller The effects of a-latrotoxin in the synapse has been described by fragments play an active role in producing the effect seen with theseveral investigators; however the mechanism by which the venom. This is in agreement with the systemic effects observed withvenom induces changes is still unclear. It is currently accepted that the venom from Chilean L. mactans (Romero et al., 2003; Romerothe interaction of a-latrotoxin with cell membranes (Krasilnikov et al., 2007) as well as with experiments in isolated organs (Romeroand Sabirov, 1992; Orlova et al., 2000; Hlubek et al., 2003) results et al., 2003). Fig. 3A shows representative traces of synaptic activityin the formation of a pore permeable to calcium and this is the in the absence or presence of TV. Neurons cultured in the presence ofmechanism by which the venom alters synaptic activity (Henkel TV display a signiﬁcant increase in synaptic events. Interestingly,and Sankaranarayanan, 1999). The presence of other small- denatured venom, boiled for 45 min at 96 8C, has no effect on themolecular weight molecules having a synergistic action have been neurons indicating a speciﬁc participation of the peptides present indescribed (Kiyatkin et al., 1992; Grishin et al., 1993). Our results, the venom (Fig. 3B). Fig. 3C and D reveal a time- and concentration-however, suggest that the fragments we studied are capable of dependent action of TV as reﬂected in an increase in the frequency ofaltering the normal functioning of isolated organs and could be events with longer incubation times and higher concentrations ofmodifying membrane conductance. Fig. 2A shows representative the venom. Both ﬁgure, suggested a pharmacology effects over thetraces of action potentials from hippocampal neurons in the synapses, because induced to think a time depend effects andabsence or presence of TEA and TV. The traces indicate that TEA concentration response, two characteristic of physiological effectalters the action potential, lengthening the repolarization phase over receptors or protein. In addition, Fig. 3C demonstrates that(Schwartzkroin and Prince, 1980) which is mediated by potassium these effects are reversible because when the venom is removed, thechannels (Woodson et al., 1978). The venom is also able to alter the synaptic frequency returns to control levels. On the contrary, higheraction potential kinetics in a manner parallel to TEA suggesting concentrations of the venom are unable to alter synaptic activitysimilar mechanisms. Fig. 2B current clamp traces, acquired in the suggesting an antagonistic effect (Fig. 3D), not explored but thepresence of TTX (100 nM) for prevented potential action, from interesting in biotechnology application. Therefore, we can suggestneurons exposed to TEA or TV. As can be seen, TEA produces a that the alteration in synaptic activity in the presence of the venomlengthening on the action potential, a similar effect found with TV. may be due to blockage of potassium channels, similar to TEAFig. 2C shows the change in membrane potential, acquired using sensible and changes in membrane potential.current clamp, for measured the changes in the potential and Noteworthy, TV from L. mactans has demonstrated to be areveals that both TEA and TV signiﬁcantly decrease the membrane potent stimulator of synaptic activity in cultured hippocampalpotential to values near the action potential threshold. These data neurons despite the fact that it lacks latrotoxin. Therefore, our datademonstrate a change in the passive properties of the membranes provides the ﬁrst evidence that polypeptides present in L. mactansin the presence of TV, similar to TEA, indicating the closing of alter synaptic activity in a manner similar to a-latrotoxin, but for apotassium channels. different mechanism. Please cite this article in press as: Parodi J, Romero F. Synaptic effects of low molecular weight components from Chilean Black Widow spider venom, Neurotoxicology (2008), doi:10.1016/j.neuro.2008.08.006
G ModelNEUTOX-899; No of Pages 64 J. Parodi, F. Romero / NeuroToxicology xxx (2008) xxx–xxxFig. 2. Effects of Chilean Latrodectus mactans venom extract on membrane potential from hippocampal neurons. The data were obtained from 12 DIV neurons exposed to acuteapplications of Chilean Latrodectus mactans venom extract (7.5 mg/ml). (A) Shows action potential traces, in the absence and presence of TEA (100 nM) or venom (7.5 mg/ml).(B) Shows membrane potential traces, with TTX (100 nM) in absence and presence of venom. (C) Graph showing membrane resistance values in the absence and presence ofvenom. The bars are mean Æ S.E. from eight different neurons (*p < 0.05).Fig. 3. Effects of Chilean Latrodectus mactans venom extract on synaptic activity. The data were obtained from 12 DIV neurons exposed to acute applications of ChileanLatrodectus mactans venom extract (7.5 mg/ml). (A) Shows synaptic currents obtained in the absence and presence of venom or boiled venom. (B) Shows miniature synapticcurrent frequency in the presence of the venom. (C) Graph of synaptic frequency at different times incubation and (D) data showing the effect of different concentrations ofthe venom. The bars are mean Æ S.E. from 19 different neurons (*p < 0.05).4. Discussion described a fractionation of the venom by HPLC–MS, and we found the absence of the higher weight component, like a-latrotoxin, this Previous report, in our laboratory and work for development a toxin, is the principal component in Black Widow venom andpatent (Under way in countries PCT) was provide information explain mostly of systemic effect secondary to this venom. A fewabout the component of the Chilean venom, form L. mactans. We report, do it description of the low weight component, present in Please cite this article in press as: Parodi J, Romero F. Synaptic effects of low molecular weight components from Chilean Black Widow spider venom, Neurotoxicology (2008), doi:10.1016/j.neuro.2008.08.006
G ModelNEUTOX-899; No of Pages 6 J. Parodi, F. Romero / NeuroToxicology xxx (2008) xxx–xxx 5the venom but not explored. The Chilean venom, not presents Conﬂict of interest: Patent pending.higher component (minor to 10 kDa, data not show) but, the Funding source: FONDEF-D02I1024 and FONDEF-D05I10416.Chilean venom can induce similar effects to another Black Widowvenom in absence of a-latrotoxin. Agreement with previous Referencesstudies (Romero et al., 2003, 2007), our data show thatpolypeptides present in the venom from Chilean Lactrodectus Ashton AC, Volynski KE, Lelianova VG, Orlova EV, Van Renterghem C, Canepari M, et al.mactans spiders increase spontaneous synaptic activity in hippo- Alpha-latrotoxin, acting via two Ca2+-dependent pathways, triggers exocytosis ofcampal neurons and changes the passive properties of the two pools of synaptic vesicles. J Biol Chem 2001;276:44695–703. Auger C, Marty A. Quantal currents at single-site central synapses. 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