Archivos Venezolanos de Farmacología y Terapéutica ISSN: 0798-0264 [email protected] Sociedad Venezolana de Farmacología Clínica y Terapéutica

Trejo, E.; Borges, A.; González de Alfonzo, R.; Lippo de Becemberg, I.; Alfonzo, M. J. zulianus and Tityus discrepans induced massive autonomic stimulation in mice Archivos Venezolanos de Farmacología y Terapéutica, vol. 31, núm. 1, enero-marzo, 2012, pp. 1-5 Sociedad Venezolana de Farmacología Clínica y Terapéutica Caracas, Venezuela

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How to cite Complete issue Scientific Information System More information about this article Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Journal's homepage in redalyc.org Non-profit academic project, developed under the open access initiative Tityus zulianus and Tityus discrepans venoms induced massive autonomic stimulation in mice E. Trejo, A. Borges, R. González de Alfonzo, I. Lippo de Becemberg and M. J. Alfonzo*. Cátedra de Patología General y Fisiopatología and Sección de Biomembranas. Instituto de Medicina Experimental. Facultad de Medicina. Universidad Central de Venezuela. Caracas. Venezuela.

E.Trejo, Magister Scientiarum en Farmacología y Doctor en Bioquímica. R. González de Alfonzo, Doctora en Bioquímica, Biología Celular y Molecular. I. Lippo de Becemberg, Doctora en Medicina M. J. Alfonzo, Doctor en Bioquímica, Biología Celular y Molecular

*Corresponding Author: Dr. Marcelo J. Alfonzo. Sección de Biomembranas. Instituto de Medicina Experimental. Facultad de Medicina Universidad Central de Venezuela. Ciudad Universitaria. Caracas. Venezuela. Teléfono: 0212-605-3654. fax: 058-212-6628877. Email: [email protected]

Running title: Massive autonomic stimulation by Venezuelan venoms. Título corto: Estimulación autonómica masiva producida por los venenos de escorpiones venezolanos.

Recibido: 06/02/0011 Aceptado: 16/09/2011

Abstract

Venezuelan scorpion envenomation is a public health problem produced in TZ mice. These clinical manifestations (diarrhea, sialorrhea, dehydra- by Tityus discrepans (TD) and Tityus zulianus (TZ) species. Patients-en- tation and intense salivation) are related to an activation of autonomic venomend by TD developed gastrointestinal and pancreatic disorders and nervous system, via an intense release of their related neurotransmitters. scorpion accidents involving TZ are associated with high mortality rate, Thus, autonomic stimulation (sympathetic) was evaluated following the which showed cardiopulmonary clinical disorders may be associated to catecholamine (Nor-Epinephrine)(NE) plasma levels in a function of en- the high levels of plasma catecholamines levels. This distinctive clinical venomation time. We found a significant increments at 1 hr, after output seems to be associated to a repertoire diversity, which has injection, in more than 640% in NE plasma levels for TZ venom while in TD been previously demonstrated. Trying to mimic the human-envenomation, group, around 520% rise in NE concentrations were detected. This mas- some toxinological studies have been performed using TD and TZ ven- sive rise in NE concentrations in TZ and TD-envenomed mice decreased oms in several biomodels such as mice and anesthetized rams. The pur- at 6 hrs but remained higher until 24 hrs for both venoms in comparison pose of this study was to evaluate, in vivo using biomodels (mice), the role with Control . However, these catecholamines plasma alterations of autonomic nervous system (sympathetic) stimulation producing some do not explain the dyspnea and hind limb spasticity and more toxinologi- of the clinical signs, via the catecholamines release, on the patho-physi- cal research should be done to understand the molecular mechanisms ology of the TZ and TD induced envenomation. Thus, a clinical signs here related to last clinical signs. 1 reported during a period of 1 hr, after a single intra-peritoneal injection Abbreviations: TD: Tityus discrepans, TTX: , TZ: Tityus zu- of sub-lethal doses of TZ or TD venom, which are related with diarrhea, lianus, NE, Nor-epinephrine, RyRs: Ryanodine receptors, IpTxA: impera- diaphoresis, intense salivation, dehydratation, dyspnea and spasticity in toxin A, ROS: reactive oxygen species, nAChR: nicotinic acetylcholine hind limbs. However, these animals did not exhibit vomiting, which is the receptor (Na+ channel), (Nav): voltage-dependent sodium channel, MCa: most frequent human-envenomed TD patients. All animals inoculated with maurocalcine, Tz1: β- toxin from Tityus zulianus. (TD or TZ) venoms developed diarrhea being more pronounced in TD group. Diaphoresis, sialorrhea and dehydratation were mainly observed Key words: Nor-epinephrine, cathecholamine release, Tityus zulianus, in TD group. Dyspnea and the hind limb spasticity were only developed Tityus discrepans, scorpion venoms.

Resumen

El escorpionismo en Venezuela es un problema actual de salud pública El propósito de este trabajo fue evaluar, “in vivo” usando un Biomode- producido por las especies de Tityus discrepans (TD) y Tityus zulianus lo (ratones), el papel de la estimulación del sistema nervioso autóno- (TZ). Los pacientes que presentan escorpionismo producido por TD de- mo (simpático) para producir algunos signos clínicos, vía la liberación sarrollan trastornos gastrointestinales y pancreáticos mientras que los de catecolaminas, en la fisiopatología del escorpionismo producido por afectados por TZ presentan una alta mortalidad y muestran una sintoma- TZ y TD. Así, los signos clínicos aquí descritos y observados durante 1 tología relacionada a desordenes cardiopulmonares, los cuales parecen hr., después de la inyección de una dosis sub-letal de los venenos de estar asociados a niveles elevados de las catecolaminas plasmáticas. TZ y TD, fueron la presencia de diarrea, diaforesis, salivación intensa, Esta clínica diferente parece estar asociada a una composición distinta deshidratación, disnea y parálisis en las extremidades posteriores. Sin de toxinas de dichos venenos, lo cual ha sido previamente demostrado. embargo, estos animales no presentaron vómitos, el cual es uno de los En un intento de mimetizar o reproducir el escorpionismo en humanos se signos más frecuentemente observado en los pacientes con accidentes han realizado estudios toxinológicos con los venenos de TZ y TD utilizan- escorpiónicos por TD. Todos los animales inyectados con los venenos de do varios biomodelos como son ratones y carneros anestesiados. TD y TZ presentaron diarrea especialmente en grupo TD. of anti-sympathetic drugs. venom scorpion this with inoculated animals in times 40 and plasma 30 betweenlevels catecholamine the in increment an to due be may rats, in nus) occita (Buthus venoms scorpion by induced alterations cardiovascular lated well with the cardiopulmonary clinical findings. On this sense, similar determined levels by HPLC.(NE) plasma These authors also in nor-epinephrine claimed that these results corre increase al. significant et a described Davila de Mazzei by reported as cat echolamines plasma human of pulmonary levels high and the to cardiovascularassociated be may some alterations TZ with envenomed patients In presents inskeletal muscle. Na the on voltage activation half-maximal the of shift leftward the was study this in described, effectrelevant most (Na channels sodium dependent voltage- mammalian of subtypes several on TZ from Tz1 β-toxin novel a of effects the described al. et Borges Later,venom. TZ mice-injected in duce a very intense pancreatic alterations related to an acute pancreatitis al. et al. Borges biomodels, et in Davila, de Mazzei by by reported death as and edema pulmonary arrest respiratory as disorders clinical cardiopulmonary showed children TZ-envenomed These old. years eight under children in rate mortality high with associated are (TZ) zulianus, 2000 involvingTityus in reported al. at Borges Interestingly; TD-envenomed rams. 2004 in al et venom.D’Suze TD by produced damage ultrastructural clinical manifestations of TD scorpion envenomation may be related to the glands vary sali of alterations ultrastructural causes venom scorpion TD that shown have 2000, al.in et Rodriguez-Acosta by reported mice in studies Other longed actionpotentials, suggestingpotassiumchannelblockage. ing sodium channels. TdF-III produced acute pancreatitis and TdF-IV pro open by membrane muscle the depolarized and dyspnea sialorrhea, as such effects muscarinic produced membrane;TdF-II postsynaptic the at venom.TD blockedthat transmission TdF-I neuromuscularclaimed They 1995 in al. et D’Suzebiomodels. severalThus, in venom (TD) discrepans Tityus al. et L. Sequera by described as disorders pancreatic developed and gastrointestinal discrepans Tityus by Patients-envenomend envenomation. scorpion of percentage high a of responsible species both being range, western-Andes Venezuelaat Táchirastates and Mérida, Zulia, at lo cated (González-Sponga) zulianus Tityus and Venezuela north-central at two mainly () sp Venezuelanspecies (Karsch) discrepans Tityus cially in Venezuela. The most serious clinical cases are produced by Tityus envenomationScorpion isapublic healthproblem intropicalareasespe Introduction estos signosclínicos. con relacionados moleculares mecanismos los entender para realizadas miembros posteriores por lo cual mas investigación toxinológica debe ser los en parálisis la y disnea la no pero observados signos los mayoríade las concentraciones plasmáticas de las catecolaminas pueden explicar la en alteraciones Estas Control. al relación con venenos ambos para hrs 24 las a elevada permaneció y hrs 6 las a descendió NE de plasmática concentración la en masivo aumento TD.Este de veneno con en grupo NE el de niveles los en 520% de aumento un y TZ con envenenados ratones los de veneno del caso el en 640% un en plasmática NE la de ficativo a 1 hr después de la inyección del veneno, en las concentraciones no.signi dramáticamenteexiste aumento que encontramosun Nosotros inyecciónla de después tiempo del función en plasmática (NE) venedel catecolaminas de concentraciones las evaluadasiguiendo fue simpática autonómica activación La correspondientes. neurotransmisores los de intensa liberaciónautónomo, la vía, nervioso sistema del activación una cas como son diarrea, diaforesis y la salivación intensa están asociadas a das en el de grupo ratones inyectados con TZ. Las manifestaciones clíni La disnea y la parálisis en los miembros posteriores fueron sólo observa 1 and Mota JV. et al. 11 6 3 . These neurotransmitter increments were reversed with the use the with reversedwere increments neurotransmitter. These reported similar respiratory distress findings using anesthetized using findings distress respiratory similar reported reported the isolation of four toxic fractions (TdF-I-IV) from the from (TdF-I-IV) fractions toxic four of isolation the reported 4 n i ln tissue lung in and

2 . Toxinological studies have been performed using 9 have reported that TZ venom is able to in to able is venom TZ that reported have 5 v n te sgetd ht oe f the of some that suggested they and ) expressed) cells 293 HEK in v . sdu canl mainly channel, sodium 1.4 7 ta sopo accidents scorpion that , 8 Ti ls group last This . 8 Moreover, . 10 .the One ------obtained from 50-60 wasscorpions reconstituted in small volume of 0.9% Zlotkin and Shulov ga ons were classified according to the criteria described by González-Spon of region Venezuela). (North-central State,MirandaAltos, Los de Scorpi Venezuela). Tityus discrepans were collected near San Antonio of and Tovar,Mora(Western de region Andes Cruz at The State, Mérida Bolivar,Santa Mesa near collected were scorpions zulianus Adult Tityus Scorpions and Venoms Materials andMethods in duced envenomation. TD and TZ the of patho-physiology the on release, catecholamines the via stimulation, (sympathetic) system nervous autonomic of role the The purpose of this study was to evaluate, in vivo using biomodels (mice), related toapheocromocytoma(acatecholaminesreleasingtumor). scribed to the ones described accidents scorpion clinical in choline, which are responsible for several symptoms and signs presented acetyl and Epineprine Nor-epinephrine, mainly neurotransmitter, nomic tissues (Na channels sodium voltage-dependent the interferewith to ability the toxinsis scorpion these effectof biological al. et Borges, by described as diversity clear-cut is there oms are single chain polypeptides, that in relation to the long-chain toxins, ven scorpion the of components main the venoms,and TZ) (TD both In tween 10 and 100 µl and NE was separated on a Supercosyl LC18 250 x Subsequently, samples were injected automatically in variable volume be 0.65 acid;mM Sodium Octyl Sulphate; citric 7% acetonitrile; 128 mM mM formic acid;EDTA; 1 pH 3.4).(1mM phase mobile in 1:2 to diluted was min.supernatant 20 The for g 17.000x at centrifuged sulphosalicilic and vortexed acid, 20% in 1:6 diluted and thawed were Samples detector. al. plasma Nor-epinephrine levels were as determinate bydescribed Lima et sure liquidchromatography (HPLC). pres high by concentrations plasma Nor-epinephrine of Determination 3000xg for 10minat4°Candplasmawas removed andstored-80°C. obtained at 0, 1, 6 and 24 hrs after injection. Whole blood was centrifuged at divided into four groups (n = 6 per group) and blood by cardiac puncture was NaCl.% 0.9 with Venom-injectedinjected werewere animals mice Control by Borgesetal. discrepans (2.51 mg/kg) and T. zulianus (1.54 mg/kg) venoms as reported T.of case discrepans the in terations al structural pancreas produce to known is concentration venom weight.Such body protein/kg mg 0.5 of dose a at venoms discrepans Tityus or zulianus Tityus with (i.p.) intraperitoneally ml) (0.2 injected were Mice Preparation ofplasmasamples was consideredas “sign ofdehydration”.time elapsed of sec 5 than More observer. the by pinched or stretched required for the skin of the mouse to time return to normal position the after, being as sign, turgor” “skin the by evaluated was which dehydration, except observation, followvisual byto easy are described here signs cal clini All observers. independent three by recorded and complied were tocon manifestationsdevelopmentclinical the of and groupexperimental or trol assigned randomly were Mice water. filter to access ad-libitum had and chow laboratory standard using fed cycle.were They light-dark 12-hour and 2°C ± 23°C of temperature ambient an with room a in cage standard in housed and bred were animals rodent All experiments. cific Experimental-UCV. Male BALB/C (20-22 g) were used throughout all spe Medicina de Instituto of guidelines Care Bioethical the byproved ap protocols the to according experiments, all in employed were Mice Bio-models. bytein standardasdescribed Lowry in determined the using supernatant bovine serum albumin (BSA) as pro was concentration protein and min, 10 for 12.000xg at centrifuged NaCl, 20 17 using a reverse phase HPLC system equipped with electrochemical with equipped system HPLC reversephase a using and their venoms were obtained by manual stimulation according to according stimulation manual by obtained werevenoms their and 13 . These sodium channel neurotoxins induce the release of auto of release the induce neurotoxins channel .sodium These 7,9 . 18 . Later, this wasmaterial lyophilized. Pools of venoms 16 14,15 for patients presenting a clinical syndrome A iia ciia otu hs en de been has output clinical similar A . 19 4 . . This dose is below the DL the below is dose .This v o te amla excitable mammalian the of )

12

. The classic The . 50 forT. ------

2 AVFT Archivos Venezolanos de Farmacología y Terapéutica Volumen 31, número 1, 2012 4 mm column with a flow velocity 1 ml/min (Isocratic pump Waters 600®). Table 1: Clinical findings in mice injected with total venom of Tity- Potential between reference and work electrode was +700 mV (Electro- us discrepans (TD) and Tityus zulianus (TZ). chemical detector Waters 464®). The results were analyzed using the ex- ternal standard method provided by Waters Breeze Program®. Clinical signs TD TZ

Statistical analysis: % % All the results are presented as the mean ± standard error. Data analysis 1. Diarrhea 100 100 performed with ANOVA and Tukey-Kramer post hoc test using the programs 2. Diaphoresis 83 50 ® ® ® Excel (Microsoft, USA), Graph Pad In Stat and Prisma (Science, USA). 3. Intense salivation 66 0 ** 4. Dehydratation 66 0 ** Results 5. Spasticity in hind limbs 0 66 ** 6. Dyspnea 0 83 ** Clinical findings: After a single intra-peritoneal injection of TZ or TD venom (0.5 mg/Kg), Clinical observations were performed after intra-peritoneal injection of 0.5 mg/kg of mice during a period of 1 hr displayed a variety of clinical signs related total venom during a period of 1 hr. with alterations in gastrointestinal, respiratory and skeletal-muscle sys- These animals were the same biomodels used for the time dependent evolution tems (Table 1). All animals inoculated with scorpion venom (TD or TZ) (chronology) of the NE plasma determinations shown in Figure 1. Percentage was developed diarrhea being more pronounced in the TD group, which in estimated from observations of three different clinical trained researchers to avoid addition, showed an anal prolapsus. Diaphoresis was more abundant in bias. n = 24 animals for each venom. (**) c2 = 77.536 (p < 0.01). TD-treated animals. Sialorrhea and dehydratation were observed only in TD group (p < 0.01). Dyspnea and the hind limb spasticity were only de- veloped in TZ mice (p <0.01). Most of these clinical manifestations are Discussion related to an intense activation of autonomic nervous system probably due to an acute release of their related neurotransmitters. The human clinical features develop as a result of envenomation with Venezuelan scorpion of Tityus ; have been reported1,2. This study, Plasma catecholamine (NE) levels during TZ and TD envenomation: here described, is an experimental model (mice), where, we systemati- cally evaluated, the appearance of more relevant clinical signs during 1 hr; The autonomic stimulation was evaluated following the onset of the cat- after the post-injection of a sub-lethal doses of these two venoms. Some echolamine (NE) plasma concentrations in a function of envenomation of these effects here described have been reported in both, human and chronology. Blood samples were taken at different times (0 hr, 1hr, 6hrs, 24 biomodels studies1,2,6,7. hrs) in the Control, TD and TZ groups. The NE circulating concentration was determined in blood samples taken from envenomed mice and a significant We decided to evaluate the early clinical output and progression of the increment (p < 0.01) was found in the NE plasma concentrations at 1 h in TD and TZ envenomation in mice due to ethical principles. It is very dif- the TZ group as shown in Figure 1. A slightly but not significant increment ficult to perform such studies, especially in scorpion-envenomed patients. in NE concentrations was observed in the Control group, but, in the TZ Thus, using mice, in a more controlled biomodel, we found that the clini- group, an increment in more than 640% in NE while under TD stimulation, cal findings observed at 1 hr being the most relevant signs: Diarrhea, around 520% rise in NE concentrations were detected. At 1hr, after ven- diaphoresis, intense salivation, dyspnea and spasticity in the hind limbs oms injection, there was a not significant difference between both venoms. as shown in Table 1. In addition, we evaluated the plasma catecholamines These augmentations in NE remained higher after 6 hrs and 24 hrs for both (NE) concentrations as an expression of peripheral sympathetic activity venoms. However, at 24 h both venoms showed values that remained sig- as shown in Figure 1. nificantly higher in comparison with the Control group (p < 0.01). Trying to understand at the molecular level, the effects of the venoms of Figure 1 scorpion of family Buthidae, producing an intense and massive release of catecholamines (NE) mainly from sympathetic neurons and adrenal gland. This biological activity might be due the fact that these venoms con- tain α and β toxins capable of slowing inactivation of voltage-dependent 3 21,22 sodium channels (Nav) that reduce the triggering threshold and pro- duces depolarization inducing the rise of cytoplasmic Ca2+, which leads to vesicle fusion to plasma membranes and the intense release of the storage-catecholamines in secretory vesicles of adrenal gland to blood stream. Our study shows a significant increment in plasma concentrations of epinephrine (NE), as a result of intense autonomic stimulation. The increase in plasma concentrations of NE is much higher (640% and 520% to TZ and TD respectively) at 1 hr before falling after 6 hrs in both ex- perimental groups. However, at 24 hrs, NE levels in the TD and TZ groups remained significantly above the Control group. Similar results have been reported by Zeghal et al.11 in studies performed in Buthus occitanus en- venomed rats, these authors reported a rise in about 300% in plasma concentrations of NE. In TZ-envenomed patients, reported high levels of plasma catecholamines are probably related with some cardiovascular and pulmonary alterations as described by Mazzei de Davila et al.8. This Time course of plasma nor-epinephrine levels (pg/ml) in mice injected with of TD last research group foun d a significant increase in NE plasma levels de- or TZ venom (0.5 mg/kg). Nor-epinephrine was determined by HPLC coupled to an termined by HPLC. Our results on the NE plasma concentrations demon- electrochemical detector as described in Methods. Each point represents the mean strated that it is possible to reproduce in biomodels (mice), at least, some ± SEM from (n = 6) animals. ANOVA. 1 hour F (2,15) = 30.73; 6 hrs F (2,15) = 11.29; clinical findings described in envenomed-human patients. 24 hrs F (2,15)= 9.87. (**) p < 0.01 vs Control. Diarrhea, Diaphoresis and intense salivation seem to be the result from ini- tial hyper-stimulation of the autonomic nervous system induced by the tox- ins present in these venoms. However, dyspnea and spasticity in the hind limbs are difficult to explain as direct effects of autonomic deregulation. Diarrhea is a sign related to a parasympathetic effect on the intestine smooth muscle, via vagal stimulation producing an increased intestinal due in part to activation of the inflammatory response leading to release to leading response inflammatory the of activation to part in due is species scorpion Tityus by envenomation following injury lung Acute cess inthe walls andalveolar spacesatlungs. pro inflammatory acute severe a of development the to associated ings deposits withfibrin neutrophil infiltration, which are histopathological find alveolar and interstitial barrier, capillary alveolar the of injury diffuse sis, venom. These developedrams TD-injected necro showingintense TD with rams-envenomed anesthetized using distress respiratory about byproduced age toxinsvenom.TD al. et D’Suze TD scorpion envenomation may be related to the ultrastructural lung dam membrane and they suggested that some of the clinical manifestations of sue, specifically denudation of epithelial cells and edema of the basement TD venom in mice (5 mg/kg), described ultrastructural changes in lung tis al. et Matos De by venom serrulatus Tityus using rats as such biomodels in described been have permeability membrane olo-capillary microscopy findings compatible with acute lung injury and increasedelectron alve and (ARDS) syndrome distress respiratory adult as symptoms omed with other species of the Tityus genus severedescribed respiratory with moderate to severe envenomation. Clinical studies in patients enven edema pulmonary of development the as findings radiographic showing al. Davilaet byde Mazzei reported as symptoms nary cardiopulmo of collection a as mainly occurs dyspnea patients, mation in this case, it is difficult or uncomfortable breathing in mice. In TZ enveno with TZ.ed dysfunction, Typically, cardiopulmonary of sign or symptom a The presence of dyspnea was observed only in the group of animals treat circulating blood volume, leadingtoslackness intheskin’s layer. dermal maintain to bed the vascular into fluid movesvolumeinterstitial depletion all diaphoresis, processes present in and TD-envenomed diarrhea mice and previously the discussed. by This depletion, volume or water of rapid loss of consequence a is sign”, turgor “skin as displayed Dehydration venomed humansandanimals. en both in salivation intense the for responsible be would findings lular granules. secretory of depletion a and reticulum endoplasmic rough cel These the of alterations severe involving glands salivary of cells acinar et Rodríguez-Acosta al. by mice TD-injected in described glands salivary in changes cellular for sis. It is possible that strong cholinergic stimulation would be responsible bahien Tityus and of serrulatus Tityus quinquestratus, species Buthus Brazilian scorpions: three of venom whole of injection intra-arterial ter sense, this In system. al et nervous Renner autonomic the of stimulation of the be result can it that shown have studies pharmacological and sp., Tityus genus the of venoms scorpion other for reported also been has vation with inoculated animals TD.in observed Ptyalism)wasonly sali Intense Polysialia, human (like salivation intense an study, this in Interestingly, diaphoretic effects. activity,sympathetic andresponsiblefor gastrointestinal be would which doses of TZ venom, suggesting that this venom possess toxins with para Borges et al. that TdF-II claimed produced muscarinic effectsvenom.They such as sialorrhea TD and the dyspnea. from (TdF-I-IV) fractions toxic 4 of lation with inoculated venom.TD al. sense,D’Suzeet this In demonstrated been has medulla adrenal the of secretion particularly sweating of nent compo adrenergic an and innervations cholinergic a by controlled are are also characterized symptom by increased sympatho-adrenal activity. a Sweat is glands (hyperhidrosis), sweating, excessive producing tion, stimula autonomic cholinergic postganglionic a of sign is Diaphoresis this biomodel. of limitation is which models, murine in develop to difficult very is which patients and similar findings have in been described Tityus serrulatus-envenomed in TD-envenomatedsign patients clinical reported most Vomitingthe is ditional signssuchasdehydration andanalprolapsedwere observed. with treated TD,mice ad in where intense more or venomsbeing TD TZ motility 23 14,24 . Diarrhea was observed in all of animals inoculated with both, with inoculated animals of all in observed was Diarrhea . Ti sg i hmn i rltd o uooi activation autonomic to related is humans in sign This . 7 27 described a similar effect in mice inoculated with sub-lethal

26 in canine models observed an intense flow of saliva af saliva of flow intense an observed models canine in . Diaphoresis was displayed at a higher rate in animals animals in rate higher a at displayed was Diaphoresis . 4 , which reported , ultrastructural which alterationsreported of the 30 and Rodriguez-Acosta et al et Rodriguez-Acosta and

28,29 . Similar lung pathology findings pathology lung Similar . 6 reported similar results similar reported 8 3 in a clinical study clinical a in reported the iso the reported 5 , in studies with studies in , 25 1,2 ------,

potentials, this effect was blocked by tetrodotoxin (TTX), implying a sodi a implying blockedeffectwastetrodotoxin(TTX), bythis potentials, and depolarization increased both miniature postsynaptic and membrane Fractionproduced fractions.Hence, TdF-II TD of effects variable de scribed muscle skeletal sartorius (frogs) amphibian of preparation isolated endplate.muscleskeletal col. mammalian y D’Suzethe at line acetylcho of release unique prolonged this a effect,of to TZ maydue be limbs.hind the possibleexplanationdevelopmentin One the spasticity of with inoculated wasanimals TZ, in only observed was that sign unique A associated with thisacuteinflammation. component neurogenic limited a and factors vasoactive by and De Matos et al. al. et D’Suze by reported as venom the by triggered is which process, indirect an inflammation acute to systemic a of target due a tissue, lung being mechanism, be to seems lungs the on toxins scorpion of effects envenomedanimals.tamulus) (Mesobuthus Apparently, scorpion the red al. et Deshpande by described as edema lung of exis tence the suggesting shown) not (data weight weight/body lung of ratio the in increase an induced venom TZ the only study our Interestingly,in complicationselicitedafterenvenomationdiorespiratory by TZ species. may be an in important vivo target and which could have a role ROS production, to play in the car intracellular neutrophil up-regulates specifically venom. No effect was observed on eosinophils, suggesting that TZ venom TD to compared neutrophils in mainly production (ROS) species oxygen ed that TZ venom induced a significantly more potent increase in reactive reactive oxygen species (ROS). In this sense, Borges et al. of cytotoxic leukocyte-derived products, including cytokines discussed. above as mechanisms molecular following the by explained be can els) (biomod mice intact in venoms zulianus Tityus and discrepans Tityus envenomationby induced to related findings summary,clinical In these muscle cells. fasciculations,these skeletalof mammalian basis in molecular especially the understand to needed is research Further TZ.with inoculated mals, the contractions, which would explain the spasticity in ani in alterations produce might discussed, above activities biological These Bungarotoxin indicatingthatanAChRs isinvolved. increase of [Ca transient a inducing cells, muscle skeletal in nAchRs with interaction ing follow activity depolarizing a displays (Bot) tunetanus occitanus Buthus skeletalmuscle.end-plate non-toxicthe Thus,the of venom (M1) fraction receptor (nAChR), which acting as a Na acetylcholine nicotinic the activate can venom (Bot) tunetanus occitanus Furthermore; Cheikh et al (RyRs) Ca venom perator im Pandinus scorpion the from (IpTxA) A imperatoxin. as such toxins, Ca induced skeletal the affect can muscle machinery specially that in the Ca venom, TZ the in toxins novel of presence Another possible mechanisms that can explain these fasciculations is the muscle. Na the on voltage tion activa half-maximal the of shift leftward the was study this in described, sodium voltage-dependent (Na channels mammalian of subtypes several on Tz1 of effects the venom.described from groupTZ β-toxinnovel Tz1 This the of al. et byBorges reported results experimental the of basis the on explained also be could TZ, with lated The development ofspasticitymentionedabove, inmiceinocu observed the molecularmechanismsresponsible for thesefasciculations. about explanation additional an without rams, anesthetized envenomed al et D’Suzeover, similar effect, being a TTX-insensitive and neuromuscular blockers. More voltage-dependent.channel um time,Fractionsame the At a has TdF-IV 37 36 , which have been shown to strongly modify Ryanodine receptors n muoacn (C) rm h sopo Muu palmatus Maurus scorpion the from (MCa) maurocalcine and 2+ 2+ channel properties.

v release in skeletal muscle as described by other scorpion scorpion other by described as muscle skeletal in release ) expressed in HEK 293 cells. One the most relevantcells.most effect293 the HEK One expressedin ) 2+ ] i , which could be blocked by a application prior of alpha- 35 34 , described the development of fasciculations in TD- in fasciculations of development the described , . These studies suggest that lung damage is caused v 1.4 sodium channel, mainly presents in skeletal in presents mainly channel, sodium 1.4 38 have that reported the yellow scorpion Buthus 10 , whom described the biological effectsbiological the described whom , 2+ release events and depolarization- + channel is able to depolarize the 32 31 n h Indian the in 6 and possibly have report 3 , using an using , 33 ------

4 AVFT Archivos Venezolanos de Farmacología y Terapéutica Volumen 31, número 1, 2012 1.-The existence of α and β�������������������������������������������������� ������������������������������������������������toxins capable of slowing inactivation of volt- tivenin administration and cardiovascular manifestations of scorpion age-dependent sodium channels (Nav) in excitable nerve cells, adrenal envenomation. Toxicon. 2002; 40: 1339-1346. glands and skeletal muscle end plates. 9.- Borges A, Trejo E, Vargas AM, Céspedes G, Hernández A, Alfonzo 2.-The existence of putative scorpion toxins like-imperatoxin A (IpTxA) MJ. Pancreatic toxicity in mice elicited by Tityus zulianus and Tityus and/or maurocalcine (MCa) that affect Ryanadine receptors Ca2+-re- discrepans scorpion venoms. Invest Clin. 2004; 45: 269-276. lease channel at the sarco(endo)plasmic reticulum in mammalian excit- able neuronal or non neuronal cells. 10.- Borges, A., Alfonzo, M., García, C., Winand, N., Leipold, E., Heine- mann, S. Isolation, molecular cloning, and functional characteriza- 3.- The existence of putative scorpion toxins like the non-toxic venom frac- tion of a novel -toxin from the Venezuelan scorpion, Tityus zulianus. tion (M1) of the Buthus occitanus tunetanus (Bot) which induces a tran- Toxicon. 2004; 43: 671–684. 2+ sient increase of [Ca ]i ,via nAchRs, at skeletal muscle cells. 11.- Zeghal K, Sahnoun Z, Guinot M, Richer C, Giudicelli JF. Character- As discussed, it is important to emphasize that the autonomic stimula- ization and mechanisms of the cardiovascular and haemodynanic tion as a single mechanism, is unable to explain all clinical findings in alterations induced by scorpion venoms in rat. Fundam Clin Pharma- both envenomed-human and biomodels. In this sense, these Venezuelan col. 2000;14:351-361. scorpions venoms might contain novel toxins, which have to be identified, isolated and whose biological activities have to be well established, to un- 12.- Borges, A., García, C.C., Lugo, E., Alfonzo, M.J., Jowers, M.J., Op derstand the molecular aspects related to the pathophysiological mecha- den Camp, H.J.M. Long-chain toxin diversity in Tityus zulianus and nisms associated with the acute inflammation process implicate in the Tityus discrepans venoms (Scorpiones, Buthidae): molecular, im- envenomation events trigger by these venoms involving human patients. munological and mass spectral analyses. Comp. Biochem. Physiol. 2006;142C, 240–252. Acknowledgments: The authors thank to the Fireman Department of San Antonio de los Altos, 13.- Gordon D, Martin-Eauclaire MF, Cestèle S, Kopeyan C, Carlier E, Miranda State for the specimens of Tityus discrepans and the community Khalifa RB, Pelhate M, Rochat H. Scorpion toxins affecting sodium councils of Santa Cruz de Mora and Tovar, Merida State, for helping in current inactivation bind to distinct homologous receptor sites on rat the collection of Tityus zulianus. Also to Dr. Lucimey De Lima and MSc. brain and insect sodium channels. J Biol Chem. 1996; 271:8034- Mary Urbina from the Laboratorio de Neuroquímica del Instituto Venezo- 8045. lano de Investigaciones Científicas (IVIC) for supporting in the plasma catecholamines determinations. This work was supported for a Grant # 14.- Amitai, Y. Clinical manifestations and management of scorpion en- CDCH PI09006088-2005 (ET) from Consejo de Desarrollo Científico y venomation. Public Health Rev. 1998; 26, 257–263. Humanístico (CDCH-UCV) and Subproyecto 2 (ET, AB, MA) del Proyec- 15.- Gueron M, Ilia R, Margulis G. poisons and the cardiovas- to Producción de antivenenos-Misión Ciencia (FONACIT # 2007000672) cular system Am J Emerg Med. 2000; 18:708-714. Review. Ministerio del Poder Popular para la Ciencia, Tecnología e Industrias In- termedias de la República Bolivariana de Venezuela. 16.- Rodriguez JM, Balsalobre M, Ponce JL, Rios A, Torregrosa NM, Te- bar J, Parilla P. Pheochromocytoma in MEN 2A syndrome. Study of References 54 patients. World J Surg. 2008; 32 :2520-2526.

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