JBPC  Vol.3 No.4 , November 2012
Tityus serrulatus venom and its toxins Ts1 and Ts5 increase cytosolic Ca2+ concentration in isolated vascular smooth muscle cells
Abstract: Voltage-gated Na+ channel (Nav channel) scorpion toxins are classified as α- and β-neurotoxins. Ts5 (α-neurotoxin) and Ts1 (β-neurotoxin) from Tityus serrulatus venom (TsV) interact with Nav channels, increasing Na+ influx and activating voltage-dependent Ca2+ channels. This study aimed to investigate the effect of TsV, Ts1 and Ts5 on the cytosolic Ca2+ concentration ([Ca2+]C) in rat aortic smooth muscle cells. Toxins were isolated by ion exchange chromatography (Ts1) followed by RP-HPLC (Ts5). The rat aortic smooth muscle cells were isolated in Hanks buffer pH 7.4 and loaded with 5 μmol/L of Fura-2AM (45 minutes at 37℃), in order to measure [Ca2+]C by fluorescence of Fura-2/AM (ratio 340/380 nm). The fluorescence was measured in one single cell (excitation: 340 and 380 nm; emission: 510 nm). TsV (100 and 500 mg/mL) and its toxins Ts1 and Ts5 (50 and 100 mg/mL each) led to a concentration-dependent increase in [Ca2+]C. Tetrodotoxin (1 mmol/L), a Nav channel blocker, and verapamil (1 mmol/L), a voltage-operated Ca2+ channel blocker, inhibited the increase in [Ca2+]C induced by TsV (500 mg/mL). In conclusion, TsV and its toxins induce a concentration-dependent increase in [Ca2+]C that probably occurs through interaction with Nav channels, thus inducing depolarization and consequent Ca2+ influx. This assumption is based on the fact that this effect is inhibited by tetrodotoxin and verapamil, showing a direct action of TsV toxins on aorta smooth muscle cells.
Cite this paper: dos A. Neto, M. , Vasconcelos, F. , M. Bendhack, L. and C. Arantes, E. (2012) Tityus serrulatus venom and its toxins Ts1 and Ts5 increase cytosolic Ca2+ concentration in isolated vascular smooth muscle cells. Journal of Biophysical Chemistry, 3, 287-294. doi: 10.4236/jbpc.2012.34035.

[1]   Brazilian Ministry of Health. Secretaria de Vigilancia em Saúde (MS/SUS). (2010) Situa??o epidemiológica das zoonoses de interesse para a saúde pública. Boletim Eletr?nico Epidemiológico, 2, 19.

[2]   Freire-Maia, L. (1995) Peripheral effects of Tityus serrulatus scorpion venom. Journal of Toxicology, Toxin reviews, 14, 423-435.

[3]   Ismail, M. (1995) The scorpion envenoming syndrome. Toxicon, 33, 825-858. doi:10.1016/0041-0101(95)00005-7

[4]   Cupo, P., Azevedo-Marques, M.M., Hering, S. (2003). Escorpionismo. In: Cardoso, J.L.C., Fran?a, F.O.S., Wen, F.H., Málaque, C.M.S., Haddad Jr, V., Eds., Animais Pe?onhentos do Brasil: Biologia, Clínica e Terapêutica dos Acidentes, Sarvier, S?o Paulo, 198-208.

[5]   Fukuhara, Y.D.M., Dellalibera-Joviliano, R., Cunha, F.Q.C., Reis, M.L. and Donadi, E.A. (2004) The kinin system in the envenomation caused by the Tityus serrulatus scorpion sting. Toxicology and Applied Pharmacology, 196, 390-395. doi:10.1016/j.taap.2003.12.026

[6]   Diaz, P., Chowell, G., Ceja, G., D’Auria, T.C., Lloyd, R.C. and Castillo-Chavez, C. (2005) Pediatric electrocardiograph abnormalities following Centruroides limpidus tecomanus scorpion envenomation. Toxicon, 45, 27-31. doi:10.1016/j.toxicon.2004.09.008

[7]   Vasconcelos, F., Lanchote, V.L., Bendhack, L.M., Giglio, R.J., Sampaio, S.V. and Arantes, E.C. (2005) Effects of voltage-gated Na+ channel toxins from Tityus serrulatus venom on rat arterial blood pressure and plasma catecholamines. Comparative Biochemistry and Physiology C, Comparative Pharmacology and Toxicology, 141, 85-92. doi:10.1016/j.cca.2005.05.012

[8]   Cusinato, D.A.C., Souza, A.M., Vasconcelos, F., Guimar?es, L.F.L., Leite, F.P., Gregório, Z.M.O., Giglio, J.R. and Arantes, E.C. (2010) Assessment of biochemical and hematological parameters in rats injected with Tityus serrulatus scorpion venom. Toxicon, 56, 1477-1486. doi:10.1016/j.toxicon.2010.09.003

[9]   Bahloul, M., Chaari, A., Dammak, H., Samet, M., Chtara, K., Chelly, H., Ben Hamida, C., Kallel, H. and Bouaziz, M. (2011) Pulmonary edema following scorpion envenomation: Mechanisms, clinical manifestations, diagnosis and treatment. International Journal of Cardiology. doi:10.1016/j.ijcard.2011.10.013

[10]   Olamendi-Portugal, T., García, B.I., López-González, I., Van Der Walt, J., Dyason, K., Ulens, C., Tytgat, J., Felix, R., Darszon, A. and Possani, L.D. (2002) Two new scorpion toxins that target voltage-gated Ca2+ and Na+ channels. Biochemical and Biophysical Research Communication, 299, 562-568. doi:10.1016/S0006-291X(02)02706-7

[11]   de la Vega, R.C.R. and Possani, L.D. (2005) Overview of scorpion toxins specific for Na+ channels and related peptides: Biodiversity, structure-function relationships and evolution. Toxicon, 46, 831-844. doi:10.1016/j.toxicon.2005.09.006

[12]   Catterall, W.A., Cestèle, S., Yarov-Yarovoy, V., Yu, F.H., Konoki, K. and Scheuer, T. (2007) Voltage-gated ion channels and gating modifier toxins. Toxicon, 49, 124-141. doi:10.1016/j.toxicon.2006.09.022

[13]   Bosmans, F. and Tytgat, J. (2007) Voltage-gated sodium channel modulation by scorpion ?-toxins. Toxicon, 49, 142-158.

[14]   Schiavon, E., Pedraza-Escalona, M., Gurrola, G.O., Olamendi-Portugal, T., Corzo, G., Wanke, E. and Possani, L.D. (2012) Negative-shift activation, current reduction and resurgent currents induced by ?-toxins from Centruroides scorpions in sodium channels. Toxicon, 59, 283-293. doi:10.1016/j.toxicon.2011.12.003

[15]   Cecchini, A.L., Vasconcelos, F., Amara, S.G., José Roberto Giglio, J.R. and Arantes, E.C. (2006) Effects of Tityus serrulatus scorpion venom and its toxin TsTX-V on neurotransmitter uptake in vitro. Toxicology and Applied Pharmacology, 217, 196-203. doi:10.1016/j.taap.2006.09.003

[16]   Gwee, M.C., Nirthanan, S., Khoo, H.E., Gopalakrishnakone. P., Kini, R.M. and Cheah, L.S. (2002) Autonomic effects of some scorpion venoms and toxins. Clinical and Experimental Pharmacology & Physiology, 29, 795-801. doi:10.1046/j.1440-1681.2002.03726.x

[17]   Arantes, E.C., Riccioppo Neto, F., Sampaio, S.V., Vieira, C.A. and Giglio, J.R. (1994) Isolation and characterization of TsTX-V, a new neurotoxin from Tityus serrulatus scorpion venom which delays the inactivation of Na+ channels. Biochimica et Biophysica Acta, 1199, 69-75. doi:10.1016/0304-4165(94)90098-1

[18]   Marangoni, S., Toyama, M.H., Arantes, E.C., Giglio, J.R., Da Silva, C.A., Carneiro, E.M., Gon?alves, A.A. and Oliveira, B. (1995) Amino acid sequence of TsTx-V, an ?-toxin from Tityus serrulatus scorpion venom, and its effect on K+ permeability of ?-cells from isolated rat is- lets of Langerhans. Biochimica et Biophysica Acta, 1243, 309-314. doi:10.1016/0304-4165(94)00142-K

[19]   Jonas, P., Vogel, W., Arantes, E.C. and Giglio, J.R. (1986) Toxin gamma of the scorpion Tityus serrulatus modifies both activation and inactivation of sodium permeability of nerve membrane. Pflügers Archiv, 407, 92-99. doi:10.1007/BF00580727

[20]   Pinheiro, C.B., Marangoni, S., Toyama, M.H. and Polikarpov, I. (2003) Structural analysis of Tityus serrulatus Ts1 neurotoxin at atomic resolution: insights into interactions with Na+ channels. Acta Crystallographica: Section D, Biological Crystallography, 59, 405-415. doi:10.1107/S090744490202111X

[21]   Cologna, C.T., Marcussi, S., Giglio, J.R., Soares, A.M. and Arantes, E.C. (2009) Tityus serrulatus scorpion venom and toxins: An overview. Protein Peptides Letters, 16, 920-932. doi:10.2174/092986609788923329

[22]   Arantes, E.C., Prado, W.A., Sampaio, S.V. and Giglio, J.R. (1989) A simplified procedure for the frationalization of Tityus serrulatus venom: Isolation and partial characterization of TsTX-IV, a new neurotoxin. Toxicon, 8, 907-916. doi:10.1016/0041-0101(89)90102-5

[23]   Zoccal, K.F., Bitencourt. C.S., Secatto, A., Sorgi, C.A., Bordon, K.C., Sampaio, S.V., Arantes, E.C. and Faccioli, L.H. (2011) Tityus serrulatus venom and toxins Ts1, Ts2 and Ts6 induce macrophage activation and production of immune mediators. Toxicon, 57, 1101-1118. doi:10.1016/j.toxicon.2011.04.017

[24]   Akaike, H., Shin, M.C., Kubo, C. and Akaike, N. (2009) Effects of scorpion toxin on excitatory and inhibitory presynaptic terminals. Toxicology, 264, 198-204. doi:10.1016/j.tox.2009.08.010

[25]   Soualmia, H., Eurin, J. and Djeridane, Y. (2009) Scorpion toxin of Androctonus australis garzonii induces neuropeptide Y release via bradykinin stimulation in rat atria and kidneys. Peptides, 30, 1553-1556. doi:10.1016/j.peptides.2009.04.022

[26]   Liu, T., Bai, Z.T., Pang, X.Y., Chai, Z.F., Jiang, F. and Ji, Y.H. (2007) Degranulation of mast cells and histamine release involved in rat pain-related behaviors and edema induced by scorpion Buthus martensi Karch venom. European Journal of Pharmacology, 57, 546-556.

[27]   Concei??o, I.M., Jurkiewicz, A., Fonseca, D.R., Opper- man, A.R., Freitas, T.A., Lebrun, I. and Garcez-do-Carmo, L. (2005) Selective release of ATP from sympathetic nerves of rat vas deferens by the toxin TsTX-I from Bra- zilian scorpion Tityus serrulatus. British Journal of Pharmacology, 144, 519-527. doi:10.1038/sj.bjp.0706062

[28]   Teixeira, C.E., de Oliveira, J.F., Baracat, J.S., Priviero, F.B.M., Okuyama, C.E., Rodrigues Netto, N. Jr., Fregonesi, A., Antunes, E. and De Nucci, G. (2004) Nitric oxide release from human corpus cavernosum induced by a purified scorpion toxin. Urology, 63, 184-189. doi:10.1016/S0090-4295(03)00785-4

[29]   Weisel-Eichler, A. and Libersat, F. (2004) Venom effects on monoaminergic systems. Journal of Comparative Physiology A, 190, 683-690. doi:10.1007/s00359-004-0526-3

[30]   Fernandes, V.M., Romano-Silva, M.A., Gomes, D.A., Prado, M.A., Santos, T.M. and Gomez, M.V. (2004) Dopamine release evoked by beta scorpion toxin, tityus gamma, in prefrontal cortical slices is mediated by intracellular calcium stores. Cell Molecular Neurobiology, 24, 757-767. doi:10.1007/s10571-004-6917-8

[31]   Teixeira Jr., A.L., Fontoura, B.F., Freire-Maia, L., Machado, C.R.S., Camargos, E.R.S. and Teixeira, M.M. (2001) Evi- dence for a direct action of Tityus serrulatus scorpion venom on the cardiac muscle. Toxicon, 39, 703-709. doi:10.1016/S0041-0101(00)00200-2

[32]   Sun, H.Y., Zhou, Z.N. and Ji, Y.H. (2005) The role of voltage-gated Na+ channels in excitation-contraction coupling of rat heart determined by BmK I, an alpha-like scorpion neurotoxin. Toxicology in Vitro, 19, 183-190. doi:10.1016/j.tiv.2004.07.005

[33]   Shinjoh, M., Nakaki, T., Otsuka, Y., Sasakawa, N. and Kato, R. (1991) Vascular smooth muscle contraction induced by Na+ channel activators, veratridine and batrachotoxin. European Journal of Pharmacology, 205, 199-202. doi:10.1016/0014-2999(91)90820-G

[34]   Neto, M.A., Lunardi, C.N., Rodrigues, G.J. and Bendhack, L.M. (2011) Vasodilatation induced by forskolin involves cyclic GMP production. Journal of Biophysical Chemistry, 2, 373-379. doi:10.4236/jbpc.2011.24042

[35]   Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248-254. doi:10.1016/0003-2697(76)90527-3

[36]   Scopes, R.K. (1974) Measurement of protein by spectrophotometry at 205 nm. Analytical Biochemistry, 59, 277-282. doi:10.1016/0003-2697(74)90034-7

[37]   Ouanes-Besbes, L., El Atrous, S., Nouira, S., Aubrey, N., Carayon, A., El Ayeb, M. and Abroug, F. (2005) Direct vs. mediated effects of scorpion venom: an experimental study of the effects of a second challenge with scorpion venom. Intensive Care Medicine, 31, 441-446. doi:10.1007/s00134-005-2555-y

[38]   Drumond Y.A., Couto, A.,S., Moraes-Santos, T., Almeida, A.P. and Freire-Maia, L. (1995) Effects of toxin Ts-gamma and tityustoxin purified from Tityus serrulatus scorpion venom on isolated rat atria. Comparative Biochemistry and Physiology. Part C, Comparative Pharmacology, Toxicology and Endocrinology, 111, 183-190.

[39]   Shannon, T.R., Ginsburg, K.S. and Bers, D.M. (2000) Potentiation of fractional sarcoplasmic reticulum calcium release by total and free intra-sarcoplasmic reticulum calcium concentration. Biophysical Journal, 78, 334-343. doi:10.1016/S0006-3495(00)76596-9

[40]   Grolleau, F., Stankiewicz, M., Kielbasiewicz, E., Martin-Eauclaire, M.-F., Lavialle, C., De Vente, J. and Lapied, B. (2006) Indirect activation of neuronal noncapacitative Ca2+ entry is the final step involved in the neurotoxic effect of Tityus serrulatus scorpion β-toxin. European Journal of Neuroscience, 23, 1465-1478. doi:10.1111/j.1460-9568.2006.04667.x

[41]   Wicher, D., Messutat, S., Lavialle, C. and Lapied, B. (2004) A new regulation of non-capacitative calcium entry in insect pacemaker neurosecretory neurons. Involvement of arachidonic acid, NO-guanylyl cyclase/cGMP, and cAMP. Journal of Biological Chemistry, 279, 50410-50419. doi:10.1074/jbc.M405800200

[42]   Moneer, Z. and Taylor, C.W. (2002) Reciprocal regulation of capacitative and non-capacitative Ca2+ entry in A7r5 vascular smooth muscle cells: Only the latter operates during receptor activation. The Biochemical Journal, 362, 13-21.

[43]   Cox, R.H., Zhou, Z. and Tulenko, T.N. (1998) Voltage- gated sodium channels in human aortic smooth muscle cells. Journal of Vascular Research, 35, 310-317.

[44]   Platoshyn, O., Remillard, C.V., Fantozzi, I., Sison, T. and Yuan, J.X.-J. (2005) Identification of functional voltage gated Na+ channels in cultured human pulmonary artery smooth muscle cells. Pflügers Archiv European Journal of Physiology, 451, 380-387. doi:10.1007/s00424-005-1478-3

[45]   Meguro, K., Iida, H., Takano, H., Morita, T., Sata, M., Nagai, R. and Nakajima, T. (2009) Function and role of voltage-gated sodium channel NaV1.7 expressed in aortic smooth muscle cells. American Journal of Physiology, Heart and Circulatory Physiology, 296, H211-219. doi:10.1152/ajpheart.00960.2008

[46]   Zhu, S. and Gao B. (2006) Molecular characterization of a possible progenitor sodium channel toxin from the Old World scorpion Mesobuthus martensii. FEBS Letters, 580, 5979-5987. doi:10.1016/j.febslet.2006.09.071