Os escorpiões são um dos grupos de animais mais antigos da Terra. Eles são artrópodes e pertecem a classe Arachinida e Ordem Scorpionida. A família Buthidae compreende as espécies responsáveis pelos acidentes graves em humanos, incluindo a espécie Tityus serrulatus, o maior responsável por esses acidentes no Brasil. A peçonha do T. serrulatus contém diversas neurotoxinas que agem especificamente em canais para sódio, potássio e cálcio da membrana plasmática de células excitáveis, causando massiva liberação de neurotransmissores.As toxinas escorpiônicas podem ser usadas como ferramentas nos estudos de estrutura e função desses canais iônicos sensíveis a voltagem e também no estudo de liberação e captação de neurotransmissores. As toxinas escôrpionicas específicas para canais para sódio sensíveis a voltagem são as principais responsáveis pelos efeitos do envenenamento por estes artrópodes e podem ser classificadas em duas classes: toxinas e . As -toxinas retardam a inativação desses canais induzindo assim um prolongamento na fase de repolarização do potencial de ação. As - toxinas alteram a dependência de voltagem de ativação dos canais para sódio para potenciais mais negativos provocando potenciais de ação espontâneos e repetitivos. As toxinas específicas para canais para potássio (KTx) são geralmente peptídeos pequenos e de caráter básico, formados por 23-43 aminoácidos estabilizados por 3-4 pontes dissulfeto. As KTx são classificadas em 4 subfamílias:, , , . Neste trabalho, uma nova neurotoxina do escorpião T. serrulatus foi isolada e caracterizada bioquímica e funcionalmente. A toxina foi testada em ampla variedade de canais incluindo 5 subtipos de canais para sódio (Nav1.4; Nav1.5; Nav1.6; Nav1.8 e DmNav1) e 12 diferentes tipos de canais para potássio (Kv1.1 a Kv1.6; Kv2.1; Kv3.1; Kv4.2; Kv4.3; Shaker IR e hERG). A peçonha bruta solúvel foi fracionada em cromatografia de troca iônica em coluna CM-Celulose-52 (2,5 cm x 63 cm), previamente equilibrada e eluída com tampão NH4HCO3 (pH 7,8). Essa primeira etapa cromatográfica permitiu a separação de 13 frações nomeadas de I XIII. A fração X foi submetida à cromatografia de fase reversa em sistema de cromatografia líquida de alta eficiência em que a toxina pura Ts15 pode ser obtida. Seu sequenciamento amino-terminal demonstrou que esse peptídeo possui 36 resíduos de aminoácidos estabilizados por 3 pontes dissulfeto. A massa molecular obtida por espectrometria de massa foi de 3956 e o pI predito pelo programa ProtParam foi de 8,86, no entanto, o pI determinado por focalização isolelétrica foi maior que 9,3. Os experimentos de eletrofisiologia utilizando as técnicas patch clamp e two microelectrode voltage clamp mostraram que a toxina Ts15 bloqueia preferencialmente os subtipos de canais para potássio Kv1.2 e Kv1.3 com IC50 de 196 ± 25 nM e 508± 67 nM respectivamente. Os ensaios de captação de neurotransmissores em sinaptosomas de cérebro de rato foram realizados adicionando 3H-GABA e 3H-Glu na presença e ausência de diferentes concentrações da toxina Ts15. Não foram observados efeitos nos canais para sódio em todas as concentrações testadas assim como na captação do GABA. Porém, foi observado aumento significante na captação do glutamato em todas as concentrações testadas, provavelmente como resultado de efeito secundário da ação da Ts15 em canais para potássio sensível a voltagem. Em conclusão, a Ts15 pode ser considerada um autêntico novo tipo de toxina escorpiônica, com afinidade para canais para potássio Kv1.2 e Kv1.3 e capaz de aumentar a captação de glutamato. Essa toxina é o único membro da nova subfamília -Ktx21 e portanto nomeada -Ktx21.1 / Scorpions are one of the most ancient groups of animals on earth. They are arthropods and belong to the class Arachinida and Order Scorpionida. The Buthidae family comprises the species that are really dangerous for human, including Tityus serrulatus that is responsible for most severe accidents in Brazil. T. serrulatus venom contains several neurotoxins that specifically act on sodium, potassium or calcium channels in excitable membranes, causing a massive release of neurotransmitters and leading to the stimulation of the autonomic nervous system. Since ion channels play important roles in many physiological processes, scorpion toxins have been used as tools for studies of the neurophysiological mechanisms involving voltage-gated ion channels and neurotransmitter release/uptake. Voltage-gated Na+ channel (Nav channel) toxins are mainly responsible of the harmful effects of scorpion venom and can be classified into two classes: and -neurotoxins. The -toxins retard Nav channel inactivation and induce a prolongation of the repolarization phase of the action potential. The -toxins shift the voltage dependence of Nav channel activation to more negative potentials that result in an increased tendency of the cell to fire spontaneously and repetitively. Voltage-gated potassium channel toxins (KTxs) are basic short chain peptides comprising 23-43 amino acid residues that can be cross-linked by 3 or 4 disulfide bridges. KTxs are classified into four large families: , , and . These peptides display varying selectivity and affinity for different Kv channel subtypes. In this work, a novel toxin from the T. serrulatus venom was isolated, biochemistry and pharmacologically characterized using a wide electrophysiological screening on 5 different subtypes of Nav channels (Nav1.4; Nav1.5; Nav1.6; Nav1.8 and DmNav1) and 12 different subtypes of Kv channels (Kv1.1 - Kv1.6; Kv2.1; Kv3.1; Kv4.2; Kv4.3; Shaker IR and hERG). The crude soluble T. serrulatus venom was fractionated by ion exchange chromatography on a CM-cellulose-52 column (2.5 cm x 63.0 cm), which was equilibrated and eluted with NH4HCO3 buffer (pH 7.8). This chromatography allowed the separation of 13 fractions which were named I to XIII. Fraction X was submitted to a reverse-phase C18 (0.46 cm x 25 cm) high performance liquid chromatography (RP-HPLC) and the pure toxin, Ts15, could be obtained. The amino acid sequence of this novel peptide showed that it contains 36 amino acids and is cross-linked by 3 disulfide bridges. The molecular mass of Ts15 (3956) was obtained by electrospray (ESI) triple-quadrupole mass spectrometry and its pI value (8,86) was predicted by ProtParam program. However, the pI determined by isoeletric focusing was greater than 9,3. Electrophysiological experiments using patch clamp and the two electrode voltage clamp technique, showed that Ts15 preferentially blocks Kv1.2 and Kv1.3 channels with IC50 value of 196 ± 25 and 508 ± 67 nM, respectively. Uptake assays were performed by adding 3H-GABA and 3H-Glu, in the absence (controls) or presence of different concentrations of Ts15, on isolated rat brain synaptosomes. No effect on Nav channels was observed, in all tested concentrations, as well as for GABA uptake. However, Ts15 induced a significant increase of the glutamate uptake, probably as a secondary effect of its action on Kv channels. In conclusion, Ts15 can be considered a bonafide novel type of scorpion toxin that presents high affinity by Kv1.2 and Kv1.3 channels and was able to increase the glutamate uptake. It is the unique member of the new -Ktx21 subfamily and therefore was named -Ktx21.1

De uma biblioteca de cDNA da glândula de veneno da cobra coral brasileira Micrurus corallinus foi isolada uma seqüência denominada NXH8. Esta seqüência de cDNA apresenta similaridade estrutural com a família de toxinas de serpentes em 'três dígitos' ricas em pontes dissulfeto. A subclasse melhor conhecida nesta família, são as alfa neurotoxinas. Uma outra seqüência distinta, denominada NXH1 e suas isoformas NXH3 e NXH7, foram isoladas anteriormente. Pertencem à mesma família de toxinas e estão presentes na mesma biblioteca. Alguns resultados da caracterização de NXH1, são utilizados neste estudo, em comparação com NXH8. Algumas características estruturais tornam a seqüência NXH8 diferente da classe usual das alfa neurotoxinas, vindo a constituir possivelmente uma nova subclasse da família. A proteína NXH8 foi expressa em diversos vetores de expressão em Escherichia coli. A proteína recombinante, expressa pelo vetor pRSET C - NXH8 foi utilizada para imunizar camundongos. O soro contra NXH8, assim como o soro anti - elapídico do Instituto Butantan, reconhece a toxina recombinante em ELISA e Western blot. O soro anti - NXH8 detecta apenas uma banda do veneno de M. corallinus em Western blot, mas apresenta reatividade cruzada com componentes do veneno de alguns elapídeos neotropicais e do velho mundo. Em contraste, dados anteriores demonstraram que o soro anti - NXH1 é específico para um componente único do veneno de M. corallinus. O veneno de M. corallinus tem alfa neurotoxinas que bloqueiam o receptor pós - sináptico nicotínico de acetilcolina nas membranas do músculo esquelético de ratos. O soro anti - NXH8 é capaz de impedir a ligação de componentes do veneno bruto a esses receptores. Já o soro contra NXH1 não apresenta a mesma capacidade inibitória. Isto indica que NXH8 tem afinidade pelo receptor nicotínico muscular de acetilcolina, ou que NXH8 compartilha de um epítopo neutralizante presente também nas alfa neurotoxinas do veneno da cobra coral M. corallinus. / A cDNA sequence encoding a putative new toxin, NXH8, was isolated from the cDNA library constructed from the venom gland of the Brazilian coral snake, Micrurus corallinus. This sequence shows a structural similarity with the snake toxin family known as 'three-fingered' toxins, a family of toxins with approximately 60 to 70 amino acids and usually 4 to 5 disulfide bonds. Irrespective of whether these proteins are functionally different, their amino acid sequences can be readily aligned, using 8 half-cystines as conserved elements, suggesting the presence of common structural features. The best known subclass of three-finger-type toxins are the curaremimetic toxins, also called alpha-neurotoxins, found in most venoms from Elapid and Sea snakes. Another toxin with a distinct sequence, known as NXH1 and its isoforms NXH3 and NXH7 had been previously isolated. They belong to the same family of toxins and were characterized from the same cDNA library. In the present study, a comparative biochemical, pharmacological and structural analyses of NXH1 and NXH8 were described. Few structural characteristics of NXH8 seem to indicate that it differs from the usual class of alpha – neurotoxins, belonging, possibly, to a new subclass of 'three-finger' toxins. The NXH8 protein was expressed in various E. coli expression vectors and the resulted recombinant toxin from pRSETC-NXH8 plasmid was used as a "toxoid" for mice immunization. The anti - NXH8 sera, as well as the anti – elapid sera from the Butantan Institute, recognized the recombinant toxin by both ELISA and Western blot assays. In contrast to the claim that anti - NXH1 sera is specific to one component of M. corallinus’s venom, the anti – NXH8 sera show cross reactivity to venom of some Neotropical and Old World elapids. The M. corallinus's venom contains alpha – toxins, which inhibit post-synaptic nicotinic acetylcholine receptor of neonatal rat skeletal muscle membrane. The anti - NXH8 serum was capable of blocking the binding of the components of the crude venom to these receptors. In contrast, the anti – NXH1 serum did not show this inhibitory effect. This indicates that either NXH8 presents affinity for muscular nicotinic acetylcholine receptor or it shares a neutralizing epitope also present in M. corallinus’s alpha – neurotoxins.

Made available in DSpace on 2014-08-20T14:37:51Z (GMT). No. of bitstreams: 1 dissertacao_luciana_aquini_fernandes_gil.pdf: 1257217 bytes, checksum: 0c4055569b5af0eb817b075544551d2f (MD5) Previous issue date: 2012-08-08 / Bovine Botulism is a lethal intoxication caused by the ingestion of the neurotoxins produced by Clostridium botulinum types C and D that inhibit the release of acetylcholine at the neuromuscular junction leading to death by flaccid paralysis. It produces important economic losses, being a major cause of casualties in cattle in several regions of Brazil. The control of the disease depends on the presence of neutralizing antibodies against botulinum neurotoxins (BONTs) in immunized cattle. Immunization is obtained inoculating toxoids produced from cultures of selected strains of C. botulinum types C and D, whose industrial production has limitations concerning efficiency and productivity. An alternative to the use of these toxoids is the production of recombinant antigens with high levels of purity and antigenicity. The C-terminal fraction of the heavy chain of botulinum neurotoxins has been the main target in the development of recombinant vaccines with promising results. In this work, two recombinant bivalent chimeras for the control of bovine botulism consisting of the neuronal receptor binding domains (NRBDs) of botulinum C and D toxins were efficiently produced in Escherichia coli. They were characterized and evaluated in mice, with promising results. Both the recombinant chimeras rLTB-C-D and rC-D were produced by cloning and expressing a synthetic gene encoding the C-terminal portion of both BONTs. The former also included the preferred codons of the E. coli heat labile enterotoxin B subunit (LTB), a potent humoral immune adjuvant. The levels of expression of the recombinant antigens were satisfactory, yielding approximately 100 mg of each recombinant antigen per liter of culture. An ELISA performed to assess the antigenicity of the molecules showed that both were recognized by sera of immunized mice suggesting the preservation of epitopes with the properties of native BONTs. Both chimeras induced high levels of neutralizing antibodies without undesirable effects. The level of neutralizing antibodies of the groups inoculated with equimolar concentrations of rLTB-C-D and rC-D containing Aluminum Hydroxide as adjuvant were similar, confirming the adjuvant properties of LTB. These results demonstrated that the recombinant chimeras were immunogenic. Sera from mice inoculated with commercial vaccines were also analyzed by ELISA using as antigens rC and rD, corroborating the neutralization. / O botulismo bovino é uma intoxicação letal causada pela ingestão da neurotoxina produzida pelo Clostridium botulinum principalmente dos tipos C e D que atua inibindo a liberação de acetilcolina na junção neuromuscular levando à morte por paralisia flácida, com grande importância econômica e sanitária, sendo uma das principais causas de morte em bovinos adultos no Brasil. O controle imunológico do botulismo bovino depende da presença de anticorpos neutralizantes contra as neurotoxinas botulínicas (NBOTs) no momento da ingestão da toxina pré-formada, por meio de imunização dos animais. Atualmente, a imunização é realizada com toxóides obtidos da detoxificação do extrato de cultivos de cepas selecionadas de C. botulinum dos tipos C e D que apresentam limitações quanto à eficiência e produção. Uma alternativa ao uso dos toxóides clássicos é a produção de vacinas recombinantes usando antígenos específicos de alta pureza e imunogenicidade. A fração C-terminal da cadeia pesada da neurotoxina botulínica tem sido o alvo principal no desenvolvimento de alternativas recombinantes a serem utilizadas como vacinas. Neste trabalho, duas quimeras recombinantes bivalentes compostas pelos domínios de ligação ao receptor neuronal (DLRNs) foram produzidas em Escherichia coli, caracterizadas e avaliadas em camundongos. As quimeras recombinantes rLTB-C-D e rC-D foram produzidas a partir da clonagem e expressão de um gene sintético que codifica a porção C-terminal das NBOTs construído com os códons preferenciais de E. coli e a subunidade B da enterotoxina termolábil de E. coli (LTB), um potente adjuvante da resposta imune humoral. O nível de expressão dos antígenos foi de aproximadamente 100mg de cada antígeno recombinante por litro de cultura. Um ELISA realizado para avaliar a antigenicidade das moléculas mostrou que ambas foram reconhecidas pelos soros padrões, sugerindo conservação de epitopos semelhantes aos dos DLRNs nativos. Ambas as quimeras foram inócuas para os camundongos, os quais não apresentaram lesões no local da inoculação bem como alteração de comportamento. Nos soros dos camundongos inoculados com as quimeras recombinantes foi possível detectar níveis de anticorpos neutralizantes. O grupo inoculado com a rLTB-C-D apresentou nível de anticorpos neutralizantes semelhante ao do grupo rC-D + hidróxido de alumínio confirmando o potencial adjuvante da LTB. As quantidades de antígenos utilizados foram equimolares. Esses resultados demonstram que as quimeras recombinantes foram imunogênicas. Os soros dos camundongos inoculados com as diferentes vacinas também foram analisados por ELISA indireto utilizando rC e rD como antígenos. Os dados obtidos neste ELISA corroboram os dados da soroneutralização.

Les venins sont des armes sophistiquées, utilisées par les organismes venimeux pour se défendre des prédateurs, ainsi que pour paralyser et tuer leurs proies. Mais dans la nature, le bien n’est jamais très loin du mal, les toxines venimeuses pouvant se révéler être des agents thérapeutiques efficaces. Les peptides de venins de fourmis ont donc été étudiés dans cette thèse afin de déterminer le potentiel de ces toxines pour la découverte de molécules thérapeutiques innovantes. A l’instar des autres venins d’insectes, les venins de fourmis restent peu étudiés, principalement en raison de la petite taille de ces insectes et des quantités limitées de venins disponibles. Cependant, les fourmis offrent l’avantage d’être des insectes sociaux très abondants dans tous les milieux terrestres. En collectant les venins de plusieurs individus, il est donc possible d’obtenir des quantités suffisantes de venin pour les analyses biochimiques et pharmacologiques.Afin d’assurer la reproductibilité des analyses, une identification taxonomique correcte est nécessaire. Dans cette optique, un outil de chimiotaxonomie a été développé durant cette thèse (permettant ainsi de regrouper les venins provenant de plusieurs colonies afin de compenser les faibles quantités de matériel biologique par individu ou par colonie).Ensuite, nous nous sommes intéressés aux facteurs écologiques impliqués dans la diversification des venins de fourmis. Pour cela, la toxicité et la composition des venins de fourmis ont été analysés en relation avec le polyéthisme, la spécialisation alimentaire et la spécialisation défensive.La diversité écologique des fourmis a amplement contribuée à la diversification des venins. En étudiant les venins de 82 espèces de fourmis, nous avons révélé la grande diversité structurale des toxines. Bien que la majorité des peptidomes sont composés par de petits peptides linéaires, des peptides structurés par des ponts disulfure ont été révélés dans de nombreux venins et constituent de nouvelles familles structurales de toxines.La purification de certains de ces peptides à ponts disulfure a permis leur caractérisation biochimique et l’évaluation de leur rôle biologique. Ainsi nous avons décrit un groupe de peptides neurotoxiques, baptisés les formicitoxines qui sont capables de bloquer les canaux calcium humains de type L. La commutatoxine est, quant à elle, un peptide avec un pont disulfure qui semble activer les récepteurs humains TRPV1 et TRPV3 et laisse supposer une implication dans l’induction de la douleur chez les mammifères.La grande diversité des peptides mise en évidence dans les venins, associée à la grande diversité écologique et taxonomique des fourmis, suggère que les venins de fourmis constituent un nouveau champ d’exploration prometteur pour la recherche de molécules thérapeutiques et insecticides. Les venins de fourmis s’ajoutent à la chimiothèque conséquente déjà représentée par les venins des autres animaux venimeux. / Venoms are sophisticated weapons employed by venomous organisms to ward off predators, as well as to subdue and kill prey. However, in nature, good is never far from bad and venom toxins may prove to be efficient therapeutic agents. Ant venom peptides were investigated in the course of this thesis to evaluate their potential in the discovery of novel drugs. Like other insect venoms, ant venoms remain understudied, mainly due to the small size of individual ants and, so, the limited a mount of venom available. The ecological diversity of ants has largely contributed to venom diversification. By studying the venom peptidomes from 82 ant species, we have revealed the great structural diversity of the toxins. Although the majority of the peptidomes are comprised of small and linear peptides, peptides structured by disulfide bonds were also brought to light in numerous venoms and constitute novel structural classes of toxins. The purification of some of these disulfided peptides permitted their biochemical characterization and the assessment oft heir biological functions. The enormous peptide diversity revealed among venoms combined with the great ecological and taxonomical diversity of ants suggests that ant venoms constitute a promising new source in the search for both novel drugs and insecticides. Ant venom augments the vast bioactive molecules library represented by venoms from other venomous animals.

[pt] O destino e os efeitos de produtos farmacêuticos recentemente se tornou uma questão de interesse ambiental embora pouco seja conhecido sobre sua entrada no ambiente. As drogas colinérgicas e colinesterásicas apresentam-se como um alvo importante da pesquisa ambiental relacionado a sua entrada no ambiente e suas interações físico-químicas e biológicas. Os ensaios com uso de à sondas protéicas são empregados para identificar a formação de complexos na presença de grupos específicos ou indicam a concentração de ligantes presente no ambiente onde se encontra a proteína. O emprego da enzima acetilcolinesterase (AChE) em biosensores para deterninação de inibidores é frequente porém poucos trabalhos exploram a fluorescência intrínseca das enzimas no desenvolvimento de metodologias para detectar espécies químicas ligantes. O objetivo deste trabalho foi propor novas sondas fluorescentes e formas de bioreconhecimento para a determinação de neurotoxinas em diferentes amostras. A proposta foi a de identificar sondas eficientes, de fácil obtenção e baixo custo para o desenvolvimento de métodos simples, sensíveis e seletivos. Para isso a detecção foi baseada na variação da fluorescência intrínseca das proteínas(por sua interação específica com os analitos) e na conjugação de fluoróforos extrínsecos específicos. Pode-se comprovar experimentalmente que a fração protéica obtida pode ser utilizada como sonda fluorescente na determinação dos analitos apresentados neste estudo. Nas determinações dos cinco analitos, os valores de LD e de LQ foram satisfatórios para a aplicação do método em amostras ambientais (solo) e clínicas (urina).As determinações de Gal apresentou desempenho satisfatório para atender diferentes tipos de amostras (LD = 1,3 x 10-9 mol L-1e LQ = 2,1 x 10-8 mol L-1).Os valores de LD e LQ para atropina (9,7 x 10-10 mol L-1 e 2,4 x 10-8 mol L-1, respectivamente) mostram-se adequados visto que outros sensores, como os eletroquimioluminescentes, apresentaram um valor de LQ de 1 × 10–7 mol L-1em amostras de urina. O método para os agrotóxicos metomil (LD = 9,5 x 10-10 mol L-1 e LQ = 2,2 x 10-8mol L-1); metamidofós (LD=1,5 x 10-9 mol L-1 e LQ=3,8 x 10-8 mol L-1) e metil paration (LD= 9,7 x 10-10 mol L-1 e LQ= 1,8 x 10-7 mol L-1) apresentarambom desempenho comparando-se à outros métodos descritos na literatura. A aplicação do método proposto em amostras de solo e urina (sem pré-tratamento) obtiveram recuperações a partir de 88 porcento (agrotóxicos em solo) e dos cinco analitos em urina. O método por fluorescência da sonda protéica não obteve diferença significativa do método analítico dos analitos por HPLC. A adaptação do método proposto à um sistema de biosensor com uso de uma sonda extrínseca (Tioflavina T) foi avaliado positivamente permitindo a imobilização da fração protéica em microplaca e o uso da Tioflavina T como sonda fluorescente da ligação AChE-inibidores. / [en] The fate and effects of pharmaceutical products has recently become a matter of environmental concern although little is known about its entry into the environment. Therefore, the cholinergic and cholinesterasicpresent a major target of environmental research related to his entry into the environment and their physico-chemical and biological interactions. Tests using the protein compounds as probes are employed where the optical properties of the complex protein-signal in the presence of analyte or specific groups indicating the concentration of ligand present in the environment. The use of acetylcholinesterase (AChE) activity in biosensors for inhibitors determination is common but few studies explore the intrinsic fluorescence of enzymes in the development of methods to detect chemical species ligands. The objective of this study was to propose new forms of fluorescent probes and bioaffinity for the determination of neurotoxins in different samples. The proposal was to identify probes efficient, easily accessible and low cost for the development of simple, sensitive and selective. For this the detection was based on the variation of the intrinsic fluorescence of proteins (by its specific interaction with the analytes) and the specific combination of extrinsic fluorophores. For the results presented in this plan can be affirmed that the protein fraction obtained can be used as a fluorescent probe in the determination of analytes of this study.Based on the five determinations of analyte, the values of LD and LQ are satisfactory for applying the method in environmental samples (ground) and clinical (urine). Measurements of Gal showed satisfactory performance to suit different types of samples (LD = 1.3 x 10-9mol L-1eLQ = 2.1 x 10-8mol L-1) values and LD LQ to atropine (9.7 x 10-10 mol L -1 and 2.4 x 10-8 mol L-1, respectively) appear to be suitable as other sensors, such as eletroquimioluminescentes showed a value of 1 LQ × 10-7 mol L-1 in urine samples. The method for pesticide methomyl (LD = 9.5 x 10-10 mol L-1 and LQ = 2.2 x 10-8mol L-1), methamidophos (LD = 1.5 x 10-9 mol L-1 and LQ = 3.8 x 10-8 mol L-1) and methyl parathion (LD = 9.7 x 10-10 mol L-1 and LQ = 1.8 x 10-7 mol L-1) comparing presents betterperformance to the other methods described in literature. The method proposed in soil samples and urine (no pretreatment) had recovered from 88% (ground pesticides) and five of analytes in urine. The t test comparison of means, applied to sets of determinations by fluorescence probe method and HPLC indicated the statistical equality in both recoveries. The adaptation of the proposed method a system of the biosensor with the use of an extrinsic probe (thioflavin T) has positively evaluated allowing immobilization of the protein in microplates and the use of thioflavin T as a fluorescent probe-connecting AChE inhibitors.

Alexandrium tamarense is a harmful algal species that can produce saxitoxins, a suite of powerful neurotoxins that bioaccumulate up the food chain and can have severe economic and health impacts. With harmful algal blooms increasing temporally and spatially, it is important for us to understand the relationship between harmful algal blooms and nutrients, particularly nitrogen from anthropogenic sources. To this end, the stable nitrogen isotopic composition (δ¹⁵N) of medium nitrate, algal cells and toxin in both nitrogen-replete and nitrogen-limited batch cultures of A. tamarense were measured in order to assess the potential for using the δ¹⁵N of the toxin as a tracer of the nitrogen source used for growth. A. tamarense cells grown under nitrate-replete conditions were depleted by 1.5‰ relative to the growth medium, and saxitoxin was depleted by 1.5‰ relative to the whole cells. Under nitrate-limiting conditions, the isotopic difference between cells and saxitoxin changed as nitrate in the growth medium was depleted, indicating uncoupling of toxin synthesis and cell growth rates under changing external nutrient conditions. Determination of the absolute magnitude of the isotopic differences between the medium nitrate and either the cells or the saxitoxin was confounded by 1) using two different nitrate sources – one nitrate source was used to grow the inoculum and a different nitrate source was used for the experimental medium - with different ‰ values and 2) the presence of an unidentified, isotopically-light, nitrogen blank in the low-nitrate medium samples. I conclude that STX nitrogen isotope values have the potential to be used as nitrogen source indicators. However, overall fractionation between whole cells and STX is unknown due to the uncoupling between cell growth and STX synthesis observed during my nitrogen-limited experiment. Based on previous research on cell growth and toxin production dynamics under different nutrient regimes, it is also reasonable to assume that the observed results here may differ if a different nitrogen source was utilized by the cells for STX production. Further research could include isotope analysis of cultures grown on different nitrogen sources, such as ammonium and urea; isotopic analysis of additional compounds, such as amino acids; or use of additional stable isotopes, such as C or O. / text

A peçonha das anêmonas do mar é uma fonte de compostos bioativos, incluindo toxinas peptídicas que são ferramentas para o estudo da estrutura e função dos canais voltagem dependentes de K+ (KV). Neste trabalho, quatro neurotoxinas foram purificadas da peçonha das anêmonas do mar Actinia bermudenesis e Bunodosoma caissarum. AbeTx1 e BcsTx4 possuem um motivo estrutural semelhante à das \"kappa-toxinas\" e análises funcionais e estruturais permitiram concluir que são os primeiros membros de um novo (tipo 5) de neurotoxinas de anêmonas do mar que atuam em canais KV. Por sua vez, a similaridade estrutural das toxinas BcsTx 1 e BcsTx2 nos permitiu inferir que estas são membros do já descrito tipo 1 (subtipo 1b) de neurotoxinas de anêmona que também atuam em canais KV. A caracterização funcional foi realizada utilizando-se diferentes subtipos de canais KV, expressos em ovócitos de Xenopus laevis e as medidas eletrofisiológicas foram feitas empregando-se a técnica de \"voltage-clamp\" com dois microelétrodos. AbeTx1, BcsTx1 e BcsTx2 (3 μM) apresentaram uma seletividade de atividade para os subtipos de KV1.1-KV1.3, KV1.6 e Shaker IR, ao passo que a BcsTx4 (3 μM) é somente capaz de bloquear a corrente dos subtipos de KV1.1, KV1.2 e KV1.6. Os mecanismos de ação envolvidos na seletividade da atividade e na potência com que estas se ligam aos seus alvos biológicos foram discutidos com base nos resultados obtidos e análises fisiológicas permitiram propor que estas toxinas atuam como \"armas\" para defesa contra predadores e/ou para captura de presas / The sea anemones venom is a rich source of bioactive compounds, including peptide toxins which are tools for studying the structure and function of voltage-dependent channels K+ (KV). In this work, four neurotoxins were purified from the venom of the sea anemones Actinia bermudenesis and Bunodosoma caissarum. AbeTx1 and BcsTx4 have a structural motif similar to that of kappa-toxins and functional and structural analysis showed that they are the first members of a new type (type 5) of sea anemone neurotoxins acting on KV channels. Moreover, the structural analysis of BcsTx1 and BcsTx2 toxins allowed us to conclude that they are members of the previously described type 1 (subtype 1b) of sea anemone neurotoxins. Functional characterization was performed by means of a wide electrophysiological screening on different KV channels using oocytes of Xenopus laevis and electrophysiological measurements were performed employing the voltage-clamp technique. AbeTx1, BcsTx1 and BcsTx2 (μM) showed a selective activity for KV1.1-KV1.3, KV1.6 and Shaker IR, while BcsTx4 (3 μM) only blocks KV1.1, KV1.2 and KV1.6. The mechanisms involved in potency and selectivity were discussed based on the results obtained and physiological analyses have provided new insights on the role of these toxins in the physiology of the sea anemones

Toxinas que atuam em canais para Na+ operados por voltagem são as principais responsáveis pelos efeitos tóxicos do envenenamento escorpiônico e podem ser divididas em duas classes: a- e b-neurotoxinas. TsTX-V e TsTX-I da peçonha de Tityus serrulatus (TsV) são, respectivamente, exemplos destas toxinas. Neste trabalho, foram avaliados os efeitos da TsV e destas toxinas sobre a pressão arterial média (PAM) e liberação de catecolaminas em ratos conscientes e não imobilizados, previamente cateterizados, bem como a captação de GABA, dopamina (DA) e glutamato (Glu) em sinaptosomas isolados de cérebro de ratos e a concentração citoplasmática de Ca+2 ([Ca+2 ]C) em células de músculo liso vascular de aorta de ratos. As toxinas foram isoladas por cromatografia de troca iônica (TsTX-I) seguida por CLAE de fase reversa (TsTX-V). As toxinas (15 e 30 g/kg) e TsV (50 e 100 g/kg) foram injetadas intravenosamente. A PAM foi monitorada continuamente através do cateter femoral. Os níveis plasmáticos de adrenalina (ADR) e noradrenalina (NA) foram determinados por CLAE de fase reversa com detector eletroquímico, em 10 min antes e 2,5, 30 e 90 min após os tratamentos. Efeitos pressores máximos foram observados em 2,5?3,5 min. TsV induziu um intenso aumento de longa duração na PAM, bem como a TsTX-I. A TsTX-V mostrou efeitos pressores menores. TsV mostrou os maiores efeitos sobre a liberação de catecolaminas, seguido pela TsTX-I e TsTX-V com um efeito máximo em 2,5 min, seguido por uma gradual redução, permanecendo, todavia, maior que os controles. Embora ambas as classes de toxinas atuem em canais para Na+, TsTX-I mostrou efeitos mais significantes e intensos sobre a liberação de catecolaminas e pressão arterial que a TsTX-V. Parece que a toxicidade da TsTX-V não está somente relacionada à sua capacidade de liberar catecolaminas, indicando que outros neutrotransmissores podem estar envolvidos em sua toxicidade. Nem a TsV ou suas toxinas foram capazes de afetar a captação de 3H-Glu. TsTXI inibiu somente a captação de 3H-DA (IC50 = 28,41 nM). Por outro lado, TsV (0,43ng/mL) inibiu a captação de 3H-GABA e 3H-DA (~50%). TsTX-V mostrou IC50 = 9,37 nM e 22,2 nM para a captação de 3H-GABA e 3HDA, respectivamente. Esses efeitos foram abolidos pelo pré-tratamento com TTX, indicando o envolvimento de canais para Na+ neste processo. Na ausência de Ca+2 e em baixas concentrações de toxinas, a redução não é tão singnificante como na presença de Ca+2. TsTX-V não reduziu a captação de 3H-GABA em células COS-7 expressando os transportadores de GABA, GAT-1 e GAT-3, sugerindo que esta toxina reduz indiretamente o transporte. A redução da captação de 3H-GABA pelos sinaptosomas pode ser devido a rápida e intensa despolarização celular, como revelado por microscopia confocal em células de glioma C6. Assim, TsTX-V causou redução da captação de 3H-GABA e 3H-DA de uma maneira independente de Ca+2, não afetando diretamente os transportadores de GABA, mas em consequencia da despolarização, envolvendo canais para Na+ operados por voltagem. TsV e suas toxinas foram capazes de aumentar a ([Ca2+ ]C , provavelmente por interargir com canais para Na+. Quando comparado aos efeitos despolarizantes do KCl 60 mM (100 %), TsV (100 e 500 g/mL) exibiu um aumento de 49,60 ± 2,58 % e 103,66 ± 5,17 %, respectivamente, enquanto que a TsTX-I e TsTX-V (50 e 100 g/mL de cada) exibiu 43,92 ± 3,06 % e 121,8 ± 8,9 %; 52,56 ± 8,33 % e 79,5 ± 6,1 % de aumento, respectivamente. TsTX-I (100 g/mL) mostrou-se mais potente nesta preparação, visto que uma dose de 100 g/mL causou efeito muito mais intenso do que a TsTX-V na mesma concentração. É possível que as diferenças observadas sobre os efeitos induzidos pela TsTX-I e TsTX-V sejam conseqüência de alterações estruturais entre canais para Na+ presentes em vários tipos de tecidos e inervações. / Voltage-gated Na+ channel toxins are mainly responsible for the toxic effects of scorpion envenoming and can be classified into two classes: a- and b-neurotoxins. TsTX-V and TsTX-I from Tityus serrulatus venom (TsV) are, respectively, examples of these toxins. In this work, were evaluate the effects of TsV and its toxins on mean arterial pressure (MAP) and catecholamines release in conscious unrestrained rats previously catheterized, as well as GABA, dopamine (DA) and glutamate (Glu) uptake in isolated rat brain synaptosomes and cytosolic Ca2+ concentration ([Ca2+ ]C) in vascular smooth muscle cells from rat aorta. Toxins were isolated by ion exchange chromatography (TsTX-I) followed by RP-HPLC (TsTX-V). The toxins (15 and 30 g/kg) and TsV (50 and 100 g/kg) were injected intravenously. MAP was continuously monitored through femoral catheter. Epinephrine (E) and norepinephrine (NE) plasma levels were determined by RP-HPLC with electrochemical detection, at 10 min before and 2.5, 30 and 90 min after treatments. Maximal pressor effects were observed at 2.5 3.5 min. TsV induced intense long lasting increase in MAP, as did TsTX-I. TsTX-V showed the lowest pressor effects. TsV showed the highest effects on catecholamines release, followed by TsTX-I and TsTX-V with maximal effect at 2.5 min, followed by a gradual reduction, however remaining higher than controls. Although both toxins act on Na+ channels, TsTX-I displayed significant and more intense effects on catecholamines release and blood pressure than TsTX-V. It seems that the toxicity of TsTX-V is not related only with its ability to release catecholamines, indicating that other neurotransmitters, may be involved in its toxicity. Neither the TsV or its toxins was capable to affect the 3H-Glu uptake. TsTX-I inhibited only 3H-DA uptake (IC50 = 28.41 nM). On the other hand, TsV (0.43ng/mL) inhibited both 3H-GABA and 3H-DA uptake (~50%). TsTX-V showed IC50 = 9.37 nM and 22.2 nM for 3H-GABA and 3H-DA uptake, respectively. These effects were abolished by pre-treatment with TTX, indicating the involvement of Na+ channels in this process. In the absence of Ca2+ and at low concentrations of toxin, the reduction is not as significant as in the presence of Ca2+. TsTX-V did not reduce 3H-GABA uptake in COS-7 cells expressing GABA transporters GAT-1 and GAT-3, suggesting that this toxin indirectly reduces the transport. The reduced 3H-GABA uptake by synaptosomes could be due to fast and intense cell depolarization as revealed by confocal microscopy of C6 glioma cells. Thus, TsTX-V causes reduction on 3H-GABA and 3H-DA uptake in a Ca2+-independent manner, not affecting directly GABA transporters, but, in consequence of depolarization, involving voltage-gated Na+ channels. TsV and its toxins were able to increase the ([Ca2+ ]C , probably by interact with Na+ channels. When compared to KCl 60 mM depolarizing effect (100 %), TsV (100 and 500 ?g/mL), showed an increase of 49.60 ± 2.58 % and 103.66 ± 5.17 %, respectively, whereas TsTX-I and TsTX-V (50 and 100?g/mL of each) showed 43.92 ± 3.06 % and 121.8 ± 8.9 %; 52.56 ± 8.33 % and 79.5 ± 6.1 %, respectively. TsTX-I (100 ?g/mL) showed most potent effects in this type of preparation, since induced most intense effect that TsTX-V in the same concentration. Thus, it is possible that the differences observed on the effects induced by both toxins are consequence of structural changes among Na+ channels present in several types of tissues and innervations .

Οι νικοτινικοί υποδοχείς της ακετυλοχολίνης (nAChRs) ανήκουν στην υπερ-οικογένεια των ιοντικών καναλιών που ενεργοποιούνται από τη δέσμευση ενός προσδέτη (LGICs) και αποτελούνται από πέντε ομόλογες υπομονάδες. Κάθε μονομερής υπομονάδα αποτελείται από μία Ν-τελική εξωκυττάρια περιοχή (ΕΚΠ), από τέσσερεις διαμεμβρανικές α-έλικες και από μία κυτταροπλασματική περιοχή. Στην ΕΚΠ βρίσκεται η χαρακτηριστική Cys-θηλιά της υπερ-οικογένειας, καθώς και οι θέσεις πρόσδεσης αγωνιστών και ανταγωνιστών του υποδοχέα. Οι γνώσεις μας γύρω από τη δομή των nAChRs προέρχονται κυρίως από κρυσταλλογραφικές δομές ομολόγων πρωτεϊνών δέσμευσης της ACh (AChBP) μαλακίων, από μια δομή του nAChR από ιχθείς του γένους Torpedo που προέρχεται από ηλεκτρονική μικροσκοπία, από την κρυσταλλογραφική δομή της α1-ΕΚΠ ποντικού σε σύμπλοκο με α-μπουγκαροτοξίνη (α-Btx) και από κρυσταλλογραφικές δομές δύο προκαρυωτικών LGICs. Παρά τη μεγάλη πρόοδο που πραγματοποιήθηκε με τα παραπάνω επιτεύγματα, ακόμη δεν έχει επιλυθεί πειραματικά η δομή ανθρώπινου υποδοχέα. Επίσης λίγα είναι γνωστά για την επίδραση της γλυκοζυλίωσης των ΕΚΠ στη λειτουργία του nAChR. Χρησιμοποιώντας ως εκμαγείο την κρυσταλλογραφική δομή του συμπλόκου α1-ΕΚΠ ποντικού/α-Btx δημιουργήθηκαν υπολογιστικά μοντέλα της ανθρώπινης α1-ΕΚΠ προσδεμένης στις τοξίνες α-μπουγκαροτοξίνη (α-Btx), α-κομπρατοξίνη (α-Cbtx), α-κωνοτοξίνη (α-Ctx) ImI και α-κωνοτοξίνη GI. Στα σύμπλοκα με α-Btx και α-Cbtx προστέθηκε η υδατανθρακική αλυσίδα, συνδεδεμένη με το κατάλοιπο Asn141, που συγκρυσταλλώθηκε μαζί με την α1-ΕΚΠ ποντικού. Για να μελετηθεί η δυναμική συμπεριφορά της αλληλεπίδρασης υποδοχέα-τοξίνης καθώς και η συνεισφορά των σακχάρων σε αυτήν πραγματοποιήθηκαν προσομοιώσεις Μοριακής Δυναμικής σε υδατικό περιβάλλον. Με τη χρήση υπολογιστικών εργαλείων για τη μελέτη των συμπλόκων προσδιορίστηκαν σε ατομικό επίπεδο οι αλληλεπιδράσεις που καθοδηγούν την πρόσδεση τοξινών στην α1-ΕΚΠ. Βρέθηκε ότι η υδατανθρακική αλυσίδα συμμετέχει δυναμικά στη δέσμευση της τοξίνης στον υποδοχέα. Τα σάκχαρα συγκλίνουν προς την προσδεμένη τοξίνη στηριζόμενα στα κατάλοιπα Ser187 και Trp184 της α1 υπομονάδας. Η τοξίνη επίσης μετακινείται φέρνοντας τη θηλιά Ι σε επαφή με τα σάκχαρα. Αναγνωρίστηκαν σημαντικές αλληλεπιδράσεις των σακχάρων με τα τοξινικά κατάλοιπα Thr6, Ser9, και Th15 της α-Btx και Thr6 και Pro7 της α-Cbtx. Επίσης επιβεβαιώθηκε η ύπαρξη μιας υδρόφιλης κοιλότητας στο εσωτερικό του υδρόφοβου πυρήνα της α1-ΕΚΠ, η οποία πιθανόν εμπλέκεται στο άνοιγμα του ιοντικού καναλιού του nAChR. Τα αποτελέσματα αυτά παρέχουν σημαντικά δεδομένα για την κατανόηση της επίδρασης της υδατανθρακικής αλυσίδας στη λειτουργία του υποδοχέα, η οποία μπορεί να αξιοποιηθεί στην αντιμετώπιση των πολλών παθολογικών καταστάσεων στις οποίες εμπλέκονται οι nAChRs. / Nicotinic acetylcholine receptors (nAChRs) belong to the superfamily of ligand-gated ion channels (LGICs). LGICs form homo- or hetero-pentamers of related subunits, and each of them consists of a N-terminal extracellular ligand-binding domain (ECD), four transmembrane α-helixes and an intracellular region. The characteristic Cys-loop of the superfamily is found in the ECD of each subunit. The ECD also contains binding sites for agonists and competitive antagonists. Our knowledge regarding the nAChR structure mainly derives from the X-ray crystal structures of the molluscan ACh-binding proteins (AChBPs), the electron microscopy structure of the Torpedo nAChR, the X-ray crystal structure of the mouse nAChR α1-ECD bound to α-bungarotoxin (α-Btx), and the X-ray crystal structures of two prokaryotic LGICs. Despite the progress made by these achievements, the determination of any human nAChR structure has not yet been accomplished. Furthermore, the effect of glycosylation on nAChR function has not yet been explored. Based on the crystal structure of the extracellular domain of the mouse nAChR α1 subunit bound to α-Btx we have generated in silico models of the human nAChR α1-ECD bound to the toxins α-bungarotoxin (α-Btx), α-cobratoxin (α-Cbtx), α-conotoxin (α-Ctx) ImI and α-conotoxin GI. In the case of the α1-ECD/α-Btx and α-Cbtx complexes, a Asn141-linked carbohydrate chain was modeled, its coordinates taken from the crystal structure of the mouse α1-ECD. To gain further insight into the structural role of glycosylation molecular dynamics (MD) simulations were carried out in explicit solvent so as to compare the conformational dynamics of the binding interface between nAChR α1 and the two toxins. The use of computational methods allowed the monitoring of the interactions that govern toxin binding. The MD simulations revealed the strengthening of the receptor-toxin interaction in the presence of the carbohydrate chain. A shift in the position of the sugars towards the bound toxin was observed. Residues Ser187 and Trp184 of nAChR act as critical anchor points for the stabilization of the sugar chain in a close position to the toxin. Toxin Finger I shifts closer to the mannoses, forming important toxin-sugar interactions that implicate residues Thr6, Ser9, and Thr15 of α-Btx, as well as Thr6 and Pro7 of α-Cbtx. Additionally the MD simulations of the human α1 ECD–toxin complexes confirmed the possible accommodation of two water molecules into a hydration cavity inside the hydrophobic core of the subunit, which may contribute to the gating mechanism of the receptor. These findings provide additional structural data that are intended to inspire biophysical studies on the functional role of glycosylation in the gating mechanism of nAChR and also guide the development of novel therapeutic agents for the treatment of nAChR-associated diseases.

Toxinas que atuam em canais para Na+ operados por voltagem são as principais responsáveis pelos efeitos tóxicos do envenenamento escorpiônico e podem ser divididas em duas classes: a- e b-neurotoxinas. TsTX-V e TsTX-I da peçonha de Tityus serrulatus (TsV) são, respectivamente, exemplos destas toxinas. Neste trabalho, foram avaliados os efeitos da TsV e destas toxinas sobre a pressão arterial média (PAM) e liberação de catecolaminas em ratos conscientes e não imobilizados, previamente cateterizados, bem como a captação de GABA, dopamina (DA) e glutamato (Glu) em sinaptosomas isolados de cérebro de ratos e a concentração citoplasmática de Ca+2 ([Ca+2 ]C) em células de músculo liso vascular de aorta de ratos. As toxinas foram isoladas por cromatografia de troca iônica (TsTX-I) seguida por CLAE de fase reversa (TsTX-V). As toxinas (15 e 30 g/kg) e TsV (50 e 100 g/kg) foram injetadas intravenosamente. A PAM foi monitorada continuamente através do cateter femoral. Os níveis plasmáticos de adrenalina (ADR) e noradrenalina (NA) foram determinados por CLAE de fase reversa com detector eletroquímico, em 10 min antes e 2,5, 30 e 90 min após os tratamentos. Efeitos pressores máximos foram observados em 2,5?3,5 min. TsV induziu um intenso aumento de longa duração na PAM, bem como a TsTX-I. A TsTX-V mostrou efeitos pressores menores. TsV mostrou os maiores efeitos sobre a liberação de catecolaminas, seguido pela TsTX-I e TsTX-V com um efeito máximo em 2,5 min, seguido por uma gradual redução, permanecendo, todavia, maior que os controles. Embora ambas as classes de toxinas atuem em canais para Na+, TsTX-I mostrou efeitos mais significantes e intensos sobre a liberação de catecolaminas e pressão arterial que a TsTX-V. Parece que a toxicidade da TsTX-V não está somente relacionada à sua capacidade de liberar catecolaminas, indicando que outros neutrotransmissores podem estar envolvidos em sua toxicidade. Nem a TsV ou suas toxinas foram capazes de afetar a captação de 3H-Glu. TsTXI inibiu somente a captação de 3H-DA (IC50 = 28,41 nM). Por outro lado, TsV (0,43ng/mL) inibiu a captação de 3H-GABA e 3H-DA (~50%). TsTX-V mostrou IC50 = 9,37 nM e 22,2 nM para a captação de 3H-GABA e 3HDA, respectivamente. Esses efeitos foram abolidos pelo pré-tratamento com TTX, indicando o envolvimento de canais para Na+ neste processo. Na ausência de Ca+2 e em baixas concentrações de toxinas, a redução não é tão singnificante como na presença de Ca+2. TsTX-V não reduziu a captação de 3H-GABA em células COS-7 expressando os transportadores de GABA, GAT-1 e GAT-3, sugerindo que esta toxina reduz indiretamente o transporte. A redução da captação de 3H-GABA pelos sinaptosomas pode ser devido a rápida e intensa despolarização celular, como revelado por microscopia confocal em células de glioma C6. Assim, TsTX-V causou redução da captação de 3H-GABA e 3H-DA de uma maneira independente de Ca+2, não afetando diretamente os transportadores de GABA, mas em consequencia da despolarização, envolvendo canais para Na+ operados por voltagem. TsV e suas toxinas foram capazes de aumentar a ([Ca2+ ]C , provavelmente por interargir com canais para Na+. Quando comparado aos efeitos despolarizantes do KCl 60 mM (100 %), TsV (100 e 500 g/mL) exibiu um aumento de 49,60 ± 2,58 % e 103,66 ± 5,17 %, respectivamente, enquanto que a TsTX-I e TsTX-V (50 e 100 g/mL de cada) exibiu 43,92 ± 3,06 % e 121,8 ± 8,9 %; 52,56 ± 8,33 % e 79,5 ± 6,1 % de aumento, respectivamente. TsTX-I (100 g/mL) mostrou-se mais potente nesta preparação, visto que uma dose de 100 g/mL causou efeito muito mais intenso do que a TsTX-V na mesma concentração. É possível que as diferenças observadas sobre os efeitos induzidos pela TsTX-I e TsTX-V sejam conseqüência de alterações estruturais entre canais para Na+ presentes em vários tipos de tecidos e inervações. / Voltage-gated Na+ channel toxins are mainly responsible for the toxic effects of scorpion envenoming and can be classified into two classes: a- and b-neurotoxins. TsTX-V and TsTX-I from Tityus serrulatus venom (TsV) are, respectively, examples of these toxins. In this work, were evaluate the effects of TsV and its toxins on mean arterial pressure (MAP) and catecholamines release in conscious unrestrained rats previously catheterized, as well as GABA, dopamine (DA) and glutamate (Glu) uptake in isolated rat brain synaptosomes and cytosolic Ca2+ concentration ([Ca2+ ]C) in vascular smooth muscle cells from rat aorta. Toxins were isolated by ion exchange chromatography (TsTX-I) followed by RP-HPLC (TsTX-V). The toxins (15 and 30 g/kg) and TsV (50 and 100 g/kg) were injected intravenously. MAP was continuously monitored through femoral catheter. Epinephrine (E) and norepinephrine (NE) plasma levels were determined by RP-HPLC with electrochemical detection, at 10 min before and 2.5, 30 and 90 min after treatments. Maximal pressor effects were observed at 2.5 3.5 min. TsV induced intense long lasting increase in MAP, as did TsTX-I. TsTX-V showed the lowest pressor effects. TsV showed the highest effects on catecholamines release, followed by TsTX-I and TsTX-V with maximal effect at 2.5 min, followed by a gradual reduction, however remaining higher than controls. Although both toxins act on Na+ channels, TsTX-I displayed significant and more intense effects on catecholamines release and blood pressure than TsTX-V. It seems that the toxicity of TsTX-V is not related only with its ability to release catecholamines, indicating that other neurotransmitters, may be involved in its toxicity. Neither the TsV or its toxins was capable to affect the 3H-Glu uptake. TsTX-I inhibited only 3H-DA uptake (IC50 = 28.41 nM). On the other hand, TsV (0.43ng/mL) inhibited both 3H-GABA and 3H-DA uptake (~50%). TsTX-V showed IC50 = 9.37 nM and 22.2 nM for 3H-GABA and 3H-DA uptake, respectively. These effects were abolished by pre-treatment with TTX, indicating the involvement of Na+ channels in this process. In the absence of Ca2+ and at low concentrations of toxin, the reduction is not as significant as in the presence of Ca2+. TsTX-V did not reduce 3H-GABA uptake in COS-7 cells expressing GABA transporters GAT-1 and GAT-3, suggesting that this toxin indirectly reduces the transport. The reduced 3H-GABA uptake by synaptosomes could be due to fast and intense cell depolarization as revealed by confocal microscopy of C6 glioma cells. Thus, TsTX-V causes reduction on 3H-GABA and 3H-DA uptake in a Ca2+-independent manner, not affecting directly GABA transporters, but, in consequence of depolarization, involving voltage-gated Na+ channels. TsV and its toxins were able to increase the ([Ca2+ ]C , probably by interact with Na+ channels. When compared to KCl 60 mM depolarizing effect (100 %), TsV (100 and 500 ?g/mL), showed an increase of 49.60 ± 2.58 % and 103.66 ± 5.17 %, respectively, whereas TsTX-I and TsTX-V (50 and 100?g/mL of each) showed 43.92 ± 3.06 % and 121.8 ± 8.9 %; 52.56 ± 8.33 % and 79.5 ± 6.1 %, respectively. TsTX-I (100 ?g/mL) showed most potent effects in this type of preparation, since induced most intense effect that TsTX-V in the same concentration. Thus, it is possible that the differences observed on the effects induced by both toxins are consequence of structural changes among Na+ channels present in several types of tissues and innervations .