Orientador: Lea Rodrigues Simioni / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-06T23:34:13Z (GMT). No. of bitstreams: 1 Barros_JosianeCristina_M.pdf: 4378084 bytes, checksum: 410e7a4f1b02b18191d0a4be7945310c (MD5) Previous issue date: 2005 / Resumo: O veneno total de Botrhops alternatus (10 mg) foi fracionado por meio de coluna m-Bondapack C18 e HPLC de fase reversa resultando em um pico ativo denominado BT II 2. Em SDS-PAGE, a toxina originou uma banda única, isolada de 14 KDa. A fração BT II 2 (0,1, 0,5, 1,0, 5,0, 10,0 e 20,0 mg/mL n=7) induziu um bloqueio irreversível da resposta contrátil, em preparações músculo biventer cervicis de pintainho, sendo esse dose-dependente. As respostas contraturantes a adição exógena de acetilcolina e cloreto de potássio não foram significativamente diferentes do controle.Os tempos necessários para se obter bloqueio neuromuscular de 50 e 90% foram de 66 _+10 para 0,1mg/mL,44_+3 para 0,5mg/mL ,para 1 mg/mL foi de 38 _+4, 54_+5 para 5mg/mL o tempo foi de 24_+1, 34_+1, para 10mg/mL foi de 22_+2, 35_+2 e para 20mg/mL foi de 24_+5,33_+6 respectivamente. Fig. 9. Alterações morfológicas foram observadas nas fibras musculares. Entretanto, a concentração da fração BTII2, 1 µg/mL, foi mais potente para induzir a hipercontração e desorganização fibrilar que o veneno bruto (100 mg/ml). Isto se deve provavelmente ao fato de que a fração está totalmente desprovida de algumas impurezas ( elementos) que pudessem eventualmente inibir este efeito no veneno total. Estes resultados sugerem que a fração BT II 2 do veneno de B. altenatus tem um importante fator neurotóxico e uma ação predominantemente pré-sináptica / Abstract: Crude venom from Bothrops alternatus (10 mg) was fractionated by reverse-phase HPLC by a C-18 m-bondapack column resulting in an active peak called BT II 2. In SDS-PAGE, the toxin migrates as a single band with a 14 kDa. In the chick biventer cervicis preparation, BT II 2 (0.1, 0.5, 1.0, 5.0, 10.0 and 20.0 mg/mL, n=7) induced a concentration-dependent and irreversible twitch-tension blockade without causing any effect on the muscle responses to ACh and KCl. The time (in min) required for producing 50% neuromuscular blockade was 65 ± 0.9; 41 ± 0.4; 35 ± 0.6; 24 ± 0.3; 22 ± 0.4 and 20 ± 0.8 respectively after a 120 min incubation. Morphological alterations was seen in muscle fibers. However, BT II 2 ( 1 mg/ml) was more potent in inducing hyprecontraction and myofibril desorganization than was the crude venom (100 mg/ml) in the same experimental conditions. Taking together, these results suggest that BT II 2 is a neurotoxic factor of the venom of B. alternatus with a preponderant presynaptic action / Mestrado / Mestre em Farmacologia

Orientadores: Angelica de Fatima de Assunção Braga, Franklin Sarmento da Silva Braga / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-12T22:04:04Z (GMT). No. of bitstreams: 1 Carvalho_VanessaHenriques_M.pdf: 2016873 bytes, checksum: ed79870ad9763d6497595ceb7fdcf4ba (MD5) Previous issue date: 2009 / Resumo: Os anestésicos locais podem interagir com os bloqueadores neuromusculares e modificar as suas propriedades farmacocinéticas e farmacodinâmicas, no entanto o mecanismo dessa interação é controverso. Este estudo experimental, realizado em preparação nervo frênico - diafragma de ratos e musculo biventer cervicis de pintainhos, teve por objetivo avaliar o efeito de diferentes anestésicos locais na transmissão neuromuscular e sua influência no bloqueio produzido pelo rocurônio. Foram avaliados os seguintes parâmetros: efeito dos anestésicos locais (lidocaína, bupivacaína racêmica, mistura em excesso enantiomérico de bupivacaína) e do rocurônio empregados isoladamente, na transmissão neuromuscular; o bloqueio produzido pelo rocurônio em preparações expostas aos anestésicos locais; a ação dos anestésicos locais na resposta contraturante da acetilcolina; seus efeitos nos potenciais de membrana e nos potenciais de placa terminal em miniatura. Os resultados foram expressos em médias e desvios padrão e analisados através dos testes t de Student, Mann-Whitney , Wilcoxon, Kruskall-Wallis e Anova com teste para comparação múltipla de Tukey. Adotou-se um nível de significância de 5% (p<0,05). Nas preparações nervo frênico -diafragma de rato, os anestésicos locais nas concentrações empregadas, não alteraram a amplitude das respostas musculares mas, potencializaram o efeito do rocurônio. Nas preparações biventer cervicis de pintainho os anestésicos locais promoveram diminuição na resposta contraturante da acetilcolina evidenciando um efeito pós-juncional. Não causaram alteração significativa nos potenciais de membrana, não demonstrando ação despolarizante na fibra muscular. A lidocaína promoveu um aumento inicial dos potenciais de placa terminal em miniatura (pptm) seguida de bloqueio e, a bupivacaína nas duas formulações causou diminuição na amplitude e na freqüência dos pptm, caracterizando um efeito pré-juncional. Os resultados obtidos neste estudo demonstram um sinergismo entre as drogas comprovado por efeitos pré e pós-juncionais / Abstract: Local anesthetics can interact with neuromuscular blockers and modify their pharmacokinetics and pharmacodynamics properties but this mechanism of interaction is very controversial. This experimental study performed in rat phrenic nerve diaphragm preparation and chick biventer cervicis muscle, had the objective to evaluate the effect of different local anesthetics in neuromuscular transmission and their influence in the block promoted by rocuronium. The parameters evaluated were that: the effect of local anesthetics (lidocaine, racemic bupivacaine, enantiomeric mixture of bupivacaine) and the rocuronium isolated in neuromuscular transmission; the block promoted by rocuronium in local anesthetics exposed preparations; the action of local anesthetics in contracture response of acetylcholine; their effects in membrane potentials and miniature end plate potentials. The results were expressed in average and standard deviation and analysed using T Student test, Mann-Whitney, Wilcoxon, Kruskall-Wallis and Anova with Tukey multiple comparative test. We adopted a level of significance of 5% (p<0,05). In rat phrenic nerv diaphragm preparation, local anesthetics in the concentrations employed did not caused alterations in the amplitude of muscle responses but potentialized the effect of rocuronium. In chick biventer cervicis preparations local anesthetics promoted a decrease in acetylcholine contracture response indicating a postjunctional effect. They did not cause significant alterations in membrane potential so they did not demonstrate depolarizing action in muscle fiber. Lidocaine promoted an initial increase in miniature end plate potentials (meps) followed by block and bupivacaine in the two formulations caused a decrease in the amplitude and frequency of meps, characterizing a prejunctional effect. The results obtained in this study demonstrate a synergism of the drugs confirmed by pre and postjunctional effects / Mestrado / Mestre em Farmacologia

Orientador: Léa Rodrigues Simioni / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-19T21:13:03Z (GMT). No. of bitstreams: 1 Ferreira_SandroRostelato_D.pdf: 2167840 bytes, checksum: f1dd1c4722a358caf196b8ef326c3b3f (MD5) Previous issue date: 2012 / Resumo: Rhinella schneideri, conhecido previamente como Bufo paracnemis, é um sapo comum em muitas regiões do Brasil. O veneno destas espécies exerce importante efeito cardiovascular em humanos e animais, mas pouco se sabe sobre sua atividade neuromuscular. Neste trabalho, nós avaliamos a neurotoxicidade do veneno de R. schneideri em preparações neuromusculares de pintainho e camundongo. Através da compressão manual das glândulas parótidas localizadas atrás dos olhos, coletou-se a secreção e então realizada a extração com metanol. O extrato metanólico foi liofilizado e testado em preparações biológicas. Preparações biventer cervicis (BC) de pintainho e nervo frênico diafragma (NFD) de camundongo foram utilizadas para o registro miográfico através de estimulação elétrica indireta para medidas eletrofisiológicas, análise morfológica e microscopia eletrônica de transmissão. Frações ativas do extrato metanólico foram obtidas submetendo-se à coluna de fase reversa Luna PFP (250 x 4,6 mm). O extrato metanólico (50 ?g/ml) causou somente facilitação da neurotransmissão em preparações NFD. Ao contrário, causou bloqueio neuromuscular significativo em preparações BC que foram concentração-dependente (3, 10 and 30 ?g/ml; a 37º C) com tempo para 50% de bloqueio, média ± erro padrão: 84±10, 51±3 e 12±0,8 min com 3, 10 e 30 ?g/ml, respectivamente; n=6-8 cada, precedido por facilitação da neurotransmissão. Não houve inibição significativa das respostas contraturantes à ACh (110 ?M) ou KCl (20 mM) após bloqueio completo em qualquer concentração testada. Em preparações BC incubadas com o extrato metanólico (10 ?g/ml) a 22º C por 70 min não observou-se qualquer alteração das respostas musculares (117±3%; n=5), mas quando a temperatura do banho foi elevada a 37º C, 50% de bloqueio ocorreu após 92±3 min (n=5; p<0.05). A incubação de preparações BC curarizadas (d-Tc, 1 ?g/ml) com o extrato metanólico (10 ?g/ml) resultou em completo e irreversível bloqueio enquanto que as preparações tratadas somente com curare mostraram a reversão completa da resposta contrátil após várias lavagens. Não houve aumento significativo nos níveis de liberação de creatinoquinase (90±21 vs. 80±15 U/l, antes e após 120 min de incubação com o extrato, respectivamente, n=5) além da ausência de alterações na morfologia das fibras musculares ou na porcentagem de danos na fibra (2.4±0.9 vs. 2.3±0.5 %, antes e após 120 min de incubação com o extrato, respectivamente, n=5). O extrato metanólico (50 ?g/ml) aumentou a resposta contrátil mas não alterou o potencial de membrana em repouso (-81±1 mV e -78±1 mV para controle e preparação tratada após 60 min). Registros eletrofisiológicos mostraram que houve um aumento progressive na frequência dos potenciais de placa terminal em miniatura (PPTM) de 34±3,5 (controle) para 88±15 (após 60 min de incubação com o extrato); houve também um aumento nos valores do conteúdo quântico, de 128±13 (controle) para 272±34 e 171±11 após 5 min e 60 min, respectivamente, em preparações tratadas com o extrato metanólico. A microscopia eletrônica de transmissão mostrou que o volume ocupado pelas vesículas sinápticas foi significativamente reduzida (32±5%; p<0.05) após 5 min mas este efeito foi reversível após 60 min de incubação para as preparações tratadas com 50 ?g/ml do extrato metanólico. Não houve dano estrutural distinguível na membrana do terminal nervoso e nas mitocôndrias das preparações tratadas com o extrato, quando comparada com as preparações controle. O pré-tratamento das preparações NFD com ouabaína (1 ?g/ml), um inibidor da bomba de Na+/K+-ATPase, por 5 min antes da incubação com o extrato, preveniu o aumento do conteúdo quântico comparado com preparações controle (118±18, 117±18 e 154±33 para preparações controle-ouabaína e tratadas com ouabaína e incubadas com o extrato por 5 min e 60 min, respectivamente). A cromatografia por HPLC do extrato metanólico resultou em 24 frações, das quais 4 (frações 20, 21, 22 e 24) causaram bloqueio neuromuscular em preparações BC. A fração 20 (3 ?g/ml) foi escolhida por ser 3 vezes mais potente que as demais e causou bloqueio neuromuscular significativo (p<0.05; tempo para 50% de bloqueio: 43±4 min; n=4) precedido por facilitação em preparações BC a 37º C. A fração 20 não inibiu as respostas contraturantes à ACh (110 ?M) ou KCl (20 mM) após completo bloqueio neuromuscular em preparações BC. Em preparações NFD, a fração (15 ?g/ml) aumentou significativamente os valores do conteúdo quântico de 117±18 (controle) para 236±44 após 5 min de incubação (n=4; p<0.05). Estes resultados indicam que o extrato metanólico do veneno de R. schneideri é capaz de interferir com a neurotransmissão por ativar e/ou bloquear a liberação da acetilcolina nos sítios pré-sinápticos, provavelmente envolvendo a bomba de Na+-K+-ATPase, sem causar qualquer dano à musculatura / Abstract: Rhinella schneideri, previously known as Bufo paracnemis, is a common toad in many regions of Brazil. The venom of this species exerts important cardiovascular effects in humans and animals, but little is known of its neuromuscular activity. In this work, we examined the neurotoxicity of R. schneideri venom in chick and mouse neuromuscular preparations. Venom was collected by manual compression of the large parotid glands behind the eyes and then extracted with methanol. The extract was lyophilized prior to testing in biological preparations. Chick biventer cervicis (BC) and mouse phrenic nerve-diaphragm (PND) preparations were mounted for conventional twitch-tension recording in response to indirect stimulation, for electrophysiological measurements, morphological analysis and transmission electronic microscope. Also, an active fraction of this methanolic extract obtained by reverse phase HPLC on a Luna PFP (250 x 4.6 mm) column. The methanolic extract (50 ?g/ml) caused facilitation but no neuromuscular blockade in PND preparations. In contrast, significant (p<0.05) concentration-dependent (3, 10 and 30 ?g/ml) neuromuscular blockade (time for 50% blockade, mean±S.E.M.: 84±10, 51±3 and 12±0.8 min with 3, 10 and 30 ?g/ml, respectively; n=6-8 each) preceded by facilitation was seen in BC preparations at 37oC. There was no inhibition of contractures to exogenous ACh (110 ?M) or KCl (20 mM) after complete blockade by any of the concentrations tested. Incubation of BC preparations with methanolic extract (10 ?g/ml) at 22oC for 70 min did not affect neuromuscular transmission (117±3%; n=5), but when the bath temperature was increased to 37oC, 50% blockade occurred within 92±3 min (n=5; p<0.05). Incubation of curarized (d-Tc, 1 ?g/ml) BC preparations with methanolic extract (10 ?g/ml) resulted in complete, irreversible blockade whereas preparations treated with curare alone showed complete reversion in the twitch-tension after washing. There was no significant increase in creatine kinase levels (90±21 vs. 80±15 U/l, before and after a 120 min incubation with extract, respectively; n=5) and no significant alterations in muscle fiber morphology or in the percentage of damaged fibers (2.4±0.9 vs. 2.3±0.5 % before and after a 120 min incubation with extract, respectively; n=5). The methanolic extract (50 ?g/ml) increased the twitch-tension but did not alter the membrane resting potential (-81±1 mV and -78±1 mV for control and poison-treated preparations after 60 min). Electrophysiological measurements showed that there was a progressive increase in the frequency of miniature end-plate potentials (MEPPs) from 34±3.5 (control) to 88±15 (after a 60 min incubation with extract); there was also an increase in the end-plate potentials (based on the quantal content) from 128±13 (control) to 272±34 and 171±11 after 5 min and 60 min, respectively, in extract-treated preparations. TEM showed that the fractional volume occupied by synaptic vesicles was significantly reduced (32±5%; p<0.05) after a 5 min but this effect was reversible after 60 min of incubation to 50 ?g/ml of methanolic extract. There was no structural damage to the membrane of the terminal boutons and the mitochondria of extract-treated preparations were indistinguishable from those of control preparations. Pretreatment of the preparations with ouabain (1 ?g/ml), a Na+/K+-ATPase pump inhibitor, for 5 min prior to incubation with methanolic extract prevented the increase in quantal content compared to preparations without extract (118±18, 117±18 and 154±33 for ouabain-treated controls and ouabain-treated preparations incubated with venom for 5 min and 60 min, respectively). HPLC of the methanolic extract resulted in 24 fractions, of which four (fractions 20, 21, 22 and 24) produced blockade in BC preparations. Fraction 20 (3 ?g/mL) was chosen because was the most potent of the four fractions and caused significant (p<0.05) neuromuscular blockade (time for 50% blockade: 43±4 min; n=4; mean±SEM) preceded by facilitation in BC preparations at 37oC. Fraction 20 did not inhibit contractures to exogenous ACh (110 ?M) or KCl (20 mM) after complete neuromuscular blockade in BC preparations. In PND preparations, fraction 20 (15 ?g/mL) significantly increased the quantal content value from 117±18 (control) to 236±44 after 5 min (n=4; p<0.05). These results indicate that the methanolic extract of R. schneideri is capable to interfere with the neurotransmission by activing and/or blocking the pre-synaptic acetylcholine release by an activity involving the Na+-K+-ATPase pump, without damaging the muscle membrane / Doutorado / Farmacologia / Doutor em Farmacologia

Congenital myasthenic syndromes (CMS) are a rare group of heterogeneous disorders, characterised by compromised neuromuscular transmission and symptoms of fatiguable muscle weakness. CMS is caused by mutations in genes that affect the structure and function of the neuromuscular junction (NMJ). In about 20% of CMS cases, patients have mutations in the gene DOK7; the protein product, DOK7, is crucial for maintaining the dense aggregation of acetylcholine receptor (AChR) clusters at the NMJ. DOK7-CMS patients do not respond to treatment with acetylcholinesterase inhibitors which are the first line treatment for most forms of CMS. Instead, a dramatic response to beta-2 adrenergic receptor (ADRB2) agonists, such as salbutamol, is observed. The aim of this project was to investigate the molecular mechanisms that underlie the beneficial effects of ADRB2 agonists. Firstly, NMJ functioning was modelled in vitro by studying AChR clusters formed on cultured C2C12 mouse myotubes in the presence of WT DOK7. Overexpression of mutant DOK7 led to a significant reduction in the number of AChR clusters, explaining the pathogenic effect of the mutation. Importantly, incubation of myotubes with salbutamol increased the number of AChR clusters and their stability. The results provide the first evidence that ADRB2 agonists directly affect proteins located at the NMJ. However, this disease model suffers from limitations. The rest of the thesis focussed on developing alternative cell culture models to explore the AChR clustering pathway. The first model combined optogenetics and fluorescence lifetime microscopy to study the effects of ADRB2 activation on AChR cluster stability in single live cells. The second used CRISPR/Cas9 genome editing tools to directly introduce Dok7 mutations to the genome of C2C12 cells, thereby overcoming some of the drawbacks associated with DOK7 overexpression. Further manipulations of these novel model systems will be used in the future to examine in more detail the molecular events underlying the pathogenic effects of DOK7 mutations and the mechanisms of ADRB2 agonists.

Repair of peripheral nerve injury often results in poor functional motor recovery. This deficit has previously been attributed to the failure of axons to regenerate into the muscle. However, we have recently reported that following nerve injury in mice, axons have regenerated to the motor end plate in animals with poor recovery. We proposed that following axonal injury, there is a critical period during which the axon must reach the muscle in order to form a functional neuromuscular junction. We have developed a mouse model of prolonged denervation, in which the sciatic nerve is crushed repeatedly every few days, preventing regenerating axons from reaching the muscle. This multiple crush model allows us to vary the period of denervation by modifying the number of crushes. Motor recovery as assessed using the toe-spreading score occurs after 3 or 4 multiple crushes every 7 days (24 or 31 days of denervation) but not after 5 crushes (38 days). Immunostaining for alpha-bungarotoxin and neurofilament confirmed end plate reinnervation. Thus following denervation > 38 days, a motor deficit persists despite end plate reinnervation. Although the mechanism for the deficit requires investigation, these results suggest that functional neuromuscular junction reestablishment more than end plate reinnervation and that there is a time limit for functional synapse reformation.

Digital averaging and analysis of miniature endplate currents (MEPCs) from mouse diaphragm was used to characterize the normal MEPC and its modification by a variety of drugs. Under normal conditions the decay of MEPCs showed consistent deviations from a simple exponential consisting in a progressive increase of rate constant, followed by a slow tail. Receptor blockade by d-tubocurarine (dTC), a-bungarotoxin, and other agents thought to occupy ACh-binding sites reduced MEPC amplitude, accelerated MEPC decay by about 30% (making it about equal to decay rate of channels opened by exogenous acetylcholine), and eliminated the early deviations from an exponential decay; dTC also abolished the late tail. Examination of the interaction of acetylcholinesterase (AChE) poisoning and receptor blockade on MEPC height and time course indicated that normally most quantal ACh is captured by receptors and, as predicted by theoretical consideration, a rather large degree of receptor blockade is necessary to reduce MEPC height. MEPC tails were exaggerated by AChE poisoning and exogenous ACh or carba-chol. The latter agents reduced MEPC height in a fashion inconsistent with blockade of ACh binding and concurrent modulation of the tail suggested an important role of desensitized receptors in tail generation. A number of other drug actions are also described quantitatively: (a) channel prolongation, typical of alcohols but also found with ketones and some amines; (b) 'channel plugging', typical of local anaesthetics but also found with many other agents, including long chain alcohols, and (c) an action to reduce MEPC size without reducing net response to exogenous agonist typical of volatile anaesthetics, associated with increase rather than decrease of ACh binding to receptor. Criteria for distinguishing different modes of modification of receptor function are discussed. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

Orientadores: Lea Rodrigues Simioni,Yoko Oshima Franco / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-07T13:41:21Z (GMT). No. of bitstreams: 1 Bueno_LilianGobbodeFreitas_M.pdf: 986839 bytes, checksum: 048ec75c1469d547de311f325aab2cc3 (MD5) Previous issue date: 2006 / Resumo: Manganês, um bloqueador neuromuscular com atividades pré e pós-sinápticas, foi utilizado neste trabalho com o objetivo de estudar a neurotoxicidade e miotoxicidade induzida pelos venenos das serpentes Crotalus durissus terrificus (Cdt) e Bothrops jararacussu (Bjssu) em preparações biventer cervicis de pintainho (BCp). O pré-tratamento das preparações com Mn2+ (0,66 e 1,6 mM) não impediu o bloqueio neuromuscular induzido pelo veneno de Cdt, reduziu parcialmente a resposta contraturante à adição exógena de ACh, mas não à de KCl. Embora o bloqueio neuromuscular induzido pelo veneno de Bjssu seja irreversível, o pré-tratamento com ambas as concentrações de Mn2+ (0,66 e 1,6 mM) tornou o bloqueio parcialmente reversível após a lavagem das preparações com solução de Krebs, e somente nas preparações pré-tratadas com 1,6 mM Mn2+ houve redução do bloqueio da resposta contraturante à adição exógena de ACh (p<0,05). Os tipos de miotoxicidade induzidos pelos venenos de Cdt e Bjssu interferiram diferentemente na resposta contrátil. Em preparações pré-tratadas com Mn2+ (1,6 mM) houve redução parcial da porcentagem de dano muscular e da atividade miotóxica (CC, U/L) induzida por ambos os venenos. Os resultados desta pesquisa indicam que: Mn2+ interfere na contratura induzida pela ACh nos receptores nicotínicos; Mn2+ não evitou a neurotoxicidade do veneno de Cdt, mas reduziu parcialmente sua miotoxicidade in vitro, por sua ação estabilizante da membrana muscular; Mn2+ reduziu parcialmente a miotoxicidade e o bloqueio neuromuscular induzido pelo veneno de Bjssu. A dupla ação do Mn2+, pré- e pós-sináptica, é considerada útil para o estudo dos venenos de serpentes, uma vez que a maioria desses venenos apresenta também estas ações. Além disso, com a utilização deste cátion foi possível resgatar a coerência entre a interpretação dos resultados experimentais e clínicos / Abstract: In this study, we examined the effects of Mn2+, a neuromuscular blocker with pre and postsynaptic actions, on the neurotoxicity and myotoxicity induced by Crotalus durissus terrificus and Bothrops jararacussu venoms in chick biventer cervicis preparations. Pretreating the preparations with Mn2+ (0.66 or 1.6 mM) did not affect the blockade induced by C. d. terrificus venom or KCl-induced contractures, but partially reduced ACh-induced contractures. On the other hand, both concentrations of Mn2+ partially prevented the blockade induced by B. jararacussu venom (seen after washing the preparations with Krebs solution), whereas only 1.6 mM Mn2+ significantly restored ACh-induced contractures. Pretreatment with Mn2+ (1.6 mM) partially prevented the muscle damage and the release of creatine kinase induced by both venoms. These results show that Mn2+ did not prevent the neurotoxicity of Cdt venom, but partially reduced its myotoxicity, whereas this metal partially attenuated the myotoxicity and neuromuscular blockade caused by B. jararacussu venom. The pre- and postsynaptic actions of Mn2+ may be useful for studying snake venoms that show one or both of these activities / Mestrado / Mestre em Farmacologia

The effect of 1,2,3,4-tetrahydro-9-aminoacridine (THA) on quantal transmitter release was examined at the frog neuromuscular junction. THA (3 μM) caused an increase in m (no. of quanta released) as measured by K+-evoked miniature endplate potential (MEPP) frequency. This was due to an increase in p (probability of release), as n (no. of functional release sites) was unchanged. The increase in p was dose-dependent over a range of 0.3-10 μM. By contrast, physostigmine (3 μM) caused a decrease in p, and neostigmine, which does not cross the nerve membrane, had no consistent effect on p. At the postsynaptic site, neostigmine produced the largest increase in MEPP size (79.2%), and THA produced the smallest (17.5%). The divergent effects of THA and physostigmine on p indicate a fundamental difference in their actions at the nerve terminal.

The release of chemical transmitter from nerve terminals is critically dependent on a transient increase in intracellular Ca2+.6,25 The increase in Ca2+ may be due to influx of Ca2+ from the extracellular fluid15 or release of Ca2+ from intracellular stores such as mitochondria.1,8,18 Whether Ca2+ utilized in transmitter release is liberated from organelles other than mitochondria is uncertain. Smooth endoplasmic reticulum is known to release Ca2+, e.g., on activation by inositol trisphosphate or cyclic adenosine diphosphate-ribose,2 so the possibility exists that Ca2+ from this source may be involved in the events leading to exocytosis. We examined this hypothesis by testing whether inositol trisphosphate and cyclic adenosine diphosphate-ribose modified transmitter release. We used liposomes to deliver these agents into the cytoplasmic compartment and binomial analysis to determine their effects on the quantal components of transmitter release. Administration of inositol trisphosphate (10-4M) caused a rapid, 25% increase in the number of quanta released. This was due to an increase in the number of functional release sites, as the other quantal parameters were unaffected. The effect was reversed with 40min of wash. Virtually identical results were obtained with cyclic adenosine diphosphate-ribose (10-4M). Inositol trisphosphate caused a 10% increase in quantal size, whereas cyclic adenosine diphosphate-ribose had no effect. The results suggest that quantal transmitter release can be increased by Ca2+ released from smooth endoplasmic reticulum upon stimulation by inositol trisphosphate or cyclic adenosine diphosphate-ribose. This may involve priming of synaptic vesicles at the release sites or mobilization of vesicles to the active zone. Inositol trisphosphate may have an additional action to increase the content of transmitter within the vesicles. These findings raise the possibility of a role of endogenous inositol phosphate and smooth endoplasmic reticulum in the regulation of cytoplasmic Ca2+ and transmitter release.

Botulinum neurotoxin (BoNT), for more than a hundred years, has been a recognized poisonous principle in spoiled food. As its chemical structure became unraveled, and as more knowledge was gained over its mechanism of toxicity, it became clear that BoNT had the potential to act therapeutically as a targeted toxin that could inactivate specific nerve populations, and thus achieve a therapeutic goal. BoNT has evolved over the past 25 years into a viable therapeutic, now being a first line treatment for dystonia, overtly altering the course of progression of this disorder. BoNT is used for hyperhidrosis and gustatory sweating syndrome, alleviation of pain, as a treatment for overactive bladder, achalasia and anal fissure; and it has gained popularity as a cosmetic aid. Many other possible uses are being explored. The greatest potential for BoNT may lie in its being a molecular Trojan Horse - able to carry a specific enzyme or specific drug to the inside of a cancer or other type of cell while bypassing other cells and thereby having little or no ill effect. BoNTs pharmaceutical potential is boundless.