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Membrane bound AChE from calf brain caudate nucleus was solubilized by use of ion-free media in presence of 10~ M EDTA and 10“5M tetracaine. The irreversible release of AChE was more effectively inhibited by divalent ions compared to monovalent ions added to the medium. EDTA appears to chelate divalent ions released from the tissue while tetracaine competes with the same ions at the membrane. The tetracaine effect is restricted to the procaine series of local anesthetics. Small amounts of soluble AChE are present in the cytosol fraction. In fresh preparations most of the enzyme appeared in a form having a molecular weight of 80.000 daltons as determined by gel filtration. The enzyme seems to be released in this form. It is proposed that this form represents the monomer form of the enzyme. In solution the AChE aggregates seemingly together with a factor that is released from the membrane in amounts stafchio- metric to the enzyme. By treatment with DEAE-Sephadex the enzyme preparation can be made non-aggregating. A highly purified nonaggregating monomeric AChE Specific activity 17150 micromoles acetylthiocholine hydrolyzed per minute at 27° C per mg protein) was obtained by affinity chromatography. Some anomalous binding phenomena was observed during the affinity chromatographic purification. The main observation was that edrophonium eluted crude enzyme preparation adsorbed to the affinity gel in a biphasic manner. It was found that in the crude preparation there is present besides unspecific material competing with the enzyme for the affinity gels a factor that increases the affinity of AChE to certain cholinergic ligands. Since the enzyme could be titrated by the factor it seems to have a very high affinity to the enzyme and the biphasic elution curve is explained by the presence of free as well as factor- bound enzyme in the preparation. In search for compounds having a similar effect it was found that fluoride ion too increased the affinity of AChE to the same ligands as the factor. The affinity of edrophonium to the site defined by the binding of AChE to MTA-CH (65x10“5m) is lower than that defined by the enzyme inhibitory constant (1.8xlO“7M). As an explanation of this finding it is proposed that the substrate induces a conformation having high affinity to edrophonium, a conformation that has a comparatively low relaxation rate. Thus acetylcholinesterase may be added to the list of enzymes that have hystere- tic properties. / <p>S. 1-54: sammanfattning, s. 55-100: 4 uppsatser</p> / digitalisering@umu

Orientador: Angelica de F. de Assunção Braga / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-06T23:53:56Z (GMT). No. of bitstreams: 1 Loyola_YolandaChristinadeSousa_D.pdf: 22412843 bytes, checksum: 64766635e73a046988de0b090a234fe6 (MD5) Previous issue date: 2006 / Resumo: Os anestésicos locais comumente empregados na prática clínica podem interagir com os bloqueadores neuromusculares e potencializar seus efeitos. Muitos autores estudaram esta interação mas o mecanismo envolvido na potencialização do bloqueio neuromuscular e a ação dos anestésicos locais nos sítios pré e pós juncionais não foram completamente elucidados. Neste trabalho através de experimentos específicos na junção neuromuscular foram estudados os seguintes parâmetros: o efeito dos anestésicos locais procaína e lidocaína na transmissão neuromuscular; a sua influência no bloqueio neuromuscular produzido pela d-tubocurarina e pelo rocurônio; a eficácia da neostigmina e da 4-aminopirídina na reversão do bloqueio produzido pela associação anestésicos locais - bloqueadores neuromusculares; os efeitos dos anestésicos locais nos potenciais de membrana e potenciais de placa terminal em miniatura. Avaliou-se também em preparações diafragma de rato cronicamente desnervado e biventer cervicis de pintainho os efeitos dos anestésicos locais na ação contraturante da acetilcolina. Os resultados foram expressos em médias e desvios padrão e analisados através dos testes de Wilcoxon e Mann-Witney, adotando-se um nível de significância de 5% (p < 0,05). Nas preparações nervo frênico - diafragma de rato, sob estimulação elétrica indireta, os anestésicos locais nas concentrações empregadas, não alteraram a amplitude das respostas musculares mas potencializaram os efeitos da d- tubocurarina e do rocurônio.. Este bloqueio foi parcialmente e totalmente revertido pela neostigmina e pela 4-aminopiridina, respectivamente. A procaína e a lidocaína não causaram alteração significativa nos potenciais de membrana, não demonstrando ação despolarizante na fibra muscular. A procaína causou diminuição na amplitude e na freqüência dos potenciais de placa terminal em miniatura (pptms) e a lidocaína, ao contrário, promoveu um aumento inicial na freqüência dos pptms seguida de bloqueio. Nas preparações biventer cervicis de pintainho e diafragma de rato cronicamente desnervado, a procaína e a lidocaína promoveram diminuição significativa na resposta contraturante da acetilcolina evidenciando um efeito pós-juncional. Os resultados obtidos demonstram um sinergismo entre as drogas devido principalmente a um efeito pós-juncional / Abstract: Local anesthetics commonly used in clinical practice can interact with neuromuscular blockers and potentiate their effects. Many authors studied this interaction, but the mechanism involved in the potentiation of neuromuscular blockers and the action of local anesthetics in the pre and postjunctional sites were not completely elucidated- In this study, in specific experiments in the neuromuscular junction the following parameters were studied: the effects of the local anesthetics procaine and lidocaine in the neuromuscular transmission; its influence on the neuromuscular blockade produced by d-tubocurarine and by rocuronium; the efficacy of neostigmine and of 4-aminopyhdine in the reversion of the blockade produced by the association of local anesthetics - neuromuscular blockers; the effects of local anesthetics on the membrane potentials and miniature end - plate potentials. It was also evaluated in chronically denervated rat diaphragm preparation and chick biventer cervicis preparation the effects of local anesthetics on acetylcholine contracture. The results were expressed in average and standard deviation and analyzed through the Wilcoxon and Mann-Witney tests, adopting a level of significance of 5% (p <0.05). In rat phrenic nerve diaphragm preparations under indirect electric stimulation, local anesthetics in the concentrations used did not change the amplitude of the muscle response but potentiated the effects of d-tubocurarine and of rocuronium. This blockade was partially and totally reverted by neostigmine and by 4-aminopyridine, respectively. Procaine and lidocaine caused no significant alteration in the membrane potentials, not demonstrating depolarizing action in the muscle fiber. Procaine caused a decrease in the amplitude and frequency of the miniature end - plate potentials {meps) and lidocaine, in contrast, promoted an initial increase in the frequency of meps followed by blockade. In chick biventer cervicis preparations and chronically denervated rat diaphragm, procaine and lidocaine promoted a significant decrease in the response to acetylcholine contracture evidencing a post-junctional effect. The results obtained demonstrate a synergism among the drugs mainly due to a postjunctional effect / Doutorado / Doutor em Farmacologia