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1.
J. venom. anim. toxins incl. trop. dis ; 26: e20190041, 2020. graf
Article in English | LILACS, VETINDEX | ID: biblio-1056676

ABSTRACT

Here, we described the presence of a neurotoxin with phospholipase A2 activity isolated from Micrurus lemniscatus venom (Mlx-8) with affinity for muscarinic acetylcholine receptors (mAChRs). Methods: The purification, molecular mass determination, partial amino acid sequencing, phospholipase A2 activity determination, inhibition of the binding of the selective muscarinic ligand [3H]QNB and inhibition of the total [3H]inositol phosphate accumulation in rat hippocampus of the Mlx-8 were determined. Results: Thirty-one fractions were collected from HPLC chromatography, and the Mlx-8 toxin was used in this work. The molecular mass of Mlx-8 is 13.628 Da. Edman degradation yielded the following sequence: NLYQFKNMIQCTNTRSWL-DFADYG-CYCGRGGSGT. The Mlx-8 had phospholipase A2 enzymatic activity. The pKi values were determined for Mlx-8 toxin and the M1 selective muscarinic antagonist pirenzepine in hippocampus membranes via [3H]QNB competition binding assays. The pKi values obtained from the analysis of Mlx-8 and pirenzepine displacement curves were 7.32 ± 0.15, n = 4 and 5.84 ± 0.18, n = 4, respectively. These results indicate that Mlx-8 has affinity for mAChRs. There was no effect on the inhibition ability of the [3H]QNB binding in hippocampus membranes when 1 µM Mlx-8 was incubated with 200 µM DEDA, an inhibitor of phospholipase A2. This suggests that the inhibition of the phospholipase A2 activity of the venom did not alter its ability to bind to displace [3H]QNB binding. In addition, the Mlx-8 toxin caused a blockade of 43.31 ± 8.86%, n = 3 and 97.42 ± 2.02%, n = 3 for 0.1 and 1 µM Mlx-8, respectively, on the total [3H]inositol phosphate content induced by 10 µM carbachol. This suggests that Mlx-8 inhibits the intracellular signaling pathway linked to activation of mAChRs in hippocampus. Conclusion: The results of the present work show, for the first time, that muscarinic receptors are also affected by the Mlx-8 toxin, a muscarinic ligand with phospholipase A2 characteristics, obtained from the venom of the Elapidae snake Micrurus lemniscatus, since this toxin was able to compete with muscarinic ligand [3H]QNB in hippocampus of rats. In addition, Mlx-8 also blocked the accumulation of total [3H]inositol phosphate induced by muscarinic agonist carbachol. Thus, Mlx-8 may be a new pharmacological tool for examining muscarinic cholinergic function.(AU)


Subject(s)
Animals , Rats , Snakes , Elapid Venoms/adverse effects , Phospholipases A2 , Inositol Phosphates , Acetylcholine , Receptors, Muscarinic/analysis , Sequence Analysis, Protein
2.
São Paulo; s.n; 1999. 100 p. ilus, tab.
Thesis in Portuguese | LILACS | ID: lil-272249

ABSTRACT

O objetivo do presente trabalho foi investigar a regulaçao dos mecanismos da neurotransmissao colinérgica em miométrio de rata, pelo estrógeno. Mais especificamente, avaliar se o tratamento agudo com estrógeno de ratas adultas afeta a afinidade, o número, os subtipos, e a sinalizaçao intracelular dos receptores muscarinicos presentes nesse tecido e, consequentemente, a responsividade miometrial, em relaçao a ratas em estro natural. Foram utilizadas ratas fêmeas, albinas, da colônia BAW com 118 a 122 dias de idade. Os animais foram divididos em dois grupos experimentais: animais em estro natural (grupo EN) e animais tratados com 25 mg de hexahidrobenzoato de estradiol, 24 horas antes dos experimentos (grupo E-25). O útero foi isolado e o músculo liso miometrial foi separado do endométrio. Os seguintes aspectos foram analisados: peso corporal e peso úmido do útero e do miométrio. Estudos de ligaçao do [3H]QNB em preparaçoes de membranas miometriais dos dois grupos experimentais. - ensaios de cinética e determinaçao da concentraçao de proteína, do tempo e da temperatura de incubaçao. - ensaios de saturaçao e determinaçao dos parâmetros de ligaçao, constante de dissociaçao (KD) e densidade de receptor (Bmax). - ensaios de competiçao e determinaçao da constante de inibiçao (Ki) e do logarítmo negativo da constante de inibiçao (Pki) . Estudo da reatividade farmacológica. curvas concentraçao efeito cumulativa de carbacoi e determinaçao dos valores de EC5O. - curvas concentraçao efeito cumulativa de carbacol, na ausência e na presença de antagonistas muscarínicos, atropina, pfHHSiD e metoctramina e determinaçao dos valores de pKB. Determinaçao da concentraçao intracelular de AMP cíclico. nível basal e efeito do forskolin. efeito do carbacol e de antagonistas muscarínicos, atropina, pfHHSiD e de metoctramina, sobre o conteúdo intracelular de AMP cíclico induzido pelo forskolin. Determinaçao da formaçao de fosfatos de inositol. nível basal e efeito do carbacol. efeito do carbacol e de antagonistas muscarínicos, atropina, pfHHSiD e de metoctramina, sobre o conteúdo intracelular de fosfatos de inositol determinado pelo carbacol. Observamos que: O tratamento estrogênico aumentou o peso uterino dos animais. Porém, nenhuma alteraçao foi observada no miométrio. o Houve urna relaçao linear entre a ligaçao específica do 3[H]QNB e a concentraçao de proteínas de membrana, a ligaçao máxima ocorreu com a concentraçao de 240 mg/ml. A concentraçao de...(au)


Subject(s)
Estrogens , Muscarinic Antagonists , Myometrium , Receptors, Muscarinic
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