Effects of Mlx-8, a phospholipase A2 from Brazilian coralsnake Micrurus lemniscatus venom, on muscarinic acetylcholine receptors in rat hippocampus

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)

Preparaçäo, estabilidade e conservaçäo do fator tecidual ativador de plasminogênio (rt-PA) para uso oftalmológico / Preparation, stability and storage of tissue plasminogen activador for ophthalmolgical purpose

Introduçäo: o fator tissular ativador do plasminogênio, um componente do sistema fibrinolítico, ativa o plasminogênio em plasmina, que degrada a rede de fibrina. O t-PA recombinante (rt-PA) tem sido utilizado em oftalmologia para diminuir as complicaçöes fibrino-proliferativas pós-operatórias. A preparaçäo comercial excede a dose preconizada para uso oftalmológico e o custo por injeçäo poderia ser reduzido se o produto fosse dividido em doses. Objetivo: avaliar se o rt-PA, para uso cardiológico, pode ser utilizado em oftalmologia, se a atividade é mantida após estocagem e, eventualmente determinar quais os melhores métodos de estocagem e de utilizaçäo. Materiais e métodos: este trabalho estudou a atividade inicial do rt-PA em placa de fibrina, a atividade residual, após 6 meses de conservaçäo em freezer à 20§ e a -70§C; e ainda a esterilidade do material. Resultados: a atividade residual após 6 meses de conservaçäo em freezer à -70§C foi 40 p/cento da atividade inicial e em congelador de geladeira à -20§C foi 34 p/cento. Se descongelado e deixado à temperatura ambiente 24 h antes de ser testado a atividade residual cai para 27 p/cento. Conclusäo: O rt-PA deve ser portanto imediata após o descongelamento