Avaliação dos efeitos de fármacos benzodiazepínicos sobre o catabolismo de nucleotídeos, nucleosídeos e acetilcolina em encéfalo de zebrafish adulto : (Danio rerio)

• Autor(a) principal: Altenhofen, Stefani
• Data de Publicação: 2012
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: Biblioteca Digital de Teses e Dissertações da PUC_RS
• Texto Completo: http://tede2.pucrs.br/tede2/handle/tede/5457
• topic: BIOLOGIA CELULAR; BIOLOGIA MOLECULAR; BENZODIAZEPINAS; ANSIEDADE; ACETILCOLINESTERASE; ADENOSINA DEAMINASE; CNPQ::CIENCIAS BIOLOGICAS::BIOLOGIA GERAL

Benzodiazepines, such as diazepam and midazolam, are a widely used class of drugs for anxiety treatment, with anxiolytic, hypnotic, and anticonvulsant properties. The use of zebrafish (Danio rerio) as a model for evaluating pharmacological mechanisms has gained importance due to their rapid development and high sensitivity to drugs. Studies have shown that behavioral parameters were altered in zebrafish after benzodiazepine treatment. Many neurotransmitter systems have been identified in this species, including purinergic and cholinergic system. Purinergic system is characterized by the action of ATP and adenosine on purinoreceptor P2 and P1, respectively. The levels of these molecules are regulated by ectonucleotidases, especially nucleoside triphosphate diphosphohydrolase (NTPDases) and ecto-5'-nucleotidase, which constitute the extracellular cascade for ATP hydrolysis to adenosine. Adenosine can be subsequently deaminated to inosine by action of adenosine deaminase (ADA). ATP is coreleased with other neurotransmitters, including acetylcholine, and has been demonstrated that adenosine can control the release of acetylcholine. Cholinergic system is characterized by the action of acetylcholine (ACh) on muscarinic and nicotinic receptors. The level of this molecule is regulated by acetylcholinesterase (AChE), which catalyzes degradation of ACh into choline and acetate. Since there are few reports relating these enzyme activities and the action mechanism of benzodiazepines, the aim of this study was evaluated the in vitro and ex vivo effects of classical benzodiazepines, such as diazepam and midazolam, on NTPDase, ecto-5'nucleotidase, ADA, and AChE activities in zebrafish brain and gene expression pattern in treatments that induced changes in enzyme activity in the ex vivo experiments. In order to elucidate whether diazepam or midazolam has direct effects on these enzymes, we performed in vitro experiments. Diazepam, at 500 μM, promoted a decrease on ATP hydrolysis (66%), whereas this drug, at 10-500 μM, reduced ADP hydrolysis (40-54%, respectively). Midazolam also decreased ATP (16-71% for 10-500 μM, respectively) and ADP hydrolysis (48-73% for 250-500 μM, respectively), and ecto-ADA activity (26-27.5% for 10-500 μM, respectively). Diazepam and midazolam did not induce significant changes on ecto-5´-nucleotidase activity at the concentrations tested. Concerning to AChE activity, 500 μM diazepam promoted a decrease on ACh hydrolysis (19%), whereas midazolam, at 50-500 μM, reduced AChE activity (18-79%, respectively). For ex vivo experiments, diazepam or midazolam exposures did not alter NTPDase activities in zebrafish brain membranes. AMP hydrolysis was decreased in animals treated with of 0.5 and 1mg/L midazolam (31.5% and 36.1%, respectively) when compared to the control group. However, diazepam was unable to alter ecto-5 -nucleotidase. Both drugs significantly decreased the ecto-ADA activity, whereas diazepam and midazolam reduced the adenosine hydrolysis at a concentration of 1.25 mg/L (30.85%) and 1 mg/L (32.8%), respectively. Diazepam did not alter cytosolic-ADA activity; however, the exposure to 0.1 mg/L midazolam induced a significant increase in cytosolic-ADA (39.9%) when compared with the control group. The gene expression pattern demonstrated that the CD73 transcript levels were increased (41.7%) after treatment with 0.5 mg/L midazolam. Moreover, the changes caused by diazepam and midazolam in the ADA activity are not related to the transcriptional control. Concerning the cholinerg signaling, diazepam decreased ACh hydrolysis at 1.25 mg/L (30.7%) when compared to the control group. Similarly, the exposure to 0.5 mg/L midazolam also changed the enzymatic activity of AChE promoting an increase in the ACh hydrolysis (36.7%). It is possible to suggest that these drugs can induce a direct effect on the enzyme activities, since we observed a decreased on nucleotide and nucleoside hydrolysis after in vitro exposure. In addition, the alteration on AMP hydrolysis, ADA and AChE activities suggest a modulation of extracellular adenosine and ACh levels induced by benzodiazepine exposure.