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Previous issue date: 2012-12-26 / 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. / F?rmacos benzodiazep?nicos, como diazepam e midazolam, s?o muito usados na pr?tica cl?nica para o tratamento da ansiedade, possuindo propriedades ansiol?ticas, hipn?ticas e anticonvulsivantes. O uso do zebrafish (Danio rerio) como modelo para avaliar mecanismos farmacol?gicos tem ganhado grande import?ncia devido ao r?pido desenvolvimento e alta sensibilidade a drogas que essa esp?cie possui. Estudos t?m demonstrado que par?metros comportamentais mostraram-se alterados em zebrafish ap?s tratamento com benzodiazep?nicos. Muitos sistemas de neurotransmiss?o foram identificados nessa esp?cie, incluindo os sistemas purin?rgico e colin?rgico. O sistema purin?rgico ? caracterizado pela a??o do ATP e adenosina (ADO) nos purinoreceptores P2 e P1, respectivamente. Os n?veis dessas mol?culas s?o regulados pela a??o das ectonucleotidases, especialmente as nucleos?deo trifosfato difosfoidrolases (NTPDases) e a ecto-5 -nucleotidase, que catalisam a hidr?lise do ATP a adenosina. A adenosina pode ser desaminada a inosina pela a??o da adenosina desaminase (ADA). O ATP ? coliberado com outros neurotransmissores, entre eles a acetilcolina, e tem sido demonstrado que a adenosina pode controlar a libera??o de acetilcolina. O sistema colin?rgico ? caracterizado pela a??o da acetilcolina (ACh) nos receptores muscar?nicos e nicot?nicos. O n?vel dessa mol?cula ? regulado pela acetilcolinesterase (AChE), que catalisa a degrada??o da ACh em colina e acetato. Uma vez que existem poucos relatos relacionando esses sistemas enzim?ticos e a a??o de f?rmacos benzodiazep?nicos, o objetivo deste estudo foi avaliar o efeito in vitro e ex vivo do tratamento com f?rmacos benzodiazep?nicos, tais como diazepam e midazolam, sobre a atividade das NTPDases, ecto-5'-nucleotidase, ADA and AChE no enc?falo de zebrafish e o padr?o de express?o g?nica nos tratamentos que induziram altera??es na atividade enzim?tica nos experimentos ex vivo. A fim de elucidar se o diazepam e o midazolam t?m efeitos diretos nessas enzimas, experimentos in vitro foram realizados. Na concentra??o de 500 μM, o diazepam diminuiu a hidr?lise de ATP (66%) e, nas concentra??es de 10-500 μM, este f?rmaco reduziu a hidr?lise de ADP (40-54%, respectivamente). O midazolam tamb?m diminuiu a hidr?lise do ATP (16-71% para 10-500 μM, respectivamente), ADP (48-73% para 250-500 μM, respectivamente) e a atividade da ecto-ADA (26-27,5% para 10-500 μM, respectivamente). Diazepam e midazolam n?o induziram altera??es significativas sobre a atividade da ecto-5?-nucleotidase nas concentra??es testadas. Com rela??o ? atividade da AChE, o diazepam, 500 μM, promoveu uma diminui??o na hidr?lise de ACh (19%) e o midazolam, nas concentra??es de 50-500 μM, reduziu a atividade da AChE (18-79%, respectivamente). Nos experimentos ex vivo, as exposi??es ao diazepam e midazolam n?o alteraram a atividade enzim?tica das NTPDases em membranas cerebrais de zebrafish. A hidr?lise do AMP diminuiu em animais tratados com 0.5 mg/L e 1 mg/L de midazolam (31.5% e 36.1%, respectivamente) quando comparados com o grupo controle. Entretanto, o diazepam foi incapaz de alterar a atividade da ecto-5 -nucleotidase. Ambos os f?rmacos diminu?ram significativamente a atividade da ecto-ADA, sendo que o diazepam e o midazolam reduziram a hidr?lise da adenosina na concentra??o de 1.25 mg/L (30.85%) e 1 mg/L (32.8%), respectivamente. O diazepam n?o alterou a atividade da ADA citos?lica, no entanto a exposi??o a 0.1 mg/L de midazolam induziu um significativo aumento na atividade dessa enzima (39.9%) quando comparado ao grupo controle. O padr?o de express?o g?nica demonstrou que os n?veis de transcritos do CD73 apresentaram-se reduzidos (41,7%) ap?s o tratamento com 0.5 mg/L de midazolam. Com rela??o a sinaliza??o colin?rgica, diazepam diminuiu a hidr?lise da ACh na concentra??o de 1.25 mg/L (30.7%) quando comparado ao grupo controle. Similarmente, a exposi??o ? concentra??o de 0.5 mg/L de midazolam tamb?m alterou a atividade enzim?tica da AChE, promovendo um aumento na hidr?lise da ACh (36.7%). ? poss?vel sugerir que essas drogas podem induzir um efeito direto na atividade enzim?tica, uma vez que foi observada uma diminui??o na hidr?lise de nucleot?deos e nucleos?deos ap?s a exposi??o in vitro. Al?m disso, as altera??es na hidr?lise do AMP e atividade da ADA e da AChE sugerem uma modula??o dos n?veis extracelulares de adenosina e acetilcolina induzidos pela exposi??o aos f?rmacos benzodiazep?nicos.
| Identifer | oai:union.ndltd.org:IBICT/oai:tede2.pucrs.br:tede/5457 |
| Date | 26 December 2012 |
| Creators | Altenhofen, Stefani |
| Contributors | Bonan, Carla Denise |
| Publisher | Pontif?cia Universidade Cat?lica do Rio Grande do Sul, Programa de P?s-Gradua??o em Biologia Celular e Molecular, PUCRS, BR, Faculdade de Bioci?ncias |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | Portuguese |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da PUC_RS, instname:Pontifícia Universidade Católica do Rio Grande do Sul, instacron:PUC_RS |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | 8198246930096637360, 600, 600, 36528317262667714 |
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