Učinci dugotrajne primjene hipnotika zolpidema u kulturi HEK 293 stanica sa stabilno eksprimiranim GABAa receptorima

master's thesis

• Title (english): The Effect of Long-Term Use of Hypnotic Zolpidem in the Culture of HEK 293 Cells with Stably Expressing GABAa Receptors
• Author: Letizia Antonella Vukorep
• Mentor: Frane Božić (mentor)
• Mentor: Jelena Šuran (mentor)
• Committee member: Frane Božić
• Committee member: Josipa Vlainić
• Committee member: Jelena Šuran
• Granter: University of Zagreb; Faculty of Veterinary Medicine; (Department of Pharmacology and Toxicology); Zagreb
• Defense date and country: 2015-06-11, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Veterinary Medicine; Basic and Preclinical Veterinary Sciences

Abstract

Hipnotik zolpidem svoje djelovanje ostvaruje vezivajući se na benzodiazepinska mjesta na α1- GABAA receptorima. Cilj ovog istraživanja bio je ispitati utjecaj trajanja primjene zolpidema i njegovog ustezanja, kao i ulogu α1 podjedinice GABAA receptora u razvoju ovisnosti i tolerancije. Naime, rekombinantni receptori koriste se za istraživanje mehanizama koji su uključeni u različite procese u mozgu a posebice pojedinih podtipova receptora. Kako bismo ispitali utjecaj kronične primjene zolpidema, HEK 293 stanice sa stabilnom ekspresijom rekombinantnih GABAA receptora izložili smo zolpidemu tijekom sedam uzastopnih dana, dok je ustezanje trajalo 24, 48, 72 i 96 sati. Vezanjem radioaktivnih liganda utvrdili smo da kroničnom primjenom zolpidema ne dolazi do promjena u broju GABAA receptora ili u ekspresiji mRNA podjedinica. Tek nakon 96- satnog ustezanja uočili smo povećani broj veznih mjesta i povećanu ekspresiju α1 i γ2S podjedinica mRNA. Prema tome, može se pretpostaviti da je ustezanje zolpidema uzrokom promijenjene ekspresije podjedinica GABAA receptora u našem eksperimentalnom sustavu. Štoviše, primjenom zolpidema i njegovim ustezanjem (kroz sve vremenske točke) došlo je do alosteričkog razdvajanja veznog mjesta za GABA i benzodiazepinskog veznog mjesta na GABAA receptorskom kompleksu kao funkcionalnog odgovora na djelovanje lijeka.
Ovo istraživanje daje uvid u molekularne i stanične mehanizme koje dovode do promjena u funkciji GABA receptora nakon kronične primjene zolpidema i njegovog ustezanja. Nađene promjene bi mogle biti povezane s tolerancijom i ovisnosti nakon dulje primjene, kako zolpidema tako i drugih GABAergičnih lijekova.

Abstract (english)

Hypnotic zolpidem produces its effects via the benzodiazepine binding site in α1-containing GABAA receptors. The aim of the study was to assess the influence of duration of zolpidem treatment and its withdrawal, as well as the role of α1-containing GABAA receptors in the development of physical dependence and tolerance. Namely, recombinant receptors can be used to characterize mechanisms involved in different processes in the brain and to delineate the contribution of specific receptor subtypes. To address the influence of chronic zolpidem treatment we exposed HEK293 cells stably expressing recombinant GABAA receptors for seven consecutive days, while withdrawal periods lasted for 24, 48, 72 and 96 hours. Using radioligand binding studies we determined that chronic zolpidem treatment did not induce changes in either GABAA receptor number or in the expression of subunit mRNAs. We observed the enhancement of binding sites and upregulated expression of subunit mRNAs only following 96-hour withdrawal. Moreover, zolpidem treatment and its withdrawal (all time points) induced functional uncoupling between GABA and benzodiazepine binding sites in the GABAA receptor complex. Accordingly, it might be assumed that zolpidem withdrawal- induced uncoupling of GABAA receptors is associated with altered GABAA receptor subunit mRNA expression. The results presented here provide an insight into molecular and cellular mechanisms probably underlying adaptive changes of GABAA receptor function in response to chronic usage and withdrawal of zolpidem and perhaps the observed molecular changes could be linked to the tolerance and dependence produced upon prolonged treatment with other GABAergic drugs.

• Keywords: zolpidem; kronična primjena i ustezanje; GABAA receptori; mRNA; radioaktivno obilježavanje veznih mjesta
• Keywords (english): zolpidem; chronic treatment i withdrawal; GABAA receptors; radioligand binding sites; mRNA
• Language: croatian
• URN:NBN: urn:nbn:hr:178:133400
• Study programme: Title: Veterinary Medicine; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: doktor/doktorica veterinarske medicine (doktor/doktorica veterinarske medicine)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Access restricted to students and staff of home institution
• Created on: 2016-01-18 09:26:51