@phdthesis{
author = "Gajić Bojić, Milica",
year = "2024",
abstract = "U posljednje vrijeme uloga GABA (eng. gamma-aminobutyric acid, GABA) i GABAA receptora (GABAAR) na periferiji postaje sve važnija. Iako su dokazi još uvijek ograničeni, sugerisano je da se hipotenzivni efekti benzodiazepina (BZD) i drugih liganada GABAAR, pored poznatih mehanizama neuromodulacije u centralnom i perifernom nervnom sistemu, ostvaruju i modulacijom "vaskularnih" GABAAR u perifernim krvnim sudovima. Pretpostavili smo da različiti pozitivni alosterni modulatori (PAM) GABAAR ispoljavaju direktne vazodilatatorne efekte preko vaskularnih GABAAR koji su eksprimirani na aorti pacova.Ispitivanja predstavljena u ovoj disertaciji sprovedena su u dva pravca. U jednom pravcu vršeno je dokazivanje prisustva α1-6 i γ2 podjedinica GABAAR u aorti pacova, primjenom RT-PCR (eng. real–time–polymerase chain reaction) i imunohistohemijske analize. U drugom pravcu vršeno je izometrijsko mjerenje kontrakcije izolovanih prstenova pacovske aorte u prisustvu različitih selektivnih i neselektivnih PAM-ova GABAAR: zolpidema (α1–selektivan), XHe–III–074 (α4–selektivan), MP–III–022, MP-III-058, GL-II-74, GL-II-73 (α5–selektivni), DK-I-56-1 (α6–selektivan), SH-I-048A, midazolama i diazepama (neselektivni). Rezultati RT-PCR analize dokazali su ekspresiju iRNK α1–5 podjedinica u homogenatu tkiva torakalne aorte pacova, dok za α6 podjedinicu nije utvrđena ekspresija iRNK. Imunohistohemijskim bojenjem histoloških presjeka pacovske aorte potvrđena je ekspresija proteina α1-5 podjedinice, kao i ekspresija proteina γ2 podjedinice, te je utvrđeno da su identifikovani proteini lokalizovani na vaskularnom glatko-mišićnom sloju aorte pacova. Testovi u kupatilu za rad sa izolovanim prstenovima aorte otkrili su značajne vazodilatacijske efekte svih ispitivanih PAM-ova (preko 50% ostvarene relaksacije prekontrahovanih preparata), pri čemu je diazepam bio najefikasniji ligand, dok je zolpidem pokazao najslabije vaskularne efekte. Flumazenil, kao antagonista BZD mjesta na GABAAR, pokazao je slabu vazoaktivnost per se, ali je značajno smanjio vazodilatacijske efekte testiranih PAM-ova, što upućuje da se njihova vazoaktivnost ostvaruje modulacijom BZD veznog mjesta na vaskularnim GABAAR. Primjenjen u koncentraciji od 10-4 M, flumazenil je značajno smanjio relaksaciju izazvanu midazolamom (P < 0,01), kao i relaksaciju izazvanu MP-III-058 (P < 0,001), i uzrokovao tako pomijeranje njihovih krivih odnosa koncentracija-odgovor udesno i naniže. Vazodilatacijski efekti diazepama nisu smanjeni u prisustvu antagonista (bikukulina, odnosno PK11195), osim onih ostvarenih pri nižim koncentracijama diazepama (10-7 M i 3x10-7 M). TSPO antagonista PK11195 ispoljio je vazodilatacijske efekte na fenilefrin (FE)- prekontrahovanim preparatima, uporedive sa efektima diazepama, što nije bio slučaj sa antagonistima bikukulinom i flumazenilom. Maksimalna efikasnost testiranih PAM-ova postignuta je pri koncentraciji (10-4 M) koju je teško postići u in vivo sistemu, osim u uslovima predoziranja i zloupotrebe lijekova. Primjenjeni u ovako visokoj koncentraciji, vazodilatacijski efekti testiranih PAM-ova bili su uporedivi sa efektima prazosina, kao referentnog antagoniste adrenergičke kontrakciije. Međutim, njihov relaksacijski potencijal značajno je niži od prazosinskog, na šta ukazuju uočene razlike u vrijednostima pEC50. Za sve ispitivane PAM-ove vazodilatacijski efekti bili su izraženiji kod FE- prekontrahovanih u odnosu na KCl- prekontrahovane preparate..., Recently, the role of GABA (gamma-aminobutyric acid) and GABAA receptors (GABAAR) in the periphery has become increasingly important. Although the evidence is still limited, it has been suggested that the hypotensive effects of benzodiazepines (BZD) and other GABAAR ligands, in addition to the known mechanisms of neuromodulation in the central and peripheral nervous system, are also achieved by the modulation of "vascular" GABAAR in peripheral blood vessels. We hypothesized that various positive allosteric modulators (PAM) of GABAAR exert direct vasodilator effects via vascular GABAAR expressed on rat aorta.The investigations presented in this dissertation were carried out in two directions. In one direction, the presence of α and γ2 subunits of the GABAAR in the rat aorta was demonstrated, using RT-PCR (real–time–polymerase chain reaction) and immunohistochemical analysis. In the second direction, isomeric measurement of contraction of isolated rat aortic rings in the presence of various selective and non-selective PAMs of GABAAR was performed: zolpidem (α1–selective), XHe-III-074 (α4–selective), MP-III-022, MP-III-058, GL-II-74, GL-II-73 (α5–selective), DK-I-56-1 (α6–selective), SH-I-048A, midazolam and diazepam (non-selective).The results of RT-PCR analysis demonstrated the expression of mRNA α1–5 subunits in the tissue homogenate of the rat thoracic aorta, while no mRNA expression was found for the α6 subunit. Immunohistochemical staining of histological sections of the rat aorta confirmed the expression of the α1-5 subunit protein, as well as the expression of the γ2 subunit protein, and it was determined that the identified proteins are localized on the vascular smooth-muscle layer of the rat aorta. Assays with isolated aortic rings revealed significant vasodilatory effects of all tested PAMs (over 50% relaxation achieved in precontracted preparation), where diazepam was the most effective ligand, while zolpidem showed the weakest vascular effects. Flumazenil exhibited weak vasoactivity per se, but significantly prevented the relaxant effects of tested PAMs. Applied at a concentration of 10-4 M, flumazenil significantly reduced midazolam-induced relaxation (P < 0.01), as well as MP-III-058-induced relaxation (P < 0.001), shifting their concentration-response curves to the right and down. The vasodilating effects of diazepam were not reduced in the presence of the antagonist (bicuculline, ie PK11195), except for those achieved at lower concentrations of diazepam (10-7 M and 3x10-7 M). The TSPO antagonist PK11195 exhibited vasodilatory effects on phenylephrine (FE)-precontracted preparations, comparable to the effects of diazepam, which was not the case with the antagonists bicuculline and flumazenil. The maximum efficiency of the tested PAMs was achieved at a concentration (10-4 M) that is difficult to achieve in the in vivo system, except in conditions of overdose and drug abuse. Applied in such a high concentration, the vasodilatory effects of the tested PAMs were comparable to the effects of prazosin, as a reference antagonist of adrenergic contraction. However, their relaxation potential is significantly lower than that of prazosin, as indicated by the observed differences in pEC50 values. For all tested PAMs, the vasodilation effects were more pronounced in FE-precontracted compared to KCl-precontracted preparations...",
publisher = "Универзитет у Београду, Фармацеутски факултет",
journal = "Универзитет у Београду",
title = "Vaskularna aktivnost pozitivnih alosternih modulatora GABAA receptora kod pacova",
url = "https://hdl.handle.net/21.15107/rcub_nardus_22506"
}
