Faculty of Pharmacy, Lj. (FFALJ)
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Vpliv gvanozintrifosfata na vezavne lastnosti histaminskih receptorjev H1 in H2 v astrocitih podgane = The influence of guanosine-tri-phosphate on binding characteristics of histamine H1 and H2 receptor subtypes in rat astrocytes ; enoviti magistrski študijski program FarmacijaKodila, KatjaEndogeni biogeni amin histamin je v različnih koncentracijah prisoten v celotnem organizmu. Do vseh bioloških sprememb, posredovanih s histaminom, pride preko štirih podtipov histaminskih ... receptorjev. Vsi histaminski receptorji spadajo v skupino receptorjev, sklopljenih s proteinom G. Po aktivaciji receptorja in sklopitvi s proteinom G, ta razpade na kompleksa alfa-GTP in beta gama. Podenota alfa, ki je hkrati tudi GTP-aza izmenja vezani gvanozindifosfat (GDP) za gvanozintrifosfat (GTP). Oba kompleksa nato sprožita nadaljnje efektorske sisteme v celici. V magistrski nalogi smo izvajali eksperimente na izoliranih membranah astrocitov novorojene podgane, ki dokazano izražajo histaminska receptorja H1 in H2. Z izvajanjem eksperimetov v izoliranih sistemih lahko določimo ključno lastnost liganda - njegovo afiniteto do vezave na receptor. Vzporedno smo izvajali poskuse z GTP in njegovim analogom Gpp(NH)p', ki ni podvržen hidrolizi. Z uporabo radioaktivno označenih ligandov (3H-mepiramin za H1 in 3H-tiotidin za H2) smo dokazali prisotnost vezavnih mest obeh histaminskih receptorjev v izoliranem sistemu membran astrocitov. Maksimalna gostota vezavnih mest za 3H-mepiramin je bila 107,9 +- 37,15 fmol/mg proteinov in 156,50 +- 45,64 fmol/mg proteinov za 3H-tiotidin. Ravnotežni konstanti disociacije za kompleks radioaktivno označen ligand:receptor sta bili 12,46 +- 7,10 nM za H1 in 9,37 +- 4,40 nM za H2. Eksperimentalno smo določili še parametre inhibicije vezave radioliganda (IC50, Ki) ob dodatku endogenega agonista in v prisotnosti GTP oziroma Gpp(NH)p'. Histamin je zaviral specifično vezavo 3H-mepiramina z inhibicijsko konstanto 35,11 nM, ki se je v prisotnosti GTP zvečala na 122,74 nM in ob dodatku Gpp(NH)p' na 221,23 nM. Nasprotno je histamin zaviral specifično vezavo 3H-tiotidina; vrednost Ki se je iz 95,20 nM za histamin znižala na 14,35 nM ob dodatku GTP in do 5,41 nM ob dodanem Gpp(NH)p'. Dodatek Gpp(NH)p' ima zaradi njegove nepodvrženosti hidrolizi močnejši vpliv na spremembo afinitete v primerjavi z GTP. Potrdili smo, da je 3H-mepiramin označil receptorsko vezavno mesto na histaminskem receptorju H1 ter ugotovili, da vezavne lastnosti histamina ustrezajo vezavnim lastnostim agonista na histaminskem receptorju H1. Ugotovili smo tudi, da se 3H-tiotidin obnaša kot inverzni agonist, ki se mogoče veže na drugo vezavno mesto kot histamin na histaminskem receptorju H2, vendar so za dokončno potrditev potrebne še nadaljnje raziskave.Type of material - master's thesis ; adult, seriousPublication and manufacture - Ljubljana : [K. Kodila], 2019Language - slovenianCOBISS.SI-ID - 4763505
Author
Kodila, Katja
Other authors
Kržan, Mojca, 1962-
Topics
Histamin |
histaminski receptor H1 |
histaminski receptor H2 |
gvanozin-5'-trifosfat |
astrociti
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Knjižnica emšf 0000000577 KODILA KATJA Vpliv 0000000736 IN: 0016246 Knjižnica emšf 577 KODILA KATJA Vpliv 736 IN: 0016246 |
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Kodila, Katja | ![]() |
Kržan, Mojca, 1962- | 08095 |
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