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Vpliv gvanozintrifosfata na vezavne lastnosti histaminskih receptorjev H1 in H2 v astrocitih podgane
Kodila, Katja (Author), Kržan, Mojca (Mentor) More about this mentor... This link opens in a new window

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Abstract
Endogeni 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 ?-GTP in ??. Podenota ?, 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.

Language:Slovenian
Keywords:histamin, histaminski receptor H1, histaminski receptor H2, GTP
Work type:Master's thesis/paper (mb22)
Organization:FFA - Faculty of Pharmacy
Year:2019
Views:106
Downloads:49
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Secondary language

Language:English
Title:The influence of guanosine-tri-phosphate on binding characteristics of histamine H1 and H2 receptor subtypes in rat astrocytes
Abstract:
Endogenous biogenic amine histamine is found in varying concentrations throughout an entire organism. It exerts its effects via histamine H1, H2, H3 and H4 receptors. The histamine receptors are a class of G protein–coupled receptors. After agonist binding to the receptor, G protein subunits dissociate; the ? subunit separates from the ?/? subunits. Subunit ? which is also a GTP-ase exchanges bonded guanosine diphosphate (GDP) for guanosine triphosphate (GTP). Both of the complexes (?-GTP, ?/?) subsequently activate second messenger systems in the cell. In the master's thesis, experiments were carried out on isolated membranes of neonatal rat astrocytes, which are proven to express histamine receptors H1 and H2. By performing experiments in isolated systems, the key feature of the ligand can be determined: its affinity for binding to the receptor. In parallel, we conducted experiments in the presence of GTP and its non-hydrolyzable analogue Gpp(NH)p'. Using the radiolabelled ligands (3H mepyramine for histamine receptor H1 and 3H-tiotidine for histamine receptor H2), the presence of both histamine receptor binding sites in the isolated membrane system of astrocytes was demonstrated. The maximum densities of binding sites were 107.9 ± 37.15 and 156.50 ± 45.64 fmol/mg protein for H1 and H2 receptor, respectively. The equilibrium dissociation constants for the complex radiolabelled ligand:receptor were 12.46 ± 7.10 nM for 3H-mepyramine binding to H1 receptor binding site; and 9.37 ± 4.40 nM for 3H-tiotidine binding to H2 receptor binding site. The inhibition binding parameters of endogenous agonist histamine (IC50, Ki) displacing either 3H-mepyramine or 3H-tiotidine binding were determined experimentally in the presence or absence of GTP or Gpp(NH)p'. Ki value of 35.11 nM for histamine inhibiting 3H-mepyramine binding increased to 122.74 nM in the presence of GTP and to 221.23 nM in the presence of Gpp (NH)p'. However, the Ki values of histamine displacing 3H-tiotidine binding decreased from 95.20 nM to 14.35 nM in the presence of GTP and to 5.41 nM in the presence of Gpp(NH)p'. In both cases Gpp(NH)p', due to its non-hydrolyzable nature, has a stronger effect on Ki value change. We confirmed that 3H-mepyramine marked the receptor binding site on the histamine H1 receptor and found that 3H-tiotidine behaved as an inverse agonist, which may bind to a different (possibly allosteric) binding site than histamine on the histamine H2 receptor. Further research is needed for definitive confirmation of the prevoius statement.

Keywords:histamine, histamine receptor H1, histamine receptor H2, GTP

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