Naslov
[3H]FLUNITRAZEPAM AND [3H]DEHYDROEPIANDROSTERONE SULFATE BINDING TO NONTRANSFECTED HUMAN EMBRYONIC KIDNEY 293 CELLS

Autori
Jazvinšćak, Maja ; Mirković, Kety ; Švob, Dubravka ; Peričić, Danka

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Fundamental & Clinical Pharmacology Vol. 13/Suppl. 1 / - Budimpešta : Elsevier, 1999

Skup
2nd European Congress of Pharmacology

Mjesto i datum
Budimpešta, Mađarska, 03.07.1999. - 07.07.1999

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
flunitrazepam; dehydroepiandrosterone sulfate; nontransfected HEK 293 cells; binding

Sažetak
Folowing transfection with cDNAs encoding for different subunits of the GABAA receptors, human embryonic kidney (HEK) 293 cells are frequently being used for biosynthesis of recombinant GABAA receptors. Subsequently, radioligand binding studies are used to evaluate the kinetic and pharmacological properties of the recombinant receptors. Therefore, it is important to know the characteristics of radioligands binding to membranes of nontransfected HEK 293 cells. The membranes were prepared as previously described ((Fuchs et al., 1995 ; Peričić et al., 1998). The benzodiazepine [3H]flunitrazepam (2 nM) and the neurosteroid [3H]dehydroepiandrosterone sulfate (DHEAS ; 10 nM) were used as radioligands. The nontransfected HEK 293 cells bound [3H]flunitrazepam with high nanomolar, and two other benzodiazepine agonists (clonazepam and diazepam) and two peripheral-type benzodiazepine ligands (4’-chlorodiazepam and PK 11195) with micromolar affinity. GABA (100 mM) and flumazenil failed to affect [3H]flunitrazepam binding. All this suggested that these binding sites are different from the central- but also from the peripheral-type benzodiazepine receptors, and that the binding of [3H]flunitrazepam does not interefere with the binding of the same radioligand to recombinant a1b2g2s GABAA receptor expressed in stably transfected HEK 293 cells (Peričić et al., in preparation). The equilibrium for specific [3H]DHEAS binding was reached after 90 min at 25 0 C. The Kd for the neurosteroid was in the micromolar range. [3H]DHEAS binding could be displaced neither by thiopental, a barbiturate supposed to interact with the neurosteroid binding site, nor by spironolactone, the antagonist of mineralocorticoid binding sites.

Izvorni jezik
Engleski

Znanstvena područja
Temeljne medicinske znanosti

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