title: Benzodiazepine modulation of recombinant alpha1beta3gamma2 GABA(A) receptor function efficacy determination using the Cytosensor microphysiometer.
abstract: Gamma-aminobutyric acid (GABA) dose dependently increased extracellular acidification rate in Ltk cells stably expressing human recombinant alpha1beta3gamma2 GABA(A) receptors but had no effect in non-transfected controls. Cells seeded at 1 x 10(5) cells/cup, with 4-5 days induction, had basal acidification rates of 105+/-2 microVs(-1) at 37 degrees C (mean+/-standard error of mean, n=37). GABA responses had a characteristic time-course with an initial alkalinisation followed by a peak of acidification, which was optimized by increasing agonist exposure from 15 s to 25-30 s. The maximum concentration of GABA tested (100 microM) produced a 40+/-2% increase over basal acidification rate (n=3), with an EC50 of 15.5 microM and a Hill slope of 1.5. Responses were specifically antagonized by bicuculline and could be modulated by benzodiazepine ligands with varying efficacies. Full benzodiazepine agonists flunitrazepam (1 microM) and zolpidem (10 microM) significantly potentiated the response to 10 microM GABA by 124+/-15% (n=7) and 117+/-23% (n=3), respectively. The partial agonist bretazenil (100 nM) produced a 45+/-13% (n=3) potentiation whilst the inverse agonist DMCM (10 microM) (methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate) inhibited the response to 20 microM GABA by 53+/-5%. The microphysiometer offers an alternative functional measure for GABA(A) receptors with the sensitivity to measure subtle modulatory effects of benzodiazepine site ligands and to determine their relative efficacy.
authors.affiliation: Merck, Sharp and Dohme Research Laboratories, Neuroscience Research Centre, Harlow, Essex, UK.
authors.forename: A J
authors.initials: AJ
authors.lastname: Smith
authors.forename: R M
authors.initials: RM
authors.lastname: McKernan
authors.forename: J R
authors.initials: JR
authors.lastname: Atack
mesh_terms: D000818:Animals; D001569:Benzodiazepines / Q000494:pharmacology*; D001570:Benzodiazepinones / Q000494:pharmacology; D015374:Biosensing Techniques; D002452:Cell Count / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D005057:Eukaryotic Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D005442:Flumazenil / Q000494:pharmacology; D005445:Flunitrazepam / Q000494:pharmacology; D018757:GABA Modulators / Q000494:pharmacology; D006801:Humans; D011725:Pyridines / Q000494:pharmacology; D011963:Receptors, GABA-A / Q000187:drug effects* / Q000502:physiology; D011994:Recombinant Proteins / Q000187:drug effects*; D013997:Time Factors; D000077334:Zolpidem; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
chemical_list: D001570:Benzodiazepinones; D018757:GABA Modulators; D011725:Pyridines; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; D001569:Benzodiazepines; D005442:Flumazenil; D005680:gamma-Aminobutyric Acid; D005445:Flunitrazepam; D000077334:Zolpidem; C054626:bretazenil