loreclezole and methyl-6-7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate

The hypothalamus influences a number of autonomic functions. The activity of hypothalamic neurons is modulated in part by release of the inhibitory neurotransmitter GABA onto these neurons. GABA(A) receptors are formed from a number of distinct subunits, designated alpha, beta, gamma, delta, epsilon, and theta, many of which have multiple isoforms. Little data exist, however, on the functional characteristics of the GABA(A) receptors present on hypothalamic neurons. To gain insight into which GABA(A) receptor subunits are functionally expressed in the hypothalamus, we used an array of pharmacologic assessments. Whole cell recordings were made from thin hypothalamic slices obtained from 1- to 14-day-old rats. GABA(A) receptor-mediated currents were detected in all neurons tested and had an average EC(50) of 20 +/- 1.6 microM. Hypothalamic GABA(A) receptors were modulated by diazepam (EC(50) = 0.060 microM), zolpidem (EC(50) = 0.19 microM), loreclezole (EC(50) = 4.4 microM), methyl-6,7-dimethoxy-4-ethyl-beta-carboline (EC(50) = 7.7 microM), and 5alpha-pregnan-3alpha-hydroxy-20-one (3alpha-OH-DHP). Conversely, these receptors were inhibited by Zn(2+) (IC(50) = 70.5 microM), dehydroepiandrosterone sulfate (IC(50) = 16.7 microM), and picrotoxin (IC(50) = 2.6 microM). The alpha4/6-selective antagonist furosemide (10-1,000 microM) was ineffective in all hypothalamic neurons tested. The results of our pharmacological analysis suggest that hypothalamic neurons express functional GABA(A) receptor subtypes that incorporate alpha1 and/or alpha2 subunits, beta2 and/or beta3 subunits, and the gamma2 subunit. Our results suggest receptors expressing alpha3-alpha6, beta1, gamma1, and delta, if present, represent a minor component of functional hypothalamic GABA(A) receptors.

Topics: Anesthetics; Animals; Animals, Newborn; Anticonvulsants; Carbolines; Convulsants; Dehydroepiandrosterone Sulfate; Diazepam; Diuretics; Female; Furosemide; GABA Agonists; GABA Modulators; gamma-Aminobutyric Acid; Hypothalamus; Male; Membrane Potentials; Organ Culture Techniques; Patch-Clamp Techniques; Picrotoxin; Pregnanolone; Pyridines; Rats; Rats, Sprague-Dawley; Receptors, GABA-A; Triazoles; Zinc; Zolpidem

CA1 pyramidal cells were voltage clamped, and GABA was applied to individual cells with a modified U-tube, rapid drug application system. With Vh = -50 mV, inward currents elicited by 10 microM GABA were inhibited by GABAA receptor (GABAR) antagonists and were baclofen insensitive, suggesting that GABA actions on isolated CA1 pyramidal cells were GABAR mediated. GABA concentration-response curves averaged from all cells were fitted best with a two-site equation, indicating the presence of at least two GABA binding sites, a higher-affinity site (EC50-1 = 11.0 microM) and a lower-affinity site (EC50-2 = 334.2 microM), on two or more populations of cells. The effects of GABAR allosteric modulators on peak concentration-dependent GABAR currents were complex and included monophasic (loreclezole) or multiphasic (diazepam) enhancement, mixed enhancement/inhibition (DMCM, zolpidem) or multiphasic inhibition (zinc). Monophasic (70% of cells) or biphasic (30% of cells) enhancement of GABAR currents by diazepam suggested three different sites on GABARs (EC50-1 =1.8 nM; EC50-2 = 75.8 nM; EC50-3 = 275.9 nM) revealing GABAR heterogeneity. The imidazopyridine zolpidem enhanced GABAR currents in 70% of cells with an EC50 = 222.5 nM, suggesting a predominance of moderate affinity alpha2 (or alpha3-) subtype-containing BZ Type IIA receptors. A small fraction of cells (10%) had a high affinity for zolpidem, something that is suggestive of alpha1 subtype-containing BZ Type I receptors. The remaining 30% of cells were insensitive to or inhibited by zolpidem, suggesting the presence of alpha5 subtype-containing BZ Type IIB receptors. Whether BZ Type I and Type II receptors coexist could not be determined. The beta-carboline methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) inhibited GABAR currents in all cells at midnanomolar concentrations, but in addition, potentiated GABAR currents in some cells at low nanomolar concentrations, characterizing two groups of cells, the latter likely due to functional assembly of alpha5betaxgamma2GABARs. In all cells, GABAR currents were moderately sensitive (EC50 = 9 microM) to loreclezole, consistent with a relatively greater beta3 subtype, than beta1 subtype, subunit mRNA expression. Two populations of cells were identified based on their sensitivities to zinc(IC50 = 28 and 182 microM), suggesting the presence of at least two GABAR isoforms including alpha5beta3gamma2 GABARs. Consistent with the heterogeneity of expression of

Topics: Allosteric Regulation; Animals; Anticonvulsants; Carbolines; Convulsants; Diazepam; Dose-Response Relationship, Drug; GABA Modulators; gamma-Aminobutyric Acid; Hippocampus; Hypnotics and Sedatives; Membrane Potentials; Patch-Clamp Techniques; Pyramidal Cells; Pyridines; Rats; Rats, Sprague-Dawley; Receptors, GABA-A; Triazoles; Zinc; Zolpidem

Human NT2 teratocarcinoma cells differentiate into neuron-like NT2-N cells when treated with retinoic acid. GABA evoked concentration-dependent whole-cell currents in NT2-N cells with an EC50 of 21.8 microM and a Hill slope of 1.2. GABAA receptor (GABAR) currents reversed at ECl- and did not display voltage-dependent rectification. GABAR single channels opened in bursts to a 23 pS main conductance level and a 19 pS subconductance level, with infrequent openings to a 27 pS conductance level. Kinetic properties of the main conductance level were similar to other native and recombinant GABAR channels. Diazepam and zolpidem enhanced GABAR currents with moderate affinity, whereas methyl-6, 7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate inhibited GABAR currents. Loreclezole enhanced GABAR currents with high affinity, but furosemide antagonized GABAR currents with low affinity. The neurosteroids alphaxalone and pregnenolone sulfate appropriately modulated GABAR currents. Zinc blocked GABAR currents with low affinity, but lanthanum did not significantly alter NT2-N GABAR currents. Reverse transcription PCR (RT-PCR) performed on RNA from NT2-N cells clearly detected transcripts encoding human alpha2, alpha3, alpha5, beta3, gamma3, and pi subtypes. The combined pharmacological and RT-PCR results are most consistent with a single or predominant GABAR isoform composed of an alpha2 and/or alpha3 subtype combined with the beta3 and gamma3 subtypes. The data do not rule out receptors containing combinations of alpha2 and/or alpha3 subtypes with the alpha5 subtype or receptors with both beta1 and beta3 subtypes. The presence or absence or the pi subunit in functionally expressed receptors could not be determined.

Topics: Anticonvulsants; Bicuculline; Carbolines; Convulsants; Diazepam; Diuretics; DNA Primers; Furosemide; GABA Modulators; GABA-A Receptor Agonists; GABA-A Receptor Antagonists; gamma-Aminobutyric Acid; Humans; Hypnotics and Sedatives; Lanthanum; Neurons; Patch-Clamp Techniques; Pentobarbital; Phenobarbital; Picrotoxin; Polymerase Chain Reaction; Pregnanediones; Pregnenolone; Pyridines; Receptors, GABA-A; RNA, Messenger; Teratocarcinoma; Triazoles; Tumor Cells, Cultured; Zinc; Zolpidem

The pharmacological properties of gamma-aminobutyric acid (GABA) type A receptor (GABAR) currents recorded from hippocampal dentate granule cells acutely dissociated from 28-35-day-old rats were characterized using the whole-cell patch-clamp technique. Granule cells were voltage-clamped to 0 mV, and GABA was applied using a modified U-tube rapid-application technique. All granule cells were moderately sensitive to GABA (EC50 = 47 microM). All granule cell GABAR currents were uniformly sensitive to Zn2+ (IC50 = 29 microM), diazepam (EC50 = 158 nM), zolpidem (EC50 = 75 nM), and dimethoxyl-4-ethyl-beta-carboline-3-carboxylate (IC50 = 60 nM). GABAR currents from only 50% of granule cells were sensitive to loreclezole (EC50 = 9 microM). These data suggest that hippocampal dentate granule cells expressed GABARs with distinctive pharmacological properties of two types: loreclezole-sensitive and -insensitive receptors. It is likely that these distinctive properties were due to the specific GABAR subtypes that assembled to produce distinct granule cell GABAR isoforms.

Topics: Allosteric Regulation; Animals; Anticonvulsants; Carbolines; Chlorides; Convulsants; Dentate Gyrus; Diazepam; Dose-Response Relationship, Drug; Female; gamma-Aminobutyric Acid; Hypnotics and Sedatives; In Vitro Techniques; Male; Membrane Potentials; Neurons; Patch-Clamp Techniques; Pyridines; Rats; Rats, Sprague-Dawley; Receptors, GABA-A; Triazoles; Zinc Compounds; Zolpidem

The benzodiazepine site on the gamma-aminobutyric acid(A) (GABAA) receptor is the principle site of action for a number of structurally diverse compounds, including the beta-carbolines, many of which bind with high affinity. The apparent reversal of inhibition and potentiation by high concentrations of methyl-6,7-dimethoxy-4-ethyl-beta-carboline (DMCM) and other beta-carbolines has been reported by several groups and is insensitive to the benzodiazepine antagonist Ro 15-1788. By using alpha 6-containing receptors, which have low affinity for benzodiazepines, we observed robust potentiation of GABAA responses by micromolar concentrations of DMCM and other beta-carbolines that is dependent on the beta subunit variant. The beta subunit-dependent potentiation by the anticonvulsant loreclezole is dependent on a single amino acid in the putative transmembrane 2 region. By using single point mutations that discriminate the loreclezole site, we show that potentiation by DMCM is also dependent on the presence of the same amino acid, Asn290, in beta 2 or beta 3 (serine in beta 1), providing evidence that the low affinity site for beta-carboline potentiation is the loreclezole site. The potentiation is independent of the alpha subunit and is more pronounced on alpha 6-containing receptors due to the lack of DMCM inhibition via the benzodiazepine site. In addition, the potentiation observed is competitive with that of loreclezole, and other beta-carbolines, such as ethyl-beta-carboline-3-carboxylate and propyl-beta-carboline-3-carboxylate, act in a similar manner. The finding that beta-carbolines can act via the loreclezole site as well as the benzodiazepine site suggests that a wider variety of compounds may act via this site and shows that compounds can interact with more than one modulatory site on the GABAA receptor.

Topics: Animals; Anticonvulsants; Benzodiazepines; Binding Sites; Carbolines; Convulsants; Drug Synergism; Female; GABA Agonists; gamma-Aminobutyric Acid; Isomerism; Macromolecular Substances; Oocytes; Point Mutation; Receptors, GABA; Triazoles; Xenopus