A FLIPR Assay for Discovery of GABAA Receptor Modulators of Natural Origin

Planta Medica ◽  
2019 ◽  
Vol 85 (11/12) ◽  
pp. 925-933 ◽  
Author(s):  
Maria Teresa Faleschini ◽  
Anne Maier ◽  
Sarah Fankhauser ◽  
Katharina Thasis ◽  
Simon Hebeisen ◽  
...  
Keyword(s):  
Gabaa Receptor ◽  
Gabaa Receptors ◽  
Chinese Hamster ◽  
Subunit Composition ◽  
Rapid Screening ◽  
Piper Nigrum ◽  
Imaging Plate ◽  
Xenopus Laevis Oocytes ◽  
Valerenic Acid ◽  
Receptor Modulators

AbstractA fluorometric imaging plate reader (FLIPR) assay utilizing Chinese hamster ovary (CHO) cells stably transfected with GABAA receptors of α 1 β 2 γ 2 subunit composition was evaluated and validated for rapid screening of plant extract libraries and efficient localization of active compounds in extracts. Validation was performed with pure compounds and extracts known to contain allosteric GABAA receptor modulators. Plants extracts that had been previously reported as active in an assay using Xenopus laevis oocytes transiently expressing GABAA receptors of α 1 β 2 γ 2 subunit composition were also active in the FLIPR assay. A protocol for HPLC-based activity profiling was developed, whereby separations of 0.4 – 1.2 mg of extracts on an analytical HPLC column were found to be sufficient for the sensitivity of the bioassay. The protocol successfully localized the activity of known GABAergic natural products, such as magnolol in Magnolia officinalis, valerenic acid in Valeriana officinalis, and piperine in Piper nigrum extract. EC50 values of compounds (magnolol: 4.81 ± 1.0 µM, valerenic acid: 12.56 ± 1.2 µM, and piperine: 5.76 ± 0.7 µM) were found to be comparable or lower than those reported using Xenopus oocyte assays.

Intravenous Anesthetics Differentially Modulate Ligand-gated Ion Channels

2000 ◽  
Vol 92 (5) ◽  
pp. 1418-1425 ◽  
Author(s):  
Pamela Flood ◽  
Matthew D. Krasowski
Keyword(s):  
Ion Channels ◽  
Gabaa Receptor ◽  
Nicotinic Acetylcholine Receptors ◽  
Gabaa Receptors ◽  
Xenopus Laevis Oocytes ◽  
Intravenous Anesthetics ◽  
Relevant Concentration ◽  
High Concentrations ◽  
Gated Ion Channels ◽  
Ligand Gated Ion Channels

Background Heteromeric neuronal nicotinic acetylcholine receptors (nAChRs) are potently inhibited by volatile anesthetics, but it is not known whether they are affected by intravenous anesthetics. Ketamine potentiates gamma-aminobutyric acid type A (GABAA) receptors at high concentrations, but it is unknown whether there is potentiation at clinically relevant concentrations. Information about the effects of intravenous anesthetics with different behavioral profiles on specific ligand-gated ion channels may lead to hypotheses as to which ion channel effect produces a specific anesthetic behavior. Methods A heteromeric nAChR composed of alpha4 and beta4 subunits was expressed heterologously in Xenopus laevis oocytes. Using the two-electrode voltage clamp technique, peak ACh-gated current was measured before and during application of ketamine, etomidate, or thiopental. The response to GABA of alpha1beta2gamma2s GABAA receptors expressed in human embryonic kidney cells and Xenopus oocytes was compared with and without coapplication of ketamine from 1 microm to 10 mm. Results Ketamine caused potent, concentration-dependent inhibition of the alpha4beta4 nAChR current with an IC50 of 0.24 microm. The inhibition by ketamine was use-dependent; the antagonist was more effective when the channel had been opened by agonist. Ketamine did not modulate the alpha1beta2gamma2s GABAA receptor response in the clinically relevant concentration range. Thiopental caused 27% inhibition of ACh response at its clinical EC50. Etomidate did not modulate the alpha4beta4 nAChR response in the clinically relevant concentration range, although there was inhibition at very high concentrations. Conclusions The alpha4beta4 nAChR, which is predominantly found in the central nervous system (CNS), is differentially affected by clinically relevant concentrations of intravenous anesthetics. Ketamine, commonly known to be an inhibitor at the N-methyl-D-aspartate receptor, is also a potent inhibitor at a central nAChR. It has little effect on a common CNS GABAA receptor in a clinically relevant concentration range. Interaction between ketamine and specific subtypes of nAChRs in the CNS may result in anesthetic behaviors such as inattention to surgical stimulus and in analgesia. Thiopental causes minor inhibition at the alpha4beta4 nAChR. Modulation of the alpha4beta4 nAChR by etomidate is unlikely to be important in anesthesia practice based on the insensitivity of this receptor to clinically used concentrations.

Isoflavonoid derivatives from Adenocarpus cincinnatus as positive GABAA receptor modulators

Planta Medica ◽  
2013 ◽  
Vol 79 (13) ◽  
Author(s):  
DC Rueda ◽  
M de Mieri ◽  
S Hering ◽  
M Hamburger
Keyword(s):  
Gabaa Receptor ◽  
Receptor Modulators

Valerenic acid potentiates and inhibits GABAA receptors: Molecular mechanism and subunit specificity

2007 ◽  
Vol 53 (1) ◽  
pp. 178-187 ◽  
Author(s):  
S. Khom ◽  
I. Baburin ◽  
E. Timin ◽  
A. Hohaus ◽  
G. Trauner ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Gabaa Receptors ◽  
Valerenic Acid

scholarly journals A possible role for a mammalian facilitative hexose transporter in the development of resistance to drugs.

1991 ◽  
Vol 11 (7) ◽  
pp. 3407-3418 ◽  
Author(s):  
J C Vera ◽  
G R Castillo ◽  
O M Rosen
Keyword(s):  
Xenopus Oocytes ◽  
Glucose Transporter ◽  
Cytochalasin B ◽  
Chinese Hamster ◽  
Multidrug Resistant ◽  
Xenopus Laevis Oocytes ◽  
Hexose Transporter ◽  
P Glycoprotein ◽  
Glut1 Protein ◽  
Facilitative Glucose Transporter

We show that D- but not L-hexoses modulate the accumulation of radioactive vinblastine in injected Xenopus laevis oocytes expressing the murine Mdr1b P-glycoprotein. We also show that X. laevis oocytes injected with RNA encoding the rat erythroid/brain glucose transport protein (GLUT1) and expressing the corresponding functional transporter exhibit a lower accumulation of [3H]vinblastine and show a greater capacity to extrude the drug than do control oocytes not expressing the rat GLUT1 protein. Cytochalasin B and phloretin, two inhibitors of the mammalian facilitative glucose transporters, can overcome the reduced drug accumulation conferred by expression of the rat GLUT1 protein in Xenopus oocytes but have no significant effect on the accumulation of drug by Xenopus oocytes expressing the mouse Mdr1b P-glycoprotein. These drugs also increase the accumulation of [3H]vinblastine in multidrug-resistant Chinese hamster ovary cells. Cytochalasin E, an analog of cytochalasin B that does not affect the activity of the facilitative glucose transporter, has no effect on the accumulation of vinblastine by multidrug-resistant Chinese hamster cells or by oocytes expressing either the mouse Mdr1b P-glycoprotein or the GLUT1 protein. In all three cases, the drug verapamil produces a profound effect on the cellular accumulation of vinblastine. Interestingly, although immunological analysis indicated the presence of massive amounts of P-glycoprotein in the multidrug-resistant cells, immunological and functional studies revealed only a minor increase in the expression of a hexose transporter-like protein in resistant versus drug-sensitive cells. Taken together, these results suggest the participation of the mammalian facilitative glucose transporter in the development of drug resistance.

In vitro and in vivo GABAA Receptor Interaction of the Propanidid Metabolite 4-(2-[Diethylamino]-2-Oxoethoxy)-3-Methoxy-Benzeneacetic Acid

Pharmacology ◽  
2018 ◽  
Vol 103 (1-2) ◽  
pp. 10-16 ◽  
Author(s):  
Alessia Cenani ◽  
Robert J. Brosnan ◽  
Heather K. Knych
Keyword(s):  
Gabaa Receptor ◽  
Gabaa Receptors ◽  
Water Solubility ◽  
Plasma Concentrations ◽  
Receptor Activation ◽  
Receptor Interaction ◽  
Dependent Manner ◽  
Benzeneacetic Acid

Background: Propanidid is a γ-aminobutyric acid type A (GABAA) receptor agonist general anesthetic and its primary metabolite is 4-(2-[diethylamino]-2-oxoethoxy)-3-methoxy-benzeneacetic acid (DOMBA). Despite having a high water solubility at physiologic pH that might predict low-affinity GABAA receptor interactions, DOMBA is reported to have no effect on GABAA receptor currents, possibly because the DOMBA concentrations studied were simply insufficient to modulate GABAA receptors. Our objectives were to measure the propanidid and DOMBA concentration responses on ­GABAA receptors and to measure the behavioral responses of DOMBA in mice at concentrations that affect GABAA receptor currents in vitro. Methods: GABAA receptors were expressed in oocytes using clones for the human GABAA α1, β2 and γ2s subunits. The effects of DOMBA (0.2–10 mmol/L) and propanidid (0.001–1 mmol/L) on oocyte GABAA currents were studied using standard 2-electrode voltage clamp techniques. Based on in vitro results, 6 mice received ­DOMBA 32 mg intraperitoneal and were observed for occurrence of neurologic effects and DOMBA plasma concentration was measured by liquid chromatography tandem mass spectrometry. Results: DOMBA both directly activates GABAA receptors and antagonizes its GABA-mediated opening in a concentration-dependent manner at concentrations between 5–10 and 0.5–10 mmol/L respectively. In vivo, DOMBA produced rapid onset sedation at plasma concentrations that correlate with direct GABAA receptor activation. Conclusion: DOMBA modulation of GABAA receptors is associated with sedation in mice. Metabolites of propanidid analogues currently in development may similarly modulate GABAA, and impaired elimination of these metabolites could produce clinically relevant neurophysiologic effects.

scholarly journals Tonic Extrasynaptic GABAA Receptor Currents Control Gonadotropin-Releasing Hormone Neuron Excitability in the Mouse

Endocrinology ◽  
2011 ◽  
Vol 152 (4) ◽  
pp. 1551-1561 ◽  
Author(s):  
Janardhan P. Bhattarai ◽  
Seon Ah Park ◽  
Jin Bong Park ◽  
So Yeong Lee ◽  
Allan E. Herbison ◽  
...  
Keyword(s):  
Gabaa Receptor ◽  
Gabaa Receptors ◽  
Brain Slice ◽  
Resting Membrane Potential ◽  
Receptor Signaling ◽  
Gaba Transporter ◽  
Gnrh Neurons ◽  
Inward Currents ◽  
Synaptic Receptors ◽  
Tonic Current

Abstract It is well established that the GABAA receptor plays an important role in regulating the electrical excitability of GnRH neurons. Two different modes of GABAA receptor signaling exist: one mediated by synaptic receptors generating fast (phasic) postsynaptic currents and the other mediated by extrasynaptic receptors generating a persistent (tonic) current. Using GABAA receptor antagonists picrotoxin, bicuculline methiodide, and gabazine, which differentiate between phasic and tonic signaling, we found that ∼50% of GnRH neurons exhibit an approximately 15-pA tonic GABAA receptor current in the acute brain slice preparation. The blockade of either neuronal (NO711) or glial (SNAP-5114) GABA transporter activity within the brain slice revealed the presence of tonic GABA signaling in ∼90% of GnRH neurons. The GABAA receptor δ subunit is only found in extrasynaptic GABAA receptors. Using single-cell RT-PCR, GABAA receptor δ subunit mRNA was identified in GnRH neurons and the δ subunit–specific agonist 4,5,6,7-tetrahydroisoxazolo [5,4-c] pyridin-3-ol was found to activate inward currents in GnRH neurons. Perforated-patch clamp studies showed that 4,5,6,7-tetrahydroisoxazolo [5,4-c] pyridin-3-ol exerted the same depolarizing or hyperpolarizing effects as GABA on juvenile and adult GnRH neurons and that tonic GABAA receptor signaling regulates resting membrane potential. Together, these studies reveal the presence of a tonic GABAA receptor current in GnRH neurons that controls their excitability. The level of tonic current is dependent, in part, on neuronal and glial GABA transporter activity and mediated by extrasynaptic δ subunit–containing GABAA receptors.

scholarly journals An association study in the Taiwan Biobank elicits the GABAA receptor genes GABRB3, GABRA5, and GABRG3 as candidate loci for sleep duration in the Taiwanese population

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Sheue-Jane Hou ◽  
Shih-Jen Tsai ◽  
Po-Hsiu Kuo ◽  
Wan-Yu Lin ◽  
Yu-Li Liu ◽  
...  
Keyword(s):  
Sleep Quality ◽  
Association Study ◽  
Gabaa Receptor ◽  
Sleep Duration ◽  
Gabaa Receptors ◽  
Aminobutyric Acid ◽  
Gamma Aminobutyric Acid ◽  
Bonferroni Correction ◽  
Taiwanese Population ◽  
Sleep Timing

Abstract Background Gamma-aminobutyric acid type A (GABAA) receptors mainly mediate the effects of gamma-aminobutyric acid, which is the primary inhibitory neurotransmitter in the central nervous system. Abundant evidence suggests that GABAA receptors play a key role in sleep-regulating processes. No genetic association study has explored the relationships between GABAA receptor genes and sleep duration, sleep quality, and sleep timing in humans. Methods We determined the association between single-nucleotide polymorphisms (SNPs) in the GABAA receptor genes GABRA1, GABRA2, GABRB3, GABRA5, and GABRG3 and sleep duration, sleep quality, and sleep timing in the Taiwan Biobank with a sample of 10,127 Taiwanese subjects. There were 10,142 subjects in the original study cohort. We excluded 15 subjects with a medication history of sedative-hypnotics. Results Our data revealed an association of the GABRB3-GABRA5-GABRG3 gene cluster with sleep duration, which has not been previously identified: rs79333046 (beta = − 0.07; P = 1.21 × 10–3) in GABRB3, rs189790076 (beta = 0.92; P = 1.04 × 10–3) in GABRA5, and rs147619342 (beta = − 0.72; P = 3.97 × 10–3) in GABRG3. The association between rs189790076 in GABRA5 and sleep duration remained significant after Bonferroni correction. A variant (rs12438141) in GABRB3 was also found to act as a potential expression quantitative trait locus. Additionally, we discovered interactions between variants in the GABRB3-GABRA5-GABRG3 gene cluster and lifestyle factors, such as tea and coffee consumption, smoking, and physical activity, that influenced sleep duration, although some interactions became nonsignificant after Bonferroni correction. We also found interactions among GABRB3, GABRA5, and GABRG3 that affected sleep duration. Furthermore, we identified an association of rs7165524 (beta = − 0.06; P = 2.20 × 10–3) in GABRA5 with sleep quality and an association of rs79465949 (beta = − 0.12; P = 3.95 × 10–3) in GABRB3 with sleep timing, although these associations became nonsignificant after Bonferroni correction. However, we detected no evidence of an association of individual SNPs in GABRA1 and GABRA2. Conclusions Our results indicate that rs189790076 in GABRA5 and gene–gene interactions among GABRB3, GABRA5, and GABRG3 may contribute to sleep duration in the Taiwanese population.

Supersensitivity to allosteric GABAA receptor modulators and alcohol in mice lacking PKCε

10.1038/14795 ◽  
1999 ◽  
Vol 2 (11) ◽  
pp. 997-1002 ◽  
Author(s):  
Clyde W. Hodge ◽  
Kristin K. Mehmert ◽  
Stephen P. Kelley ◽  
Thomas McMahon ◽  
Ashley Haywood ◽  
...  
Keyword(s):  
Gabaa Receptor ◽  
Receptor Modulators

Allosteric modulation of GABAA receptors in acutely dissociated neurons of the suprachiasmatic nucleus

1996 ◽  
Vol 270 (6) ◽  
pp. C1726-C1734 ◽  
Author(s):  
M. Shimura ◽  
N. Harata ◽  
M. Tamai ◽  
N. Akaike
Keyword(s):  
Gabaa Receptor ◽  
Suprachiasmatic Nucleus ◽  
Gabaa Receptors ◽  
Response Curve ◽  
Equilibrium Potential ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Gabaa Receptor Antagonist ◽  
Inward Currents ◽  
Concentration Response Curve

The gamma-aminobutyric acid (GABA)-induced response was investigated in acutely dissociated suprachiasmatic nucleus (SCN) neurons of 11- to 14-day-old rats, under the voltage-clamp condition of nystatin-perforated patch recording. At a holding potential of -40 mV, application of GABA induced inward currents in a concentration-dependent manner. Pentobarbital and 5 beta-pregnan-3 alpha-ol-20-one (pregnanolone) similarly induced inward currents. GABA-induced inward currents were suppressed in a concentration-dependent manner by pretreating neurons with a GABAA receptor antagonist, bicuculline. Bicuculline (3 x 10(-6) M) shifted the concentration-response curve of GABA to the left in a competitive manner. Reversal potential of the GABA response (EGABA) was -3.4 +/- 0.7 mV, close to the theoretical Cl- equilibrium potential of -4.1 mV. Pretreating SCN neurons with diazepam, pentobarbital, and pregnanolone enhanced the 3 x 10(-6) M GABA response. Diazepam (3 x 10(-8) M), pentobarbital (3 x 10(-5) M), and pregnanolone (10(-7) M) shifted the concentration-response curve of GABA to the left without changing the maximal amplitude of GABA responses. EGABA in the presence of diazepam, pentobarbital, or pregnanolone was the same as that in their absence. These results show that the GABA response in acutely dissociated SCN neurons is mediated by the GABAA receptor. Because the GABAA receptor of SCN neurons is allosterically augmented by diazepam, pentobarbital, and pregnanolone, similarly as in other regions of the central nervous system, the present study opens up ways to functionally modulate the GABAA receptors in SCN.

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