142 research outputs found
Modifications of diflunisal and meclofenamate carboxyl groups affect their allosteric effects on GABAA receptor ligand binding
Gamma-aminobutyric acid type A receptors (GABAAR) are allosterically modulated by the nonsteroidal anti-inflammatory drugs diflunisal and fenamates. The carboxyl group of these compounds is charged at physiological pH and therefore penetration of the compounds into the brain is low. In the present study we have transformed the carboxyl group of diflunisal and meclofenamate into non-ionizable functional groups and analyzed the effects of the modifications on stimulation of [(3)H]muscimol binding and on potentiation of γ-aminobutyric acid-induced displacement of 4'-ethenyl-4-n-[2,3-(3)H]propylbicycloorthobenzoate. N-Butylamide derivative of diflunisal modulated radioligand binding with equal or higher potency than the parent compound, while diflunisalamide showed reduced allosteric effect as compared to diflunisal. Amide derivative of meclofenamate equally affected radioligand binding parameters, while both diflunisal and meclofenamate methyl esters were less active than the parent compounds. Our study clearly demonstrates that an intact carboxyl group in diflunisal and meclofenamate is not indispensable for their positive GABAAR modulation. Further derivatization of the compound might yield compounds with higher selectivity for GABAARs that could be utilized in drug development.</p
Enhancement of GABAergic activity:neuropharmacological effects of benzodiazepines and therapeutic use in anaesthesiology
GABA is the major inhibitory neurotransmitter in the central nervous
system (CNS). The type A GABA receptor (GABAAR) system is the primary
pharmacological target for many drugs used in clinical anesthesia. The
ι1, β2, and γ2 subunit-containing GABAARs located in the various parts
of CNS are thought to be involved in versatile effects caused by
inhaled anesthetics and classic benzodiazepines (BZD), both of which are
widely used in clinical anesthesiology. During the past decade, the
emergence of tonic inhibitory conductance in extrasynaptic GABAARs has
coincided with evidence showing that these receptors are highly
sensitive to the sedatives and hypnotics used in anesthesia. Anesthetic
enhancement of tonic GABAergic inhibition seems to be preferentially
increased in regions shown to be important in controlling memory,
awareness, and sleep. This review focuses on the physiology of the
GABAARs and the pharmacological properties of clinically used BZDs.
Although classic BZDs are widely used in anesthesiological practice,
there is a constant need for new drugs with more favorable
pharmacokinetic and pharmacodynamic effects and fewer side effects. New
hypnotics are currently developed, and promising results for one of
these, the GABAAR agonist remimazolam, have recently been published.</p
Humulone Modulation of GABAA Receptors and Its Role in Hops Sleep-Promoting Activity
Humulus lupulus L. (hops) is a major constituent of beer. It exhibits neuroactive properties that make it useful as a sleeping aid. These effects are hypothesized to be mediated by an increase in GABAA receptor function. In the quest to uncover the constituents responsible for the sedative and hypnotic properties of hops, recent evidence revealed that humulone, a prenylated phloroglucinol derivative comprising 35â70% of hops alpha acids, may act as a positive modulator of GABAA receptors at low micromolar concentrations. This raises the question whether humulone plays a key role in hops pharmacological activity and potentially interacts with other modulators such as ethanol, bringing further enhancement in GABAA receptor-mediated effects of beer. Here we assessed electrophysiologically the positive modulatory activity of humulone on recombinant GABAA receptors expressed in HEK293 cells. We then examined humulone interactions with other active hops compounds and ethanol on GABA-induced displacement of [3H]EBOB binding to native GABAA receptors in rat brain membranes. Using BALB/c mice, we assessed humuloneâs hypnotic behavior with pentobarbital- and ethanol-induced sleep as well as sedation in spontaneous locomotion with open field test. We demonstrated for the first time that humulone potentiates GABA-induced currents in Îą1β3Îł2 receptors. In radioligand binding to native GABAA receptors, the inclusion of ethanol enhanced humulone modulation of GABA-induced displacement of [3H]EBOB binding in rat forebrain and cerebellum as it produced a leftward shift in [3H]EBOB displacement curves. Moreover, the additive modulatory effects between humulone, isoxanthohumol and 6-prenylnaringenin were evident and corresponded to the sum of [3H]EBOB displacement by each compound individually. In behavioral tests, humulone shortened sleep onset and increased the duration of sleep induced by pentobarbital and decreased the spontaneous locomotion in open field at 20 mg/kg (i.p.). Despite the absence of humulone effects on ethanol-induced sleep onset, sleep duration was increased dose-dependently down to 10 mg/kg (i.p.). Our findings confirmed humuloneâs positive allosteric modulation of GABAA receptor function and displayed its sedative and hypnotic behavior. Humulone modulation can be potentially enhanced by ethanol and hops modulators suggesting a probable enhancement in the intoxicating effects of ethanol in hops-enriched beer.</p
The Influence of AA29504 on GABA A Receptor Ligand Binding Properties and Its Implications on Subtype Selectivity
The unique pharmacological properties of δ-containing γ-aminobutyric acid type A receptors (δ-GABAARs) make them an attractive target for selective and persistent modulation of neuronal excitability. However, the availability of selective modulators targeting δ-GABAARs remains limited. AA29504 ([2-amino-4-(2,4,6-trimethylbenzylamino)-phenyl]-carbamic acid ethyl ester), an analog of K+ channel opener retigabine, acts as an agonist and a positive allosteric modulator (Ago-PAM) of δ-GABAARs. Based on electrophysiological studies using recombinant receptors, AA29504 was found to be a more potent and effective agonist in δ-GABAARs than in γ2-GABAARs. In comparison, AA29504 positively modulated the activity of recombinant δ-GABAARs more effectively than γ2-GABAARs, with no significant differences in potency. The impact of AA29504's efficacy- and potency-associated GABAAR subtype selectivity on radioligand binding properties remain unexplored. Using [3H]4'-ethynyl-4-n-propylbicycloorthobenzoate ([3H]EBOB) binding assay, we found no difference in the modulatory potency of AA29504 on GABA- and THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol)-induced responses between native forebrain GABAARs of wild type and δ knock-out mice. In recombinant receptors expressed in HEK293 cells, AA29504 showed higher efficacy on δ- than γ2-GABAARs in the GABA-independent displacement of [3H]EBOB binding. Interestingly, AA29504 showed a concentration-dependent stimulation of [3H]muscimol binding to γ2-GABAARs, which was absent in δ-GABAARs. This was explained by AA29504 shifting the low-affinity γ2-GABAAR towards a higher affinity desensitized state, thereby rising new sites capable of binding GABAAR agonists with low nanomolar affinity. Hence, the potential of AA29504 to act as a desensitization-modifying allosteric modulator of γ2-GABAARs deserves further investigation for its promising influence on shaping efficacy, duration and plasticity of GABAAR synaptic responses
Stereospecific modulation of GABA(A) receptor function by urocanic acid isomers
A deamination product of histidine, urocanic acid, accumulates in the
skin of mammals as trans-urocanic acid. Ultraviolet (UV) irradition
converts it to the cis-isomer that is an important mediator in
UV-induced immunosuppression. We have recently shown that urocanic acid
interferes with the agonist binding to GABAA receptors. We now report
that the effects of urocanic acid on binding of a convulsant ligand
(t-butylbicyclo[35S]phosphorothionate) to GABAA receptors in brain
membrane homogenates are dependent on pH of the incubation medium, the
agonistic actions being enhanced at the normal pH of the skin (5.5).
Using Xenopus laevis oocytes expressing recombinant rat
alpha1beta1gamma2S GABAA receptors, the low pH potentiated the direct
agonistic action of trans-urocanic acid under two-electrode
voltage-clamp, whereas cis-urocanic acid retained its low efficacy both
at pH 5.5 and 7.4. The results thus indicate clear differences between
urocanic acid isomers in functional activity at one putative receptor
site of immunosuppression, the GABAA receptor, the presence of which
in the skin remains to be demonstrated.</p
Extrasynaptic 뫉GABAA receptors are high affinity muscimol receptors
Muscimol, the major psychoactive ingredient in the mushroom Amanita muscaria, has been regarded as a universal nonâselective GABAâsite agonist. Deletion of the GABAA receptor (GABAAR) δ subunit in mice (δKO) leads to a drastic reduction in high affinity muscimol binding in brain sections and loss of behavioral low dose muscimol effects. Here we use forebrain and cerebellar brain homogenates from WT and δKO mice to show that deletion of the δ subunit leads to a >50% loss of high affinity 5 nM [3H]muscimol binding sites despite the relatively low abundance of δâcontaining GABAARs (δâGABAAR) in the brain. By subtracting residual high affinity binding in δKO mice and measuring the slow association and dissociation rates we show that native δâGABAARs in WT mice exhibit high affinity [3H]muscimol binding sites (KD ~1.6 nM on Îą4βδ receptors in the forebrain and ~1 nM on Îą6βδ receptors in the cerebellum at room temperature). Coâexpression of the δ subunit with Îą6 and β2 or β3 in recombinant (HEK 293) expression leads to the appearance of a slowly dissociating [3H]muscimol component. In addition, we compared muscimol currents in recombinant Îą4β3δ and Îą4β3 receptors and show that δ subunit coâexpression leads to highly muscimolâsensitive currents with an estimated EC50 of around 1â2 nM and slow deactivation kinetics. These data indicate that δ subunit incorporation leads to a dramatic increase of GABAAR muscimol sensitivity. We conclude that biochemical and behavioral low dose muscimol selectivity for δ subunitâcontaining receptors is due to low nanomolar binding affinity on δâGABAARs.</p
Hops compounds modulatory effects and 6-prenylnaringenin dual mode of action on GABAA receptors
Hops (Humulus lupulus L.), a major component of beer, contain potentially neuroactive compounds that made it useful in traditional medicine as a sleeping aid. The present study aims to investigate the individual components in hops acting as allosteric modulators in GABAA receptors and bring further insight into the mode of action behind the sedative properties of hops. GABA-potentiating effects were measured using [3H]ethynylbicycloorthobenzoate (EBOB) radioligand binding assay in native GABAA receptors. Flumazenil sensitivity of GABA-potentiating effects, [3H]Ro 15â4513, and [3H]flunitrazepam binding assays were used to examine the binding to the classical benzodiazepines site. Humulone (alpha acid) and 6-prenylnaringenin (prenylflavonoid) were the most potent compounds displaying a modulatory activity at low micromolar concentrations. Humulone and 6-prenylnaringenin potentiated GABA-induced displacement of [3H]EBOB binding in a concentration-dependent manner where the IC50 values for this potentiation in native GABAA receptors were 3.2 ΟM and 3.7 ΟM, respectively. Flumazenil had no significant effects on humulone- or 6-prenylnaringenin-induced displacement of [3H]EBOB binding. [3H]Ro 15â4513 and [3H]flunitrazepam displacements were only minor with humulone but surprisingly prominent with 6-prenylnaringenin despite its flumazenil-insensitive modulatory activity. Thus, we applied molecular docking methods to investigate putative binding sites and poses of 6-prenylnaringenin at the GABAA receptor Îą1β2Îł2 isoform. Radioligand binding and docking results suggest a dual mode of action by 6-prenylnaringenin on GABAA receptors where it may act as a positive allosteric modulator at Îą+β- binding interface as well as a null modulator at the flumazenil-sensitive Îą+Îł2- binding interface.</ul
GammaâAminobutyric Acid System GenesâNo Evidence for a Role in Alcohol Use and Abuse in a CommunityâBased Sample
Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/106921/1/acer12352.pd
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