Selective antagonism of 5alpha-reduced neurosteroid effects at GABA(A) receptors

Although neurosteroids have rapid effects on GABA(A) receptors, study of steroid actions at GABA receptors has been hampered by a lack of pharmacological antagonists. In this study, we report the synthesis and characterization of a steroid analog, (3alpha,5alpha)-17-phenylandrost-16-en-3-ol (17PA), that selectively antagonized neurosteroid potentiation of GABA responses. We examined 17PA using the alpha1beta2gamma2 subunit combination expressed in Xenopus laevis oocytes. 17PA had little or no effect on baseline GABA responses but antagonized both the response augmentation and the direct gating of GABA receptors by 5alpha-reduced potentiating steroids. The effect was selective for 5alpha-reduced potentiating steroids; 5beta-reduced potentiators were only weakly affected. Likewise, 17PA did not affect barbiturate and benzodiazepine potentiation. 17PA acted primarily by shifting the concentration response for steroid potentiation to the right, suggesting the possibility of a competitive component to the antagonism. 17PA also antagonized 5alpha-reduced steroid potentiation and gating in hippocampal neurons and inhibited anesthetic actions in X. laevis tadpoles. Analogous to benzodiazepine site antagonists, the development of neurosteroid antagonists may help clarify the role of GABA-potentiating neurosteroids in health and disease.     

Panic induction with cholecystokinin-tetrapeptide (CCK-4) Increases plasma concentrations of the neuroactive steroid 3alpha, 5alpha tetrahydrodeoxycorticosterone (3alpha, 5alpha-THDOC) in healthy volunteers.

3alpha-reduced neuroactive steroids such as 3alpha, 5alpha-tetrahydroprogesterone (3alpha, 5alpha-THP) and 3alpha, 5alpha-tetrahydrodeoxycorticosterone (3alpha, 5alpha-THDOC) are potent positive allosteric modulators of gamma-aminobutyric acid type A (GABAA) receptors and display pronounced anxiolytic activity in animal models. Experimental panic induction with cholecystokinin-tetrapeptide (CCK-4) and sodium lactate is accompanied by a decrease in 3alpha, 5alpha-THP concentrations in patients with panic disorder, but not in healthy controls. However, no data are available on 3alpha, 5alpha-THDOC concentrations during experimental panic induction. Therefore, we quantified 3alpha, 5alpha-THDOC concentrations in 10 healthy volunteers (nine men, one woman) before and after panic induction with CCK-4 by means of a highly sensitive and specific gas chromatography/mass spectrometry analysis. CCK-4 elicited a strong panic response as assessed by the Acute Panic Inventory. This was accompanied by an increase in 3alpha, 5alpha-THDOC, ACTH and cortisol concentrations. This increase in 3alpha, 5alpha-THDOC might be a consequence of hypothalamic-pituitary-adrenal (HPA) axis activation following CCK-4-induced panic, and might contribute to the termination of the anxiety/stress response following challenge with CCK-4 through enhancement of GABAA receptor function.     

Multiple structural features of steroids mediate subtype-selective effects on human alpha4beta3delta GABAA receptors

Neurosteroids have been shown to mediate some of their physiological effects via a modulatory site on type A inhibitory gamma-aminobutyric acid (GABAA) receptors. In particular, recent evidence has implicated selective potentiation of the delta subunit of GABAA receptors as an important mediator of in vitro and in vivo neurosteroid activity. However, this has been demonstrated for only a very small number of steroids, so both the generality of this finding, and the structural features of steroids which mediate functional delta-selectivity, are unclear. We have used a potentiometric assay based on fluorescence resonance energy transfer to measure GABA-activated responses in L(tk-) cells stably transfected with human GABAA receptor alpha4beta3delta and alpha4beta3gamma2 receptor subtypes. A set of 28 steroids were evaluated on these subtypes to characterise their functional potency and efficacy in modulating GABA responses. For most compounds there was a clear separation of their efficacy profiles between the receptor subtypes, with a substantially larger maximal response at the alpha4beta3delta receptor. 5beta-Pregnan-3beta-ol-20-one, 5beta-pregnane-3alpha,20beta-diol and 5beta-pregnane-3alpha,17alpha-diol-11,20-dione showed particularly high efficacy for alpha4beta3delta. No compounds were identified that simply inhibited responses at delta-containing receptors. However, 5beta-pregnane-3alpha,17alpha,20beta-triol, prednisolone 21-acetate, 4-pregnene-17alpha,20alpha-diol-3-one-20-acetate, 4-pregnen-20alpha-ol-3-one, and 5beta-pregnane-3alpha,17alpha,21-triol-20-one inhibited, though did not abolish, GABA responses at the alpha4beta3gamma2 subtype, while evoking modest-amplitude potentiation of alpha4beta3delta responses. Molecular modelling on this compound series using principal components analysis indicates that several structural features of steroids underlie their relative functional selectivity for potentiation of delta-containing GABAA receptors.     

Auto-modulation of neuroactive steroids on GABA A receptors: a novel pharmacological effect

GABA(A) receptor function is modulated by various important drugs including neuroactive steroids that act on allosteric modulatory sites and can directly activate GABA(A) receptor channels at high concentrations. We used whole cell patch-clamp recordings and rapid applications of the neuroactive steroid alphaxalone to investigate repetitive steroid effects. Alphaxalone potentiation of submaximal GABA-evoked currents was enhanced significantly by repetitive coapplications at all investigated recombinant isoforms (alpha1beta3delta, alpha1beta3gamma2L, alpha6beta3delta, alpha6beta3gamma2L) and at GABA(A) receptors of differentiated human NT2 neurons. A similar increase of current amplitudes was induced by repetitive applications of a high steroid concentration without GABA. We refer to these reversible effects as auto-modulation because repeated interactions of steroids enhanced their own pharmacological impact at the receptor sites in a time and concentration dependent manner without affecting GABA controls. Pronounced auto-modulatory actions were also measured using the neurosteroid 5alpha-THDOC in contrast to indiplon, THIP, and pentobarbital indicating a steroid specificity. Protein kinase A inhibition significantly reduced alphaxalone auto-modulation at alpha1beta3gamma2L, alpha6beta3gamma2L, and alpha6beta3delta subtypes while it enhanced potentiation at alpha1beta3delta isoforms suggesting a crucial influence of receptor subunit composition and phosphorylation for steroid actions. Especially at extrasynaptic GABA(A) receptor sites containing the delta subunit steroid auto-modulation may have a critical role in enhancing potentiation of GABA-induced currents.     

Substrate specificity of human 3(20)alpha-hydroxysteroid dehydrogenase for neurosteroids and its inhibition by benzodiazepines

In this report, we compared kinetic constants and products in the reduction of the neurosteroids, 3alpha,5alpha-tetrahydroprogesterone (3alpha,5alpha THP) and 3alpha,5alpha-tetrahydrodeoxycorticosterone (3alpha,5alpha-THDOC), and their precursors, 5alpha-dihydroprogesterone (5alpha-DHP), 5alpha-dihydrodeoxycorticosterone (5alpha-DHDOC) and progesterone, by three isoenzymes (AKR1C1, AKR1C2 and AKR1C3) of human 3alpha-hydroxysteroid dehydrogenase. AKR1C1 efficiently reduced 3alpha,5alpha-THP, 5alpha-DHP and progesterone to their 20alpha-hydroxy metabolites, and slowly converted 5alpha-DHDOC to 3alpha,5alpha-THDOC. AKR1C2 exhibited low 20-ketoreductase activity for 3alpha,5alpha-THP and moderate 3-ketoreductase activity for 5alpha-DHP and 5alpha-DHDOC. 3alpha,5alpha-THDOC was not reduced by the two isoenzymes. No significant activity for the steroids was detected with AKR1C3. The results suggest that AKR1C2 is involved in the neurosteroid synthesis, but AKR1C1 decreases the neurosteroid concentrations in human brain by inactivating 3alpha,5alpha-THP and eliminating the precursors from the synthetic pathways. In addition, we found that the several benzodiazepines inhibited the three isoenzymes noncompetitively with respect to the substrate. Although cloxazolam was a potent and specific inhibitor of AKR1C3, diazepam, estazolam, flunitrazepam, medazepam and nitrazepam, that inhibited AKR1C1 and AKR1C2, may influence the neurosteroid metabolism.     

Pentobarbital differentially modulates alpha1beta3delta and alpha1beta3gamma2L GABAA receptor currents

GABAA receptors are modulated by a variety of compounds, including the neurosteroids and barbiturates. Although the effects of barbiturates on alphabetagamma isoforms, thought to dominate phasic (synaptic) GABAergic inhibition, have been extensively studied, the effects of pentobarbital on kinetic properties of alphabetadelta GABAA receptors, thought to mediate tonic (extra- or perisynaptic) inhibition, are unknown. Using ultrafast drug delivery and single channel recording techniques, we demonstrate isoform-specific pentobarbital modulation of low-efficacy, minimally desensitizing alpha1beta3 currents and high-efficacy, rapidly desensitizing alpha1beta3gamma2L currents. Specifically, with saturating concentrations of GABA, pentobarbital substantially potentiated peak alpha1beta3delta receptor currents but failed to potentiate peak alpha1beta3gamma2L receptor currents. Also, pentobarbital had opposite effects on the desensitization of alpha1beta3delta (increased) and alpha1beta3gamma2L (decreased) receptor currents evoked by saturating GABA. Pentobarbital increased steady-state alpha1beta3delta receptor single channel open duration primarily by introducing a longer duration open state, whereas for alpha1beta3gamma2L receptor channels, pentobarbital increased mean open duration by increasing the proportion and duration of the longest open state. The data support previous suggestions that GABA may be a partial agonist at alphabetadelta isoforms, which may render them particularly sensitive to allosteric modulation. The remarkable increase in gating efficacy of alpha1beta3delta receptors suggests that alphabetadelta isoforms, and by inference tonic forms of inhibition, may be important targets for barbiturates.     

Conserved site for neurosteroid modulation of GABA A receptors

This study addresses whether the potentiation site for neurosteroids on GABA(A) receptors is conserved amongst different GABA(A) receptor isoforms. The neurosteroid potentiation site was previously identified in the alpha1beta2gamma2S receptor by mutation of Q241 to methionine or leucine, which reduced the potentiation of GABA currents by the naturally occurring neurosteroids, allopregnanolone or tetrahydrodeoxycorticosterone (THDOC). By using heterologous expression of GABA(A) receptors in HEK cells, in combination with whole-cell patch clamp recording methods, a relatively consistent potentiation by allopregnanolone of GABA-activated currents was evident for receptors composed of one alpha subunit isoform (alpha2-5) assembled with beta3 and gamma2S subunits. Using mutant alphabetagamma receptors, the neurosteroid potentiation was universally dependent on the conserved glutamine residue in M1 of the respective alpha subunit. Studying wild-type and mutant receptors composed of alpha4beta3delta subunits revealed that the delta subunit is unlikely to contribute to the neurosteroid potentiation binding site and probably affects the efficacy of potentiation. Thus, in keeping with the ability of neurosteroids to potentiate GABA currents via a broad variety of GABA(A) receptor isoforms in neurons, the potentiation site is structurally highly conserved on this important neurotransmitter receptor family.     

Endogenous gamma-aminobutyric acid (GABA)(A) receptor active neurosteroids and the sedative/hypnotic action of gamma-hydroxybutyric acid (GHB): a study in GHB-S (sensitive) and GHB-R (resistant) rat lines

In the rat brain, gamma-hydroxybutyric-acid (GHB) increases the concentrations of 3alpha-hydroxy,5alpha-pregnan-20-one (allopregnanolone, 3alpha,5alpha-THP) and 3alpha,21-dihydroxy,5alpha-pregnan-20-one (allotetrahydrodeoxycorticosterone/3alpha,5alphaTHDOC), two neurosteroids acting as positive allosteric modulators of gamma-aminobutyric acid (GABA)(A) receptors. This study was aimed at assessing whether neurosteroids play a role in GHB-induced loss of righting reflex (LORR). Basal and GHB-stimulated brain concentrations of endogenous 3alpha,5alpha-THP and 3alpha,5alpha-THDOC were analyzed in two rat lines, GHB-sensitive (GHB-S) and GHB-resistant (GHB-R), selectively bred for opposite sensitivity to GHB-induced sedation/hypnosis. Basal neurosteroid concentrations were similar in brain cortex of the two rat lines. However, in male GHB-S rats, administration of GHB (1000 mg/kg, i.p., 30 min) increased brain cortical concentrations of 3alpha,5alpha-THP and 3alpha,5alpha-THDOC 7- and 2.5-fold, respectively, whilst male GHB-R animals displayed only a 4- and 2-fold increase, respectively. In GHB-S rats this increase lasted up to 90 min and declined 180 min following GHB administration, a time course that matches LORR onset and duration. In contrast, in GHB-R rats, which failed to show GHB-induced LORR, brain cortical 3alpha,5alpha-THP and 3alpha,5alpha-THDOC had returned to control values within 90 min. At onset of LORR, a similar increase in brain cortical levels of 3alpha,5alpha-THP and 3alpha,5alpha-THDOC (2-3-fold) was observed in GHB-S female rats and in the few female GHB-R rats that lost the righting reflex after GHB administration, but not in female GHB-R rats failing to show LORR. Sub-hypnotic doses (7.5 and 12.5 mg/kg, i.p.) of pregnanolone, administered 10 min before GHB, dose-dependently facilitated the expression of GHB-induced LORR in GHB-R male rats. These results suggest that the GHB-induced increases of brain 3alpha,5alpha-THP and 3alpha,5alpha-THDOC concentrations are implicated in the eliciting of the sedative/hypnotic action of GHB.     

High affinity, heterogeneous displacement of [3H]EBOB binding to cerebellar GABA A receptors by neurosteroids and GABA agonists

Heterogeneous binding interactions of cerebellar GABA(A) receptors were investigated with GABA agonists and neurosteroids. GABA(A) receptors of rat cerebellum were labelled with [(3)H]ethynylbicycloorthobenzoate (EBOB), a convulsant radioligand. Saturation analysis revealed a homogenous, nanomolar population of [(3)H]EBOB binding. Both GABA and 5alpha-tetrahydrodeoxycorticosterone (5alpha-THDOC) displaced [(3)H]EBOB binding heterogeneously, with nanomolar and micromolar potencies. The nanomolar phase of displacement by GABA was selectively abolished by 100 microM furosemide. Physiological concentrations of allopregnanolone (8 nM) and 5alpha-THDOC (20 nM) increased the displacing effects of nanomolar GABA. GABA (0.3 microM ) and 5alpha-THDOC (0.3 microM ) potentiated the micromolar population of displacement by the other. Taurine inhibited [(3)H]EBOB binding also heterogeneously, with micromolar and millimolar potencies, and 0.3 microM 5alpha-THDOC potentiated this inhibition. 5beta-THDOC did not affect [(3)H]EBOB binding significantly but in 1 microM it antagonised selectively the nanomolar displacement by 5alpha-THDOC. [(3)H]EBOB binding to hippocampal GABA(A) receptors was inhibited by GABA and allopregnanolone with low (micromolar) potencies and with slope values higher than unity referring to allosteric interaction. High affinity displacement of cerebellar [(3)H]EBOB binding by GABA agonists and neurosteroids can be associated with constitutively open alpha(6)betadelta GABA(A) receptors, tonic GABAergic inhibitory neurotransmission and its modulation by physiological concentrations of neurosteroids.     

Receptor subtype-dependent positive and negative modulation of GABA(A) receptor function by niflumic acid,a nonsteroidal anti-inflammatory drug

In addition to blocking cyclooxygenases, members of the fenamate group of nonsteroidal anti-inflammatory drugs have been proposed to affect brain GABAA receptors. Using quantitative autoradiography with GABAA receptor-associated ionophore ligand [35S]t-butylbicyclophosphorothionate (TBPS) on rat brain sections, one of the fenamates, niflumate, at micromolar concentration was found to potentiate GABA actions in most brain areas, whereas being in the cerebellar granule cell layer an efficient antagonist similar to furosemide. With recombinant GABAA receptors expressed in Xenopus laevis oocytes, we found that niflumate potentiated 3 microM GABA responses up to 160% and shifted the GABA concentration-response curve to the left in alpha1beta2gamma2 receptors, the predominant GABAA receptor subtype in the brain. This effect needed the gamma2 subunit, because on alpha1beta2 receptors, niflumate exhibited solely an antagonistic effect at high concentrations. The potentiation was not abolished by the specific benzodiazepine site antagonist flumazenil. Niflumate acted as a potent antagonist of alpha6beta2 receptors (with or without gamma2 subunit) and of alphaXbeta2gamma2 receptors containing a chimeric alpha1 to alpha6 subunit, which suggests that niflumate antagonism is dependent on the same transmembrane domain 1- and 2-including fragment of the alpha6 subunit as furosemide antagonism. This antagonism was noncompetitive because the maximal GABA response, but not the potency, was reduced by niflumate. These data show receptor subtype-dependent positive and negative modulatory actions of niflumate on GABAA receptors at clinically relevant concentrations, and they suggest the existence of a novel positive modulatory site on alpha1beta2gamma2 receptors that is dependent on the gamma2 subunit but not associated with the benzodiazepine binding site.     

Effects of subunit types of the recombinant GABAA receptor on the response to a neurosteroid.

When vertebrate brain poly(A)+ RNA is expressed in Xenopus oocytes the response of the GABA receptors formed is found to be inhibited allosterically by a neurosteroid, pregnenolone sulphate (PS). This negative modulation was reproduced after expressing RNAs encoding bovine GABAA receptor subunits in the combinations alpha i + beta 1, or alpha i + beta 1 + gamma 2 (where i = 1, 2 or 3). The characteristics of this inhibition vary significantly with the type of the alpha subunit (alpha 1, alpha 2, or alpha 3) used. When the bovine gamma 2L alternate form of the gamma 2 subunit was replaced by the human gamma 2S subunit, the behaviour was unchanged: the human gamma 2S subunit used is a newly-cloned form, which encodes a polypeptide with two amino acid differences from the human gamma 2 subunit previously described. The results of co-application of PS and 3 alpha-hydroxy-5 alpha-pregnan-ol-20-one, a neurosteroid which is a positive modulator of the GABAA receptor, indicate that these act at different sites on the receptor. PS also increases the desensitisation of the receptor by GABA. This effect, also, is alpha-subunit-type dependent and occurs by an acceleration of the fast phase of desensitisation.     

Mechanisms of potentiation of the mammalian GABAA receptor by the marine cembranoid eupalmerin acetate

BACKGROUND AND PURPOSE: Eupalmerin acetate (EPA) is a marine diterpene compound isolated from the gorgonian octocorals Eunicea succinea and Eunicea mammosa. The compound has been previously shown to modulate muscle-type and neuronal nicotinic acetylcholine receptors, which are inhibited in the presence of low micromolar concentrations of EPA. In this study, we examined the effect of EPA on another transmitter-gated ion channel, the GABA(A) receptor. EXPERIMENTAL APPROACH: Whole-cell and single-channel recordings were made from HEK 293 cells transiently expressing rat wild-type and mutant alpha1beta2gamma2L GABA(A) receptors. KEY RESULTS: Our findings demonstrate that, at micromolar concentrations, EPA potentiates the rat alpha1beta2gamma2L GABA(A) receptor. The analysis of single-channel currents recorded in the presence of EPA showed that the kinetic mode of action of EPA is similar to that of neuroactive steroids. Mutations to residues alpha1Q241 and alpha1N407/Y410, previously shown to affect receptor modulation by neurosteroids, also diminished potentiation by EPA. Exposure to a steroid antagonist, (3alpha,5alpha)-17-phenylandrost-16-en-3-ol, reduced potentiation by EPA. Additionally, exposure to EPA led to potentiation of GABA(A) receptors activated by very high concentrations (1-10 microM) of allopregnanolone. In tadpole behavioural assays, EPA caused loss of righting reflex and loss of swimming reflex. CONCLUSIONS AND IMPLICATIONS: We conclude that EPA either interacts with the putative neurosteroid binding site on the GABA(A) receptor or shares with neurosteroids the key transduction elements involved in channel potentiation by steroids. The results indicate that cembranoids represent a novel class of GABA(A) receptor modulators.     

Cultured Hippocampal Pyramidal Neurons Express Two Kinds of GABAA Receptors

We combined a study of the subcellular distribution of the alpha1, alpha2, alpha4, beta1, beta2/3, gamma2, and delta subunits of the GABAA receptor with an electrophysiological analysis of GABAA receptor currents determine the to types of receptors expressed on cultured hippocampal pyramidal neurons. The immunocytochemistry study demonstrated that alpha1, alpha2, beta2/3, and gamma2 subunits formed distinct clusters of various sizes, which were colocalized with clusters of glutamate decarboxylase (GAD) immunoreactivity at rates ranging from 22 to 58%. In contrast, alpha4, beta1, and delta subunits were distributed diffusely over the cell soma and neuronal processes of cultured neurons and did not colocalize with the synaptic marker GAD. Whole-cell GABA receptor currents were moderately sensitive to GABAA and were modulated by diazepam. The whole-cell currents were also enhanced by the neurosteroid allopregnanolone (10 nM). Tonic currents, measured as changes in baseline current and noise, were sensitive to Zn2+, furosemide, and loreclezole; they were insensitive to diazepam. These studies suggest that two kinds of GABAA receptors are expressed on cultured hippocampal neurons. One kind of receptor formed clusters, which were present at GABAergic synapses and in the extrasynaptic membrane. The alpha1, alpha2, beta2/3, and gamma2 subunits were contained in clustered receptors. The second kind was distributed diffusely in the extrasynaptic membrane. The alpha4, beta1, and delta subunits were contained in these diffusely distributed receptors. The properties of tonic currents recorded from these neurons were similar to those from recombinant receptors containing alpha4, beta1, and delta subunits.     

A comparison of the pharmacological properties of recombinant human and rat alpha(1)beta(2)gamma(2L) GABA(A) receptors in Xenopus oocytes

In the present study, we compared the pharmacology, particularly neurosteroid modulation of the GABA(A) receptor, between human and rat alpha(1)beta(2)gamma(2)(L) GABA(A) receptors and between human receptors containing the long (L) and short (S) forms of the gamma(2)-subunit. We observed that maximum responses to GABA were significantly higher with the human alpha(1)beta(2)gamma(2)(L) receptor compared with the rat receptor. In terms of neurosteroid modulation, increases in the EC(15) response to GABA induced by 3alpha-OH-5beta-pregnan-20-one (3alpha5betaP), 5alpha-androstane-3alpha,17beta-diol (3alpha5alphaADL) and 5alpha-pregnane-3alpha,20beta-diol (3alpha5alpha-diol) were significantly greater for the rat compared with the human receptor. Responses to 30 micromol/L GABA were inhibited by 3beta-OH-5alpha-pregnan-20-one (UC1010) and 5beta-pregnan-3beta,20(R)-diol (UC1020) to a greater degree for human and rat receptors, respectively. Responses to GABA + 3alpha5alphaTHDOC were inhibited by 5alpha-pregnan-3beta,20(S)-diol (UC1019) and pregnenolone sulphate to a greater degree for human and rat receptors, respectively. The GABA dose-response curves for human alpha(1)beta(2)gamma(2)(S) and alpha(1)beta(2)gamma(2)(L) receptors were identical. However, the maximum GABA-evoked current, the direct gating effect of pentobarbital and the allosteric potentiation of the GABA EC(15) response by 3alpha5alphaTHDOC and 3alpha5betaP were significantly higher with alpha(1)beta(2)gamma(2)(S) than alpha(1)beta(2)gamma(2)(L) receptors. Inhibition of the response to 30 micromol/L GABA by UC1010 and UC1020 was greater for a(1)beta(2)gamma(2)(L) and alpha(1)beta(2)gamma(2)(S) receptors, respectively. Inhibition of responses to 3alpha5alphaTHDOC + GABA by UC1019 and UC1010 was significantly higher for alpha(1)beta(2)gamma(2)(L) receptors. In conclusion, the site of activation by GABA and neurosteroid modulation differ between human and rat alpha(1)beta(2)gamma(2)(L) receptors, as well as between human receptors containing the L and S splice variants of the gamma(2)-subunit.     

Pharmacological properties of alpha 9 alpha 10 nicotinic acetylcholine receptors revealed by heterologous expression of subunit chimeras

The nicotinic acetylcholine receptor (nAChR) alpha9 and alpha10 subunits are expressed primarily within hair cells of the inner ear and have been implicated in auditory processing. Although functional recombinant nAChRs generated by the coexpression of alpha9 and alpha10 in Xenopus laevis oocytes have been described previously, there have been no reports of the successful heterologous expression of alpha9alpha10 nAChRs in cultured cell lines. In this study, subunit chimeras (alpha9chi and alpha10chi) have been constructed that contain the extracellular, ligand binding domain of the alpha9 or alpha10 subunits fused to the C-terminal domain of the 5-hydroxytryptamine type 3A (5HT3A) subunit. Specific high-affinity binding of the nicotinic radioligand [3H]methyllycaconitine was detected in membrane preparations of mammalian cells transfected with alpha9chi or alpha10chi alone, but significantly higher levels of binding were detected when alpha9chi and alpha10chi were cotransfected, providing evidence of a requirement for coassembly of alpha9 and alpha10 for the efficient formation of a nicotinic binding site. The pharmacological profile of alpha9chialpha10chi receptors, determined by equilibrium radioligand binding studies, is broadly similar to that determined previously by electrophysiological studies conducted with native and recombinant alpha9alpha10 nAChRs. In agreement with evidence that alpha9alpha10 nAChRs exhibit an atypical pharmacological profile, we have identified specific high-affinity binding of several non-nicotinic ligands including strychnine (a glycine receptor antagonist), bicuculline (a GABAA receptor antagonist), and atropine (a muscarinic acetylcholine receptor antagonist). Results have also been compared with radioligand binding data conducted with a previously described alpha7/5HT3A (alpha7chi) subunit chimera.     

Modulation of GABAA receptors and neuropeptide secretion by the neurosteroid allopregnanolone in posterior and intermediate pituitary

A number of neurosteroids bind to GABAA receptors and alter their responsiveness to neurotransmitters. Considerable effort has been devoted to understanding how this form of receptor modulation alters inhibitory synaptic function. Neurosteroid-sensitive GABAA receptors have also been demonstrated in many endocrine cells, but little is known about how neurosteroids modulate the release of hormones. Here, the action of allopregnanolone, a neurosteroid that enhances GABAA receptor-mediated responses, was investigated in posterior pituitary nerve terminals and intermediate pituitary endocrine cells. Patch clamp recordings showed that GABA-evoked currents were enhanced to similar degrees and with similar concentration dependences in both locations. An organ bath preparation of the neurointermediate lobe was used to investigate drug effects on secretion of vasopressin and alpha-melanocyte stimulating hormone. GABA increased the basal release of vasopressin and alpha-melanocyte stimulating hormone from the posterior and intermediate pituitary lobe, respectively, an effect that could be blocked by picrotoxinin. Vasopressin release evoked by electrical stimulation was also examined, and a small statistically significant inhibition by 5 microM GABA was observed. Allopregnanolone increased the basal release of vasopressin, and this effect was blocked by the GABAA receptor antagonist picrotoxinin. Allopregnanolone had no effect in conjunction with GABA. In contrast to the posterior lobe, allopregnanolone had no effect on release from the intermediate lobe. Thus, allopregnanolone in physiological relevant concentrations modulates GABAA receptors in both the posterior and intermediate lobes, but only affects hormone release in the posterior lobe.     

Studies of molecular pharmacophore/receptor models for GABAA/benzodiazepine receptor subtypes: binding affinities of substituted beta-carbolines at recombinant alpha x beta 3 gamma 2 subtypes and quantitative structure-activity relationship studies via a comparative molecular field analysis.

Binding affinities of a series of 44 beta-carbolines with various substituents at the 3-, 4-, 6- and 7-positions are reported at 5 distinct recombinant GABAA/benzodiazepine receptor (BzR) subtypes [alpha x beta 3 gamma 2 (x = 1-3, 5, 6)]. Many of these ligands displayed better selectivity for the alpha 1 containing GABAA isoform. The most selective BCCT 2 and SPH 195 (17) displayed potent affinity (Ki = 0.72 and 7.2 nM for the alpha 1 beta 3 gamma 2 receptor subtype, respectively) and an overall selectivity of 20 and 23 fold, respectively, for the alpha 1 beta 3 gamma 2 receptor subtype. These are the most selective ligands in vitro for the alpha 1 containing GABAA/Bz receptor isoform reported to date to our knowledge. QSAR studies of these ligands for each receptor subtype have been carried out via a Comparative Molecular Field Analysis (CoMFA) and an included volume analysis. Geometries and charge distributions of these ligands have been optimized using ab initio methods (J. Med. Chem., 1992, 35, 4001-4010). Active conformations of flexible 3-alkoxylated beta-carbolines have been examined via a CoMFA approach. QSAR studies via CoMFA support the previous hypothesis that beta-carbolines with different intrinsic activities may follow an alternative alignment rule when they bind into the pharmacophore/receptor site of the BzR. Examination of binding affinities of beta-carbolines by this modeling strategy has established some of the differences, in particular, topologic differences between the lipophilic pockets in the alpha 1 beta 3 gamma 2, alpha 2 beta 3 gamma 2, alpha 3 beta 3 gamma 2, alpha 5 beta 3 gamma 2 and alpha 6 beta 3 gamma 2 subtypes as well as some of the similarities among the pharmacophore/receptor models of these five distinct GABAA/Bz receptor subtypes.     

Honokiol and magnolol selectively interact with GABAA receptor subtypes in vitro.

Honokiol and magnolol have been identified as modulators of the GABAA receptors in vitro. Our previous study suggested a possible selectivity of honokiol and magnolol on GABAA receptor subtypes. This possibility was examined in the current study by 3H-muscimol and 3H-flunitrazepam binding assays on various rat brain membrane preparations and human recombinant GABA(A) receptor subunit combinations expressed by the Sf-9/baculovirus system. Generally, honokiol and magnolol have a similar enhancing effect on (3)H-muscimol binding to various membrane preparations in nonsaturation binding assays. Honokiol and magnolol preferentially increased (3)H-muscimol binding to hippocampus compared to cortex and cerebellum (with a maximum enhancement of 400% of control). As for subunit combinations, honokiol and magnolol have a more potent enhancing effect on alpha2 subunit containing combinations (with a maximum enhancement of 400-450% of control). This action was independent of the gamma subunit. In saturation binding assays, magnolol affected either the number of binding sites (ca. 4-fold on alpha2 containing combinations) or the binding affinity (on alpha1 containing combinations) of (3)H-muscimol binding to various GABAA receptor subunit combinations. In contrast, honokiol increased only binding sites on alpha2beta3gamma2s and alpha2beta3 combinations, but both the number of binding sites and the binding affinity on alpha1beta2gamma2S and alpha(1)beta2 combinations. These results indicate that honokiol and magnolol have some selectivity on different GABAA receptor subtypes. The property of interacting with GABAA receptors and their selectivity could be responsible for the reported in vivo effects of these two compounds.     

Interaction of beta-carboline inverse agonists for the benzodiazepine site with another site on GABAA receptors.

1. We examined the effects of methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM), a beta-carboline inverse agonist for the benzodiazepine site, on gamma-aminobutyric acid (GABA)-induced Cl-currents in several cloned rat GABAA receptor subtypes expressed in human embryonic kidney cells. The Cl- currents were measured in the whole cell configuration of patch clamp techniques. 2. DMCM at low concentrations (< 0.5 microM) occupying only the benzodiazepine site decreased GABA-induced Cl currents in the alpha 1 beta 2 gamma 2 and alpha 3 beta 2 gamma 2 subtypes as expected from an inverse agonist, but produced no change in the alpha 6 beta 2 gamma 2 subtype (perhaps a neutral antagonist). The drug at higher concentrations (> 0.5 microM) enhanced Cl- currents in all the subtypes with a half maximal concentration of 6 to 20 microM, depending on the alpha isoform. In the alpha 1 beta 2 subtype, which is without the benzodiazepine site, DMCM monophasically increased Cl- currents with a half maximal concentration of 1.9 microM. 3. Ro 15-1788 (a classical benzodiazepine antagonist) had no effect on Cl- current enhancement by DMCM and, in fact, increased the current level through blocking current inhibition by DMCM via the benzodiazepine site. Also, Cl- current enhancement by pentobarbitone or by 3 alpha, 21-dihydroxy-5 alpha-pregnan-20-one was additive to that by DMCM at saturating doses. It appears that the agonist site for DMCM is distinct from those for benzodiazepines, barbiturates and neurosteroids. 4. Among beta-carboline analogues, methyl-beta-carboline-3-carboxylate and propyl-beta-carboline-3-carboxylate markedly enhanced GABA-induced Cl currents in the alpha 1 beta 2 gamma 2 subtype, while N-methyl-beta-carboline-3-carboxamide and 1-methyl-7-methoxy-3,4-dihydro-beta-carboline did not. It appears that the 3-carboxyl ester moiety is necessary for beta-carbolines to interact with a novel site on GABAA receptors as agonists.     

Tonically active GABAA receptors in hippocampal pyramidal neurons exhibit constitutive GABA-independent gating

Phasic and tonic inhibitory currents of hippocampal pyramidal neurons exhibit distinct pharmacological properties. Picrotoxin and bicuculline methiodide inhibited both components, consistent with a role for GABAA receptors; however, gabazine, at a concentration that abolished miniature GABAergic inhibitory postsynaptic currents and responses to exogenous GABA, had no effect on tonic currents. Because all GABA-activated GABAA receptors in pyramidal neurons are gabazine-sensitive, it follows that tonic currents are not GABA-activated. Furthermore, picrotoxin-sensitive spontaneous single-channel events recorded from outside-out patches had the same chord conductance as GABA-activated channels and were gabazine-resistant. Therefore, we hypothesize that GABAA receptors, constitutively active in the absence of GABA, mediate tonic current; the failure of gabazine to block tonic current reflects a lack of negative intrinsic efficacy of the antagonist. We compared the negative efficacies of bicuculline and gabazine using the general anesthetic propofol to directly activate GABAA receptors native to pyramidal neurons or alpha1beta3gamma2 receptors recombinantly expressed in human embryonic kidney 293 cells. Propofol activated gabazine-resistant, bicuculline-sensitive currents when applied to either preparation. Although gabazine had negligible efficacy as an inhibitor of propofol-activated currents, it prevented inhibition by bicuculline, which acts as an inverse agonist inhibiting GABA-independent gating. Recombinant alpha1beta1/3gamma2 receptors also mediated agonist-independent tonic currents that were resistant to gabazine and inhibited by bicuculline. Thus, gabazine is a competitive antagonist with negligible negative efficacy and is therefore unable to inhibit GABAA receptors that are active in the absence of GABA because of either anesthetic or spontaneous gating. Moreover, spontaneously active GABAA receptors mediate gabazine-resistant tonic currents in pyramidal neurons.