Interaction of caffeine with the GABAA receptor complex: alterations in receptor function but not ligand binding

Behavioral and neurochemical evidence indicates interactions between caffeine and other adenosine receptor ligands and the gamma-aminobutyric acid (GABA)-benzodiazepine system. To assess the effects of caffeine on binding and function at the GABAA receptor, we studied the effects of behaviorally-active doses of caffeine on benzodiazepine and Cl- channel binding and on overall function of the GABAA receptor as measured by Cl- uptake. There was no effect of caffeine on benzodiazepine receptor binding in cortical synaptosomal membranes at concentrations of 1-100 microM. No effects on benzodiazepine binding were found ex vivo in mice treated with caffeine, 20 and 40 mg/kg. At the putative Cl- channel site labeled by t-butylbicyclophosphorothionate (TBPS), binding was unchanged in vitro after caffeine treatment (1 and 10 microM) in washed and unwashed membranes. However, in ex vivo studies caffeine (20 and 40 mg/kg) increased numbers of TBPS sites in unwashed but not washed membranes. Muscimol-stimulated Cl- uptake into cortical synaptoneurosomes was decreased in mice treated with caffeine, 20 and 40 mg/kg. Similar results were observed in in vitro preparations treated with 50 microM but not 100 microM caffeine. These results indicate that caffeine administration significantly alters the Cl- transport function of the GABAA receptor complex.     

Neuroactive flavonoids interacting with GABAA receptor complex

Classical benzodiazepines (BZs) are the most widely prescribed drugs acting on the central nervous system (CNS). They exert their therapeutic effects via binding to the BZ-site of GABAA receptors, and allosterically modulating the chloride flux through the ion channel complex. Given the multiple actions of classical BZs, the serious limitations to their usefulness have directed much research into development of novel ligands for the BZ-site with retained therapeutic effectiveness and minimal side effects. From the studies of CNS-active chemical constituents of medicinal herbs, some members of the family of flavonoids were demonstrated to have moderate binding affinities for the BZ-site. In vivo studies revealed that these compounds were mostly partial agonists of GABAA receptors, and only a few flavonoids were shown to possess antagonistic activities. At effective anxiolytic doses, the actions of partial agonistic flavonoids were often not accompanied by sedative and myorelaxant side effects. Based on structure-activity relationship (SAR) studies, incorporation of electronegative groups to the C6 and C3' on the flavone backbone was found to yield significant increases in the binding affinities for the BZ-site. It was also shown that 2'-hydroxyl was a critical moiety on flavonoids with regard to BZ-site binding. These have guided the identification of several synthetic flavonoids with high BZ-site binding affinity and in vivo activity, and further quantitative SAR studies resulted in the development of several pharmacophore models. This review attempts to summarize these findings, which has led to the establishment of flavonoids as potential therapeutics for GABAA receptor-mediated disorders.     

Caprolactam-barbiturate interaction at the GABAA receptor complex in the guinea-pig intestine

4,6,6-Trimethylcaprolactam antagonised GABAA receptor-mediated contractile responses in guinea-pig isolated ileum, displacing the GABA dose-response curve to the right in a non-parallel manner, and causing a depression of the maximum response. Pentobarbitone not only potentiated the GABAA receptor-mediated contractions but also reversed this non-competitive antagonism by 4,6,6-trimethylcaprolactam, shifting the dose-response curve for GABA to the left and restoring the maximum response. It is conclude that this caprolactam acts at the picrotoxin-barbiturate site on the Cl(-)-ionophore complex.     

Biphasic effect of GABAA receptor agonists on prolactin secretion: evidence for two types of GABAA receptor complex on lactotrophes

The effects of GABA receptor agonists on prolactin secretion in vitro was examined using a rapid superfusion system. GABA and muscimol caused a biphasic effect on prolactin secretion, both components of which were antagonised by bicuculline methiodide, while baclofen had no effect on basal or stimulated secretion, demonstrating the GABAA receptor specificity of both components. Homocarnosine caused only inhibition of secretion, and a range of partly rigid GABA analogues were relatively poor at causing stimulation of secretion. Both effects of muscimol were antagonised by low-chloride medium and the anion channel blocker DIDS, but strychnine and picrotoxinin were both potent and selective antagonists of the stimulatory effect. These results demonstrate a novel biphasic effect of GABAA agonists or prolactin secretion, the two components of which appear to be independent and mediated by different types or states of GABAA receptor/chloride channel complex.     

Differential alterations in striatal dopamine receptor sensitivity induced by repeated administration of clinically equivalent doses of haloperidol,sulpiride or clozapine in rats

Rats received therapeutically equivalent doses of either haloperidol (1.7–1.9 mg/kg/day), sulpiride (112–116 mg/kg/day) or clozapine (30–35 mg/kg/day) continuously for 4 weeks. Treatment with haloperidol, but not sulpiride or clozapine, caused inhibition of stereotyped behaviour induced by apomorphine (0.125–0.25 mg/kg SC). Following drug withdrawal for up to 7 days, haloperidol and sulpiride, but not clozapine treatment caused an exaggeration of stereotyped behaviour induced by apomorphine.B_max values for striatal ³H-spiperione binding were erevated in animals treated for 2 and 4 weeks with haloperidol, but not with sulpiride or clozapine. Following drug withdrawal, haloperidol, but not sulpiride or clozapine, treatment caused an increase in B_max for striatal ³H-piperone binding.B_max values for striatal ³H-NPA binding revealed no change during haloperidol or clozapine treatment. Sulpiride treatment for 1 week caused an increase in B_max for ³H-NPA binding, which returned to control levels at 2 and 4 weeks. Following drug withdrawal, there was an increase in B_max for ³H-NPA binding in rats treated with haloperidol and sulpiride, but not clozapine.On continuous treatment and following withdrawal from haloperidol, sulpiride, or clozapine the ability of dopamine to stimulate striatal adenylate cyclase activity did not differ from that in control animals.Repeated administration of sulpiride or clozapine may not induce striatal dopamine receptor supersensitivity when given in clinically relevant doses, although haloperidol does.     

The GABAA receptor antagonist picrotoxin attenuates most sleep changes induced by progesterone

Progesterone has been shown to exert benzodiazepine-like effects on sleep, which suggests that they are mediated by an agonistic modulation of GABA_A receptor functioning. To assess the involvement of GABA_A receptors, we investigated the sleep responses to one dose of the GABA_A antagonist picrotoxin (1.5 mg/kg) and progesterone (90 mg/kg), administered IP to eight rats alone and in combination, during the first 4 post-injection hours. Compared with vehicle, picrotoxin significantly delayed the latency to non-rapid eye movement sleep (non-REMS) and thereby decreased all sleep states, but barely affected the EEG activity within non-REMS. Progesterone significantly shortened non-REMS latency, increased pre-REMS, depressed low-frequency EEG activity (≤8 Hz) and augmented EEG activity in the higher frequencies within non-REMS. Except for the changes in high-frequency EEG activity, picrotoxin attenuated all effects of progesterone. These findings support the notion that GABA_A receptors play an important role in the sleep effects of progesterone. Received: 27 May 1998/Final version: 26 June 1998     

GABAA receptor complex function in frontal cortex membranes from control and neurological patients.

The functional integrity of the GABAA receptor-benzodiazepine (BZ) recognition site-Cl- ionophore complex was assessed by means of [35S]TBPS (t-butylbicyclophosphorothionate) binding to frontal cortex membranes prepared from frozen postmortem brain tissue taken from control (n = 4), Alzheimer (n = 7), Parkinson (n = 3) and Huntington's chorea (n = 2) patients. Specific [35S]TBPS binding was similar in control, Parkinson's disease and Huntington's chorea brains, but was significantly reduced (78% control, P less than 0.01) in frontal cortex membranes from Alzheimer's patients. The linkage between the BZ recognition sites and the GABAA receptor-linked Cl- ionophore was functionally intact in these membranes as BZ site agonists (zolpidem, alpidem, flunitrazepam and clonazepam) enhanced [35S]TBPS binding under the conditions used (well-washed membranes in the presence of 1.0 M NaCl). Zolpidem (BZ1 selective) exhibited a biphasic enhancement in control membranes whereas the other compounds induced a bell-shaped concentration-response curve. The enhancement of [35S]TBPS binding by alpidem, flunitrazepam and clonazepam was greater in frontal cortex membranes from Alzheimer's patients than in controls whereas it tended to be reduced in membranes from the brains of Huntington's chorea patients. These studies demonstrate the functional integrity of the GABAA receptor macromolecular complex and also the usefulness of [35S]TBPS binding in the study of human postmortem tissue.     

A four-ligand hypercube model to quantify allosteric interactions within the GABAA receptor complex

The aim of this study was to investigate the couplings between various binding sites on the GABA(A) receptor complex. We investigated combinations of three test compounds: (1) GABA (gamma-aminobutyric acid), (2) Org 20549 [(2 beta 3 alpha 5 alpha)-21hydroxy-3Hydroxy-2(4morpholinyl)pregnan-20one methane-sulphonate)], a neuroactive steroid and (3) retigabine (D-23129, N-(2-amino-4-(4-fluorobenzylamino)-phenyl) carbamic acid ethyl ester), a new antiepileptic drug. Receptor-binding assays were conducted using rat brain membranes. [3H]TBOB ([3H]-t-butyl-bicyclo-ortho-benzoate) was the tracer ligand. All three test compounds inhibited the binding of [3H]TBOB with EC(50)'s of 4.0, 98 and 23 microM, respectively. Isobolic analysis of the combination data showed that the three compounds act in synergy in displacing [3H]TBOB. These interactions could be described and quantified by a hypercube model in which each of the three test compounds and [3H]TBOB bind to different, allosterically coupled sites such that each of the test compounds allosterically displaces the tracer [3H]TBOB and allosterically enhances the affinity of any other test compound by a factor 4.4. The simultaneous binding of any two ligands enhances the affinity of the third by a factor 9. These results may contribute to the understanding of individual variability in drug responses and to the discussion about rational polytherapy.     

Modulation of the GABAA receptor complex by steroids in slices of rat cuneate nucleus.

1. Several derivatives of pregnane and androstane that have hypnotic properties have been investigated for their ability to potentiate responses to the GABAA receptor agonist muscimol and to reduce the effect of the non-competitive GABAA antagonist picrotoxin. 2. Depolarizing responses to muscimol in slices of rat cuneate nucleus were potentiated most potently by 3 alpha-hydroxy,5 alpha-pregnane-11,20-dione (alphaxalone), which gave half-maximal potentiation at 0.15 microM. The 5 beta isomer of alphaxalone had little effect up to 3 microM but in analogues lacking an 11-keto substituent (pregnanolones), both the 5 alpha- and 5 beta-isomers potentiated with potencies 20 and 10 times lower, respectively, than that of alphaxalone. The alpha configuration of the 3-hydroxy was essential in alphaxalone, the 3 beta-hydroxy isomer being inactive. However, there was little difference between the potencies of the 3 alpha- and 3 beta-hydroxy configurations in the pregnanolones, although the maximal effects of the 3 beta-hydroxy isomers were rather lower than those of the 3 alpha-hydroxy isomers. 3. Reductions in the effect of picrotoxin as an antagonist of muscimol were caused most potently by the 3 alpha-hydroxy pregnanolones, with a ten fold reduction in picrotoxin potency at 1 microM concentrations of these steroids. Alphaxalone and its 5 beta-isomer were about half as potent. Androsterone was about 10 times less potent and the 3 beta-hydroxy pregnanolones were ineffective. 4. This difference in the structure-activity relationships for steroidal potentiation of muscimol and reduction in picrotoxin antagonism of muscimol is reminiscent of an analogous distinction found with the barbiturates.     

Synergy between retigabine and GABA in modulating the convulsant site of the GABAA receptor complex

The molecular mechanism underlying the activity of the novel antiepileptic drug retigabine is not yet fully understood. The aim of this study was to investigate whether retigabine interacts directly with the GABA(A) receptor complex (gamma-aminobutyric acid). Receptor-binding assays were conducted using rat brain membranes. [3H]-t-Butyl-bicyclo-orthobenzoate ([3H]TBOB) was used as a tracer ligand. We determined the effects of GABA and retigabine in the presence of several concentrations of GABA on the binding of [3H]TBOB. GABA inhibited [3H]TBOB binding with an EC(50) of 4.8 microM. In the absence of GABA, retigabine inhibited [3H]TBOB with an EC(50) of 124 microM and an EC(50) of 42 microM in the presence of 2.5 microM GABA. Isobolic analysis revealed that retigabine acts in synergy with GABA in displacing [3H]TBOB. This synergy could be quantified by a molecular model in which GABA and retigabine both allosterically displace [3H]TBOB, and retigabine allosterically enhances the binding of GABA and vice versa with a factor of 4. In summary, we found that retigabine does indeed interact with a site on the GABA(A) receptor complex, and this site is positively allosterically coupled with the GABA site. This GABA-positive effect may well contribute to the clinical anticonvulsive effects of retigabine.     

GABA and the GABAA receptor

The neurotransmitter gamma-aminobutyric acid (GABA) inhibits the activity of signal-receiving neurons by interacting with the GABAA receptor on these cells. The GABAA receptor is a channel-forming protein that allows the passage of chloride ions into the cells. Excessive GABAA activation may play a role in mediating the sedative effects of alcohol and other sedating and anesthetic agents. For example, alcohol enhances the GABAA-mediated chloride flow into cells and may thereby enhance neuronal inhibition. Alcohol's effects on the GABAA-receptor function likely involve other molecules (e.g., other neurotransmitters and proteins that add phosphate groups to the receptor [i.e., protein kinases]). Several experimental approaches also have suggested that changes in GABAA-receptor function contribute to the tolerance to and dependence on alcohol. Finally, individual differences in the GABA system may play a role in determining a person's susceptibility to developing alcohol dependence.     

GABAA and NMDA receptor density alterations and their behavioral correlates in the gestational methylazoxymethanol acetate model for schizophrenia

Hippocampal hyperactivity driven by GABAergic interneuron deficits and NMDA receptor hypofunction is associated with the hyperdopaminergic state often observed in schizophrenia. Furthermore, previous research in the methylazoxymethanol acetate (MAM) rat model has demonstrated that repeated peripubertal diazepam administration can prevent the emergence of adult hippocampal hyperactivity, dopamine-system hyperactivity, and associated psychosis-relevant behaviors. Here, we sought to characterize hippocampal GABA_A and NMDA receptors in MAM-treated rats and to elucidate the receptor mechanisms underlying the promising effects of peripubertal diazepam exposure. Quantitative receptor autoradiography was used to measure receptor density in the dorsal hippocampus CA1, ventral hippocampus CA1, and ventral subiculum. Specifically, [³H]-Ro15-4513 was used to quantify the density of α5GABA_A receptors (α5GABA_AR), [³H]-flumazenil to quantify α1-3;5GABA_AR, and [³H]-MK801 to quantify NMDA receptors. MAM rats exhibited anxiety and schizophrenia-relevant behaviors as measured by elevated plus maze and amphetamine-induced hyperlocomotion (AIH), although diazepam only partially rescued these behaviors. α5GABA_AR density was reduced in MAM-treated rats in all hippocampal sub-regions, and negatively correlated with AIH. Ventral hippocampus CA1 α5GABA_AR density was positively correlated with anxiety-like behavior. Dorsal hippocampus CA1 NMDA receptor density was increased in MAM-treated rats, and positively correlated with AIH. [³H]-flumazenil revealed no significant effects. Finally, we found no significant effect of diazepam treatment on receptor densities, potentially related to the only partial rescue of schizophrenia-relevant phenotypes. Overall, our findings provide first evidence of α5GABA_AR and NMDA receptor abnormalities in the MAM model, suggesting that more selective pharmacological agents may become a novel therapeutic mechanism in schizophrenia.Subject terms: Psychosis, Schizophrenia, Experimental models of disease, Schizophrenia, Psychosis     

GABAA receptor-associated protein regulates GABAA receptor cell-surface number in Xenopus laevis oocytes

GABA(A) receptor-associated protein (GABARAP) was isolated previously in a yeast two-hybrid screen using the intracellular loop of the gamma2 subunit of the GABA(A) receptor as bait. GABARAP has been shown to participate in the membrane-clustering and intracellular-trafficking of GABA(A) receptors, including a stimulation of the surface expression of GABA(A) receptors. To assess this quantitatively, we used Xenopus laevis oocytes expressing alpha1beta2gamma2S-containing GABA(A) receptors to demonstrate that coexpression of GABARAP increased net surface levels of GABA(A) receptors as shown by both increased GABA currents and surface-expressed protein. This GABARAP stimulation of GABA currents required the receptor gamma2 subunit and full-length GABARAP: deletion of the microtubule-binding domain (amino acids 1-22) or disrupting the polymerization of microtubules abolished the enhancement, indicating that the effect of GABARAP was derived from the interaction with microtubules. GABARAP coexpression did not alter the general properties of GABA(A) receptors such as sensitivity to GABA or benzodiazepines, but it increased surface levels of receptor protein in oocytes. Rather, it seems to supplement inadequate amounts of endogenous GABARAP to support optimum trafficking and/or stabilization of surface GABA(A) receptors.     

A progesterone metabolite enhances the activity of the GABAA receptor complex at the pituitary level

The interaction of 5 alpha-pregnane-3 alpha-ol-20-one (5 alpha 3 alpha P), a progesterone metabolite, with the GABAA receptor chloride channel complex was investigated at the pituitary level. In nanomolar concentrations this steroid potentiated the inhibitory effect of muscimol (a GABAA agonist) on prolactin release from rat pituitary cells in culture. In micromolar concentrations 5 alpha 3 alpha P had a direct inhibitory effect, similar to that of muscimol, with an IC50 value of 370 nM. This effect was antagonized by bicuculline, a GABAA antagonist, and by picrotoxin, a chloride ion channel blocker. Its reduced isomer, 5 alpha 3 beta P, and progesterone (Pg) were devoid of activity. Using [35S]t-butylbicyclophosphorothionate ([35S]TBPS) as a ligand, we demonstrated, for the first time, specific barbiturate sites on pituitary membranes, similar to those of the central nervous system, with a Kd value of 25 nM and a Bmax value of 62 fmol/mg protein. 5 alpha 3 alpha P inhibited the binding of [35S]TBPS. In contrast, its 3 beta isomer was inactive. These data show that 5 alpha 3 alpha P enhanced the activity of the GABAA receptor complex at the pituitary level and suggest that its inhibitory effect on prolactin release might be mediated by the barbiturate site or by a closely related site.     

Characterization of U-97775 as a GABAA receptor ligand of dual functionality in cloned rat GABAA receptor subtypes.

1. U-97775 (tert-butyl 7-chloro-4,5-dihydro-5-[(1-(3,4,5-trimethyl)piperazino)carbonyl]- imidazo[1,5-a])quinoxaline-3-carboxylate) is a novel GABAA receptor ligand of dual functionality and was characterized for its interactions with cloned rat GABAA receptors expressed in human embryonic kidney cells. 2. The drug produced a bell-shaped dose-response profile in the alpha 1 beta 2 gamma 2 receptor subtype as monitored with GABA-induced Cl- currents in the whole cell patch-clamp technique. At low concentrations (< 0.5 microM), U-97775 enhanced the currents with a maximal increase of 120% as normalized to 5 microM GABA response (control). An agonist interaction of U-97775 with the benzodiazepine site is suggested, because Ro 15-1788 (an antagonist at the benzodiazepine site) abolished the current increase and [3H]-flunitrazepam binding was inhibited by U-97775 with a Ki of 1.2 nM. 3. The enhancement of GABA currents progressively disappeared as the U-97775 concentration was raised above 1 microM, and the current amplitude was reduced to 40% below the control at 10 microM U-97775. The current inhibition by U-97775 (10 microM) was not affected by Ro 15-1788. It appears that U-97775 interacts with a second site on GABA receptors, distinct from the benzodiazepine site, to reverse its agonistic activity on the benzodiazepine site and also to inhibit GABA currents. 4. U-97775 at low concentrations reduced and at high concentrations enhanced [35S]-TBPS binding. Ro 15-1788 selectively blocked the effect of U-97775 at low concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

The GABAA receptor complex in hepatic encephalopathy. Autoradiographic evidence for the presence of elevated levels of a benzodiazepine receptor ligand

Autoradiographic analysis was used to examine radioligand binding to benzodiazepine (BZ) and GABAA receptors in the brains of rabbits with hepatic encephalopathy (HE). Thin sections of whole brain from normal rabbits and rabbits with HE were mounted on slides and subdivided into two groups. One group was washed before incubation with radioligand, while the second group was not prewashed. [3H]Flunitrazepam binding to BZ receptors was decreased by 22% to 42% (p less than 0.05) in the cerebral cortex, superior and inferior colliculi, and cerebellum of unwashed sections from rabbits with HE compared to all other groups. The binding of [3H]Ro 15-1788 to unwashed sections from rabbits with HE was reduced by a similar degree (18% to 37%, p less than 0.05) in the cerebral cortex, hippocampus, superior colliculus, and cerebellar cortex. Incubation of sections with the GABA-mimetic muscimol and NaCl produced an additional decrease in [3H]flunitrazepam binding to the cortex and hippocampus (25% to 31%, p less than 0.05) in unwashed HE rabbit brain, but increased radioligand binding (27% to 71%, p less than 0.05) to several regions in control rabbits. No changes in radioligand binding to either GABAA or peripheral benzodiazepine receptors was observed between HE and control rabbit sections. These findings are consistent with previous electrophysiologic and neurochemical observations indicating no significant changes in either the function or density of GABAA or BZ receptors in this model of HE. Further, they indicate that a reversible BZ receptor ligand with agonist properties is present in the brain in HE. This substance may contribute to the enhancement of GABAergic tone observed in this syndrome.     

Involvement of GABAA receptor sites in diazepam hypothermia

1. Intraperitoneal (i.p.) injection of diazepam (1.5-6 mg/kg) decreased the core body temperature (BT) of the rats. The effect was dose-dependent. 2. The hypothermia produced by diazepam (6 mg/kg, i.p.) was decreased in animals pretreated with high doses of bicuculline (BIC, 3 mg/kg, i.p.), while low doses of BIC (1.5 mg/kg, i.p.) potentiated the hypothermia. 3. Picrotoxin (PIC, 1 and 2 mg/kg, i.p.) pretreatment also decreased the hypothermic effect of diazepam. 4. Pretreatment of animals with atropine (AT, 10 mg/kg, i.p.) or propranolol (PRO, 10 mg/kg, i.p.) potentiated the hypothermic response of diazepam. Phenoxybenzamine (PHEN, 0.5 mg/kg, i.p.), methergoline (METH, 0.5 mg/kg, i.p.) or pimozide (PIM, 0.5 mg/kg, i.p.) did not change the diazepam hypothermia. 5. Single administration of BIC, PIC, PRO, PIM or METH also induced hypothermia. 6. One can postulate that diazepam hypothermia may be induced through GABAA receptor sites. However, further studies will clarify this hypothesis.