Comparison of the distribution of convulsant/barbiturate and benzodiazepine receptors using light microscopic autoradiography

Some convulsant drugs elicit CNS excitation by blocking neuronal activity at GABAergic synapses whereas depressant compounds may result in the enhancement of GABAergic transmission. These effects are thought to involve drug actions at a multireceptor complex involving a benzodiazepine receptor, GABA receptor, picrotoxin receptor and a chloride ionophore. A radiolabeled convulsant, [35S]t-butylbicyclophosphorothionate [( 35S]-TBT) has been developed and used to characterize the binding to the "picrotoxin" or convulsant/barbiturate site. The microscopic distribution of the convulsant/barbiturate sites are reported in this communication, as demonstrated by receptor autoradiography after labeling tissue sections with [35S]-TBT. Comparison of the distribution of these sites with those of the benzodiazepine receptors show a close regional correlation in many areas. The convulsant/barbiturate sites and the benzodiazepine receptors, however, are unevenly distributed in the rat cerebellum and exist in separate lamina.     

Effect of ergot alkaloids on 3H-flunitrazepam binding to mouse brain GABAA receptors

In vitro effects of dihydroergotoxine, dihydroergosine, dihydroergotamine, alpha-dihydroergocriptine (ergot alkaloids), diazepam, methyl-beta-Carboline-3-carboxilate (beta-CCM), flumazenil (benzodiazepines), gamma-amino butyric acid (GABA) and thiopental (barbiturate) were studied on mouse brain (cerebrum minus cerebral cortex) benzodiazepine binding sites labeled with 3H-flunitrazepam. Specific, high affinity (affinity constant, Kd = 57.7 8.6 nM) binding sites for 3H-flunitrazepam on mouse brain membranes were identified. All benzodiazepine drugs inhibited 3H-flunitrazepam binding with nanomolar potencies. In contrast to benzodiazepines, all ergot drugs, GABA and thiopental produced an enhancement of 3H-flunitrazepam binding to its binding site at the GABAA receptor of the mouse brain. The rank order of potency was: neurotransmitter (GABA) > dihydroergotoxine > thiopental > alpha-dihydroergocriptine > dihydroergosine > dihydroergotamine. The results suggest that dihydrogenated ergot derivatives do not bind to the brain benzodiazepine binding sites labeled with 3H-flunitrazepam. However, an enhancement of 3H-flunitrazepam binding by all ergot drugs tested, clearly identifies an allosteric interaction with the benzodiazepine binding sites of GABAA receptors.     

Potentiation of GABAA receptors expressed in Xenopus oocytes by perfume and phytoncid.

To study the effects of perfume and phytoncid on GABAA receptors, ionotropic GABAA receptors were expressed in Xenopus oocytes by injecting mRNAs that had been prepared from rat whole brain. Essential oil, perfume and such phytoncid as leaf alcohol, hinokitiol, pinene, eugenol, citronellol and citronellal potentiated the response in the presence of GABA at low concentrations (10 and 30 microM), possibly because they bound to the potentiation-site in GABAA receptors and increased the affinity of GABA to the receptors. Since it is known that the potentiation of GABAA receptors by benzodiazepine, barbiturate, steroids and anesthetics induces the anxiolytic, anticonvulsant and sedative activity or anesthetic effect, these results suggest the possibility that the intake of perfume or phytoncid through the lungs, the skin or the intestines modulates the neural transmission in the brain through ionotropic GABAA receptors and changes the frame of the human mind, as alcohol or tobacco does.     

The effects of barbiturates upon the hemodynamic responses to intravenous methionine-enkephalin in dogs: modulation by the GABA complex

In conscious animals, the intravenous administration of enkephalins increases heart rate (HR) and mean systemic arterial blood pressure (MAP); however, when given during barbiturate anesthesia, enkephalins reduce HR and MAP. We have investigated the potential role of the gamma-aminobutyric acid (GABA) complex (consisting of chloride-ion channel and binding sites for GABA, benzodiazepine, and barbiturate/picrotoxin) as the site of modulation of enkephalin responses by certain anesthetic agents in our chronically instrumented dog model. In our model, methionine-enkephalin (Met5-ENK) (35 micrograms/kg intravenously) increased HR and MAP, but following induction of general anesthesia with barbiturate (pentobarbital) or of sedation with benzodiazepine (diazepam), Met5-ENK produced vasodepressor responses despite differing levels of consciousness in the treated animals. Subsequent administration of picrotoxin restored pressor responses to Met5-ENK in the barbiturate-treated dogs, but not in those treated with benzodiazepine; picrotoxin did not alter the level of consciousness. Picrotoxin had no effect upon Met5-ENK responses in the conscious state. In contrast, alpha-chloralose, a convulsive anesthetic agent which does not appear to alter GABA complex activity, blunted but did not reverse pressor responses to Met5-ENK, despite causing a level of anesthesia similar to that produced by barbiturate. The observed pressor response to Met5-ENK during alpha-chloralose anesthesia was totally inhibited by naloxone, indicating that this response was still mediated by opiate receptors. Our data are compatible with modulation of enkephalin responses by GABA complex activity. Systemic enkephalins may generate afferent signals which may subsequently undergo GABA complex processing; the state of activation of the GABA complex may then determine whether systemic enkephalin signals are translated as vasopressor or vasodepressor responses.     

Prevalence between different alpha subunits performing the benzodiazepine binding sites in native heterologous GABA(A) receptors containing the alpha2 subunit

The presence of two heterologous alpha subunits and a single benzodiazepine binding site in the GABA(A) receptor implicates the existence of pharmacologically active and inactive alpha subunits. This fact raises the question of whether a particular alpha subtype could predominate performing the benzodiazepine binding site. The hippocampal formation expresses high levels of alpha subunits with different benzodiazepine binding properties (alpha1, alpha2 and alpha5). Thus, we first demonstrated the existence of alpha2-alpha1 (36.3 +/- 5.2% of the alpha2 population) and alpha2-alpha5 (20.2 +/- 2.1%) heterologous receptors. A similar alpha2-alpha1 association was observed in cortex. This association allows the direct comparison of the pharmacological properties of heterologous native GABA(A) receptors containing a common (alpha2) and a different (alpha1 or alpha5) alpha subunit. The alpha2 subunit pharmacologically prevailed over the alpha1 subunit in both cortex and hippocampus (there was an absence of high-affinity binding sites for Cl218,872, zolpidem and [3H]zolpidem). This prevalence was directly probed by zolpidem displacement experiments in alpha2-alpha1 double immunopurified receptors (K(i) = 295 +/- 56 nM and 200 +/- 8 nM in hippocampus and cortex, respectively). On the contrary, the alpha5 subunit pharmacologically prevailed over the alpha2 subunit (low- and high-affinity binding sites for zolpidem and [3H]L-655,708, respectively). This prevalence was probed in alpha2-alpha5 double immunopurified receptors. Zolpidem displayed a single low-affinity binding site (K(i) = 1.73 +/- 0.54 microM). These results demonstrated the existence of a differential dominance between the different alpha subunits performing the benzodiazepine binding sites in the native GABA(A) receptors.     

Effect of thiopental on hippocampal GABA receptors in rat brain

The effect of thiopental on rat brain hippocampal GABA receptors was studied in slice preparations and membrane fractions. In slice preparations, thiopental at a concentration of 10⁻⁵ M enhanced the GABA (1−5 × 10⁻⁴M) inhibition of the field potentials evoked in pyramidal neurons by stratum radiatum stimulation. In hippocampal slices obtained from chronically barbital-administered (100 mg/kg, b.i.d., 10 days) rats, less enhancement of thiopental on GABA inhibition of the field potentials was observed. In binding experiments, two components of specific [³H]GABA binding to membrane fractions were observed. While thiopental was without effect on high-affinity sites, [³H]GABA binding with low affinity was enhanced by 80% in the presence of 10⁻⁵ M thiopental. The results are discussed in relation to the phenomena underlying chronic barbiturate administration.     

Differential effects of zinc on native GABA(A) receptor function in rat hippocampus and cerebellum.

Hippocampal noradrenergic and cerebellar glutamatergic granule cell axon terminals possess GABA(A) receptors mediating enhancement of noradrenaline and glutamate release, respectively. The hippocampal receptor is benzodiazepine-sensitive, whereas the cerebellar one is not affected by benzodiazepine agonists, indicating the presence of an alpha6 subunit. We tested here the effects of Zn2+ on these two native GABA(A) receptor subtypes using superfused rat hippocampal and cerebellar synaptosomes. In the cerebellum, zinc ions strongly inhibited (IC50 approximately 1 microM) the potentiation of the K(+)-evoked [3H]D-aspartate release induced by GABA. In contrast, the GABA-evoked release of [3H]noradrenaline from hippocampal synaptosomes was much less sensitive to Zn2+ (IC50 > 30 microM). The effects of Zn2+ were then studied in two rat lines selected for high (ANT) and low (AT) alcohol sensitivity because granule cell GABA(A) receptors in ANT, but not AT, rats respond to benzodiazepine agonists due to a critical mutation in the alpha6 subunit. GABA increased the K(+)-evoked release of [3H]DCNS REGIONS-aspartate from cerebellar synaptosomes of AT and ANT rats, an effect prevented by the GABAA selective antagonist bicuculline. In AT rat cerebellum, the effect of GABA was strongly inhibited by Zn2+ (IC50 < or = 1 microM), whereas in ANT rats, the divalent cation was about 100-fold less potent. Thus, native benzodiazepine-sensitive GABAA receptors appear largely insensitive to functional inhibition by Zn2+ and vice versa. Changes in sensitivity to Zn2+ inhibition consequent to mutations in cerebellar granule cell GABA(A) receptor subunits may lead to changes in glutamate release from parallel fibers onto Purkinje cells and may play important roles in cerebellar dysfunctions.     

Characterization of the interaction between fenamates and hippocampal neuron GABA(A) receptors

Fenamate NSAIDs have several central effects, including anti-epileptic and neuroprotective actions. The underlying mechanism(s) of these actions are not presently understood. In this study, the effects of five members of the fenamate NSAID group were investigated on native ligand-gated ion channels expressed in cultured rat hippocampal neurons. All fenamates tested (1-100 microM) dose-dependently potentiated GABA-evoked currents; mefenamic acid (MFA) was the most potent and efficacious and was found to shift the GABA dose-response curve to the left without effect on the maximum amplitude or the GABA Hill Slope. The modulation of GABA receptors by MFA was not reduced in the presence of the benzodiazepine antagonist, flumazenil (10 microM) and was moderately voltage-dependent. MFA at concentrations >or=10 microM evoked dose-dependent currents in the absence of GABA. These currents were potentiated by diazepam (1 microM) and blocked by bicuculline (10 microM). The MFA (50 microM) current-voltage relationship and reversal potential were similar to that evoked by GABA. MFA (1-100 microM) had no effects on sub-maximal glycine, glutamate or NMDA evoked currents. These data show that fenamate NSAIDs are a highly effective class of GABA(A) receptor modulator and activators.     

Semisynthetic preparation of amentoflavone: A negative modulator at GABA(A) receptors

Amentoflavone is found in a number of plants with medicinal properties, including Ginkgo biloba and Hypericum perforatum (St. John's Wort). We have developed a rapid and economic semi-synthetic preparation of amentoflavone from biflavones isolated from autumnal Ginkgo biloba leaves. Several studies have shown that amentoflavone binds to benzodiazepine receptors. Using two electrode voltage-clamp methodology, amentoflavone has been shown to be a negative modulator of GABA at GABA(A) alpha(1)beta(2)gamma(2L) receptors expressed in Xenopus laevis oocytes This action appears to be independent of the flumazenil-sensitive benzodiazepine modulatory sites on the GABA(A) receptor.     

Benzodiazepine receptors on human blood platelets

Binding studies conducted on membrane preparation from human platelets using (3H) Ro5-4864 and (3H) diazepam showed specific and saturable binding. Scatchard analysis revealed a single class of binding sites with KD = 10.8 +/- 0.9 nM and Bmax = 775 +/- 105 fmol/mg protein for (3H) Ro5-4864 and KD = 10.5 +/- 1.1 nM and Bmax = 133 +/- 19 fmol/mg for (3H) diazepam. We were unable to detect any GABA binding site on crude membrane preparation, nor did GABA enhance the binding of (3H) Ro5-4864 or (3H) diazepam. This suggests that benzodiazepine receptors are uncoupled to GABA system on human platelets. Ro15-1788, a specific antagonist for "central type" benzodiazepine (BDZ) binding sites was inactive in displacing (3H) Ro5-4864 from membrane receptors, while PK 11195 (a specific ligand for the "peripheral type" receptor) was the most potent of the drugs tested in inhibiting (3H) Ro5-4864 binding. These results indicate that human blood platelets bear "peripheral-type" BDZ receptor. Moreover, we could not detect any (3H) propyl beta carboline specific binding on platelet membranes. Results on benzodiazepine receptors on human circulating lymphocytes are also reported and similarity in pharmacological properties with platelet benzodiazepine receptors is suggested.     

Development and SAR of functionally selective allosteric modulators of GABAA receptors

Positive modulators at the benzodiazepine site of α2- and α3-containing GABA(A) receptors are believed to be anxiolytic. Through oocyte voltage clamp studies, we have discovered two series of compounds that are positive modulators at α2-/α3-containing GABA(A) receptors and that show no functional activity at α1-containing GABA(A) receptors. We report studies to improve this functional selectivity and ultimately deliver clinical candidates. The functional SAR of cinnolines and quinolines that are positive allosteric modulators of the α2- and α3-containing GABA(A) receptors, while simultaneously neutral antagonists at α1-containing GABA(A) receptors, is described. Such functionally selective modulators of GABA(A) receptors are expected to be useful in the treatment of anxiety and other psychiatric illnesses.     

Solubilization by CHAPS detergent of barbiturate-enhanced benzodiazepine-GABA receptor complex

Abstract: Barbiturates enhance the binding of [³H]flunitrazepam to benzodiazepine receptors solubilized with the detergent 3-[(3-cholamidopropyl)-dimethylammonio]propanesulfonate (CHAPS) from bovine cortex. The enhancement by the barbiturates is seen as a decrease in the dissociation constant, K _d , for specific benzodiazepine binding, with no effect on the number of binding sites. The effect of the barbiturates is facilitated by chloride ions, is concentration-dependent, and has a specificity that correlates well with the anesthetic potency of barbiturates. [³H]Flunitrazepam binding activity is stable with storage at 4°C., but barbiturate enhancement of soluble benzodiazepine binding activity decayed rapidly ( t _1/2= 48 h). [³H]Muscimol binding (GABA receptor) activity was also enhanced by barbiturates. Agarose gel filtration column chromatography of the CHAPS-solubilized receptor proteins showed the same elution profile as receptors solubilized with sodium deoxycholate, and enhancement by barbiturates was observed for both the benzodiazepine and GABA binding activities.     

Soluble benzodiazepine receptors: Gabaergic regulation

Benzodiazepine receptor binding has been measured in soluble brain extracts with ³H-flunitrazepam as a ligand. Binding to soluble receptors is enhanced by GABAergic agonists with potencies and maximal augmentation essentially the same as on membrane bound benzodiazepine receptors. The GABA induced increase of binding to soluble receptors is reversed by the GABA antagonist bicuculline.     

Effect of Chronic Ethanol Treatment on the γ-Aminobutyric Acid-Mediated Enhancement of [3H]Flunitrazepam Binding in Rat Cortex and Hippocampus

The mechanism by which ethanol affects the gamma-aminobutyric acid (GABA)/benzodiazepine complex is not clear. It is known that ethanol enhances the Cl- influx mediated by the GABAA receptor complex, and although chronic ethanol administration does not change the KD or Bmax for [3H]flunitrazepam binding, some reports have suggested that it could modify the modulation of benzodiazepine binding produced by GABA. In the present work, we studied the effect of chronic ethanol treatment on the modulation by GABA of [3H]flunitrazepam binding, using light microscopic autoradiography. This technique allows the measurement of densities of benzodiazepine receptors in different brain areas, the visual cortex and hippocampus, which appear to constitute the anatomical support for the behavioral and physiological responses affected by ethanol. We found enhancement of benzodiazepine binding by GABA at concentrations of greater than 10(-6) M for the various cortical and hippocampal areas studied from both control and ethanol-treated animals; this enhancement peaked at 10(-4) M GABA but decreased at 10(-3) M GABA. We found a clear effect of ethanol treatment on the modulatory properties of GABAA receptor, in both cortex and hippocampus, although only in cortex were the differences statistically significant between control and ethanol-treated animals.     

Two neighboring residues of loop A of the alpha1 subunit point towards the benzodiazepine binding site of GABAA receptors

Benzodiazepines are widely used drugs exerting sedative, anxiolytic, muscle relaxant, and anticonvulsant effects by acting through specific high affinity binding sites on some GABA(A) receptors. It is important to understand how these ligands are positioned in this binding site. We are especially interested here in the conformation of loop A of the alpha(1)beta(2)gamma(2) GABA(A) receptor containing a key residue for the interaction of benzodiazepines: alpha(1)H101. We describe a direct interaction of alpha(1)N102 with a diazepam- and an imidazobenzodiazepine-derivative. Our observations help to better understand the conformation of this region of the benzodiazepine pocket in GABA(A) receptor.     

The lack of CB1 receptors prevents neuroadapatations of both NMDA and GABA(A) receptors after chronic ethanol exposure

As the contribution of cannabinoid (CB1) receptors in the neuroadaptations following chronic alcohol exposure is unknown, we investigated the neuroadaptations induced by chronic alcohol exposure on both NMDA and GABA(A) receptors in CB1-/- mice. Our results show that basal levels of hippocampal [(3)H]MK-801 ((1)-5-methyl-10,11-dihydro-5Hdibenzo[a,d]cyclohepten-5,10-imine) binding sites were decreased in CB1-/- mice and that these mice were also less sensitive to the locomotor effects of MK-801. Basal level of both hippocampal and cerebellar [(3)H]muscimol binding was lower and sensitivity to the hypothermic effects of diazepam and pentobarbital was increased in CB1-/- mice. GABA(A)alpha1, beta2, and gamma2 and NMDA receptor (NR) 1 and 2B subunit mRNA levels were altered in striatum of CB1-/- mice. Our results also showed that [(3)H]MK-801 binding sites were increased in cerebral cortex and hippocampus after chronic ethanol ingestion only in wild-type mice. Chronic ethanol ingestion did not modify the sensitivity to the locomotor effects of MK-801 in both genotypes. Similarly, chronic ethanol ingestion reduced the number of [(3)H]muscimol binding sites in cerebral cortex, but not in cerebellum, only in CB1+/+ mice. We conclude that lifelong deletion of CB1 receptors impairs neuroadaptations of both NMDA and GABA(A) receptors after chronic ethanol exposure and that the endocannabinoid/CB1 receptor system is involved in alcohol dependence.     

Developing dual functional allosteric modulators of GABA(A) receptors

Positive modulators at benzodiazepine sites of α2- and α3-containing GABA(A) receptors are believed to be anxiolytic. Negative allosteric modulators of α5-containing GABA(A) receptors enhance cognition. By oocyte two-electrode voltage clamp and subsequent structure-activity relationship studies, we discovered cinnoline and quinoline derivatives that were both positive modulators at α2-/α3-containing GABA(A) receptors and negative modulators at α5-containing GABA(A) receptors. In addition, these compounds showed no functional activity at α1-containing GABA(A) receptors. Such dual functional modulators of GABA(A) receptors might be useful for treating comorbidity of anxiety and cognitive impairments in neurological and psychiatric illnesses.     

Kinetic properties of GABA rho1 homomeric receptors expressed in HEK293 cells

The rho1 subunit of the ionotropic GABA receptors is thought to contribute to the formation of the GABA(C) receptors with pharmacological and physiological properties distinct from those of GABA(A) receptors. Previous characterization of this subunit expressed in the Xenopus oocytes revealed an ion channel with slow activation and deactivation and no desensitization, quite different from the properties of GABA(C) receptors observed in native cells. We expressed the human rho1 subunit in human embryonic kidney (HEK) 293 cells and quantitatively characterized the kinetic properties of these receptors using a rapid drug application device. The rho1 subunit expressed in HEK293 cells exhibited pharmacological and kinetic properties qualitatively identical to those described when rho1 was expressed in the oocytes. An apparent desensitizing current observed during a constant GABA application was determined to be secondary to an E(Cl) shift. Detailed kinetic analyses and parameter estimation for a five-state kinetic model revealed that the channel is best described by a set of rate constants with a notably faster GABA unbinding K(off) rate compared to the parameters proposed for the same subunit expressed in the oocytes. The same subunit expressed in hippocampal neurons showed activation and deactivation kinetics identical to the current characterized in HEK293 cells. The kinetic properties of rho1 subunit expressed in a nonoocyte model system may be better described quantitatively by the rate constants presented here.     

Protection of soluble benzodiazepine receptors from heat inactivation by GABAergic ligands

Benzodiazepine receptors were solubilized from calf brain cortex by the ionic detergent deoxycholate and by the nonionic detergent Triton X-100. Approximately 90% of the soluble benzodiazepine receptors of both preparations were heat inactivated within 30 min at 55 degrees C. 100 microM of gamma-aminobutyric acid (GABA) protected 80% of Triton X-100 solubilized benzodiazepine receptors and 56% of the deoxycholate soluble benzodiazepine receptors from heat inactivation. Time course of heat inactivation showed that the deoxycholate soluble receptors are more sensitive to heat than the Triton X-100 soluble receptors.     

Distinct patterns of expression and regulation of GABA receptors containing the delta subunit in cerebellar granule and hippocampal neurons

Neuronal plasticity is achieved by regulation of the expression of genes for neurotransmitter receptors such as the type A receptor (GABA(A)R) for gamma-aminobutyric acid. We now show that two different rat neuronal populations in culture manifest distinct patterns of GABA(A)R plasticity in response to identical stimuli. Whereas prolonged exposure to ethanol had no effect on expression of the delta subunit of GABA(A)Rs at the mRNA or protein level in cerebellar granule neurons, it increased the abundance of delta subunit mRNA and protein in hippocampal neurons. Subsequent ethanol withdrawal transiently down-regulated delta subunit expression in cerebellar granule neurons and gradually normalized that in hippocampal neurons. These effects of ethanol exposure and withdrawal were accompanied by corresponding functional changes in GABA(A)Rs. GABA(A)Rs containing the delta subunit were also distributed differentially in the cerebellar and hippocampal neurons. These findings reveal complex and distinct mechanisms of regulation of the expression of GABA(A)Rs that contain the delta subunit in different neuronal types.