Benzodiazepine Ro 15-1788: Electrophysiological evidence for partial agonist activity

doi:10.1016/0304-3940(83)90208-2

Neuroscience Letters

Volume 43, Issues 2–3, 30 December 1983, Pages 321-326

https://doi.org/10.1016/0304-3940(83)90208-2 Get rights and content

Abstract

The effects of diazepam and Ro 15-1788 were assessed upon responses of mouse spinal cord (SC) neurons in cell culture to the amino acid neurotransmitters 4-aminobutyric acid (GABA) and S-glutamic acid. Diazepam (100 nM) enhanced GABA responses by 65 ± 3% (113 cells), while Ro 15-1788 (100 nM) failed to alter GABA responses but reduced their enhancement by diazepam. Higher Ro 15-1788 concentrations (1 μM or 10 μM) enhanced GABA responses to a moderate extent, while blocking further enhancement of GABA by diazepam. Neither diazepam nor Ro 15-1788 affected glutamate responses or resting membrane potential or conductance of spinal cord neurons. These results provide electrophysiological support for partial agonist, rather than pure antagonist, activity of Ro 15-1788.

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Cited by (41)

Endogenous Positive Allosteric Modulation of GABA<inf>A</inf> Receptors by Diazepam binding inhibitor
2013, Neuron
Citation Excerpt :
Similarly, FLZ has been observed to reduce evoked inhibitory postsynaptic potential (IPSP) amplitude in hippocampal CA1 pyramidal neurons (King et al., 1985) and eIPSC duration in dissociated cortical neurons (Vicini et al., 1986). Some reports have indicated agonist effects of FLZ (Skerritt and Macdonald, 1983; De Deyn and Macdonald, 1987; Weiss et al., 2002), including at receptors carrying α3 subunits (Ramerstorfer et al., 2010). This would not explain the reductions in duration and decay time observed here, although FLZ increased sIPSC amplitude and slightly increased charge transfer in both WT and α3(H126R) nRT cells, potentially indicating a nonspecific effect representing actions on presynaptic terminals (Table S1).
Benzodiazepines (BZs) allosterically modulate γ-aminobutyric acid type-A receptors (GABA_ARs) to increase inhibitory synaptic strength. Diazepam binding inhibitor (DBI) protein is a BZ site ligand expressed endogenously in the brain, but functional evidence for BZ-mimicking positive modulatory actions has been elusive. We demonstrate an endogenous potentiation of GABAergic synaptic transmission and responses to GABA uncaging in the thalamic reticular nucleus (nRT) that is absent in both nm1054 mice, in which the Dbi gene is deleted, and mice in which BZ binding to α3 subunit-containing GABA_ARs is disrupted. Viral transduction of DBI into nRT is sufficient to rescue the endogenous potentiation of GABAergic transmission in nm1054 mice. Both mutations enhance thalamocortical spike-and-wave discharges characteristic of absence epilepsy. Together, these results indicate that DBI mediates endogenous nucleus-specific BZ-mimicking (“endozepine”) roles to modulate nRT function and suppress thalamocortical oscillations. Enhanced DBI signaling might serve as a therapy for epilepsy and other neurological disorders.
The point mutation γ2F77I changes the potency and efficacy of benzodiazepine site ligands in different GABA<inf>A</inf> receptor subtypes
2010, European Journal of Pharmacology
Benzodiazepine site agonists or inverse agonists enhance or reduce γ-aminobutyric acid_A (GABA_A) receptor-mediated inhibition of neurons, respectively. Recently, it was demonstrated that the point mutation γ2F77I causes a drastic change in the affinity of a variety of benzodiazepine agonists or inverse agonists in receptor binding studies. Here we investigated the potency and efficacy of 10 benzodiazepine site ligands from 6 structural classes in wild-type and γ2F77I point mutated recombinant GABA_A receptors composed of α1β3γ2, α2β3γ2, α3β3γ2, α4β3γ2, α5β3γ2, and α6β3γ2 subunits. Results indicate that the effects of the benzodiazepine site ligands zolpidem, zopiclone, Cl218872, L-655,708 and DMCM were nearly completely eliminated in all mutated receptors up to a 1 μM concentration. The effects of bretazenil, Ro15-1788 or abecarnil were eliminated in some, but not all mutated receptors, suggesting that the γ2F77I mutation differentially influences the actions of these ligands in different receptor subtypes. In addition, this point mutation also influences the efficacy of diazepam for enhancing GABA-induced chloride flux, suggesting that the amino acid residue γ2F77 might also be involved in the transduction of the effect of benzodiazepines from binding to gating. The application of these drugs in a novel mouse model is discussed.
Enhancement of benzodiazepine binding sites following chronic treatment with flumazenil
2005, European Journal of Pharmacology
The aim of this study was to improve our knowledge of the mechanisms leading to adaptive changes in γ-aminobutyric acid_A (GABA_A) receptors following chronic drug treatment. Exposure (48 h) of human embryonic kidney (HEK 293) cells stably expressing recombinant α₁β₂γ_2S GABA_A receptors to the antagonist of benzodiazepine binding sites, flumazenil (5 μM), enhanced the maximum number (B_max) and the equilibrium dissociation constant (K_d) of [³H]flunitrazepam binding sites. The flumazenil-induced enhancement in B_max was potentiated by GABA (50 μM) and reduced by the GABA_A receptor antagonist, bicuculline (100 μM). Flumazenil-induced enhancement in K_d was affected by neither of these treatments. GABA (50 μM) enhanced the density of [³H]flunitrazepam binding sites, and this enhancement was greater in the presence of diazepam (1 μM). The results suggest that chronic flumazenil treatment up-regulates in a bicuculline-sensitive manner benzodiazepine binding sites at stably expressed GABA_A receptors.
Pharmacologic characteristics of excitatory γ-amino-butyric acid (GABA) receptors in a snail neuron
1997, General Pharmacology
- 1.
  1. The pharmacologic characteristics of excitatory γ-aminobutyric acid (GABA) receptors, termed muscimol II type GABA receptors, found in a giant neuron type, v-LCDN (ventral-left cerebral distinct neuron), of an African giant snail (Achatina fulica Férussac), were studied using the mammalian GABA receptor agonists, antagonists and synergists and GABA uptake inhibitor using the voltage clamp technique.
- 2.
  2. GABA and its agonists, ejected by brief pressure, produced an inward current (I_in) of the following order of potency: trans-t-aminocrotonic acid (TACA)>GABA>muscimol>isoguvacine> 5-aminopentanoic acid and cis-4-aminocrotonic acid (CACA). (±)-Baclofen and 3-aminopropylphosphonic acid (APPA) were ineffective. The I_in values produced by GABA, TACA, isoguvacine and CACA were stable for at least 60 min, whereas the I_in induced by muscimol was not.
- 3.
  3. According to the dose-response curves of GABA, TACA, isoguvacine and CACA, measured by the varied pressure duration method, the ED₅₀ value of CACA was larger than those of the other compounds, and E_max of TACA was larger than that of GABA, whereas E_max values of isoguvacine and CACA were smaller.
- 4.
  4. The perfusion of β-alanine, pentobarbital and 5-aminopentanoic acid inhibited the I_in induced by GABA, whereas (-)-bicuculline, pitrazepin, diazepam and 2-hydroxysaclofen had no effect.
- 5.
  5. From the effects of β-alanine on the dose-response curves of GABA, measured by the varied pressure duration method, β-alanine competitively inhibited the I_in caused by GABA. According to the effects of pentobarbital on the dose-response curves of GABA, this drug noncompetitively inhibited the I_in using the varied pressure duration method, and partly competitively and partly noncompetitively using the Y-tube method. The effects of 5-aminopentanoic acid on the dose-response curves of GABA indicated that this drug noncompetitively inhibited the I_in using the varied pressure duration method, and partly noncompetitively and partly uncompetitively using the Y-tube method.
- 6.
  6. The pharmacologic features of the Achatina muscimol II type GABA receptors were similar to those of mammalian GABA_C (GABA_pl) receptors, except for the effects of pentobarbital.
Ligand-dependent effects of ethanol and diethylether at brain benzodiazepine receptors
1992, Pharmacology, Biochemistry and Behavior
The GABA_A receptor chloride channel complex interacts with various categories of sedatives, including the benzodiazepines, and possibly ethanol and volatile general anesthetics. Thus, specific binding of tritiated derivatives of a benzodiazepine antagonist, flumazenil, and an agonist, flunitrazepam, to rat brain membrane fragments was monitored at equilibrium in the presence and absence of anesthetizing concentrations of ethanol and diethylether. Ethanol produced a concentration-dependent inhibition of [³H]flumazenil binding, which was not reversed by the GABA_A receptor competitive antagonist bicuculline, but had no effect on [³H]flunitrazepam binding. Both ethanol and diethylether decreased the affinity of the benzodiazepine site for [³H]flumazenil. These data indicate that ethanol and diethylether have GABA-independent effects at the benzodiazepine sites of the GABA_A receptor. These findings are inconsistent with a two-state functional model of the benzodiazepine site and, instead, support a model containing a specific, antagonist-favored conformation.
Suppressant effects of midazolam on responses of spinal dorsal horn neurones in rabbits
1992, Neuropharmacology
Recordings of noxious intra-arterial bradykinin (BK)-induced chemonociceptive and spontaneous activity from 30 single spinal lamina V neurones of the dorsal horns in non-anaesthetized and decerebrated rabbits, were performed with tungsten microelectrodes. Intravenous injection of midazolam (0.2 $mg kg$ ; 7 neurones) depressed BK-induced neural discharges by 55.0 ± 6.2% (P < 0.05) and 57.9 ± 8.4% (P < 0.05) 5 and 25 min after administration, respectively. Treatment with flumazenil (0.2 $mg kg$ , i.V.; 7 neurones), administered 20 min after midazolam, completely reversed the inhibition by midazolam of the BK-induced spinal lamina V neural responses and spontaneous neuronal activity. In contrast, a large dose of naloxone (1.0 $mg kg$ , i.V.; 6 neurones), administered 20 min after midazolam, failed to alter the midazolam-induced depressant effects on the nociceptive responses, at the spinal dorsal horn. Treatments with flumazenil (5 neurones) and naloxone (5 neurones) did not influence either the spontaneous or the BK-induced neuronal discharges, recorded in spinal lamina V cells. Midazolam depressed the nociceptive responses probably through its agonistic activity on the binding to the GABA-benzodiazepine-barbiturate system in the spinal dorsal horn.

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Benzodiazepine Ro 15-1788: Electrophysiological evidence for partial agonist activity

Abstract

Life Sci.

Europ. J. Pharmacol.

Life Sci.

Brain Res.

Brain Res.

Neurosci. Lett.

Europ. J. Pharmacol.

Europ. J. Pharmacol.

Brain Res.

Chlordiazepoxide selectively augments GABA action in spinal cord cell cultures

Nature (Lond.)