Two metabolites of the steroid hormones progesterone and deoxycorticosterone, 3 alpha-hydroxy-5 alpha-dihydroprogesterone and 3 alpha, 5 alpha-tetrahydrodeoxycorticosterone, are potent barbiturate-like ligands of the gamma-aminobutyric acid (GABA) receptor-chloride ion channel complex. At concentrations between 10(-7) and 10(-5)M both steroids inhibited binding of the convulsant t-butylbicyclophosphorothionate to the GABA-receptor complex and increased the binding of the benzodiazepine flunitrazepam; they also stimulated chloride uptake (as measured by uptake of 36Cl-) into isolated brain vesicles, and potentiated the inhibitory actions of GABA in cultured rat hippocampal and spinal cord neurons. These data may explain the ability of certain steroid hormones to rapidly alter neuronal excitability and may provide a mechanism for the anesthetic and hypnotic actions of naturally occurring and synthetic anesthetic steroids.

https://science.sciencemag.org/content/sci/232/4753/1004.full.pdf

Steroid Hormone Metabolites are Barbiturate-Like Modulators of the GABA Receptor

The physiology of excitatory amino acids in the vertebrate central nervous system.

The compound MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine maleate)] is a potent anticonvulsant that is active after oral administration and whose mechanism of action is unknown. We have detected high-affinity (Kd = 37.2 +/- 2.7 nM) binding sites for [3H]MK-801 in rat brain membranes. These sites are heat-labile, stereoselective, and regionally specific, with the hippocampus showing the highest density of sites, followed by cerebral cortex, corpus striatum, and medulla-pons. There was no detectable binding in the cerebellum. MK-801 binding sites exhibited a novel pharmacological profile, since none of the major neurotransmitter candidates were active at these sites. The only compounds that were able to compete for [3H]MK-801 binding sites were substances known to block the responses of excitatory amino acids mediated by the N-methyl-D-aspartate (N-Me-D-Asp) receptor subtype. These comprised the dissociative anesthetics phencyclidine and ketamine and the sigma-type opioid N-allylnormetazocine (SKF 10,047). Neurophysiological studies in vitro, using a rat cortical-slice preparation, demonstrated a potent, selective, and noncompetitive antagonistic action of MK-801 on depolarizing responses to N-Me-D-Asp but not to kainate or quisqualate. The potencies of phencyclidine, ketamine, SKF 10,047, and the enantiomers of MK-801 as N-Me-D-Asp antagonists correlated closely (r = 0.99) with their potencies as inhibitors of [3H]MK-801 binding. This suggests that the MK-801 binding sites are associated with N-Me-D-Asp receptors and provides an explanation for the mechanism of action of MK-801 as an anticonvulsant.

https://www.pnas.org/content/pnas/83/18/7104.full.pdf

The anticonvulsant MK-801 is a potent N-methyl-D-aspartate antagonist.

The current studies further investigated the effects, in animal models of anxiety, of novel putative anxiolytic and anxiogenic compounds believed to induce their effects by actions at the GABA-benzodiazepine receptor complex. It was expected that the results would also provide further validation for a novel test of anxiety based on the ratio of open to closed arm entries in an elevated plus maze in the rat. The novel putative anxiolytics CL 218,872 (10–20 mg/kg) and tracazolate (5 mg/kg) significantly elevated the percentage of time spent on the open arms of an elevated plus-maze, consistent with their anxiolytic activity in several other animal tests. Also consistent with results from other animal tests, no anxiolytic activity was observed for the phenylquinoline PK 8165 (10–25 mg/kg), the 3,4-benzodiazepine tofisopam (25–50 mg/kg), or buspirone (0.5–20 mg/kg). The benzodiazepine receptor inverse agonists FG 7142 (1–5 mg/kg) and CGS 8216 (3–10 mg/kg) had anxiogenic activity in this test, as did the atypical benzodiazepine Ro 5-4864 (1–5 mg/kg). Interestingly, however, the benzodiazepine receptor antagonists Ro 15-1788 (10–20 mg/kg) and ZK 93426 (5–10 mg/kg) had no anxiogenic activity in this test.

Anxiolytic and anxiogenic drug effects on exploratory activity in an elevated plus-maze: a novel test of anxiety in the rat

Magnesium. An update on physiological, clinical and analytical aspects.

Modulation of the GABA receptor complex by a steroid anaesthetic.

Coronal sections of rat brain (500 micron thick) were trimmed to form 'wedges' of tissue consisting of cerebral cortex and corpus callosum. When these slices were placed in a two-compartment bath, the cortical tissue could be depolarized, relative to the corpus callosum, by superfusions of high K+, or by amino acids such as L-glutamate, L-aspartate, quisqualate, kainate and N-methyl D-aspartate (NMDA). Responses to NMDA were reduced by magnesium ions, by the organic antagonists (-)-2-amino 5-phosphonovalerate (APV) and 2-amino 7-phosphonoheptanoate (APH), and by the dissociative anaesthetic ketamine. In this preparation, all these antagonists shifted the NMDA dose-response curve to the right in a parallel manner. A Schild plot for Mg2+ had a slope significantly less than unity, indicative of a non-competitive action, whilst Schild plots for (-)-APV, APH and ketamine appeared linear and had slopes of approximately 1. Analysis of the results of combination experiments suggested that the presumed competitive antagonists, (-)-APV and APH, share a common site of action as NMDA antagonists, and that this site is distinct from that at which ketamine exerts its action. The action of Mg2+ is clearly different from that of either (-)-APV or ketamine. It is concluded that ketamine is a non-competitive antagonist of NMDA and may act at an allosteric site on the NMDA receptor complex to influence its function.

Quantitative studies on some antagonists of N-methyl D-aspartate in slices of rat cerebral cortex.

Effects of membrane cholesterol on the sensitivity of the GABAA receptor to GABA in acutely dissociated rat hippocampal neurones

Classification of some GABA antagonists with regard to site of action and potency in slices of rat cuneate nucleus.

Differential antagonism by epipregnanolone of alphaxalone and pregnanolone potentiation of [3H]flunitrazepam binding suggests more than one class of binding site for steroids at GABAA receptors.

Michael A. Simmonds

Papers

Modulation of the GABA receptor complex by a steroid anaesthetic.

Quantitative studies on some antagonists of N-methyl D-aspartate in slices of rat cerebral cortex.

Effects of membrane cholesterol on the sensitivity of the GABAA receptor to GABA in acutely dissociated rat hippocampal neurones

Classification of some GABA antagonists with regard to site of action and potency in slices of rat cuneate nucleus.

Differential antagonism by epipregnanolone of alphaxalone and pregnanolone potentiation of [3H]flunitrazepam binding suggests more than one class of binding site for steroids at GABAA receptors.