The effects of a competitive antagonist on GABA-evoked currents in the presence of sedative-hypnotic agents.

BACKGROUND: Many sedative-hypnotic agents are thought to act by positively modulating γ-aminobutyric acid type A (GABAA) receptors. However, for many agents, the location(s) of the binding site(s) responsible for such receptor modulation is uncertain. We previously developed a low efficacy ligand (naphthalene-etomidate) that binds within a homologous set of hydrophobic cavities located at GABAA receptor subunit interfaces in the transmembrane domain, and thus acts as a competitive antagonist for higher efficacy sedative-hypnotics that also bind to these sites. In this report, we describe studies using this compound as a pharmacological screening tool to test whether sedative-hypnotics representing a range of chemical classes can modulate GABAA receptors by binding within these receptor cavities. METHODS: The impact of naphthalene-etomidate on GABA-evoked currents that were mediated by oocyte-expressed α1ß3γ2L GABAA receptors and potentiated by muscimol, alphaxalone, 2,2,2-trichloroethanol, isoflurane, AA29504, loreclezole, or diazepam was quantified using electrophysiological techniques. RESULTS: Naphthalene-etomidate (300 µM) significantly reduced GABAA receptor currents potentiated by alphaxalone (by 22 ± 11%), 2,2,2-trichloroethanol (by 23 ± 6%), isoflurane (by 32 ± 10%), AA29504 (by 41 ± 6%), loreclezole (by 43 ± 9%), but significantly increased those potentiated by muscimol (by 26 ± 11%). Naphthalene-etomidate significantly increased currents potentiated by a low (1 µM) diazepam concentration (by 56 ± 14%) while reducing those potentiated by a high (100 µM) diazepam concentration (by 11 ± 7%). CONCLUSIONS: Our results suggest that many (but not all) sedative-hypnotics are capable of positively modulating the GABAA receptor by binding within a common set of hydrophobic cavities.

Metabolic studies of synaptamide in an immortalized dopaminergic cell line.

INTRODUCTION: Synaptamide, the N-acylethanolamine of docosahexaenoic acid (DHA), is structurally similar to the endocannabinoid N-arachidonoylethanolamine, anandamide. It is an endogenous ligand at the orphan G-protein coupled receptor 110 (GPR110; ADGRF1), and induces neuritogenesis and synaptogenesis in hippocampal and cortical neurons, as well as neuronal differentiation in neural stem cells. PURPOSE: Our goal was to characterize the metabolic fate (synthesis and metabolism) of synaptamide in a dopaminergic cell line using immortalized fetal mesencephalic cells (N27 cells). Both undifferentiated and differentiating N27 cells were used in this study in an effort to understand synaptamide synthesis and metabolism in developing and adult cells. METHODS: Radiotracer uptake and hydrolysis assays were conducted in N27 cells incubated with [1-14C]DHA or with one of two radioisotopomers of synaptamide: [α,ß-14C2]synaptamide and [1-14C-DHA]synaptamide. RESULTS: Neither differentiated nor undifferentiated N27 cells synthesized synaptamide from radioactive DHA, but both rapidly incorporated radioactivity from exogenous synaptamide into membrane phospholipids, regardless of which isotopomer was used. Pharmacological inhibition of fatty acid amide hydrolase (FAAH) reduced formation of labeled phospholipids in undifferentiated but not differentiated cells. CONCLUSIONS: In undifferentiated cells, synaptamide uptake and metabolism is driven by its enzymatic hydrolysis (fatty acid amide hydrolase; FAAH), but in differentiating cells, the process seems to be FAAH independent. We conclude that differentiated and undifferentiated N27 cells utilize synaptamide via different mechanisms. This observation could be extrapolated to how different mechanisms may be in place for synaptamide uptake and metabolism in developing and adult dopaminergic cells.

Etomidate and Etomidate Analog Binding and Positive Modulation of γ-Aminobutyric Acid Type A Receptors: Evidence for a State-dependent Cutoff Effect.

WHAT WE ALREADY KNOW ABOUT THIS TOPIC: WHAT THIS ARTICLE TELLS US THAT IS NEW: BACKGROUND:: Naphthalene-etomidate, an etomidate analog containing a bulky phenyl ring substituent group, possesses very low γ-aminobutyric acid type A (GABAA) receptor efficacy and acts as an anesthetic-selective competitive antagonist. Using etomidate analogs containing phenyl ring substituents groups that range in volume, we tested the hypothesis that this unusual pharmacology is caused by steric hindrance that reduces binding to the receptor's open state. METHODS: The positive modulatory potencies and efficacies of etomidate and phenyl ring-substituted etomidate analogs were electrophysiology defined in oocyte-expressed α1ß3γ2L GABAA receptors. Their binding affinities to the GABAA receptor's two classes of transmembrane anesthetic binding sites were assessed from their abilities to inhibit receptor labeling by the site-selective photolabels [H]azi-etomidate and tritiated R-5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid. RESULTS: The positive modulatory activities of etomidate and phenyl ring-substituted etomidate analogs progressively decreased with substituent group volume, reflecting significant decreases in both potency (P = 0.005) and efficacy (P < 0.0001). Affinity for the GABAA receptor's two ß - α anesthetic binding sites similarly decreased with substituent group volume (P = 0.003), whereas affinity for the receptor's α - ß/γ - ß sites did not (P = 0.804). Introduction of the N265M mutation, which is located at the ß - α binding sites and renders GABAA receptors etomidate-insensitive, completely abolished positive modulation by naphthalene-etomidate. CONCLUSIONS: Steric hindrance selectively reduces phenyl ring-substituted etomidate analog binding affinity to the two ß - α anesthetic binding sites on the GABAA receptor's open state, suggesting that the binding pocket where etomidate's phenyl ring lies becomes smaller as the receptor isomerizes from closed to open.

N-Docosahexaenoylethanolamine (synaptamide): Carbon-14 radiolabeling and metabolic studies.

N-Docosahexaenoylethanolamine (synaptamide) is structurally similar to the endocannabinoid N-arachidonoylethanolamine (anandamide), but incorporates the omega-3 22:6 fatty acid docosahexaenoic acid (DHA) in place of the omega-6 20:4 fatty acid arachidonic acid (AA). Some brain membrane lipid effects may be mediated via synaptamide. In competition experiments with mouse brain homogenate in vitro, we found that synaptamide was an order-of-magnitude poorer inhibitor of radioactive anandamide hydrolysis than was anandamide itself. Also, enzyme-mediated hydrolysis of synaptamide was observed to occur at a slower rate than for anandamide. We have synthesized synaptamide radiolabeled with carbon-14 in both the ethanolamine ([α,ß-14C2]synaptamide) and in the DHA ([1-14C]synaptamide) moieties. The brain penetration, distribution, and metabolism of radiolabeled synaptamide were studied in mice in vivo relative to anandamide, DHA, and AA. Brain uptake of labeled synaptamide was greater than for labeled DHA, consistent with previous studies of labeled anandamide and AA in our laboratory. After administering either isotopomer of radiolabeled synaptamide, radiolabeled phospholipids were found in mouse brain. Pretreatment of mice with PF3845, a potent, specific inhibitor of fatty acid amide hydrolase (FAAH), eliminated formation of labeled phospholipids measured after 15min, suggesting that synaptamide is hydrolyzed nearly exclusively by FAAH, though it is a poorer substrate for FAAH than anandamide.