Alcohol dependence potentiates substance P/neurokinin-1 receptor signaling in the rat central nucleus of amygdala.

Behavioral and clinical studies suggest a critical role of substance P (SP)/neurokinin-1 receptor (NK-1R) signaling in alcohol dependence. Here, we examined regulation of GABA transmission in the medial subdivision of the central amygdala (CeM) by the SP/NK-1R system, and its neuroadaptation following chronic alcohol exposure. In naïve rats, SP increased action potential-dependent GABA release, and the selective NK-1R antagonist L822429 decreased it, demonstrating SP regulation of CeM activity under basal conditions. SP induced a larger GABA release in alcohol-dependent rats accompanied by decreased NK-1R expression compared to naïve controls, suggesting NK-1R hypersensitivity which persisted during protracted alcohol withdrawal. The NK-1R antagonist blocked acute alcohol-induced GABA release in alcohol-dependent and withdrawn but not in naïve rats, indicating that dependence engages the SP/NK-1R system to mediate acute effects of alcohol. Collectively, we report long-lasting CeA NK-1R hypersensitivity corroborating that NK-1Rs are promising targets for the treatment of alcohol use disorder

Efficient modulation of of γ-aminobutyric acid type A (GABAA) receptors by piperine derivatives

[Image: see text] Piperine activates TRPV1 (transient receptor potential vanilloid type 1 receptor) receptors and modulates γ-aminobutyric acid type A receptors (GABA(A)R). We have synthesized a library of 76 piperine analogues and analyzed their effects on GABA(A)R by means of a two-microelectrode voltage-clamp technique. GABA(A)R were expressed in Xenopus laevis oocytes. Structure–activity relationships (SARs) were established to identify structural elements essential for efficiency and potency. Efficiency of piperine derivatives was significantly increased by exchanging the piperidine moiety with either N,N-dipropyl, N,N-diisopropyl, N,N-dibutyl, p-methylpiperidine, or N,N-bis(trifluoroethyl) groups. Potency was enhanced by replacing the piperidine moiety by N,N-dibutyl, N,N-diisobutyl, or N,N-bistrifluoroethyl groups. Linker modifications did not substantially enhance the effect on GABA(A)R. Compound 23 [(2E,4E)-5-(1,3-benzodioxol-5-yl)-N,N-dipropyl-2,4-pentadienamide] induced the strongest modulation of GABA(A) (maximal GABA-induced chloride current modulation (I(GABA-max) = 1673% ± 146%, EC(50) = 51.7 ± 9.5 μM), while 25 [(2E,4E)-5-(1,3-benzodioxol-5-yl)-N,N-dibutyl-2,4-pentadienamide] displayed the highest potency (EC(50) = 13.8 ± 1.8 μM, I(GABA-max) = 760% ± 47%). Compound 23 induced significantly stronger anxiolysis in mice than piperine and thus may serve as a starting point for developing novel GABA(A)R modulators

GABA Receptors and the Pharmacology of Sleep

Current GABAergic sleep-promoting medications were developed pragmatically, without making use of the immense diversity of GABAA receptors. Pharmacogenetic experiments are leading to an understanding of the circuit mechanisms in the hypothalamus by which zolpidem and similar compounds induce sleep at α2βγ2-type GABAA receptors. Drugs acting at more selective receptor types, for example, at receptors containing the α2 and/or α3 subunits expressed in hypothalamic and brain stem areas, could in principle be useful as hypnotics/anxiolytics. A highly promising sleep-promoting drug, gaboxadol, which activates αβδ-type receptors failed in clinical trials. Thus, for the time being, drugs such as zolpidem, which work as positive allosteric modulators at GABAA receptors, continue to be some of the most effective compounds to treat primary insomnia

Alcohol dependence potentiates substance P/neurokinin-1 receptor signaling in the rat central nucleus of amygdala.

Behavioral and clinical studies suggest a critical role of substance P (SP)/neurokinin-1 receptor (NK-1R) signaling in alcohol dependence. Here, we examined regulation of GABA transmission in the medial subdivision of the central amygdala (CeM) by the SP/NK-1R system, and its neuroadaptation following chronic alcohol exposure. In naïve rats, SP increased action potential-dependent GABA release, and the selective NK-1R antagonist L822429 decreased it, demonstrating SP regulation of CeM activity under basal conditions. SP induced a larger GABA release in alcohol-dependent rats accompanied by decreased NK-1R expression compared to naïve controls, suggesting NK-1R hypersensitivity which persisted during protracted alcohol withdrawal. The NK-1R antagonist blocked acute alcohol-induced GABA release in alcohol-dependent and withdrawn but not in naïve rats, indicating that dependence engages the SP/NK-1R system to mediate acute effects of alcohol. Collectively, we report long-lasting CeA NK-1R hypersensitivity corroborating that NK-1Rs are promising targets for the treatment of alcohol use disorder

HPLC-based activity profiling approach for the discovery of GABAA receptor ligands using a functional assay with Xenopus oocytes

An approach for rapid HPLC-based profiling for new GABAA ligands of natural origin has been developed. Active extracts are separated by a single injection of 3 - 10 mg of extract onto a semi-preparative (150 × 10 mm i. d.) HPLC column with gradient elution and time-based fractionation. The microfractions are tested in an automated two-microelectrode voltage-clamp assay on Xenopus oocytes expressing recombinant GABAA channels composed of α1, β2 and γ2S subunits. The protocol has been validated by spiking experiments with inactive extract and the GABAA receptor ligand magnolol, and by profiling of active extracts such as valerian extract containing the known GABAA receptor ligand valerenic acid. For dereplication of GABA containing extracts, we established a rapid and simple procedure by which GABA is analyzed as OPA derivative by reversed-phase HPLC. This dereplication protocol was validated with plant and fungal extracts which had been previously tested active or inactive in the oocyte assay and with spiking experiments

Identification of the putative binding pocket of valerenic acid on GABAA receptors using docking studies and site-directed mutagenesis

Background and Purpose β2/3-subunit-selective modulation of GABAA receptors by valerenic acid (VA) is determined by the presence of transmembrane residue β2/3N265. Currently, it is not known whether β2/3N265 is part of VA's binding pocket or is involved in the transduction pathway of VA's action. The aim of this study was to clarify the localization of VA's binding pocket on GABAA receptors. Experimental Approach Docking and a structure-based three-dimensional pharmacophore were employed to identify candidate amino acid residues that are likely to interact with VA. Selected amino acid residues were mutated, and VA-induced modulation of the resulting GABAA receptors expressed in Xenopus oocytes was analysed. Key Results A binding pocket for VA at the β+/α- interface encompassing amino acid β3N265 was predicted. Mutational analysis of suggested amino acid residues revealed a complete loss of VA's activity on β3M286W channels as well as significantly decreased efficacy and potency of VA on β3N265S and β3F289S receptors. In addition, reduced efficacy of VA-induced IGABA enhancement was also observed for α1M235W, β3R269A and β3M286A constructs. Conclusions and Implications Our data suggest that amino acid residues β3N265, β3F289, β3M286, β3R269 in the β3 subunit, at or near the etomidate/propofol binding site(s), form part of a VA binding pocket. The identification of the binding pocket for VA is essential for elucidating its pharmacological effects and might also help to develop new selective GABAA receptor ligands