γ-Hydroxybutyric acid (GHB) binding to brain-specific high-affinity sites is well-established and proposed to explain both physiological and pharmacological actions. However, the mechanistic links between these lines of data are unknown. To identify molecular targets for specific GHB high-affinity binding, we undertook photolinking studies combined with proteomic analyses and identified several GABA A receptor subunits as possible candidates. A subsequent functional screening of various recombinant GABA A receptors in Xenopus laevis oocytes using the two-electrode voltage clamp technique showed GHB to be a partial agonist at αβδ-but not αβγ-receptors, proving that the δ-subunit is essential for potency and efficacy. GHB showed preference for α4 over α(1,2,6)-subunits and preferably activated α4β1δ (EC 50 = 140 nM) over α4β (2/3)δ (EC 50 = 8.41/1.03 mM). Introduction of a mutation, α4F71L, in α4β1(δ)-receptors completely abolished GHB but not GABA function, indicating nonidentical binding sites. Radioligand binding studies using the specific GHB radioligand [ 3 H](E,RS)-(6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene)acetic acid showed a 39% reduction (P = 0.0056) in the number of binding sites in α4 KO brain tissue compared with WT controls, corroborating the direct involvement of the α4-subunit in high-affinity GHB binding. Our data link specific GHB forebrain binding sites with α4-containing GABA A receptors and postulate a role for extrasynaptic α4δ-containing GABA A receptors in GHB pharmacology and physiology. This finding will aid in elucidating the molecular mechanisms behind the proposed function of GHB as a neurotransmitter and its unique therapeutic effects in narcolepsy and alcoholism.γ-hydroxybutyric acid receptor | γ-hydroxybutyric acid high-affinity binding sites | α4-subunit knockout | photoaffinity ligand T he GABA metabolite γ-Hydroxybutyric acid (GHB) is present in micromolar concentrations in the mammalian brain, where it has been proposed to act as a neurotransmitter (1). Additionally, GHB is a drug of abuse (Fantasy) and a registered drug for treating narcolepsy (2) and alcoholism (3). GHB binds to at least two distinct populations of low-and high-affinity binding sites in the brain (4). When GHB is ingested in high doses and reaches millimolar concentrations in the brain, it induces behavioral effects such as sedation, motor incoordination and hypothermia (3). These actions are largely mediated by metabotropic GABA B receptors, because effects are prevented by GABA B receptor antagonist pretreatment (5) and completely abolished in GABA B(1)

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BackgroundThe fruit of Elaeagnus umbellata has high medicinal values and is an excellent source of phytochemicals. This study was aimed to evaluate the antioxidant, enzyme inhibitory and antidiabetic potential of Elaeagnus umbellata.MethodsThe antioxidant potential of the crude extract and subfractions of E. umbellata fruit were determined using DPPH (2, 20-diphenyl-1-picrylhydrazyl) and ABTS (2, 2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid) assays. The enzyme inhibitory potentials of extracts against α-amylase and α-glucosidase enzymes were also determined. The in vivo anti-hyperglycemic effects of the extract in STZ-induced type 2 diabetes were determined using Sprague Dawley adult rats. HPLC system (Agilent 1260) was used for the identification of bioactive compounds present in extracts. Molecular docking was used to identify and compare the interaction between the compounds (active constituents) and standard inhibitor acarbose with the α-amylase and α-glucosidase active sites.ResultsThe chloroform, ethyl acetate, and butanol fractions showed significant antioxidant potential with IC50 values of 40, 45 and 60 μg/mL against DPPH and 57, 70 and 120 μg/mL against ABTS free radicals respectively. The chloroform and ethyl acetate were highly active against α-amylase and α-glucosidase (IC50 values 58 and 200 μg/ml against α-amylase 60 and 140 μg/ml against α-glucosidase. The crude extract, chloroform, and ethyl acetate fractions were more potent in controlling the hyperglycemia in STZ-induced type 2 diabetes in rats and considerable reduction of glucose level was observed compared to the non-treated group. Furthermore, the extracts were also found useful in controlling the secondary complications associated with type 2 diabetes mellitus which was evident from the observed substantial reduction in the blood level of serum glutamate oxaloacetate transaminase, serum glutamate pyruvate transaminase, alkaline phosphatase, total cholesterol, low-density lipoproteins, and triglycerides. The molecular docking approach indicated the favorable inhibitory interaction between the docked compounds and the active sites of the α-amylase and α-glucosidase. All docked compounds occupied the same binding site as occupied by acarbose.ConclusionIt was concluded that E. umbellata can be used in the treatment of type 2 diabetes and oxidative stress. The extracts were also found to be effective in relieving the secondary complications associated with type 2 diabetes.Graphical abstract Electronic supplementary materialThe online version of this article (10.1186/s12906-018-2381-8) contains supplementary material, which is available to authorized users.

GABAA receptors are members of the ligand-gated ion channel superfamily that mediate inhibitory neurotransmission in the central nervous system. They are thought to be composed of 2 alpha (α), 2 beta (β) subunits and one other such as a gamma (γ) or delta (δ) subunit. The potency of GABA is influenced by the subunit composition. However, there are no reported systematic studies that evaluate GABA potency on a comprehensive number of subunit combinations expressed in Xenopus oocytes, despite the wide use of this heterologous expression system in structure-function studies and drug discovery. Thus, the aim of this study was to conduct a systematic characterization of the potency of GABA at 43 human recombinant GABA(A) receptor combinations expressed in Xenopus oocytes using the two-electrode voltage clamp technique. The results show that the α-subunits and to a lesser extent, the β-subunits influence GABA potency. Of the binary and ternary combinations with and without the γ2L subunit, the α6/γ2L-containing receptors were the most sensitive to GABA, while the β2- or β3-subunit conferred higher sensitivity to GABA than receptors containing the β1-subunit with the exception of the α2β1γ2L and α6β1γ2L subtypes. Of the δ-subunit containing GABA(A) receptors, α4/δ-containing GABA(A) receptors displayed highest GABA sensitivity, with mid-nanomolar concentrations activating α4β1δ and α4β3δ receptors. At α4β2δ, GABA had low micromolar activity.

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Purpose. Rosmarinus officinalis, traditionally known as rosemary, has been widely used in traditional medicines and has long been known as the herb of remembrance. However, few studies have investigated the effects of non-volatile components of rosemary on central nervous system function. Methods. Fractionation of R. officinalis led to the isolation of salvigenin, rosmanol and cirsimaritin, which were investigated in mouse models of acute toxicity, antinociception (tail immersion and hot plate tests), depression (tail suspension and forced swim tests) and anxiety (elevated plus maze and light/dark box paradigms). Results. Rosmanol, cirsimaritin and salvigenin were not found to exhibit any signs of acute toxicity (50-200 mg/kg), but elicited antinociceptive, antidepressant and anxiolytic activities. Conclusion. Rosmanol, cirsimaritin and salvigenin, all previously shown to have biphasic modulation of GABAA receptors, demonstrated CNS activity in mouse models of antinociception, antidepressant and anxiolysis. The anxiolytic activity of all three compounds was not ameliorated by flumazenil, but was inhibited by pentylenetetrazol, suggesting a mode of action via GABAA receptors at a site other than the high affinity benzodiazepine binding site. This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page.

Triterpenes possess anti-inflammatory and anti-nociceptive effects. In this study anti-inflammatory activities of Asparacosin A were evaluated' using in-vitro cyclooxygenases 1 and 2 (COX-1/2) inhibition assays. Moreover, anti-nociceptive activities were assessed in-vivo by carrageenan-induced paw edema test, xylene-induced ear edema tests, and acetic acid-induced writhing and formalin tests. Additionally molecular docking was conducted to elucidate the binding mechanism of the compound and to correlate the in-vitro findings with the in-silico data. Oral administration of Asparacosin A at the doses of 10, 20, and 40 mg/kg induced significant anti-inflammatory effects ( * p < 0.05, ** p < 0.01, and *** p < 0.001) in a dose-dependent manner in both models. Asparacosin A also inhibited the human recombinant COX-2 enzyme and caused a dose-dependent decrease in the levels of TNF-α, IL-1β, and PGE2 in the carrageenan-induced paws. Moreover, Asparacosin A displayed significant anti-nociceptive effects ( * p < 0.05, ** p < 0.01, *** p < 0.001) at the doses of 10, 20, and 40 mg/kg in acetic-acid induced writhing test. However, in formalin test, Asparacosin A (10–40 mg/kg, p.o) produced anti-nociceptive effects only in the late phase, similar to the effect observed with the reference drug celecoxib (50 mg/kg, p.o). Molecular docking was carried out on both COX-1 and COX-2 structures which revealed that Asparacosin A targets allosteric binding site similar to the binding mode of the selective COX inhibitor. In conclusion, Asparacosin A exhibits anti-inflammatory and peripheral anti-nociceptive activities which are likely mediated via inhibition of COX-2 enzyme and inflammatory cytokines. Furthermore, Asparacosin A can serve as a model to obtain new and more selective potent anti-inflammatory and anti-nociceptive drugs.