Non-competitive Action Research Articles

Molecular Basis for Non-Covalent, Non-Competitive FAAH Inhibition

Fatty acid amide hydrolase (FAAH) plays a key role in the control of cannabinoid signaling and it represents a promising therapeutic strategy for the treatment of a wide range of diseases, including neuropathic pain and chronic inflammation. Starting from kinetics experiments carried out in our previous work for the most potent inhibitor 2-amino-3-chloropyridine amide (TPA14), we have investigated its non-competitive mechanism of action using molecular dynamics, thermodynamic integration and QM-MM/GBSA calculations. The computational studies highlighted the impact of mutations on the receptor binding pockets and elucidated the molecular basis of the non-competitive inhibition mechanism of TPA14, which prevents the endocannabinoid anandamide (AEA) from reaching its pro-active conformation. Our study provides a rationale for the design of non-competitive potent FAAH inhibitors for the treatment of neuropathic pain and chronic inflammation.

Games in Rigged Economies

Modern economies evolved from simpler human exchanges into very convoluted systems. Today, a multitude of aspects can be regulated, tampered with, or left to chance; these are economic {\em degrees of freedom} which together shape the flow of wealth. Economic actors can exploit them, at a cost, and bend that flow in their favor. If intervention becomes widespread, microeconomic strategies of different actors can collide or resonate, building into macroeconomic effects. How viable is a `rigged' economy, and how is this viability affected by growing economic complexity and wealth? Here we capture essential elements of `rigged' economies with a toy model. Nash equilibria of payoff matrices in simple cases show how increased intervention turns economic degrees of freedom from minority into majority games through a dynamical phase. These stages are reproduced by agent-based simulations of our model, which allow us to explore scenarios out of reach for payoff matrices. Increasing economic complexity is then revealed as a mechanism that spontaneously defuses cartels or consensus situations. But excessive complexity enters abruptly into a regime of large fluctuations that threaten the system's viability. This regime results from non-competitive efforts to intervene the economy coupled across degrees of freedom, becoming unpredictable. Thus non-competitive actions can result in negative spillover due to sheer economic complexity. Simulations suggest that wealth must grow faster than linearly with economic complexity to avoid this regime and keep economies viable in the long run. Our work provides testable conclusions and phenomenological charts to guide policing of `rigged' economic systems.

Insight into the Mechanism of 17β-Hydroxysteroid Dehydrogenase Type 3 Inhibition by the Androsterone Derivative RM-532-105

Background: The last step in the production of androgen testosterone from 4-androstene- 3,17-dione (4-dione) in testis involves the 17β-hydroxysteroid dehydrogenase type 3 (17β-HSD3). Blocking this microsomal enzyme with an inhibitor would lower the level of testosterone and, consequently, could be an approach for the treatment of androgen-dependent diseases. RM-532-105 was developed as a steroidal inhibitor of 17β-HSD3, but its mechanism of action is not yet known. Objective: To identify potential binding sites of the 17β-HSD3 substrate 4-dione, cofactor NADPH, as well as inhibitor RM-532-105. Methods: Since there is no crystal structure of 17β-HSD3 available, complexed or not with a ligand, a homology model was prepared followed by molecular docking, and enzymatic assay experiments were performed. Results: Transfected LNCaP prostate cancer cells were used as a source of 17β-HSD3 activity for the transformation of 4-dione into testosterone in the presence of varying concentrations of a substrate, a cofactor or an inhibitor. Molecular modeling experiments and enzymatic assays with these cells suggest a competitive action of RM-532-105 with the cofactor and a non-competitive action with the substrate 4-dione. Conclusion: These results allow the selection of one inhibitor orientation in the enzyme binding site, from the two possibilities predicted by the docking experiments, and appear to be in agreement with previous structure-activity relationships.

Modeling Epac1 interactions with the allosteric inhibitor AM-001 by co-solvent molecular dynamics

The exchange proteins activated by cAMP (EPAC) are implicated in a large variety of physiological processes and they are considered as promising targets for a wide range of therapeutic applications. Several recent reports provided evidence for the therapeutic effectiveness of the inhibiting EPAC1 activity cardiac diseases. In that context, we recently characterized a selective EPAC1 antagonist named AM-001. This compound was featured by a non-competitive mechanism of action but the localization of its allosteric site to EPAC1 structure has yet to be investigated. Therefore, we performed cosolvent molecular dynamics with the aim to identify a suitable allosteric binding site. Then, the docking and molecular dynamics were used to determine the binding of the AM-001 to the regions highlighted by cosolvent molecular dynamics for EPAC1. These analyses led us to the identification of a suitable allosteric AM-001 binding pocket at EPAC1. As a model validation, we also evaluated the binding poses of the available AM-001 analogues, with a different biological potency. Finally, the complex EPAC1 with AM-001 bound at the putative allosteric site was further refined by molecular dynamics. The principal component analysis led us to identify the protein motion that resulted in an inactive like conformation upon the allosteric inhibitor binding.

Effects of Cycloleucine in the Nucleus Accumbens Septi on the Elevated plus Maze Test in Rats

Introduction: In recent years, an important number of studies have emphasized the psychopharmacological actions of cycloleucine (1-aminocyclopentanecarboxylic acid) acting on the NR1 subunit (glycine allosteric site) of NMDA (N-methyl-D-aspartic acid) receptor. We studied the effects of its injection in an anxiety test. Methods: The elevated plus maze test was used. Male rats bilaterally cannulated into the nucleus accumbens septi (NAS) were employed. Rats were divided into 5 groups that received either 1 µL injections of saline or cycloleucine (0.5, 1, 2, or 4 µg) 15 min before testing. Results: Time spent in the open arm was significantly increased by cycloleucine treatment with all doses (1 and 2 µg, p < 0.05; 0.5 and 4 µg, p < 0.01), like number of extreme arrivals (0.5 and 1 µg, p < 0.05; 2 µg, p < 0.01; and 4 µg, p < 0.001). Open arm entries were increased by the highest dose only (4 µg, p < 0.01). Discussion/Conclusion: Present results show no difference between all doses in the time spent in the open arm, suggesting an indirect, noncompetitive action of the drug. The increase in extreme arrivals and open arm entries suggests a dose influence in these parameters. We conclude that cycloleucine influence on the NMDA receptors within NAS leads to anxiolytic-like effects and behavioral disinhibition, which once more confirms the involvement of NAS in anxiety processing.

Identification of aurintricarboxylic acid as a potent allosteric antagonist of P2X1 and P2X3 receptors

The homotrimeric P2X3 receptor, one of the seven members of the ATP-gated P2X receptor family, plays a crucial role in sensory neurotransmission. P2X3 receptor antagonists have been identified as promising drugs to treat chronic cough and are suggested to offer pain relief in chronic pain such as neuropathic pain. Here, we analysed whether compounds affect P2X3 receptor activity by high-throughput screening of the Spectrum Collection of 2000 approved drugs, natural products and bioactive substances. We identified aurintricarboxylic acid (ATA) as a nanomolar-potency antagonist of P2X3 receptor-mediated responses. Two-electrode voltage clamp electrophysiology-based concentration–response analysis and selectivity profiling revealed that ATA strongly inhibits the rP2X1 and rP2X3 receptors (with IC50 values of 8.6 nM and 72.9 nM, respectively) and more weakly inhibits P2X2/3, P2X2, P2X4 or P2X7 receptors (IC50 values of 0.76 μM, 22 μM, 763 μM or 118 μM, respectively). Patch-clamp analysis of mouse DRG neurons revealed that ATA inhibited native P2X3 and P2X2/3 receptors to a similar extent than rat P2X3 and P2X2/3 receptors expressed in Xenopus oocytes. In a radioligand binding assay, up to 30 μM ATA did not compete with [3H]-ATP for rP2X3 receptor binding, indicating a non-competitive mechanism of action. Molecular docking studies, site-directed mutagenesis and concentration–response analysis revealed that ATA binds to the negative allosteric site of the hP2X3 receptor. In summary, ATA as a drug-like pharmacological tool compound is a nanomolar-potency, allosteric antagonist with selectivity towards αβ-methylene-ATP-sensitive P2X1 and P2X3 receptors.

Non-Competitive but pH-Dependent Action of SB-366791 on Proton-Induced Activation of TRPV1 Receptors

The transient receptor potential vanilloid type 1 (TRPV1) is a nonselective cation channel gated by numerous chemical and physical stimuli (protons, capsaicin, heat, etc). TRPV1 receptors are important integrators of multiple noxious and inflammatory signals in vertebrates. Modulation of TRPV1 receptors activity is considered to be a promising strategy for pain treatment. SB-366791 is a TRPV1 antagonist that demonstrates good analgesic effects in various models of pain. Molecular mechanisms of the SB-366791 action on TRPV1 are not clear. It antagonizes capsaicin activation in a competitive manner, but the data on its action in the case of activation by protons are controversial. Here we studied effects of SB-366791 when TRPV1 receptors are activated by acidification. We carried out patch-clamp experiments (voltage-clamp mode) on cultured CHO cells stably expressing rat TRPV1 receptors. The whole-cell proton-evoked currents were reduced in the presence of SB-366791. Concentration dependencies of the inhibitory effect of SB-366791 were studied at different pH values. Stronger acidification reduced the maximum effect of SB-366791, while the IC50 values were virtually unaffected. Thus, SB-366791 acts in a non-competitive but pH-dependent way. Probably, there is an allosteric interplay between proton- and capsaicin-binding sites.

Novel Non-carboxylate Benzoylsulfonamide-Based Protein Tyrosine Phosphatase 1B Inhibitors with Non-competitive Actions.

A novel series of benzoylsulfonamide derivatives were synthesized and biologically evaluated. Among them, 4-(biphenyl-4-ylmethylsulfanylmethyl)-N-(hexane-1-sulfonyl)benzamide (compound 18K) was identified as a protein tyrosine phosphatase 1B (PTP1B) inhibitor with potent and selective inhibitory activity against PTP1B (IC50=0.25 µM). Compound 18K functioned as a non-competitive inhibitor and bound to the allosteric site of PTP1B. It also showed high oral absorption in mice (the maximum drug concentration (Cmax)=45.5 µM at 30 mg/kg), rats (Cmax=53.6 µM at 30 mg/kg), and beagles (Cmax=37.8 µM at 10 mg/kg), and significantly reduced plasma glucose levels at 30 mg/kg/d (per os (p.o.)) for one week with no side effects in db/db mice. In conclusion, the substituted benzoylsulfonamide was shown to be a novel scaffold of a non-competitive and allosteric PTP1B inhibitor, and compound 18K has potential as an efficacious and safe anti-diabetic drug as well as a useful tool for investigations of the physiological and pathophysiological effects of allosteric PTP1B inhibition.

Muscle-Type Nicotinic Receptor Modulation by 2,6-Dimethylaniline, a Molecule Resembling the Hydrophobic Moiety of Lidocaine.

To identify the molecular determinants responsible for lidocaine blockade of muscle-type nAChRs, we have studied the effects on this receptor of 2,6-dimethylaniline (DMA), which resembles lidocaine’s hydrophobic moiety. Torpedo marmorata nAChRs were microtransplanted to Xenopus oocytes and currents elicited by ACh (IACh), either alone or co-applied with DMA, were recorded. DMA reversibly blocked IACh and, similarly to lidocaine, exerted a closed-channel blockade, as evidenced by the enhancement of IACh blockade when DMA was pre-applied before its co-application with ACh, and hastened IACh decay. However, there were marked differences among its mechanisms of nAChR inhibition and those mediated by either the entire lidocaine molecule or diethylamine (DEA), a small amine resembling lidocaine’s hydrophilic moiety. Thereby, the IC50 for DMA, estimated from the dose-inhibition curve, was in the millimolar range, which is one order of magnitude higher than that for either DEA or lidocaine. Besides, nAChR blockade by DMA was voltage-independent in contrast to the increase of IACh inhibition at negative potentials caused by the more polar lidocaine or DEA molecules. Accordingly, virtual docking assays of DMA on nAChRs showed that this molecule binds predominantly at intersubunit crevices of the transmembrane-spanning domain, but also at the extracellular domain. Furthermore, DMA interacted with residues inside the channel pore, although only in the open-channel conformation. Interestingly, co-application of ACh with DEA and DMA, at their IC50s, had additive inhibitory effects on IACh and the extent of blockade was similar to that predicted by the allotopic model of interaction, suggesting that DEA and DMA bind to nAChRs at different loci. These results indicate that DMA mainly mimics the low potency and non-competitive actions of lidocaine on nAChRs, as opposed to the high potency and voltage-dependent block by lidocaine, which is emulated by the hydrophilic DEA. Furthermore, it is pointed out that the hydrophobic (DMA) and hydrophilic (DEA) moieties of the lidocaine molecule act differently on nAChRs and that their separate actions taken together account for most of the inhibitory effects of the whole lidocaine molecule on nAChRs.

‘Look at what we can do with all the broken stuff!’ Youth agency and sporting creativity in sites of war, conflict and disaster

Too often youth are considered ‘victims’ in sites of war, conflict and disaster, with little consideration for their perspectives and ability to develop culturally specific responses to such conditions and events. Thus, unique forms of youth agency, creativity and resourcefulness continue to be overlooked. This paper adopts an interdisciplinary, multi-scale approach to reveal examples of youth agency and sporting creativity within local contexts of conflict and post-disaster, and signals important considerations for Sport for Development and Peace organisations working in such locations. Examining the different scales at which youth are engaging with informal, non-competitive action sports (i.e. skateboarding, parkour) in sites of conflict and disaster, this paper consists of three parts: (1) youth agency in sites of conflict, (2) youth agency in sites of post-disaster, and (3) transnational corporations use of action sports for ‘cause marketing’ in contexts of ‘disaster capitalism’. Each section offers insights from a series of on-going case studies (including interviews, media analysis and digital ethnography) in the post-disaster zones of Christchurch and New Orleans, and conflict zone of Palestine. In so doing, this paper offers a critical examination of the fluid and dynamic nature of youth agency and everyday politics in disrupted and dangerous geographies, but ultimately advocates the need to locate such initiatives within the broader flows of power, which are never neutral, equal or consistent.

ABSOLUTELY VOID COMPETITIVE ACTIONS IN INTERNATIONAL COMPETITION LAW, INCLUDING PRICE FIXING, MARKET SHARING AND RESTRICTION ON PRODUCTION

To guarantee and ensure the proper function of the market, competition law is necessary. Its goal is to condemn non-competitive behaviors. This paper not only seeks to address the source of competition law and its mechanism, but also explains absolutely void competitive actions including price fixing, market sharing and restriction on production and their impacts on market. In fact, today competition law which is a combination of behavioral and structural regulations offers a combination of behavior-oriented and result-oriented constraints that provide a different regulation approach for different types of anti-competitive behaviors. Three main forms of competition law formed to control absolutely void competitive actions including mergers laws, antitrust laws and coordinated behavior laws. This paper explains that while the market sharing is in contrast with antitrust laws, can lead to another type of anti-competitive action which is restriction on production. This eventually leads to low efficiency and prevent domestic market integration. Price fixing can also make difficulties in span of pricing competition and thus makes harmful effects for customers. Anti-competitive action of restriction on production may cause price fixing. As a result a void competitive action can reproduce other non-competitive actions and these void activities are repeated regularly. This makes clearer the need for enforcement of competition laws. Finally, some measures are mentioned such as fines, imprisonment and collective sanctions of the colluding companies that considered competition laws to restrict anti-competitive activities.

O066. Kynurenine pathway metabolites in cluster headache.

Cluster headache is a severe, disabling disorder with pain that ranks among the most severe known to humans. It is associated with accompanying autonomic symptoms ipsilateral to the pain and a sense of restlessness or agitation. Patients with cluster headaches have few therapeutic options, and a further 10-20% develop drug-resistant attacks. The often brief duration of cluster attacks makes abortive therapy a challenge, and preventive medications are almost always provided to patients. Although NMDA-R activation by glutamate has been hypothesized to play a role in the pathophysiology of primary headache disorders, its role is still not fully understood. In fact, the trigeminovascular nociceptive transmission from primary afferents through the trigeminal nucleus caudalis and on to other parts of the CNS involves both the NMDA and non-NMDA glutamate receptors. The kynurenine pathway (KP), accounting for more than 90% of the tryptophan metabolism, generates neuroactive compounds that are able to interact with glutamate receptors both in the central and in the peripheral nervous systems. Among the KP metabolites, Kynurenic Acid (KYNA) and Quinolinic Acid (QUINA) have been shown to interact with ionotropic glutamate receptors. QUINA acts as an orthosteric agonist at the GluN2 subunits of NMDA receptors and it might regulate endogenous glutamate release and uptake inhibition and lipid peroxidation. In contrast, KYNA acts as a competitive antagonist at the glycine site on the GluN1 subunit of NMDA receptors, thereby inhibiting NMDA receptor function. KYNA also inhibits the kainate and the amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) glutamate receptors, and has a non-competitive inhibitory action on the a7-nicotinic acetylcholine receptors[1]. In this preliminary study we enrolled 14 cluster headache patients (CH, 13 males) and 15 age-matched healthy controls (HC, 14 males). We developed a HPLC tandem mass spectrometry method to assess the serum concentrations of KYNA, QUINA, Anthranilic acid and Kynurenine. Kynurenine serum levels resulted not significantly different between the groups (HC 0.33±0.1 and CH 0.35±0.14; Z= -0.53, p = 0.597), while both QUINA, KYNA and Anthranilic Acid were significantly reduced in cluster headache patients with respect to healthy controls (QUINA: HC 18.94±5.24 and CH 3.14±4.87; Z= -4.38, p < 0.001; KYNA: HC 3.53±1.33 and CH 2.53±1.20; Z= -12.45, p = 0.041; Anthranilic Acid: HC 1.28±1.14 and CH 0.17±0.06; Z= -4.46, p < 0.001). These results highlight that the endogenous regulation of the glutamatergic transmission in cluster headache might play an important role in its pathophysiology. Written informed consent to publication was obtained from the patient(s).

Meroterpenoid Chrodrimanins Are Selective and Potent Blockers of Insect GABA-Gated Chloride Channels.

Meroterpenoid chrodrimanins, produced from Talaromyces sp. YO-2, are known to paralyze silkworm (Bombyx mori) larvae, but their target is unknown. We have investigated the actions of chrodrimanin B on ligand-gated ion channels of silkworm larval neurons using patch-clamp electrophysiology. Chrodrimanin B had no effect on membrane currents when tested alone at 1 μM. However, it completely blocked the γ-aminobutyric acid (GABA)-induced current and showed less pronounced actions on acetylcholine- and L-glutamate-induced currents, when delivered at 1 μM for 1 min prior to co-application with transmitter GABA. Thus, chrodrimanins were also tested on a wild-type isoform of the B. mori GABA receptor (GABAR) RDL using two-electrode voltage-clamp electrophysiology. Chrodrimanin B attenuated the peak current amplitude of the GABA response of RDL with an IC50 of 1.66 nM. The order of the GABAR-blocking potency of chrodrimanins B > D > A was in accordance with their reported insecticidal potency. Chrodrimanin B had no open channel blocking action when tested at 3 nM on the GABA response of RDL. Co-application with 3 nM chrodrimanin B shifted the GABA concentration response curve to a higher concentration and further increase of chrodrimanin B concentration to10 nM; it reduced maximum current amplitude of the GABA response, pointing to a high-affinity competitive action and a lower affinity non-competitive action. The A282S;T286V double mutation of RDL, which impairs the actions of fipronil, hardly affected the blocking action of chrodrimanin B, indicating a binding site of chrodrimanin B distinct from that of fipronil. Chrodrimanin B showed approximately 1,000-fold lower blocking action on human α1β2γ2 GABAR compared to RDL and thus is a selective blocker of insect GABARs.

Allosteric Noncompetitive Small Molecule Selective Inhibitors of CD45 Tyrosine Phosphatase Suppress T-Cell Receptor Signals and Inflammation In Vivo

CD45 is a receptor-like member of the protein tyrosine phosphatase (PTP) family. We screened in silico for small molecules binding at a predicted allosteric pocket unique to the CD45 intracellular domain, and validated inhibitors by in vitro phosphatase assays. Compound 211 exhibited a CD45 IC50 value of 200 nM and had >100-fold selectivity over six related PTPs. The relevance of the allosteric pocket was verified through site-directed mutagenesis. Compound 211 has a noncompetitive mechanism of action, and it is extremely effective at preventing dephosphorylation of substrate Lck phosphotyrosine (pY)-505 versus preventing dephosphorylation of Lck pY-393. In cultured primary T cells, compound 211 prevents T-cell receptor-mediated activation of Lck, Zap-70, and mitogen-activated protein kinase, and interleukin-2 production. In a delayed-type hypersensitivity reaction in vivo, compound 211 abolished inflammation. This work demonstrates a novel approach to develop effective allosteric inhibitors that can be expanded to target the corresponding allosteric domains of other receptor PTPs.

Copper, aluminum, iron and calcium inhibit human acetylcholinesterase in vitro

Acetylcholinesterase (AChE) is an important part of cholinergic nerves where it participates in termination of neurotransmission. AChE can be inhibited by e.g. some Alzheimer disease drugs, nerve agents, and secondary metabolites. In this work, metal salts aluminum chloride, calcium chloride, cupric chloride, ferric chloride, potassium chloride, magnesium chloride and sodium chloride were tested for their ability to inhibit AChE. Standard Ellman assay based on human recombinant AChE was done and inhibition was measured using Dixon plot. No inhibition was proved for sodium, potassium and magnesium ions. However, aluminum, cupric, ferric and calcium ions were able to inhibit AChE via noncompetitive mechanism of inhibition. Though the inhibition is much weaker when compared to e.g. drugs with noncompetitive mechanism of action, biological relevance of the findings can be anticipated.

Pivaloylcodeine, a new codeine derivative, for the inhibition of morphine glucuronidation. An in vitro study in the rat

We have previously found that phenanthrenic opioids, including codeine, modulate morphine glucuronidation in the rat. Here codeine and five of its derivatives were compared in their effects on the synthesis of morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) from morphine by rat liver microsomal preparations, and by primary cultures of rat hepatocytes previously incubated for 72h with either codeine or its derivatives. Acetylcodeine and pivaloylcodeine shared the capability of the parent compound of inhibiting the synthesis of M3G by liver microsomes through a noncompetitive mechanism of action. Their IC50 were 3.25, 2.27, and 4.32μM, respectively. Dihydrocodeine, acetyldihydrocodeine, and lauroylcodeine were ineffective. In all the experimental circumstances M6G was undetectable in the incubation medium. In primary hepatocyte cultures codeine only inhibited M3G formation, but with a lower efficacy than that observed with microsomes (IC50 20.91 vs 4.32μM). Preliminary results show that at micromolar concentrations codeine derivatives exhibit a low rate of affinity for μ opiate receptors. In conclusion, acetyl and pivaloyl derivatives of codeine noncompetitively inhibit liver glucuronidation of morphine interacting with microsomes. This study further strengths the notion that phenanthrenic opioids can modulate morphine glucuronidation independently from their effects on μ opiate receptors.

Ginger and its pungent constituents non-competitively inhibit activation of human recombinant and native 5-HT3 receptors of enteric neurons.

Beneficial effects of ginger in the treatment of gastrointestinal (GI) problems and chemotherapy-induced nausea and vomiting are well accepted. In rodents, the action of ginger seems to be mediated by the inhibition of 5-HT3 receptors, which are established targets to combat emesis and irritable bowel syndrome. Heterologously expressed human 5-HT3 A or 5-HT3 AB receptors were characterized by means of Ca(2+) influx studies using HEK293 cells. Complementing Ca(2+) measurements in Fluo-4-AM-stained whole-mount preparations of the human submucous plexus were carried out. Furthermore, [3H]GR65630 binding assays were performed to reveal the mode of action of ginger and its pungent compounds. We show for the first time that ginger extracts and its pungent arylalkane constituents concentration-dependently inhibit activation of human 5-HT3 receptors. Ginger extracts inhibited both receptors with increasing content of pungent compounds, confirming that these are part of ginger's active principle. Inhibition potencies of the arylalkanes 6-gingerol and 6-shogaol on both receptors were in the low micromolar range. A lipophilic ginger extract and 6-gingerol had no influence on 5-HT potency, but reduced the 5-HT maximum effect, indicating non-competitive inhibition. The non-competitive action was confirmed by [(3) H]GR65630 binding, showing that the ginger extract did not displace the radioligand from 5-HT3 A and 5-HT3 AB receptors. The potential relevance of the inhibitory action of ginger on native 5-HT3 receptors in the gut was confirmed in whole-mount preparations of the human submucous plexus. While a general neurotoxic effect of 6-gingerol was ruled out, it inhibited the 2-methyl-5-HT-mediated activation of 5-HT3 receptors residing on enteric neurons. Our findings may encourage the use of ginger extracts to alleviate nausea in cancer patients receiving chemotherapy and to treat functional GI disorders.

Pain‐releasing action of Platelet‐activating factor (PAF) antagonists in neuropathic pain animal models and the mechanisms of action

Platelet-activating factor (PAF) has been implicated in the pathology of neuropathic pain. Previous studies reported that PAF receptor (PAF-R) antagonists have varied anti-allodynia effects by route of administration and nerve injury models in rats. The present study elucidated the effectiveness of PAF antagonists against neuropathic pain in four different models of peripheral nerve injury and provided insights into the mode of anti-allodynia action. PAF antagonists, TCV-309, BN 50739 and WEB 2086 by intravenous (i.v.) and oral administration have potent and long-lasting anti-allodynia action in mice neuropathic pain models. Treatment with PAF antagonists before surgery delayed the initiation of allodynia until the effects of these treatments were abolished. Intrathecal (i.t.) injection of the PAF antagonists and siRNA against PAF receptor ameliorated allodynia. I.t. injection of the glycine receptor (GlyR)α3 siRNA reduced the anti-allodynia effect of PAF antagonists. This evidence suggests that the anti-allodynia effect of PAF antagonists is at least in part mediated by spinal relief of PAF-induced dysfunction of GlyRα3. An analysis of the mode of anti-allodynia action of TCV-309 in vivo revealed a competitive action against PAF shortly after the injection of TCV-309, converting to a non-competitive action later. The present results revealed the effectiveness in anti-allodynia of PAF antagonists in different nerve injury models, and the unique mode of action; long-lasting anti-allodynia effects mediated by spinal GlyRα3 with a competitive manner at the initial stage and the following non-competitive manner of inhibition.

Inhibitors of Glutamate Dehydrogenase Block Sodium-Dependent Glutamate Uptake in Rat Brain Membranes

We recently found evidence for anatomic and physical linkages between the astroglial Na+-dependent glutamate transporters (GLT-1/EAAT2 and GLAST/EAAT1) and mitochondria. In these same studies, we found that the glutamate dehydrogenase (GDH) inhibitor, epigallocatechin-monogallate (EGCG), inhibits both glutamate oxidation and Na+-dependent glutamate uptake in astrocytes. In the present study, we extend this finding by exploring the effects of EGCG on Na+-dependent l-[3H]-glutamate (Glu) uptake in crude membranes (P2) prepared from rat brain cortex. In this preparation, uptake is almost exclusively mediated by GLT-1. EGCG inhibited l-[3H]-Glu uptake in cortical membranes with an IC50 value of 230 μM. We also studied the effects of two additional inhibitors of GDH, hexachlorophene (HCP) and bithionol (BTH). Both of these compounds also caused concentration-dependent inhibition of glutamate uptake in cortical membranes. Pre-incubating with HCP for up to 15 min had no greater effect than that observed with no pre-incubation, showing that the effects occur rapidly. HCP decreased the Vmax for glutamate uptake without changing the Km, consistent with a non-competitive mechanism of action. EGCG, HCP, and BTH also inhibited Na+-dependent transport of d-[3H]-aspartate (Asp), a non-metabolizable transporter substrate, and [3H]-γ-aminobutyric acid (GABA). In contrast to the forebrain, glutamate uptake in crude cerebellar membranes (P2) is likely mediated by GLAST (EAAT1). Therefore, the effects of these compounds were examined in cerebellar membranes. In this region, none of these compounds had any effect on uptake of either l-[3H]-Glu or d-[3H]-Asp, but they all inhibited [3H]-GABA uptake. Together these studies suggest that GDH is preferentially required for glutamate uptake in forebrain as compared to cerebellum, and GDH may be required for GABA uptake as well. They also provide further evidence for a functional linkage between glutamate transport and mitochondria.

The Mechanistic Basis for Noncompetitive Ibogaine Inhibition of Serotonin and Dopamine Transporters

Ibogaine, a hallucinogenic alkaloid proposed as a treatment for opiate withdrawal, has been shown to inhibit serotonin transporter (SERT) noncompetitively, in contrast to all other known inhibitors, which are competitive with substrate. Ibogaine binding to SERT increases accessibility in the permeation pathway connecting the substrate-binding site with the cytoplasm. Because of the structural similarity between ibogaine and serotonin, it had been suggested that ibogaine binds to the substrate site of SERT. The results presented here show that ibogaine binds to a distinct site, accessible from the cell exterior, to inhibit both serotonin transport and serotonin-induced ionic currents. Ibogaine noncompetitively inhibited transport by both SERT and the homologous dopamine transporter (DAT). Ibogaine blocked substrate-induced currents also in DAT and increased accessibility of the DAT cytoplasmic permeation pathway. When present on the cell exterior, ibogaine inhibited SERT substrate-induced currents, but not when it was introduced into the cytoplasm through the patch electrode. Similar to noncompetitive transport inhibition, the current block was not reversed by increasing substrate concentration. The kinetics of inhibitor binding and dissociation, as determined by their effect on SERT currents, indicated that ibogaine does not inhibit by forming a long-lived complex with SERT, but rather binds directly to the transporter in an inward-open conformation. A kinetic model for transport describing the noncompetitive action of ibogaine and the competitive action of cocaine accounts well for the results of the present study.