Structure optimization of positive allosteric modulators of GABAB receptors led to the unexpected discovery of antagonists/potential negative allosteric modulators.

Positive allosteric modulators (PAMs) of GABAB receptor represent an interesting alternative to receptor agonists such as baclofen, as they act on the receptor in a more physiological way and thus are devoid of the side effects typically exerted by the agonists. Based on our interest in the identification of new GABAB receptor PAMs, we followed a merging approach to design new chemotypes starting from selected active compounds, such as GS39783, rac-BHFF, and BHF177, and we ended up with the synthesis of four different classes of compounds. The new compounds were tested alone or in the presence of 10 µM GABA using [35S]GTPγS binding assay to assess their functionality at the receptor. Unexpectedly, a number of them significantly inhibited GABA-stimulated GTPγS binding thus revealing a functional switch with respect to the prototype molecules. Further studies on selected compounds will clarify if they act as negative modulators of the receptor or, instead, as antagonists at the orthosteric binding site.

Identification and biochemical analyses of selective CB2 agonists.

Cannabinoid CB1 and CB2 receptors are activated by Δ9-tetrahydrocannabinol, a psychoactive component of marijuana. The cannabinoid CB1 receptor is primarily located in the brain and is responsible for the psychoactive side effects, whereas the cannabinoid CB2 receptor is located in immune cells and is an attractive target for immune-related maladies. We identify small molecules that selectively bind to the cannabinoid CB2 receptor and can be further developed into therapeutics. The affinity of three molecules, ABK5, ABK6, and ABK7, to the cannabinoid CB2 receptor was determined with radioligand competition binding. The potency of G-protein coupling was determined with GTPγS binding. The three compounds bound selectively to the cannabinoid CB2 receptor, and no binding to the cannabinoid CB1 receptor was detected up to 10 µM. Immunoblotting studies show that the amount of ERK1/2 and MEK phosphorylation increased in a Gi/o-dependent manner. Furthermore, an immune cell line (Jurkat cells) was treated with ABK5, and as a result, inhibited cell proliferation. These three compounds are novel cannabinoid CB2 receptor agonists and hold promise to be further developed to treat inflammation and the often-associated pain.

Alterations in the Activity of Spinal and Thalamic Opioid Systems in a Mice Neuropathic Pain Model.

Clinical studies have reported lower effectivity of opioid drugs in therapy of neuropathic pain. Therefore, to determine the changes in endogenous opioid systems in this pain more precisely, we have studied the changes in the pain-related behavior on days 1, 14, and 28 following a chronic constriction injury (CCI) to the sciatic nerve in mice. In parallel, we have studied the changes of µ-(MOP), δ-(DOP) and κ-(KOP) receptors, proenkephalin (PENK) and prodynorphin (PDYN) mRNA levels, as well as GTPγS binding of opioid receptors on the ipsi- and contralateral parts of the spinal cord and thalamus on the 14th day following CCI, as on this day the greatest manifestation of pain-related behavior was observed. On ipsilateral spinal cord, the decrease in MOP/DOP/KOP receptor and increase in PDYN/PENK mRNA expression was observed. In thalamus, MOP/DOP/KOP receptor expression decreased contralaterally. On ipsilateral side, there were no changes in PDYN/PENK or DOP/KOP receptor expression, but MOP mRNA decreased. The spinal GTPγS binding of MOP/DOP/KOP receptor ligands decreased on the ipsilateral side, yet the effect was less pronounced for DOP receptor ligands. In thalamus, a decrease was observed on the contralateral side for all opioid receptor ligands, especially for DOP ligand. A less pronounced decrease in GTPγS binding of spinal DOP ligands may indicate a weaker stimulation of ascending nociceptive pathways, which could explain the absence of decreased activity of DOP receptor ligands in neuropathy. These findings may suggest that drugs with a higher affinity for the DOP receptor will perform better in neuropathic pain.

Placement of Hydroxy Moiety on Pendant of Peptidomimetic Scaffold Modulates Mu and Kappa Opioid Receptor Efficacy.

In an effort to expand the structure-activity relationship (SAR) studies of a series of mixed-efficacy opioid ligands, peptidomimetics that incorporate methoxy and hydroxy groups around a benzyl or 2-methylindanyl pendant on a tetrahydroquinoline (THQ) core of the peptidomimetics were evaluated. Compounds containing a methoxy or hydroxy moiety in the o- or m-positions increased binding affinity to the kappa opioid receptor (KOR), whereas compounds containing methoxy or hydroxy groups in the p-position decreased KOR affinity and reduced or eliminated efficacy at the mu opioid receptor (MOR). The results from a substituted 2-methylindanyl series aligned with the findings from the substituted benzyl series. Our studies culminated in the development of 8c, a mixed-efficacy MOR agonist/KOR agonist with subnanomolar binding affinity for both MOR and KOR.

Stratification of Cannabinoid 1 Receptor (CB1R) Agonist Efficacy: Manipulation of CB1R Density through Use of Transgenic Mice Reveals Congruence between In Vivo and In Vitro Assays.

Synthetic cannabinoids (SCs) are an emerging class of abused drugs that differ from each other and the phytocannabinoid ∆9-tetrahydrocannabinol (THC) in their safety and cannabinoid-1 receptor (CB1R) pharmacology. As efficacy represents a critical parameter to understanding drug action, the present study investigated this metric by assessing in vivo and in vitro actions of THC, two well-characterized SCs (WIN55,212-2 and CP55,940), and three abused SCs (JWH-073, CP47,497, and A-834,735-D) in CB1 (+/+), (+/-), and (-/-) mice. All drugs produced maximal cannabimimetic in vivo effects (catalepsy, hypothermia, antinociception) in CB1 (+/+) mice, but these actions were essentially eliminated in CB1 (-/-) mice, indicating a CB1R mechanism of action. CB1R efficacy was inferred by comparing potencies between CB1 (+/+) and (+/-) mice [+/+ ED50 /+/- ED50], the latter of which has a 50% reduction of CB1Rs (i.e., decreased receptor reserve). Notably, CB1 (+/-) mice displayed profound rightward and downward shifts in the antinociception and hypothermia dose-response curves of low-efficacy compared with high-efficacy cannabinoids. In vitro efficacy, quantified using agonist-stimulated [35S]GTPγS binding in spinal cord tissue, significantly correlated with the relative efficacies of antinociception (r = 0.87) and hypothermia (r = 0.94) in CB1 (+/-) mice relative to CB1 (+/+) mice. Conversely, drug potencies for cataleptic effects did not differ between these genotypes and did not correlate with the in vitro efficacy measure. These results suggest that evaluation of antinociception and hypothermia in CB1 transgenic mice offers a useful in vivo approach to determine CB1R selectivity and efficacy of emerging SCs, which shows strong congruence with in vitro efficacy.

Mu-Opioid (MOP) receptor mediated G-protein signaling is impaired in specific brain regions in a rat model of schizophrenia.

Schizophrenia is a complex mental health disorder. Clinical reports suggest that many patients with schizophrenia are less sensitive to pain than other individuals. Animal models do not interpret schizophrenia completely, but they can model a number of symptoms of the disease, including decreased pain sensitivities and increased pain thresholds of various modalities. Opioid receptors and endogenous opioid peptides have a substantial role in analgesia. In this biochemical study we investigated changes in the signaling properties of the mu-opioid (MOP) receptor in different brain regions, which are involved in the pain transmission, i.e., thalamus, olfactory bulb, prefrontal cortex and hippocampus. Our goal was to compare the transmembrane signaling mediated by MOP receptors in control rats and in a recently developed rat model of schizophrenia. Regulatory G-protein activation via MOP receptors were measured in [(35)S]GTPγS binding assays in the presence of a highly selective MOP receptor peptide agonist, DAMGO. It was found that the MOP receptor mediated activation of G-proteins was substantially lower in membranes prepared from the 'schizophrenic' model rats than in control animals. The potency of DAMGO to activate MOP receptor was also decreased in all brain regions studied. Taken together in our rat model of schizophrenia, MOP receptor mediated G-proteins have a reduced stimulatory activity compared to membrane preparations taken from control animals. The observed distinct changes of opioid receptor functions in different areas of the brain do not explain the augmented nociceptive threshold described in these animals.

Small Molecule CXCR3 Antagonists.

Chemokines and their receptors are known to play important roles in disease. More than 40 chemokine ligands and 20 chemokine receptors have been identified, but, to date, only two small molecule chemokine receptor antagonists have been approved by the FDA. The chemokine receptor CXCR3 was identified in 1996, and nearly 20 years later, new areas of CXCR3 disease biology continue to emerge. Several classes of small molecule CXCR3 antagonists have been developed, and two have shown efficacy in preclinical models of inflammatory disease. However, only one CXCR3 antagonist has been evaluated in clinical trials, and there remain many opportunities to further investigate known classes of CXCR3 antagonists and to identify new chemotypes. This Perspective reviews the known CXCR3 antagonists and considers future opportunities for the development of small molecules for clinical evaluation.

Discovery of tripeptide-derived multifunctional ligands possessing delta/mu opioid receptor agonist and neurokinin 1 receptor antagonist activities.

Several bifunctional peptides were synthesized and characterized based on the pentapeptide-derived ligand NP30 (1: Tyr-DAla-Gly-Phe-Gly-Trp-O-[3',5'-Bzl(CF3)2]). Modification and truncation of amino acid residues were performed, and the tripeptide-derived ligand NP66 (11: Dmt-DAla-Trp-NH-[3',5'-(CF3)2-Bzl]) was obtained based on the overlapping pharmacophore concept. The Trp(3) residue of ligand 11 works as a message residue for both opioid and NK1 activities. The significance lies in the observation that the approach of appropriate truncation of peptide sequence could lead to a tripeptide-derived chimeric ligand with effective binding and functional activities for both mu and delta opioid and NK1 receptors with agonist activities at mu and delta opioid and antagonist activity at NK1 receptors, respectively.

Characterization of kappa opioid receptor mediated, dynorphin-stimulated [35S]GTPγS binding in mouse striatum for the evaluation of selective KOR ligands in an endogenous setting.

Differential modulation of kappa opioid receptor (KOR) signaling has been a proposed strategy for developing therapies for drug addiction and depression by either activating or blocking this receptor. Hence, there have been significant efforts to generate ligands with diverse pharmacological properties including partial agonists, antagonists, allosteric modulators as well as ligands that selectively activate some pathways while not engaging others (biased agonists). It is becoming increasingly evident that G protein coupled receptor signaling events are context dependent and that what may occur in cell based assays may not be fully indicative of signaling events that occur in the naturally occurring environment. As new ligands are developed, it is important to assess their signaling capacity in relevant endogenous systems in comparison to the performance of endogenous agonists. Since KOR is considered the cognate receptor for dynorphin peptides we have evaluated the selectivity profiles of dynorphin peptides in wild-type (WT), KOR knockout (KOR-KO), and mu opioid receptor knockout (MOR-KO) mice using [35S]GTPγS binding assay in striatal membrane preparations. We find that while the small molecule KOR agonist U69,593, is very selective for KOR, dynorphin peptides promiscuously stimulate G protein signaling in striatum. Furthermore, our studies demonstrate that norBNI and 5'GNTI are highly nonselective antagonists as they maintain full potency and efficacy against dynorphin signaling in the absence of KOR. Characterization of a new KOR antagonist, which may be more selective than NorBNI and 5'GNTI, is presented using this approach.

Use of radiolabeled antagonist assays for assessing agonism at D2 and D3 dopamine receptors: comparison with functional GTPγS assays.

BACKGROUND: Cell-based drug screening assays are essential tools for drug discovery and development targeting G protein-coupled receptors, which include dopamine D3 receptors. D3 is notorious for its poor coupling to G protein in most heterologous cell lines, and therefore D3 agonist-stimulated binding of [(35)S]GTPγS to G protein cannot be observed in many "non-functional" D3 expressing cell lines. NEW METHOD: The present work explores the use of an alternate method for assessing agonist activity, consisting of measuring the difference in agonist competition between [(3)H]spiperone bound to low-affinity states of the receptor and that with radioligand bound to high-affinity states (GTP shift assay). COMPARISON WITH EXISTING METHOD: The current study describes the determination of GTP shifts in [(3)H]spiperone binding assays for the assessment of agonists' potencies (at D2 and D3) and efficacies (at D3). Compared with GTPγ(35)S binding assays, the new method removes the cumbersome need of functional D3 cell lines and limited project duration due to short half-life of isotope (35)S. CONCLUSION: The new method allows the estimation of potency (D2 and D3) and efficacy (D3) at the level of receptor and G protein activation in a simple fashion from shifts in monophasic-inhibition curves. Moreover, it does not require [(35)S]GTPγS binding assays with functional D3 cells. This method will have wide applicability for D3-selective agonist screening. It may also be useful for other GPCRs circumventing the need for functional assays and offering the ability to detect agonist activity regardless of the particular signaling pathway.

Dimeric carbamoylguanidine-type histamine H2 receptor ligands: A new class of potent and selective agonists.

The bioisosteric replacement of the acylguanidine moieties in dimeric histamine H2 receptor (H2R) agonists by carbamoylguanidine groups resulted in compounds with retained potencies and intrinsic activities, but considerably improved stability against hydrolytic cleavage. These compounds achieved up to 2500 times the potency of histamine when studied in [(35)S]GTPγS assays on recombinant human and guinea pig H2R. Unlike 3-(imidazol-4-yl)propyl substituted carbamoylguanidines, the corresponding 2-amino-4-methylthiazoles revealed selectivity over histamine receptor subtypes H1R, H3R and H4R in radioligand competition binding studies. H2R binding studies with three fluorescent compounds and one tritium-labeled ligand, synthesized from a chain-branched precursor, failed due to pronounced cellular accumulation and high non-specific binding. However, the dimeric H2R agonists proved to be useful pharmacological tools for functional studies on native cells, as demonstrated for selected compounds by cAMP accumulation and inhibition of fMLP-stimulated generation of reactive oxygen species in human monocytes.

Methanol extract of Ficus platyphylla ameliorates seizure severity, cognitive deficit and neuronal cell loss in pentylenetetrazole-kindled mice.

Decoctions of Ficus plathyphylla are used in Nigeria's folk medicine to manage epilepsy for many years and their efficacies are widely acclaimed among the rural communities of Northern Nigeria. In this study, we examined the ameliorative effects of the standardized methanol extract of Ficus platyphylla (FP) stem bark on seizure severity, cognitive deficit and neuronal cell loss in pentylenetetrazole-kindled mice. The (35)S-GTPγS, glutamate and γ-aminobutyric acid receptors binding properties of the extract were also evaluated. Male CD-1 mice were kindled with an initial subeffective dose of pentylenetetrazole (PTZ, 37.5mg/kg, i.p.) for a total of 13 convulsant injections and the treatment groups concurrently received FP (100 and 200mg/kg). Control animals received the same number of saline injections. Twenty-four h after kindling completion the animals' learning performance was tested in a two-way shuttle-box. The animals were challenged with another subeffective dose of PTZ (32.5mg/kg, i.p.) on day 7 after kindling completion. Animals were sacrificed a day after the challenged experiment and the brains were processed for histological investigation. FP ameliorates seizure severity, cognitive deficits and neuronal cell loss in PTZ kindled mice. Components of the extract showed affinity for GABAergic and glutamatergic receptors. Glutamate release was diminished and the (35)S-GTPγS binding assay revealed no intrinsic activity at glutamatergic receptors. Our results revealed that FP contains psychoactive secondary metabolites with anticonvulsant properties, thus supporting the isolation and development of the biologically active components of this medicinal plant as antiepileptic agents.

Potency enhancement of the κ-opioid receptor antagonist probe ML140 through sulfonamide constraint utilizing a tetrahydroisoquinoline motif.

Optimization of the sulfonamide-based kappa opioid receptor (KOR) antagonist probe molecule ML140 through constraint of the sulfonamide nitrogen within a tetrahydroisoquinoline moiety afforded a marked increase in potency. This strategy, when combined with additional structure-activity relationship exploration, has led to a compound only six-fold less potent than norBNI, a widely utilized KOR antagonist tool compound, but significantly more synthetically accessible. The new optimized probe is suitably potent for use as an in vivo tool to investigate the therapeutic potential of KOR antagonists.

Differential effects of exercise on brain opioid receptor binding and activation in rats.

Physical exercise stimulates the release of endogenous opioid peptides supposed to be responsible for changes in mood, anxiety, and performance. Exercise alters sensitivity to these effects that modify the efficacy at the opioid receptor. Although there is evidence that relates exercise to neuropeptide expression in the brain, the effects of exercise on opioid receptor binding and signal transduction mechanisms downstream of these receptors have not been explored. Here, we characterized the binding and G protein activation of mu opioid receptor, kappa opioid receptor or delta opioid receptor in several brain regions following acute (7 days) and chronic (30 days) exercise. As regards short- (acute) or long-term effects (chronic) of exercise, overall, higher opioid receptor binding was observed in acute-exercise animals and the opposite was found in the chronic-exercise animals. The binding of [(35) S]GTPγS under basal conditions (absence of agonists) was elevated in sensorimotor cortex and hippocampus, an effect more evident after chronic exercise. Divergence of findings was observed for mu opioid receptor, kappa opioid receptor, and delta opioid receptor receptor activation in our study. Our results support existing evidence of opioid receptor binding and G protein activation occurring differentially in brain regions in response to diverse exercise stimuli. We characterized the binding and G protein activation of mu, kappa, and delta opioid receptors in several brain regions following acute (7 days) and chronic (30 days) exercise. Higher opioid receptor binding was observed in the acute exercise animal group and opposite findings in the chronic exercise group. Higher G protein activation under basal conditions was noted in rats submitted to chronic exercise, as visible in the depicted pseudo-color autoradiograms.

Effect of Iboga alkaloids on µ-opioid receptor-coupled G protein activation.

OBJECTIVE: The iboga alkaloids are a class of small molecules defined structurally on the basis of a common ibogamine skeleton, some of which modify opioid withdrawal and drug self-administration in humans and preclinical models. These compounds may represent an innovative approach to neurobiological investigation and development of addiction pharmacotherapy. In particular, the use of the prototypic iboga alkaloid ibogaine for opioid detoxification in humans raises the question of whether its effect is mediated by an opioid agonist action, or if it represents alternative and possibly novel mechanism of action. The aim of this study was to independently replicate and extend evidence regarding the activation of µ-opioid receptor (MOR)-related G proteins by iboga alkaloids. METHODS: Ibogaine, its major metabolite noribogaine, and 18-methoxycoronaridine (18-MC), a synthetic congener, were evaluated by agonist-stimulated guanosine-5´-O-(γ-thio)-triphosphate ([(35)S]GTPγS) binding in cells overexpressing the recombinant MOR, in rat thalamic membranes, and autoradiography in rat brain slices. RESULTS AND SIGNIFICANCE: In rat thalamic membranes ibogaine, noribogaine and 18-MC were MOR antagonists with functional Ke values ranging from 3 uM (ibogaine) to 13 uM (noribogaine and 18MC). Noribogaine and 18-MC did not stimulate [(35)S]GTPγS binding in Chinese hamster ovary cells expressing human or rat MORs, and had only limited partial agonist effects in human embryonic kidney cells expressing mouse MORs. Ibogaine did not did not stimulate [(35)S]GTPγS binding in any MOR expressing cells. Noribogaine did not stimulate [(35)S]GTPγS binding in brain slices using autoradiography. An MOR agonist action does not appear to account for the effect of these iboga alkaloids on opioid withdrawal. Taken together with existing evidence that their mechanism of action also differs from that of other non-opioids with clinical effects on opioid tolerance and withdrawal, these findings suggest a novel mechanism of action, and further justify the search for alternative targets of iboga alkaloids.

Repeated low-dose administration of the monoacylglycerol lipase inhibitor JZL184 retains cannabinoid receptor type 1-mediated antinociceptive and gastroprotective effects.

The monoacylglycerol lipase (MAGL) inhibitor 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184) produces antinociceptive and anti-inflammatory effects. However, repeated administration of high-dose JZL184 (40 mg/kg) causes dependence, antinociceptive tolerance, cross-tolerance to the pharmacological effects of cannabinoid receptor agonists, and cannabinoid receptor type 1 (CB1) downregulation and desensitization. This functional CB1 receptor tolerance poses a hurdle in the development of MAGL inhibitors for therapeutic use. Consequently, the present study tested whether repeated administration of low-dose JZL184 maintains its antinociceptive actions in the chronic constriction injury of the sciatic nerve neuropathic pain model and protective effects in a model of nonsteroidal anti-inflammatory drug-induced gastric hemorrhages. Mice given daily injections of high-dose JZL184 (≥16 mg/kg) for 6 days displayed decreased CB1 receptor density and function in the brain, as assessed in [(3)H]SR141716A binding and CP55,940 [(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol]-stimulated guanosine 5'-O-(3-[(35)S]thio)triphosphate binding assays, respectively. In contrast, normal CB1 receptor expression and function were maintained following repeated administration of low-dose JZL184 (≤8 mg/kg). Likewise, the antinociceptive and gastroprotective effects of high-dose JZL184 underwent tolerance following repeated administration, but these effects were maintained following repeated low-dose JZL184 treatment. Consistent with these observations, repeated high-dose JZL184, but not repeated low-dose JZL184, elicited cross-tolerance to the common pharmacological effects of Δ(9)-tetrahydrocannabinol. This same pattern of effects was found in a rimonabant [(5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide)]-precipitated withdrawal model of cannabinoid dependence. Taken together, these results indicate that prolonged, partial MAGL inhibition maintains potentially beneficial antinociceptive and anti-inflammatory effects, without producing functional CB1 receptor tachyphylaxis/tolerance or cannabinoid dependence.

Yokukansan inhibits morphine tolerance and physical dependence in mice: the role of α2A-adrenoceptor.

Yokukansan (YKS) is a traditional Japanese medicine consisting of seven medicinal herbs that is used for the treatment of neurosis, insomnia, and the behavioral/psychological symptoms of dementia. This study examined the effects of YKS on morphine tolerance and physical dependence in mice. Daily oral administration of YKS (0.5 or 1.0 g/kg) for 3 weeks significantly attenuated morphine tolerance and naloxone-precipitated morphine withdrawal signs (jumps and body weight loss) without affecting the analgesic effect of morphine. The inhibitory effect of YKS on withdrawal jumps in morphine-dependent mice was blocked by a single pretreatment with an α(2)-adrenoceptor antagonist, yohimbine, but not by an α(1)-adrenoceptor antagonist, prazosin. A similar inhibitory effect on withdrawal jumps was observed by repeated administration of yohimbine. The membrane expression of α(2A)-adrenoceptors in the pons/medulla was decreased in morphine withdrawn animals; this reduction was prevented by repeated administration of YKS or yohimbine. Competitive radioligand and [(35)S]guanosine-5'-O-(3-thiotriphosphate) binding assays revealed that YKS and its constituent herbs, Glycyrrhiza (GR) and Uncaria hook (UH), had specific binding affinity for and antagonist activity against the α(2A)-adrenoceptor. Certain chemical constituents, including GR -derived glycyrrhizin and its metabolite, 18ß-glycyrrhetinic acid, and UH-derived geissoschizine methyl ether (GME), shared such activities. Repeated administration of GR, UH, glycyrrhizin or GME significantly inhibited morphine withdrawal signs. These results suggest that YKS and its active constituents inhibit morphine tolerance and physical dependence, and that the latter is due at least in part to the prevention of the decreased membrane expression of the α(2A)-adrenoceptor in the brainstem by its prolonged blockade.

Involvement of NOX1/NADPH oxidase in morphine-induced analgesia and tolerance.

The involvement of reactive oxygen species (ROS) in morphine-induced analgesia and tolerance has been suggested, yet how and where ROS take part in these processes remains largely unknown. Here, we report a novel role for the superoxide-generating enzyme NOX1/NADPH oxidase in the regulation of analgesia and acute analgesic tolerance. In mice lacking Nox1 (Nox1(-/Y)), the magnitude of the analgesia induced by morphine was significantly augmented. More importantly, analgesic tolerance induced by repeated administration of morphine was significantly suppressed compared with that in the littermates, wild-type Nox1(+/Y). In a membrane fraction obtained from the dorsal spinal cord, no difference was observed in morphine-induced [(35)S]GTPγS-binding between the genotypes, whereas morphine-stimulated GTPase activity was significantly attenuated in Nox1(-/Y). At 2 h after morphine administration, a significant decline in [(35)S]GTPγS-binding was observed in Nox1(+/Y) but not in Nox1(-/Y). No difference in the maximal binding and affinity of [(3)H]DAMGO was observed between the genotypes, but the translocation of protein kinase C isoforms to the membrane fraction following morphine administration was almost completely abolished in Nox1(-/Y). Finally, the phosphorylation of RGS9-2 and formation of a complex by Gαi2/RGS9-2 with 14-3-3 found in morphine-treated Nox1(+/Y) were significantly suppressed in Nox1(-/Y). Together, these results suggest that NOX1/NADPH oxidase attenuates the pharmacological effects of opioids by regulating GTPase activity and the phosphorylation of RGS9-2 by protein kinase C. NOX1/NADPH oxidase may thus be a novel target for the development of adjuvant therapy to retain the beneficial effects of morphine.

Essential role for orbitofrontal serotonin 1B receptors in obsessive-compulsive disorder-like behavior and serotonin reuptake inhibitor response in mice.

BACKGROUND: Perseveration and sensorimotor gating deficits are core features of obsessive-compulsive disorder (OCD). Serotonin 1B receptor (5-HT1BR) agonists exacerbate OCD symptoms in patients and induce perseveration and sensorimotor gating deficits in mice. Serotonin reuptake inhibitors (SRIs), but not noradrenaline reuptake inhibitors (NRIs), reduce OCD symptoms following 4 to 8 weeks of treatment. Using mice, we compared the effects of chronic SRI versus NRI treatment on 5-HT1BR-induced OCD-like behavior and 5-HT1BR sensitivity in orbitofrontal-subcortical OCD circuits. Furthermore, we localized the 5-HT1BR population that mediates OCD-like behavior. METHODS: Mice chronically received the SRI clomipramine or the NRI desipramine and were examined for 5-HT1BR-induced OCD-like behavior or 5-HT1BR binding and G-protein coupling in caudate putamen, nucleus accumbens, and orbitofrontal cortex. Separate mice were tested for OCD- or depression-like behavior following 4, 14, 21, 28, or 56 days of SRI treatment. Finally, OCD-like behavior was assessed following intra-orbitofrontal 5-HT1BR agonist infusion or intra-orbitofrontal 5-HT1BR antagonist infusion coupled with systemic 5-HT1BR agonist treatment. RESULTS: Effective, but not ineffective, OCD treatments reduced OCD-like behavior in mice with a time course that parallels the delayed therapeutic onset in OCD patients and downregulated 5-HT1BR expression in the orbitofrontal cortex. Intra-orbitofrontal 5-HT1BR agonist infusion induced OCD-like behavior, and intra-orbitofrontal 5-HT1BR antagonist infusion blocked OCD-like effects of systemic 5-HT1BR agonist treatment. CONCLUSIONS: These results indicate that orbitofrontal 5-HT1BRs are necessary and sufficient to induce OCD-like behavior in mice and that SRI pharmacotherapy reduces OCD-like behavior by desensitizing orbitofrontal 5-HT1BRs. Our findings suggest an essential role for orbitofrontal 5-HT1BRs in OCD pathophysiology and treatment.

Chronic neuropathic pain in mice reduces µ-opioid receptor-mediated G-protein activity in the thalamus.

Neuropathic pain is a debilitating condition that is often difficult to treat using conventional pharmacological interventions and the exact mechanisms involved in the establishment and maintenance of this type of chronic pain have yet to be fully elucidated. The present studies examined the effect of chronic nerve injury on µ-opioid receptors and receptor-mediated G-protein activity within the supraspinal brain regions involved in pain processing of mice. Chronic constriction injury (CCI) reduced paw withdrawal latency, which was maximal at 10 days post-injury. [d-Ala2,(N-Me)Phe4,Gly5-OH] enkephalin (DAMGO)-stimulated [(35)S]GTPγS binding was then conducted at this time point in membranes prepared from the rostral ACC (rACC), thalamus and periaqueductal grey (PAG) of CCI and sham-operated mice. Results showed reduced DAMGO-stimulated [(35)S]GTPγS binding in the thalamus and PAG of CCI mice, with no change in the rACC. In thalamus, this reduction was due to decreased maximal stimulation by DAMGO, with no difference in EC(50) values. In PAG, however, DAMGO E(max) values did not significantly differ between groups, possibly due to the small magnitude of the main effect. [(3)H]Naloxone binding in membranes of the thalamus showed no significant differences in B(max) values between CCI and sham-operated mice, indicating that the difference in G-protein activation did not result from differences in µ-opioid receptor levels. These results suggest that CCI induced a region-specific adaptation of µ-opioid receptor-mediated G-protein activity, with apparent desensitization of the µ-opioid receptor in the thalamus and PAG and could have implications for treatment of neuropathic pain.