Pharmacological characterization of novel synthetic opioids (NSO) found in the recreational drug marketplace.

Novel synthetic opioids (NSO) are increasingly encountered in illicit heroin and counterfeit pain pills. Many NSO are resurrected from older biomedical literature or patent applications, so limited information is available about their biological effects. Here we examined the pharmacology of three structurally-distinct NSO found in the recreational drug market: N-(1-(2-phenylethyl)-4-piperidinyl)-N-phenylbutyramide (butyrylfentanyl), 3,4-dichloro-N-[(1R,2R)-2-(dimethylamino)cyclohexyl]-N-methylbenzamide (U-47700) and 1-cyclohexyl-4-(1,2-diphenylethyl)piperazine (MT-45). Radioligand binding and GTPγS functional assays were carried out in cells transfected with murine mu- (MOR-1), delta- (DOR-1) or kappa-opioid receptors (KOR-1). Antinociceptive effects were determined using the radiant heat tail flick technique in mice, and opioid specificity was assessed with the mu-opioid antagonist naloxone. Butyrylfentanyl, U-47700 and MT-45 displayed nM affinities at MOR-1, but were less potent than morphine, and had much weaker effects at DOR-1 and KOR-1. All NSO exhibited agonist actions at MOR-1 in the GTPγS assay. Butyrylfentanyl and U-47700 were 31- and 12-fold more potent than morphine in the tail flick assay, whereas MT-45 was equipotent with morphine. Analgesic effects were reversed by naloxone and absent in genetically-engineered mice lacking MOR-1. Our findings confirm that butyrylfentanyl, U-47700 and MT-45 are selective MOR-1 agonists with in vitro affinities less than morphine. However, analgesic potencies vary more than 30-fold across the compounds, and in vitro binding affinity does not predict in vivo potency. Taken together, our findings highlight the risks to humans who may unknowingly be exposed to these and other NSO when taking adulterated heroin or counterfeit pain medications. This article is part of the Special Issue entitled 'Designer Drugs and Legal Highs.'

Functional Characterization of a Novel Series of Biased Signaling Dopamine D3 Receptor Agonists.

Dopamine receptors play an integral role in controlling brain physiology. Importantly, subtype selective agonists and antagonists of dopamine receptors with biased signaling properties have been successful in treating psychiatric disorders with a low incidence of side effects. To this end, we recently designed and developed SK609, a dopamine D3 receptor (D3R) selective agonist that has atypical signaling properties. SK609 has shown efficacy in reversing akinesia and reducing L-dopa-induced dyskinesia in a hemiparkinsonian rats. In the current study, we demonstrate that SK609 has high selectivity for D3R with no binding affinity on D2R high- or low-affinity state when tested at a concentration of 10 µM. In addition, SK609 and its analogues do not induce desensitization of D3R as determined by repeated agonist treatment response in phosphorylation of ERK1/2 functional assay. Most significantly, SK609 and its analogues preferentially signal through the G-protein-dependent pathway and do not recruit ß-arrestin-2, suggesting a functional bias toward the G-protein-dependent pathway. Structure-activity relationship (SAR) studies using analogues of SK609 demonstrate that the molecules bind at the orthosteric site by maintaining the conserved salt bridge interactions with aspartate 110 on transmembrane 3 and aryl interactions with histidine 349 on transmembrane 6, in addition to several hydrophobic interactions with residues from transmembranes 5 and 6. The compounds follow a strict SAR with reference to the three pharmacophore elements: substituted phenyl ring, length of the linker connecting phenyl ring and amine group, and orientation and hydrophobic branching groups at the amine among SK609 analogues for efficacy and functional selectivity. These features of SK609 and the analogues suggest that biased signaling is an inherent property of this series of molecules.

Anticonvulsant effects of structurally diverse GABA(B) positive allosteric modulators in the DBA/2J audiogenic seizure test: Comparison to baclofen and utility as a pharmacodynamic screening model.

The GABA(B) receptor has been indicated as a promising target for multiple CNS-related disorders. Baclofen, a prototypical orthosteric agonist, is used clinically for the treatment of spastic movement disorders, but is associated with unwanted side-effects, such as sedation and motor impairment. Positive allosteric modulators (PAM), which bind to a topographically-distinct site apart from the orthosteric binding pocket, may provide an improved side-effect profile while maintaining baclofen-like efficacy. GABA, the major inhibitory neurotransmitter in the CNS, plays an important role in the etiology and treatment of seizure disorders. Baclofen is known to produce anticonvulsant effects in the DBA/2J mouse audiogenic seizure test (AGS), suggesting it may be a suitable assay for assessing pharmacodynamic effects. Little is known about the effects of GABA(B) PAMs, however. The studies presented here sought to investigate the AGS test as a pharmacodynamic (PD) screening model for GABA(B) PAMs by comparing the profile of structurally diverse PAMs to baclofen. GS39783, rac-BHFF, CMPPE, A-1295120 (N-(3-(4-(4-chloro-3-fluorobenzyl)-6-methoxy-3,5-dioxo-4,5-dihydro-1,2,4-triazin-2(3H)-yl)phenyl)acetamide), and A-1474713 (N-(3-(4-(4-chlorobenzyl)-3,5-dioxo-4,5-dihydro-1,2,4-triazin-2(3H)-yl)phenyl)acetamide) all produced robust, dose-dependent anticonvulsant effects; a similar profile was observed with baclofen. Pre-treatment with the GABA(B) antagonist SCH50911 completely blocked the anticonvulsant effects of baclofen and CMPPE in the AGS test, indicating such effects are likely mediated by the GABA(B) receptor. In addition to the standard anticonvulsant endpoint of the AGS test, video tracking software was employed to assess potential drug-induced motor side-effects during the acclimation period of the test. This analysis was sensitive to detecting drug-induced changes in total distance traveled, which was used to establish a therapeutic index (TI = hypoactivity/anticonvulsant effects). Calculated TIs for A-1295120, CMPPE, rac-BHFF, GS39783, and A-1474713 were 5.31x, 5.00x, 4.74x, 3.41x, and 1.83x, respectively, whereas baclofen was <1. The results presented here suggest the DBA/2J mouse AGS test is a potentially useful screening model for detecting PD effects of GABA(B) PAMs and can provide an initial read-out on target-related motor side-effects. Furthermore, an improved TI was observed for PAMs compared to baclofen, indicating the PAM approach may be a viable therapeutic alternative to baclofen.

Isotopic Discrimination in the Double-Crested Cormorant (Phalacrocorax auritus).

The diet-tissue discrimination factor is the amount by which a consumer's tissue varies isotopically from its diet, and is therefore a key element in models that use stable isotopes to estimate diet composition. In this study we measured discrimination factors in blood (whole blood, red blood cells and plasma), liver, muscle and feathers of Double-crested Cormorants (Phalacrocorax auritus) for stable isotope ratios of carbon, nitrogen and sulfur. Cormorants exhibited discrimination factors that differed significantly among tissue types (for carbon and nitrogen), and differed substantially (in the context of the isotopic variation among relevant prey species) from those observed in congeneric species. The Double-crested Cormorant has undergone rapid population expansion throughout much of its historic range over the past three decades, leading to both real and perceived conflicts with fisheries throughout North America, and this study provides an essential link for the use of stable isotope analysis in researching foraging ecology, diet, and resource use of this widespread and controversial species.

Enhanced Functional Activity of the Cannabinoid Type-1 Receptor Mediates Adolescent Behavior.

Adolescence is characterized by drastic behavioral adaptations and comprises a particularly vulnerable period for the emergence of various psychiatric disorders. Growing evidence reveals that the pathophysiology of these disorders might derive from aberrations of normal neurodevelopmental changes in the adolescent brain. Understanding the molecular underpinnings of adolescent behavior is therefore critical for understanding the origin of psychopathology, but the molecular mechanisms that trigger adolescent behavior are unknown. Here, we hypothesize that the cannabinoid type-1 receptor (CB1R) may play a critical role in mediating adolescent behavior because enhanced endocannabinoid (eCB) signaling has been suggested to occur transiently during adolescence. To study enhanced CB1R signaling, we introduced a missense mutation (F238L) into the rat Cnr1 gene that encodes for the CB1R. According to our hypothesis, rats with the F238L mutation (Cnr1(F238L)) should sustain features of adolescent behavior into adulthood. Gain of function of the mutated receptor was demonstrated by in silico modeling and was verified functionally in a series of biochemical and electrophysiological experiments. Mutant rats exhibit an adolescent-like phenotype during adulthood compared with wild-type littermates, with typical high risk/novelty seeking, increased peer interaction, enhanced impulsivity, and augmented reward sensitivity for drug and nondrug reward. Partial inhibition of CB1R activity in Cnr1(F238L) mutant rats normalized behavior and led to a wild-type phenotype. We conclude that the activity state and functionality of the CB1R is critical for mediating adolescent behavior. These findings implicate the eCB system as an important research target for the neuropathology of adolescent-onset mental health disorders. SIGNIFICANCE STATEMENT: We present the first rodent model with a gain-of-function mutation in the cannabinoid type-1 receptor (CB1R). Adult mutant rats exhibit an adolescent-like phenotype with typical high risk seeking, impulsivity, and augmented drug and nondrug reward sensitivity. Adolescence is a critical period for suboptimal behavioral choices and the emergence of neuropsychiatric disorders. Understanding the basis of these disorders therefore requires a comprehensive knowledge of how adolescent neurodevelopment triggers behavioral reactions. Our behavioral observations in adult mutant rats, together with reports on enhanced adolescent CB1R signaling, suggest a pivotal role for the CB1R in an adolescent brain as an important molecular mediator of adolescent behavior. These findings implicate the endocannabinoid system as a notable research target for adolescent-onset mental health disorders.

Activation of Neuropeptide Y Receptors Modulates Retinal Ganglion Cell Physiology and Exerts Neuroprotective Actions In Vitro.

Neuropeptide Y (NPY) is expressed in mammalian retina but the location and potential modulatory effects of NPY receptor activation remain largely unknown. Retinal ganglion cell (RGC) death is a hallmark of several retinal degenerative diseases, particularly glaucoma. Using purified RGCs and ex vivo rat retinal preparations, we have measured RGC intracellular free calcium concentration ([Ca2+]i) and RGC spiking activity, respectively. We found that NPY attenuated the increase in the [Ca2+]i triggered by glutamate mainly via Y1 receptor activation. Moreover, (Leu31, Pro34)-NPY, a Y1/Y5 receptor agonist, increased the initial burst response of OFF-type RGCs, although no effect was observed on RGC spontaneous spiking activity. The Y1 receptor activation was also able to directly modulate RGC responses by attenuating the NMDA-induced increase in RGC spiking activity. These results suggest that Y1 receptor activation, at the level of inner or outer plexiform layers, leads to modulation of RGC receptive field properties. Using in vitro cultures of rat retinal explants exposed to NMDA, we found that NPY pretreatment prevented NMDA-induced cell death. However, in an animal model of retinal ischemia-reperfusion injury, pretreatment with NPY or (Leu31, Pro34)-NPY was not able to prevent apoptosis or rescue RGCs. In conclusion, we found modulatory effects of NPY application that for the first time were detected at the level of RGCs. However, further studies are needed to evaluate whether NPY neuroprotective actions detected in retinal explants can be translated into animal models of retinal degenerative diseases.

Maturational alterations in constitutive activity of medial prefrontal cortex kappa-opioid receptors in Wistar rats.

Opioid receptors can display spontaneous agonist-independent G-protein signaling (basal signaling/constitutive activity). While constitutive κ-opioid receptor (KOR) activity has been documented in vitro, it remains unknown if KORs are constitutively active in native systems. Using [(35) S] guanosine 5'-O-[gamma-thio] triphosphate coupling assay that measures receptor functional state, we identified the presence of medial prefrontal cortex KOR constitutive activity in young rats that declined with age. Furthermore, basal signaling showed an age-related decline and was insensitive to neutral opioid antagonist challenge. Collectively, the present data are first to demonstrate age-dependent alterations in the medial prefrontal cortex KOR constitutive activity in rats and changes in the constitutive activity of KORs can differentially impact KOR ligand efficacy. These data provide novel insights into the functional properties of the KOR system and warrant further consideration of KOR constitutive activity in normal and pathophysiological behavior. Opioid receptors exhibit agonist-independent constitutive activity; however, kappa-opioid receptor (KOR) constitutive activity has not been demonstrated in native systems. Our results confirm KOR constitutive activity in the medial prefrontal cortex (mPFC) that declines with age. With the ability to presynaptically inhibit multiple neurotransmitter systems in the mPFC, maturational or patho-logical alterations in constitutive activity could disrupt corticofugal glutamatergic pyramidal projection neurons mediating executive function. Regulation of KOR constitutive activity could serve as a therapeutic target to treat compromised executive function.

Differential Effect of Neuropeptides on Excitatory Synaptic Transmission in Human Epileptic Hippocampus.

Development of novel disease-modifying treatment strategies for neurological disorders, which at present have no cure, represents a major challenge for today's neurology. Translation of findings from animal models to humans represents an unresolved gap in most of the preclinical studies. Gene therapy is an evolving innovative approach that may prove useful for clinical applications. In animal models of temporal lobe epilepsy (TLE), gene therapy treatments based on viral vectors encoding NPY or galanin have been shown to effectively suppress seizures. However, how this translates to human TLE remains unknown. A unique possibility to validate these animal studies is provided by a surgical therapeutic approach, whereby resected epileptic tissue from temporal lobes of pharmacoresistant patients are available for neurophysiological studies in vitro. To test whether NPY and galanin have antiepileptic actions in human epileptic tissue as well, we applied these neuropeptides directly to human hippocampal slices in vitro. NPY strongly decreased stimulation-induced EPSPs in dentate gyrus and CA1 (up to 30 and 55%, respectively) via Y2 receptors, while galanin had no significant effect. Receptor autoradiographic binding revealed the presence of both NPY and galanin receptors, while functional receptor binding was only detected for NPY, suggesting that galanin receptor signaling may be impaired. These results underline the importance of validating findings from animal studies in human brain tissue, and advocate for NPY as a more appropriate candidate than galanin for future gene therapy trials in pharmacoresistant TLE patients.

4'-O-methylhonokiol increases levels of 2-arachidonoyl glycerol in mouse brain via selective inhibition of its COX-2-mediated oxygenation.

BACKGROUND AND PURPOSE: 4'-O-methylhonokiol (MH) is a natural product showing anti-inflammatory, anti-osteoclastogenic, and neuroprotective effects. MH was reported to modulate cannabinoid CB2 receptors as an inverse agonist for cAMP production and an agonist for intracellular [Ca2+]. It was recently shown that MH inhibits cAMP formation via CB2 receptors. In this study, the exact modulation of MH on CB2 receptor activity was elucidated and its endocannabinoid substrate-specific inhibition (SSI) of cyclooxygenase-2 (COX-2) and CNS bioavailability are described for the first time. METHODS: CB2 receptor modulation ([35S]GTPγS, cAMP, and ß-arrestin) by MH was measured in hCB2-transfected CHO-K1 cells and native conditions (HL60 cells and mouse spleen). The COX-2 SSI was investigated in RAW264.7 cells and in Swiss albino mice by targeted metabolomics using LC-MS/MS. RESULTS: MH is a CB2 receptor agonist and a potent COX-2 SSI. It induced partial agonism in both the [35S]GTPγS binding and ß-arrestin recruitment assays while being a full agonist in the cAMP pathway. MH selectively inhibited PGE2 glycerol ester formation (over PGE2) in RAW264.7 cells and significantly increased the levels of 2-AG in mouse brain in a dose-dependent manner (3 to 20 mg kg(-1)) without affecting other metabolites. After 7 h from intraperitoneal (i.p.) injection, MH was quantified in significant amounts in the brain (corresponding to 200 to 300 nM). CONCLUSIONS: LC-MS/MS quantification shows that MH is bioavailable to the brain and under condition of inflammation exerts significant indirect effects on 2-AG levels. The biphenyl scaffold might serve as valuable source of dual CB2 receptor modulators and COX-2 SSIs as demonstrated by additional MH analogs that show similar effects. The combination of CB2 agonism and COX-2 SSI offers a yet unexplored polypharmacology with expected synergistic effects in neuroinflammatory diseases, thus providing a rationale for the diverse neuroprotective effects reported for MH in animal models.

Stable isotope turnover and half-life in animal tissues: a literature synthesis.

Stable isotopes of carbon, nitrogen, and sulfur are used as ecological tracers for a variety of applications, such as studies of animal migrations, energy sources, and food web pathways. Yet uncertainty relating to the time period integrated by isotopic measurement of animal tissues can confound the interpretation of isotopic data. There have been a large number of experimental isotopic diet shift studies aimed at quantifying animal tissue isotopic turnover rate λ (%·day(-1), often expressed as isotopic half-life, ln(2)/λ, days). Yet no studies have evaluated or summarized the many individual half-life estimates in an effort to both seek broad-scale patterns and characterize the degree of variability. Here, we collect previously published half-life estimates, examine how half-life is related to body size, and test for tissue- and taxa-varying allometric relationships. Half-life generally increases with animal body mass, and is longer in muscle and blood compared to plasma and internal organs. Half-life was longest in ecotherms, followed by mammals, and finally birds. For ectotherms, different taxa-tissue combinations had similar allometric slopes that generally matched predictions of metabolic theory. Half-life for ectotherms can be approximated as: ln (half-life) = 0.22*ln (body mass) + group-specific intercept; n = 261, p<0.0001, r2 = 0.63. For endothermic groups, relationships with body mass were weak and model slopes and intercepts were heterogeneous. While isotopic half-life can be approximated using simple allometric relationships for some taxa and tissue types, there is also a high degree of unexplained variation in our models. Our study highlights several strong and general patterns, though accurate prediction of isotopic half-life from readily available variables such as animal body mass remains elusive.

Inhibition of opioid receptor mediated G-protein activity after chronic administration of kynurenic acid and its derivative without direct binding to opioid receptors.

There is an increasing number of evidence showing analgesic properties of the kynurenic acid (KYNA), and also some studies demonstrate that kynurenine might interact with the opioid system. Therefore in this study, for the first time we investigated the direct binding affinity of KYNA and its structural analog KYNA-1 towards mu, kappa and delta opioid receptor in competition binding experiments applying opioid receptor specific radioligands. The binding affinity measurements were performed in Chinese hamster ovary cell lines overexpressing the corresponding opioid receptor (mu and kappa opioid receptor were rat, delta opioid receptor were mouse sequence). Additionally we also examined the chronic effect of these compounds on mu, kappa and delta opioid receptor and also nociceptin peptide receptor mediated G-protein activity in [(35)S]GTPγS binding assays performed in mouse cortex and striatum membranes. Our results showed that KYNA and KYNA-1 had no affinity towards any of the three classic opioid receptors. On the other hand the compounds significantly decreased opioid and nociceptin receptor mediated G-protein activity or in some cases enhanced the potency of the activating ligand. Moreover, the alterations were receptor and brain region specific. Accordingly, we conclude that KYNA and KYNA-1 do not interact directly with the opioid receptors, but more likely alter the receptor functions intracellularly.

Exploring pharmacological activities and signaling of morphinans substituted in position 6 as potent agonists interacting with the µ opioid receptor.

BACKGROUND: Opioid analgesics are the most effective drugs for the treatment of moderate to severe pain. However, they also produce several adverse effects that can complicate pain management. The µ opioid (MOP) receptor, a G protein-coupled receptor, is recognized as the opioid receptor type which primarily mediates the pharmacological actions of clinically used opioid agonists. The morphinan class of analgesics including morphine and oxycodone are of main importance as therapeutically valuable drugs. Though the natural alkaloid morphine contains a C-6-hydroxyl group and the semisynthetic derivative oxycodone has a 6-carbonyl function, chemical approaches have uncovered that functionalizing position 6 gives rise to a range of diverse activities. Hence, position 6 of N-methylmorphinans is one of the most manipulated sites, and is established to play a key role in ligand binding at the MOP receptor, efficacy, signaling, and analgesic potency. We have earlier reported on a chemically innovative modification in oxycodone resulting in novel morphinans with 6-acrylonitrile incorporated substructures. RESULTS: This study describes in vitro and in vivo pharmacological activities and signaling of new morphinans substituted in position 6 with acrylonitrile and amido functions as potent agonists and antinociceptive agents interacting with MOP receptors. We show that the presence of a 6-cyano group in N-methylmorphinans has a strong influence on the binding to the opioid receptors and post-receptor signaling. One 6-cyano-N-methylmorphinan of the series was identified as the highest affinity and most selective MOP agonist, and very potent in stimulating G protein coupling and intracellular calcium release through the MOP receptor. In vivo, this MOP agonist showed to be greatly effective against thermal and chemical nociception in mice with marked increased antinociceptive potency than the lead molecule oxycodone. CONCLUSION: Development of such novel chemotypes by targeting position 6 provides valuable insights on ligand-receptor interaction and molecular mode of action, and may aid in identification of opioid therapeutics with enhanced analgesic properties and fewer undesirable effects.

Pharmacological properties of AC-3933, a novel benzodiazepine receptor partial inverse agonist.

We investigated in this study the pharmacological properties of AC-3933 (5-(3-methoxyphenyl)-3-(5-methyl-1,2,4-oxadiazol-3-yl)-1,6-naphthyridin-2(1H)-one), a novel benzodiazepine receptor (BzR) partial inverse agonist. AC-3933 potently inhibited [3H]-flumazenil binding to rat whole brain membrane with a Ki value of 5.15 ± 0.39 nM and a GABA ratio of 0.84 ± 0.03. AC-3933 exhibited almost no affinity for the other receptors, transporters and ion channels used in this study. In addition, AC-3933, in the presence of GABA (1 µM), gradually but significantly increased [³5S] tert-butylbicyclophosphorothionate binding to rat cortical membrane to 117.1% of the control (maximum increase ratio) at 3000 nM. However, this increase reached a plateau at 30 nM with hardly any change at a concentration range of 100-3000 nM (from 115.2% to 117.1%). AC-3933 (0.1-10 µM) significantly enhanced KCl-evoked acetylcholine (ACh) release from rat hippocampal slices in a concentration-dependent manner. Moreover, in vivo brain microdialysis showed that intragastric administration of AC-3933 at the dose of 10 mg/kg significantly increased extracellular ACh levels in the hippocampus of freely moving rats (area under the curve (AUC0₋2 h) of ACh level; 288.3% of baseline). These results indicate that AC-3933, a potent and selective BzR inverse agonist with low intrinsic activity, might be useful in the treatment of cognitive disorders associated with degeneration of the cholinergic system.

Inflammation enhances Y1 receptor signaling, neuropeptide Y-mediated inhibition of hyperalgesia, and substance P release from primary afferent neurons.

Neuropeptide Y (NPY) is present in the superficial laminae of the dorsal horn and inhibits spinal nociceptive processing, but the mechanisms underlying its anti-hyperalgesic actions are unclear. We hypothesized that NPY acts at neuropeptide Y1 receptors in the dorsal horn to decrease nociception by inhibiting substance P (SP) release, and that these effects are enhanced by inflammation. To evaluate SP release, we used microdialysis and neurokinin 1 receptor (NK1R) internalization in rat. NPY decreased capsaicin-evoked SP-like immunoreactivity in the microdialysate of the dorsal horn. NPY also decreased non-noxious stimulus (paw brush)-evoked NK1R internalization (as well as mechanical hyperalgesia and mechanical and cold allodynia) after intraplantar injection of carrageenan. Similarly, in rat spinal cord slices with dorsal root attached, [Leu(31), Pro(34)]-NPY inhibited dorsal root stimulus-evoked NK1R internalization. In rat dorsal root ganglion neurons, Y1 receptors colocalized extensively with calcitonin gene-related peptide (CGRP). In dorsal horn neurons, Y1 receptors were extensively expressed and this may have masked the detection of terminal co-localization with CGRP or SP. To determine whether the pain inhibitory actions of Y1 receptors are enhanced by inflammation, we administered [Leu(31), Pro(34)]-NPY after intraplantar injection of complete Freund's adjuvant (CFA) in rat. We found that [Leu(31), Pro(34)]-NPY reduced paw clamp-induced NK1R internalization in CFA rats but not uninjured controls. To determine the contribution of increased Y1 receptor-G protein coupling, we measured [(35)S]GTPγS binding simulated by [Leu(31), Pro(34)]-NPY in mouse dorsal horn. CFA inflammation increased the affinity of Y1 receptor G-protein coupling. We conclude that Y1 receptors contribute to the anti-hyperalgesic effects of NPY by mediating the inhibition of SP release, and that Y1 receptor signaling in the dorsal horn is enhanced during inflammatory nociception.

Cannabinoids in disguise: Δ9-tetrahydrocannabinol-like effects of tetramethylcyclopropyl ketone indoles.

Synthetic indole-derived cannabinoids have become commonly used recreational drugs and continue to be abused despite their adverse consequences. As compounds that were identified early in the epidemic (e.g., naphthoylindoles) have become legally banned, new compounds have appeared on the drug market. Two tetramethylcyclopropyl ketone indoles, UR-144 [(1-pentyl-1H-indol-3-yl)-(2,2,3,3-tetramethylcyclopropyl)methanone] and XLR-11 [(1-(5-fluoropentyl)-1H-indol-3-yl)-(2,2,3,3-tetramethylcyclopropyl)methanone], recently have been identified in confiscated products. These compounds are structurally related to a series of CB2-selective compounds explored by Abbott Labs. The purpose of the present study was to evaluate the extent to which UR-144 and XLR-11 shared cannabinoid effects with Δ9-tetrahydrocannabinol (Δ9-THC). Indices of in vitro and in vivo activity at cannabinoid receptors were assessed. Similar to other psychoactive cannabinoid agonists, XLR-11 and UR-144 showed low nanomolar (<30) affinity for CB1 and CB2 receptors, activated these receptors as full agonists, and produced dose-dependent effects that were blocked by rimonabant in mice, including antinociception, hypothermia, catalepsy and suppression of locomotor activity. The potency of both compounds was several-fold greater than Δ9-THC. XLR-11 and UR-144 also substituted for Δ9-THC in a Δ9-THC discrimination procedure in mice, effects that were attenuated by rimonabant. Analysis of urine from mice treated with the compounds revealed that both were extensively metabolized, with predominant urinary excretion as glucuronide conjugates. Together, these results demonstrate that UR-144 and XLR-11 share a pharmacological profile of in vitro and in vivo effects with Δ9-THC and other abused indole-derived cannabinoids and would be predicted to produce Δ9-THC-like subjective effects in humans.

GABAB receptor activation attenuates the stimulant but not mesolimbic dopamine response to ethanol in FAST mice.

Neural processes influenced by γ-aminobutyric acid B (GABA(B)) receptors appear to contribute to acute ethanol sensitivity, including the difference between lines of mice bred for extreme sensitivity (FAST) or insensitivity (SLOW) to the locomotor stimulant effect of ethanol. One goal of the current study was to determine whether selection of the FAST and SLOW lines resulted in changes in GABA(B) receptor function, since the lines differ in sensitivity to the GABA(B) receptor agonist baclofen and baclofen attenuates the stimulant response to ethanol in FAST mice. A second goal was to determine whether the baclofen-induced reduction in ethanol stimulation in FAST mice is associated with an attenuation of the mesolimbic dopamine response to ethanol. In Experiment 1, the FAST and SLOW lines were found to not differ in GABA(B) receptor function (measured by baclofen-stimulated [(35)S]GTPγS binding) in whole brain or in several regional preparations, except in the striatum in one of the two replicate sets of selected lines. In Experiment 2, baclofen-induced attenuation of the locomotor stimulant response to ethanol in FAST mice was not accompanied by a reduction in dopamine levels in the nucleus accumbens, as measured by microdialysis. These data suggest that, overall, GABA(B) receptor function does not play an integral role in the genetic difference in ethanol sensitivity between the FAST and SLOW lines. Further, although GABA(B) receptors do modulate the locomotor stimulant response to ethanol in FAST mice, this effect does not appear to be due to a reduction in tonic dopamine signaling in the nucleus accumbens.

Application of in vitro [³5S]GTPγ-S autoradiography in studies of growth hormone effects on opioid receptors in the male rat brain.

Chronic treatment with opiates may inhibit cell growth and trigger apoptosis. On the contrary, growth hormone (GH) has been demonstrated to stimulate neurogenesis and counteract apoptosis. We recently demonstrated that recombinant human GH (rhGH) may reverse opiate-induced apoptosis in cells derived from prenatal mouse hippocampus. Thus, GH might be able to prevent the impaired cognitive capabilities that may occur in both humans and other mammals in connection to chronic opiate treatment. In order to explore the mechanism by which GH exerts its beneficial effects we here examined the impact of GH treatment on the levels of delta and mu opioid peptide (DOP and MOP, respectively) receptors in the male rat brain. The rats were treated with rhGH (Genotropin®) at two different doses (0.07 and 0.7 IU/kg), twice daily, during 7 days. Following decapitation, the levels of DOP and MOP receptor functionality were determined using [³5S]GTPγS autoradiography. The results demonstrate that rhGH affects the levels of the MOP receptor functionality in certain areas of the brain. These alterations were seen in e.g. amygdala and thalamus, i.e. regions that recently have been implicated in learning and memory. The activity level of DOP receptors was not affected. Thus, the data support that the beneficial effect of GH on counteracting apoptosis might involve a direct or indirect effect on the MOP but not the DOP receptor.

Statistical parametric mapping reveals regional alterations in cannabinoid CB1 receptor distribution and G-protein activation in the 3D reconstructed epileptic rat brain.

PURPOSE: The endocannabinoid system is known to modulate seizure activity in several in vivo and in vitro models, and CB(1) -receptor activation is anticonvulsant in the rat pilocarpine model of acquired epilepsy (AE). In these epileptic rats, a unique redistribution of the CB(1) receptor occurs within the hippocampus; however, an anatomically inclusive analysis of the effect of status epilepticus (SE)-induced AE on CB(1) receptors has not been thoroughly evaluated. Therefore, statistical parametric mapping (SPM), a whole-brain unbiased approach, was used to study the long-term effect of pilocarpine-induced SE on CB(1) -receptor binding and G-protein activation in rats with AE. METHODS: Serial coronal sections from control and epileptic rats were cut at equal intervals throughout the neuraxis and processed for [(3) H]WIN55,212-2 (WIN) autoradiography, WIN-stimulated [(35) S]GTPγS autoradiography, and CB(1) -receptor immunohistochemistry (IHC). The autoradiographic techniques were evaluated with both region of interest (ROI) and SPM analyses. KEY FINDINGS: In rats with AE, regionally specific increases in CB(1) -receptor binding and activity were detected in cortex, discrete thalamic nuclei, and other regions including caudate-putamen and septum, and confirmed by IHC. However, CB(1) receptors were unaltered in several brain regions, including substantia nigra and cerebellum, and did not exhibit regional decreases in rats with AE. SIGNIFICANCE: This study provides the first comprehensive evaluation of the regional distribution of changes in CB(1) -receptor expression, binding, and G-protein activation in the rat pilocarpine model of AE. These regions may ultimately serve as targets for cannabinomimetic compounds or manipulation of the endocannabinoid system in epileptic brain.

The presumed atypical chemokine receptor CXCR7 signals through G(i/o) proteins in primary rodent astrocytes and human glioma cells.

SDF-1/CXCL12 binds to the chemokine receptors, CXCR4 and CXCR7, and controls cell proliferation and migration during development, tumorigenesis, and inflammatory processes. It is currently assumed that CXCR7 would represent an atypical or scavenger chemokine receptor which modulates the function of CXCR4. Contrasting this view, we demonstrated recently that CXCR7 actively mediates SDF-1 signaling in primary astrocytes. Here, we provide evidence that CXCR7 affects astrocytic cell signaling and function through pertussis toxin-sensitive G(i/o) proteins. SDF-1-dependent activation of G(i/o) proteins and subsequent increases in intracellular Ca(2+) concentration persisted in primary rodent astrocytes with depleted expression of CXCR4, but were abolished in astrocytes with depleted expression of CXCR7. Moreover, CXCR7-mediated effects of SDF-1 on Erk and Akt signaling as well as on astrocytic proliferation and migration were all sensitive to pertussis toxin. Likewise, pertussis toxin abolished SDF-1-induced activation of Erk and Akt in CXCR7-only expressing human glioma cell lines. Finally, consistent with a ligand-biased function of CXCR7 in astrocytes, the alternate CXCR7 ligand, I-TAC/CXCL11, activated Erk and Akt through ß-arrestin. The demonstration that SDF-1-bound CXCR7 activates G(i/o) proteins in astrocytes could help to explain some discrepancies previously observed for the function of CXCR4 and CXCR7 in other cell types.

Effect of ΔFosB overexpression on opioid and cannabinoid receptor-mediated signaling in the nucleus accumbens.

The stable transcription factor ΔFosB is induced in the nucleus accumbens (NAc) by chronic exposure to several drugs of abuse, and transgenic expression of ΔFosB in the striatum enhances the rewarding properties of morphine and cocaine. However, the mechanistic basis for these observations is incompletely understood. We used a bitransgenic mouse model with inducible expression of ΔFosB in dopamine D(1) receptor/dynorphin-containing striatal neurons to determine the effect of ΔFosB expression on opioid and cannabinoid receptor signaling in the NAc. Results showed that mu opioid-mediated G-protein activity and inhibition of adenylyl cyclase were enhanced in the NAc of mice that expressed ΔFosB. Similarly, kappa opioid inhibition of adenylyl cyclase was enhanced in the ΔFosB expressing mice. In contrast, cannabinoid receptor-mediated signaling did not differ between mice overexpressing ΔFosB and control mice. These findings suggest that opioid and cannabinoid receptor signaling are differentially modulated by expression of ΔFosB, and indicate that ΔFosB expression might produce some of its effects via enhanced mu and kappa opioid receptor signaling in the NAc.