Deep brain stimulation of the periaqueductal gray releases endogenous opioids in humans.

Deep brain stimulation (DBS) of the periaqueductal gray (PAG) is used in the treatment of severe refractory neuropathic pain. We tested the hypothesis that DBS releases endogenous opioids to exert its analgesic effect using [11C]diprenorphine (DPN) positron emission tomography (PET). Patients with de-afferentation pain (phantom limb pain or Anaesthesia Dolorosa (n=5)) who obtained long-lasting analgesic benefit from DBS were recruited. [11C]DPN and [15O]water PET scanning was performed in consecutive sessions; first without, and then with PAG stimulation. The regional cerebral tracer distribution and kinetics were quantified for the whole brain and brainstem. Analysis was performed on a voxel-wise basis using statistical parametric mapping (SPM) and also within brainstem regions of interest and correlated to the DBS-induced improvement in pain score and mood. Brain-wide analysis identified a single cluster of reduced [11C]DPN binding (15.5% reduction) in the caudal, dorsal PAG following DBS from effective electrodes located in rostral dorsal/lateral PAG. There was no evidence for an accompanying focal change in blood flow within the PAG. No correlation was found between the change in PAG [11C]DPN binding and the analgesic effect or the effect on mood (POMSSV) of DBS. The analgesic effect of DBS in these subjects was not altered by systemic administration of the opioid antagonist naloxone (400ug). These findings indicate that DBS of the PAG does indeed release endogenous opioid peptides focally within the midbrain of these neuropathic pain patients but we are unable to further resolve the question of whether this release is responsible for the observed analgesic benefit.

Differential control of opioid antinociception to thermal stimuli in a knock-in mouse expressing regulator of G-protein signaling-insensitive Gαo protein.

Regulator of G-protein signaling (RGS) proteins classically function as negative modulators of G-protein-coupled receptor signaling. In vitro, RGS proteins have been shown to inhibit signaling by agonists at the µ-opioid receptor, including morphine. The goal of the present study was to evaluate the contribution of endogenous RGS proteins to the antinociceptive effects of morphine and other opioid agonists. To do this, a knock-in mouse that expresses an RGS-insensitive (RGSi) mutant Gαo protein, Gαo(G184S) (Gαo RGSi), was evaluated for morphine or methadone antinociception in response to noxious thermal stimuli. Mice expressing Gαo RGSi subunits exhibited a naltrexone-sensitive enhancement of baseline latency in both the hot-plate and warm-water tail-withdrawal tests. In the hot-plate test, a measure of supraspinal nociception, morphine antinociception was increased, and this was associated with an increased ability of opioids to inhibit presynaptic GABA neurotransmission in the periaqueductal gray. In contrast, antinociception produced by either morphine or methadone was reduced in the tail-withdrawal test, a measure of spinal nociception. In whole-brain and spinal cord homogenates from mice expressing Gαo RGSi subunits, there was a small loss of Gαo expression and an accompanying decrease in basal G-protein activity. Our results strongly support a role for RGS proteins as negative regulators of opioid supraspinal antinociception and also reveal a potential novel function of RGS proteins as positive regulators of opioid spinal antinociceptive pathways.

Quantification of opioid receptor availability following spontaneous epileptic seizures: correction of [11C]diprenorphine PET data for the partial-volume effect.

Previous positron emission tomography (PET) studies in refractory temporal lobe epilepsy (TLE) using the non-selective opioid receptor antagonist [(11)C]diprenorphine (DPN) did not detect any changes in mesial temporal structures, despite known involvement of the hippocampus in seizure generation. Normal binding in smaller hippocampi is suggestive of increased receptor concentration in the remaining grey matter. Correction for partial-volume effect (PVE) has not been used in previous DPN PET studies. Here, we present PVE-corrected DPN-PET data quantifying post-ictal and interictal opioid receptor availability in humans with mTLE. Eight paired datasets of post-ictal and interictal DPN PET scans and eleven test/retest control datasets were available from a previously published study on opioid receptor changes in TLE following seizures (Hammers et al., 2007a). Five of the eight participants with TLE had documented hippocampal sclerosis. Data were re-analyzed using regions of interest and a novel PVE correction method (structural functional synergistic-resolution recovery (SFS-RR); (Shidahara et al., 2012)). Data were denoised, followed by application of SFS-RR, with anatomical information derived via precise anatomical segmentation of the participants' MRI (MAPER; (Heckemann et al., 2010)). [(11)C]diprenorphine volume-of-distribution (VT) was quantified in six regions of interest. Post-ictal increases were observed in the ipsilateral fusiform gyri and lateral temporal pole. A novel finding was a post-ictal increase in [(11)C]DPN VT relative to the interictal state in the ipsilateral parahippocampal gyrus, not observed in uncorrected datasets. As for voxel-based (SPM) analyses, correction for global VT values was essential in order to demonstrate focal post-ictal increases in [(11)C]DPN VT. This study provides further direct human in vivo evidence for changes in opioid receptor availability in TLE following seizures, including changes that were not evident without PVE correction. Denoising, resolution recovery and precise anatomical segmentation can extract valuable information from PET studies that would be missed with conventional post-processing procedures.

RACK1 identified as the PCBP1-interacting protein with a novel functional role on the regulation of human MOR gene expression.

Poly C binding protein 1 (PCBP1) is an expressional regulator of the mu-opioid receptor (MOR) gene. We hypothesized the existence of a PCBP1 co-regulator modifying human MOR gene expression by protein-protein interaction with PCBP1. A human brain cDNA library was screened using the two-hybrid system with PCBP1 as the bait. Receptor for activated protein kinase C (RACK1) protein, containing seven WD domains, was identified. PCBP1-RACK1 interaction was confirmed via in vivo validation using the two-hybrid system, and by co-immunoprecipitation with anti-PCBP1 antibody and human neuronal NMB cell lysate, endogenously expressing PCBP1 and RACK1. Further co-immunoprecipitation suggested that RACK1-PCBP1 interaction occurred in cytosol alone. Single and serial WD domain deletion analyses demonstrated that WD7 of RACK1 is the key domain interacting with PCBP1. RACK1 over-expression resulted in a dose-dependent decrease of MOR promoter activity using p357 plasmid containing human MOR promoter and luciferase reporter gene. Knock-down analysis showed that RACK1 siRNA decreased the endogenous RACK1 mRNA level in NMB, and elevated MOR mRNA level as indicated by RT-PCR. Likewise, a decrease of RACK1 resulted in an increase of MOR proteins, verified by (3) H-diprenorphine binding assay. Collectively, this study reports a novel role of RACK1, physically interacting with PCBP1 and participating in the regulation of human MOR gene expression in neuronal NMB cells.

Betulinic acid, derived from the desert lavender Hyptis emoryi, attenuates paclitaxel-, HIV-, and nerve injury-associated peripheral sensory neuropathy via block of N- and T-type calcium channels.

The Federal Pain Research Strategy recommended development of nonopioid analgesics as a top priority in its strategic plan to address the significant public health crisis and individual burden of chronic pain faced by >100 million Americans. Motivated by this challenge, a natural product extracts library was screened and identified a plant extract that targets activity of voltage-gated calcium channels. This profile is of interest as a potential treatment for neuropathic pain. The active extract derived from the desert lavender plant native to southwestern United States, when subjected to bioassay-guided fractionation, afforded 3 compounds identified as pentacyclic triterpenoids, betulinic acid (BA), oleanolic acid, and ursolic acid. Betulinic acid inhibited depolarization-evoked calcium influx in dorsal root ganglion (DRG) neurons predominantly through targeting low-voltage-gated (Cav3 or T-type) and CaV2.2 (N-type) calcium channels. Voltage-clamp electrophysiology experiments revealed a reduction of Ca, but not Na, currents in sensory neurons after BA exposure. Betulinic acid inhibited spontaneous excitatory postsynaptic currents and depolarization-evoked release of calcitonin gene-related peptide from lumbar spinal cord slices. Notably, BA did not engage human mu, delta, or kappa opioid receptors. Intrathecal administration of BA reversed mechanical allodynia in rat models of chemotherapy-induced peripheral neuropathy and HIV-associated peripheral sensory neuropathy as well as a mouse model of partial sciatic nerve ligation without effects on locomotion. The broad-spectrum biological and medicinal properties reported, including anti-HIV and anticancer activities of BA and its derivatives, position this plant-derived small molecule natural product as a potential nonopioid therapy for management of chronic pain.

Post-endocytic fates of delta-opioid receptor are regulated by GRK2-mediated receptor phosphorylation and distinct beta-arrestin isoforms.

Once internalized, some G protein-coupled receptors (GPCRs) can recycle back to the cell surface, while some of them are delivered to lysosomes for degradation. Because recycling and degradation represent two opposing receptor fates, understanding the mechanisms that determine post-endocytic fate of GPCRs is of great importance. Our recent work has verified that agonist-induced internalization of delta-opioid receptor (DOR) employs both phosphorylation-dependent and -independent mechanisms in HEK293 cells. To investigate whether these two internalization mechanisms work differently in receptor regulation, we monitored receptor post-endocytic fates using flow cytometry, surface receptor biotinylation and radioligand binding assays. Results showed that the internalized wild type DOR could either recycle to the cell surface or be degraded. Mutant DOR M4/5/6, which lacks all three G protein-coupled receptor kinase 2 (GRK2) phosphorylation sites, could also internalize upon agonist challenge although in a reduced level as compared with the wild type counterpart. However, the internalized mutant DOR could not recycle back to the cell surface and all mutant DOR was degraded after internalization. Inhibition of GRK2 expression by GRK2 RNAi also strongly attenuated recycling of DOR. Furthermore, overexpression of GRK2, which significantly increased receptor phosphorylation and internalization, also targeted more internalized receptors to the recycling pathway. These data suggest that GRK2-catalyzed receptor phosphorylation is critically involved in DOR internalization and recycling, and the phosphorylation-independent internalization leads to receptor degradation. Data obtained from beta-arrestin1 and beta-arrestin2 RNAi experiments indicated that both beta-arrestin1 and beta-arrestin2 participate in phosphorylation-dependent internalization and the subsequent recycling of DOR. However, phosphorylation-independent internalization and degradation of DOR were strongly blocked by beta-arrestin2 RNAi, but not beta-arrestin1 RNAi. Taken together, these data demonstrate for the first time that GRK2 phosphorylation-dependent internalization mediated by both beta-arrestin1 and beta-arrestin2 leads DOR to recycle, whereas GRK2-independent internalization mediated by beta-arrestin2 alone leads to receptor degradation. Thus, the post-endocytic fate of internalized DOR can be regulated by GRK2-catalyzed receptor phosphorylation as well as distinct beta-arrestin isoforms.

A combined [11C]diprenorphine PET study and fMRI study of acupuncture analgesia.

Functional neuroimaging studies suggest that a lateral network in the brain is associated with the sensory aspects of pain perception while a medial network is associated with affective aspects. The highest concentration of opioid receptors is in the medial network. There is significant evidence that endogenous opioids are central to the experience of pain and analgesia. We applied an integrative multimodal imaging approach during acupuncture. We found functional magnetic resonance imaging signal changes in the orbitofrontal cortex, insula, and pons and [11C]diprenorphine positron emission tomography signal changes in the orbitofrontal cortex, medial prefrontal cortex, insula, thalamus, and anterior cingulate cortex. These findings include brain regions within both the lateral and medial pain networks.

Residues W320 and Y328 within the binding site of the µ-opioid receptor influence opiate ligand bias.

The development of G protein-biased agonists for the µ-opioid receptor (MOR) offers a clear drug discovery rationale for improved analgesia and reduced side-effects of opiate pharmacotherapy. However, our understanding of the molecular mechanisms governing ligand bias is limited, which hinders our ability to rationally design biased compounds. We have investigated the role of MOR binding site residues W320 and Y328 in controlling bias, by receptor mutagenesis. The pharmacology of a panel of ligands in a cAMP and a ß-arrestin2 assay were compared between the wildtype and mutated receptors, with bias factors calculated by operational analysis using ΔΔlog(τ/KA) values. [3H]diprenorphine competition binding was used to estimate affinity changes. Introducing the mutations W320A and Y328F caused changes in pathway bias, with different patterns of change between ligands. For example, DAMGO increased relative ß-arrestin2 activity at the W320A mutant, whilst its ß-arrestin2 response was completely lost at Y328F. In contrast, endomorphin-1 gained activity with Y328F but lost activity at W320A, in both pathways. For endomorphin-2 there was a directional shift from cAMP bias at the wildtype towards more ß-arrestin2 bias at W320A. We also observe clear uncoupling between mutation-driven changes in function and binding affinity. These findings suggest that the mutations influenced the balance of pathway activation in a ligand-specific manner, thus identifying residues in the MOR binding pocket that govern ligand bias. This increases our understanding of how ligand/receptor binding interactions can be translated into agonist-specific pathway activation.

Gender dependent rate of metabolism of the opioid receptor-PET ligand [18F]fluoroethyldiprenorphine.

AIM: The morphinane-derivate 6-O-(2-[(18)F]fluoroethyl)-6-O-desmethyldiprenorphine ([(18)F]FDPN) is a nonselective opioid receptor ligand currently used in positron emission tomography (PET). Correction for plasma metabolites of the arterial input function is necessary for quantitative measurements of [(18)F]FDPN binding. A study was undertaken to investigate if there are gender dependent differences in the rate of metabolism of [(18)F]FDPN. METHODS: The rate of metabolism of [(18)F]FDPN was mathematically quantified by fitting a bi-exponential function to each individual's dynamic metabolite data. RESULTS: No statistically significant gender differences were found for age, weight, body mass index or dose. However, significant differences (p < 0.01) in two of the four kinetic parameters describing the rate of metabolism were found between the two groups, with women metabolizing [(18)F]FDPN faster than men. These differences were found in the contribution of the fast and slow kinetic components of the model describing the distribution of radioactive species in plasma, indicating a higher rate of enzyme-dependent degradation of [(18)F]FDPN in women than in men. CONCLUSION: The findings reinforce the need for individualized metabolite correction during [(18)F]FDPN-PET scans and also indicate that in certain cases, grouping according to gender could be performed in order to minimize methodological errors of the input function prior to kinetic analyses.

Striatal opioid receptor availability is related to acute and chronic pain perception in arthritis: does opioid adaptation increase resilience to chronic pain?

The experience of pain in humans is modulated by endogenous opioids, but it is largely unknown how the opioid system adapts to chronic pain states. Animal models of chronic pain point to upregulation of opioid receptors (OpR) in the brain, with unknown functional significance. We sought evidence for a similar relationship between chronic pain and OpR availability in humans. Using positron emission tomography and the radiotracer (11)C-diprenorphine, patients with arthritis pain (n = 17) and healthy controls (n = 9) underwent whole-brain positron emission tomography scanning to calculate parametric maps of OpR availability. Consistent with the upregulation hypothesis, within the arthritis group, greater OpR availability was found in the striatum (including the caudate) of patients reporting higher levels of recent chronic pain, as well as regions of interest in the descending opioidergic pathway including the anterior cingulate cortex, thalamus, and periaqueductal gray. The functional significance of striatal changes were clarified with respect to acute pain thresholds: data across patients and controls revealed that striatal OpR availability was related to reduced pain perception. These findings are consistent with the view that chronic pain may upregulate OpR availability to dampen pain. Finally, patients with arthritis pain, compared with healthy controls, had overall less OpR availability within the striatum specifically, consistent with the greater endogenous opioid binding that would be expected in chronic pain states. Our observational evidence points to the need for further studies to establish the causal relationship between chronic pain states and OpR adaptation.

Compartmental analysis of diprenorphine binding to opiate receptors in the rat in vivo and its comparison with equilibrium data in vitro.

The regional binding of the opiate receptor ligand diprenorphine has been examined in rat brain both in vivo and in vitro. The time course of total label in specific brain regions was followed up to 2 h after intravenous bolus injection of [3H]diprenorphine, with or without a pulse chase of unlabelled diprenorphine at 30 min. In addition, total label was measured 30 min after injection of labelled diprenorphine at nontracer concentrations over a range of specific activities. Total data sets for each region were fitted simultaneously to a compartmental model to give estimates of maximal binding capacity (Bmax), the second-order apparent association rate constant, and the first-order dissociation rate constant of the receptor-ligand complex. The model incorporated the use of a reference region with low specific binding (cerebellum). The binding of diprenorphine to rat brain homogenates was measured in vitro under equilibrium conditions at 37 degrees C, pH 7.4, in the presence and absence of naloxone, to give corresponding regional estimates of Bmax and the half-saturation constant Kd. The results showed a close correlation between in vitro and in vivo regional estimates of Bmax over a wide range. There were no significant interregional differences either in Kd in vitro or in the Kd derived from the in vivo analysis, although in vitro and in vivo estimates differed by an order of magnitude. This work was carried out as part of a validation study with a view to the application of the compartmental model to data obtained in vivo in humans using positron emission tomography, when successive studies over a range of specific activities are not feasible.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantification of human opiate receptor concentration and affinity using high and low specific activity [11C]diprenorphine and positron emission tomography.

[11C]Diprenorphine, a weak partial opiate agonist, and positron emission tomography were used to obtain noninvasive regional estimates of opiate receptor concentration (Bmax) and affinity (Kd) in human brain. Different compartmental models and fitting strategies were compared statistically to establish the most reliable method of parameter estimation. Paired studies were performed in six normal subjects using high (769-5,920 Ci/mmol) and low (27-80 Ci/mmol) specific activity (SA) [11C]diprenorphine. Two subjects were studied a third time using high SA [11C]diprenorphine after a pretreatment with 1-1.5 mg/kg of the opiate antagonist naloxone. After the plasma radioactivity was corrected for metabolites, the brain data were analyzed using a three-compartment model and nonlinear least-squares curve fitting. Linear differential equations were used to describe the high SA (low receptor occupancy) kinetics. The k3/k4 ratio varied from 1.0 +/- 0.2 (occipital cortex) to 8.6 +/- 1.6 (thalamus). Nonlinear differential equations were used to describe the low SA (high receptor occupancy) kinetics and the curve fits provided the konf2 product. The measured free fraction of [11C]diprenorphine in plasma (f1) was 0.30 +/- 0.03, the average K1/k2 ratio from the two naloxone studies was 1.1 +/- 0.2, and the calculated free fraction of [11C]diprenorphine in the brain (f2) was 0.3. Using the paired SA studies, the estimated kinetic parameters, and f2, separate estimates of Bmax and Kd were obtained. Bmax varied from 2.3 +/- 0.5 (occipital cortex) to 20.6 +/- 7.3 (cingulate cortex) nM. The average Kd (eight brain regions) was 0.85 +/- 0.17 nM.

N-(3-[18F]fluoropropyl)-N-nordiprenorphine: synthesis and characterization of a new agent for imaging opioid receptors with positron emission tomography.

A series of N-fluoroalkyl (1-5) and N-alkyl (6-8) analogues of the high-affinity opioid receptor antagonist diprenorphine (9) has been synthesized and evaluated with in vitro binding assays. Three of the N-fluoroalkyl compounds were prepared with the positron-emitting radionuclide 18F (1a, 2a, 5a), and their biodistribution was determined in rats. Compounds 2a and 5a were made by using a two-step labeling procedure, [18F]fluoride displacement of an iodoalkyl triflate followed by N-alkylation, that required 2 h and proceeded in 4-6% overall radiochemical yield at the end of synthesis. The effective specific activity of compounds 2a and 5a, determined by competitive receptor binding assay, was 840-1820 Ci/mmol. Compound 1a was made by the same two-step procedure, with the bromoalkyl triflate, in 0.3-0.6% radiochemical yield at an effective specific activity of 106-264 Ci/mmol. Specificity of binding in vivo was measured as the percent injected dose/gram of striatal tissue divided by the percent injected dose/gram of cerebellar tissue. The best striatum to cerebellum ratio (3.32 +/- 0.74 at 30 min) was achieved with N-(3-[18F]-fluoropropyl)-N-nordiprenorphine (2a, [18F]FPND). The high specific binding demonstrated by this compound indicates that it may be useful for in vivo imaging of opioid receptors with positron emission tomography.

Endogenous opioids implicated in the dynamics of experimental drug addiction: an in vivo autoradiographic analysis.

Endogenous opioids have been implicated in the neurobiological mechanisms underlying drug addiction. Although some information is available concerning effects of abused drugs on the endogenous opioid systems, the interpretation of these effects is hampered because data on the actual changes in the endogenous opioids during the dynamics of the drug addiction are lacking. The present report deals with changes in endogenous opioid activity before and after the daily self-administration session in rats offered cocaine or ethanol, using an in vivo autoradiographic receptor occupancy procedure. In separate saline-controlled experiments drug-naive rats were allowed to intravenously self-administer cocaine (30 microg/infusion) and ethanol (0.05%) for five consecutive daily sessions of 6 h. Immediately following the last session on day 5 or just before a scheduled next daily session on day 6, the rats were injected with [3H]diprenorphine and subsequently prepared for autoradiography. Decreased [3H]diprenorphine binding was observed throughout the subcortical brain after the daily session in cocaine, but hardly in animals self-administering ethanol. These changes are thought to reflect a direct or an indirect effect of the drug on endogenous opioid systems. Before the daily session, the [3H]diprenorphine binding was decreased in restricted areas of the mesocorticolimbic system and of the thalamus in both cocaine and ethanol self-administering animals. These data suggest that release of endogenous opioids at the time the desire for cocaine or ethanol is high, which may be pertinent for drug-induced craving and relapse of drug addicts.

6-O-(2-[18F]fluoroethyl)-6-O-desmethyldiprenorphine ([18F]DPN): synthesis, biologic evaluation, and comparison with [11C]DPN in humans.

UNLABELLED: 6-O(2-[18F]fluoroethyl)-6- -desmethyldiprenorphine ([18F]DPN) was developed and biologically evaluated. Results of animal experiments, binding studies in vivo, and a human PET study are reported and compared with those of [11C]DPN. METHODS: [18F]DPN was obtained by 18F-fluoroethylation of 3-O-trityl-6-O-desmethyldiprenorphine and subsequent deprotection in good radiochemical yields (23% +/- 7%; 100 min; 37 TBq/mmol). Binding of [18F]DPN to mu, kappa, and delta opioid receptors was shown by autoradiography studies on rat brain slices. Quantification of cerebral opioid receptor binding in men was performed by spectral analysis of a dynamic PET scan (25 frames, 90 min) after intravenous application of 63 MBq [18F]DPN (36 GBq/micromol) and correction for metabolites. RESULTS: [18F]DPN shows high affinity to opioid receptors. Parametric images (impulse response function at 60 min) of this human study showed a binding pattern of [18F]DPN equal to that of a control group (n = 9 healthy volunteers) after administration of [11C]DPN. CONCLUSION: The advantage of the longer half-life of 18F will allow extended scanning periods, more flexible interventions (e.g., displacement studies), and DPN to be available to PET centers without an on-site cyclotron.

Sex-dependent differences in N-(3-[18F]fluoropropyl)-N-nordiprenorphine biodistribution and metabolism.

In our work with the new opioid receptor ligand, N-(3-[18F]fluoropropyl)-N-nordiprenorphine, ([18F]FPND), we have noted significant sex-dependent differences in metabolism and distribution. In female rats, metabolism of this ligand proceeds without significant P-450-mediated oxidation of the N-fluoroalkyl side chain, while in male rats, this is the dominant metabolic pathway. In biodistribution experiments, striatal uptake of this ligand is higher in female than in male rats, but no difference in cerebellar uptake is observed. In male rats, no metabolites of [18F]FPND are produced that cross the blood-brain barrier. In contrast, female rats produce a metabolite that both crosses the blood-brain barrier and exhibits opioid receptor-specific binding superior to that of the parent compound. These studies demonstrate that the possibility of sex-dependent metabolism must be considered when the rat is employed to screen new radiopharmaceuticals.

Inhibition of morphine withdrawal by the association of RB 101, an inhibitor of enkephalin catabolism, and the CCKB antagonist PD-134,308.

1. The effects induced in rats on naloxone-precipitated morphine withdrawal syndrome by the new mixed inhibitor of enkephalin catabolism able to cross the blood-brain barrier RB 101 (N-((R,S)-2-benzyl-3[(S)(2-amino-4-methylthio)butyl dithio]-1-ox-opropyl-L-phenylalanine benzyl ester) given alone or associated with the selective CCKB antagonist, PD-134,308, were investigated. 2. The systemic administration of RB 101 (5, 10 and 20 mg kg-1, i.v.) elicited a significant decrease in 8 of the 14 withdrawal signs evaluated. PD-134,308 (3 mg kg-1, i.p.) did not modify the expression of morphine abstinence when given alone, but induced a strong facilitation of RB 101 responses (12 of 14 withdrawal signs were decreased). This potentiation was particularly intense in peripherally mediated withdrawal signs. 3. In order to clarify the biochemical mechanisms implicated in these responses, the effects induced by the association of RB 101 and PD-134,308 on the occupation of brain opioid receptors by endogenous enkephalins were also investigated in mice. PD-134,308, as well as RB 101, inhibited [3H]-diprenorphine binding to opioid receptors. These results suggest that an increase in endogenous enkephalin levels induced by PD-134,308 could participate in the facilitation of RB 101 behavioural responses. 4. RB 101 has a promising potential role in the management of the opiate withdrawal syndrome. CCKB antagonists, such as PD-134,308 may be useful in potentiating this anti-withdrawal effect.

Human interleukin-2 could bind to opioid receptor and induce corresponding signal transduction.

Interleukin-2 (IL-2) was found to have an analgesic effect in the peripheral nervous system. Both electrophysiological and behavioural experiments suggested an anti-noceiceptive effect of IL-2 in animals. Biochemical experiments revealed that IL-2 could displace diprenorphine binding in both NG 108-15 cells and HEK 293 cells transfected with pCDNA3-DOR (delta opioid receptor). Furthermore, IL-2 significantly inhibited cAMP production stimulated by forskolin in both NG 108-15 and transfected HEK 293 cells, indicating that the binding of IL-2 to the delta opioid receptor is functional.

Quantitation of [11C]diprenorphine cerebral kinetics in man acquired by PET using presaturation, pulse-chase and tracer-only protocols.

The quantitation of regional cerebral in vivo opioid receptor rate constants using [11C]diprenorphine and positron emission tomography (PET) using 3 types of protocol (presaturation, pulse-chase naloxone displacement and tracer-only protocols) together with measurements of regional cerebral blood flow is described in normal volunteers. Arterial blood was sampled continuously for radioactivity and was corrected for metabolites and plasma/blood partition of radioactivity to provide a continuous plasma input function. A compartmental model involving 3 tissue compartments was used to describe the regional cerebral pharmacokinetics of the tracer. The compartments comprised: (1) free plus rapidly exchanging non-specifically bound ligand, (2) specifically bound, naloxone displaceable ligand, and (3) a kinetically distinguishable non-specifically bound pool. Regional estimates of fractional rate constants relating to specific binding were obtained using naloxone in a pulse-chase design of tracer displacement. Less precise estimates of these rate constraints were obtained from single-tracer-only studies, but when binding was expressed as the tissue total volume of distribution relative to plasma there was good correlation with regional values obtained from pulse-chase studies performed in the same individuals. The application of these protocols to the measurement of indices of regional-specific opioid receptor binding in the human brain is discussed.

Distinct changes in the behavioural effects of morphine and naloxone in CCK2 receptor-deficient mice.

The effects of morphine, mu-opioid receptor agonist, and naloxone, a non-selective opioid receptor antagonist, in the locomotor activity and place conditioning tests were studied in the CCK(2) receptor-deficient male mice. The exposure of mice to the motility boxes for 3 consecutive days induced a significant inhibition of locomotor activity in the wild-type (+/+) mice compared to homozygous (-/-) animals. The administration of naloxone (10 mg/kg i.p.) to animals, adapted to the motility boxes, induced a significant reduction of locomotor activity in the homozygous (-/-), but not in the wild-type (+/+) mice. Treatment of habituated mice with morphine (10 mg/kg i.p.) caused a stronger increase of locomotor activity in the wild-type (+/+) mice compared to the homozygous (-/-) littermates. In the place preference test the pairing of the preferred side with naloxone (1 and 10 mg/kg i.p.) induced a dose-dependent place aversion in the wild-type (+/+) mice. The treatment with naloxone was less effective in the homozygous (-/-) mice, because the high dose of naloxone (10 mg/kg) tended to shift the preference. The pairing of morphine (3 mg/kg i.p.) injections with the non-preferred side induced a significant place preference both in the wild-type (+/+) and homozygous (-/-) mice. The increased density of opioid receptors was established in the striatum of homozygous (-/-) mice, but not in the other forebrain structures. In conclusion, the targeted invalidation of CCK(2) receptors induces a dissociation of behavioural effects of morphine and naloxone. Morphine-induced place preference remained unchanged, whereas hyper-locomotion was less pronounced in the mutant mice compared to the wild-type (+/+) littermates. By contrast, naloxone-induced place aversion was weaker, but naloxone caused a stronger inhibition of locomotor activity in the homozygous (-/-) mice than in the wild-type (+/+) animals. These behavioural alterations can be explained in the light of data that the targeted mutation of CCK(2) receptors induces distinct changes in the properties of opioid receptors in various brain structures.