DAMGO Binding Research Articles

Novel Receptor-Binding-Based Assay for the Detection of Opioids in Human Urine Samples.

Consumption of opioids is a growing global health problem. The gold standard for drugs of abuse screening is immunochemical assays. However, this method comes with some disadvantages when screening for a wide variety of opioids. Detection of the binding of a compound at the human μ-opioid receptor (MOR) offers a promising alternative target. Here, we set up a urine assay to allow for detection of compounds that bind at the MOR, thus allowing the assay to be utilized as a screening tool for opioid intake. The assay is based on the incubation of MOR-containing cell membranes with the selective MOR-ligand DAMGO and urine. After filtration, the amount of DAMGO in the eluate is analyzed by liquid chromatography tandem mass spectroscopy (LC-MS/MS). The absence of DAMGO in the eluate corresponds to a competing MOR ligand in the urine sample, thus indicating opiate/opioid intake by the suspect. Sensitivity and specificity were determined by the analysis of 200 consecutive forensic routine casework urine samples. A pronounced displacement of DAMGO was observed in 29 of the 35 opiate/opioid-positive samples. Detection of fentanyl intake proved to be the most challenging aspect. Applying a cut-off value of, e.g., 10% DAMGO binding would lead to a sensitivity of 83% and a specificity of 95%. Consequently, the novel assay proved to be a promising screening tool for opiate/opioid presence in urine samples. The nontargeted approach and possible automation of the assay make it a promising alternative to conventional methods.

Biased Activation Mechanism Induced by GPCR Heterodimerization: Observations from μOR/δOR Dimers.

GPCRs regulate multiple intracellular signaling cascades. Biasedly activating one signaling pathway over the others provides additional clinical utility to optimize GPCR-based therapies. GPCR heterodimers possess different functions from their monomeric states, including their selectivity to different transducers. However, the biased signaling mechanism induced by the heterodimerization remains unclear. Motivated by the issue, we select an important GPCR heterodimer (μOR/δOR heterodimer) as a case and use microsecond Gaussian accelerated molecular dynamics simulation coupled with potential of mean force and protein structure network (PSN) to probe mechanisms regarding the heterodimerization-induced constitutive β-arrestin activity and efficacy change of the agonist DAMGO. The results show that only the lowest energy state of the μOR/δOR heterodimer, which adopts a slightly outward shift of TM6 and an ICL2 conformation close to the receptor core, can selectively accommodate β-arrestins. PSN further reveals important roles of H8, ICL1, and ICL2 in regulating the constitutive β-arrestin-biased activity for the apo μOR/δOR heterodimer. In addition, the heterodimerization can allosterically alter the binding mode of DAMGO mainly by means of W7.35. Consequently, DAMGO transmits the structural signal mainly through TM6 and TM7 in the dimer, rather than TM3 similar to the μOR monomer, thus changing the efficacy of DAMGO from a balanced agonist to the β-arrestin-biased one. On the other side, the binding of DAMGO to the heterodimer can stabilize μOR/δOR heterodimers through a stronger interaction of TM1/TM1 and H8/H8, accordingly enhancing the interaction of μOR with δOR and the binding affinity of the dimer to the β-arrestin. The agonist DAMGO does not change main compositions of the regulation network from the dimer interface to the transducer binding pocket of the μOR protomer, but induces an increase in the structural communication of the network, which should contribute to the enhanced β-arrestin coupling. Our observations, for the first time, reveal the molecular mechanism of the biased signaling induced by the heterodimerization for GPCRs, which should be beneficial to more comprehensively understand the GPCR bias signaling.

A novel receptor binding based assay to screen for opiates and (synthetic) opioids in human urine samples

Up to date there are, without knowledge of the compounds structure or utilization of antibodies, almost no comprehensive screening methods for the detection (synthetic) opioid uptake in urine samples available. Consequently, appearance of new opioids on the drug market require a tedious effort to update the present methods (e.g. ELISA or LC-MS/MS methods). Detection of the binding of a compound at the human μ-opioid receptor (MOR) offers a promising alternative analytical target as almost all of the opioids on the drug market take effect through binding at this receptor. Recently, we demonstrated that it is possible to accurately determine the binding of a compound (Ki value) at the MOR by LC MS/MS analysis rather than by radioactive detection methods. Here we set up the assay to allow for a detection of compounds that bind at the MOR in urine samples, thus allowing the assay to be utilised as a screening tool for opioid uptake. The assay is performed in a 96-well format and is based on the incubation of readily available cell membranes (prepared from CHO-K1 cells stably transfected with MOR) with the selective MOR-ligand DAMGO ([D-Ala2, N-MePhe4, Gly5-ol]-enkephalin) and 100 μL of urine for 60 minutes. After filtration and washing steps the amount of DAMGO in the eluate is analysed by a rapid and highly sensitive LC-MS/MS method on a QTRAP 6500+ system. Absence of DAMGO in the eluate corresponds to a competing MOR-ligand in the urine sample thus indicating opiate/opioid uptake by the suspect. Sensitivity and specificity was determined by the analysis 200 consecutive forensic routine casework urine samples (with known presence/absence of opiates and opioids determined by immunochemical assays, GC-MS and LC-Q-ToF screenings). DAMGO binding in the positive samples ranged from < 1% to 59% (Mean: 7%, Median: 3%). Twelve different opiates/opioids and/or there metabolites were present in 35 positive samples. A pronounced displacement (<10% DAMGO binding) was observed in 29 of the 35 samples. Detection of fentanyl uptake proved to be the most challenging analyte with 5 out of 10 positive samples showing an insufficient DAMGO displacement (14%–30% DAMGO binding, fentanyl concentration in these five sample: 1.0–5.7 ng/mL. Additionally, a urine sample containing 91 ng/mL tramadol and 42 ng/mL O-desmethyltramadol showed a poor displacement of DAMGO at the MOR. This can be explained by the low Ki values of the compounds at the MOR of 6014 and 40 nM, respectively. Among the nine false positive samples, no cluster of a particular compound was observed. Consequently, a cut-off value of 10% DAMGO binding would lead to a sensitivity of 83% and a specificity of 95% while a sensitivity of > 90% and a specificity of 88% would be achieved by applying a cut-off value of 17% DAMGO binding. The novel assay proved to be a suitable screening tool for opiate/opioid presence in urine samples. The non-targeted approach and possible automation of the assay make it a promising high throughput-capable alternative to conventional methods.

Pharmacological characterization of kratom alkaloids at opiate receptors: binding affinities, in vitro and in vivo functional assessments

Kratom (Mitragyna speciosa) is consumed by 10‐15 million individuals in the US for its mood‐elevating effects, as an alternative to traditional opiates for pain relief and to attenuate opiate withdrawal symptoms. Complex natural products, such as kratom, contain a variety of chemicals whose combined effects can only be understood by the meticulous identification and characterization of the individual chemicals and their effects in preclinical model systems. Twenty‐five indole and oxindole kratom alkaloids were evaluated for mu (MOR), delta (DOR) and kappa (KOR) opiate receptors binding affinities, opiate receptor mediated G‐protein activation, inhibition of forskolin stimulated cAMP accumulation and b‐arrestin2 recruitment. Several indole and oxindole alkaloids exhibited partial agonism at the MOR (7‐hydroxymitragynine, corynoxine A &gt; epiallo‐isopaynantheine, isospeciofoline, speciociliatine, corynoxine B &gt; mitragynine, 3‐epicorynoxine B &gt; paynantheine and speciogynine) – all of which also exhibited strong G protein bias. Mitraciliatine and isopaynantheine were weak MOR antagonists. Speciophylline exhibited positive allosteric modulatory activity at the MOR as evidenced by increased affinity of DAMGO binding and potentiation of [met]‐enkephalin‐induced inhibition of forskolin stimulated cAMP activity in the absence of intrinsic activity. Corynoxine A induced displayed similar affinity at the mu opiate receptor as 7 hydroxymitragynine but was less potent in the nociceptive hot plate assay and the mechanical allodynia von Frey test. Corynoxine did not significantly alter respiration or heart rate or induce a conditioned place preference. Results demonstrate the pharmacological complexity of kratom alkaloids and have important implications for determining the combined effects of less abundant indole and oxindole alkaloids with regard to the therapeutic as well as potential harmful effects of kratom.

Chronic aerobic exercise: Autoradiographic assessment of GABA(a) and mu-opioid receptor binding in adult rats

Exercise programs have shown great potential for both the prevention and treatment of substance use disorder (SUD). As exercise has been shown to have potent effects on physical and psychological health, it is reasonable to examine the mechanism of how exercise can be used as an adjunct treatment for addiction. The present study examined the effects of chronic aerobic (treadmill) exercise on both GABA(a) and mu-opioid receptor levels in the brains of male and female rats. GABA(a) receptor binding, measured by [3H] Flunitrazepam, was increased in the cingulate cortex following exercise, but only in females. Mu-opioid receptor expression, measured by [3H] ([D-Ala2, N-MePhe4, Gly-ol]-enkephalin) (DAMGO), showed no effect of exercise while showing an effect of sex, with increased [3H] DAMGO binding in the brains of sedentary males compared to that of sedentary females. Our findings support the potential role for GABA(a) signaling in the cingulate cortex as part of the mechanism of action of aerobic exercise. These data, along with prior reports, aid our understanding of the neurochemical impact and mechanism of chronic aerobic exercise on neuropsychiatric disease, particularly regarding addiction.

Methocinnamox Produces Sustained Antagonism of the Antinociceptive Effects of Morphine and Persistent Decreases in [3H]DAMGO Binding to μ Opioid Receptors in Rat Cortex

The opioid crisis continues in the US despite the availability of pharmacotherapies that are effective in some patients. A novel μ opioid receptor antagonist, methocinnamox (MCAM), has a long duration of action in blocking the effects of opioids, which could provide a substantial advantage over currently available medications, particularly for overdose. MCAM has been shown to attenuate the behavioral effects of μ, but not k, opioid receptor agonists, and this antagonism, which can last for several weeks, is thought to be due to pseudoirreversible binding of MCAM to μ opioid receptors. In the current study, a warm water tail withdrawal procedure was used to examine sustained antagonism by MCAM in one group of 8 rats. This study showed that 10 mg/kg MCAM attenuated the antinociceptive effects of morphine (1.78–17.8 mg/kg) 5 and 13 days after MCAM administration with antagonism beginning to wane 21 days after MCAM. In separate groups of 8 rats, that same dose of MCAM decreased [3H]DAMGO binding to μ opioid receptors in rat cortical homogenates measured 1, 5 and 21 days after MCAM injection, indicating that MCAM occupied μ opioid receptors at all time points tested. For example, maximal binding (Bmax) was 28.51 ± 3.33 and 30.96 ± 1.73 fmol/mg protein 5 and 21 days, respectively, after vehicle administration, and 16.03 ± 4.15 and 12.50 ± 1.27 fmol/mg protein 5 and 21 days, respectively, after administration of 10 mg/kg MCAM. Binding affinity (KD) was not markedly changed after MCAM administration. Thus, MCAM produces a sustained decrease in [3H]DAMGO binding to μ opioid receptors in rat cortex that parallels antagonism of the antinociceptive effects of μ opioid receptor agonists. This finding supports the notion that MCAM blocks the behavioral effects of μ opioid receptor agonists by binding pseudoirreversibly to these receptors. The unique binding profile of MCAM to μ opioid receptors could increase the usefulness of MCAM as a treatment for opioid abuse and overdose.Support or Funding InformationSupported by NIH (R01DA005018 and R01048417) and the Welch Foundation (AQ‐0039).

Modulation of μ-opioid receptor activation by acidic pH is dependent on ligand structure and an ionizable amino acid residue.

Background and PurposeAdverse side effects of conventional opioids can be avoided if ligands selectively activate peripheral opioid receptors in injured tissue. Injury and inflammation are typically accompanied by acidification. In this study, we examined influences of low pH and mutation of the ionizable amino acid residue H2976.52 on μ‐opioid receptor binding and signalling induced by the μ‐opioid receptor ligands fentanyl, DAMGO, and naloxone.Experimental ApproachHEK 293 cells stably transfected with μ‐opioid receptors were used to study opioid ligand binding, [35S]‐GTPγS binding, and cAMP reduction at physiological and acidic pH. We used μ‐opioid receptors mutated at H2976.52 to A (MOR‐H2976.52A) to delineate ligand‐specific interactions with H2976.52.Key ResultsLow pH and the mutant receptor MOR‐H2976.52A impaired naloxone binding and antagonism of cAMP reduction. In addition, DAMGO binding and G‐protein activation were decreased under these conditions. Fentanyl‐induced signalling was not influenced by pH and largely independent of H2976.52.Conclusions and ImplicationsOur investigations indicate that low pH selectively impairs μ‐opioid receptor signalling modulated by ligands capable of forming hydrogen bonds with H2976.52. We propose that protonation of H2976.52 at acidic pH reduces binding and subsequent signalling of such ligands. Novel agonists targeting opioid receptors in injured tissue might benefit from lack of hydrogen bond formation with H2976.52.

Roux-en-Y gastric bypass in rat reduces mu-opioid receptor levels in brain regions associated with stress and energy regulation.

Roux-en-Y gastric bypass surgery (RYGB) is the most common and effective weight loss procedure for severe obesity. However, a significant increase in addictive behaviors and new-onset substance use disorder (SUD) are sometimes observed post-surgery. The endogenous opioid system is known to play a major role in motivated behavior and reward, as well as the abuse of substances, including alcohol, tobacco, opioids and highly palatable foods. Here, we examined the effects of RYGB on mu-opioid receptor levels in the brain. Male Sprague-Dawley rats were assigned to one of four groups: standard diet with sham surgery (control), ad libitum high-energy high-fat (HF) diet with sham surgery, calorie restricted HF diet with sham surgery (Sham-FR), or HF diet with RYGB surgery. Control and HF groups were fed their respective diets for 8 weeks, with surgery performed on the eighth week. After 9 weeks on their respective diets post-surgery, animals were sacrificed for mu-opioid receptor autoradiography using the [3H] [D-Ala2,N-Me-Phe4-Gly5-ol]- enkephalin (DAMGO) ligand. Rats with RYGB showed reduced DAMGO binding in the central amygdala compared to sham-operated HF diet controls, and in the hypothalamus compared to high-fat fed Sham-FR. Diet alone did not change [3H] DAMGO binding in any region. These findings show that RYGB surgery, independent of diet or caloric restriction, decreases mu opioid signaling in specific regions important for stress and energy regulation. Thus, RYGB surgery may lead to greater stress sensitivity via downregulated mu opioid signaling in the central amygdala, which may contribute to the observed increased risk in some subjects for addictive behavior.

Endomorphins and DAMGO Utilize Different Mechanisms to Produce Analgesia

There is significant variability among animals in their response to mu opioid receptor (MOR1) analgesics. The mu opioid receptor gene (OPRM1) undergoes extensive alternative splicing, producing several splice variants with 1, 6, and 7 transmembrane (TM) domains. Variants containing 6 TMs lack TM1 and are associated with the exon 11 promoter. We previously showed that mice lacking exon 11 variants respond normally to methadone and morphine, but fentanyl, heroin, and morphine‐6‐glucuronide potency is significantly reduced (Pan et al, 2009). Here, we report that in exon 11 KO mice the DAMGO response is intact both supraspinally and spinally but endomorphin analgesiais significantly reduced. [3H]DAMGO binding in exon 11 KO brain and spinal cord was not significantly altered, but binding of the endomorphin 2 analog 125I‐DAPP (dmt(125I)‐D‐Ala‐Phe‐Phe‐NH2) was reduced.No binding of [3H]DAMGO or 125I‐DAPP was detected in mice lacking both exon 1 7TM MOR variants and exon 11 6TM variants. In [35S]GTPγS studies of brain and spinal cord binding, max stimulation by DAMGO was equal in wild type and exon 11 KO mice whereas IDAPP stimulation was reduced in exon 11 KO mice. Overall, our results indicate that DAMGO does not recruit 6TM exon 11 variants for binding and analgesia, whereas endomorphins require a mechanism including 6TM exon 11 variants for analgesia and a significant portion of binding.

An altered spinal serotonergic system contributes to increased thermal nociception in an animal model of depression

The olfactory bulbectomized (OB) rat, an animal model of chronic depression with comorbid anxiety, exhibits a profound dysregulation of the brain serotonergic signalling, a neurotransmission system involved in pain transmission and modulation. We here report an increased nociceptive response of OB rats in the tail flick test which is reverted after chronic, but not acute, administration of fluoxetine. Autoradiographic studies demonstrated down-regulation of 5-HT transporters ([(3)H]citalopram binding) and decreased functionality of 5-HT1A receptors (8-OH-DPAT-stimulated [(35)S]GTPγS binding) in the dorsal horn of the lumbar spinal cord in OB rats. Acute administration of fluoxetine (5-40 mg/kg i.p.) did not modify tail flick latencies in OB rats. However, chronic fluoxetine (10 mg/kg/day s.c., 14 days; osmotic minipumps) progressively attenuated OB-associated thermal hyperalgesia, and a total normalization of the nociceptive response was achieved at the end of the treatment with the antidepressant. In these animals, autoradiographic studies revealed further down-regulation of 5-HT transporters and normalization in the functionality of 5-HT1A receptors on the spinal cord. On the other hand, acute morphine (0.5-10 mg/kg s.c.) produced a similar analgesic effect in OB and sham and OB rats, and no changes were detected in the density ([(3)H]DAMGO binding) and functionality (DAMGO-stimulated [(35)S]GTPγS binding) of spinal μ-opioid receptors in OB rats before and after chronic fluoxetine. Our findings demonstrate the participation of the spinal serotonergic system in the increased thermal nociception exhibited by the OB rat and the antinociceptive effect of chronic fluoxetine in this animal model of depression.

Functional Characteristics of the Naked Mole Rat μ-Opioid Receptor

While humans and most animals respond to µ-opioid receptor (MOR) agonists with analgesia and decreased aggression, in the naked mole rat (NMR) opioids induce hyperalgesia and severe aggression. Single nucleotide polymorphisms in the human mu-opioid receptor gene (OPRM1) can underlie altered behavioral responses to opioids. Therefore, we hypothesized that the primary structure of the NMR MOR may differ from other species. Sequencing of the NMR oprm1 revealed strong homology to other mammals, but exposed three unique amino acids that might affect receptor-ligand interactions. The NMR and rat oprm1 sequences were cloned into mammalian expression vectors and transfected into HEK293 cells. Radioligand binding and 3'-5'-cyclic adenosine monophosphate (cAMP) enzyme immunoassays were used to compare opioid binding and opioid-mediated cAMP inhibition. At normalized opioid receptor protein levels we detected significantly lower [3H]DAMGO binding to NMR compared to rat MOR, but no significant difference in DAMGO-induced cAMP inhibition. Strong DAMGO-induced MOR internalization was detectable using radioligand binding and confocal imaging in HEK293 cells expressing rat or NMR receptor, while morphine showed weak or no effects. In summary, we found minor functional differences between rat and NMR MOR suggesting that other differences e.g. in anatomical distribution of MOR underlie the NMR's extreme reaction to opioids.

Time-course of neuropathic pain in mice deficient in neuronal or inducible nitric oxide synthase

Nitric oxide which is synthesised by nitric oxide synthase (NOS) is involved in processes related to regeneration after nerve injury and neuropathic pain. Here we investigated functional aspects of the nociceptive system. For that purpose, the chronic constriction injury (CCI) model induced by loose ligation of the sciatic nerve was employed in C57Bl/6J wild-type (WT), nNOS and iNOS knock-out (−/−) mice. Their thermal and mechanical pain thresholds were then measured over a period of six weeks. In addition, 3H-DAMGO, 3H-CP 55.940, and 3H-l-glutamate binding, and neuronal (NeuN-immunostained) and astroglial (GFAP-immunostained) cell composition were studied. There were no significant differences in cell composition between the three strains used. Significant differences between CCI and sham-operated animals were found in nNOS−/− after day 6, in WT mice after day 10, and in iNOS−/− after day 17 post surgery. The mechanical pain threshold was normalised after day 45 post surgery in WT mice only. There were no changes in DAMGO and glutamate binding. However, we found significant differences in CP 55.940 binding in the spinal cord. It was concluded that NOS–cannabinoid interaction contributes to differences in nociceptive behaviour.

Potentiation of morphine-induced mechanical antinociception by σ1 receptor inhibition: Role of peripheral σ1 receptors

We studied the modulation of morphine-induced mechanical antinociception and side effects by σ1 receptor inhibition. Both wild-type (WT) and σ1 receptor knockout (σ1-KO) mice showed similar responses to paw pressure (100–600 g). The systemic (subcutaneous) or local (intraplantar) administration of σ1 antagonists (BD-1063, BD-1047, NE-100 and S1RA) was devoid of antinociceptive effects in WT mice. However, σ1-KO mice exhibited an enhanced mechanical antinociception in response to systemic morphine (1–16 mg/kg). Similarly, systemic treatment of WT mice with σ1 antagonists markedly potentiated morphine-induced antinociception, and its effects were reversed by the selective σ1 agonist PRE-084. Although the local administration of morphine (50–200 μg) was devoid of antinociceptive effects in WT mice, it induced dose-dependent antinociception in σ1-KO mice. This effect was limited to the injected paw. Enhancement of peripheral morphine antinociception was replicated in WT mice locally co-administered with σ1 antagonists and the opioid. None of the σ1 antagonists tested enhanced morphine-antinociception in σ1-KO mice, confirming a σ1-mediated action. Morphine-induced side-effects (hyperlocomotion and inhibition of gastrointestinal transit) were unaltered in σ1-KO mice. These results cannot be explained by a direct interaction of σ1 ligands with μ-opioid receptors or adaptive changes of μ-receptors in σ1-KO mice, given that [3H]DAMGO binding in forebrain, spinal cord, and hind-paw skin membranes was unaltered in mutant mice, and none of the σ1 drugs tested bound to μ-opioid receptors. These results show that σ1 receptor inhibition potentiates morphine-induced mechanical analgesia but not its acute side effects, and that this enhanced analgesia can be induced at peripheral level.

Differential activation of the μ‐opioid receptor by oxycodone and morphine in pain‐related brain regions in a bone cancer pain model

Bone cancer pain is chronic and often difficult to control with opioids. However, recent studies have shown that several opioids have distinct analgesic profiles in chronic pain. To clarify the mechanisms underlying these distinct analgesic profiles, functional changes in the μ-opioid receptor were examined using a mouse femur bone cancer (FBC) model. In the FBC model, the B(max) of [(3) H]-DAMGO binding was reduced by 15-45% in the periaqueductal grey matter (PAG), region ventral to the PAG (vPAG), mediodorsal thalamus (mTH), ventral thalamus and spinal cord. Oxycodone (10(-8) -10(-5) M) and morphine (10(-8) -10(-5) M) activated [(35) S]-GTPγS binding, but the activation was significantly attenuated in the PAG, vPAG, mTH and spinal cord in the FBC model. Interestingly, the attenuation of oxycodone-induced [(35) S]-GTPγS binding was quite limited (9-26%) in comparison with that of morphine (46-65%) in the PAG, vPAG and mTH, but not in the spinal cord. Furthermore, i.c.v. oxycodone at doses of 0.02-1.0 μg per mouse clearly inhibited pain-related behaviours, such as guarding, limb-use abnormalities and allodynia-like behaviour in the FBC model mice, while i.c.v. morphine (0.05-2.0 μg per mouse) had only partial or little analgesic effect on limb-use abnormalities and allodynia-like behaviour. These results show that μ-opioid receptor functions are attenuated in several pain-related regions in bone cancer in an agonist-dependent manner, and suggest that modification of the μ-opioid receptor is responsible for the distinct analgesic effect of oxycodone and morphine.

Mu opioid receptor availability in people with psychiatric disorders who died by suicide: a case control study

BackgroundMu opioid receptors have previously been shown to be altered in people with affective disorders who died as a result of suicide. We wished to determine whether these changes were more widespread and independent of psychiatric diagnoses.MethodsMu receptor levels were determined using [3 H]DAMGO binding in BA24 from 51 control subjects; 38 people with schizophrenia (12 suicides); 20 people with major depressive disorder (15 suicides); 13 people with bipolar disorder (5 suicides) and 9 people who had no history of psychiatric disorders but who died as a result of suicide. Mu receptor levels were further determined in BA9 and caudate-putamen from 38 people with schizophrenia and 20 control subjects using [3 H]DAMGO binding and, in all three regions, using Western blots. Data was analysed using one-way ANOVAs with Bonferroni’s Multiple Comparison Test or, where data either didn’t approximate to a binomial distribution or the sample size was too small to determine distribution, a Kruskal-Wallis test with Dunn’s Multiple Comparison Test.Results[3 H]DAMGO binding density was lower in people who had died as a result of suicide (p<0.01). People with schizophrenia who had died as a result of suicide had lower binding than control subjects (p<0.001), whilst people with bipolar disorder (non- suicide) had higher levels of binding (p<0.05). [3 H]DAMGO binding densities, but not mu protein levels, were significantly decreased in BA9 from people with schizophrenia who died as a result of suicide (p<0.01).ConclusionsOverall these data suggest that mu opioid receptor availability is decreased in the brains of people with schizophrenia who died as a result of suicide, which would be consistent with increased levels of endogenous ligands occupying these receptors.

Reduced expression of the mu opioid receptor in some, but not all, brain regions in mice with OPRM1 A112G

OPRM1 A118G is a common single nucleotide polymorphism (SNP) in the coding region of the human mu opioid receptor (MOPR) gene OPRM1. This SNP is associated with higher morphine doses required for postoperative analgesia as well as a variety of drug addiction phenotypes. A mouse model possessing the equivalent substitution (A112G) in the Oprm1 gene was generated to facilitate mechanistic studies. Mice homozygous for the G112 allele (G/G) displayed lower antinociception to morphine compared with those homozygous for A112 allele (A/A), similar to humans, suggesting that the mice are a good model to further characterize underlying factors contributing to phenotypes associated with this SNP. Here, we compared [3H]DAMGO binding to the MOPR in the brains of A/A and G/G mice using quantitative in vitro autoradiography. A/A mice exhibited higher [3H]DAMGO binding than G/G in the cingulate, motor, and insular cortices, nucleus accumbens core and shell, hypothalamus, thalamus, amygdala, periaqueductal gray, superficial gray of superior colliculus, and ventral tegmental area. No genotype differences were observed in somatosensory cortex, caudate putamen, and hippocampus. When males and females were examined separately, A/A mice showed higher [3H]DAMGO binding than G/G mice in more brain regions in males than in females. Radioligand binding using brain membranes also showed higher [3H]DAMGO binding in the cortex and thalamus in A/A mice than G/G mice but no genotype differences in the caudate putamen or hippocampus. Thus, the A112G SNP is associated with reduced MOPR expression in some, but not all, brain regions, and appears to have some sex differences. The elevated MOPR expression in periaqueductal gray and thalamus in A/A mice are consistent with their higher antinociceptive responses to morphine. The higher MOPR levels in nucleus accumbens and/or ventral tegmental area of A/A mice is consistent with the higher morphine-induced hyperactivity and locomotor sensitization observed in these mice. Thus, these results provide some insights into the observed decreased clinical opioid potency in humans with the A118G SNP.

Hesperidin induces antinociceptive effect in mice and its aglicone, hesperetin, binds to μ-opioid receptor and inhibits GIRK1/2 currents

This paper extended the evaluation of the depressant and antinociceptive activities of hesperidin in order to determine its effectiveness by the intraperitoneal and oral routes, its pharmacological interaction with diverse pathways of neurotransmission and the role of its aglycone, hesperetin. The capacity of hesperidin and hesperetin to bind to μ-opioid receptor and their actions on μ-opioid receptor co-expressed with GIRK1/GIRK2 channels (G protein-activated inwardly rectifying K+ channels) in Xenopus laevis oocytes were also determined.Hesperidin exhibited a depressant activity in the hole board and locomotor activity tests, antinociceptive activities in the abdominal writhing and hot plate tests and no motor incoordination in the inverted screen and rotarod assays, only by the intraperitoneal route. Hesperetin did not show any effects in vivo in mice in these models, but in vitro it displaced the [3H]DAMGO binding with low-affinity and inhibited inward currents through the expressed GIRK1/2 channels. Although hesperidin actions in vivo demonstrated to be mediated by an opioid mechanism of action, it failed to directly bind to and activate the μ-opioid receptor or produce any change on inward GIRK1/2 currents in vitro. However, it should be considered that hesperidin may be metabolized, possibly resulting in crucial changes in its biological activity.

Agonist-selective Dynamic Compartmentalization of Human Mu Opioid Receptor as Revealed by Resolutive FRAP Analysis

Techniques for analyzing the membrane diffusion of molecules are the most promising methods for investigating the compartmentalization of G-protein-coupled receptors, particularly as relevant to receptor signaling processes. Here, we report fluorescence recovery after photobleaching (FRAP) measurements performed at variable spot radius for human mu opioid (hMOP) receptors on SH-SY5Y neuroblastoma cells in the presence of ligands. Although an antagonist did not affect the behavior of the receptors compared with the basal state, two different agonists, DAMGO and morphine, caused markedly different changes to receptor diffusion. Like receptors in the absence of ligand, receptors bound to morphine exhibited diffusion confined to joined semipermeable domains, but with smaller domain size and diffusion coefficient. This effect was inhibited by pertussis toxin, strongly suggesting that this dynamic behavior is associated with early steps of signaling. In the presence of DAMGO, half of the receptors displayed free long-range diffusion and the other half were confined to smaller isolated domains. Hypertonic sucrose buffer suppressed this effect, which we attribute to receptor entry into clathrin-coated pits. It is likely that the observation of distinct receptor dynamics in the presence of DAMGO and morphine involves the agonist-selective phosphorylation of the receptor.

Ethanol exposure selectively alters β-endorphin content but not [3H]-DAMGO binding in discrete regions of the rat brain

The dopaminergic mesocorticolimbic system plays an important role in the reinforcing effects of ethanol. Opioid peptides modulate the activity of this system and have been suggested to mediate, at least in part, the reinforcing properties of ethanol. Thus, beta-endorphin (beta-END) could participate in the development of ethanol reinforcement and addiction. The aim of this work was to investigate the acute and chronic ethanol effects on beta-END content in regions of the mesolimbic system and to examine if chronic ethanol treatment alters ligand binding to mu opioid receptor (muOR). Male Wistar rats received a single acute ethanol dose of 2.5 g/kg or water by intra-gastric administration. For chronic ethanol treatment experiments, one group of rats was given ethanol (10% v/v solution) to drink, two groups were given equivalent volumes of sucrose (14.14% isocaloric solution) or water, respectively, and a fourth group had ad libitum access to food and water. Treatment was followed for 4 weeks. Beta-endorphin content in brain regions was quantified by radioimmunoassay and ligand binding studies to muOR were performed by quantitative autoradiography using 8 nM [(3)H]-DAMGO as radioligand. Acute ethanol decreased beta-END content in the hypothalamus (26%) 1h after administration. No ethanol effects were observed in the midbrain, ventral tegmental area, substantia nigra, nucleus accumbens, nucleus accumbens-septum and prefrontal cortex. Chronic ethanol treatment neither changed beta-END levels nor [(3)H]-DAMGO binding to mu opioid receptors in any of the regions studied. However, beta-END levels in the sucrose group were significantly increased in the nucleus accumbens and substantia nigra, in comparison to all other groups. These findings suggest that different neural mechanisms and specific brain regions may be involved in the reinforcing effects of ethanol and sucrose.

Discovery of Dermorphin-Based Affinity Labels with Subnanomolar Affinity for Mu Opioid Receptors

A series of potent electrophilic affinity labels (IC(50) = 0.1-5 nM) containing either a bromoacetamide or isothiocyanate based on the mu opioid receptor (MOR) selective peptide dermorphin were prepared. All four analogues exhibited wash resistant inhibition of [(3)H]DAMGO binding at subnanomolar to nanomolar concentrations, suggesting that these analogues bind covalently to MOR. To our knowledge, these peptides are the highest affinity peptide-based affinity labels for MOR reported to date.