Brief Opioid Exposure Paradoxically Augments Primary Afferent Input to Spinal Excitatory Neurons via α2δ-1-Dependent Presynaptic NMDA Receptors.

Treatment with opioids not only inhibits nociceptive transmission but also elicits a rebound and persistent increase in primary afferent input to the spinal cord. Opioid-elicited long-term potentiation (LTP) from TRPV1-expressing primary afferents plays a major role in opioid-induced hyperalgesia and analgesic tolerance. Here, we determined whether opioid-elicited LTP involves vesicular glutamate transporter-2 (VGluT2) or vesicular GABA transporter (VGAT) neurons in the spinal dorsal horn of male and female mice and identified underlying signaling mechanisms. Spinal cord slice recordings revealed that µ-opioid receptor (MOR) stimulation with DAMGO initially inhibited dorsal root-evoked EPSCs in 87% VGluT2 neurons and subsequently induced LTP in 49% of these neurons. Repeated morphine treatment increased the prevalence of VGluT2 neurons displaying LTP with a short onset latency. In contrast, DAMGO inhibited EPSCs in 46% VGAT neurons but did not elicit LTP in any VGAT neurons even in morphine-treated mice. Spinal superficial laminae were densely innervated by MOR-containing nerve terminals and were occupied by mostly VGluT2 neurons and few VGAT neurons. Furthermore, conditional Grin1 knockout in dorsal root ganglion neurons diminished DAMGO-elicited LTP in lamina II neurons and attenuated hyperalgesia and analgesic tolerance induced by repeated treatment with morphine. In addition, DAMGO-elicited LTP in VGluT2 neurons was abolished by protein kinase C inhibition, gabapentin, Cacna2d1 knockout, or disrupting the α2δ-1-NMDA receptor interaction with an α2δ-1 C terminus peptide. Thus, brief MOR stimulation distinctively potentiates nociceptive primary afferent input to excitatory dorsal horn neurons via α2δ-1-coupled presynaptic NMDA receptors, thereby causing hyperalgesia and reducing analgesic actions of opioids.SIGNIFICANCE STATEMENT Opioid drugs are potent analgesics for treating severe pain and are commonly used during general anesthesia. However, opioid use often induces pain hypersensitivity, rapid loss of analgesic efficacy, and dose escalation, which can cause dependence, addiction, and even overdose fatality. This study demonstrates for the first time that brief opioid exposure preferentially augments primary sensory input to genetically identified glutamatergic excitatory, but not GABAergic/glycinergic inhibitory, neurons in nociceptive dorsal horn circuits. This opioid-elicited synaptic plasticity is cell type specific and mediated by protein kinase C-dependent and α2δ-1-dependent activation of NMDA receptors at primary sensory nerve terminals. These findings elucidate how intraoperative use of opioids for preemptive analgesia paradoxically aggravates postoperative pain and increases opioid consumption and suggest new strategies to improve opioid analgesic efficacy.

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 ß-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 ß-arrestins. PSN further reveals important roles of H8, ICL1, and ICL2 in regulating the constitutive ß-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 ß-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 ß-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 ß-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.

The rostral medial frontal cortex is crucial for engagement in consummatory behavior.

The medial frontal cortex (MFC) in rodents emits rhythmic activity that is entrained to the animal's licking cycle during consumption and encodes the value of consumed fluids. These signals are especially prominent in the rostral half of the MFC. This region is located above an orbitofrontal region where mu-opioid receptors regulate intake and reversible inactivation reduces behavioral measures associated with the incentive value and palatability of liquid sucrose. Here, we examined the effects of reversible inactivation and stimulation of mu-opioid receptors in rostral MFC on behavior in an incentive contrast licking task. Adult male rats licked to receive access to liquid sucrose, which alternated between high (16%) and low (4%) values over 30 s periods. Bilateral infusion of muscimol reduced the total number of licks over the 30 min test sessions, the time spent actively consuming sucrose, and the ratio of licks for the higher and lower value fluids. Inactivation did not alter licking frequency or variability or microstructural measures such as the duration of licking bouts that are classically associated with the palatability of a liquid reward. Infusions of [d-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO; 1 µg/µL) at the same sites had inconsistent behavioral effects across different subjects. Our findings suggest that the rostral MFC has a distinct role in the control of consummatory behavior and contributes to persistent consumption and not to the expression of palatability. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Sucrose drinking mimics effects of nucleus accumbens µ-opioid receptor stimulation on fat intake and brain c-Fos-expression.

Objectives: We have previously shown that the combined consumption of fat and a sucrose solution induces overeating, and there is evidence indicating that sucrose drinking directly stimulates fat intake. One neurochemical pathway by which sucrose may enhance fat intake is through the release of endogenous opioids in the nucleus accumbens (NAC).Methods: To test this hypothesis, we provided rats with a free-choice high-fat diet for two weeks. During the second week, rats had access to an additional bottle of water or a 30% sucrose solution for five minutes per day. After these two weeks, we infused vehicle or the µ-opioid receptor agonist [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) into the NAC 30 min after their daily access to the additional bottle of water or the sucrose solution.Results: Sucrose drinking had two effects, (1) it stimulated fat intake in the absence of DAMGO infusion, (2) it diminished sensitivity to DAMGO, as it prevented the rapid increase in fat intake typically seen upon DAMGO infusion in the nucleus accumbens. In a second experiment, we confirmed that these results are not due to the ingested calories of the sucrose solution. Lastly, we investigated which brain areas are involved in the observed effects on fat intake by assessing c-Fos-expression in brain areas previously linked to DAMGO's effects on food intake. Both intra-NAC DAMGO infusion and sucrose consumption in the absence of DAMGO infusion had no effect on c-Fos-expression in orexin neurons and the central amygdala but increased c-Fos-expression in the NAC as well as the basolateral amygdala.Discussion: In conclusion, we confirm that sucrose drinking stimulates fat intake, likely through the release of endogenous opioids.

Regulation of Opioid Receptors by Their Endogenous Opioid Peptides.

Activation of µ, δ, and κ opioid receptors by endogenous opioid peptides leads to the regulation of many emotional and physiological responses. The three major endogenous opioid peptides, ß-endorphin, enkephalins, and dynorphins result from the processing of three main precursors: proopiomelanocortin, proenkephalin, and prodynorphin. Using a knockout approach, we sought to determine whether the absence of endogenous opioid peptides would affect the expression or activity of opioid receptors in mice lacking either proenkephalin, ß-endorphin, or both. Since gene knockout can lead to changes in the levels of peptides generated from related precursors by compensatory mechanisms, we directly measured the levels of Leu-enkephalin and dynorphin-derived peptides in the brain of animals lacking proenkephalin, ß-endorphin, or both. We find that whereas the levels of dynorphin-derived peptides were relatively unaltered, the levels of Leu-enkephalin were substantially decreased compared to wild-type mice suggesting that preproenkephalin is the major source of Leu-enkephalin. This data also suggests that the lack of ß-endorphin and/or proenkephalin does not lead to a compensatory change in prodynorphin processing. Next, we examined the effect of loss of the endogenous peptides on the regulation of opioid receptor levels and activity in specific regions of the brain. We also compared the receptor levels and activity in males and females and show that the lack of ß-endorphin and/or proenkephalin leads to differential modulation of the three opioid receptors in a region- and gender-specific manner. These results suggest that endogenous opioid peptides are important modulators of the expression and activity of opioid receptors in the brain.

Mechanism of ß-arrestin recruitment by the µ-opioid G protein-coupled receptor.

Agonists to the µ-opioid G protein-coupled receptor (µOR) can alleviate pain through activation of G protein signaling, but they can also induce ß-arrestin activation, leading to such side effects as respiratory depression. Biased ligands to µOR that induce G protein signaling without inducing ß-arrestin signaling can alleviate pain while reducing side effects. However, the mechanism for stimulating ß-arrestin signaling is not known, making it difficult to design optimum biased ligands. We use extensive molecular dynamics simulations to determine three-dimensional (3D) structures of activated ß-arrestin2 stabilized by phosphorylated µOR bound to the morphine and D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) nonbiased agonists and to the TRV130 biased agonist. For nonbiased agonists, we find that the ß-arrestin2 couples to the phosphorylated µOR by forming strong polar interactions with intracellular loop 2 (ICL2) and either the ICL3 or cytoplasmic region of transmembrane (TM6). Strikingly, Gi protein makes identical strong bonds with these same ICLs. Thus, the Gi protein and ß-arrestin2 compete for the same binding site even though their recruitment leads to much different outcomes. On the other hand, we find that TRV130 has a greater tendency to bind the extracellular portion of TM2 and TM3, which repositions TM6 in the cytoplasmic region of µOR, hindering ß-arrestin2 from making polar anchors to the ICL3 or to the cytosolic end of TM6. This dramatically reduces the affinity between µOR and ß-arrestin2.

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.

DAMGO-induced µ opioid receptor internalization and recycling restore morphine sensitivity in tolerant rat.

This study investigated the effect of DAMGO-induced µ opioid receptor (MOR) internalization on morphine tolerance. Male Sprague-Dawley rats (200-250 g) aged 6-8 weeks were administered morphine via intrathecal (i.t.) injection (15 µg/10 µl twice daily for 6 days) to induce antinociceptive tolerance, which was evaluated using the tail-flick and paw-withdrawal tests. Response latency was calculated as the percentage of maximum possible effect (%MPE). A bolus of DAMGO was administered by i.t. injection on day 6, and the tail-flick and paw-withdrawal tests were carried out 24, 48, and 72 h later. Membrane and cytosolic MOR expression was assessed by western blotting. HEK293 cells were transfected with MOR-FLAG plasmid and after 6 days of morphine treatment (10 µM), the cells were treated with 1 µM DAMGO, and MOR localization was examined by immunofluorescence analysis 30 and 60 min later. Repeated morphine treatment induced tolerance after 5 days; however, i.t. DAMGO administration restored morphine sensitivity and enhanced acute morphine-induced antinociception after 24, 48, and 72 h. In HEK293 cells, DAMGO treatment stimulated MOR internalization after 30 min and MOR recycling to the membrane after 1 h. Membrane and cytoplasmic MOR expression in vivo was unchanged 24, 48, and 72 h after i.t. DAMGO injection. Morphine does not cause significant MOR internalization or downregulation, and can readily induce tolerance. DAMGO counters this effect by enhancing receptor endocytosis, thereby reversing morphine-induced antinociceptive tolerance and restoring its analgesic efficacy.

Biased agonism of clinically approved µ-opioid receptor agonists and TRV130 is not controlled by binding and signaling kinetics.

Binding and signaling kinetics have previously proven important in validation of biased agonism at GPCRs. Here we provide a comprehensive kinetic pharmacological comparison of clinically relevant µ-opioid receptor agonists, including the novel biased agonist oliceridine (TRV130) which is in clinical trial for pain management. We demonstrate that the bias profile observed for the selected agonists is not time-dependent and that agonists with dramatic differences in their binding kinetic properties can display the same degree of bias. Binding kinetics analyses demonstrate that buprenorphine has 18-fold higher receptor residence time than oliceridine. This is thus the largest pharmacodynamic difference between the clinically approved drug buprenorphine and the clinical candidate oliceridine, since their bias profiles are similar. Further, we provide the first pharmacological characterization of (S)-TRV130 demonstrating that it has a similar pharmacological profile as the (R)-form, oliceridine, but displays 90-fold lower potency than the (R)-form. This difference is driven by a significantly slower association rate. Finally, we show that the selected agonists are differentially affected by G protein-coupled receptor kinase 2 and 5 (GRK2 and GRK5) expression. GRK2 and GRK5 overexpression greatly increased µ-opioid receptor internalization induced by morphine, but only had modest effects on buprenorphine and oliceridine-induced internalization. Overall, our data reveal that the clinically available drug buprenorphine displays a similar pharmacological bias profile in vitro compared to the clinical candidate drug oliceridine and that this bias is independent of binding kinetics suggesting a mechanism driven by receptor-conformations. This article is part of the Special Issue entitled 'New Vistas in Opioid Pharmacology'.

Agonist binding of human mu opioid receptors expressed in the yeast Pichia pastoris: Effect of cholesterol complementation.

This study compared pharmacological profiles between human mu opioid receptors (hMOR) overexpressed in the SH-SY5Y neuroblastoma cell line (SH-hMOR) and the methylotrophic yeast Pichia pastoris (Pp-hMOR). Affinity determinations were performed by direct binding with the tritiated agonist DAMGO and antagonist diprenorphine (DIP). Additionally, displacement of these drugs with agonists (morphine and DAMGO) and antagonists (ß-funaltrexamine, naloxone and diprenorphine) was examined. Tritiated DAMGO could bind to membranes prepared from Pp-hMOR, although the receptor was not coupled with G-proteins. The data obtained with this yeast strain suggested that only 7.5% of receptors were in a high-affinity-state conformation. This value was markedly less than that estimated in SH-hMOR membranes, which reached 50%. Finally, to understand the pharmacological discrepancies between Pp-hMOR and SH-hMOR, the role of sterols was evaluated. The major sterol in P. pastoris is ergosterol, while hMOR naturally functions in a cholesterol-containing membrane environment. Cell membranes were sterol-depleted or cholesterol-loaded with methyl-ß-cyclodextrine. The results indicated that cholesterol must be present to ensure Pp-hMOR function. The proportion of high-affinity-state conformation was reversibly increased by cholesterol complementation.

Ligand-dependent spatiotemporal signaling profiles of the µ-opioid receptor are controlled by distinct protein-interaction networks.

Ligand-dependent differences in the regulation and internalization of the µ-opioid receptor (MOR) have been linked to the severity of adverse effects that limit opiate use in pain management. MOR activation by morphine or [d-Ala2,N-MePhe4, Gly-ol]enkephalin (DAMGO) causes differences in spatiotemporal signaling dependent on MOR distribution at the plasma membrane. Morphine stimulation of MOR activates a Gαi/o-Gßγ-protein kinase C (PKC) α phosphorylation pathway that limits MOR distribution and is associated with a sustained increase in cytosolic extracellular signal-regulated kinase (ERK) activity. In contrast, DAMGO causes a redistribution of the MOR at the plasma membrane (before receptor internalization) that facilitates transient activation of cytosolic and nuclear ERK. Here, we used proximity biotinylation proteomics to dissect the different protein-interaction networks that underlie the spatiotemporal signaling of morphine and DAMGO. We found that DAMGO, but not morphine, activates Ras-related C3 botulinum toxin substrate 1 (Rac1). Both Rac1 and nuclear ERK activity depended on the scaffolding proteins IQ motif-containing GTPase-activating protein-1 (IQGAP1) and Crk-like (CRKL) protein. In contrast, morphine increased the proximity of the MOR to desmosomal proteins, which form specialized and highly-ordered membrane domains. Knockdown of two desmosomal proteins, junction plakoglobin or desmocolin-1, switched the morphine spatiotemporal signaling profile to mimic that of DAMGO, resulting in a transient increase in nuclear ERK activity. The identification of the MOR-interaction networks that control differential spatiotemporal signaling reported here is an important step toward understanding how signal compartmentalization contributes to opioid-induced responses, including anti-nociception and the development of tolerance and dependence.

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.

The HECT ubiquitin E3 ligase Smurf2 degrades µ-opioid receptor 1 in the ubiquitin-proteasome system in lung epithelial cells.

Opioids are widely used for relieving clinical acute or chronic pain. The biological effects of opioids are through activating µ-opioid receptor 1 (MOR1). Most studies have focused on the consequences of agonist-induced MOR1 phosphorylation, ubiquitination, and internalization. Agonist-mediated MOR1 degradation, which is crucial for receptor stability and responsiveness, has not been well studied. E3 ubiquitin-protein ligase SMURF2 (Smurf2), a homolog to E6AP carboxy terminus (HECT) ubiquitin E3 ligase, has been shown to regulate MOR1 ubiquitination and internalization; however, its role in MOR1 degradation has not been studied. Here, we demonstrate that Smurf2 mediates [d-Ala2,N-MePhe4,Gly5-ol]-enkephalin (DAMGO, an agonist of MOR1)-induced MOR1 ubiquitination and degradation. DAMGO decreased MOR1 levels in the ubiquitin-proteasome system. MOR1 was modified by a Lys48-linked polyubiquitin chain. Overexpression of Smurf2 induced MOR1 ubiquitination and accelerated DAMGO-induced MOR1 degradation, whereas downregulation of Smurf2 attenuated MOR1 degradation. Furthermore, DAMGO increased lung epithelial cell migration and proliferation, and the effect was attenuated by overexpressing Smurf2. Collectively, these data unveil that Smurf2 negatively regulates MOR1 activity by reducing its stability. We also demonstrate an unrevealed biological function of MOR1 in lung epithelial cells. DAMGO-MOR1 promote cell migration and proliferation in lung epithelial cells, suggesting a potential effect of DAMGO in lung repair and remodeling after lung injury.

Synthesis and Biological Evaluation of Fentanyl Analogues Modified at Phenyl Groups with Alkyls.

A series of fentanyl analogues modified at the phenyl group of the phenethyl with alkyl and/or hydroxyl and alkoxy, and the phenyl group in the anilido moiety replaced with benzyl or substituted benzyl, were synthesized. The in vitro opioid receptor functional activity of these compounds was evaluated by assessment of their ability to modulate forskolin-stimulated cAMP accumulation and by their ability to induce ß-arrestin2 recruitment. Compound 12 is a potent µ-opioid (MOP) receptor agonist, a potent κ-opioid (KOP) receptor antagonist with weak ß-arrestin2 recruitment activity. Compounds 10 and 11 are potent MOP receptor agonists with weak δ-opioid (DOP) receptor antagonist activity and moderate KOP receptor antagonist activity as well as weak ß-arrestin2 recruitment activity at the MOP receptor. These compounds are promising leads for discovery of potent opioid analgesics with reduced side effects relative to clinically available strong opioid analgesics.

A Genetically Encoded Biosensor Reveals Location Bias of Opioid Drug Action.

Opioid receptors (ORs) precisely modulate behavior when activated by native peptide ligands but distort behaviors to produce pathology when activated by non-peptide drugs. A fundamental question is how drugs differ from peptides in their actions on target neurons. Here, we show that drugs differ in the subcellular location at which they activate ORs. We develop a genetically encoded biosensor that directly detects ligand-induced activation of ORs and uncover a real-time map of the spatiotemporal organization of OR activation in living neurons. Peptide agonists produce a characteristic activation pattern initiated in the plasma membrane and propagating to endosomes after receptor internalization. Drugs produce a different activation pattern by additionally driving OR activation in the somatic Golgi apparatus and Golgi elements extending throughout the dendritic arbor. These results establish an approach to probe the cellular basis of neuromodulation and reveal that drugs distort the spatiotemporal landscape of neuronal OR activation.

Verification of a genetic locus for methamphetamine intake and the impact of morphine.

A quantitative trait locus (QTL) on proximal chromosome (Chr) 10 accounts for > 50% of the genetic variance in methamphetamine (MA) intake in mice selectively bred for high (MAHDR) and low (MALDR) voluntary MA drinking. The µ-opioid receptor (MOP-r) gene, Oprm1, resides at the proximal end of Chr 10, and buprenorphine reduces MA intake in MAHDR mice. However, this drug has only partial agonist effects at MOP-r. We investigated the impact of a full MOP-r agonist, morphine, on MA intake and saccharin intake, measured MOP-r density and affinity in several brain regions of the MA drinking lines and their C57BL/6J (B6) and DBA/2J (D2) progenitor strains, and measured MA intake in two congenic strains of mice to verify the QTL and reduce the QTL interval. Morphine reduced MA intake in the MAHDR line, but also reduced saccharin and total fluid intake. MOP-r density was lower in the medial prefrontal cortex of MAHDR, compared to MALDR, mice, but not in the nucleus accumbens or ventral midbrain; there were no MOP-r affinity differences. No significant differences in MOP-r density or affinity were found between the progenitor strains. Finally, Chr 10 congenic results were consistent with previous data suggesting that Oprm1 is not a quantitative trait gene, but is impacted by the gene network underlying MA intake. Stimulation of opioid pathways by a full agonist can reduce MA intake, but may also non-specifically affect consummatory behavior; thus, a partial agonist may be a better pharmacotherapeutic.

T394A Mutation at the µ Opioid Receptor Blocks Opioid Tolerance and Increases Vulnerability to Heroin Self-Administration in Mice.

The etiology and pathophysiology underlying opioid tolerance and dependence are still unknown. Because mu opioid receptor (MOR) plays an essential role in opioid action, many vulnerability-related studies have focused on single nucleotide polymorphisms of MOR, particularly on A118G. In this study, we found that a single-point mutation at the MOR T394 phosphorylation site could be another important susceptive factor in the development of opioid tolerance and dependence in mice. T394A mutation, in which a threonine at 394 was replaced by an alanine, did not alter agonist binding to MOR and opioid analgesia, but resulted in loss of etorphine-induced MOR internalization in spinal dorsal horn neurons and opioid analgesic tolerance induced by either morphine or etorphine. In addition, this mutation also caused an increase in intravenous heroin self-administration and in nucleus accumbens dopamine response to heroin. These findings suggest that T394 phosphorylation following MOR activation causes MOR internalization and desensitization, which subsequently contributes to the development of tolerance in both opioid analgesia and opioid reward. Accordingly, T394A mutation blocks opioid tolerance and leads to an increase in brain dopamine response to opioids and in opioid-taking behavior. Thus, the T394 may serve as a new drug target for modulating opioid tolerance and the development of opioid abuse and addiction. SIGNIFICANCE STATEMENT: The mechanisms underlying opioid tolerance and susceptibility to opioid addiction remain unclear. The present studies demonstrate that a single-point mutation at the T394 phosphorylation site in the C-terminal of mu opioid receptor (MOR) results in loss of opioid tolerance and enhanced vulnerability to heroin self-administration. These findings suggest that modulation of the MOR-T394 phosphorylation or dephosphorylation status may have therapeutic potential in management of pain, opioid tolerance, and opioid abuse and addiction. Accordingly, MOR-T394 mutation or polymorphisms could be a risk factor in developing opioid abuse and addiction and therefore be used as a new biomarker in prediction and prevention of opioid abuse and addiction.

Biased µ-opioid receptor agonists diversely regulate lateral mobility and functional coupling of the receptor to its cognate G proteins.

There are some indications that biased µ-opioid ligands may diversely affect µ-opioid receptor (MOR) properties. Here, we used confocal fluorescence recovery after photobleaching (FRAP) to study the regulation by different MOR agonists of receptor movement within the plasma membrane of HEK293 cells stably expressing a functional yellow fluorescent protein (YFP)-tagged µ-opioid receptor (MOR-YFP). We found that the lateral mobility of MOR-YFP was increased by (D-Ala2,N-MePhe4,Gly5-ol)-enkephalin (DAMGO) and to a lesser extent also by morphine but decreased by endomorphin-2. Interestingly, cholesterol depletion strongly enhanced the ability of morphine to elevate receptor mobility but significantly reduced or even eliminated the effect of DAMGO and endomorphin-2, respectively. Moreover, the ability of DAMGO and endomorphin-2 to influence MOR-YFP movement was diminished by pertussis toxin treatment. The results obtained by agonist-stimulated [35S]GTPγS binding assays indicated that DAMGO exhibited higher efficacy than morphine and endomorphin-2 did and that the efficacy of DAMGO, contrary to the latter agonists, was enhanced by cholesterol depletion. Overall, our study provides clear evidence that biased MOR agonists diversely affect receptor mobility in plasma membranes as well as MOR/G protein coupling and that the regulatory effect of different ligands depends on the membrane cholesterol content. These findings help to delineate the fundamental properties of MOR regarding their interaction with biased MOR ligands and cognate G proteins.

Exploring Factors Causing Low Brain Penetration of the Opioid Peptide DAMGO through Experimental Methods and Modeling.

To advance the development of peptide analogues for improved treatment of pain, we need to learn more about the blood-brain barrier transport of these substances. A low penetration into the brain, with an unbound brain to blood ratio, Kp,uu, of 0.08, is an important reason for the lack of effect of the enkephalin analogue DAMGO (H-Tyr-d-Ala-Gly-MePhe-Gly-ol) according to earlier findings. The aim of this study was to investigate the role of efflux transporters, metabolism in the brain, and/or elimination through interstitial fluid bulk flow for the brain exposure of DAMGO. The in vivo brain distribution of DAMGO was evaluated using microdialysis in the rat. Data were analyzed with population modeling which resulted in a clearance into the brain of 1.1 and an efflux clearance 14 µL/min/g_brain. The efflux clearance was thus much higher than the bulk flow known from the literature. Coadministration with the efflux transporter inhibitors cyclosporin A and elacridar in vivo did not affect Kp,uu. The permeability of DAMGO in the Caco-2 assay was very low, of the same size as mannitol. The efflux ratio was <2 and not influenced by cyclosporin A or elacridar. These results indicate that the well-known efflux transporters Pgp and Bcrp are not responsible for the higher efflux of DAMGO, which opens up for an important role of other transporters at the BBB.

[THE INFLUENCE OF OPIOID PEPTIDES DSLET AND DAGO ON LIPID PEROXIDATION AND THE ACTIVITY OF ANTIOXIDANT ENZYMES IN THE LIVER TISSUE OF RATS IN THE STRESS OF DIFFERENT DURATION].

It was established that immobilization stress of different duration (3, 6 or 12 hours) causes the activation of lipid peroxidation in liver tissue of rats 39 hours after stressor action. Activation of lipid peroxidation within 7 days was observed in animals exposed to 6 or 12 hours stress. The activation of superoxide dismutase and catalase activity was revealed in rats after 3 hours immobility, but 12 hours stress was accompanied by superoxide dismutase inhibition. Agonists of different opioid receptors were shown to afford the antioxidant effects inhibiting the accumulation of malondialdehyde and acylhydroperoxides in the liver tissue and stimulating the antioxidant enzyme activity. Opioid peptides had a stimulatory effect on superoxide dismutase activity and less one on catalase activity in animal exposed to immobilization of different duration. A selective agonist of opioid delta-receptors DSLET manifested more expressed antioxidant effect, causing the inhibition of LPO metabolites production and the stimulation of superoxide dismutase and catalase activity.