Activation Of Μ-opioid Receptors Research Articles

Change in functional selectivity of morphine with the development of antinociceptive tolerance

Opioids, such as morphine, are the most effective treatment for pain but their efficacy is diminished with the development of tolerance following repeated administration. Recently, we found that morphine activated ERK in opioid-tolerant but not in naïve rats, suggesting that morphine activation of μ-opioid receptors is altered following repeated morphine administration. Here, we have tested the hypothesis that μ-opioid receptor activation of ERK in the ventrolateral periaqueductal gray (vlPAG) is dependent on dynamin, a protein implicated in receptor endocytosis. Rats were made tolerant to repeated microinjections of morphine into the vlPAG. The effects of dynamin on ERK activation and antinociception were assessed by microinjecting myristoylated dominant-negative dynamin peptide (Dyn-DN) or a scrambled control peptide into the vlPAG. Microinjection of a fluorescent dermorphin analogue (DERM-A594) into the vlPAG was used to monitor μ-opioid receptor internalization. Morphine did not activate ERK and Dyn-DN administration had no effect on morphine-induced antinociception in saline-pretreated rats. In contrast, morphine-induced ERK activation in morphine-pretreated rats that was blocked by Dyn-DN administration. Dyn-DN also inhibited morphine antinociception. Finally, morphine reduced DERM-A594 internalization only in morphine-tolerant rats indicating that μ-opioid receptors were internalized and unavailable to bind DERM-A594. Repeated morphine administration increased μ-opioid receptor activation of ERK signalling via a dynamin-dependent mechanism. These results demonstrate that the balance of agonist signalling to G-protein and dynamin-dependent pathways is altered, effectively changing the functional selectivity of the agonist-receptor complex. This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2.

New opioids.

Despite the skilled use of opioid analgesics, which is crucial to the relief of cancer pain, there is a lack of evidence to support many aspects of current clinical practice. Therefore, there is a significant need for more effective treatment options. New opioids have been marketed in the past years, including hydrocodone and oxymorphone. Moreover, mixed opioids with combined mechanisms of action have been developed; one such agent, tapentadol, is a centrally acting oral analgesic that possesses a combined mechanism of action: μ-opioid receptor activation with norepinephrine reuptake inhibition. Drug development strategies involving naloxone have been initiated to reduce peripheral opioid-related adverse effects. The rationale is based on the local antagonist activity of naloxone in intestinal opioid receptors and the negligible oral bioavailability of naloxone, particularly in a prolonged-release formulation. New delivery systems have been developed to provide rapid analgesia with potent opioid drugs such as fentanyl. Despite the upcoming availability of these new drugs and technologies that will add to existing types of opioid medication, their benefits and liabilities will ultimately need to be determined by the individual physician and individual patient experiencing pain.

New insights into mechanisms of opioid inhibitory effects on capsaicin-induced TRPV1 activity during painful diabetic neuropathy

Painful diabetic neuropathy is a disease of the peripheral sensory neuron with impaired opioid responsiveness. Since μ-opioid receptor (MOR) activation can inhibit the transient receptor potential vanilloid 1 (TRPV1) activity in peripherally sensory neurons, this study investigated the mechanisms of impaired opioid inhibitory effects on capsaicin-induced TRPV1 activity in painful diabetic neuropathy. Intravenous injection of streptozotocin (STZ, 45 mg/kg) in Wistar rats led to a degeneration of insulin producing pancreatic β-cells, elevated blood glucose, and mechanical hypersensitivity (allodynia). In these animals, local morphine's inhibitory effects on capsaicin-induced nocifensive behavior as well as on capsaicin-induced TRPV1 current in dorsal root ganglion cells were significantly impaired. These changes were associated with a loss in MOR but not TRPV1 in peripheral sensory neurons. Intrathecal delivery of nerve growth factor in diabetic animals normalized sensory neuron MOR and subsequently rescued morphine's inhibitory effects on capsaicin-induced TRPV1 activity in vivo and in vitro. These findings identify a loss in functional MOR on sensory neurons as a contributing factor for the impaired opioid inhibitory effects on capsaicin-induced TRPV1 activity during advanced STZ-induced diabetes. Moreover, they support growing evidence of a distinct regulation of opioid responsiveness during various painful states of disease (e.g. arthritis, cancer, neuropathy) and may give novel therapeutic incentives.

Enhancement of antinociceptive effect of morphine by antidepressants in diabetic neuropathic pain model

BackgroundRecent studies have shown that influence of antidepressants on analgesic action of opioids is heterogeneous. The aim of this study was to investigate the effect of acute and repeated (21 days) antidepressant (amitriptyline, moclobemide and reboxetine) treatment on the antinociceptive action of morphine, an opioid agonist, in streptozotocin (STZ)-induced neuropathic pain model. MethodsThe studies were performed on the male Wistar rats. The changes in nociceptive thresholds were determined by using mechanical stimuli (the Randall–Selitto and the von Frey tests). Diabetes was induced by intramuscular administration of STZ. ResultsIn this work we report that acute as well as repeated per os administration of antidepressants (amitriptyline, moclobemide and reboxetine) significantly potentiated the antihyperalgesic effect of morphine in STZ-induced neuropathic pain model. ConclusionCombination therapy, such as classical antidepressants (amitriptyline, moclobemide) with opioids, or agents with noradrenaline reuptake inhibition and μ-opioid receptor activation could be a new target for research into treatment of painful diabetic neuropathy.

Basal μ-opioid receptor availability in the amygdala predicts the inhibition of pain-related brain activity during heterotopic noxious counter-stimulation

The aim of this study was to investigate the association between the magnitude of anti-nociceptive effects induced by heterotopic noxious counter-stimulation (HNCS) and the basal μ-opioid receptor availability in the amygdala. In 8 healthy volunteers (4 females and 4 males), transcutaneous electrical stimulation was applied to the right sural nerve to produce the nociceptive flexion reflex (RIII-reflex), moderate pain, and scalp somatosensory evoked potentials (SEPs). Immersion of the left hand in cold water for 20min was used as HNCS. In a separate session, basal μ-opioid receptor availability was measured using positron emission tomography with the radiotracer [11C]carfentanil. HNCS produced a reduction of the P260 amplitude (p<0.05), a late component of SEP that reflects activity in the anterior cingulate cortex. This reduction was associated with higher basal μ-opioid receptor availability in the amygdala on the right (R2=0.55, p=0.03) with a similar trend on the left (R2=0.24, p=0.22). Besides, HNCS did not induce significant changes in pain and RIII-reflex amplitude (p>0.05). These results suggest that activation of μ-opioid receptors in the amygdala may contribute to the anti-nociceptive effects of HNCS. The lack of RIII-reflex modulation further suggests that μ-opioid receptor activation in the amygdala contributes to decrease pain-related brain activity through a cerebral mechanism independent of descending modulation.

A Real-Time, Fluorescence-Based Assay for Measuring µ-Opioid Receptor Modulation of Adenylyl Cyclase Activity in Chinese Hamster Ovary Cells

Inhibition of adenylyl cyclase (AC) activity is frequently used to measure µ-opioid receptor (MOR) activation. We sought to develop a simple, rapid assay of AC activity in whole cells that could be used to study MOR signaling. Chinese hamster ovary cells expressing human MOR (CHO-MOR cells) were grown in 96-well plates and loaded with membrane potential–sensitive fluorescent dye. CHO-MOR cells were treated with the AC activator forskolin (FSK), with or without simultaneous application of MOR agonists, and the resulting change in fluorescence was measured. CHO-MOR cells hyperpolarized in response to application of FSK (pEC50, 7.3) or calcitonin (pEC50, 9.4). A submaximally effective concentration of FSK (300 nM) caused a 52% ± 2% decrease in fluorescence. Simultaneous application of the opioids DAMGO (pEC50, 7.4; Emax, 56%), morphine (pEC50, 7.0; Emax, 61%); and buprenorphine (pEC50, 8.6; Emax, 24%) inhibited the FSK response in a dose-dependent manner while having no effect by themselves. The effects of DAMGO were blocked by pertussis toxin. This assay represents a simple, robust method for real-time observation of AC inhibition by MOR in CHO cells. It represents an appealing alternative to end-point assays that rely on cAMP accumulation and can avoid potential confounds associated with rapid desensitization of MOR signaling.

The Contribution of Gi/o Protein to Opioid Antinociception in an Oxaliplatin-Induced Neuropathy Rat Model

Oxaliplatin is a chemotherapeutic agent that induces chronic refractory neuropathy. To determine whether opioids effectively relieve this chronic neuropathy, we investigated the efficacies of morphine, oxycodone, and fentanyl, and the mechanisms underlying opioid antinociception, in oxaliplatin-induced neuropathy in rats. Rats exhibited significant mechanical allodynia following 2 weeks of chronic oxaliplatin administration. Within the range of doses that did not induce sedation and/or muscle rigidity, morphine (3 mg/kg, subcutaneously, s.c.) and oxycodone (0.3-0.56 mg/kg, s.c.) completely reversed oxaliplatin-induced mechanical allodynia, whereas fentanyl (0.017-0.03 mg/kg, s.c.) showed partial antinociception. The antinociception of the optimal doses of morphine and oxycodone were completely inhibited by pertussis toxin (PTX; 0.5 μg/rat, i.c.v.), a Gi/o protein inhibitor, while the partial effect of fentanyl was not affected in the oxaliplatin model. In the [(35)S]-GTPγS binding assay, activation of μ-opioid receptor by fentanyl, but not by morphine or oxycodone, in the mediodorsal thalamus was significantly reduced in oxaliplatin-treated rats. These results indicate that the lower antinociceptive potency of fentanyl in the oxaliplatin model might in part result from the loss of PTX-sensitive Gi/o protein activation, and the degree of Gi/o protein activation might be related to the potency of antinociception by opioids in this model.

Endogenous analgesia, dependence, and latent pain sensitization.

Endogenous activation of µ-opioid receptors (MORs) provides relief from acute pain. Recent studies have established that tissue inflammation produces latent pain sensitization (LS) that is masked by spinal MOR signaling for months, even after complete recovery from injury and re-establishment of normal pain thresholds. Disruption with MOR inverse agonists reinstates pain and precipitates cellular, somatic, and aversive signs of physical withdrawal; this phenomenon requires N-methyl-D-aspartate receptor-mediated activation of calcium-sensitive adenylyl cyclase type 1 (AC1). In this review, we present a new conceptual model of the transition from acute to chronic pain, based on the delicate balance between LS and endogenous analgesia that develops after painful tissue injury. First, injury activates pain pathways. Second, the spinal cord establishes MOR constitutive activity (MORCA) as it attempts to control pain. Third, over time, the body becomes dependent on MORCA, which paradoxically sensitizes pain pathways. Stress or injury escalates opposing inhibitory and excitatory influences on nociceptive processing as a pathological consequence of increased endogenous opioid tone. Pain begets MORCA begets pain vulnerability in a vicious cycle. The final result is a silent insidious state characterized by the escalation of two opposing excitatory and inhibitory influences on pain transmission: LS mediated by AC1 (which maintains the accelerator) and pain inhibition mediated by MORCA (which maintains the brake). This raises the prospect that opposing homeostatic interactions between MORCA analgesia and latent NMDAR-AC1-mediated pain sensitization creates a lasting vulnerability to develop chronic pain. Thus, chronic pain syndromes may result from a failure in constitutive signaling of spinal MORs and a loss of endogenous analgesic control. An overarching long-term therapeutic goal of future research is to alleviate chronic pain by either (a) facilitating endogenous opioid analgesia, thus restricting LS within a state of remission, or (b) extinguishing LS altogether.

Natriorexigenic effect of DAMGO is decreased by blocking AT1 receptors in the central nucleus of the amygdala

μ-Opioid receptor (μ-OR) activation with agonist [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO) in the central nucleus of the amygdala (CeA) induces sodium (0.3M NaCl) intake in rats. The purpose of this study was to examine the effects of pre-injections of losartan (AT1 angiotensin receptor antagonist) into the CeA on 0.3M NaCl and water intake induced by DAMGO injected bilaterally in the same area in rats submitted to water deprivation–partial rehydration (WD–PR) and in rats treated with the diuretic furosemide (FURO) combined with a low dose of the angiotensin-converting enzyme inhibitor captopril (CAP) injected subcutaneously (FURO/CAP). Male Sprague–Dawley rats with stainless steel cannulas implanted bilaterally into the CeA were used. In WD–PR rats, bilateral injections of DAMGO (2nmol in 0.5μL) into the CeA induced 0.3M NaCl and water intake, and pre-treatment with losartan (108nmol in 0.5μL) injected into the CeA reduced 0.3M NaCl and water intake induced by DAMGO. In FURO/CAP rats, pre-treatment with losartan (108nmol in 0.5μL) injected into the CeA attenuated the increase in 0.3M NaCl and water intake induced by DAMGO (2nmol in 0.5μL) injected into the same site. The results suggest that the natriorexigenic effect of DAMGO injected into the CeA is facilitated by endogenous angiotensin II acting on AT1 receptors in the CeA, which drives rats to ingest large amounts of hypertonic NaCl.

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

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

Constitutive μ-Opioid Receptor Activity Leads to Long-Term Endogenous Analgesia and Dependence

Opioid receptor antagonists increase hyperalgesia in humans and animals, which indicates that endogenous activation of opioid receptors provides relief from acute pain; however, the mechanisms of long-term opioid inhibition of pathological pain have remained elusive. We found that tissue injury produced μ-opioid receptor (MOR) constitutive activity (MOR(CA)) that repressed spinal nociceptive signaling for months. Pharmacological blockade during the posthyperalgesia state with MOR inverse agonists reinstated central pain sensitization and precipitated hallmarks of opioid withdrawal (including adenosine 3',5'-monophosphate overshoot and hyperalgesia) that required N-methyl-D-aspartate receptor activation of adenylyl cyclase type 1. Thus, MOR(CA) initiates both analgesic signaling and a compensatory opponent process that generates endogenous opioid dependence. Tonic MOR(CA) suppression of withdrawal hyperalgesia may prevent the transition from acute to chronic pain.

Effects of signalled reward type, food status and a μ-opioid receptor antagonist on cue-induced anticipatory behaviour in laying hens (Gallus domesticus)

Studies using classical conditioning have shown that hens display high frequencies of dopamine-controlled cue-induced anticipatory behaviours in the cue-reward interval when signalling mealworm rewards. However, it is not known whether anticipatory behaviours are reward specific, and whether the opioid system is involved in their control. The purpose of the present study was to investigate (1) effect of incentive value of rewards, and (2) the involvement of μ-opioid receptor activation, on the expression of cue-induced anticipatory behaviours in laying hens. Incentive value was manipulated by reward type (mealworm and whole wheat) and by physiological state (sated and fasted hens). Hens (n=14) were trained to associate a cue (green or red light) with a reward (whole wheat or mealworms). Blue light served as an unrewarded control stimulus. Cue-induced anticipatory head movements (latency to first head movement after cue presentation, and frequency of head movements in the cue-reward interval), steps (frequency), and pecking at reward (latency), were registered in sated and fasted hens during a 25s cue-reward interval. An involvement of the opioid system in mediating cue-induced anticipatory behaviours was tested by intraperitoneal injection of the μ-opioid receptor antagonist naloxone at 5.0mgkg−1. Saline served as control. Injections were administered 30min before the light cues. Individual hens were tested on all treatment combinations: sated/saline, fasted/saline, sated/naloxone, and fasted/naloxone. Incentive value of signalled reward was differentially reflected by the frequency of cue-induced head movements (P<0.0001). Hens displayed more head movements in response to signalled mealworms (33.1±0.9) than signalled whole wheat (28.5±1.1) and unrewarded cue (17.2±1.0). The frequency of steps was higher in response to the cue signalling mealworms (12.2±1.0) and whole wheat (9.9±0.7) than to the unrewarded cue (6.3±0.7, P<0.0001 and P=0.0003, respectively), but there was no difference between the rewarded cues. Latency to initiate the first head movement was longer in response to the unrewarded cue than to cues signalling whole wheat and mealworms (P=0.051 (tendency) and P=0.0017). Hunger amplified the frequency of head movements (P<0.002) and tended to affect frequency of steps (P<0.072). No effects of treatment with naloxone were found as tested here. In conclusion, cue-induced anticipatory head movements reflect incentive value of food rewards in laying hens. The role of opioid regulation of reward processes in hens needs to be further investigated.

Orexigenic effects of endomorphin-2 (EM-2) related to decreased CRH gene expression and increased dopamine and norepinephrine activity in the hypothalamus

Endomorphin-1 (EM-1) and endomorphin-2 (EM-2) are opioid peptides which are selective partial agonists of μ-opioid receptor. We studied the effects of EM-2 injected into the arcuate nucleus (ARC) of the hypothalamus on feeding behavior and gene expression of orexigenic [agouti-related peptide (AgRP), neuropeptide Y (NPY) and orexin-A] and anorexigenic [cocaine and amphetamine-regulated transcript (CART), corticotrophin releasing hormone (CRH) and proopiomelanocortin (POMC)] peptides in male Wistar rats fed a standard laboratory diet. Furthermore, we evaluated the effects of EM-2 on dopamine (DA), norepinephrine (NE) and serotonin (5-hydroxytryptamine, 5-HT) steady state concentrations, in the hypothalamus. 64 rats (16 for each group of treatment) were injected into the ARC, at 9.00am, with either vehicle or EM-2 (0.50–0.75μmol/kg) or EM-2 (0.50μmol/kg) plus β-funaltrexamine (0.20μmol/kg). Food intake was recorded through 24h following injection, and hypothalamic DA, NE, 5-HT levels and neuropeptide gene expression were evaluated 24h after EM-2 administration. Compared to vehicle, EM-2 significantly increased food intake, throughout 24h post-injection. Furthermore, EM-2 treatment led to a significant increase of DA and NE concentrations and a decrease of CRH mRNA levels. On the other hand, β-funaltrexamine administration reverted both feeding stimulatory and neuromodulatory effects induced by EM-2. We can conclude that the orexigenic effect of μ-opioid receptor activation by EM-2 could be related to both inhibition of CRH and stimulation of dopamine and norepinephrine levels, in the hypothalamus.

Gz- and not Gi-proteins are coupled to pre-junctional μ-opioid receptors in bovine airways

We investigated the signal transmission pathway by which activation of μ-opioid receptors attenuates acetylcholine (ACh) release in bovine trachealis. Electrical stimulation (ES)-induced [3H]-ACh release was determined in bovine tracheal smooth muscle strips pre-incubated with either the Gi-protein inhibitor pertussis toxin (PTX, 500ng/ml and 1μg/ml) or the Gz-protein specific inhibitor arachidonic acid (AA, 10−6M and 10−5M) and then treated with DAMGO (D-Ala2,N-MePhe4,Gly-ol5-enkephalin) 10−5M. Indomethacin 10−5M was used to block AA cascade. The inhibitory effect of DAMGO on ES-induced [3H]-ACh release was PTX-insensitive, but, by contrast, ablated by AA in a concentration-dependent manner. AA 10−5M alone reduced [3H]-ACh release, an effect that was prevented by iberiotoxin 10−7M, suggesting an involvement of Ca2+-activated K+-channels. Western blot analysis consistently showed immunoreactive bands against a specific antibody anti-Gz-α subunit at ∼40kDa, consistent with the presence of Gz-protein. The present findings suggest that in isolated bovine trachealis, activation of μ-opioid receptors inhibits ACh-release through a signal transmission pathway involving Gz-protein rather than Gi-protein.

Treatment of Opioid-Induced Constipation: Focus on the Peripheral μ-Opioid Receptor Antagonist Methylnaltrexone

Most prescribed opioids exert their analgesic effects via activation of central μ-opioid receptors. However, μ-opioid receptors are also located in the gastrointestinal (GI) tract, and activation of these receptors by opioids can lead to GI-related adverse effects, in particular opioid-induced constipation (OIC). OIC has been associated with increased use of healthcare resources, increased healthcare costs, and decreased quality of life for patients. Nonpharmacologic (e.g., increased fiber uptake) and pharmacologic agents (e.g., laxatives) may be considered for the treatment and prevention of OIC. However, many interventions, such as laxatives alone, are generally insufficient to reverse OIC because they do not target the underlying cause of OIC, opioid activation of μ-opioid receptors in the GI tract. Therefore, there has been keen interest in antagonism of the μ-opioid receptor in the periphery to inhibit the effects of opioids in the GI tract. In this review, currently available pharmacologic therapies for the treatment and prevention of OIC are summarized briefly, with a primary focus on the administration of the peripheral μ-opioid receptor antagonist methylnaltrexone bromide in patients with OIC and advanced illness who are receiving palliative care. Also, clinical trial data of methylnaltrexone treatment in patients with OIC and other pain conditions (i.e., chronic noncancer pain and pain after orthopedic surgery) are reviewed. Data support that methylnaltrexone is efficacious for the treatment of OIC and has a favorable tolerability profile.

Involvement of supraspinal and peripheral naloxonazine-insensitive opioid receptor sites in the expression of μ-opioid receptor agonist-induced physical dependence

Withdrawal syndrome after the cessation of μ-opioid receptor agonists remains an obstacle in the clinical treatment of pain. There is limited information available on the mechanisms that underlie the expression of the withdrawal signs of opioids, and especially regarding the involvement of μ-opioid receptor subtypes and the location of the responsible opioid receptors. Therefore, the present study was designed to determine the mechanism of the expression of withdrawal signs in μ-opioid receptor agonist-dependent mice. Morphine-, oxycodone- and fentanyl-dependent mice showed a marked loss of body-weight and other signs of withdrawal after a naloxone challenge. Interestingly, the phenotype of the withdrawal signs for morphine and oxycodone was different from that of fentanyl. Furthermore, pretreatment with naloxonazine (so-called μ1-opioid receptor antagonist), did not significantly alter the withdrawal signs precipitated by naloxone in these μ-opioid receptor agonist-dependent mice, whereas the peripherally limited opioid receptor antagonist naloxone methiodide significantly increased the loss of body-weight accompanied by diarrhea, indicating that a peripheral naloxonazine-insensitive site for opioid receptors, as an adaptation mechanism, plays an important role in the expression of at least the loss of body-weight. On the other hand, i.c.v. treatment with naloxone methiodide potently induced jumping behavior and trembling in morphine-dependent mice. These results indicate that the prolonged activation of supraspinal μ-opioid receptors plays a role in most of the physical dependence induced by μ-opioid receptor agonists in mice. Thus, the withdrawal symptoms observed after the cessation of μ-opioid receptor agonists are distinctly regulated though supraspinal and peripheral naloxonazine-insensitive sites of μ-opioid receptors.

A Novel Membrane Estrogen Receptor Activated by STX Induces Female Sexual Receptivity through an Interaction with mGluR1a

Membrane initiated estradiol signaling has been shown to be vital for multiple physiological processes. Several receptors have been proposed to mediate the actions of estradiol at the membrane. Here, we examined the ability of STX, an agonist of a novel putative membrane estrogen receptor, to activate sexually receptive behavior in the female rat. Infusions of STX into the arcuate nucleus of the hypothalamus resulted in the activation and internalization of μ-opioid receptors in the medial preoptic nucleus, an action that is required for lordosis behavior. Indeed, STX was able to augment sexual receptivity in female rats given a sub-behavioral dose of estradiol. However, if the mGluR1a antagonist, LY367,385, was administered prior to STX, its circuit-activating effects, the internalization of μ-opioid receptors, were lost. This suggests that the receptor stimulated by STX activates rapid membrane-initiated signaling through an interaction with mGluR1a - an effect previously described for estrogen receptor-α at the membrane.

Effects of μ-opoiod receptor on proliferation and migration of human epidermal stem cells in vitro

Objective To investigate the affects of μ-opioid receptor activation on proliferation and migration of epidermal stem cells (ESCs) in vitro.Methods Primary human ESCs (hESCs) were isolated and cultured,and marked by both integrin β31 and eytokeratin 19 (CK19) to be identified.The hESCs of the second generation were cultured with K-SFM supplemented with 1 nmol/L β-endorphins in group A,with K-SFM supplemented with 10 nmol/L naloxone and 1 nmol/L β-endorphins in group B,and with K-SFM supplemented with no drugs in group C.The proliferative ability of hESCs in each group was determined by using methyl thiazol tetrazolium (MTT) assay for 5 consecutive days.The 100 μm scratch wounds were created by scraping confluent hESCs plated on Petri-dishes with a sterile pipette tip in vitro.The pictures of migrating cells from the wound edge were taken at 24,48,72 and 96 h after incubarton with different media.The wound healing rate was calculated by Image-Pro Plus software at 72 h.Results Cultured primary hESCs could adhere to the wall and showed cobblestone-like shape.Immunofluorescence staining showed the positive results for integrin β1 and CK19.The proliferation rate of hESCs detected hy using MTT assay was significantly higher in group A than in groups C,visibly higher in group C than in group B during the day 2 to day 5,and the difference between each two groups was statistically significant (P ＜ 0.05).In group A,the number of cells migrating the wound edge was greater than in group C,and less in group B than in group C at 24 h.The wound healing rate in groups A,B and C at 72 h was (91.80 ± 0.05) ％,(79.40 ± 0.06) ％,and (86.30 ± 0.75) ％ respectively,showing significant differences among groups (P ＜ 0.05).Conclusion The activation of μ-opioid receptor can promote the proliferation and migration of hESCs in vitro.The μ-opioid receptor may be involved in some modutory processes of skin metabolisms. Key words: Epidermal stem cells ; Opioid receptor ; Proliferation ; Migration

A Continuous, Fluorescence-based Assay of µ-Opioid Receptor Activation in AtT-20 Cells

Opioids are widely prescribed analgesics, but their use is limited due to development of tolerance and addiction, as well as high variability in individual response. The development of improved opioid analgesics requires high-throughput functional assays to assess large numbers of potential opioid ligands. In this study, we assessed the ability of a proprietary “no-wash” fluorescent membrane potential dye to act as a reporter of µ–opioid receptor (MOR) activation and desensitization via activation of G-protein-coupled inwardly rectifying potassium channels. AtT-20 cells stably expressing mouse MOR were assayed in 96-well plates using the Molecular Devices FLIPR membrane potential dye. Dye emission intensity decreased upon membrane hyperpolarization. Fluorescence decreased in a concentration-dependent manner upon application of a range of opioid ligands to the cells, with high-efficacy agonists producing a decrease of 35% to 40% in total fluorescence. The maximum effect of morphine faded in the continued presence of agonist, reflecting receptor desensitization. The effects of opioids were prevented by prior treatment with pertussis toxin and blocked by naloxone. We have demonstrated this assay to be an effective method for assessing ligand signaling at MOR, which may potentially be scaled up as an additional high-throughput screening technique for characterizing novel opioid ligands.

Design and functional evaluation of an optically active μ-opioid receptor

The use of opioids, which achieve therapeutic analgesia through activation of μ-opioid receptors, are limited in the management of chronic pain by adverse effects including tolerance and addiction. Optogenetics is an emerging approach of designing molecular targets that can produce cell-specific receptor-mediated analgesia with minimal side effects. Here we report the design and functional characterization of a chimeric μ-opioid receptor that could be photoactivated to trigger intracellular signaling. A prototype optoactive μ-opioid receptor (optoMOR) was designed by replacing the intracellular domains from rhodopsin with those of the native μ-opioid receptor and was transiently expressed in human embryonic kidney (HEK293) cells. Expression and distribution of the protein were confirmed by immunocytochemistry. The signal-transduction mechanisms induced by photoactivation of the optoMOR were evaluated and compared with the native μ-opioid receptor stimulation by an agonist, D-Ala2, N-MePhe4, Gly-ol-enkephalin (DAMGO). Cells were depolarized by extracellular potassium and the depolarization-induced calcium (Ca2+) influx was quantified by using Fura-2 imaging. The forskolin-stimulated adenylate cyclase/cAMP cascade was evaluated by ELISA or western blotting of brain-derived neurotrophic factor (BDNF) and the phosphorylation of cAMP response element binding protein (CREB). The optoMOR protein distribution was observed intracellularly and on the plasma membrane similar to the native μ-opioid receptor in HEK293 cells. Photoactivation of optoMOR decreased the Ca2+ influx and inhibited the forskolin-induced cAMP generation, activation of CREB, and BDNF levels in optoMOR-expressing cells similar to the activation of native μ-opioid receptor by DAMGO. Thus the current study has accomplished the design of a prototype optoMOR and characterized the cellular signaling mechanisms activated by light stimulation of this receptor.