Total-body imaging of mu-opioid receptors with [11C]carfentanil in non-human primates.

PURPOSE: Mu-opioid receptors (MORs) are widely expressed in the central nervous system (CNS), peripheral organs, and immune system. This study measured the whole body distribution of MORs in rhesus macaques using the MOR selective radioligand [11C]carfentanil ([11C]CFN) on the PennPET Explorer. Both baseline and blocking studies were conducted using either naloxone or GSK1521498 to measure the effect of the antagonists on MOR binding in both CNS and peripheral organs. METHODS: The PennPET Explorer was used for MOR total-body PET imaging in four rhesus macaques using [11C]CFN under baseline, naloxone pretreatment, and naloxone or GSK1521498 displacement conditions. Logan distribution volume ratio (DVR) was calculated by using a reference model to quantitate brain regions, and the standard uptake value ratios (SUVRs) were calculated for peripheral organs. The percent receptor occupancy (%RO) was calculated to establish the blocking effect of 0.14 mg/kg naloxone or GSK1521498. RESULTS: The %RO in MOR-abundant brain regions was 75-90% for naloxone and 72-84% for GSK1521498 in blocking studies. A higher than 90% of %RO were observed in cervical spinal cord for both naloxone and GSK1521498. It took approximately 4-6 min for naloxone or GSK1521498 to distribute to CNS and displace [11C]CFN from the MOR. A smaller effect was observed in heart wall in the naloxone and GSK1521498 blocking studies. CONCLUSION: [11C]CFN total-body PET scans could be a useful approach for studying mechanism of action of MOR drugs used in the treatment of acute and chronic opioid use disorder and their effect on the biodistribution of synthetic opioids such as CFN. GSK1521498 could be a potential naloxone alternative to reverse opioid overdose.

Presynaptic inhibition of excitatory synaptic transmission from the calcitonin gene-related peptide-containing parabrachial neurons to the central amygdala in mice - unexpected influence of systemic inflammation thereon.

The monosynaptic connection from the lateral parabrachial nucleus (LPB) to the central amygdala (CeA) serves as a fundamental pathway for transmitting nociceptive signals to the brain. The LPB receives nociceptive information from the dorsal horn and spinal trigeminal nucleus and sends it to the "nociceptive" CeA, which modulates pain-associated emotions and nociceptive sensitivity. To elucidate the role of densely expressed mu-opioid receptors (MORs) within this pathway, we investigated the effects of exogenously applied opioids on LPB-CeA synaptic transmission, employing optogenetics in mice expressing channelrhodopsin-2 in LPB neurons with calcitonin gene-related peptide (CGRP). A MOR agonist ([D-Ala2,N-Me-Phe4,Glycinol5]-enkephalin, DAMGO) significantly reduced the amplitude of light-evoked excitatory postsynaptic currents (leEPSCs), in a manner negatively correlated with an increase in the paired-pulse ratio. An antagonist of MORs significantly attenuated these effects. Notably, this antagonist significantly increased leEPSC amplitude when applied alone, an effect further amplified in mice subjected to lipopolysaccharide injection 2 h before brain isolation, yet not observed at the 24-h mark. We conclude that opioids could shut off the ascending nociceptive signal at the LPB-CeA synapse through presynaptic mechanisms. Moreover, this gating process might be modulated by endogenous opioids, and the innate immune system influences this modulation.

Differential impact of two critical respiratory centres in opioid-induced respiratory depression in awake mice.

KEY POINTS: The main cause of death from opioid overdose is respiratory depression due to the activation of µ-opioid receptors (MORs). We conditionally deleted MORs from neurons in two key areas of the brainstem respiratory circuitry (the Kölliker-Fuse nucleus (KF) and pre-Bötzinger complex (preBötC)) to determine their role in opioid-induced respiratory disturbances in adult, awake mice. Deletion of MORs from KF neurons attenuated respiratory rate depression at all doses of morphine. Deletion of MORs from preBötC neurons attenuated rate depression at the low dose, but had no effect on rate following high doses of morphine. Instead, high doses of morphine increased the occurrence of apnoeas. The results indicate that opioids affect distributed key areas of the respiratory network in a dose-dependent manner and countering the respiratory effects of high dose opioids via the KF may be an effective approach to combat overdose. ABSTRACT: The primary cause of death from opioid overdose is respiratory failure. High doses of opioids cause severe rate depression and increased risk of fatal apnoea, which correlate with increasing irregularities in breathing pattern. µ-Opioid receptors (MORs) are widely distributed throughout the brainstem respiratory network, but the mechanisms underlying respiratory depression are poorly understood. The medullary pre-Bötzinger complex (preBötC) and the pontine Kölliker-Fuse nucleus (KF) are considered critical for inducing opioid-related respiratory disturbances. We used a conditional knockout approach to investigate the roles and relative contribution of MORs in KF and preBötC neurons in opioid-induced respiratory depression in awake adult mice. The results revealed dose-dependent and region-specific opioid effects on the control of both respiratory rate and pattern. Respiratory depression induced by an anti-nociceptive dose of morphine was significantly attenuated following deletion of MORs from either the KF or the preBötC, suggesting cumulative network effects on respiratory rate control at low opioid doses. Deletion of MORs from KF neurons also relieved rate depression at near-maximal respiratory depressant doses of morphine. Meanwhile, deletion of MORs from the preBötC had no effect on rate following administration of high doses of morphine. Instead, a severe ataxic breathing pattern emerged with many apnoeas. We conclude that opioids affect distributed areas of the respiratory network and opioid-induced respiratory depression cannot be attributed to only one area in isolation. However, countering the effects of near maximal respiratory depressant doses of opioids in the KF may be a powerful approach to combat opioid overdose.

Mu opioid receptors inhibit GABA release from parvalbumin interneuron terminals onto CA1 pyramidal cells.

Selectively activating (by optogenetics) parvalbumin-expressing (PV) interneurons induces GABA release onto CA1 pyramidal cells. Here we report that this release was attenuated by presynaptic mu opioid receptors (MORs) activation. On the other hand, conventional electric shock, presumably activating non-selectively presynaptic GABAergic terminals, also induced GABA release; however, this release showed relatively limited depression by MORs activation. The data suggest that MORs specifically inhibit GABA release from PV terminals and therefore, further support the idea that MORs contribute to homeostasis in CA1 neuro-circuit.

Behavioral Pharmacology of Drugs Acting at Mu Opioid Receptors.

Despite the therapeutic utility of opioids for relieving pain, other behavioral effects, including their potential for abuse and overdose, can be quite detrimental to individuals as well as society and have contributed to the ongoing opioid crisis. The dramatic escalation in overdose deaths over the last 15 years was initially driven by abuse of prescription opioids, although abuse of heroin, fentanyl, and fentanyl analogs has been increasing, largely due to increased availability and lower cost compared with prescription opioids. All of these opioids share pharmacological properties, acting as agonists at mu opioid receptors, and produce similar behavioral effects, including abuse-related, pain-relieving, dependence-producing, and respiratory-depressant effects. Despite their similarities, opioids are not pharmacologically identical. In fact, drugs that act at mu opioid receptors, including abused opioids, can vary on a number of dimensions, including pharmacological efficacy, drug-receptor interactions, receptor selectivity, and pharmacokinetics. Overall, these differences impact behavioral effects of drugs acting at mu opioid receptors, and this chapter describes variations in those behavioral effects and how these differences continue to provide new strategies that can be developed to address the ongoing opioid epidemic.

Central Amygdala Circuits Mediate Hyperalgesia in Alcohol-Dependent Rats.

Alcohol withdrawal symptoms contribute to excessive alcohol drinking and relapse in alcohol-dependent individuals. Among these symptoms, alcohol withdrawal promotes hyperalgesia, but the neurological underpinnings of this phenomenon are not known. Chronic alcohol exposure alters cell signaling in the central nucleus of the amygdala (CeA), and the CeA is implicated in mediating alcohol dependence-related behaviors. The CeA projects to the periaqueductal gray (PAG), a region critical for descending pain modulation, and may have a role in alcohol withdrawal hyperalgesia. Here, we tested the roles of (1) CeA projections to PAG, (2) CeA melanocortin signaling, and (3) PAG µ-opioid receptor signaling in mediating thermal nociception and alcohol withdrawal hyperalgesia in male Wistar rats. Our results demonstrate that alcohol dependence reduces GABAergic signaling from CeA terminals onto PAG neurons and alters the CeA melanocortin system, that CeA-PAG projections and CeA melanocortin signaling mediate alcohol withdrawal hyperalgesia, and that µ-opioid receptors in PAG filter CeA effects on thermal nociception.SIGNIFICANCE STATEMENT Hyperalgesia is commonly seen in individuals with alcohol use disorder during periods of withdrawal, but the neurological underpinnings behind this phenomenon are not completely understood. Here, we tested whether alcohol dependence exerts its influence on pain modulation via effects on the limbic system. Using behavioral, optogenetic, electrophysiological, and molecular biological approaches, we demonstrate that central nucleus of the amygdala (CeA) projections to periaqueductal gray mediate thermal hyperalgesia in alcohol-dependent and alcohol-naive rats. Using pharmacological approaches, we show that melanocortin receptor-4 signaling in CeA alters alcohol withdrawal hyperalgesia, but this effect is not mediated directly at synaptic inputs onto periaqueductal gray-projecting CeA neurons. Overall, our findings support a role for limbic influence over the descending pain pathway and identify a potential therapeutic target for treating hyperalgesia in individuals with alcohol use disorder .

The role of the opioid system in decision making and cognitive control: A review.

The opioid system regulates affective processing, including pain, pleasure, and reward. Restricting the role of this system to hedonic modulation may be an underestimation, however. Opioid receptors are distributed widely in the human brain, including the more "cognitive" regions in the frontal and parietal lobes. Nonhuman animal research points to opioid modulation of cognitive and decision-making processes. We review emerging evidence on whether acute opioid drug modulation in healthy humans can influence cognitive function, such as how we choose between actions of different values and how we control our behavior in the face of distracting information. Specifically, we review studies employing opioid agonists or antagonists together with experimental paradigms of reward-based decision making, impulsivity, executive functioning, attention, inhibition, and effort. Although this field is still in its infancy, the emerging picture suggests that the mu-opioid system can influence higher-level cognitive function via modulation of valuation, motivation, and control circuits dense in mu-opioid receptors, including orbitofrontal cortex, basal ganglia, amygdalae, anterior cingulate cortex, and prefrontal cortex. The framework that we put forward proposes that opioids influence decision making and cognitive control by increasing the subjective value of reward and reducing aversive arousal. We highlight potential mechanisms that might underlie the effects of mu-opioid signaling on decision making and cognitive control and provide directions for future research.

The mu-opioid receptor is a molecular marker for poor prognosis in hepatocellular carcinoma and represents a potential therapeutic target.

BACKGROUND: Opioid receptors are implicated in cancer progression and long-term patient outcomes. However, the prognostic significance, underlying mechanisms, and therapeutic value of mu-opioid receptor (MOP) in hepatocellular carcinoma (HCC) remain unclear. METHODS: MOP expression in human biopsy HCC samples was evaluated using RNA microarrays, quantitative real-time polymerase chain reaction (qRT-PCR), and immunochemical analyses. Molecular and cellular techniques, including siRNA-mediated depletion and lentiviral vector-mediated overexpression, were used to elucidate the functions and mechanisms of MOP. The effect of the MOP agonist morphine in HCC was evaluated both in vitro and in vivo. The therapeutic value of MOP inhibitors in HCC progression and metastasis was investigated with in vitro experiments and subcutaneous and orthotopic HCC mouse models in vivo. RESULTS: Through microarray analysis and qRT-PCR, we identified that MOP is highly expressed in human HCC tumours. High MOP expression in HCC tumours was confirmed by immunocytochemistry and correlated with aggressive clinicopathological features and a worse prognosis. Depletion of MOP suppressed cell proliferation, migration, and invasion, whereas overexpression of MOP promoted cell growth and metastasis in human HCC cell lines. Both clinical and biological evidence revealed that MOP-mediated epithelial-mesenchymal transition promotes HCC metastasis and poor prognosis. Morphine promotes cell proliferation, migration, and invasion in vitro and in vivo in mouse models. More importantly, MOP inhibitors suppressed cell growth, invasion, and metastasis in vitro and in the subcutaneous and orthotopic xenograft models. CONCLUSIONS: MOP plays a key oncogenic function in hepatocarcinogenesis. Its overexpression is associated with poor prognosis in patients with HCC. Furthermore, MOP inhibitors may be a promising strategy for HCC therapy.

Effect of electromagnetic field on cyclic adenosine monophosphate (cAMP) in a human mu-opioid receptor cell model.

During the cell communication process, endogenous and exogenous signaling affect normal as well as pathological developmental conditions. Exogenous influences such as extra-low-frequency electromagnetic field (EMF) have been shown to effect pain and inflammation by modulating G-protein receptors, down-regulating cyclooxygenase-2 activity, and affecting the calcium/calmodulin/nitric oxide pathway. Investigators have reported changes in opioid receptors and second messengers, such as cyclic adenosine monophosphate (cAMP), in opiate tolerance and dependence by showing how repeated exposure to morphine decreases adenylate cyclase activity causing cAMP to return to control levels in the tolerant state, and increase above control levels during withdrawal. Resonance responses to biological systems using exogenous EMF signals suggest that frequency response characteristics of the target can determine the EMF biological response. In our past research we found significant down regulation of inflammatory markers tumor necrosis factor alpha (TNF-α) and nuclear factor kappa B (NFκB) using 5 Hz EMF frequency. In this study cAMP was stimulated in Chinese Hamster Ovary (CHO) cells transfected with human mu-opioid receptors, then exposed to 5 Hz EMF, and outcomes were compared with morphine treatment. Results showed a 23% greater inhibition of cAMP-treating cells with EMF than with morphine. In order to test our results for frequency specific effects, we ran identical experiments using 13 Hz EMF, which produced results similar to controls. This study suggests the use of EMF as a complementary or alternative treatment to morphine that could both reduce pain and enhance patient quality of life without the side-effects of opiates.

Association of smoking with µ-opioid receptor availability before and during naltrexone blockade in alcohol-dependent subjects.

Persons with a history of alcohol dependence are more likely to use tobacco and to meet criteria for nicotine dependence compared with social drinkers or non-drinkers. The high levels of comorbidity of nicotine and alcohol use and dependence are thought to be related to interactions between nicotinic, opioid and dopamine receptors in mesolimbic regions. The current study examined whether individual differences in regional µ-opioid receptor (MOR) availability were associated with tobacco use, nicotine dependence and level of nicotine craving in 25 alcohol-dependent (AD) subjects. AD subjects completed an inpatient protocol, which included medically supervised alcohol withdrawal, monitored alcohol abstinence, transdermal nicotine maintenance (21 mg/day) and Positron Emission Tomography (PET) imaging using the MOR agonist [(11) C]-carfentanil (CFN) before (basal scan) and during treatment with 50 mg/day naltrexone (naltrexone scan). Subjects who had higher scores on the Fagerström Nicotine Dependence Test had significantly lower basal scan binding potential (BPND ) across mesolimbic regions, including the amygdala, cingulate, globus pallidus, thalamus and insula. Likewise, the number of cigarettes per day was negatively associated with basal scan BPND in mesolimbic regions. Higher nicotine craving was significantly associated with lower BPND in amygdala, globus pallidus, putamen, thalamus and ventral striatum. Although blunted during naltrexone treatment, the negative association was maintained for nicotine dependence and cigarettes per day, but not for nicotine craving. These findings suggest that intensity of cigarette smoking and severity of nicotine dependence symptoms are systematically related to reduced BPND across multiple brain regions in AD subjects.

Dermorphin [D-Arg2, Lys4] (1-4) Amide Alleviates Frostbite-Induced Pain by Regulating TRP Channel-Mediated Microglial Activation and Neuroinflammation.

Cold injury or frostbite is a common medical condition that causes serious clinical complications including sensory abnormalities and chronic pain ultimately affecting overall well-being. Opioids are the first-choice drug for the treatment of frostbite-induced chronic pain; however, their notable side effects, including sedation, motor incoordination, respiratory depression, and drug addiction, present substantial obstacle to their clinical utility. To address this challenge, we have exploited peripheral mu-opioid receptors as potential target for the treatment of frostbite-induced chronic pain. In this study, we investigated the effect of dermorphin [D-Arg2, Lys4] (1-4) amide (DALDA), a peripheral mu-opioid receptor agonist, on frostbite injury and hypersensitivity induced by deep freeze magnet exposure in rats. Animals with frostbite injury displayed significant hypersensitivity to mechanical, thermal, and cold stimuli which was significant ameliorated on treatment with different doses of DALDA (1, 3, and 10 mg/kg) and ibuprofen (100 mg/kg). Further, molecular biology investigations unveiled heightened oxido-nitrosative stress, coupled with a notable upregulation in the expression of TRP channels (TRPA1, TRPV1, and TRPM8), glial cell activation, and neuroinflammation (TNF-α, IL-1ß) in the sciatic nerve, dorsal root ganglion (DRG), and spinal cord of frostbite-injured rats. Treatment with DALDA leads to substantial reduction in TRP channels, microglial activation, and suppression of the inflammatory cascade in the ipsilateral L4-L5 DRG and spinal cord of rats. Overall, findings from the present study suggest that activation of peripheral mu-opioid receptors mitigates chronic pain in rats by modulating the expression of TRP channels and suppressing glial cell activation and neuroinflammation.

Peripheral mu-opioid receptor activation by dermorphin [D-Arg2, Lys4] (1-4) amide alleviates behavioral and neurobiological aberrations in rat model of chemotherapy-induced neuropathic pain.

Paclitaxel, a frequently utilized chemotherapeutic agent, often gives rise to severe and distressing sensory neuropathy in patients undergoing chemotherapy. Unfortunately, current therapeutics for chemotherapy-induced neuropathic pain (CINP) demonstrate limited effectiveness and are burdened with the potential for central side effects such as sedation, respiratory depression, cognitive impairment, and addiction, posing substantial clinical challenges. In light of these limitations, present study is designed to investigate the therapeutic potential of Dermorphin [D-Arg2, Lys4] (1-4) amide (DALDA), a preferential peripherally acting mu-opioid receptor agonist, in rat model of CINP. The primary objective was to assess the analgesic properties of DALDA and elucidate the underlying mechanisms governing its therapeutic activity. Our findings revealed that DALDA treatment significantly ameliorated paclitaxel-induced evoked and spontaneous ongoing pain in rats without causing drug addiction and other central side effects. Molecular analyses further unveiled that paclitaxel administration resulted in increased expression of TRP channels, NR2B, voltage-gated sodium channels (VGSCs) and neuroinflammatory markers in both the dorsal root ganglion (DRG) and the spinal cord (L4-L5 region) of rats. DALDA treatment significantly downregulated ion channels (TRPs, VGSCs) and NR2B expressions, concomitant with the inhibition of microglial activation, resulting in the suppression of oxido-nitrosative stress and neuroinflammatory cascade. Findings from the current study suggests that peripheral mu-opioid receptors may offer a potential target for the treatment of patients suffering from CINP, offering new avenues for improved pain relief while minimizing central side effects.

Neuroprotective effects of naltrexone in a mouse model of post-traumatic seizures.

Traumatic Brain Injury (TBI) induces neuroinflammatory response that can initiate epileptogenesis, which develops into epilepsy. Recently, we identified anti-convulsive effects of naltrexone, a mu-opioid receptor (MOR) antagonist, used to treat drug addiction. While blocking opioid receptors can reduce inflammation, it is unclear if post-TBI seizures can be prevented by blocking MORs. Here, we tested if naltrexone prevents neuroinflammation and/or seizures post-TBI. TBI was induced by a modified Marmarou Weight-Drop (WD) method on 4-week-old C57BL/6J male mice. Mice were placed in two groups: non-telemetry assessing the acute effects or in telemetry monitoring for interictal events and spontaneous seizures both following TBI and naltrexone. Molecular, histological and neuroimaging techniques were used to evaluate neuroinflammation, neurodegeneration and fiber track integrity at 8 days and 3 months post-TBI. Peripheral immune responses were assessed through serum chemokine/cytokine measurements. Our results show an increase in MOR expression, nitro-oxidative stress, mRNA expression of inflammatory cytokines, microgliosis, neurodegeneration, and white matter damage in the neocortex of TBI mice. Video-EEG revealed increased interictal events in TBI mice, with 71% mice developing post-traumatic seizures (PTS). Naltrexone treatment ameliorated neuroinflammation, neurodegeneration, reduced interictal events and prevented seizures in all TBI mice, which makes naltrexone a promising candidate against PTS, TBI-associated neuroinflammation and epileptogenesis in a WD model of TBI.

Single-neuron projectome-guided analysis reveals the neural circuit mechanism underlying endogenous opioid antinociception.

Endogenous opioid antinociception is a self-regulatory mechanism that reduces chronic pain, but its underlying circuit mechanism remains largely unknown. Here, we showed that endogenous opioid antinociception required the activation of mu-opioid receptors (MORs) in GABAergic neurons of the central amygdala nucleus (CEA) in a persistent-hyperalgesia mouse model. Pharmacogenetic suppression of these CEAMOR neurons, which mimics the effect of MOR activation, alleviated the persistent hyperalgesia. Furthermore, single-neuron projection analysis revealed multiple projectome-based subtypes of CEAMOR neurons, each innervating distinct target brain regions. We found that the suppression of axon branches projecting to the parabrachial nucleus (PB) of one subtype of CEAMOR neurons alleviated persistent hyperalgesia, indicating a subtype- and axonal-branch-specific mechanism of action. Further electrophysiological analysis revealed that suppression of a distinct CEA-PB disinhibitory circuit controlled endogenous opioid antinociception. Thus, this study identified the central neural circuit that underlies endogenous opioid antinociception, providing new insight into the endogenous pain modulatory mechanisms.

Association of the OPRM1 variant rs1799971 (A118G) and clinical manifestations in tramadol poisoned patients: a cross-sectional study.

INTRODUCTION: The opioid receptor mu1 is a protein coding gene that can have different codes for a protein and may have variations (polymorphisms) affecting how opioids work. The aim of this study was to investigate the prevalence of the most common opioid receptor mu1 polymorphism (A118G) and any relationship between this polymorphism and features following tramadol overdose. MATERIALS AND METHODS: This was a cross-sectional study of patients admitted with tramadol poisoning to an Iranian hospital. These patients were not taking any other drugs or medications and had no history of seizures. RESULTS: The results showed that among the 83 patients included in the study, 57 (69 per cent) had the AA genotype, 25 (30 per cent) had the AG genotype, and one (1 per cent) had the GG genotype for the opioid receptor mu1 A118G polymorphism. Nausea and/or vomiting occurred in nine (11 per cent) patients and dizziness in 38 (46 per cent) patients. Serious adverse events included seizures in 51 (60 per cent) patients and respiratory failure requiring mechanical ventilation in 21 (25 per cent) patients. However, there was no significant association between the opioid receptor mu1 A118G polymorphism and these adverse events. DISCUSSION: In our study, the frequency of the A allele was greater than the G allele, and the AA genotype was more prevalent than AG. The GG genotype was the least common among the polymorphisms of opioid receptor mu1 rs1799971. There was no significant association between the opioid receptor mu1 A118G polymorphism and symptoms in tramadol-poisoned patients. Although these allele proportions are similar to the results reported in other Caucasian populations, they are dissimilar to the findings in Chinese and Singaporean populations. In these Asian studies, the predominant allele was the G allele. It has been suggested that a mutated G allele will decrease the production of opioid receptor mu1-related messenger ribonucleic acid and related proteins, leading to fewer mu-opioid receptors in the brain. CONCLUSIONS: This study found no significant association between the opioid receptor mu1 A118G polymorphism and adverse outcomes in tramadol-poisoned patients. However, more research is needed to draw more definitive conclusions due to the limited evidence and variability of opioid receptor mu1 polymorphisms in different populations.

Paradoxical potentiation of the exercise pressor reflex by endomorphin 2 in the presence of naloxone.

When contracting muscles are freely perfused, the acid-sensing ion channel 3 (ASIC3) on group IV afferents plays a minor role in evoking the exercise pressor reflex. We recently showed in isolated dorsal root ganglion neurons innervating the gastrocnemius muscles that two mu opioid receptor agonists, namely endomorphin 2 and oxycodone, potentiated the sustained inward ASIC3 current evoked by acidic solutions. This in vitro finding prompted us to determine whether endomorphin 2 and oxycodone, when infused into the arterial supply of freely perfused contracting hindlimb muscles, potentiated the exercise pressor reflex. We found that infusion of endomorphin 2 and naloxone in decerebrated rats potentiated the pressor responses to contraction of the triceps surae muscles. The endomorphin 2-induced potentiation of the pressor responses to contraction was prevented by infusion of APETx2, an ASIC3 antagonist. Specifically, the peak pressor response to contraction averaged 19.3 ± 5.6 mmHg for control (n = 10), 27.2 ± 8.1 mmHg after naloxone and endomorphin 2 infusion (n = 10), and 20 ± 8 mmHg after APETx2 and endomorphin 2 infusion (n = 10). Infusion of endomorphin 2 and naloxone did not potentiate the pressor responses to contraction in ASIC3 knockout rats (n = 6). Partly similar findings were observed when oxycodone was substituted for endomorphin 2. Oxycodone infusion significantly increased the exercise pressor reflex over its control level, but subsequent APETx2 infusion failed to restore the increase to its control level (n = 9). The peak pressor response averaged 23.1 ± 8.6 mmHg for control (n = 9), 33.2 ± 11 mmHg after naloxone and oxycodone were infused (n = 9), and 27 ± 8.6 mmHg after APETx2 and oxycodone were infused (n = 9). Our data suggest that after opioid receptor blockade, ASIC3 stimulation by the endogenous mu opioid, endomorphin 2, potentiated the exercise pressor reflex.NEW & NOTEWORTHY This paper provides the first in vivo evidence that endomorphin 2, an endogenous opioid peptide, can paradoxically increase the magnitude of the exercise pressor reflex by an ASIC3-dependent mechanism even when the contracting muscles are freely perfused.

Opioid requirement, opioid receptor expression, and clinical outcomes in patients with advanced prostate cancer.

BACKGROUND: Preclinical studies show that opioids stimulate angiogenesis and tumor progression through the mu opioid receptor (MOR). Although MOR is overexpressed in several human malignancies, the effect of chronic opioid requirement on cancer progression or survival has not been examined in humans. METHODS: We performed a retrospective analysis on 113 patients identified in the Minneapolis VA Tumor Registry (test cohort) and 480 patients from the national VA Central Cancer Registry (validation cohort) who had been diagnosed with stage IV prostate cancer between 1995 and 2010 to examine whether MOR expression or opioid requirement is associated with disease progression and survival. All opioids were converted to oral morphine equivalents for comparison. Laser scanning confocal microscopy was used to analyze MOR immunoreactivity in prostate cancer biopsies. The effects of variables on outcomes were analyzed in univariable and multivariable models. RESULTS: In patients with metastatic prostate cancer, MOR expression and opioid requirement were independently associated with inferior progression-free survival (hazard ratio [HR] 1.65, 95% confidence interval [CI] 1.33-2.07, P<.001 and HR 1.08, 95% CI 1.03-1.13, P<.001, respectively) and overall survival (HR 1.55, 95% CI 1.20-1.99, P<.001 and HR 1.05, 95% CI 1.00-1.10, P = .031, respectively). The validation cohort confirmed that increasing opioid requirement was associated with worse overall survival (HR 1.005, 95% CI 1.002-1.008, P = .001). CONCLUSION: Higher MOR expression and greater opioid requirement are associated with shorter progression-free survival and overall survival in patients with metastatic prostate cancer. Nevertheless, clinical practice should not be changed until prospective randomized trials show that opioid use is associated with inferior clinical outcomes, and that abrogation of the peripheral activities of opioids ameliorates this effect.

Multiple episodes of sodium depletion in the rat: a remodeling of the electrical properties of median preoptic nucleus neurons.

In rat brain, the detection and integration of chemosensory and neural signals are achieved, inter alia, by the median preoptic nucleus (MnPO) during a disturbance of the hydromineral balance. This is allowed through the presence of the sodium (Na(+) ) sensor neurons. Interestingly, enkephalins and mu-opioid receptors (µ-ORs) are known for their role in ingestive behaviors and have previously been shown to regulate the excitability of MnPO neurons following a single Na(+) depletion. However, little is known about the role of these µ-ORs in the response enhancement following repeated Na(+) challenge. Therefore, we used whole-cell recordings in acute brain slices to determine neuronal plasticity in the electrical properties of the MnPO Na(+) sensor-specific neuronal population following multiple Na(+) depletions. Our results show that the population of Na(+) sensor neurons was represented by 80% of MnPO neurons after a single Na(+) depletion and was reduced after three Na(+) depletions. Interestingly, the subpopulation of Na(+) sensors responding to D-Ala(2) ,N-MePhe(4) ,Gly-ol-enkephalin (DAMGO), a specific µ-OR agonist, represented 11% of MnPO neurons after a single Na(+) depletion and the population doubled after three Na(+) depletions. Moreover, Na(+) sensor neurons displayed modifications in the discharge pattern distribution and shape of calcium action potentials after three Na(+) depletions but these changes did not occur in Na(+) sensors responding to DAMGO. Thus, the reinforced µ-OR functionality in Na(+) sensors might take place to control the neuronal hyperexcitability and this plasticity in opioid-sensitive and Na(+) detection MnPO networks might sustain the enhanced salt ingestion induced by repeated exposure to Na(+) depletion.

CB1 and CB2 receptors are novel molecular targets for Tamoxifen and 4OH-Tamoxifen.

Tamoxifen (Tam) is classified as a selective estrogen receptor modulator (SERM) and is used for treatment of patients with ER-positive breast cancer. However, it has been shown that Tam and its cytochrome P450-generated metabolite 4-hydroxy-Tam (4OH-Tam) also exhibit cytotoxic effects in ER-negative breast cancer cells. These observations suggest that Tam and 4OH-Tam can produce cytotoxicity via estrogen receptor (ER)-independent mechanism(s) of action. The molecular targets responsible for the ER-independent effects of Tam and its derivatives are poorly understood. Interestingly, similar to Tam and 4OH-Tam, cannabinoids have also been shown to exhibit anti-proliferative and apoptotic effects in ER-negative breast cancer cells, and estrogen can regulate expression levels of cannabinoid receptors (CBRs). Therefore, this study investigated whether CBRs might serve as novel molecular targets for Tam and 4OH-Tam. We report that both compounds bind to CB1 and CB2Rs with moderate affinity (0.9-3 µM). Furthermore, Tam and 4OH-Tam exhibit inverse activity at CB1 and CB2Rs in membrane preparations, reducing basal G-protein activity. Tam and 4OH-Tam also act as CB1/CB2R-inverse agonists to regulate the downstream intracellular effector adenylyl cyclase in intact cells, producing concentration-dependent increases in intracellular cAMP. These results suggest that CBRs are molecular targets for Tam and 4OH-Tam and may contribute to the ER-independent cytotoxic effects reported for these drugs. Importantly, these findings also indicate that Tam and 4OH-Tam might be used as structural scaffolds for development of novel, efficacious, non-toxic cancer drugs acting via CB1 and/or CB2Rs.

Loperamide, a peripheral Mu-Opioid receptor agonist, attenuates chemotherapy-induced neuropathic pain in rats.

Opioids are employed in the management of chemotherapy-induced neuropathic pain (CINP) when other pain management approaches have failed and proven ineffective. However, their use in CINP is generally considered as a second-line or adjunctive therapy owing to their central side effects and development of tolerance with their long-term usage. Targeting peripheral sites may offer several advantages over the conventional CNS-based approaches as peripheral targets modulate pain signals at their source, thereby relieving pain with higher specificity, efficacy and minimizing adverse effects associated with off-site CNS actions. Therefore, present study was designed with an aim to investigate the effect of loperamide, a peripherally acting mu-opioid receptor agonist, on paclitaxel-induced neuropathic pain in rats and elucidate its underlying mechanism. Loperamide treatment significantly attenuated mechanical, and cold hypersensitivity and produced significant place preference behaviour in neuropathic rats indicating its potential to treat both evoked and spontaneous pain. More importantly, loperamide treatment in naïve rats did not produce place preference to drug-paired chamber pointing towards its non-addictive analgesic potential. Further, molecular investigations revealed increased expression of ion channels such as TRPA1, TRPM8; voltage-gated sodium channels (VGSCs) and neuroinflammatory markers in the dorsal root ganglion (DRG) and lumbar (L4-L5) spinal cord of neuropathic rats, which was significantly downregulated upon loperamide treatment. These findings collectively suggest that activation of peripheral mu-opioid receptors contributes to the amelioration of both evoked and spontaneous pain in neuropathic rats by downregulating TRP channels and VGSCs along with suppression of oxido-nitrosative stress and neuroinflammatory cascade.