Expression profile and immunomodulatory roles of methionine-enkephalin and delta opioid receptor in Octopus ocellatus.

In this study, the expressions and distributions of methionine-enkephalin (Met-enk) and δ opioid receptor in the nervous system of Octopus ocellatus, and the immune regulatory mechanisms of Met-enk on O. ocellatus were explored. The distributions and expressions of Met-enk and δ opioid receptor were assessed by immunohistochemistry and enzyme-linked immunosorbent assay. UV-spectrophotometer, microplate reader, and flow cytometer were used to examine the effects of different concentrations of Met-enk on phagocytosis, antioxidant effects, and body surface mucus immunity of O. ocellatus hemocytes. The data were used to study the mechanisms of Met-enk immunity regulation in O. ocellatus. According to the results, the expression levels of Met-enk and δ opioid receptor in O. ocellatus lymphocytes were higher than those in hemocytes. The expression levels of Met-enk in the ganglia of O. ocellatus decreased in the following order: pedal ganglia > cerebral ganglia > visceral ganglia > optic ganglia > stellate ganglia. Moreover, the phagocytic activity of O. ocellatus hemocytes was enhanced with increasing Met-enk concentration. With increasing Met-enk concentration, the expressions of nitric oxide, total nitric oxide synthase, inducible nitric oxide synthase, catalase, hydrogen peroxide, myeloperoxidase, reduced glutathione, α-naphthy acetate esterase, and methionine aminopeptidases decreased in serums of O. ocellatus in the experimental group compared to the blank group. Similarly, the content of reduced glutathione in the hemocytes of O. ocellatus was also lower in the experimental group than in the blank group; however, the expressions of other substances were higher compared to the blank group. Furthermore, α-naphthy acetate esterase, myeloperoxidase, and hydrogen peroxide expressions in mucus immunity trials of the body surface were lower in the experimental group compared to the blank group. These results indicate that the distributions and expressions of Met-enk and δ opioid receptor in the nervous system of O. ocellatus were related to axoplasmic transport and immune regulation mechanisms. Met-enk participates in cellular immunity, humoral immunity, and mucus immunity in the form of neurotransmitters, thereby regulating the immune response of O. ocellatus.

Digital nets conformational sampling (DNCS) - an enhanced sampling technique to explore the conformational space of intrinsically disordered peptides.

We propose digital nets conformational sampling (DNCS) - an enhanced sampling technique to explore the conformational ensembles of peptides, especially intrinsically disordered peptides (IDPs). The DNCS algorithm relies on generating history-dependent samples of dihedral variables using bitwise XOR operations and binary angle measurements (BAM). The algorithm was initially studied using met-enkephalin, a highly elusive neuropeptide. The DNCS method predicted near-native structures and the energy landscape of met-enkephalin was observed to be in direct correlation with earlier studies on the neuropeptide. Clustering analysis revealed that there are only 24 low-lying conformations of the molecule. The DNCS method has then been tested for predicting optimal conformations of 42 oligopeptides of length varying from 3 to 8 residues. The closest-to-native structures of 86% of cases are near-native and 24% of them have a root mean square deviation of less than 1.00 Å with respect to their crystal structures. The results obtained reveal that the DNCS method performs well, that too in less computational time.

HDAC6 Inhibition Reverses Cisplatin-Induced Mechanical Hypersensitivity via Tonic Delta Opioid Receptor Signaling.

Peripheral neuropathic pain induced by the chemotherapeutic cisplatin can persist for months to years after treatment. Histone deacetylase 6 (HDAC6) inhibitors have therapeutic potential for cisplatin-induced neuropathic pain since they persistently reverse mechanical hypersensitivity and spontaneous pain in rodent models. Here, we investigated the mechanisms underlying reversal of mechanical hypersensitivity in male and female mice by a 2 week treatment with an HDAC6 inhibitor, administered 3 d after the last dose of cisplatin. Mechanical hypersensitivity in animals of both sexes treated with the HDAC6 inhibitor was temporarily reinstated by a single injection of the neutral opioid receptor antagonist 6ß-naltrexol or the peripherally restricted opioid receptor antagonist naloxone methiodide. These results suggest that tonic peripheral opioid ligand-receptor signaling mediates reversal of cisplatin-induced mechanical hypersensitivity after treatment with an HDAC6 inhibitor. Pointing to a specific role for δ opioid receptors (DORs), Oprd1 expression was decreased in DRG neurons following cisplatin administration, but normalized after treatment with an HDAC6 inhibitor. Mechanical hypersensitivity was temporarily reinstated in both sexes by a single injection of the DOR antagonist naltrindole. Consistently, HDAC6 inhibition failed to reverse cisplatin-induced hypersensitivity when DORs were genetically deleted from advillin+ neurons. Mechanical hypersensitivity was also temporarily reinstated in both sexes by a single injection of a neutralizing antibody against the DOR ligand met-enkephalin. In conclusion, we reveal that treatment with an HDAC6 inhibitor induces tonic enkephalin-DOR signaling in peripheral sensory neurons to suppress mechanical hypersensitivity.SIGNIFICANCE STATEMENT Over one-fourth of cancer survivors suffer from intractable painful chemotherapy-induced peripheral neuropathy (CIPN), which can last for months to years after treatment ends. HDAC6 inhibition is a novel strategy to reverse CIPN without negatively interfering with tumor growth, but the mechanisms responsible for persistent reversal are not well understood. We built on evidence that the endogenous opioid system contributes to the spontaneous, apparent resolution of pain caused by nerve damage or inflammation, referred to as latent sensitization. We show that blocking the δ opioid receptor or its ligand enkephalin unmasks CIPN in mice treated with an HDAC6 inhibitor (latent sensitization). Our work provides insight into the mechanisms by which treatment with an HDAC6 inhibitor apparently reverses CIPN.

Methionine enkephalin(MENK) upregulated memory T cells in anti-influenza response.

Novel prophylactic drugs and vaccination strategies for protection against influenza virus should induce specific effector T-cell immune responses in pulmonary airways and peripheral lymphoid organs. Designing approaches that promote T-cell-mediated responses and memory T-cell differentiation would strengthen host resistance to respiratory infectious diseases. The results of this study showed that pulmonary delivery of MENK via intranasal administration reduced viral titres, upregulated opioid receptor MOR and DOR, increased the proportions of T-cell subsets including CD8+ T cells, CD8+ TEM cells, NP/PA-effector CD8+ TEM cells in bronchoalveolar lavage fluid and lungs, and CD4+/CD8+ TCM cells in lymph nodes to protect mice against influenza viral challenge. Furthermore, we demonstrated that, on the 10th day of infection, the proportions of CD4+ TM and CD8+ TM cells were significantly increased, which meant that a stable TCM and TEM lineage was established in the early stage of influenza infection. Collectively, our data suggested that MENK administered intranasally, similar to the route of natural infection by influenza A virus, could exert antiviral activity through upregulating T-cell-mediated adaptive immune responses against influenza virus.

The influence of the opioid pentapeptide methionine-enkephalin on seasonal and FSH-induced ovarian recrudescence in the gecko Hemidactylus frenatus.

Although methionine-enkephalin (M-ENK) is implicated in the regulation of reproductive functions in vertebrates, its function in reptiles is little understood. This study aims to elucidate the role of M-ENK on seasonal and follicle stimulating hormone (FSH)-induced ovarian recrudescence in the gecko Hemidactylus frenatus. In the first experiment, administration of 5 µg M-ENK did not affect germinal bed activity or follicular developmental stages I, II, and III (previtellogenic) and IV (vitellogenic), but there were no stage V (vitellogenic) follicles in the ovary. However, there was a significant decrease in the mean numbers of oogonia and primary oocytes in the germinal bed associated with the complete absence of stage IV and V follicles in 25 µg M-ENK-treated lizards in contrast to experimental controls. Furthermore, there was a significant decrease in gonadotropin-releasing hormone - immunoreactive (GnRH-ir) content in the median eminence (ME) and pars distalis (PD) of the pituitary gland and sparse labelling of hypothalamic GnRH-ir neurons in 25 µg M-ENK-treated lizards. In the second experiment, treatment with FSH during the regression phase of the ovarian cycle resulted in the appearance of stage IV and V follicles, in contrast to their absence in the initial controls and treatment controls. However, treatment with 25 µg M-ENK + FSH did not result in the appearance of these follicles, indicating the inhibitory effect of M-ENK on FSH-induced ovarian recrudescence. These findings suggest that M-ENK inhibits the germinal bed and vitellogenic follicular growth in a dose-dependent manner, possibly mediated through the suppression of GnRH release in the ME and PD. In addition, M-ENK may also act at the level of the ovary in the gecko.

Proenkephalin+ regulatory T cells expanded by ultraviolet B exposure maintain skin homeostasis with a healing function.

Regulatory T (Treg) cells, expressing CD25 (interleukin-2 receptor α chain) and Foxp3 transcription factor, maintain immunological self-tolerance and suppress various immune responses. Here we report a feature of skin Treg cells expanded by ultraviolet B (UVB) exposure. We found that skin Treg cells possessing a healing function are expanded by UVB exposure with the expression of an endogenous opioid precursor, proenkephalin (PENK). Upon UVB exposure, skin Treg cells were expanded with a unique TCR repertoire. Also, they highly expressed a distinctive set of genes enriched in "wound healing involved in inflammatory responses" and the "neuropeptide signaling pathway," as indicated by the high expression of Penk. We found that not only was PENK expression at the protein level detected in the UVB-expanded skin Treg (UVB-skin Treg) cells, but that a PENK-derived neuropeptide, methionine enkephalin (Met-ENK), from Treg cells promoted the outgrowth of epidermal keratinocytes in an ex vivo skin explant assay. Notably, UVB-skin Treg cells also promoted wound healing in an in vivo wound closure assay. In addition, UVB-skin Treg cells produced amphiregulin (AREG), which plays a key role in Treg-mediated tissue repair. Identification of a unique function of PENK+ UVB-skin Treg cells provides a mechanism for maintaining skin homeostasis.

Timing of treatment with an endogenous opioid alters immune response and spinal cord pathology in female mice with experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is a progressive disease of the central nervous system (CNS) that primarily affects women during the second or third decade of life. The mechanism is hypothesized to involve unregulated peripheral inflammation resulting in blood-brain barrier damage, and eventual axonal damage and demyelination. Based on this understanding, the animal model of MS, experimental autoimmune encephalomyelitis (EAE), often is utilized to study lymphocyte activation. Therapeutic paradigms of exogenous opioid growth factor (OGF) or low-dose naltrexone (LDN) treatment can modulate EAE, but little is reported regarding OGF or LDN effects on peripheral inflammation, microglia activation, and/or macrophage proliferation. Moreover, little is known about differential responses to LDN or OGF relative to the duration and timing of treatment. Utilizing a female mouse model of EAE, two treatment regimens were established to investigate differences between prophylactic treatment and traditional therapy initiated at the time of disease presentation. Prophylactic OGF or LDN treatment delayed the onset of behavior, suppressed neutrophil replication, and curtailed lymphocyte proliferation which ultimately improved behavioral outcome. Traditional therapy with OGF or LDN reversed behavioral deficits, restored OGF and IL-17 serum levels, and inhibited microglial activation within 8 days. Reduced serum OGF levels in untreated EAE mice correlated with increased microglia activation within lumbar spinal cords. Both treatment regimens of OGF or LDN reduced activated microglia, whereas only prophylactic treatment prevented CNS macrophage aggregation. These data demonstrate that the timing of LDN or OGF treatment initiation alters outcomes and can prevent or reverse behavioral deficits, cytokine activation, and spinal cord pathology.

Iterative Landmark-Based Umbrella Sampling (ILBUS) Protocol for Sampling of Conformational Space of Biomolecules.

Computer simulations of biomolecules such as molecular dynamics often suffer from insufficient sampling. Due to limited computational resources, insufficient sampling prevents obtaining proper equilibrium distributions of observed properties. To deal with this problem, we proposed a simulation protocol for efficient resampling of collected off-equilibrium trajectories. These trajectories are utilized for the initial mapping of the conformational space, which is later properly resampled by the introduced Iterative Landmark-Based Umbrella Sampling (ILBUS) method. Reconstruction of static equilibrium properties is achieved by the multistate Bennett acceptance ratio (MBAR) method, which enables efficient use of simulated data. The ILBUS protocol is geometry-based and does not demand any additional collective variable or a dimensional-reduction technique. The only requirement is a set of suitably spaced reference conformations, which serve as landmarks in the mapped conformational space. Additionally, the ILBUS protocol encompasses an iterative process that optimizes the force constant used in the umbrella sampling simulation. Such tuning is an inherent feature of the protocol and does not need to be performed by the user in advance. Furthermore, even the simulations with suboptimal force constants can be used in estimates by MBAR. We demonstrate the feasibility and the performance of this approach in the study of the conformational landscape of the alanine dipeptide, met-enkephalin, and adenylate kinase.

Crotalphine Modulates Microglia M1/M2 Phenotypes and Induces Spinal Analgesia Mediated by Opioid-Cannabinoid Systems.

Pain is a worldwide public health problem and its treatment is still a challenge since clinically available drugs do not completely reverse chronic painful states or induce undesirable effects. Crotalphine is a 14 amino acids synthetic peptide that induces a potent and long-lasting analgesic effect on acute and chronic pain models, peripherally mediated by the endogenous release of dynorphin A and the desensitization of the transient receptor potential ankyrin 1 (TRPA1) receptor. However, the effects of crotalphine on the central nervous system (CNS) and the signaling pathway have not been investigated. Thus, the central effect of crotalphine was evaluated on the partial sciatic nerve ligation (PSNL)-induced chronic neuropathic pain model. Crotalphine (100 µg/kg, p.o.)-induced analgesia on the 14th day after surgery lasting up to 24 h after administration. This effect was prevented by intrathecal administration of CB1 (AM251) or CB2 (AM630) cannabinoid receptor antagonists. Besides that, crotalphine-induced analgesia was reversed by CTOP, nor-BNI, and naltrindole, antagonists of mu, kappa, and delta-opioid receptors, respectively, and also by the specific antibodies for ß-endorphin, dynorphin-A, and met-enkephalin. Likewise, the analgesic effect of crotalphine was blocked by the intrathecal administration of minocycline, an inhibitor of microglial activation and proliferation. Additionally, crotalphine decreased the PSNL-induced IL-6 release in the spinal cord. Importantly, in vitro, crotalphine inhibited LPS-induced CD86 expression and upregulated CD206 expression in BV-2 cells, demonstrating a polarization of microglial cells towards the M2 phenotype. These results demonstrated that crotalphine, besides activating opioid and cannabinoid analgesic systems, impairs central neuroinflammation, confirming the neuromodulatory mechanism involved in the crotalphine analgesic effect.

'Is there any point in me doing this?' Views and experiences of women in the Diabetes and Antenatal Milk Expressing (DAME) trial.

The Diabetes and Antenatal Milk Expressing (DAME) randomised controlled trial (RCT) was conducted in 2011-2015, at six sites in Melbourne, Australia to explore the effect of advising women with diabetes in pregnancy to express breast milk from 36 weeks gestation. Infants whose mothers were randomised to express in pregnancy were more likely to be exclusively breast milk fed during their hospital stay, and there was no evidence of harm. This paper explores women's views and experiences of antenatal expressing. In this two-arm RCT, 635 women with diabetes in pregnancy who were otherwise of low medical risk were randomised at 36-37 weeks gestation to usual care (not expressing, n = 316), or the intervention, where women were advised to hand express for 10 min twice daily until birth (n = 319). Semistructured face-to-face interviews were conducted with 10 women who expressed antenatally. They were asked about their experiences of antenatal expressing, including how they felt about the overall experience, the amount of breast milk they expressed, making time to express, and their experience of breastfeeding. Thematic analysis of the in-depth interviews identified six themes: (1) learning and adapting expressing, (2) feelings and sensations associated with expressing, (3) support, (4) dis/empowerment, (5) health, and (6) the value of breast milk. Women had both positive and negative experiences of antenatal expressing. If health professionals are advising antenatal expressing to women, it is important they understand the range of outcomes and experiences.

Sequential assignment of NMR spectra of peptides at natural isotopic abundance with zero- and ultra-low-field total correlation spectroscopy (ZULF-TOCSY).

A novel method dubbed ZULF-TOCSY results from the combination of Zero and Ultra-Low Field (ZULF) with high-field, high-resolution NMR, leading to a generalization of the concept of total correlation spectroscopy (TOCSY). ZULF-TOCSY is a new building block for NMR methods, which has the unique property that the polarization is evenly distributed among all NMR-active nuclei such as 1H, 13C, 15N, 31P, etc., provided that they belong to the same coupling network, and provided that their relaxation is not too fast at low fields, as may occur in macromolecules. Here, we show that ZULF-TOCSY correlations can be observed for peptides at natural isotopic abundance, such as the protected hexapeptide Boc-Met-enkephalin. The analysis of ZULF-TOCSY spectra readily allows one to make sequential assignments, thus offering an alternative to established heteronuclear 2D experiments like HMBC. For Boc-Met-enkephalin, we show that ZULF-TOCSY allows one to observe all expected cross-peaks between carbonyl carbons and α-CH protons, while the popular HMBC method provides insufficient information.

Group 2 innate lymphoid cells promote beiging of white adipose tissue and limit obesity.

Obesity is an increasingly prevalent disease regulated by genetic and environmental factors. Emerging studies indicate that immune cells, including monocytes, granulocytes and lymphocytes, regulate metabolic homeostasis and are dysregulated in obesity. Group 2 innate lymphoid cells (ILC2s) can regulate adaptive immunity and eosinophil and alternatively activated macrophage responses, and were recently identified in murine white adipose tissue (WAT) where they may act to limit the development of obesity. However, ILC2s have not been identified in human adipose tissue, and the mechanisms by which ILC2s regulate metabolic homeostasis remain unknown. Here we identify ILC2s in human WAT and demonstrate that decreased ILC2 responses in WAT are a conserved characteristic of obesity in humans and mice. Interleukin (IL)-33 was found to be critical for the maintenance of ILC2s in WAT and in limiting adiposity in mice by increasing caloric expenditure. This was associated with recruitment of uncoupling protein 1 (UCP1)(+) beige adipocytes in WAT, a process known as beiging or browning that regulates caloric expenditure. IL-33-induced beiging was dependent on ILC2s, and IL-33 treatment or transfer of IL-33-elicited ILC2s was sufficient to drive beiging independently of the adaptive immune system, eosinophils or IL-4 receptor signalling. We found that ILC2s produce methionine-enkephalin peptides that can act directly on adipocytes to upregulate Ucp1 expression in vitro and that promote beiging in vivo. Collectively, these studies indicate that, in addition to responding to infection or tissue damage, ILC2s can regulate adipose function and metabolic homeostasis in part via production of enkephalin peptides that elicit beiging.

Sialorphin Potentiates Effects of [Met5]Enkephalin without Toxicity by Action other than Peptidase Inhibition.

This dose-response study investigated the effects of sialorphin on [Met5]enkephalin (ME)-induced inhibition of contractions in mouse vas deferens and antinociception in male rats. Differences were compared among combinations of three chemical peptidase inhibitors: amastatin, captopril, and phosphoramidon. The ratio of potencies of ME in mouse vas deferens pretreated with both sialorphin (100 µM) and a mixture of the three peptidase inhibitors (1 µM each) was higher than that with the mixture of peptidase inhibitors alone at any dose. Intrathecal administration of sialorphin (100-400 nmol) significantly and dose dependently increased ME (3 nmol)-induced antinociception with the mixture of three peptidase inhibitors (10 nmol each). The degree of antinociception with a combination of any two of the peptidase inhibitors (10 nmol each) in the absence of sialorphin was less than that in the presence of sialorphin (200 nmol). Pretreatment with both sialorphin (200 nmol) and the mixture of three peptidase inhibitors (10 nmol each) produced an approximately 100-fold augmentation in ME (10 nmol)-induced antinociception, but without signs of toxicity such as motor dysfunction in rats. Radioligand receptor binding assay revealed that sialorphin did not affect either binding affinity or maximal binding capacity of [d-Ala2,N-MePhe4,Gly-ol5]enkephalin. These results indicate that sialorphin potentiates the effects of ME without toxicity by a mechanism other than peptidase inhibition and with no effect on its affinity to µ-opioid receptors. SIGNIFICANCE STATEMENT: Sialorphin is regarded as an endogenous peptidase inhibitor that interacts with enkephalin-degrading enzymes. The results of these in vitro and in vivo studies confirm that sialorphin potentiates the effects of [Met5]enkephalin without toxicity by an action other than peptidase inhibition. This suggests that sialorphin offers the advantage of reducing or negating the side effects of opioid drugs and endogenous opioid peptides.

Near UV and Visible Light Induce Iron-Dependent Photodegradation Reactions in Pharmaceutical Buffers: Mechanistic and Product Studies.

Near UV (λ = 320-400 nm) and visible light (λ = 400-800 nm) can lead to the oxidation of pharmaceutical proteins, which can affect efficiency and promote immunogenicity. However, no concise mechanism has been established for the photo-oxidation of pharmaceutical proteins under near UV and visible light. Here, we show that carboxylic acid buffer-Fe3+ complexes can function as photosensitizers, causing peptide degradation via the formation of various radicals and oxidants. Three pharmaceutical relevant carboxylic acid buffers (citrate, acetate, and succinate) were tested under near UV and visible light. Oxidation reactions were monitored for model peptides containing readily oxidizable amino acids, such as methionine- or leucine-enkephalin and proctolin peptide. Oxidation products were evaluated by RP-HPLC coupled to UV or fluorescent detection and RP-HPLC-MS/MS. Specifically for citrate buffer, the light-induced formation of H2O2, •OH, •CO2-, and formaldehyde was demonstrated. The peptides displayed oxidation of Met, hydroxylation of Tyr and Phe, as well as the formation of novel products from Tyr. Experiments with 18O2 resulted in the incorporation of 18O into various reaction products, consistent with a metal-catalyzed activation of O2 into reactive oxygen species. The addition of EDTA and DTPA did not prevent the oxidation of the peptides and, in some cases, enhanced the oxidation. Our results demonstrate that pharmaceutical buffer-Fe3+ complexes, exposed to UV and visible light, can promote various pathways of oxidation reactions in pharmaceutical formulations.

Glutathione and Glutathione-Like Sequences of Opioid and Aminergic Receptors Bind Ascorbic Acid, Adrenergic and Opioid Drugs Mediating Antioxidant Function: Relevance for Anesthesia and Abuse.

Opioids and their antagonists alter vitamin C metabolism. Morphine binds to glutathione (l-γ-glutamyl-l-cysteinyl-glycine), an intracellular ascorbic acid recycling molecule with a wide range of additional activities. The morphine metabolite morphinone reacts with glutathione to form a covalent adduct that is then excreted in urine. Morphine also binds to adrenergic and histaminergic receptors in their extracellular loop regions, enhancing aminergic agonist activity. The first and second extracellular loops of adrenergic and histaminergic receptors are, like glutathione, characterized by the presence of cysteines and/or methionines, and recycle ascorbic acid with similar efficiency. Conversely, adrenergic drugs bind to extracellular loops of opioid receptors, enhancing their activity. These observations suggest functional interactions among opioids and amines, their receptors, and glutathione. We therefore explored the relative binding affinities of ascorbic acid, dehydroascorbic acid, opioid and adrenergic compounds, as well as various control compounds, to glutathione and glutathione-like peptides derived from the extracellular loop regions of the human beta 2-adrenergic, dopamine D1, histamine H1, and mu opioid receptors, as well as controls. Some cysteine-containing peptides derived from these receptors do bind ascorbic acid and/or dehydroascorbic acid and the same peptides generally bind opioid compounds. Glutathione binds not only morphine but also naloxone, methadone, and methionine enkephalin. Some adrenergic drugs also bind to glutathione and glutathione-like receptor regions. These sets of interactions provide a novel basis for understanding some ways that adrenergic, opioid and antioxidant systems interact during anesthesia and drug abuse and may have utility for understanding drug interactions.

Separation of Acute Desensitization and Long-Term Tolerance of µ-Opioid Receptors Is Determined by the Degree of C-Terminal Phosphorylation.

Phosphorylation of sites on the C terminus of the µ-opioid receptor (MOR) results in the induction of acute desensitization that is thought to be a precursor for the development of long-term tolerance. Alanine mutations of all 11 phosphorylation sites on the C terminus of MORs almost completely abolished desensitization and one measure of tolerance in locus coeruleus neurons when these phosphorylation-deficient MORs were virally expressed in MOR knockout rats. In the present work, we identified specific residues that underlie acute desensitization, receptor internalization, and tolerance and examined four MOR variants with different alanine or glutamate mutations in the C terminus. Alanine mutations in the sequence between amino acids 375 and 379 (STANT-3A) and the sequence between amino acids 363 and 394 having four additional alanine substitutions (STANT + 7A) reduced desensitization and two measures of long-term tolerance. After chronic morphine treatment, alanine mutations in the sequence between 354 and 357 (TSST-4A) blocked one measure of long-term tolerance (increased acute desensitization and slowed recovery from desensitization) but did not change a second (decreased sensitivity to morphine). With the expression of receptors having glutamate substitutions in the TSST sequence (TSST-4E), an increase in acute desensitization was present after chronic morphine treatment, but the sensitivity to morphine was not changed. The results show that all 11 phosphorylation sites contribute, in varying degrees, to acute desensitization and long-term tolerance. That acute desensitization and tolerance are not necessarily linked illustrates the complexity of events that are triggered by chronic treatment with morphine. SIGNIFICANCE STATEMENT: In this work, we showed that the degree of phosphorylation on the C terminus of the µ-opioid receptor alters acute desensitization and internalization, and in measures of long-term tolerance to morphine. The primary conclusion is that the degree of phosphorylation on the 11 possible sites of the C terminus has different roles for expression of the multiple adaptive mechanisms that follow acute and long-term agonist activation. Although the idea that acute desensitization and tolerance are intimately linked is generally supported, these results indicate that disruption of one phosphorylation cassette of the C terminus TSST (354-357) distinguishes the two processes.

Defining the caudal hypothalamic arcuate nucleus with a focus on anorexic excitatory neurons.

KEY POINTS: Excitatory glutamate neurons are sparse in the rostral hypothalamic arcuate nucleus (ARC), the subregion that has received the most attention in the past. In striking contrast, excitatory neurons are far more common (by a factor of 10) in the caudal ARC, an area which has received relatively little attention. These glutamate cells may play a negative role in energy balance and food intake. They can show an increase in phosphorylated Stat-3 in the presence of leptin, are electrically excited by the anorectic neuromodulator cholecystokinin, and inhibited by orexigenic neuromodulators neuropeptide Y, met-enkephalin, dynorphin and the catecholamine dopamine. The neurons project local axonal connections that excite other ARC neurons including proopiomelanocortin neurons that can play an important role in obesity. These data are consistent with models suggesting that the ARC glutamatergic neurons may play both a rapid and a slower role in acting as anorectic neurons in CNS control of food intake and energy homeostasis. ABSTRACT: Here we interrogate a unique class of excitatory neurons in the hypothalamic arcuate nucleus (ARC) that utilizes glutamate as a fast neurotransmitter using mice expressing GFP under control of the vesicular glutamate transporter 2 (vGluT2) promoter. These neurons show a unique distribution, synaptic characterization, cellular physiology and response to neuropeptides involved in energy homeostasis. Although apparently not previously appreciated, the caudal ARC showed a far greater density of vGluT2 cells than the rostral ARC, as seen in transgenic vGluT2-GFP mice and mRNA analysis. After food deprivation, leptin induced an increase in phosphorylated Stat-3 in vGluT2-positive neurons, indicating a response to hormonal cues of energy state. Based on whole-cell recording electrophysiology in brain slices, vGluT2 neurons were spontaneously active with a spike frequency around 2 Hz. vGluT2 cells were responsive to a number of neuropeptides related to energy homeostasis; they were excited by the anorectic peptide cholecystokinin, but inhibited by orexigenic neuropeptide Y, dynorphin and met-enkephalin, consistent with an anorexic role in energy homeostasis. Dopamine, associated with the hedonic aspect of enhancing food intake, inhibited vGluT2 neurons. Optogenetic excitation of vGluT2 cells evoked EPSCs in neighbouring neurons, indicating local synaptic excitation of other ARC neurons. Microdrop excitation of ARC glutamate cells in brain slices rapidly increased excitatory synaptic activity in anorexigenic proopiomelanocortin neurons. Together these data support the perspective that vGluT2 cells may be more prevalent in the ARC than previously appreciated, and play predominantly an anorectic role in energy metabolism.

Isolation stress impacts Met-enkephalin in the hypothalamo-pituitary-adrenocortical axis in growing Polish Mountain sheep: a possible role of the opioids in modulation of HPA axis.

It was hypothesized that there is cross-talk between the classical constituents of the hypothalamo-pituitary-adrenocortical axis (HPA) and Met-enkephalin in the HPA axis. The study examined effects of isolation stress, sex, and age on concentrations of native Met-enkephalin and pro-enkephalin (PENK) gene expression in tissues of the HPA (hypothalamus, pituitary gland and adrenal cortex) in 3-, 6- and 9-month old female and male lambs. In addition, the effects of isolation stress on in vitro release Met-enkephalin from fragments of the hypothalamus or adrenal cortex were examined. Isolation stress was followed by decreases in the concentration of Met-enkephalin in both the pituitary gland and adrenal cortex. There were also increases in the hypothalamic concentration of Met-enkephalin together with increases in PENK gene expression in the HPA in 6- and 9-months old females and males. There were reductions in release of Met-enkephalin from hypothalamic and adrenocortical tissue in vitro after isolation stress. In the presence of naltrexone, there were increases in basal release in vitro of Met-enkephalin from hypothalamic tissue from control and stressed female lambs but a decrease in tissue from stressed male lambs. In a somewhat similar manner, the presence of naltrexone was associated with increases in the basal release of Met-enkephalin from adrenocortical tissue from control female lambs but a decrease with tissue from stressed female and both stressed and control male lambs. Lay summary The present studies examine the impact of isolation stress on Met-enkephalin in growing female and male lambs. The results clearly showed the involvement of Met-enkephalin modulation of the psychological stress response in growing female and male lambs.

1-Substituted sialorphin analogues-synthesis, molecular modelling and in vitro effect on enkephalins degradation by NEP.

Rat sialorphin (Gln-His-Asn-Pro-Arg) is a natural blocker of neprilysin (NEP) that belongs to the family of endogenous opioid peptide-degrading enzymes. Studies have confirmed the efficiency of sialorphin in blocking the activity of NEP, both in vitro and in vivo. It has been demonstrated that this inhibitor has a strong analgesic, anti-inflammatory, immunological and metabolic effect either directly or indirectly by affecting the level of Met/Leu-enkephalins. In this work, sialorphin and their 12 analogues were synthesised using the solid-phase method. The effect of the peptides on the degradation of Met-enkephalin by NEP and metabolic degradation in human plasma was investigated in vitro. We show that the change in the N-terminal amino acid configuration from L to D in almost all peptides, except D-Arg-His-Asn-Pro-Arg (peptide XI), led to the abolition of their inhibitory activity. With molecular modelling technique we explained the structural properties of the L and D-arginine located on the N-terminal part of the peptide. The detailed analysis of the protein binding pocket allowed us to explain why D-arginine is so unique among all D residues. Peptide XI showed the highest stability among the tested peptides in human plasma. For instance sialorphin after a 2-hour incubation in human plasma was almost completely decomposed, while the level of peptide XI dropped to 45% after 48 h under these conditions.

Synthesis and biological evaluation of quercetin and resveratrol peptidyl derivatives as potential anticancer and antioxidant agents.

Quercetin and resveratrol are polyphenolic compounds, members of the flavonoid and the stilbene family, respectively, both medicinally important as dietary anticancer and antioxidant agents. They are present in a variety of foods-including fruits, vegetables, tea, wine, as well as other dietary supplements-and are responsible for various health benefits. Different quercetin and resveratrol esters of Leu/Met-enkephalin and tetrapeptide Leu-Ser-Lys-Leu (LSKL) were synthesized as model systems for monitoring the influence of the peptides on biological activity of resveratrol and quercetin. General formula of the main peptidyl-quercetin derivatives is 2-[3-(aa)n-4-hydroxyphenyl]-3,5,7-tri-hydroxy-4H-1-benzopyran-4-on, and the general formula of the main peptidyl-resveratrol derivatives is (E)-5-[4-(aa)n)styryl]benzene-1,3-diol. The antioxidant and anticancer activities of prepared compounds were investigated. Significant anticancer activity was obtained for the LSKL-based both quercetin and resveratrol derivatives. All prepared compounds exhibit antioxidant activity, in particular quercetin derivative containing Met-enkephalin.