Met-enkephalin Research Articles

Effects of Restraint Stress on Circulating Corticosterone and Met Enkephalin in Chickens: Induction of Shifts in Insulin Secretion and Carbohydrate Metabolism.

This study examined the effects of acute restraint stress in the presence or absence of naltrexone on the circulating concentrations of insulin, glucose, Met-enkephalin and corticosterone in 14-week-old chickens [design: 2 sex × 2 stress/non-stress × 2 +/- naltrexone]. In chickens (five male and five females per treatment) subjected to restraint for 30 min, there were increases in the plasma concentrations of corticosterone and Met-enkephalin. The plasma concentrations of insulin and glucose were also increased in the chickens during restraint. Moreover, there were increases in the plasma concentrations of insulin and glucose in the chickens. The patterns of expression of the proenkephalin gene (PENK) in both the anterior pituitary gland and the adrenal gland were very similar to that of plasma Met-enkephalin. There were relationships between the plasma concentrations of corticosterone, Met-enkephalin, insulin and glucose after 30 min of restraint. The effects of naltrexone treatment on both untreated and stressed chickens were also examined, with naltrexone attenuating the stress-induced increases in the plasma concentrations of corticosterone, Met-enkephalin and glucose but not in those of insulin. The present study demonstrates that stress increases insulin secretion in chickens but also induces insulin resistance.

Current pharmacotherapeutic properties of low-dose naltrexone therapy in humans and possible therapeutic and prophylactic indications in cats and dogs

Naltrexone was first developed in 1963 as an opioid antagonist for therapeutic use in opioid-dependent individuals (alcohol, narcotics, and tobacco) with a 50 mg/day dose approved by the Food and Drug Administration (FDA). In 1985, Dr. Bernard Bihari explained that when naltrexone is taken in very low doses (1-5 mg/day) before bedtime, it blocks opioid receptors for 2- 4 hours and leads to the release of endorphins in the early morning hours, serving as an opioid agonist and immunomodulatory agent. This extra endorphin secretion has been proven to enhance the immune system response in AIDS patients. Endogenous opioids that affect cell development are referred to as opioid growth factor (OGF) and met-enkephalin (ME). Low-dose-naltrexone (LDN) pharmacotherapeutically possesses immunomodulatory, anticarcinogenic, antiviral, antibacterial, antiparasitic, and antifungal properties. LDN also increases the production and sensitivity of OGF, ME, and OGF receptor (OGFr) in the bloodstream, thereby enhancing quality of life. Based on these pharmacotherapeutic properties, LDN application is believed to be effective in the treatment or prophylaxis of various infectious diseases in cats and dogs, particularly vasculitis-related diseases such as feline infectious peritonitis (FIP) and infectious canine hepatitis (ICH), hypothyroidism, gastrointestinal disorders, spondylosis, rheumatoid arthritis, autoimmune diseases, pneumonia, atopic and allergic dermatitis, pyodermas, resistant pyodermas, methicillin-resistant Staphylococcus aureus (MRSA) or various gastrointestinal disorders, alopecia, stubborn dermatomycosis, stubborn demodicosis, various geriatric and oncogenic diseases, and deep depressions or obsessive-compulsive disorders. Thus, this review aims to evaluate the pharmacotherapeutic properties of LDN therapy usage and mechanisms of action in detail and assess the potential indications in cats and dogs based on these mechanisms.

The opioid peptide met-enkephalin modulates thalamo-cortical excitation inhibition balance in a medial thalamus-anterior cingulate cortex circuit

Activation of opioid receptors in the anterior cingulate cortex (ACC) mediates aspects of analgesia induced by both exogenous and endogenous opioids. We have previously shown that opioid signaling disrupts both afferent excitatory and indirect inhibitory synaptic transmission from the medial thalamus (MThal) to the ACC, but the effects of non-selective opioid peptides such as [Met]5-enkephalin (ME) within this circuit remain poorly understood. The goal of the current study was to understand how ME modulates thalamic-driven excitatory and inhibitory synaptic transmission onto layer V pyramidal neurons in the ACC. We used pharmacology, brain slice electrophysiology and optogenetic stimulation to study opioid-mediated modulation of optically evoked glutamatergic and GABAergic transmission. The results revealed that bath perfused ME inhibited both AMPA-mediated excitatory and GABA-mediated inhibitory synaptic transmission in the ACC. However, inhibitory transmission was more potently inhibited than excitatory transmission by ME. This preferential reduction in GABAA-mediated synaptic transmission was primarily due to the activation of delta opioid receptors by ME and resulted in a net disinhibition of MThal-ACC excitatory pathway. These results suggest that moderate concentrations of ME can lead to a net increase in excitatory drive of ACC circuitry and that analgesia may be associated with disinhibition rather than inhibition of ACC subcircuits.

Transcutaneous electrical nerve stimulation effects on pain-intensity and endogenous opioids levels among chronic low-back pain patients: A randomised controlled trial.

Transcutaneous electrical nerve stimulation (TENS) is a promising non-pharmacological modality for the management of chronic low back pain (CLBP), but its efficacy and mode of action have not been clearly established. To evaluate the responses of plasma beta-endorphin (βE), met-enkephalin (ME), and pain intensity (PI) among patients with CLBP exposed to TENS or sham-TENS. This double-blind trial involved 62 participants (aged 53.29 ± 5.07 years) randomised into TENS group (frequency 100 Hz, burst-rate 2 Hz, burst-width 150 μs, intensity 40 mA, duration 30 min), and sham-TENS group. The PI and plasma concentrations of βE and ME were measured at baseline, immediately (0 hr), 1 hr, 24 hrs, and 48 hrs post-intervention. Data were analysed using general linear model repeated measures, ordinal regression, one-way analysis of variance, Kruskal-Wallis test, independent and paired samples t-tests, Mann-Whitney U test, Wilcoxon signed-rank test, and Kendall's tau coefficient. There was a significant temporal difference in PI between groups, F (1, 58) = 18.83, p< 0.001; the TENS group had better pain relief. The relative analgesic effect of TENS started immediately after the intervention (median difference [M⁢D] =-3, p< 0.001), peaked at 1 hr (M⁢D=-4, p< 0.001), and worn out by 24 hrs (M⁢D=-1, p= 0.029). However, there was no significant difference in βE and ME between the groups from 0 hr to 24 hrs post interventions, and no significant correlation between the PI, and βE, or ME. TENS significantly reduced PI up to 24 hrs after treatment.

Assessment of Plasma-Endogenous Opioid Neuropeptide Levels and Psychometric Properties of Non-Suicidal Self-Injury in Adolescents

Objective Non-suicidal self-injury (NSSI) is one of the most common mental health problems and growing public-health issues, coupled with a significant population-level burden among adolescents in both developed and developing countries. We aimed to assess the role of endogenous opioid system–emotion regulation circuitry in NSSI through measurement of plasma beta-endorphin (β-EP), met-enkephalin (MENK) levels, and determination of psychometric features of Turkish adolescent subjects. Method In this research, we measured plasma β-EP and MENK levels of 49 adolescents with NSSI and 39 control subjects without NSSI between the ages of 12–18 years. All adolescent subjects were observed in the outpatient clinic, and their clinical and sociodemographic characteristics were examined. All subjects were assessed using the Brief Symptom Inventory (BSI) and Inventory of Statements About Self Injury (ISAS). Results Plasma β-EP levels were statistically lower in adolescents with NSSI than control group, whereas there was no statistically significant difference in MENK levels. β-EP levels showed a negative correlation with depression severity. The data obtained from BSI and ISAS were not found to be associated with both β-EP and MENK levels, while subscale scores exhibited versatile correlations. Conclusion Our findings supported the salient role of β-EP in NSSI behavior. Also, decreased plasma β-EP could be assessed as a reliable indicator for NSSI. However, it is possible that measurement of basal plasma levels of neuropeptides might also bring many confounders and could cause bias. Therefore, repeated measurements of plasma-endogenous opioid neuropeptides in a time-dependent manner—concomitant to engage of NSSI behavior—might give more reliable results.

Comprehensive Signaling Profiles Reveal Unsuspected Functional Selectivity of δ-Opioid Receptor Agonists and Allow the Identification of Ligands with the Greatest Potential for Inducing Cyclase Superactivation.

Prolonged exposure to opioid receptor agonists triggers adaptations in the adenylyl cyclase (AC) pathway that lead to enhanced production of cyclic adenosine monophosphate (cAMP) upon withdrawal. This cellular phenomenon contributes to withdrawal symptoms, hyperalgesia and analgesic tolerance that interfere with clinical management of chronic pain syndromes. Since δ-opioid receptors (DOPrs) are a promising target for chronic pain management, we were interested in finding out if cell-based signaling profiles as generated for drug discovery purposes could inform us of the ligand potential to induce sensitization of the cyclase path. For this purpose, signaling of DOPr agonists was monitored at multiple effectors. The resulting signaling profiles revealed marked functional selectivity, particularly for Met-enkephalin (Met-ENK) whose signaling bias profile differed from those of synthetic ligands like SNC-80 and ARM390. Signaling diversity among ligands was systematized by clustering agonists according to similarities in Emax and Log(τ) values for the different responses. The classification process revealed that the similarity in Gα/Gβγ, but not in β-arrestin (βarr), responses was correlated with the potential of Met-ENK, deltorphin II, (d-penicillamine2,5)-enkephalin (DPDPE), ARM390, and SNC-80 to enhance cAMP production, all of which required Ca2+ mobilization to produce this response. Moreover, superactivation by Met-ENK, which was the most-effective Ca2+ mobilizing agonist, required Gαi/o activation, availability of Gβγ subunits at the membrane, and activation of Ca2+ effectors such as calmodulin and protein kinase C (PKC). In contrast, superactivation by (N-(l-tyrosyl)-(3S)-1,2,3,4-tetrahydroisoquinoline-3-carbonyl)-l-phenylalanyl-l-phenylalanine (TIPP), which was set in a distinct category through clustering, required activation of Gαi/o subunits but was independent of the Gβγ dimer and Ca2+ mobilization, relying instead on Src and Raf-1 to induce this cellular adaptation.

Effects of Sleep Deprivation by Olfactorily Induced Sexual Arousal Compared to Immobilization Stress and Manual Sleep Deprivation on Neuromessengers and Time Keeping Genes in the Suprachiasmatic Nuclei and Other Cerebral Entities of Syrian Hamsters-An Immunohistochemical Study.

We investigated the effects of sexual arousal induced by olfactory stimuli on the expression of neuromodulators, neurotransmitters and sexual steroid receptors in the suprachiasmatic nucleus (SCN, the circadian pacemaker of mammals) and other cerebral entities of Syrian hamsters (Mesocricetus auratus) compared to manual sleep deprivation and immobilization stress. The hamsters kept under a 12:12 hours (h) light:dark cycle were deprived of sleep by sexual stimulation, gentle manual handling or immobilization stress for 1 h at the beginning of the light phase and subsequently sacrificed at zeitgeber time 01:00, respectively; for comparison, hamsters were manually sleep deprived for 6 or 20 h or sacrificed after completing a full sleep phase. As demonstrated by immunohistochemistry, apart from various alterations after manual sleep deprivation, sexual stimulation caused down-regulation of arginine-vasopressin (AVP), vasointestinal peptide (VIP), serotonin (5-HT), substance P (SP), and met-enkephalin (ME) in the SCN. Somatostatin (SOM) was diminished in the medial periventricular nucleus (MPVN). In contrast, an increase in AVP was observed in the PVN, that of oxytocin (OXY) in the supraoptic nucleus (SON), of tyrosine-hydroxylase (TH) in the infundibular nucleus (IN), and dopamine beta-hydroxylase (DBH) in the A7 neuron population of the brain stem (A7), respectively. Testosterone in plasma was increased. The results indicate that sexual arousal extensively influences the neuropeptide systems of the SCN, suggesting an involvement of the SCN in reproductive behavior.

Chronic Morphine Exposure and μ‐opioid Receptor Phosphorylation Deficiency Enhances Opioid Sensitivity of Thalamo‐Striatal Synapses

The pain relieving and rewarding properties of opioids are widely attributed to action at the μ-opioid receptor (MOR). Prolonged opioid administration results in analgesic tolerance, where increasing doses of drug are required to achieve the same effect. Tolerance develops to some opioid effects, such as analgesia and reward, more than others, such as respiratory depression and constipation. These effects are mediated through separate neural pathways, indicating MORs might be differentially regulated depending on where they are present. MOR signaling is regulated by receptor phosphorylation; upon activation by an agonist, MORs are subsequently phosphorylated at the carboxyl (C)-terminal tail and flagged for internalization, terminating signaling via associated G-proteins. Glutamatergic projections from the mediodorsal thalamus (MD) to the striatum are involved in processing the affective dimension of pain, or the negative emotion associated with perception of painful stimuli. MORs are present at these synapses, but how MOR signaling here is regulated is not well understood. The aim of this work was to determine how chronic opioid exposure alters opioid sensitivity at MORs present on thalamic terminals in the striatum, and whether opioid sensitivity is enhanced in MOR phosphorylation-deficient mice. We recorded optically evoked excitatory postsynaptic currents (EPSCs) in striatal medium spiny neurons in mouse brain slices. Opioid sensitivity was determined by measuring EPSC inhibition induced by MOR agonists morphine and met-enkephalin (ME). Chronic treatment with morphine enhanced EPSC inhibition by morphine, but not ME, at these receptors in wild-type (WT) mice. Drug-naïve MOR C-terminal phosphorylation-deficient mice showed enhanced EPSC inhibition by opioids compared to WT mice. The finding that chronic morphine exposure induced sensitization, rather than tolerance, to morphine at glutamatergic thalamo-striatal terminals suggests a synapse specific effect that contributes to tolerance overall. While MORs present at thalamo-striatal terminals showed increased, rather than decreased, opioid sensitivity, this effect may still be important in contributing to analgesic tolerance at the behavioral level. Future studies will investigate whether the enhanced opioid sensitivity following chronic opioid treatment is phosphorylation-dependent and how chronic morphine treatment alters signaling of downstream effectors of MOR.

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.

Effects of Intracerebroventricular and Intra-Arcuate Nucleus Injection of Ghrelin on Pain Behavioral Responses and Met-Enkephalin and β-Endorphin Concentrations in the Periaqueductal Gray Area in Rats.

Ghrelin is an endogenous ligand for orphan growth hormone secretagogue receptors. Ghrelin receptors have been found in central nervous system (CNS) areas responsible for pain modulation and transmission. This study investigated the effects of intracerebroventricular (ICV) and intra-arcuate nucleus (ARC) injection of ghrelin on pain behavioral responses and levels of β-endorphin (β-EP) and met-enkephalin (MENK) in the periaqueductal gray area (PAG) during the formalin test in rats. Thirty-five male rats were studied in five groups. Ghrelin was injected into the left lateral ventricle (ICV, 5 µL) or into the ARC (1 µL). After 15 min, formalin (2.5%) was subcutaneously injected into the left hind paw. Behavioral nociceptive scores were recorded for 60 min. MENK and β-EP were collected by microdialysis in the PAG and determined by high-performance liquid chromatography (HPLC). ICV and ARC injection of ghrelin significantly reduced pain in all phases of the formalin test (p < 0.001). Dialysate concentrations of MENK and β-EP in the PAG increased in all the phases (p < 0.01). In conclusion, the present study shows that the ARC nucleus and the endogenous opioid system are involved in ghrelin-induced pain modulation.

Met-enkephalin inhibits ROS production through Wnt/β-catenin signaling in the ZF4 cells of zebrafish

Opioid neuropeptides are developed early in the course of a long evolutionary process. As the endogenous messengers of immune system, opioid neuropeptides participate in regulating immune response. In this study, the mechanism that Met-enkephalin (M-ENK) inhibits ROS production through Wnt/β-catenin signaling was investigated in the ZF4 cells of zebrafish. ZF4 cells were exposed to 0, 10, 20, 40, 80, and 160 μM Met-enkephalin (M-ENK) for 24 h, and the cell viability was detected with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. The cell viability was significantly increased by 10, 20, 40, 80, and 160 μM M-ENK. After ZF4 cells were exposed to 0, 20, 40, and 80 μM M-ENK for 24 h, the mRNA expression of Wnt10b, β-catenin, and CCAAT/enhancer binding protein α (C/EBPα) was significantly increased by 40 and 80 μM M-ENK. However, the mRNA and protein expression of GSK-3β was significantly decreased by 40 and 80 μM M-ENK. The protein expression of β-catenin was significantly induced by 40 and 80 μM M-ENK, while the protein expression of p-β-catenin was significantly decreased by 20, 40, and 80 μM M-ENK. In addition, the mRNA expression of CAT, SOD, and GSH-PX was significantly increased by 40 and 80 μM M-ENK. The levels of H2O2, ·OH, and O2·- were significantly decreased, but the activity of CAT, SOD, and GSH-PX was significantly increased by 40 and 80 μM M-ENK. The fluorescence intensity of reactive oxygen species (ROS) was decreased, and that of mitochondrial membrane potential (MMP) was increased with the increase of M-ENK concentration in ZF4 cells. The results showed that M-ENK could induce Wnt/β-catenin signaling, which further inhibited ROS production through the induction of C/EBPα, MMP, and the activities of antioxidant enzymes.

Enhancement of μ-opioid receptor desensitization by orexin-A in rat locus coeruleus neurons

Opioids have always been used in clinical practice for pain management. However, development of tolerance to their effects following long term administration, seriously restricts further clinical use of these drugs. In this regard, μ-opioid receptor (MOR) desensitization, as an initial step in development of opioid tolerance, is of particular significance. Previous studies support the involvement of orexinergic system in development of opioid tolerance. Locus coeruleus (LC) nucleus has been shown to modulate pain and development of tolerance. Opioid receptors (particularly μ) are densely expressed within the LC. Moreover, it receives widespread orexinergic inputs and orexin type 1 receptors (OX1Rs) are also highly expressed in this brain region. In the present study, the effect of orexin-A (OXA) on met-enkephalin (ME)-induced MOR desensitization was investigated in locus coeruleus neurons of male Wistar rats (2–3weeks of age). ME (30μM), as a potent MOR agonist, was applied for 10min and the outward K+ current was recorded using whole cell patch clamp recording. The percentage of decrease in ME-induced K+ current was considered as the degree of MOR desensitization. Results indicated that OXA (100nM) enhances ME-induced MOR desensitization via affecting OX1Rs in rat locus coeruleus neurons and this effect is mediated by a protein kinase C dependent mechanism within the LC. The activity of orexinergic system might potentiate the signaling pathways underlying opioid-induced receptor desensitization.

Abstract 5527: Oral administration of a nano-enabled form of Met-Enkephalin peptide controls pancreatic cancer growth

Abstract Introduction Peptides are a potential source of highly specific bioactive molecules useful for the treatment of various conditions, including cancer. Their wider application is limited by their poor oral bioavailability due to e.g. rapid degradation in the gut or in the blood. Unfavorable physical-chemical properties can further limit their applicability. For example, Met-enkephalin (MENK) is an endogenous opioid peptide having anti-tumor activity against a diverse range of cancers. While previous reports describe anti-cancer effects of MENK after parenteral administration in humans this approach has significant limitations because only low drug concentrations are achievable due to the peptide's poor aqueous solubility. Here, we demonstrate that a nano-enabled form of MENK created by combining MENK with the amphiphilic polymer GCPQ to form ‘molecular envelopes’, is orally bioavailable and active in murine models of pancreatic cancer. Method Nano-enabled MENK (10 mg/ml) formulation was prepared by encapsulating the peptide in GCPQ nanoparticles (50 mg/ml). A MENK solution was prepared in water (10 mg/ml). Swiss nude mice bearing tumor xenografts of pancreatic cancer cells (MIA PaCa-2) were randomized in to three groups (n = 5), each receiving MENK (50 mg/kg), nano-enabled MENK formulation (50 mg/kg) or water. All formulations were orally administered every two days over a period of 29 days once tumors were fully established. Throughout the study the tumor volumes were measured using calipers and the body weight of the mice monitored. Results For mice treated with the nano-enabled MENK formulation at a dose of 50 mg/kg, a complete suppression in the tumor growth was observed within two weeks of the start of treatment which continued while treatment continued and for two weeks after withdrawal of the treatment on 4th week. In contrast, no statistically significant delay in tumor growth was observed for mice treated with the MENK solution (50 mg/kg) compared with the untreated control. Overall the treatment was well tolerated as suggested by the limited impact of therapy on animal body weight. In vivo pharmacodynamics studies demonstrated significantly higher anti tumor efficacy for nano-enabled MENK formulation compared to free MENK in established MIA PaCa-2 tumor xenograft model. Conclusion The current study demonstrates that the MENK peptide when administered orally in the form of nano-enabled ‘molecular envelopes’ is able to control the growth of MIA PaCa-2 pancreatic tumour xenograft tumors over a period of four weeks. The above results suggest that a nano-enabled oral form of this endogenous peptide could have wider application in cancer therapy. Furthermore, it suggests that other peptides with anti-cancer activity could be utilized as oral peptide pill for cancer therapy. Citation Format: Preethi Marimuthu, Ijeoma F. Uchegbu, Andreas G. Schätzlein. Oral administration of a nano-enabled form of Met-Enkephalin peptide controls pancreatic cancer growth. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5527. doi:10.1158/1538-7445.AM2015-5527

Orexin type 1 receptor antagonism in rat locus coeruleus prevents the analgesic effect of intra-LC met-enkephalin microinjection

Long-term administration of opiates leads to development of tolerance to analgesic effects. This in turn compromise clinical use of these drugs for pain management. Although extensive studies have been conducted, the involved cellular mechanisms are still poorly understood. The nucleus locus coeruleus (LC), which is a dense homogenous cluster of noradrenergic neurons in brainstem, has been reported to be involved in mediating opiate effects including analgesia and tolerance. LC neurons express a high density of opioid receptors. On the other hand, orexinergic neurons send widespread projections to the LC region. Among the two types of orexin receptors (OX1R and OX2R), OX1R is highly expressed in LC neurons. It has been shown that orexin-A is involved in modulation of nociceptive behavior. Also, previous studies have demonstrated the involvement of OX1R in the development of morphine induced analgesia and tolerance. In the present study, the involvement of OX1R in development of met-enkephalin (ME) analgesic tolerance was investigated in LC nucleus. The tail flick test was used to evaluate the analgesic effect of intra-LC microinjection of ME in male Wistar rats (250–300g). Analgesic responses were reported as the percentage of maximum possible effect (% of MPE). Also, SB-334867 was used as a selective OX1R antagonist. Results indicate that intra-LC microinjection of ME (5μg/100nL) results in development of analgesic tolerance in 3days. Also, OX1R antagonism in LC nucleus significantly prevents the analgesic effect of intra-LC met-enkephalin microinjection. It appears that the analgesic effect of ME in LC neurons is mediated by orexinergic system.

Mucoadhesive nano-sized supramolecular assemblies for improved pre-corneal drug residence time

Context: Mucoadhesive nanoparticles were compared with non-aggregated constituent polymers for effect on pre-corneal residence of dexamethasone phosphate (DP) or met-enkephalin (ME), administered by eye-drops to rabbits.Objective: Deepening the knowledge of ophthalmic nanoparticulate systems in terms of ability to prolong pre-corneal residence.Materials and methods: Medicated nanoparticles resulted from gelation of quaternary ammonium–chitosan conjugate or its thiolated derivative with hyaluronan in the presence of drug. Particles were analyzed by light scattering. Dialysis removed non-encapsulated drug, dynamic dialysis measured drug–polymer interactions, and lyophilization-stabilized product. Dispersions were regenerated from lyophilized products. Also solutions of non-thiolated or thiolated chitosan derivative were administered. Mean drug residence time (MRT) in tears was determined by collecting samples from lower marginal tear strip of albino rabbits using capillaries.Results and discussion: Nanoparticle size of regenerated dispersions was 400–430 nm (DP-systems), 360–370 nm (ME-systems); the drug content was 2.5 mg/mL (DP) or 0.3 mg/mL (ME). The MRT for DP nanoparticles from non-thiolated derivative was higher than that for non-aggregated polymer, due to stronger concurrent interactions of positively charged nanoparticles with ocular surface and drug. Thiolated polymer nanoparticles and non-aggregated parent polymer, both interacting weakly with DP, showed similar MRT values. The MRT of ME could only be enhanced by protecting drug from enzymatic hydrolysis. This was done by nanoparticle systems, whereas non-aggregated polymers were ineffective.Conclusion: Developing a nanoparticle system rather than a solution of mucoadhesive polymer, for prolonging pre-corneal residence, is convenient, provided nanoparticles interact strongly with both ocular surface and drug, or protect drug from metabolic degradation.

The enkephalinergic nervous system and its immunomodulation on the developing immune system during the ontogenesis of oyster Crassostrea gigas

Enkephalinergic neuroendocrine-immune regulatory system is one of the most important neuroendocrine-immune systems in both vertebrates and invertebrates for its significant role in the immune regulation. In the present study, the early onset of enkephalinergic nervous system and its immunomodulation on the developing immune system during the ontogenesis of oyster Crassostrea gigas were investigated to illustrate the function of neural regulation on the innate immune system in oyster larvae. [Met5]-enkephalin (Met-ENK) was firstly observed on the marginal of the dorsal half of D-hinged larvae. Six immune-related molecules, including four PRRs (CgCTL-1, CgCTL-2, CgCTL-4, CgNatterin-3) and two immune effectors (CgTNF-1 and CgEcSOD) were detected in the early developmental stages of trochophore, D-hinged and umbo larvae of oyster. After incubated with [Met5]-enkephalin, the mRNA expression level of all the PRRs changed significantly (p < 0.05). In trochophore larvae, the expression level of CgNatterin-3 decreased dramatically (p < 0.05) at 6 h, and the expression level of CgCTL-4 was significantly down-regulated at 3 h and 6 h (p < 0.05), respectively. In D-hinged and umbo larvae, only CgCTL-1 was significantly down-regulated and the differences were significant at 3 h and 6 h (p < 0.05), while the expression level of CgCTL-2 and CgCTL-4 increased significantly at 3 h after treatment (p < 0.05). Moreover, the expression levels of immune effectors were up-regulated significantly at 3 h and 6 h in trochophore larvae (p < 0.05). The expression level of CgTNF-1 in both blank and experiment groups was up-regulated but there was no significant difference in D-hinged larvae stage. On the contrary, the expression level of CgEcSOD in D-hinged larvae decreased dramatically at 3 h and 6 h after [Met5]-enkephalin incubation (p < 0.05). In umbo larvae, the expression level of CgTNF-1 and CgEcSOD in the experiment group increased significantly at 6 h after [Met5]-enkephalin treatment (p < 0.05), while no significant difference was found in the blank group. In addition, the anti-bacterial activities of the total protein extract from trochophore, D-hinged and umbo larvae increased significantly (p < 0.05) at both 3 h and 6 h after [Met5]-enkephalin incubation compared to that in the blank group, and PO activities of both D-hinged and umbo larvae total protein extract increased significantly (p < 0.05) while no significant difference was observed in trochophore larvae. The PO activities of the total protein extract in all the experiment groups decreased after the treatment with [Met5]-enkephalin for 6 h, but no significant difference was observed when compared to the blank group. Furthermore, after incubation for 6 h, the concentration of both CgTNF-1 and CgIL17-5 increased dramatically compared to that in the blank group (p < 0.05). These results together indicated that the enkephalinergic nervous system of oyster was firstly appeared in D-hinged larvae, while the primitive immune defense system existed in the region of prototroch in trochophore larvae and developed maturely after D-hinged larvae. The developing immune system could be regulated by the neurotransmitter [Met5]-enkephalin released by the neuroendocrine system in oyster C. gigas.

Analgesic roles of peripheral intrinsic met-enkephalin and dynorphin A in long-lasting inflammatory pain induced by complete Freund's adjuvant in rats.

Previous studies have focused on strategies for pain relief based on the peripheral opioid system. However, little is known with regard to the profile of the peripheral opioid system in long-lasting inflammatory pain. In the current study, the intrinsic changes of the peripheral opioids were investigated in long-lasting inflammatory pain. A rat model of complete Freund's adjuvant (CFA)-induced inflammatory pain was established. Paw swelling and thermal hyperalgesia (paw withdrawal latency, PWL) were analyzed until day 18 after the CFA injection. The levels of peripheral opioids and their upstream inducers, corticotrophin-releasing factor (CRF) and interleukin (IL)-1β, were measured, and validation experiments were performed using opioid receptor antagonists. Long-lasting inflammatory pain was successfully induced in the rats, as shown by the significantly increased paw swelling and decreased PWLs. On day 18 after the CFA injection, the IL-1β levels were significantly elevated, while CRF remained at a normal level in the paw inflammatory tissue. In addition, met-enkephalin (Met-ENK) and dynorphin A (DYN A) levels were significantly increased, while the β-endorphin level remained normal. Local intraplantar administration of δ- and κ-opioid receptor antagonists resulted in more substantial pain, but did not significantly affect the PWLs of the normal control rats. Therefore, the results indicated that the increased levels of local Met-ENK and DYN A in CFA-induced long-lasting inflammatory pain may be involved in peripheral intrinsic analgesia.

Desensitization of mu opioid receptors on Kölliker‐Fuse neurons

Tolerance to the respiratory depressant effects of opioids develops less readily than tolerance to analgesia, creating a dosing limit in chronic pain patients. Desensitization of mu opioid receptors is considered an initial step in the development of tolerance. Currently, most information about desensitization has been obtained in locus coeruleus (LC) neurons. Given the lack of respiratory tolerance, this study examined desensitization of mu opioid receptors on respiratory‐controlling Kölliker‐Fuse (KF) neurons. In voltage‐clamp recordings from KF neurons in rat brain slice, the opioid receptor agonist [Met5]enkephalin (ME) caused an outward current in a population of KF neurons due to activation of GIRK conductance. Compared to ME, the partial opioid agonist morphine produced a current that was proportionally larger than the morphine‐mediated current in LC brain slice preparations, indicating that KF neurons may have a large receptor reserve and thus be resistant to desensitization. Desensitization was induced by prolonged (10 min) perfusion of a high concentration of ME (30 µM). Desensitization of the ME‐mediated GIRK current was significantly less in KF neurons than in LC neurons. In contrast to LC, desensitization was not enhanced by activation of PKC or in slices from morphine‐treated rats. Thus, a lack of desensitization of mu opioid receptors on KF neurons may contribute to the relative lack of tolerance to the respiratory depressant effect of opioids. This will be tested using in situ and in vivo preparations of morphine‐treated rats. Supported by DA038069 (ESL) and DA08163 (JTW).

A high performance system to study the influence of temperature in on-line solid-phase extraction capillary electrophoresis

A novel high performance system to control the temperature of the microcartridge in on-line solid phase extraction capillary electrophoresis (SPE–CE) is introduced. The mini-device consists in a thermostatic bath that fits inside of the cassette of any commercial CE instrument, while its temperature is controlled from an external circuit of liquid connecting three different water baths. The circuits are controlled from a switchboard connected to an array of electrovalves that allow to rapidly alternate the water circulation through the mini-thermostatic-bath between temperatures from 5 to 90°C. The combination of the mini-device and the forced-air thermostatization system of the commercial CE instrument allows to optimize independently the temperature of the sample loading, the clean-up, the analyte elution and the electrophoretic separation steps.The system is used to study the effect of temperature on the C18-SPE–CE analysis of the opioid peptides, Dynorphin A (Dyn A), Endomorphin1 (END) and Met-enkephalin (MET), in both standard solutions and in spiked plasma samples. Extraction recoveries demonstrated to depend, with a non-monotonous trend, on the microcartridge temperature during the sample loading and became maximum at 60°C. Results prove the potential of temperature control to further enhance sensitivity in SPE–CE when analytes are thermally stable.

Effects of Enteromyxum scophthalmi experimental infection on the neuroendocrine system of turbot, Scophthalmus maximus (L.)

Enteromyxum scophthalmi is an intestinal myxosporean parasite responsible for serious outbreaks in turbot Scophthalmus maximus (L.) culture, in North-western Spain. The disease affects the digestive tract, provokes severe catarrhal enteritis, emaciation and high rates of mortality. The digestive parasitization triggers a response with the coordinate participation of immune and neuroendocrine systems through the action of peptides released by enteroendocrine cells and present in nervous elements, acting as neuro-immune modulators. The present study was designed to assess the response of the turbot neuroendocrine system against E. scophthalmi infection. Immunohistochemical tests were applied to sections of the gastrointestinal tract of uninfected and E. scophthalmi-infected turbot to characterize the presence of bombesin (BOM), glucagon (GLUC), somatostatin (SOM), leu-enkephalin (LEU) and met-enkephalin (MET). The occurrence of E. scophthalmi in the turbot gastrointestinal tract increased the number of enteroendocrine cells immunoreactive to SOM, LEU and MET. On the other hand, BOM and GLUC immunoreactive cells were less numerous in the gastrointestinal tract of the parasitized turbot. Scarce immunoreactivity to BOM, GLUC and SOM was observed in nerve fibres and neurons of the myenteric plexus of control and infected fish. The results indicate that E. scophthalmi infection in turbot induced changes in the neuroendocrine system, with the diminution of the anorexigenic peptides BOM and GLUC; the increase of enkephalins, related to pro-inflammatory processes; and the increase of SOM, which may cause inhibitory effects on the immune response, constituting a compensatory mechanism to the exacerbated response observed in E. scophthalmi-infected turbot.