Opioid Peptide Levels Research Articles

Major clinical evidence on the use of low-dose naltrexone in the treatment of cancer: a systematic review

Introduction: Opioid receptors are groups of receptors (γ-, κ-, δ-, and ζ-opioid receptors) that are widely distributed in nerve cells in the brain, spinal cord, and digestive tract. Naltrexone is a type of general opioid receptor antagonist. It has been used to treat chronic pain syndrome, autoimmune diseases, and cancer at a dose of 5 mg/day, which is generally called low-dose naltrexone (LDN). Objective: It was to analyze the pharmacological functions of low-dose naltrexone, especially in anti-inflammation and immunoregulation, and its therapeutic potential against cancer. Methods: The research and development of the work were carried out from June to July 2024 in the Scopus, PubMed, Science Direct, and Scielo databases, using scientific articles from the last 15 years, following the PRISMA rules. The quality of the studies was based on the GRADE and AMSTAR2 instruments, and the risk of bias was assessed using the Cochrane instrument (Funnel Plot). Results and Conclusion: Fifteen studies were included in the systematic review out of 30. Most studies showed homogeneity in their results, with X2=94.5%>50%. Low-dose naltrexone has immunomodulatory and therapeutic effects. Low-dose naltrexone regulates the production of inflammatory cytokines, influencing the level of endogenous opioid peptides in the body. Furthermore, low-dose naltrexone has an antitumor effect and can modulate the neuroblastoma tumor response, delaying the onset and reducing the incidence rate of tumors, significantly decreasing tumor volume and weight, and DNA synthesis in cancer.

Effects of lamb sex and ewe parity on suckling-related neonatal behaviors and weaning weight of small-tailed han lambs

Suckling behavior is essential for the growth, health, and welfare of the lamb. The factors that influence the suckling-related behavior varied across breeds, and they remain unclear in Small-tailed Han lamb. This study aims to investigate the effects of ewe parity, lamb sex, and birth order on the suckling-related behaviors of Small-tailed Han lamb. The neonatal behaviors of 36 lambs were observed during the first week after birth using focal-animal sampling methods, and the levels of cholecystokinin (CCK), endogenous opioid peptides and growth hormone (GH) were measured with ELISA methods before weaning. Results showed that male lambs began standing and suckling behaviors earlier than female lambs, and the duration of each suckling bout was longer in male lambs (P < 0.05). Lambs born to multiparous ewes more frequently suckled and attempted to stand earlier than lambs from primiparous ewes (P < 0.05). The weaning weights of male lambs and lambs from multiparous ewes were heavier, and the serum levels of CCK, opioid peptides, and GH were sometimes higher in these lambs before weaning (P < 0.05). Meanwhile, lamb birth order has no significant effect on the suckling-related behaviors of lambs (P > 0.05). In addition, the suckling-related behaviors of male lamb and lambs born to multiparous ewes were found to strongly correlate with each other. In conclusion, lamb sex and ewe parity could affect the suckling-related behaviors and weaning weights of Small-tailed Han lambs. Moreover, male lambs and lambs born to multiparous ewes seemed to have better suckling-related behaviors. Many of these behaviors were highly connected with one another. These phenomena may be related to the higher serum levels of CCK, opioid peptides, and GH. The effects of lamb sex on suckling-related behaviors were different from those of other breeds in previous research. The male lambs were observed to initiate sucking-related behaviors earlier in this study. Our findings will also help in extensively understanding the variability in the effects of these factors on suckling-related behavior across sheep breeds.

Chronic stress differentially altersmRNAexpression of opioid peptides and receptors in the dorsal hippocampus of female and male rats

Chronic immobilization stress (CIS) results in sex-dependent changes in opioid peptide levels and receptor subcellular distributions within the rat dorsal hippocampus, which are paralleled with an inability for males to acquire conditioned place preference (CPP) to oxycodone. Here, RNAScope in situ hybridization was used to determine the expression of hippocampal opioid peptides and receptors in unstressed (US) and CIS estrus female and male adult (∼2.5 months old ) Sprague Dawley rats. In all groups, dentate granule cells expressed PENK and PDYN; additionally, numerous interneurons expressed PENK. OPRD1 and OPRM1 were primarily expressed in interneurons, and to a lesser extent, in pyramidal and granule cells. OPRK1-was expressed in sparsely distributed interneurons. There were few baseline sex differences: US females compared to US males had more PENK-expressing and fewer OPRD1-expressing granule cells and more OPRM1-expressing CA3b interneurons. Several expression differences emerged after CIS. Both CIS females and males compared to their US counterparts had elevated: (1) PENK-expressing dentate granule cells and interneurons in CA1 and CA2/3a; (2) OPRD1 probe number and cell expression in CA1, CA2/3a and CA3b and the dentate gyrus; and (3) OPRK1-expressing interneurons in the dentate hilus. Also, CIS males compared to US males had elevated: (1) PDYN expression in granule cells; (2) OPRD1 probe and interneuron expression in CA2/3a; (3) OPRM1 in granule cells; and (4) OPRK1 interneuron expression in CA2/3a. The sex-specific changes in hippocampal opioid gene expression may impact network properties and synaptic plasticity processes that may contribute to the attenuation of oxycodone CPP in CIS males.

Cannabinoid Receptor-2 Activation in Keratinocytes Contributes to Elevated Peripheral β-Endorphin Levels in Patients With Obstructive Jaundice.

Cholestatic diseases are often accompanied by elevated plasma levels of endogenous opioid peptides, but it is still unclear whether central or peripheral mechanisms are involved in this process, and little is known about the change of pain threshold in these patients. The purpose of this study was to determine the preoperative pain threshold, postoperative morphine consumption, and central and peripheral β-endorphin levels in patients with obstructive jaundice. This study also tests the hypothesis that activation of the cannabinoid receptor-2 (CB2R) in skin keratinocytes by endocannabinoids is the mechanism underlying circulating β-endorphin elevation in patients with obstructive jaundice. The electrical pain thresholds, 48-hour postoperative morphine consumption, concentrations of β-endorphin in plasma and cerebrospinal fluid, skin and liver β-endorphin expression, and plasma levels of endocannabinoids were measured in jaundiced (n = 32) and control (n = 32) patients. Male Sprague-Dawley rats and human keratinocytes (human immortalized keratinocyte cell line [HaCaT]) were used for the in vivo and in vitro experiments, respectively. Mechanical and thermal withdrawal latency, plasma level, and skin expression of β-endorphin were measured in CB2R-antagonist-treated and control bile duct-ligated (BDL) rats. In cultured keratinocytes, the effect of CB2R agonist AM1241-induced β-endorphin expression was observed and the phosphorylation of extracellular-regulated protein kinases 1/2, p38, and signal transducer and activator of transcription (STAT) pathways were investigated. This study found (1) the plasma level of β-endorphin (mean ± standard error of the mean [SEM]) was 193.9 ± 9.6 pg/mL in control patients, while it was significantly increased in jaundiced patients (286.6 ± 14.5 pg/mL); (2) the electrical pain perception threshold and the electrical pain tolerance threshold were higher in patients with obstructive jaundice compared with controls, while the 48-hour postoperative morphine consumption was lower in the jaundiced patients; (3) there was no correlation between plasma β-endorphin levels, electrical pain thresholds, and 48-hour postoperative morphine consumption in patients with obstructive jaundice; (4) the plasma level of the endogenous cannabinoid anandamide was increased in the jaundiced patients; (5) CB2R antagonist treatment of the BDL rats reduced β-endorphin levels in plasma and skin keratinocytes, while it did not alter the nociceptive thresholds in BDL and control rats; (6) the endocannabinoid anandamide-induced β-endorphin synthesis and release via CB2R in cultured keratinocytes; and (7) phosphorylation of extracellular-regulated protein kinases 1/2 is involved in the CB2R-agonist-induced β-endorphin expression in keratinocytes. CB2R activation in keratinocytes by the endocannabinoid anandamide may play an important role in the peripheral elevation of β-endorphin during obstructive jaundice.

Phase II double-blind randomised controlled trial of exogenous administration of melatonin in chronic pain (DREAM—CP): a study protocol

IntroductionChronic pain is prevalent, and approximately half of patients with chronic pain experience sleep disturbance. Exogenous melatonin is licensed to treat primary insomnia and there is some evidence for analgesic...

Activation of Kappa Opioid Receptor Regulates the Hypothermic Response to Calorie Restriction and Limits Body Weight Loss.

Mammals maintain a nearly constant core body temperature (Tb) by balancing heat production and heat dissipation. This comes at a high metabolic cost that is sustainable if adequate calorie intake is maintained. When nutrients are scarce or experimentally reduced such as during calorie restriction (CR), endotherms can reduce energy expenditure by lowering Tb [1-6]. This adaptive response conserves energy, limiting the loss of body weight due to low calorie intake [7-10]. Here we show that this response is regulated by the kappa opioid receptor (KOR). CR is associated with increased hypothalamic levels of the endogenous opioid Leu-enkephalin, which is derived from the KOR agonist precursor dynorphin [11]. Pharmacological inhibition of KOR, but not of the delta or the mu opioid receptor subtypes, fully blocked CR-induced hypothermia and increased weight loss during CR independent of calorie intake. Similar results were seen with DIO mice subjected to CR. In contrast, inhibiting KOR did not change Tb in animals fed ad libitum (AL). Chemogenetic inhibition of KOR neurons in the hypothalamic preoptic area reduced the CR-induced hypothermia, whereas chemogenetic activation of prodynorphin-expressing neurons in the arcuate or the parabrachial nucleus lowered Tb. These data indicate that KOR signaling is a pivotal regulator of energy homeostasis and can affect body weight during dieting by modulating Tb and energy expenditure.

Endogenous peripheral pain regulatory systems in orofacial pain patients

IntroductionInitiation of pulpal pain begins with the release of bacterial toxins and inflammatory mediators that either activate or sensitize nociceptors thereby activating the trigeminal pain pathway. In addition, pain perception quickly triggers inhibitory signals that are often mediated by endogenously produced analgesic substances such as opioid peptides. The balance between inflammatory mediators and endogenously released analgesics may contribute to clinical conditions such as symptomatic versus asymptomatic pulpitis. We hypothesized that patients with asymptomatic periradicular pathosis have increased levels of the opioid peptides compared to patients with symptomatic periradicular pathosis. In addition, we also proposed to study the immune cell type that is responsible for the release the opioid peptides in periradicular lesions. Material and methodFifty tissue biopsy samples obtained from patients who have received endodontic microsurgery and assigned to either symptomatic and asymptomatic groups (n = 25/group) based on their pre-operative clinical periradicular testings. Immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and Fluorescence-Activated Cell Sorting (FACS) were performed to determine the expressions, levels and the cellular sources of opioid peptides, namely; β-endorphin (END), met-enkephalin (ENK) and dynorphin A (DYN), respectively. Radioimmunoassay (RIA) was performed to determine the functional role of conditioned media (CM) from asymptomatic and symptomatic patients on regulation of trigeminal (TG) neuronal activity in vitro. ResultsCompared to symptomatic group, patients with asymptomatic lesions showed 3-fold increase in END levels only. Macrophages showed the highest expression of END, DYN and ENK. This study shows that the application of CM from asymptomatic and symptomatic patients triggered differential regulation of TG neuronal activity. ConclusionEndogenous opioid peptides are present in periapical lesions. The higher level of endogenous opioid peptides in asymptomatic periapical lesions could explain the lack of symptoms in asymptomatic lesions.

Episodic Ethanol Exposure in Adolescent Rats Causes Residual Alterations in Endogenous Opioid Peptides.

Adolescent binge drinking is associated with an increased risk of substance use disorder, but how ethanol affects the central levels of endogenous opioid peptides is still not thoroughly investigated. The aim of this study was to examine the effect of repeated episodic ethanol exposure during adolescence on the tissue levels of three different endogenous opioid peptides in rats. Outbred Wistar rats received orogastric (i.e., gavage) ethanol for three consecutive days per week between 4 and 9 weeks of age. At 2 h and 3 weeks, respectively, after the last exposure, beta-endorphin, dynorphin B and Met-enkephalin-Arg6Phe7 (MEAP) were analyzed with radioimmunoassay. Beta-endorphin levels were low in the nucleus accumbens during ethanol intoxication. Remaining effects of adolescent ethanol exposure were found especially for MEAP, with low levels in the amygdala, and high in the substantia nigra and ventral tegmental area three weeks after the last exposure. In the hypothalamus and pituitary, the effects of ethanol on beta-endorphin were dependent on time from the last exposure. An interaction effect was also found in the accumbal levels of MEAP and nigral dynorphin B. These results demonstrate that repeated episodic exposure to ethanol during adolescence affected opioid peptide levels in regions involved in reward and reinforcement as well as stress response. These alterations in opioid networks after adolescent ethanol exposure could explain, in part, the increased risk for high ethanol consumption later in life.

РІВЕНЬ ГОРМОНІВ СТРЕС-ЛІМІТУЮЧОЇ СИСТЕМИ У ВАГІТНИХ З ПСИХОЕМОЦІЙНИМИ ПОРУШЕННЯМИ, ОБУМОВЛЕНИМИ ТРИВОЖНІСТЮ

Мета дослідження – визначити рівень гормонів стрес-лімітуючої системи у вагітних з урахуванням їх психоемоційного стану.Матеріали та методи. Обстежено 86 жінок у ІІ та на початку ІІІ триместру вагітності. До основної групи включені 60 вагітних із середнім та високим рівнями ситуативної тривожності, а також 26 вагітних із рівнем тривоги 30 балів і нижче, що свідчить про низький рівень СТ (група порівняння). Для оцінки ситуативної тривожності та особистісної тривожності використовували шкалу Ч. Д. Спілбергера, яка адаптована Ю. Л. Ханіним. Кількісну оцінку концентрації гормонів у плазмі крові визначали методом імуноферментного аналізу на апараті «SIRIO S». Для визначення рівня пролактину (Пр) використовували тест-систему Monobind INC (США), кортизолу (К) та інсуліну (Ін) – тест-системи DRG (Німеччина). Рівень ендогенних опіоїдних пептидів (β-ендорфіну) визначали з використанням тест-системи Peninsula Laboratories, LLC (США).Результати дослідження та їх обговорення. Групи дослідження були розділені залежно від рівня СТ. Встановлено, що середній показник СТ у групі порівняння склав (26,3±1,2) бала та був статистично достовірно нижчим (p&lt;0,05) за середній показник в основній групі ((37,9±1,4) бала). Досліджуючи концентрацію Пр, встановлено, що взаємовплив із тривожністю підтверджується наявністю позитивного кореляційного зв’язку між його рівнем та СТ (r=+0,690, p&lt;0,05), а також менш значиму з ОТ (r=+0,254, p&lt;0,05) відповідно. При оцінці рівня Пр було встановлено статистично достовірно вищий (p&lt;0,05) серед вагітних із середньо-високим рівнем тривожності 136,2 (118,0; 151,6) нг/мл порівняно з жінками з низьким її рівнем 93,7 (67,4; 110,7) нг/мл. При порівнянні концентрації К у вагітних із середньо-високим рівнем СТ – 416,0 (368,2; 560,0) нг/мл встановлено статистично достовірне (p&lt;0,05) його переважання порівняно з жінками, які мали низький її рівень – 343,2 (304,6; 370,5) нг/мл. Взаємозв’язок рівня СТ та К у вагітних із високим та середнім її рівнями підтверджується також наявністю позитивного кореляційного зв’язку (r=+0,704, p&lt;0,05). У вагітних із низьким рівнем СТ таких зв’язків не встановлено. При оцінці рівня Ін встановлено, що він не мав зв'язку з тривожністю, а також із терміном гестації та віком вагітних. Так, рівень Ін серед вагітних із середньо-високим рівнем тривожності склав 11,5 (9,1; 16,8) мкл Од/мл, а в групі жінок із низьким її рівнем – 12,2 (9,8; 21,9) мкл Од/мл та був статистично не достовірним (p&gt;0,05). Однак розрахунок К/Ін індексу, як маркера стресу, дозволив встановити його статистично достовірне (p&lt;0,05) переважання в групі вагітних із середньо-високим рівнем 39,4 (23,2; 54,9) порівняно з жінками з низьким 24,6 (16,02; 34,1) її рівнем. При оцінці β-ендорфіну не було встановлено статистично достовірної різниці (p&gt;0,05) між показниками у вагітних із середньо-високим – 1,2 (0,6; 3,6) нг/мл та низьким – 1,4 (0,7; 3,0) нг/мл рівнями СТ. Вірогідно, це свідчить про відсутність виразного впливу означеного нейропептиду на формування емоційного стану в жінок під час вагітності.Висновки. Оцінка рівня пролактину, як ендогенного анксіолітика, дозволила встановити статистично достовірне (р&lt;0,05) його зростання у вагітних із середньо-високим рівнем СТ у порівнянні з групою жінок із низьким її рівнем. Зростання рівня пролактину у вагітних із низьким рівнем СТ залежить від терміну гестації (r=+0,665, p&lt;0,05) та не має зв’язку як з рівнем СТ, так і ОТ. Серед вагітних із середнім та високим рівнями СТ мала місце протилежна картина – негативний зв’язок із терміном вагітності (r = -0,338, p&gt;0,05) та позитивний – із рівнем СТ (r = +0,576, p&lt;0,05). Порівняння рівня інсуліну та β-ендорфіну у вагітних з урахуванням рівня СТ не дозволило встановити статистично значимої різниці (p&gt;0,05). Однак визначення К/Ін індексу, як маркера стресу, дозволило встановити його статистично достовірне (p&lt;0,05) переважання в групі вагітних із середньо-високим рівнем, порівнюючи з вагітними, які мали низький рівень СТ.

Genetic predictors of celiac disease, lactose intolerance, and vitamin D function and presence of peptide morphins in urine of children with neurodevelopmental disorders

Gastrointestinal disturbances, nutritional deficiencies, and food intolerances are frequently observed in children with neurodevelopmental disorders (NDD). To reveal possible association of celiac disease risk variants (HLA-DQ), lactose intolerance associated variant (LCT-13910C>T) as well as variant associated with vitamin D function (VDR FokI) with NDD, polymerase chain reaction-based methodology was used. Additionally, intestinal peptide permeability was estimated in NDD patients and healthy children by measuring the level of peptides in urine using high-performance liquid chromatography. Levels of opioid peptides, casomorphin 8, and gluten exorphin C were significantly elevated in urine samples of NDD patients (P = 0.004 and P = 0.005, respectively), but no association of genetic risk variants for celiac disease and lactose intolerance with NDD was found. Our results indicate that increased intestinal peptide permeability observed in analyzed NDD patients is not associated with genetic predictors of celiac disease or lactose intolerance. We have also found that FF genotype of VDR FokI and lower serum levels of vitamin D (25-OH) showed association with childhood autism (CHA), a subgroup of NDD. We hypothesize that vitamin D might be important for the development of CHA.

Intra- and interregional coregulation of opioid genes: broken symmetry in spinal circuits.

Regulation of the formation and rewiring of neural circuits by neuropeptides may require coordinated production of these signaling molecules and their receptors that may be established at the transcriptional level. Here, we address this hypothesis by comparing absolute expression levels of opioid peptides with their receptors, the largest neuropeptide family, and by characterizing coexpression (transcriptionally coordinated) patterns of these genes. We demonstrated that expression patterns of opioid genes highly correlate within and across functionally and anatomically different areas. Opioid peptide genes, compared with their receptor genes, are transcribed at much greater absolute levels, which suggests formation of a neuropeptide cloud that covers the receptor-expressed circuits. Surprisingly, we found that both expression levels and the proportion of opioid receptors are strongly lateralized in the spinal cord, interregional coexpression patterns are side specific, and intraregional coexpression profiles are affected differently by left- and right-side unilateral body injury. We propose that opioid genes are regulated as interconnected components of the same molecular system distributed between distinct anatomic regions. The striking feature of this system is its asymmetric coexpression patterns, which suggest side-specific regulation of selective neural circuits by opioid neurohormones.-Kononenko, O., Galatenko, V., Andersson, M., Bazov, I., Watanabe, H., Zhou, X. W., Iatsyshyna, A., Mityakina, I., Yakovleva, T., Sarkisyan, D., Ponomarev, I., Krishtal, O., Marklund, N., Tonevitsky, A., Adkins, D. L., Bakalkin, G. Intra- and interregional coregulation of opioid genes: broken symmetry in spinal circuits.

Adoptive transfer of M2 macrophages reduces neuropathic pain via opioid peptides

BackgroundDuring the inflammation which occurs following nerve damage, macrophages are recruited to the site of injury. Phenotypic diversity is a hallmark of the macrophage lineage and includes pro-inflammatory M1 and anti-inflammatory M2 populations. Our aim in this study was to investigate the ability of polarized M0, M1, and M2 macrophages to secrete opioid peptides and to examine their relative contribution to the modulation of neuropathic pain.MethodsMouse bone marrow-derived cells were cultured as unstimulated M0 macrophages or were stimulated into an M1 phenotype using lipopolysaccharide and interferon-γ or into an M2 phenotype using interleukin-4. The macrophage phenotypes were verified using flow cytometry for surface marker analysis and cytokine bead array for cytokine profile assessment. Opioid peptide levels were measured by radioimmunoassay and enzyme immunoassay. As a model of neuropathic pain, a chronic constriction injury (CCI) of the sciatic nerve was employed. Polarized M0, M1, and M2 macrophages (5 × 105 cells) were injected perineurally twice, on days 14 and 15 following CCI or sham surgery. Mechanical and heat sensitivity were measured using the von Frey and Hargreaves tests, respectively. To track the injected macrophages, we also transferred fluorescently stained polarized cells and analyzed the surface marker profile of endogenous and injected cells in the nerves ex vivo.ResultsCompared to M0 and M1 cells, M2 macrophages contained and released higher amounts of opioid peptides, including Met-enkephalin, dynorphin A (1–17), and β-endorphin. M2 cells transferred perineurally at the nerve injury site reduced mechanical, but not heat hypersensitivity following the second injection. The analgesic effect was reversed by the perineurally applied opioid receptor antagonist naloxone methiodide. M2 cells did not affect sensitivity following sham surgery. Neither M0 nor M1 cells altered mechanical and heat sensitivity in CCI or sham-operated animals. Tracing the fluorescently labeled M0, M1, and M2 cells ex vivo showed that they remained in the nerve and preserved their phenotype.ConclusionsPerineural transplantation of M2 macrophages resulted in opioid-mediated amelioration of neuropathy-induced mechanical hypersensitivity, while M1 macrophages did not exacerbate pain. Therefore, rather than focusing on macrophage-induced pain generation, promoting opioid-mediated M2 actions may be more relevant for pain control.

Can the pathophysiology of autism be explained by the nature of the discovered urine peptides and dietary antigens?

PurposeA: 1. To develop the urine analysis for exorphins for routine use in blood and cerebrospinal fluid (CSF).2. Disorders where patient related validation must be carried out: schizophrenia, depression (uni- and bipolar) and autism.MethodA: HPLC-MS/MS (fragmentation mass spectrometry) technology.With both a specific HPLC retention time and MS/MS (fragmentation) this method is close to an absolute technique for peptide recognition.B: ELISA against specific proteins (gliadin, gluten and casein and transglutaminase 6) (Table 1 og 2).BackgroundA: schizophrenia: increased opioid peptide levels have been found in Schizophrenia using HPLC, immune assay and behavioral tests. [1–6] as part of a general peptide increase in urine. Since peptides are signaling compounds inhibition of peptidases during transport and work up of samples is critical to prevent break down, which is as expected fast at room temperature.Strongly supporting is view is the data on postpartum psychoses (a very symptom rich psychosis) where also amino acid sequence of human casomorphin found increased, has been done [7–8]. The opioids can explain most of the symptoms of the psychotic schizophrenic state [6]. It is of paramount importance then to measure these peptides in carefully diagnosed patients on and without medication, in urine, blood and spinal fluid.As can be seen in Table 1, it is important to measure IgA and IgG antibodies against the precursor proteins for the exorphins, which are found increased by several groups, and also have direct effects on the nervous system [9].B. In depression increase levels of peptides has been found [18,28,29] and also opioid levels measured as opium receptor binding peptides [28]. In schizoaffective psychosis MS/MS exact detection of exorphins have been published [6]. Also in this syndrome it is critical to be able to measure the exorphins in blood and CSF, especially since the peptidases involved in break down of exorphins are decreased in depressions [30,31]. Inflammatory interleukins are also increased in depressions both uni- and bipolar [32] indicative of inflammatory processes probably in the gut. Inflammatory interleukins increase the permeability of epithelial membranes [33].C. Autism. Considerable work has been done using HPLC with UV detection and co-chromatography [12,34–40]. However, with HPLC–MS/MS we can ensure that we are measuring only the exorphins and not chromatographically similar peaks that hide inside the main peak [41–43]. We therefore need to validate the new method in autism for both urine, blood and CSF (CSF collected only when spinal tap has to be done in any case).Inhibition of break down in urine, blood and cerebrospinal fluid (CSF)After extensive testing we have been left with three inhibitors. Citric acid 0.2 M; acetic acid 0.2 M and aprotenine [44,45].These body fluids will be provided by Prof Dr E. Severance and Prof Dr R. Yolken (Johns Hopkins Univ.) and Prof Dr. Cunningham (Uppsala Univ. Sweden). Lab 1 provides monovettes with citric acid as peptidase inhibitor for urine collection. Blood will be collected in EDTA – aprotenin vacuum test tubes (Vacutainer) as will be CSF.HPLC and MS/MS detection.The amount of urine analyzed on the HPLC after work up = 250 nanomles creatinine. To pick out generally active peptides in any one disorder, five and five autistic children or schizophrenic derived and depressive derived urines are mixed, creatinine re-determined and rerun. Peaks that are common to all patients increase or remain the same, while individual peaks of material on the HPLC runs are diluted out.The complete procedure is published in detail [48]. If we use reporter ions we do not have to match all the peaks as shown in attached figures. On Fig. 1, synthetic bovine β-casomorphine 1-4 (Y-P-F-P) is compared to biologically isolated compound from a batch of five autistic children. On Fig. 2, the faster routine analysis using reporter ions is shown for bovine β-casomorhne 1-4. Top trace is synthetic casomorphin 1-4 and bottom trace is biologically isolated compound. The complete analysis for a series of opioids is published [48].Program is then in sequence:– A: further validation of method for urine in the different disorders;– B: validation of method for blood in the same disorders;– C: validation of method for CSF (spinal fluid) in schizophrenics and depressive patients.NB.To avoid overlooking new compounds a complete HPLC run with UV 215 nm (peptide bonds); 280 nm (aromatic groups) and 325 nm (Indolyl-acryloid) shall be run for urines. If sufficient serum is available and spinal fluid these will also be run on HPLC in addition to MS/MS detection.Antibody assays will be done at Johns Hopkins using ELISA, Transglutaminase 6 antibodies at Lab 1 also using ELISA assay.Figures and references not available in the abstract.Disclosure of interestThe authors have not supplied their declaration of competing interest.

A ghrelin receptor (GHS-R1A) antagonist attenuates the rewarding properties of morphine and increases opioid peptide levels in reward areas in mice

Gut-brain hormones such as ghrelin have recently been suggested to have a role in reward regulation. Ghrelin was traditionally known to regulate food intake and body weight homoeostasis. In addition, recent work has pin-pointed that this peptide has a novel role in drug-induced reward, including morphine-induced increase in the extracellular levels of accumbal dopamine in rats. Herein the effect of the ghrelin receptor (GHS-R1A) antagonist, JMV2959, on morphine-induced activation of the mesolimbic dopamine system was investigated in mice. In addition, the effects of JMV2959 administration on opioid peptide levels in reward related areas were investigated. In the present series of experiment we showed that peripheral JMV2959 administration, at a dose with no effect per se, attenuates the ability of morphine to cause locomotor stimulation, increase the extracellular levels of accumbal dopamine and to condition a place preference in mice. JMV2959 administration significantly increased tissue levels of Met-enkephalin-Arg6Phe7 in the ventral tegmental area, dynorphin B in hippocampus and Leu-enkephalin-Arg6 in striatum. We therefore hypothesise that JMV2959 prevents morphine-induced reward via stimulation of delta receptor active peptides in striatum and ventral tegmental areas. In addition, hippocampal peptides that activate kappa receptor may be involved in JMV2959׳s ability to regulate memory formation of reward. Given that development of drug addiction depends, at least in part, of the effects of addictive drugs on the mesolimbic dopamine system the present data suggest that GHS-R1A antagonists deserve to be elucidated as novel treatment strategies of opioid addiction.

Role of Endogenous Opioid System in Ischemic-Induced Late Preconditioning.

BackgroundOpioid receptors (OR) are involved in myocardial late preconditioning (LPC) induced by morphine and δ1-opioid receptor (δ1-OR) agonists. The role of OR in ischemic-induced LPC is unknown. We investigated whether 1) OR are involved in the trigger and/or mediation phase of LPC and 2) a time course effect on the expression of different opioid receptors and their endogenous ligands exists.MethodsMale Wistar rats were randomly allocated to four groups (each group n = 8). Awake animals were ischemic preconditioned by a 5 minutes coronary occlusion. 24 hours later, anesthetized animals underwent 25 minutes coronary occlusion followed by 2 hours of reperfusion. The role of OR was investigated by treatment with intraperitoneal naloxone (Nal) 10 minutes prior to LPC (Nal-LPC; trigger phase) or 10 min prior to sustained ischemia (LPC-Nal; mediation phase).ResultsLPC reduced infarct size from 61±10% in controls to 25±9% (P<0.001). Naloxone during trigger or mediation phase completely abolished LPC-induced cardioprotection (59±9% and 62±9%; P<0.001 vs. LPC). 8, 12 and 24 hours after the ischemic stimulus, expression of δ-OR in the heart was increased, whereas μ-opioid receptor (μ-OR) and κ-opioid receptor (κ-OR) were not. Plasma concentrations of β-endorphin and leu-enkephalin but not dynorphin were increased by LPC.ConclusionIschemic LPC is triggererd and mediated by OR. Expression of δ-OR and plasma levels of endogenous opioid peptides are increased after ischemic LPC.

Application of cross-species PET imaging to assess neurotransmitter release in brain.

RationaleThis review attempts to summarize the current status in relation to the use of positron emission tomography (PET) imaging in the assessment of synaptic concentrations of endogenous mediators in the living brain.ObjectivesAlthough PET radioligands are now available for more than 40 CNS targets, at the initiation of the Innovative Medicines Initiative (IMI) “Novel Methods leading to New Medications in Depression and Schizophrenia” (NEWMEDS) in 2009, PET radioligands sensitive to an endogenous neurotransmitter were only validated for dopamine. NEWMEDS work-package 5, “Cross-species and neurochemical imaging (PET) methods for drug discovery”, commenced with a focus on developing methods enabling assessment of changes in extracellular concentrations of serotonin and noradrenaline in the brain.ResultsSharing the workload across institutions, we utilized in vitro techniques with cells and tissues, in vivo receptor binding and microdialysis techniques in rodents, and in vivo PET imaging in non-human primates and humans. Here, we discuss these efforts and review other recently published reports on the use of radioligands to assess changes in endogenous levels of dopamine, serotonin, noradrenaline, γ-aminobutyric acid, glutamate, acetylcholine, and opioid peptides. The emphasis is on assessment of the availability of appropriate translational tools (PET radioligands, pharmacological challenge agents) and on studies in non-human primates and human subjects, as well as current challenges and future directions.ConclusionsPET imaging directed at investigating changes in endogenous neurochemicals, including the work done in NEWMEDS, have highlighted an opportunity to further extend the capability and application of this technology in drug development.

Alcohol-induced changes in opioid peptide levels in adolescent rats are dependent on housing conditions.

BackgroundEndogenous opioids are implicated in the mechanism of action of alcohol and alcohol affects opioids in a number of brain areas, although little is known about alcohol's effects on opioids in the adolescent brain. One concern, in particular when studying young animals, is that alcohol intake models often are based on single housing that may result in alcohol effects confounded by the lack of social interactions. The aim of this study was to investigate short- and long-term alcohol effects on opioids and the influence of housing conditions on these effects.MethodsIn the first part, opioid peptide levels were measured after one 24-hour session of single housing and 2-hour voluntary alcohol intake in adolescent and adult rats. In the second part, a model with a cage divider inserted during 2-hour drinking sessions was tested and the effects on opioids were examined after 6 weeks of adolescent voluntary intake in single-and pair-housed rats, respectively.ResultsThe effects of single housing were age specific and affected Met-enkephalin-Arg6Phe7 (MEAP) in particular. In adolescent rats, it was difficult to distinguish between effects induced by alcohol and single housing, whereas alcohol-specific effects were seen in dynorphin B (DYNB), beta-endorphin (BEND), and MEAP levels in adults. Voluntary drinking affected several brain areas and the majority of alcohol-induced effects were not dependent on housing. However, alcohol effects on DYNB and BEND in the amygdala were dependent on housing. Housing alone affected MEAP in the cingulate cortex.ConclusionsAge-specific housing- and alcohol-induced effects on opioids were found. In addition, prolonged voluntary alcohol intake under different housing conditions produced several alcohol-induced effects independent of housing. However, housing-dependent effects were found in areas implicated in stress, emotionality, and alcohol use disorder. Housing condition and age may therefore affect the reasons and underlying mechanisms for drinking and could potentially affect the outcome of a number of end points in research on alcohol intake.

Sex and estrogen receptor expression influence opioid peptide levels in the mouse hippocampal mossy fiber pathway

The opioid peptides, dynorphin (DYN) and enkephalin (L-ENK) are contained in the hippocampal mossy fiber pathway where they modulate synaptic plasticity. In rats, the levels of DYN and L-ENK immunoreactivity (-ir) are increased when estrogen levels are elevated (Torres-Reveron et al., 2008, 2009). Here, we used quantitative immunocytochemistry to examine whether opioid levels are similarly regulated in wildtype (WT) mice over the estrous cycle, and how these compared to males. Moreover, using estrogen receptor (ER) alpha and beta knock-out mice (AERKO and BERKO, respectively), the present study examined the role of ERs in rapid, membrane-initiated (6h), or slower, nucleus-initiated (48h) estradiol effects on mossy fiber opioid levels. Unlike rats, the levels of DYN and L-ENK-ir did not change over the estrous cycle. However, compared to males, females had higher levels of DYN-ir in CA3a and L-ENK-ir in CA3b. In WT and BERKO ovariectomized (OVX) mice, neither DYN- nor L-ENK-ir changed following 6 or 48h estradiol benzoate (EB) administration. However, DYN-ir significantly increased 48h after EB in the dentate gyrus (DG) and CA3b of AERKO mice only. These findings suggest that cyclic hormone levels regulate neither DYN nor L-ENK levels in the mouse mossy fiber pathway as they do in the rat. This may be due to species-specific differences in the mossy fiber pathway. However, in the mouse, DYN levels are regulated by exogenous EB in the absence of ERα possibly via an ERβ-mediated pathway requiring new gene transcription.

Role of endogenous opioid peptides in the infarct size-limiting effect of adaptation to chronic continuous hypoxia

AimsThe objective of this study was to examine the involvement of endogenous opioid peptides and opioid receptor (OR) subtypes in the cardioprotective effect of adaptation to chronic hypoxia in rats. Main methodsRats were exposed to continuous normobaric hypoxia (CNH; 12% oxygen) for 3 weeks. Myocardial ischemia was induced by 20-min coronary artery occlusion followed by 3-h reperfusion in anesthetized open-chest animals. Various OR antagonists were administered to rats prior to ischemia. The size of myocardial infarction and the incidence of ischemic ventricular arrhythmias were assessed. Myocardial and plasma concentrations of opioid peptides (met-enkephalin, β-endorphin, and endomorphins) were determined. Key findingsAdaptation to CNH significantly increased myocardial and plasma concentrations of opioids, potentiated their further elevation by ischemia/reperfusion, and reduced myocardial infarct size, but it did not affect the incidence of ischemic arrhythmias. The infarct size-limiting effect of CNH was abolished by OR antagonists naltrexone (non-selective), naloxone methiodide (non-selective peripherally acting), TIPP[ψ] (δ-OR), naltriben (δ2-OR), or CTAP (μ-OR), while BNTX (δ1-OR) and nor-binaltorphimine (κ-OR) had no effect. SignificanceThe results suggest that the infarct size-limiting effect afforded by adaptation to CNH is mediated by activation of peripheral δ2- and μ-ORs by elevated levels of endogenous opioid peptides.

Morphine treatment selectively regulates expression of rat pituitary POMC and the prohormone convertases PC1/3 and PC2

The prohormone convertases, PC1/3 and PC2 are thought to be responsible for the activation of many prohormones through processing including the endogenous opioid peptides. We propose that maintenance of hormonal homeostasis can be achieved, in part, via alterations in levels of these enzymes that control the ratio of active hormone to prohormone. In order to test the hypothesis that exogenous opioids regulate the endogenous opioid system and the enzymes responsible for their biosynthesis, we studied the effect of short-term morphine or naltrexone treatment on pituitary PC1/3 and PC2 as well as on the level of pro-opiomelanocortin (POMC), the precursor gene for the biosynthesis of the endogenous opioid peptide, β-endorphin. Using ribonuclease protection assays, we observed that morphine down-regulated and naltrexone up-regulated rat pituitary PC1/3 and PC2 mRNA. Immunofluorescence and Western blot analysis confirmed that the protein levels changed in parallel with the changes in mRNA levels and were accompanied by changes in the levels of phosphorylated cyclic-AMP response element binding protein. We propose that the alterations of the prohormone processing system may be a compensatory mechanism in response to an exogenous opioid ligand whereby the organism tries to restore its homeostatic hormonal milieu following exposure to the opioid, possibly by regulating the levels of multiple endogenous opioid peptides and other neuropeptides in concert.