Endogenous Opioid Signaling in the Mouse Retina Modulates Pupillary Light Reflex.

Opioid peptides and their receptors are expressed in the mammalian retina; however, little is known about how they might affect visual processing. The melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs), which mediate important non-image-forming visual processes such as the pupillary light reflex (PLR), express ß-endorphin-preferring, µ-opioid receptors (MORs). The objective of the present study was to elucidate if opioids, endogenous or exogenous, modulate pupillary light reflex (PLR) via MORs expressed by ipRGCs. MOR-selective agonist [D-Ala2, MePhe4, Gly-ol5]-enkephalin (DAMGO) or antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) was administered via intravitreal injection. PLR was recorded in response to light stimuli of various intensities. DAMGO eliminated PLR evoked by light with intensities below melanopsin activation threshold but not that evoked by bright blue irradiance that activated melanopsin signaling, although in the latter case, DAMGO markedly slowed pupil constriction. CTAP or genetic ablation of MORs in ipRGCs slightly enhanced dim-light-evoked PLR but not that evoked by a bright blue stimulus. Our results suggest that endogenous opioid signaling in the retina contributes to the regulation of PLR. The slowing of bright light-evoked PLR by DAMGO is consistent with the observation that systemically applied opioids accumulate in the vitreous and that patients receiving chronic opioid treatment have slow PLR.

Lemairamin, isolated from the Zanthoxylum plants, alleviates pain hypersensitivity via spinal α7 nicotinic acetylcholine receptors.

Lemairamin (also known as wgx-50), is isolated from the pericarps of the Zanthoxylum plants. As an agonist of α7 nicotinic acetylcholine receptors (α7nAChRs), it can reduce neuroinflammation in Alzheimer's disease. This study evaluated its antinociceptive effects in pain hypersensitivity and explored the underlying mechanisms. The data showed that subcutaneous lemairamin injection dose-dependently inhibited formalin-induced tonic pain but not acute nociception in mice and rats, while intrathecal lemairamin injection also dose-dependently produced mechanical antiallodynia in the ipsilateral hindpaws of neuropathic and bone cancer pain rats without affecting mechanical thresholds in the contralateral hindpaws. Multiple bi-daily lemairamin injections for 7 days did not induce mechanical antiallodynic tolerance in neuropathic rats. Moreover, the antinociceptive effects of lemairamin in formalin-induced tonic pain and mechanical antiallodynia in neuropathic pain were suppressed by the α7nAChR antagonist methyllycaconitine. In an α7nAChR antagonist-reversible manner, intrathecal lemairamin also stimulated spinal expression of IL-10 and ß-endorphin, while lemairamin treatment induced IL-10 and ß-endorphin expression in primary spinal microglial cells. In addition, intrathecal injection of a microglial activation inhibitor minocycline, anti-IL-10 antibody, anti-ß-endorphin antiserum or µ-opioid receptor-preferred antagonist naloxone was all able to block lemairamin-induced mechanical antiallodynia in neuropathic pain. These data demonstrated that lemairamin could produce antinociception in pain hypersensitivity through the spinal IL-10/ß-endorphin pathway following α7nAChR activation.

Transgenic increase in the ß-endorphin concentration in cerebrospinal fluid alleviates morphine-primed relapse behavior through the µ opioid receptor in rats.

BACKGROUND: Opioid-primed relapse is a global burden. Although current strategies have improved, optimal therapy is urgently needed. METHODS: A recombinant adenovirus (Ad-NEP) expressing ß-endorphin (ß-EP) was designed and injected intracerebroventricularly (icv) into the right lateral ventricle in rats. Spatial and temporal ß-EP expression in the lateral ventricle wall, subventricular zone and adjacent choroid plexus and the ß-EP concentration in the cerebrospinal fluid (CSF) were observed during a 21-day period. A morphine priming-induced conditioned place preference (CPP) rat model was established. The ß-EP-ir neuron counts, CSF ß-EP concentration, and CPP score, which were used to evaluate morphine-primed reinstatement following extinction, were recorded 7 days after the icv injection. Additionally, the rats were pretreated with the irreversible µ opioid receptor antagonist ß-funaltrexamine (ß-FNA) and the selective κ opioid receptor antagonist nor-binaltorphimine (nor-BNI) to identify the receptor-dependent mechanism. RESULTS: Both peak ß-EP expression in target neurons and the peak CSF ß-EP concentration occurred 7 to 8 days after Ad-NEP icv injection. The sustainable increase in the CSF ß-EP concentration was correlated with a decrease in the CPP score 7 days after the Ad-NEP icv injection. Furthermore, reinstatement was almost reversed by ß-FNA pretreatment 24 hours before the behavioral test, but nor-BNI had little effect. CONCLUSION: The increasing cerebrospinal fluid ß-endorphin concentrations showed that the therapeutic effect on opioid relapse occurred predominantly through a µ opioid receptor-dependent mechanism. The Ad-NEP adenovirus can be considered an alternative therapy for opioid relapse.

Sensitivity of T-Lymphocytes to Hormones of the Anterior Pituitary Gland.

The review provides information about the features of the sensitivity of thymocytes, lymphoid organs' cells and T-lymphocytes of peripheral blood to the hormones secreted by anterior pituitary gland's cells: growth hormone, thyrotropin, adrenocorticotropic hormone, prolactin and ß-endorphin. Some aspects of the T-lymphocytes's response to humoral signals from the hypophysis are shown in the article. Also the pituitary hormones' role in the regulation of proliferation, differentiation, and cytokine production of T-lymphocytes in normal and pathological conditions of the organism being discussed.

Autocrine Interleukin-10 Mediates Glucagon-Like Peptide-1 Receptor-Induced Spinal Microglial ß-Endorphin Expression.

The glucagon-like peptide-1 (GLP-1) receptor agonist exenatide stimulates microglial ß-endorphin expression and subsequently produces neuroprotection and antinociception. This study illustrated an unrecognized autocrine role of IL-10 in mediation of exenatide-induced ß-endorphin expression. Treatment with exenatide in cultured primary spinal microglia concentration dependently stimulated the expression of the M2 microglial markers IL-10, IL-4, Arg 1, and CD206, but not the M1 microglial markers TNF-α, IL-1ß, IL-6, or CD68. Intrathecal exenatide injection also significantly upregulated spinal microglial expression of IL-10, IL-4, Arg 1, and CD206, but not TNF-α, IL-1ß, IL-6, or CD68. Intrathecal injection of exenatide stimulated spinal microglial expression of IL-10 and ß-endorphin in neuropathic rats. Furthermore, treatment with IL-10 (but not IL-4) stimulated ß-endorphin expression in cultured primary microglia, whereas treatment with ß-endorphin failed to increase IL-10 expression. The IL-10-neutralizing antibody entirely blocked exenatide-induced spinal microglial expression of ß-endorphin in vitro and in vivo and fully blocked exenatide mechanical antiallodynia in neuropathic rats. Moreover, specific cAMP/PKA/p38 signal inhibitors and siRNA/p38ß, but not siRNA/p38α, completely blocked exenatide-induced IL-10 expression in cultured primary microglia. Knock-down of IL-10 receptor-α mRNA using siRNA fully inhibited exenatide-induced spinal microglial ß-endorphin expression and mechanical antiallodynia in neuropathy. Exenatide also markedly stimulated phosphorylation of the transcription factor STAT3 in cultured primary microglia and ß-endorphin stimulation was completely inhibited by the specific STAT3 activation inhibitor. These results revealed that IL-10 in microglia mediated ß-endorphin expression after GLP-1 receptor activation through the autocrine cAMP/PKA/p38ß/CREB and subsequent IL-10 receptor/STAT3 signal pathways.SIGNIFICANCE STATEMENT Activation of GLP-1 receptors specifically and simultaneously stimulates the expression of anti-inflammatory cytokines IL-10 and IL-4, as well as the neuroprotective factor ß-endorphin from microglia. GLP-1 receptor agonism induces ß-endorphin expression and antinociception through autocrine release of IL-10. Activation of GLP-1 receptors stimulates IL-10 and ß-endorphin expression subsequently through the Gs-cAMP/PKA/p38ß/CREB and IL-10/IL-10 receptor-α/STAT3 signal transduction pathways.

B Lymphocytes Express Pomc mRNA, Processing Enzymes and ß-Endorphin in Painful Inflammation.

Immune cell-derived beta-endorphin (END) and other opioid peptides elicit potent and clinically relevant inhibition of pain (analgesia) in inflamed tissue by activation of peripheral opioid receptors. Pro-opiomelanocortin (POMC) is the polypeptide precursor of END and is processed by prohormone convertases (PCs). This study aims to decipher the processing of POMC in lymphocyte subsets in a rat model of unilateral painful hindpaw inflammation. Lymphocytes, isolated from popliteal lymph nodes, were separated into B-cells, T-cells, T-helper cells and cytotoxic T-cells using magnetic cell sorting, and were examined by polymerase chain reaction, immunofluorescence and radioimmunoassay. At 2 h of inflammation, POMC exon 2-3 mRNA was mostly expressed in B- but not in T-cells. Prohormone convertase 1 (PC1) mRNA and protein were upregulated in B-cells and T-helper cells. Prohormone convertase 2 (PC2) was expressed in T- and B-cells, both in inflamed and non-inflamed lymph nodes. END was expressed in B- but not in T-cells. We conclude that POMC, its processing enzymes and END are predominantly expressed in B-lymphocytes at 2 h of paw inflammation.

60 YEARS OF POMC: Purification and biological characterisation of melanotrophins and corticotrophins.

The remarkable conservation of the primary structures and anatomical location of dogfish α-melanocyte-stimulating hormone (MSH), corticotrophin-like intermediate lobe peptide (CLIP) and adrenocorticotrophic hormone (ACTH) compared with mammals reinforced the tissue-specific processing hypothesis of ACTH peptides in the pituitary gland. The cloning of dogfish pro-opiomelanocortin (POMC) led to the identification of δ-MSH and simultaneously revealed the high conservation of the γ-MSH sequence during evolution. These studies have also shown that ß-MSH is much less conserved during evolution and in some species is not even processed from ß-LPH. Human pro-γ-MSH potentiates the corticosteroidogenic activity of ACTH and peptides generated from its N-terminal, in particular big-γ-MSH, appear to have adrenal mitogenic activity. Human big-γ-MSH (from the zona intermedia) may also cause the adrenache. The review finishes with a cautionary note with regard to the misdiagnosis of the ectopic ACTH syndrome in which partial processing of ACTH can result in large concentrations of α-MSH and CLIP, which can interfere in the performance of two-site immunoassays, and the problem of the correct disulphide bridge arrangement in synthetic N-POMC peptides is also discussed.

Skin ß-endorphin mediates addiction to UV light.

UV light is an established carcinogen, yet evidence suggests that UV-seeking behavior has addictive features. Following UV exposure, epidermal keratinocytes synthesize proopiomelanocortin (POMC) that is processed to melanocyte-stimulating hormone, inducing tanning. We show that, in rodents, another POMC-derived peptide, ß-endorphin, is coordinately synthesized in skin, elevating plasma levels after low-dose UV. Increases in pain-related thresholds are observed and reversed by pharmacologic opioid antagonism. Opioid blockade also elicits withdrawal signs after chronic UV exposure. This effect was sufficient to guide operant behavioral choices to avoidance of opioid withdrawal (conditioned place aversion). These UV-induced nociceptive and behavioral effects were absent in ß-endorphin knockout mice and in mice lacking p53-mediated POMC induction in epidermal keratinocytes. Although primordial UV addiction, mediated by the hedonic action of ß-endorphin and anhedonic effects of withdrawal, may theoretically have enhanced evolutionary vitamin D biosynthesis, it now may contribute to the relentless rise in skin cancer incidence in humans.

Effect of intravenous general anaesthesia with epidural block on the expression of pre-endogenous opioid peptide genes.

OBJECTIVE: To measure the plasma concentrations of three endogenous opioid peptides and the levels of preproenkephalin (PPE) and preprodynorphin (PPD) mRNA in peripheral blood lymphocytes of patients during scheduled surgery performed under intravenous general anaesthesia combined with an epidural block. METHODS: Patients were anaesthetized and arterial blood was collected at 0 (baseline), 20, 40, 60, and 80 min during surgery. The plasma concentrations of ß-endorphin, leucine-enkephalin and dynorphin A were measured using radioimmunoassay. Reverse transcription-polymerase chain reaction was used to measure the levels of PPD and PPE mRNA in peripheral blood lymphocytes collected during surgery. RESULTS: Fifteen patients participated in this prospective study. The plasma concentrations of ß-endorphin were significantly lower at all time-points compared with the baseline value. The plasma concentrations of leucine-enkephalin and dynorphin A were significantly lower at 40, 60, and 80 min compared with baseline. The PPD/ß-actin ratio was significantly lower at 80 min compared with baseline, while the PPE/ß-actin ratio showed no significant change. CONCLUSION: The level of mRNA from two pre-endogenous opioid peptide genes either decreased or remained unchanged during surgery under intravenous general anaesthesia with epidural block, suggesting that patients remained pain free during surgery.

Suppression of acute morphine withdrawal syndrome by adenovirus-mediated ß-endorphin in rats.

BACKGROUND: Endogenous ß-endorphin (ß-EP) in the central nervous system (CNS) is decreased upon opioid addiction. The current study examined whether exogenous ß-EP, delivered using an adenoviral vector into the CNS could attenuate morphine withdrawal syndrome in rats. METHODS: The model of opioid-dependent rats was set up by receiving subcutaneous injection of morphine using an escalating regimen for 6days (5, 10, 20, 40, 50, 60mg/kg, three times/day). The adenovirus mediated ß-EP gene was constructed based on our previous work. The ilea of opioid-dependent rats were isolated and treated with the supernatant of Ad-NEP. The basic and naloxone-induced (4µm/l) contractions of dependent ilea were recorded. The Ad-NEP was injected into the left lateral ventricle of the addition rats. The expression of the ß-EP gene was verified by radioimmunoassay of the cerebrospinal fluid (CSF) and immunocytochemistry for ß-EP. Withdrawal syndrome was evaluated after intraperitoneal injection of naloxone. RESULTS: The contractions of dependent ilea were attenuated with supernatant containing ß-EP expressed by Ad-NEP. Injection of the Ad-NEP resulted in significant increases in ß-EP level in the CSF and ß-EP-positive neurons. Rats receiving adenovirus carrying the ß-EP gene had significantly less severe withdrawal symptoms upon naloxone challenge. CONCLUSIONS: Exogenous ß-EP mediated by adenovirus could attenuate withdrawal syndrome in morphine-dependent rats.

The novel role of cathepsin L for neuropeptide production illustrated by research strategies in chemical biology with protease gene knockout and expression.

Neuropeptides are essential for cell-cell communication in the nervous and endocrine systems. Production of active neuropeptides requires proteolytic processing of proneuropeptide precursors in secretory vesicles that produce, store, and release neuropeptides that regulate physiological functions. This review describes research strategies utilizing chemical biology combined with protease gene knockout and expression to demonstrate the key role of cathepsin L for production of neuropeptides in secretory vesicles. Cathepsin L was discovered using activity-based probes and mass spectrometry to identify proenkephalin cleaving activity as cathepsin L. Significantly, in vivo protease gene knockout and expression approaches illustrate the key role of cathepsin L for neuropeptide production. Notably, cathepsin L is colocalized with neuropeptide secretory vesicles, the major site of proteolytic processing of proneuropeptides to generate active neuropeptides. Cathepsin L participates in producing opioid neuropeptides consisting of enkephalin, ß-endorphin, and dynorphin, as well as in generating the POMC-derived peptide hormones ACTH and α-MSH. In addition, NPY, CCK, and catestatin neuropeptides utilize cathepsin L for their biosynthesis. The role of cathepsin L for neuropeptide production indicates its unique biological role in secretory vesicles, which contrasts with its role in lysosomes for protein degradation. Interesting evaluations of protease gene knockout studies in mice that lack cathepsin L compared to the PC1/3 and PC2 (PC, prohormone convertase) indicate the significant role of cathepsin L in neuropeptide production. Thus, dual cathepsin L and prohormone convertase protease pathways participate in neuropeptide production. These recent new findings indicate cathepsin L as a novel 'proprotein convertase' for production of neuropeptides that mediate cell-cell communication in health and disease.

Binding of synthetic peptide TPLVTLFK to nonopioid beta-endorphin receptor on rat brain membranes.

The synthetic peptide TPLVTLFK corresponding to the sequence 12-19 of beta-endorphin (referred to as octarphin) was found to bind to high-affinity naloxone-insensitive binding sites on membranes isolated from the rat brain cortex (K(d) = 2.6 +/- 0.2 nM). The binding specificity study revealed that these binding sites were insensitive not only to naloxone but also to alpha-endorphin, gamma-endorphin, [Met(5)]enkephalin, and [Leu(5)]enkephalin, as well. The [(3)H]octarphin specific binding with brain membranes was inhibited by unlabeled beta-endorphin (K(i) = 2.4 +/- 0.2 nM) and a selective agonist of nonopioid beta-endorphin receptor decapeptide immunorphin SLTCLVKGFY (K(i) = 2.9 +/- 0.2 nM). At the same time, unlabeled octarphin completely (by 100%) inhibited the specific binding of [(3)H]immunorphin with membranes (K(i) = 2.8 +/- 0.2 nM). Thus, octarphin binds with a high affinity and specificity to nonopioid receptor of beta-endorphin on rat brain cortex membranes.

Age-related changes in pro-opiomelanocortin (POMC) and related receptors in human epidermis.

SYNOPSIS: Much effort has been placed in cosmetic research for better understanding of the effects of ageing on skin's appearance, structure, mechanical properties and function. It is now of common knowledge that UV radiations induce pre-mature skin ageing notably in the epidermis where UV radiations induce keratinocyte differentiation. As UV radiations have also been shown to regulate the pro-opiomelanocortin (POMC) peptide family in the skin and because no study has been conducted so far to investigate the age-related changes in POMC and related receptors, we analysed POMC, MC-1R, MC-2R and MOR-1 at mRNA level and MC-1R, MC-2R and MOR-1 at protein level too in primary cultures of normal human keratinocytes obtained from female donors aged from 17 to 75 years old. Regarding the gene expressions, we observed that MC-1R, MC-2R and MOR-1 suffered a dramatic decrease after 50 years of age, whereas POMC increased five-fold. Western blot analysis confirmed these results except for MOR-1 whose expression appeared to decrease at older age, around 70 years old. Immunostainings specific to MC-1R, MC-2R and MOR-1 performed on full-thickness skin biopsies also revealed an intense staining in the basal and spinous layers of a 30-year-old donor, whereas no reactivity could be observed in a 60-year-old one. We conclude that POMC and POMC-related receptors suffer a dramatically disturbed balance with ageing and that this may be implicated in the general process of skin ageing.

A monomeric variant of insulin degrading enzyme (IDE) loses its regulatory properties.

BACKGROUND: Insulin degrading enzyme (IDE) is a key enzyme in the metabolism of both insulin and amyloid beta peptides. IDE is unique in that it is subject to allosteric activation which is hypothesized to occur through an oligomeric structure. METHODOLOGY/PRINCIPAL FINDINGS: IDE is known to exist as an equilibrium mixture of monomers, dimers, and higher oligomers, with the dimer being the predominant form. Based on the crystal structure of IDE we deleted the putative dimer interface in the C-terminal region, which resulted in a monomeric variant. Monomeric IDE retained enzymatic activity, however instead of the allosteric behavior seen with wild type enzyme it displayed Michaelis-Menten kinetic behavior. With the substrate Abz-GGFLRKHGQ-EDDnp, monomeric IDE retained approximately 25% of the wild type activity. In contrast with the larger peptide substrates beta-endorphin and amyloid beta peptide 1-40, monomeric IDE retained only 1 to 0.25% of wild type activity. Unlike wild type IDE neither bradykinin nor dynorphin B-9 activated the monomeric variant of the enzyme. Similarly, monomeric IDE was not activated by polyphosphates under conditions in which the activity of wild type enzyme was increased more than 50 fold. CONCLUSIONS/SIGNIFICANCE: These findings serve to establish the dimer interface in IDE and demonstrate the requirement for an oligomeric form of the enzyme for its regulatory properties. The data support a mechanism where the binding of activators to oligomeric IDE induces a conformational change that cannot occur in the monomeric variant. Since a conformational change from a closed to a more open structure is likely the rate-determining step in the IDE reaction, the subunit induced conformational change likely shifts the structure of the oligomeric enzyme to a more open conformation.

Peripheral non-viral MIDGE vector-driven delivery of beta-endorphin in inflammatory pain.

BACKGROUND: Leukocytes infiltrating inflamed tissue produce and release opioid peptides such as beta-endorphin, which activate opioid receptors on peripheral terminals of sensory nerves resulting in analgesia. Gene therapy is an attractive strategy to enhance continuous production of endogenous opioids. However, classical viral and plasmid vectors for gene delivery are hampered by immunogenicity, recombination, oncogene activation, anti-bacterial antibody production or changes in physiological gene expression. Non-viral, non-plasmid minimalistic, immunologically defined gene expression (MIDGE) vectors may overcome these problems as they carry only elements needed for gene transfer. Here, we investigated the effects of a nuclear localization sequence (NLS)-coupled MIDGE encoding the beta-endorphin precursor proopiomelanocortin (POMC) on complete Freund's adjuvant-induced inflammatory pain in rats. RESULTS: POMC-MIDGE-NLS injected into inflamed paws appeared to be taken up by leukocytes resulting in higher concentrations of beta-endorphin in these cells. POMC-MIDGE-NLS treatment reversed enhanced mechanical sensitivity compared with control MIDGE-NLS. However, both effects were moderate, not always statistically significant or directly correlated with each other. Also, the anti-hyperalgesic actions could not be increased by enhancing beta-endorphin secretion or by modifying POMC-MIDGE-NLS to code for multiple copies of beta-endorphin. CONCLUSION: Although MIDGE vectors circumvent side-effects associated with classical viral and plasmid vectors, the current POMC-MIDGE-NLS did not result in reliable analgesic effectiveness in our pain model. This was possibly associated with insufficient and variable efficacy in transfection and/or beta-endorphin production. Our data point at the importance of the reproducibility of gene therapy strategies for the control of chronic pain.

The obesity susceptibility gene Cpe links FoxO1 signaling in hypothalamic pro-opiomelanocortin neurons with regulation of food intake.

Reduced food intake brings about an adaptive decrease in energy expenditure that contributes to the recidivism of obesity after weight loss. Insulin and leptin inhibit food intake through actions in the central nervous system that are partly mediated by the transcription factor FoxO1. We show that FoxO1 ablation in pro-opiomelanocortin (Pomc)-expressing neurons in mice (here called Pomc-Foxo1(-/-) mice) increases Carboxypeptidase E (Cpe) expression, resulting in selective increases of alpha-melanocyte-stimulating hormone (alpha-Msh) and carboxy-cleaved beta-endorphin, the products of Cpe-dependent processing of Pomc. This neuropeptide profile is associated with decreased food intake and normal energy expenditure in Pomc-Foxo1(-/-) mice. We show that Cpe expression is downregulated by diet-induced obesity and that FoxO1 deletion offsets the decrease, protecting against weight gain. Moreover, moderate Cpe overexpression in the arcuate nucleus phenocopies features of the FoxO1 mutation. The dissociation of food intake from energy expenditure in Pomc-Foxo1(-/-) mice represents a model for therapeutic intervention in obesity and raises the possibility of targeting Cpe to develop weight loss medications.

Serotonin 5-HT2C receptor-independent expression of hypothalamic NOR1, a novel modulator of food intake and energy balance, in mice.

NOR1, Nur77 and Nurr1 are orphan nuclear receptors and members of the NR4A subfamily. Here, we report that the expression of hypothalamic NOR1 was remarkably decreased in mildly obese beta-endorphin-deficient mice and obese db/db mice with the leptin receptor mutation, compared with age-matched wild-type mice, whereas there were no genotypic differences in the expression of hypothalamic Nur77 or Nurr1 in these animals. The injection of NOR1 siRNA oligonucleotide into the third cerebral ventricle significantly suppressed food intake and body weight in mice. On the other hand, the decreases in hypothalamic NOR1 expression were not found in non-obese 5-HT2C receptor-deficient mice. Moreover, systemic administration of m-chlorophenylpiperazine (mCPP), a 5-HT2C/1B receptor agonist, had no effect on hypothalamic NOR1 expression, while suppressing food intake in beta-endorphin-deficient mice. These findings suggest that 5-HT2C receptor-independent proopiomelanocortin-derived peptides regulate the expression of hypothalamic NOR1, which is a novel modulator of feeding behavior and energy balance.

Nicotine anxiogenic and rewarding effects are decreased in mice lacking beta-endorphin.

The endogenous opioid system plays an important role in the behavioral effects of nicotine. Thus, micro-opioid receptor and the endogenous opioids derived from proenkephalin are involved in the central effects of nicotine. However, the role played by the different endogenous opioid peptides in the acute and chronic effects of nicotine remains to be fully established. Mice lacking beta-endorphin were acutely injected with nicotine at different doses to evaluate locomotor, anxiogenic and antinociceptive responses. The rewarding properties of nicotine were evaluated by using the conditioned place-preference paradigm. Mice chronically treated with nicotine were acutely injected with mecamylamine to study the behavioral expression of nicotine withdrawal. Mice lacking beta-endorphin exhibited a spontaneous hypoalgesia and hyperlocomotion and a reduction on the anxiogenic and rewarding effects induced by nicotine. Nicotine induced similar antinociception and hypolocomotion in both genotypes and no differences were found in the development of physical dependence. The dissociation between nicotine rewarding properties and physical dependence suggests a differential implication of beta-endorphin in these addictive related responses.

Major role of cathepsin L for producing the peptide hormones ACTH, beta-endorphin, and alpha-MSH, illustrated by protease gene knockout and expression.

The pituitary hormones adrenocorticotropic hormone (ACTH), beta-endorphin, and alpha-melanocyte stimulating hormone (alpha-MSH) are synthesized by proteolytic processing of their common proopiomelanocortin (POMC) precursor. Key findings from this study show that cathepsin L functions as a major proteolytic enzyme for the production of POMC-derived peptide hormones in secretory vesicles. Specifically, cathepsin L knock-out mice showed major decreases in ACTH, beta-endorphin, and alpha-MSH that were reduced to 23, 18, and 7% of wild-type controls (100%) in pituitary. These decreased peptide levels were accompanied by increased levels of POMC consistent with proteolysis of POMC by cathepsin L. Immunofluorescence microscopy showed colocalization of cathepsin L with beta-endorphin and alpha-MSH in the intermediate pituitary and with ACTH in the anterior pituitary. In contrast, cathepsin L was only partially colocalized with the lysosomal marker Lamp-1 in pituitary, consistent with its extralysosomal function in secretory vesicles. Expression of cathepsin L in pituitary AtT-20 cells resulted in increased ACTH and beta-endorphin in the regulated secretory pathway. Furthermore, treatment of AtT-20 cells with CLIK-148, a specific inhibitor of cathepsin L, resulted in reduced production of ACTH and accumulation of POMC. These findings demonstrate a prominent role for cathepsin L in the production of ACTH, beta-endorphin, and alpha-MSH peptide hormones in the regulated secretory pathway.