ABSTRACT
Morphine, codeine, morphine-6-glucuronide, and morphine-3-glucuronide are synthesized de novo in mammalian cells and in the central nervous system. Knowledge on endogenous morphine-like compound distribution in the adult mouse brain has been recently improved, and new hypotheses have been suggested about the potential implications in brain physiology. Endogenous morphine-like compounds have been shown to be synthesized in the spinal cord, but their localization is unknown. Here we describe the distribution of endogenous morphine-like compounds (morphine and/or its glucuronides and/or codeine) in the adult mouse spinal cord using a well-validated antibody. By using different microscopy approaches, we found the presence of morphine, codeine, or morphine glucuronides in γ-aminobutyric acid (GABA)-ergic neurons and astrocytes of the spinal cord. Whereas GABAergic neurons containing endogenous morphine-like compounds were located primarily in the ventral horn, astrocytes that were labeled for morphine-like compounds were found throughout the gray matter and the white matter. Our study demonstrates the possibility that endogenous morphine-like compounds in the central nervous system have other functions beyond their analgesic functions.
Subject(s)
Astrocytes/metabolism , GABAergic Neurons/metabolism , Morphine Derivatives/analysis , Spinal Cord/metabolism , Animals , Codeine/analysis , Codeine/biosynthesis , Immunohistochemistry , Male , Mice , Mice, Inbred C57BL , Microscopy, Confocal , Microscopy, Electron, Transmission , Morphine/analysis , Morphine/biosynthesis , Morphine Derivatives/metabolismABSTRACT
Morphine is endogenously synthesized in the central nervous system and endogenous dopamine is thought to be necessary for endogenous morphine formation. As Parkinson's disease results from the loss of dopamine and is associated with central pain, we considered how endogenous morphine is regulated in the untreated and l-DOPA-treated parkinsonian brain. However, as the cellular origin and overall distribution of endogenous morphine remains obscure in the pathological adult brain, we first characterized the distribution of endogenous morphine-like compound immunoreactive cells in the rat striatum. We then studied changes in the endogenous morphine-like compound immunoreactivity of medium spiny neurons in normal, Parkinson's disease-like and l-DOPA-treated Parkinson's disease-like conditions in experimental (rat and monkey) and human Parkinson's disease. Our results reveal an unexpected dramatic upregulation of neuronal endogenous morphine-like compound immunoreactivity and levels in experimental and human Parkinson's disease, only partially normalized by l-DOPA treatment. Our data suggest that endogenous morphine formation is more complex than originally proposed and that the parkinsonian brain experiences a dramatic upregulation of endogenous morphine immunoreactivity. The functional consequences of such endogenous morphine upregulation are as yet unknown, but based upon the current knowledge of morphine signalling, we hypothesize that it is involved in fatigue, depression and pain symptoms experienced by patients with Parkinson's disease.
Subject(s)
Brain/metabolism , Parkinsonian Disorders/metabolism , 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine/pharmacology , Aged , Analysis of Variance , Animals , Brain/drug effects , Brain/pathology , Brain/ultrastructure , Choline O-Acetyltransferase/metabolism , Chromatography, High Pressure Liquid/methods , Dendrites/metabolism , Dendrites/ultrastructure , Disease Models, Animal , Dopamine/metabolism , Dopamine Agents/pharmacology , Dopamine Plasma Membrane Transport Proteins/deficiency , Dopamine Plasma Membrane Transport Proteins/metabolism , Dopamine and cAMP-Regulated Phosphoprotein 32/metabolism , Enzyme Inhibitors/pharmacology , Enzyme-Linked Immunosorbent Assay/methods , Female , Functional Laterality , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , Glial Fibrillary Acidic Protein/metabolism , Glutamate Decarboxylase/metabolism , Humans , Levodopa/pharmacology , Macaca fascicularis , Male , Medial Forebrain Bundle/drug effects , Medial Forebrain Bundle/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microscopy, Immunoelectron/methods , Middle Aged , Nerve Growth Factors/metabolism , Organic Chemicals/metabolism , Oxidopamine/adverse effects , Parkinsonian Disorders/chemically induced , Parkinsonian Disorders/drug therapy , Parkinsonian Disorders/pathology , Postmortem Changes , Rats , Rats, Sprague-Dawley , Receptors, Dopamine D2/metabolism , S100 Calcium Binding Protein beta Subunit , S100 Proteins/metabolism , Tandem Mass Spectrometry , alpha-Methyltyrosine/pharmacologyABSTRACT
Endogenous morphine, morphine-6-glucuronide, and codeine, which are structurally identical to vegetal alkaloids, can be synthesized by mammalian cells from dopamine. However, the role of brain endogenous morphine and its derivative compounds is a matter of debate, and knowledge about its distribution is lacking. In this study, by using a validated antibody, we describe a precise mapping of endogenous morphine-like compounds (morphine and/or its glucuronides and/or codeine) in the mouse brain. First, a mass spectrometry approach confirmed the presence of morphine and codeine in mouse brain, but also, of morphine-6-glucuronide and morphine-3-glucuronide representing two metabolites of morphine. Second, light microscopy allowed us to observe immunopositive cell somas and cytoplasmic processes throughout the mouse brain. Morphine-like immunoreactivity was present in various structures including the hippocampus, olfactory bulb, band of Broca, basal ganglia, and cerebellum. Third, by using confocal microscopy and immunofluroscence co-localization, we characterized cell types containing endogenous opiates. Interestingly, we observed that morphine-like immunoreactivity throughout the encephalon is mainly present in γ-aminobutyric acid (GABA)ergic neurons. Astrocytes were also labeled throughout the entire brain, in the cell body, in the cytoplasmic processes, and in astrocytic feet surrounding blood vessels. Finally, ultrastructural localization of morphine-like immunoreactivity was determined by electron microscopy and showed the presence of morphine-like label in presynaptic terminals in the cerebellum and postsynaptic terminals in the rest of the mouse brain. In conclusion, the presence of endogenous morphine-like compounds in brain regions not usually involved in pain modulation opens the exciting opportunity to extend the role and function of endogenous alkaloids far beyond their analgesic functions.
Subject(s)
Astrocytes/chemistry , Brain/anatomy & histology , Morphine/chemistry , Neurons/chemistry , gamma-Aminobutyric Acid/metabolism , Animals , Astrocytes/ultrastructure , Brain/metabolism , Brain Chemistry , Codeine/metabolism , Dopamine/metabolism , Humans , Immunohistochemistry , Male , Mice , Mice, Inbred C57BL , Morphine Derivatives/metabolism , Neurons/ultrastructure , Patch-Clamp TechniquesABSTRACT
BACKGROUND: Mice deficient for the stable tubule only peptide (STOP) display altered dopaminergic neurotransmission associated with severe behavioural defects including disorganized locomotor activity. Endogenous morphine, which is present in nervous tissues and synthesized from dopamine, may contribute to these behavioral alterations since it is thought to play a role in normal and pathological neurotransmission. RESULTS: In this study, we showed that STOP null brain structures, including cortex, hippocampus, cerebellum and spinal cord, contain high endogenous morphine amounts. The presence of elevated levels of morphine was associated with the presence of a higher density of mu opioid receptor with a higher affinity for morphine in STOP null brains. Interestingly, STOP null mice exhibited significantly lower nociceptive thresholds to thermal and mechanical stimulations. They also had abnormal behavioural responses to the administration of exogenous morphine and naloxone. Low dose of morphine (1 mg/kg, i.p.) produced a significant mechanical antinociception in STOP null mice whereas it has no effect on wild-type mice. High concentration of naloxone (1 mg/kg) was pronociceptive for both mice strain, a lower concentration (0.1 mg/kg) was found to increase the mean mechanical nociceptive threshold only in the case of STOP null mice. CONCLUSIONS: Together, our data show that STOP null mice displayed elevated levels of endogenous morphine, as well as an increase of morphine receptor affinity and density in brain. This was correlated with hypernociception and impaired pharmacological sensitivity to mu opioid receptor ligands.
Subject(s)
Microtubule-Associated Proteins/deficiency , Morphine/pharmacology , Pain/physiopathology , Analgesics, Opioid , Animals , Mice , Mice, Knockout , Microtubule-Associated Proteins/physiology , Morphine/analysis , Naloxone/administration & dosage , Naloxone/pharmacology , Narcotic Antagonists , Nerve Tissue Proteins , Opiate Alkaloids , Pain/drug therapy , Receptors, Opioid, mu/analysis , Receptors, Opioid, mu/metabolismABSTRACT
BACKGROUND: Mammalian cells synthesize morphine and the respective biosynthetic pathway has been elucidated. Human neutrophils release this alkaloid into the media after exposure to morphine precursors. However, the exact role of endogenous morphine in inflammatory processes remains unclear. We postulate that morphine is released during infection and can be determined in the serum of patients with severe infection such as sepsis. METHODOLOGY: The presence and subcellular immunolocalization of endogenous morphine was investigated by ELISA, mass spectrometry analysis and laser confocal microscopy. Neutrophils were activated with Interleukin-8 (IL-8) or lipopolysaccharide (LPS). Morphine secretion was determined by a morphine-specific ELISA. mu opioid receptor expression was assessed with flow cytometry. Serum morphine concentrations of septic patients were determined with a morphine-specific ELISA and morphine identity was confirmed in human neutrophils and serum of septic patients by mass spectrometry analysis. The effects of the concentration of morphine found in serum of septic patients on LPS-induced release of IL-8 by human neutrophils were tested. PRINCIPAL FINDINGS: We confirmed the presence of morphine in human neutrophil extracts and showed its colocalisation with lactoferrin within the secondary granules of neutrophils. Morphine secretion was quantified in the supernatant of activated human polymorphonuclear neutrophils in the presence and absence of Ca(2+). LPS and IL-8 were able to induce a significant release of morphine only in presence of Ca(2+). LPS treatment increased mu opioid receptor expression on neutrophils. Low concentration of morphine (8 nM) significantly inhibited the release of IL-8 from neutrophils when coincubated with LPS. This effect was reversed by naloxone. Patients with sepsis, severe sepsis and septic shock had significant higher circulating morphine levels compared to patients with systemic inflammatory response syndrome and healthy controls. Mass spectrometry analysis showed that endogenous morphine from serum of patient with sepsis was identical to poppy-derived morphine. CONCLUSIONS: Our results indicate that morphine concentrations are increased significantly in the serum of patients with systemic infection and that morphine is, at least in part, secreted from neutrophils during sepsis. Morphine concentrations equivalent to those found in the serum of septic patients significantly inhibited LPS-induced IL-8 secretion in neutrophils.
Subject(s)
Morphine/blood , Neutrophils/physiology , Sepsis/blood , Blotting, Western , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Humans , Immunohistochemistry , Interleukin-8/pharmacology , Lipopolysaccharides/pharmacology , Lymphocyte Activation/drug effects , Microscopy, Confocal , Neutrophils/drug effects , Receptors, Opioid, mu/metabolism , Tandem Mass SpectrometryABSTRACT
Morphine was first identified in opium from Papaver somniferum, and is still one of the strongest known analgesic compounds used in hospital. Since the beginning of the 80s, endogenous morphine, with an identical structure to that of morphine isolated from poppies, has been characterised in numerous mammalian cells and tissues. In mammals, the biosynthesis of endogenous morphine is associated with dopamine, as demonstrated in the SH-SY5Y human neuronal catecholamine-producing cell line. More recently, morphine andmorphine-6-glucuronide has been shown to be present in the humanneuro blastoma SH-SY5Y cell line and that morphine is secreted from the large dense core vesicles in response to nicotine stimulation via a Ca2+-dependent mechanism suggesting its implication in neurotransmission. An increasing number of publications have demonstrated its presence and implication in different biological processes at the central and peripheral levels. The present review reports the major data concerning endogenous morphine presence and implication in physiological processes (AU)
Morfina endógena a nivel central y periférico. La morfina se identificó por primera vez en el opio procedentede Papaver somniferum, y sigue siendo uno de los analgésicos más potentes conocidos empleados en los hospitales. Desde comienzos dela década de los 80s, la morfina endógena, con una estructura idéntica a la morfina aislada de las amapolas, se caracterizado en numerosas células y tejidos de mamíferos. En mamíferos, la biosíntesis dela morfina endógena está asociada a la dopamina, como se ha demostrado en la línea celular neuronal humana productora de catecolaminasSH-SY5Y. Más recientemente, se ha demostrado la presencia de morfina y mofina-6-glucorónido en la línea celular de neuroblastoma humano SH-SY5Y y que esta morfina es secretada desde vesículas densas en respuesta a estimulación con nicotina vía un mecanismo dependiente de Ca2+ sugiriendo su implicación en l la neurotransmisión. Un número cada vez mayor de publicaciones han demostrado su presencia e implicación en diferentes procesos biológicos a niveles central y periférico. La presente revisión recoge los datos más importantes sobre la presencia e implicación en procesos fisiológicos de la morfina endógena (AU)
Subject(s)
Humans , Morphine/isolation & purification , Alkaloids/isolation & purification , Analgesics/pharmacokinetics , Dopamine , Analgesia/methods , Receptors, Opioid/analysisABSTRACT
BACKGROUND: The phosphatidylethanolamine-binding protein (PEBP/RKIP), initially found to bind phosphatidylethanolamine (PE), has been shown to be associated with morphine derivatives. Our recent study on bovine primary chromaffin cells showed that inside secretory granules, PEBP is noncovalently associated to endogenous morphine-6-glucuronide (M6G), a highly analgesic morphine metabolite. During stress, M6G-PEBP complexes may be released into circulation to target peripheral opioid receptors. We now report the investigation of PEBP binding properties towards morphine and morphine analogs. MATERIAL/METHODS: Noncovalent electrospray ionization mass spectrometry (ESI-MS) was used to investigate bovine and human PEBP binding properties towards morphine and morphine-glucuronides. RESULTS: We describe for the first time that: (i) PEBP directly interacts with morphine glucuronides (M3G and M6G) but not with morphine, (ii) that the presence of a glucuronide group either on the 3rd or the 6th morphine's carbon does not affect these interactions, (iii) that M6G binds PEBP in a similar manner as the reference ligand PE and (iv) that PEBP displays a similar affinity for PE, M6G and M3G. CONCLUSIONS: Our results suggest that PEBP might protect M6G following its secretion into blood, leading to a longer half life. This study highlights the potentialities of ESI-MS to validate / invalidate the formation of protein: ligand noncovalent complexes when low affinity binders (i.e., compounds with affinities lower than 10(3) M(-1)) are concerned.
Subject(s)
Mass Spectrometry , Morphine Derivatives/metabolism , Phosphatidylethanolamine Binding Protein/metabolism , Animals , Cattle , Humans , Morphine Derivatives/chemistry , Phosphatidylethanolamines/metabolism , TitrimetryABSTRACT
BACKGROUND: Morphine, the principal active agent in opium, is not restricted to plants, but is also present in different animal tissues and cell types, including the mammalian brain. In fact, its biosynthetic pathway has been elucidated in a human neural cell line. These data suggest a role for morphine in brain physiology (e.g., neurotransmission), but this hypothesis remains a matter of debate. Recently, using the adrenal neuroendocrine chromaffin cell model, we have shown the presence of morphine-6-glucuronide (M6G) in secretory granules and their secretion products, leading us to propose that these endogenous alkaloids might represent new neuroendocrine factors. Here, we investigate the potential function of endogenous alkaloids in the central nervous system. METHODOLOGY AND PRINCIPAL FINDINGS: Microscopy, molecular biology, electrophysiology, and proteomic tools were applied to human neuroblastoma SH-SY5Y cells (i) to characterize morphine and M6G, and (ii) to demonstrate the presence of the UDP-glucuronyltransferase 2B7 enzyme, which is responsible for the formation of M6G from morphine. We show that morphine is secreted in response to nicotine stimulation via a Ca(2+)-dependent mechanism involving specific storage and release mechanisms. We also show that morphine and M6G at concentrations as low as 10(-10) M are able to evoke specific naloxone-reversible membrane currents, indicating possible autocrine/paracrine regulation in SH-SY5Y cells. Microscopy and proteomic approaches were employed to detect and quantify endogenous morphine in the mouse brain. Morphine is present in the hippocampus, cortex, olfactory bulb, and cerebellum at concentration ranging from 1.45 to 7.5 pmol/g. In the cerebellum, morphine immunoreactivity is localized to GABA basket cells and their termini, which form close contacts on Purkinje cell bodies. CONCLUSIONS/SIGNIFICANCE: The presence of morphine in the brain and its localization in particular areas lead us to conclude that it has a specific function in neuromodulation and/or neurotransmission. Furthermore, its presence in cerebellar basket cell termini suggests that morphine has signaling functions in Purkinje cells that remain to be discovered.
