Endogenous morphine and its metabolites in mammals: History, synthesis, localization and perspectives

Neuroscience 233 (2013) 95–117

REVIEW ENDOGENOUS MORPHINE AND ITS METABOLITES IN MAMMALS: HISTORY, SYNTHESIS, LOCALIZATION AND PERSPECTIVES A. LAUX-BIEHLMANN, J. MOUHEICHE, J. VE´RIE`PE AND Y. GOUMON *

The biosynthetic pathway of endogenous opiates in mammals: a comparison with plants 98 L-tyrosine to dopamine 98 The formation of norlaudanosoline and norcoclaurine 98 Three O-methylation steps to form (R)-reticuline 100 (R)-reticuline to thebaine 100 Thebaine to morphine: two parallel pathways 100 Conclusion: conservation throughout evolution 100 Investigation of new endogenous opiate compounds of interest 100 Precursors 100 Morphine catabolites 101 In humans 101 In other mammals 103 Localization of endogenous opiates in mammals 103 The central nervous system 103 Endogenous morphine in astrocytes 103 Endogenous morphine in GABAergic cells 103 In the periphery 105 Different cellular partners may be involved in endogenous opiate biosynthesis 106 Is EM biosynthesis restricted to catecholaminergic cells? 106 The precursor-uptake hypothesis 106 Localization of enzymes involved in endogenous morphine biosynthesis 107 What are the roles of endogenous opiates? 107 Endogenous opiate levels 107 At the periphery: stress and immune responses 107 Endogenous opiates in the adrenal medulla 107 Endogenous opiates in the immune system 107 Proposed roles in the CNS 108 Do endogenous opiates act as neurotransmitters or neuromediators? 108 Endogenous opiates and control of nociception 108 Endogenous opiates and memory 108 Endogenous opiates and addiction 108 Endogenous opiates, neurogenesis and structural plasticity 109 Endogenous opiates and neuroinflammation 109 Endogenous opiates and CNS pathologies 109 Schizophrenia 109 A ‘‘hypermorphinergic’’ pathology? 109 Endogenous morphine, schizophrenia and nociception 110 Parkinson’s disease 110 Conclusion 110 Acknowledgements 110 References 110

Nociception and Pain Department, Institut des Neurosciences Cellulaires et Inte´gratives, CNRS UPR3212 and Universite´ de Strasbourg, F-67084 Strasbourg, France

Abstract—Morphine derived from Papaver somniferum is commonly used as an analgesic compound for pain relief. It is now accepted that endogenous morphine, structurally identical to vegetal morphine–alkaloid, is synthesized by mammalian cells from dopamine. Morphine binds mu opioid receptor and induces antinociceptive effects. However, the exact role of these compounds is a matter of debate although different links with infection, sepsis, inflammation, as well as major neurological pathologies (Parkinson’s disease, schizophrenia) have been proposed. The present review describes endogenous morphine and morphine derivative discovery, synthesis, localization and potential implications in physiological and pathological processes. Ó 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

Key words: morphine, morphine-glucuronide, dopamine, opioid receptor, analgesia. Contents General introduction and nomenclature 96 Endogenous opiates: a history of their discovery 96 Discovery of endogenous morphine-like compounds 96 Identification of endogenous opiates 96 Evidence of de novo morphine biosynthesis in mammals 98

*Corresponding author. E-mail address: [email protected] (Y. Goumon). Abbreviations: 4-HPAA, 4-hydroxyphenylacetaldehyde; 4-HPP, 4hydroxyphenylpyruvate; AADC, aromatic L-amino acid decarboxylase; COMT, catechol-O-methyltransferase; CSF, cerebrospinal fluid; CYP2D6, cytochrome P450 2D6; DOPAL, 3,4-dihydroxyphenylacetaldehyde; DOR, delta opioid receptor; EM, endogenous morphine; EMM, endogenous morphine metabolites; HPLC, high-performance liquid chromatography; KOR, kappa opioid receptor; M3G, morphine-3glucuronide; M3S, morphine-3-sulfate; M6G, morphine-6-glucuronide; M6S, morphine-6-sulfate; MAO, monoamine oxidase; MD2, myeloid differentiation protein 2; MLC, morphine-like compound; MOR, mu opioid receptor; MS, mass spectrometry; NCS, norcoclaurine synthase; PNMT, phenylethanolamine N-methyltransferase; RIA, radioimmunoassay; TH, tyrosine hydroxylase; THP, tetrahydropapaveroline; TLR4, toll-like receptor 4; TYDC, tyrosine decarboxylase; TyrAT, tyrosine aminotransferase; UGT, UDP-glucuronosyl-transferase enzymes. 0306-4522/12 $36.00 Ó 2012 IBRO. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuroscience.2012.12.013 95

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GENERAL INTRODUCTION AND NOMENCLATURE In the early 1950s, the presence of specific morphinebinding receptors was hypothesized on the basis of morphine’s analgesic effects. In the 1970s, three different receptors for opioid and opiate compounds were discovered: the Mu (l), Delta (d) and Kappa (j) opioid receptors (MOR, DOR and KOR, respectively) (Pert et al., 1973; Pert and Snyder, 1973). More recently, a fourth opioid receptor named nociceptin/ orphanin FQ (NOR) was cloned. These opioid receptors have seven transmembrane domains coupled to G proteins (for review: Kieffer and Evans, 2002, 2009; Trescot et al., 2008; Dietis et al., 2011; Al-Hasani and Bruchas, 2012; Feng et al., 2012). The presence of opioid receptors has led to the characterization of several endogenous ligands called opioids due to their peptidic nature: enkephalins (Hughes et al., 1975a,b; Simantov and Snyder, 1976), b-endorphin (Bradbury et al., 1976; Graf et al., 1976; Lazarus et al., 1976; Li and Chung, 1976; Li et al., 1976), dynorphin (Cox et al., 1975; Goldstein et al., 1979; Lowney et al., 1979), nociceptin/orphanin FQ (Meunier et al., 1995; Reinscheid et al., 1995) and endomorphins (Hackler et al., 1997; Zadina et al., 1997). In addition to endogenous opioid peptides, endogenous morphine-like molecules, which are known as endogenous opiates due to their alkaloid nature have been discovered (Gintzler et al., 1976a,b; Blume et al., 1977; Shorr et al., 1978). Nevertheless, these molecules remain unfamiliar to most scientists, with only a small number of laboratories focusing on this particular research area. The data on endogenous opiates are scattered among the thousands of articles on ‘‘exogenous morphine’’ and endogenous opioid peptides. Furthermore, confusion arises when scientists use the term ‘‘opiates’’ instead of ‘‘opioids’’ for endogenous opioid peptides. Because no clear consensus exists, the following conventions will be used in this review: ‘‘opioids’’ will refer to peptides having an affinity for opioid receptors, whereas ‘‘opiates’’ will refer to natural or synthetic morphine-derived alkaloids (morphine, codeine, morphine-glucuronides. . .). The present review summarizes the findings from the available literature on endogenous opiates (endogenous morphine and endogenous morphine metabolites). Their history, synthesis in mammals, tissue/cellular localization and potential functions in physiological and pathological states will be addressed in the following paragraphs. We will also discuss exciting fundamental and therapeutic perspectives that have recently emerged from cutting-edge research.

Endogenous opiates: a history of their discovery Discovery of endogenous morphine-like compounds. In 1903, the French scientist Dr. Mavrojannis made the observation that morphine injections in rats led to symptoms related to catalepsy and subsequently

hypothesized that such symptoms in mammals are the consequences of endogenous ‘‘morphine-like’’ compounds (Mavrojannis, 1903). In 1970, Davis and Walsh (1970) were the first to propose the presence of ‘‘true’’ morphine in mammals that potentially arose from a synthetic pathway similar to the biosynthetic pathway described in plants. Around the time of the discovery of the first endogenous opioid peptides, the group of Pr. S. Spector demonstrated the existence of an endogenous non-peptide ‘‘morphine-like compound’’ (MLC) (Gintzler et al., 1976a,b, 1978). This MLC was detected in rabbit and cat brains using a radioimmunoassay (RIA) directed against morphine. Once extracted, this compound displayed pharmacological properties identical to those of morphine. Furthermore, this MLC, which bound opioid receptors, was resistant to peptidase/protease treatments (Gintzler et al., 1976a,b, 1978). In parallel, other studies described the presence of a MLC in guinea-pig blood and small intestines (Schulz et al., 1977) as well as in human blood (Pert et al., 1976). A year later, Blume et al. (1977) described an antimorphine antibody that was able to bind specifically to MLC; the fact that it did not interfere with endogenous opioid peptides ruled out any possible artifacts. This group postulated that MLC has a molecular structure similar to that of morphine and hypothesized the existence of two families of compounds (peptide and non-peptide) that bind opioid receptors. The presence of MLC was described in the mouse brain and immunolocalized in neuronal perikarya and processes of the cerebellum and the raphe nuclei (Gintzler et al., 1978). Its presence was also described in the cerebrospinal fluid (CSF), urine and brain extracts of patients naı¨ ve for exogenous morphine or morphine derivatives, suggesting its endogenous origin (Shorr et al., 1978; Wuster et al., 1978). In 1981, Killian et al. (1981) confirmed the existence of a non-peptide compound immunoprecipitated by anti-morphine antibodies in the calf brain, with an analgesic activity that could be blocked by naloxone, an opioid receptor antagonist. Identification of endogenous opiates. In 1985, Goldstein and collaborators described the presence of four different MLCs in the bovine brain and adrenal gland. Using a NMR (nuclear magnetic resonance) approach, they demonstrated that one of the four MLCs was structurally identical to morphine extracted from poppies (Goldstein et al., 1985). Morphine endogenously present in mammals was named ‘‘endogenous morphine’’ (EM) as opposed to morphine from plants (exogenous morphine). The same group subsequently described in the bovine hypothalamus, the presence of endogenous codeine, a morphine precursor (Weitz et al., 1986). In the years that followed, several studies focused on the presence of EM and endogenous codeine using high-performance liquid chromatography (HPLC), NMR and mass spectrometry (MS) approaches in tissues from different species (for review: Meijerink et al., 1999; Stefano et al., 2000).

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Fig. 1. Morphine biosynthetic pathway in mammals and plants: from L-tyrosine to reticuline. Common intermediates (in italic) present in both kingdoms are in black font. Specific compounds present in plants appear in green whereas mammals’ ones are in red font. Identified enzymes (i.e., cloned) appear in bold font in green for plants and in red for mammals. Unknown or putative enzymes are in black font. References: 1, Facchini and De Luca (1994); 2, Wassenberg et al. (2010); 3, Lee and Facchini (2011); 4, Bilkova et al. (2005); 5, Florang et al. (2007); 6, Deitrich and Erwin (1980); 7 , Samanani and Facchini (2002); 8, Morishige et al. (2000); 9, Loeffler et al. (1995); 10, Ziegler et al. (2005); 11, Silber et al. (1991); 12, Facchini and Park (2003); 13, Morishige et al. (2000); 14, Grobe et al. (20110); 15, Ziegler et al. (2009); 16, Hirata et al. (2004). TYDC, tyrosine/dopa decarboxylase; 4-HPP, 4-hydroxyphenylpyruvate; 4-HPAA, 4-hydroxyphenylacetaldehyde; L-DOPA, 3,4-dihydroxy-L-phenylalanine; DOPAL, 3,4-dihydroxyphenylacetaldehyde; NCS, norcoclaurine synthase; AO, amine oxydase; CNMT, (S)-coclaurine-N-methyl-transferase; 6-OMT, 6-O-methyltransferase; DRS, 1,2-dehydroreticuline synthase; DRR, 1,2-dehydroreticuline reductase; 40 -OMT, 40 -O-methyltransferase; NMCH, (S)-N-methylcoclaurine-30 hydroxylase; TH, tyrosine hydroxylase; AADC, aromatic L-amino acid decarboxylase; MAO, monoamine oxydase; NMT, N-methyltransferase; TyrAT, tyrosine aminotransferase; THP, tetrahydropapaveroline; COMT, catechol-O-methyltransferase. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Evidence of de novo morphine biosynthesis in mammals. In 1970, Davis and Walsh (1970) proposed that morphine biosynthesis occurred in mammals through a biosynthetic pathway similar to that described in plants. In 1986, three precursors of plant morphine synthesis (salutaridine, thebaine and codeine) were shown to increase EM levels in the rat brain, revealing the presence of enzymes involved in the de novo biosynthesis of morphine in mammals (Donnerer et al., 1986). The conversion of reticuline to salutaridine, which is regarded as the key step in the formation of the morphine skeleton, was later demonstrated in vivo in the rat liver (Weitz et al., 1987). Additionally, the presence of thebaine, a morphine precursor, was described in the bovine brain (Kodaira et al., 1989). Conclusive evidence of de novo morphine synthesis in mammals was provided by the laboratory of Pr. M. H. Zenk. By cultivating the human neuroblastoma cell line SHSY5Y in the presence of 18O2, this group was able to isolate different radiolabeled intermediates of morphine biosynthesis along with radiolabeled morphine, which together account for the entire synthetic pathway of morphine in this human cell line (Poeaknapo et al., 2004; Boettcher et al., 2005). More recently, an in vivo study showed that mice with a disrupted tyrosine hydroxylase gene were unable to synthesize EM in the brain (Neri et al., 2008). As tyrosine hydroxylase is the key enzyme for dopamine formation, these results indicate that morphine synthesis requires dopamine. Finally, the discovery of EM and several precursors in mouse urine confirmed the synthesis of morphine in animals in vivo (Grobe et al., 2010).

L-tyrosine

is hydroxylated to L-DOPA by tyrosine hydroxylase (TH), and L-DOPA is subsequently decarboxylated by the aromatic L-amino acid decarboxylase (AADC) to form dopamine. Cytochrome P450 2D6 (CYP2D6) has been shown to catalyze the hydroxylation of tyramine to form dopamine and thus represents an alternative biosynthetic pathway for dopamine formation (Hiroi et al., 1998a,b; Bromek et al., 2011). In mammals, DOPAL formation is catalyzed by monoamine oxidase (MAO) from dopamine (Wachtel and Abercrombie, 1994). In opium poppy, the same intermediates are involved in the formation of dopamine. However, only tyrosine decarboxylase (TYDC), which catalyzes the same reactions as AADC, has been characterized in plants. Plants can form DOPAL, but it is its monohydroxylated homolog, 4-hydroxyphenylacetaldehyde (4-HPAA), that is involved in morphine biosynthesis. 4-HPAA is synthesized from 4-hydroxyphenylpyruvate (4-HPP) formed by the transamination of L-tyrosine by the tyrosine aminotransferase (TyrAT) (Lee and Facchini, 2011). The 4-HPAA intermediate exists in mammals and may represent a new intermediate in EM biosynthesis. Indeed, in humans, neutrophils and macrophages produce 4HPAA from L-tyrosine via a myeloperoxidase (Hazen et al., 1999; Whitman et al., 1999; Exner et al., 2001). Moreover, the production of 4-HPAA by these cells occurs under specific physiological or pathological conditions that have been shown to increase EM blood levels (Hazen et al., 2000; Heller et al., 2000). The formation of norlaudanosoline and norcoclaurine

THE BIOSYNTHETIC PATHWAY OF ENDOGENOUS OPIATES IN MAMMALS: A COMPARISON WITH PLANTS The morphine synthetic pathway in mammals appears to be highly similar to the pathway described in opium poppy on the basis of common intermediates. The synthetic pathway of morphine in opium poppy has been well characterized in the literature (for review: Kirby, 1967). However, the discovery of enzymes involved in morphine synthesis in plants has recently occurred, and only half of these enzymes have been cloned and characterized during the last decade. It is therefore not surprising that only a few enzymes involved in the mammalian morphine synthetic pathway have been described and that their discovery was based on sequence similarities. This chapter presents, in five steps, the morphine biosynthetic pathway (Figs. 1 and 2), highlighting the similarities and differences between the pathways in mammals (Poeaknapo et al., 2004; Boettcher et al., 2005) and opium poppy (for review: Facchini, 2001; Ziegler et al., 2009). L-tyrosine

to dopamine

In mammals, morphine biosynthesis begins with the formation of two compounds, dopamine and 3,4dihydroxyphenylacetaldehyde (DOPAL) from L-tyrosine.

In mammals, the production of morphine has been found to derive from norlaudanosoline, which is also known as tetrahydropapaveroline (THP). Norlaudanosoline, considered as dopamine catabolite, is formed by a spontaneous Picted–Spengler condensation reaction between dopamine and DOPAL (for review: Marchitti et al., 2007). It is important to note that both enantiomers, (R)-norlaudanosoline and (S)norlaudanosoline, are present in mammals (Cashaw et al., 1983; Cashaw, 1993b,a; Sango et al., 2000). Surprisingly, high levels of THP have been associated with pathological states such as Parkinson’s disease and alcoholism. In plants, it was originally hypothesized that (S)norlaudanosoline was the precursor of morphinan alkaloids. However, only (S)-norcoclaurine was found to be involved in morphine biosynthesis. (S)-norcoclaurine is yielded by the condensation of dopamine with 4HPAA, which is catalyzed by (S)-norcoclaurine synthase (NCS) (Ilari et al., 2009). In mammals, the involvement of norcoclaurine cannot be excluded from the EM biosynthetic pathway. Hence, 4HPAA is produced in human neutrophils and macrophages from L-tyrosine via a myeloperoxidase (Hazen et al., 1999; Whitman et al., 1999; Exner et al., 2001). Accordingly, endogenously formed norcoclaurine has been detected in the brains of rats treated with alcohol (Haber et al., 1997).

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Fig. 2. Morphine biosynthetic pathway in mammals and plants: from reticuline to morphine. Common intermediates (in italic) present in both kingdoms are in black font. Specific compounds present in plant appear in green whereas mammals’ ones are in red font. Identified enzymes (i.e., cloned) appear in bold font in green for plants and in red for mammals. References: 1, Gesell et al. (2009); 2, Grobe et al. (2009); 3, Gerardy and Zenk (1993); 4, Grothe et al. (2001); 5, Hagel and Facchini (2010); 6, Lenz and Zenk (1995); 7, Zhu (20080. SalSyn, salutaridine synthase; SalR, salutaridine 7-oxidoreductase; SalAT, salutaridinol-7-O-acetate; COR, codeine reductase; T6ODM, thebaine-6-O-demethylase; CODM, codeine-Odemethylase. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Three O-methylation steps to form (R)-reticuline In mammals, two pathways may lead to the formation of (R)-reticuline. (i) In 2005, Boettcher et al. (2005) demonstrated the presence of (S)-norlaudanosoline and (S)-reticuline and discussed their possible roles in EM biosynthesis. The enzymes involved in these three methylation steps are still unknown, although it has been proposed that COMT (catechol-O-methyltransferase) is one of these enzymes (Mantione et al., 2008). (S)-reticuline is finally converted to the (R)enantiomer by an unknown enzyme. (ii) The second possible pathway involves the formation of (R)-norlaudanosoline and its conversion into (R)-reticuline. Indeed, N-methyltransferase (NMT; E.C. 2.1.1.49) was recently found to perform the last (R)-norlaudanosoline methylation step in a manner specific to the enantiomer configuration (Grobe et al., 2011). This second pathway is more interesting from an evolutionary point of view, and the formation of an (R)-enantiomer of norlaudanosoline requires fewer intermediates to form (R)reticuline. In plants, (S)-norcoclaurine is converted into (R)reticuline after three methylations and one hydroxylation. The four enzymes implicated and the order of these steps have been described and characterized in the literature (Fig. 1). (R)-reticuline to thebaine The conversion of (R)-reticuline to salutaridine has been shown to occur in vivo in the rat liver (Weitz et al., 1987). This reaction is the critical step in the formation of the morphine skeleton and therefore represents the limiting step. This step is likely performed by the CYP2D6 enzyme in humans and CYP3A4 in rats (Grobe et al., 2009). Salutaridine is then converted to thebaine through several identified intermediates. However, the enzymes involved in these reactions remain unknown in mammals. In plants, identical intermediates have been observed, and the different enzymes implicated have already been described and characterized in the literature (Fig. 2). Thebaine to morphine: two parallel pathways As in plants, two parallel pathways exist in mammals for the formation of morphine from thebaine. Furthermore, all of the intermediates that lead to morphine are present in both kingdoms. The enzymes involved in this pathway were only recently characterized in plants (Lenz and Zenk, 1995; Hagel and Facchini, 2010). In mammals, only CYP2D6, which demethylates thebaine to oripavine and codeine to morphine, is known. Conclusion: conservation throughout evolution A comparison between the morphine synthetic pathways described in mammals and in plants indicates a strong

conservation throughout evolution (Meijerink et al., 1999; Kream et al., 2007). In addition to its existence in plants and mammals, morphine is also found in other vertebrates, such as eels (Epple et al., 1993) and toads (Oka et al., 1985). Surprisingly, EM is also present in invertebrates, including molds (Stefano et al., 1993) (Zhu et al., 2001a), mollusks (Goumon et al., 2001; Sonetti et al., 2005), arthropods (Casares et al., 2005), annelids (Pryor and Elizee, 2000), nematodes (Goumon et al., 2000b) and flatworms (Pryor and Elizee, 2000; Zhu et al., 2002). This conservation of morphine biosynthesis throughout evolution suggests not only a conservation of the enzymatic machinery but also a crucial physiological role of EM.

INVESTIGATION OF NEW ENDOGENOUS OPIATE COMPOUNDS OF INTEREST The presence of the EM biosynthetic pathway in mammals poses numerous questions. Indeed, mammalian cells and tissues may be exposed to EM precursors and/or derivatives. Most of the studies cited in this section describe the pharmacological properties of morphine precursors and catabolites without considering the endogenously produced molecules. Nevertheless, these studies help introduce the possible effects and pharmacological properties of these compounds.

Precursors The formation of THP (also named norlaudanosoline), a dopamine catabolite, has been well characterized and corresponds to the first steps of the EM biosynthetic pathway. The neurotoxic effect of norlaudanosoline has been found to involve the inhibition of mitochondrial respiration and increased reactive oxygen species (ROS) production (for review: Surh and Kim, 2010). Norlaudanosoline decreases dopamine biosynthesis by inhibiting TH (Kim et al., 2005; Yao et al., 2010) and inhibits dopamine uptake by acting on its plasma membrane transporter (Okada et al., 1998). Through a blockade of L-type Ca++ channels, reticuline induces central depressant effects (Morais et al., 1998), anti-platelet aggregation (Chen et al., 2000) and hypotensive effects (Dias et al., 2004; Medeiros et al., 2009). Furthermore, two studies have characterized salutaridine as a partial agonist of GABA(A) receptors (Kardos et al., 1984; Eisenreich et al., 2003). Thebaine induces antinociceptive effects (Aceto et al., 1999), whereas oripavine, codeinone and morphinone have analgesic effects but are potentially toxic (Yeh, 1981; Fang et al., 1984; Aceto et al., 1989). More recently, intermediates identified between thebaine and morphine were found to bind opioid receptors and induce G protein activation (Nikolaev et al., 2007; Zhang et al., 2012).

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Morphine catabolites

In humans. Morphine catabolism mainly occurs in the liver (Fig. 3), but it has also been reported in the brain and kidneys. In the liver, up to 60% of morphine is converted into morphine-3-glucuronide (M3G), 10% into morphine6-glucuronide (M6G) and less than 1% into morphine3,6-diglucuronide (Yeh et al., 1977a; Osborne et al., 1990; Hasselstrom and Sawe, 1993; Lotsch et al., 1996). In mammals, the glucuronidation of morphine is dependent on the family of UDP-glucuronosyltransferase enzymes (UGT). In humans, 31 members of the UGT family have been characterized (Mackenzie et al., 1997, 2005), and of these, the UGT1A1, 1A3, 1A6, 1A8, 1A9, 1A10, 2A1 and 2B7 isoforms have been found to be involved in morphine glucuronidation (Jedlitschky et al., 1999; Stone et al., 2003). These UGT isoforms form M3G, whereas only the UGT2B7

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isoform appears to produce both M3G and M6G. In addition to their localization in the liver, the UGT1A6 and 2B7 isoforms are also expressed in neurons and astrocytes, suggesting that morphine can be glucuronidated in CNS cells (Suleman et al., 1998; King et al., 1999; Nagano et al., 2000; Yamada et al., 2003; Buckley and Klaassen, 2007). Concerning their activities, M6G is 1–600 times more analgesic than morphine, whereas M3G has long been considered devoid of any activity (reviewed in: (Lotsch and Geisslinger, 2001; Coller et al., 2009)). However, it was shown that M3G counteracts the analgesic effects of morphine (Smith et al., 1990; Gong et al., 1992) and induces allodynia after intrathecal administration (Lewis et al., 2010). This effect occurs through its binding to toll-like receptor 4 (TLR4) and, more importantly, to its accessory protein myeloid differentiation protein 2 (MD2) (Komatsu et al., 2009; Hutchinson et al., 2010a; Lewis et al., 2010, 2012). Besides, morphine binds both MOR

Fig. 3. Morphine catabolites in humans. Synthetic representation of compounds detected in the blood or urine of humans after morphine administration.

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Fig. 4. Morphine catabolites found in other mammals. These compounds are present in the blood or urine of rodents after morphine administration. None of these molecules has been found so far in humans.

and TLR4 (Komatsu et al., 2009; Lewis et al., 2012; Stevens et al., 2012). Alternatively, morphine can be converted into hydromorphone (morphinone) (Yeh et al., 1977b; Oyler et al., 2000; Cone et al., 2006, 2008; McDonough et al., 2008; Wasan et al., 2008; Hughes et al., 2012) and exhibits high analgesic activities through its binding to MOR (Kumar et al., 2008; Volpe et al., 2011). The

sulfoconjugated morphine-derived compounds morphine-3-sulfate (M3S) and morphine-6-sulfate (M6S) have also been described. However, only M3S is detected in humans after morphine administration (Yeh et al., 1977a,b,c). Several articles have studied phenolsulphotransferase enzymes that may be involved in morphine sulfo-derivative formation (Foldes and Meek, 1973; Rein et al., 1982; Donnerer et al., 1987;

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Nagano et al., 2000). M6S, but not M3S, has been described as a more potent analgesic than morphine after intracerebroventricular injections (Brown et al., 1985; Zuckerman et al., 1999), an effect that may be related to its ability to bind and activate MOR (Frolich et al., 2011). A recent article reported the presence of 3.47% of morphine-3-glucoside and 0.4% of morphine-6-glucoside in the urine of morphine-treated cancer patients (Chen et al., 2003). However, there is no available data on the formation or potential activities of these compounds. Morphine has also been shown to be demethylated into normorphine. Still, only 1% of this compound is found in urine (Yeh, 1975; Yeh et al., 1977a,b). Normorphine can also be detected in two glucuronidated forms in urine: normorphine-3-glucuronide and normorphine-6-glucuronide (Chen et al., 2003). Normorphine binds and activates MOR and therefore exhibits analgesic activity (Sullivan et al., 1989; Frolich et al., 2011). In other mammals. In addition to morphine derivatives found in humans, other morphine catabolism products have been reported in non-human mammalian species (Daly et al., 1965; Misra et al., 1974a,b; Yeh et al., 1979; Frolich et al., 2011) (summarized in Fig. 4). It has been proposed that an O-methylation of morphine subsequently yields codeine (Borner and Abbott, 1973), but such a conversion is still a matter of debate (Yeh, 1974). In the bovine brain, endogenous 6acetylmorphine has been reported, suggesting a possible acetylation of EM (Weitz et al., 1988). The enzymes implicated and the ratios of the compounds formed remain unknown.

LOCALIZATION OF ENDOGENOUS OPIATES IN MAMMALS The central nervous system Immunohistochemistry, HPLC, MS and radioimmuno assay (RIA) approaches have improved the characterization of EM in the mammalian CNS. Several studies have specifically focused on the quantification of endogenous opiate levels in different areas of the CNS (Table 1, adapted from (Perea-Sasiaı´ n, 2008)). At the cellular level, Bianchi and collaborators provided evidence for the immunolocalization of endogenous opiates to the hippocampus, striatum, cortex and cerebellum cells (Bianchi et al., 1994). More recently, a precise mapping of the immunoreactivity of endogenous opiates was performed in the mouse CNS (Fig. 5), revealing their localization in both astrocytes and GABA neurons (Fig. 6). In the brain, endogenous opiate immunoreactivity has been observed in various structures, including the hippocampus, olfactory bulb, band of Broca, basal ganglia and cerebellum. In the spinal cord, endogenous opiates are mainly found in GABA neurons in the gray matter of the ventral horn and in astrocytes. While these compounds are present in presynaptic terminals in the cerebellum, they are

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found in postsynaptic terminals in the rest of the CNS (Laux et al., 2011, 2012). Endogenous morphine in astrocytes. Astrocytes do not express TH (Jaeger, 1985) and are therefore unable to synthesize dopamine or, by extension, endogenous opiates de novo. Two hypotheses have been proposed to explain the presence of endogenous opiates in astrocytes (Laux et al., 2011, 2012). (i) The first proposes that astrocytes can uptake morphine precursors and thus accomplish morphine biosynthesis. However, further studies of uptake mechanisms and enzyme expression are required to confirm this hypothesis. (ii) The second hypothesis proposes that astrocytes are key cells involved in neuromediator recycling from the extracellular space and may therefore be involved in morphine catabolism. Accordingly, transporters may mediate an uptake of endogenous opiates from the extracellular space. Opiate (endogenous or exogenous) transport mechanisms in the CNS remain poorly understood, and only a few transporters are known to carry morphine. The uptake of morphine by astrocytes is further supported by their expression of the P-glycoprotein (PgP) transporter (Gaillard et al., 2000; Calatozzolo et al., 2005; Chen and Sommer, 2009), which transports morphine across the blood–brain barrier (Dagenais et al., 2001; Seelbach et al., 2007). It is important to note that astrocytes express UGT1A6 and that the formation of endogenous M3G may therefore occur in these specific cells (Suleman et al., 1998; Heurtaux et al., 2006). Moreover, astrocytes express multidrug-resistant proteins (MRP)-1, -3, -4 and -5, as well as the glucose transporter (GLUT), which are potentially involved in morphine/M6G/M3G/ codeine uptake/efflux (Hirrlinger et al., 2002; Bourasset et al., 2003; Somogyi et al., 2007; van de Wetering et al., 2007). Hence, astrocytes may excrete glucuronidated derivatives upon the glucuronidation of morphine by UGTs. The present hypothesis of morphine catabolism (endogenous and exogenous) by astrocytes presents several avenues for future research, particularly in the field of tolerance/addiction to opiates. It is also important to note that endogenous opiates are present in astrocytic foot processes around blood vessels, suggesting a possible capture and/or release of morphine and its derivatives in the blood (Laux et al., 2011, 2012). Endogenous morphine in GABAergic cells. In addition to astrocytes, endogenous opiates are present in GABAergic cells throughout the CNS (Laux et al., 2011, 2012). However, not all GABAergic neurons are concerned, indicating that only a subpopulation of these cells is able to produce EM or its derivatives. In the CNS, GABAergic cells represent the most widespread cell population. As an example, more than 16 subtypes of GABAergic neurons have been characterized in the hippocampus (Somogyi and Klausberger, 2005). No information is currently available, however, regarding the neurochemical characteristics of GABAergic cells containing endogenous opiates. Nevertheless,

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Table 1. Amounts of EM, codeine, M3G and M6G present in CNS. ELISA, enzyme-linked immunosorbent assay; GC–MS, gas chromatography–mass spectrometry; HPLC, high-performance liquid chromatography; IP, immunoprecipitation; LC-MS/MS, liquid chromatography tandem mass-spectrometry; QTOF-MS/MS, quadrupole time-of-flight tandem mass spectrometry; RIA, radioimmunoassay. Adapted from Perea-Sasiaı´ n (2008) Structure

Species

Amounts

References

Detection techniques

Donnerer et al. (1986) Guarna et al. (1998) Molina et al. (1995a) Goumon et al. (2000) Guarna et al. (2002) Laux et al. (2011) Laux et al. (2011)

HPLC, RIA HPLC, GC/MS HPLC, RIA HPLC, GC/MS HPLC, GC/MS IP, LC/MS-MS HPLC, LC/MS-MS

Charlet et al. (2010) Horak et al. (1993)

ELISA HPLC, RIA

Monkey

26 ± 6 fmol/g 79 ± 16 fmol/g 0.23 ± 0.013 ng/g 329 ± 93 fmol/g 31 ± 9 fmol/g 7 ± 3.2 ng/g 0.31 ± 0.012 ng/g 6.3 fmol 17.1 fmol 1.01 ± 0.31 pmol/g 0.61 ± 0.22 pmol/g M6G: 0.25 ± 0.15 pmol/g/M3G: 0.21 ± 0.22 pmol/g 0.25 ± 0.23 ng/g 0.39 pmol/g (9 h) 1.16 pmol/g (17 h) 0.11 ng/g 0.04 ng/g

Neri et al. (2004)

GC/MS

Temporal lobe

Human

3.4 ng/g

Fricchione et al. (2008)

HPLC, RIA

Cortex

Rat

0.62 ± 0.32 pmol/g 0.43 ± 0.09 pmol/g 1 pmol/g 1.47 pmol/g 0.62 fmol/mg

HPLC, HPLC, HPLC, ELISA HPLC, RIA HPLC, RIA HPLC,

Morphine Brain (global)

Rat

Mouse

Codeine

Bovine Cat Dog

0.05 ± 0.01 pmol/g 1.5 ± 0.7 ng/g 0.15 pmol/g

Donnerer et al. (1987) Lee and Spector (1991) Meijerink et al. (1999) Muller et al. (2008) Laux et al. (2011) Gintzler et al. (1976a) Oka et al. (1985) Gintzler et al. (1976a) Meijerink et al. (1999)

Hippocampus

Mouse Rabbit Cat

7.46 pmol/g 8 ± 3 ng/g 5 ± 2 ng/g

Muller et al. (2008) Gintzler et al. (1976a) Gintzler et al. (1976a)

ELISA RIA RIA

Basal ganglia

Rabbit Calf Cat

33 ± 10 ng/g 14 ng/g 24 ± 7 ng/g

Gintzler et al. (1976a) Killian et al. (1981) Gintzler et al. (1976a)

RIA IP RIA

Olfactory bulb

Mouse

2.27 pmol/g 2 fmol/mg

Muller et al. (2008) Laux et al. (2011)

ELISA HPLC, LC/MS-MS

Hypothalamus

Rat Rabbit Bovine Cat Dog

7.22 pmol/g 14 ± 4 ng/g 0.25 a` 4.9 pmol/g 12 ± 6 ng/g 0.22 pmol/g

Meijerink et al. (1999) Gintzler et al. (1976a) Goldstein et al. (1985) Gintzler et al. (1976a) Meijerink et al. (1999)

HPLC, RIA RIA HPLC, RIA RIA HPLC, RIA

Cerebellum

Rat

0.63 ± 0.28 pmol/g 0.37 ± 0.07 pmol/g 1.474 pmol/g 1.13 ± 0.59 ng/g 1.48 pmol/g 12 ± 3 ng/g 0.09 pmol/g 14 ± 4 ng/g 0.134 pmol/g

0.80 ± 0.26 pmol/g 0.48 ± 0.08 pmol/g

Donnerer et al. (1987) Lee and Spector (1991) Meijerink et al. (1999) Charlet et al. (2010) Muller et al. (2008) Gintzler et al. (1976a) Oka et al. (1985) Gintzler et al. (1976a) Meijerink et al. (1999)

HPLC, HPLC, HPLC, ELISA ELISA RIA HPLC, RIA HPLC,

0.93 ± 0.36 pmol/g 0.52 ± 0.07 pmol/g 4 ± 1 ng/g 2.7 ± 1 ng/g

1.84 ± 0.57 pmol/g 0.38 ± 0.09 pmol/g

Donnerer et al. (1987) Lee and Spector (1991) Gintzler et al. (1976a) Gintzler et al. (1976a)

HPLC, RIA, MS HPLC, RIA RIA RIA

0.67 ± 0.18 pmol/g 0.59 ± 0.10 pmol/g 0.346 pmol/g 0.54 ± 0.44 ng/g 3.39 pmol/g

1.20 ± 0.27 pmol/g 0.47 ± 0.08 pmol/g

Donnerer et al. (1987) Lee and Spector (1991) Meijerink et al. (1999) Charlet et al. (2010) Muller et al. (2008)

HPLC, RIA, MS HPLC, RIA HPLC, RIA ELISA ELISA

Mouse

Mouse Rabbit Bovine Cat Dog Midbrain

Rat Rabbit Cat

Brainstem

Rat

Mouse

0.81 ± 0.11 pmol/g 0.63 ± 0.08 pmol/g

RIA, MS RIA QTOF MS/MS LC/MS-MS RIA RIA

RIA, MS RIA QTOF MS/MS

RIA RIA

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A. Laux-Biehlmann et al. / Neuroscience 233 (2013) 95–117 Table 1 (continued) Structure

Species

Amounts Morphine

Spinal cord

Rabbit Dog

4 ± 1 ng/g 0.388 pmol/g

Rat Rat Mouse Dog

0.13 pmol/g 1.64 ± 0.49 pmol/g 0.05 ± 0.01 ng/g 0.16 pmol/g

References

Detection techniques

Gintzler et al. (1976a) Meijerink et al. (1999)

RIA HPLC, RIA

Meijerink et al. (1999) Donnerer et al. (1987) Charlet et al. (2010) Meijerink et al. (1999)

HPLC, RIA HPLC, RIA, MS ELISA HPLC, RIA

Codeine

3.22 ± 1.24 pmol/g

Fig. 5. Localization of endogenous morphine and/or metabolites in adult mouse brain and spinal cord. ACB, accumbens nucleus; AH, Ammon’s horn; AON, anterior olfactory nucleus; BST, bed nucleus of the stria terminalis; CBX, cerebellum; CP, caudate putamen; CTX, cortex; DCO, dorsal cochlear nucleus; DG, dentate gyrus; DR, dorsal raphe nucleus; FN, fastigial nucleus; FS, fundus striati; gl, glomerular layer; GP, Globus pallidus; gr, granular cell layer; HY, hypothalamus; IC, inferior colliculus; INC, interstitial nucleus of Cajal; LV, lateral ventricle; MB, midbrain; mi, mitral cell layer; ml, molecular layer; MR, median raphe nucleus; MY, medulla; ND, nucleus of Darkschewitsch; NDB, nucleus of the diagonal band; NLL, nucleus of the lateral lemniscus; NOT, nucleus of the optic tract; OB, olfactory bulb; OT, olfactory tubercle; P, pons; PAG, periaqueductal gray; PB, parabrachial nucleus; PIR, piriform cortex; pl, purkinje layer; PON, periolivary nucleus; PVT, paraventricular thalamic nucleus; RN, red nucleus; RT, reticular thalamic nucleus; SC, superior colliculus; sgz, subgranular zone; SI, substantia innominata; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata; STN, subthalamic nucleus; SUB, subiculum; TH, thalamus; TSN, triangular septal nucleus; VN, vestibular nucleus; VTN, ventral tegmental nucleus; ZI, zona Incerta. In the spinal cord (lower left), laminae are labeled using roman number.

endogenous opiates specifically occur in a well-defined morphological subpopulation of GABAergic cells corresponding to the basket cells of the cerebellum and the sub-granular layer of the dentate gyrus (Muller et al., 2008; Laux et al., 2011, 2012). This neuronal population may therefore have the transporters and enzymes necessary for the capture of morphine precursors (THP. . .) and completion of the EM synthesis reactions. Interestingly, pluricellular interaction is required for morphine biosynthesis in opium poppy, and three specific cell types are implicated in this process (Bird et al., 2003). Therefore, the identification and study of this subpopulation could provide important information about the EM synthesis pathway.

In the periphery In mammals, EM and endogenous morphine metabolites (EMM) have been detected in the adrenal gland (Oka et al., 1985; Donnerer et al., 1987; Molina et al., 1995a; Goumon and Stefano, 2000), which is one of the major organs implicated in stress responses (Goumon et al., 2009). Moreover, EM has been found in tumoral rat pheochomocytoma PC-12 (Goumon et al., 2000c; Poeaknapo et al., 2004), while the presence of M6G has been reported in the secretory granules of bovine primary chromaffin cells (Goumon et al., 2006). EM has also been detected in lung cells (dog and human (Munjal et al., 1995)); rat and human hepatocytes

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Fig. 6. Immunolocalization of endogenous opiates in adult mouse cortex. An immunolabel is observed in GABAergic neurons (black arrow) and astrocytes (white arrow). The antibody recognizes endogenous codeine, morphine and its glucuronidated derivatives (Laux et al., 2011, 2012). Scale bar = 20 lm.

(Donnerer et al., 1986, 1987; Molina et al., 1995b; Poeaknapo et al., 2004); and the rat spleen, thymus and kidneys (Molina et al., 1995b). Morphine and its derivatives have been reported in the rat pancreas (Lee and Spector, 1991), and the formation of morphine intermediates such as THP and reticuline has been observed in the human pancreatic cell line DAN-G (Poeaknapo et al., 2004). Surprisingly, rabbit (Oka et al., 1985), rat (Donnerer et al., 1987), toad (Oka et al., 1985), and human skin cells (Weinstein et al., 2002), as well as the human keratinocyte cell line HaCaT (Poeaknapo et al., 2004) contain EM. EM has also been detected in the mouse (Horak et al., 1993), rat (Donnerer et al., 1987) and human hearts (Zhu et al., 2001b) as well as in granulocytes (Zhu et al., 2005; Boettcher et al., 2006; Glattard et al., 2010) and mononuclear cells (Boettcher et al., 2006). The reported amounts of EM in these different tissues and cells vary from one study to another. Such variation may result from several physiological conditions known to increase EM levels in plasma or tissues. In mice models in particular, the EM levels detected in the brain, heart and spleen have been found to be up to six times higher in the afternoon (16–17 h) than in the morning (9–10 h), indicating a circadian influence on EM synthesis (Horak et al., 1993). Many of the other conditions discussed in the following paragraphs also increase EM levels in the plasma or tissues. These conditions are described in some detail to help determine the roles of EM. Different cellular partners may be involved in endogenous opiate biosynthesis

Is EM biosynthesis restricted to catecholaminergic cells? Since the characterization in 2005 of the EM biosynthetic pathway in the catecholaminergic human neuroblastoma SH-SY5Y (Poeaknapo et al., 2004; Boettcher et al., 2005), it has been postulated that EM

synthesis occurs in dopaminergic/catecholaminergic cells specifically at the periphery surrounded by chromaffin (Goumon and Stefano, 2000; Goumon et al., 2000c; Goumon et al., 2006), polymorphonuclear (PMN) (Zhu et al., 2005) and mononuclear (MN) cells (Boettcher et al., 2006). Moreover, dopamine is necessary for EM formation (Neri et al., 2008). As described in this chapter, endogenous opiates are present in non-catecholaminergic cells; more specifically, they are found in the CNS in astrocytes and GABAergic cells (Muller et al., 2008; Laux et al., 2011, 2012). Hence, the synthesis of EM within the CNS appears to be more complex than expected and cannot simply be extrapolated from data limited to the neuroblastoma cell line SH-SY5Y (Muller et al., 2008; Boettcher et al., 2005). The precursor-uptake hypothesis. In contrast to the periphery, where EM synthesis appears to occur in single dopaminergic and/or catecholaminergic cells, the presence of endogenous alkaloids in non-dopaminergic cells of the CNS suggests a possible uptake of EM precursors. Indeed, various precursors involved in different stages of the EM synthetic pathway could be taken up from the extracellular space, cerebrospinal fluid or blood by cells expressing the enzymes necessary to complete the synthesis of EM. One possibility is that in the CNS, astrocytes and GABAergic neurons uptake morphine precursors to finalize the synthesis of EM. Indeed, the main dopamine transporter DAT is found at the synaptic terminals of dopaminergic neurons but is also expressed by astrocytes (Takeda et al., 2002) and non-dopaminergic neurons (Ciliax et al., 1995; Freed et al., 1995). This dopamine uptake may also rely on OCT-3 (organic cation transporter 3), which is expressed by astrocytes (Gasser et al., 2009) and able to transport dopamine to non-dopaminergic cells (Chemuturi and Donovan, 2007). The PMAT (plasma membrane monoamine transporter), which can also transport dopamine, is

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present in many CNS neurons (Dahlin et al., 2007). Finally, EMT (extraneuronal monoamine transporter) and NET (norepinephrine transporter) are also involved in dopamine uptake by astrocytes (Inazu et al., 2003a,b) and may contribute to precursor uptake. Until now, only one study has reported an interaction between THP and DAT in which THP blocks dopamine transport (Okada et al., 1998). It is also unclear whether THP may itself be transported by DAT. Unfortunately, no information is available concerning the possible transport of other precursors of EM, and the possibility of morphine capture by GABAergic neurons cannot be excluded. Localization of enzymes involved in endogenous morphine biosynthesis. In mammals, the presence of enzymes involved in dopamine synthesis (TH and AADC) is necessary for EM formation. Nevertheless, these enzymes, which are mainly present in dopaminergic neurons, are not expressed by ‘‘morphinergic’’ neurons or astrocytes (Jaeger, 1985; Laux et al., 2011, 2012). The capture of precursors described in the previous paragraph therefore seems to represent an essential step for EM synthesis in CNS cells. The formation of DOPAL from dopamine by an MAO is a crucial step in the formation of THP and the consequent generation of EM. Two types of MAOs (MAO-A and MAOB) are expressed by neurons and astrocytes in many CNS structures (Ekblom et al., 1993; Ikemoto et al., 1997a,b; Jahng et al., 1997), including nondopaminergic cells, and may thus allow cells to produce THP. Following THP formation, three methylation steps are necessary for the formation of reticuline. In the past few years, it has been proposed, albeit without demonstration, that catechol-O-methyltransferase (COMT) and phenylethanolamine N-methyltransferase (PNMT) may be involved in at least one of these steps (Mantione et al., 2008). It was also recently reported that N-methyltransferase (NMT) transforms (R)norreticuline to (R)-reticuline in mammals, while PNMT has no activity (Grobe et al., 2011). NMT was initially reported to catalyze the N-methylation of tryptamine (Thompson et al., 1999) present within the CNS (Boarder and Rodnight, 1976). Nevertheless, the cellular localization of NMT remains unknown. Among the members of the cytochrome P450 enzyme family, which is involved in many enzymatic reactions, CYP2D6 is widely expressed in CNS neurons (Norris et al., 1996; Siegle et al., 2001) but not expressed in human astrocytes (Siegle et al., 2001). Because CYP2D6 catalyzes four steps of the EM biosynthetic pathway, the presence of this enzyme in cells appears to be crucial for the de novo synthesis of EM. The absence of this enzyme in astrocytes therefore suggests that this synthesis does not occur in astrocytes. However, the existence of another p450 enzyme involved in morphine biosynthesis cannot be ruled out. At the present time, none of these enzymes can be used for a mouse knock-out model depleted for endogenous opiates because they are implicated in a

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number of crucial physiological processes. Additional investigations are required for the discovery of specific EM biosynthesis enzymes and the generation of a relevant animal model with disrupted EM synthesis.

WHAT ARE THE ROLES OF ENDOGENOUS OPIATES? Endogenous opiate levels The effects of exogenous morphine on the CNS at concentrations commonly used for many therapeutic purposes (lM range) have been extensively detailed in the literature. These concentrations are far higher than those detected for EM (pM-nM). It is therefore difficult to extrapolate the potential effects of EM from the effects observed for exogenous morphine (Perea-Sasiaı´ n, 2008). Under healthy conditions in humans, peripheral EM concentrations are barely detectable (pM). In contrast, EM blood levels increase to the nM range under pathological conditions, particularly under states of infection or inflammation (see next paragraph) (Glattard et al., 2010; Laux-Biehlmann et al., 2012). Within the CNS, EM has been found to increase from fmoles to pmoles per gram of tissue, making it difficult to determine the effective local concentrations. The effects of EM in the CNS have not been well documented and are often extrapolated from results obtained with low amounts of administered exogenous morphine. At the periphery: stress and immune responses Endogenous opiates in the adrenal medulla. As mentioned above, EM and endogenous M6G are present in chromaffin cell granules of the adrenal medulla, a major stress response organ, by releasing catecholamines (e.g., adrenaline) into the blood. Studies have shown that primary bovine chromaffin cell cultures contain M6G in their secretory granules, which is secreted upon nicotine stimulation (Goumon et al., 2005, 2006). The involvement of these cells in response to stress suggests that M6G is co-secreted with catecholamines and could therefore be involved in stress responses. As no studies have been performed in human chromaffin cells, the presence of EM or endogenous M6G in these cells is unknown. Endogenous opiates in the immune system. EM synthesis has been demonstrated in immune cells including human monocytes and polymorphonuclear cells, suggesting an involvement of EM in immune responses (Boettcher et al., 2005; Zhu et al., 2005; Glattard et al., 2010; Laux-Biehlmann et al., 2012). Accordingly, EM levels were found to be significantly higher in the blood of patients and pigs having had coronary bypass or laparotomy surgeries compared to healthy donors and control animals. For example, a coronary bypass surgery performed in humans increased blood concentrations of EM from 0.28 to 3.9 nM (Brix-Christensen et al., 1997). Similarly, coronary bypass surgery in piglets induced EM blood

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levels of 10 nM, whereas no EM was detected in the control group (Brix-Christensen et al., 2000). Interestingly, two studies carried out in humans have shown that open cholecystectomy significantly increased EM levels in the blood (0.2 nM) compared to a laparoscopic procedure (0.018 nM) (Yoshida et al., 2000; Madbouly et al., 2010). Together, these data suggest that this EM increase induces particular effects as the blood concentrations during these states are compatible with MOR affinity (nM). Alternatively, lipopolysaccharide injections in rats or injections of muramyl dipeptide in mice to mimic bacterial infection have been shown to increase EM levels in tissues (Horak et al., 1993; Goumon et al., 2000a). Other stresses such as fasting cause a similar increase in EM concentrations in the blood and the spinal cord (Molina et al., 1995a; Meijerink et al., 1999; Goumon et al., 2000a). The immunosuppressive effects of morphine via NO production by immune cells inhibit the synthesis of proinflammatory cytokines, chemotaxis and phagocytosis (Stefano et al., 2000). In addition, morphine-induced NO production by the endothelium reduces the expression of adhesion molecules, allowing for the recruitment of immune cells toward inflammation sites and activating the hypothalamic–pituitary–adrenal axis, which promotes glucocorticoid release (Mellon and Bayer, 1998a). Morphine also inhibits immune stem cell differentiation into lymphocytes, T-cell proliferation (Mellon and Bayer, 1998b) and the cytolytic activity of natural killer cells (Mellon and Bayer, 1999). It is important to note that EM levels during sepsis (8 nM) significantly inhibit the secretion of IL-8 in vitro from human polymorphonuclear cells primed with LPS (Glattard et al., 2010). The release of EM in the blood under stress conditions may represent a physiological mechanism that reduces inflammatory responses (i.e., anti-inflammatory effects). Proposed roles in the CNS

Do endogenous opiates act as neurotransmitters or neuromediators? The role of EM as a neurotransmitter has been considered for many years (for review: Guarna et al., 2005). In fact, EM satisfies several neurotransmitter criteria: neuronal synthesis, presynaptic vesicular localization, release upon potassium depolarization (Guarna et al., 1998) or nicotine stimulation (Muller et al., 2008) and reuptake by neurons (Bianchi et al., 1993). However, recent studies have shown that EM and EMM are primarily localized in the postsynaptic compartment, except in the cerebellum, where they are present in the presynaptic terminals of basket cells (BC) innervating the cell bodies of Purkinje neurons (Muller et al., 2008; Laux et al., 2011). This particular location, which is likely exclusive to the cerebellum, casts doubt on the hypothesis that morphine functions as a neurotransmitter. However, it is possible that only a minority of neurons, such as basket cells, are capable of EM storage in their presynaptic terminals (Bianchi et al., 1994; Muller et al., 2008; Laux et al., 2011).

In neurons, where EM is located in the postsynaptic compartment, another role for this alkaloid warrants consideration. While neurotransmission ‘‘traditionally’’ occurs by the release of neurotransmitters from the presynaptic terminal, some postsynaptic terminals release neurotransmitters or neuromediators in a ‘‘retrograde’’ manner to activate presynaptic receptors (examples include nitric oxide and endocannabinoids; for review: Ludwig and Pittman, 2003). The presence of EM and its derivatives in dendrites and the postsynaptic endings of many neurons throughout the CNS is not trivial and may be indicative of potential retrograde release. Moreover, the presence of opioid receptors, especially MORs, in the presynaptic regions of some neurons strengthen this idea (Drake and Milner, 1999; Svingos et al., 2001a,b; Zhang and Pan, 2010). Endogenous opiates and control of nociception. Based on the analgesic effects of exogenous morphine, one of the first roles that comes to mind for its endogenous counterpart is the control of nociception processes. Accordingly, multiple studies have suggested that EM may be involved in these processes. First, immunohistochemical approaches have indicated the presence of EM in the neurons and processes of CNS areas involved in the control of nociception, including the rostroventral medulla, periaqueductal gray matter, raphe magnus nuclei and superficial laminae of the spinal cord (Stefano et al., 2000; Laux et al., 2011, 2012). Moreover, when anti-morphine antibodies were injected into the CSF of mice, Guarna et al. (2002) observed reduced levels of cerebral EM. This treatment reduced the latency of paw licking, indicating that thermal hyperalgesia occurred when the EM level was reduced. On the other hand, KO mice for MOR have an increased sensitivity to thermal nociceptive stimuli but not mechanical stimuli (Kieffer and Gaveriaux-Ruff, 2002). Endogenous opiates and memory. EM has been described in neurons and astrocytes in the hippocampus, suggesting a possible role of EM in memory processes. More precisely, EM is present in GABAergic neurons of Amon’s horn and in GABAergic basket cells of the subgranular zone (SGZ) of the dentate gyrus (Laux et al., 2011). A study using injections of anti-morphine antibodies into the CSF of mice suggested a link between EM and memory (Guarna et al., 2004). In this experiment, EM CNS levels were significantly elevated after 12 h of fasting compared to control animals. When these animals were tested for memory, they showed impaired memory acquisition and consolidation due to increased EM levels. Moreover, this impairment was abolished when the EM was immunodepleted. These data suggest that EM might, under stress conditions such as fasting, inhibit memory processes. Endogenous opiates and addiction. The role of dopamine, a precursor of EM, as a key neurotransmitter for addictive processes prompted the consideration of a possible link between EM and addiction. Work

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performed on two invertebrate models, Mytilus edulis and Homarus americanus, showed that exposure to addictive substances (ethanol, nicotine or cocaine) increases EM release by a factor of two in the nervous systems of these organisms (Zhu et al., 2006a,b). In mammals, a release of EM by human immune cells after an administration of cocaine, alcohol or nicotine has been reported (Zhu et al., 2006a). Interestingly, alcoholism induces an overexpression of CYP2D6, an enzyme involved in four steps of the EM synthesis pathway (Miksys et al., 2002), in different cerebral structures. Finally, it has been shown that alcoholism dramatically increases circulating levels of THP, which is a precursor of EM synthesis in mammals (Haber et al., 1997; Sango et al., 2000; McCoy et al., 2003). On the basis of these findings, a link between EM and addiction has been proposed (Stefano et al., 2007). Endogenous opiates, neurogenesis and structural plasticity. In the adult mammalian brain, the subventricular zone (SVZ) bordering the lateral ventricle and the SGZ of the hippocampus dentate gyrus are both adult neurogenesis niches. Exogenously administered morphine regulates the survival and production of neural progenitors and also affects neuronal plasticity. Indeed, morphine inhibits neurogenesis in the SGZ of mice and adult rats through its action on MOR (Harburg et al., 2007; Arguello et al., 2008; Fischer et al., 2008; Sargeant et al., 2008). Morphine causes structural changes in different areas of the CNS (e.g., the nucleus accumbens), affecting dendrites and the dendritic spine structure (Harston et al., 1980; Ikeda et al., 2000, 2010; Badiani and Robinson, 2004; Robinson and Kolb, 2004). Morphine has also been found to promote axonal and synaptic regeneration in the spinal cord of injured mice (Zeng et al., 2007). Together, these effects of exogenously administered morphine on neurogenesis and structural plasticity highlight a potential role for EM. Additionally, EM and EMM are present in the cells of the SGZ and the SVZ. In both cases, EM and its derivatives are notably present in radial astrocytes (Laux et al., 2012). Using nestin, an immunohistochemical marker for radial glialike progenitors (von Bohlen Und Halbach, 2007), unpublished data from our lab have confirmed EM localization in these cells. The presence of EM in radial glia-like progenitors of the SGZ and SVZ suggest an involvement of this endogenous alkaloid in neurogenesis. Furthermore, these cells express MOR (Persson et al., 2003a,b), and the action of endogenous opioids on MOR has been found to inhibit neurogenesis induced by an ischemia in the SGZ (Kolodziej et al., 2008). Interestingly, the dentate gyrus of MOR-KO mice contains a greater quantity of immature postmitotic neurons (Harburg et al., 2007). Exogenous morphine has an effect on structural plasticity in vitro. A high morphine concentration (1 mM) inhibits the neurite outgrowth of hippocampal neurons or of granular cerebellum neurons in primary culture

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(Brailoiu et al., 2004). Conversely, a low concentration (<1 pM) stimulates the neurite outgrowth of PC-12 cells and spinal cord or cortex neurons independently of MOR (Tenconi et al., 1996; Brailoiu et al., 2004). In the absence of direct evidence, EM or EMM control of neurogenesis and brain plasticity cannot be excluded, particularly during pathological states leading to increased biosynthesis. Endogenous opiates and neuroinflammation. Recent data suggest that morphine and M3G bind TLR4 and, more specifically, its accessory protein MD2 (Hutchinson et al., 2010b,c). TLR4 is a pathogenassociated molecular pattern receptor involved in neuroinflammatory responses to various CNS stimuli through the activation of microglial cells and astrocytes. TLR4 is mainly present in immune cells and microglia. However, its presence has been reported on other CNS cells including astrocytes and neurons (for review: Buchanan et al., 2010). The action of morphine and M3G on TLR4 presents new research perspectives in the area of endogenous opiates. It would be particularly interesting, for example, to investigate the effect of endogenous opiates (morphine, M6G and M3G ratios) on neuroinflammation. EM may act on both MOR and TLR4 present on astrocytes and microglial cells, whereas the action of endogenous M3G would be restricted to the TLR4 receptor. Surprisingly, activated astrocytes over-express UGT1A6 under infectious states (Heurtaux et al., 2006) and accordingly, the EM/ M3G ratio would be altered during a neuroinflammatory state and directly activate MOR/TLR4.

ENDOGENOUS OPIATES AND CNS PATHOLOGIES Schizophrenia A ‘‘hypermorphinergic’’ pathology? Schizophrenia is a psychosis manifested by signs of mental dissociation, emotional discordance and delusional activity. It is a major cause of disability worldwide (Murray and Lopez, 1996). The dopamine deregulation hypothesis for schizophrenia is often proposed as the underlying neurobiological mechanism of this disease. This hypothesis postulates that an excess of dopamine in the mesolimbic pathway is responsible for some schizophrenia symptoms (for review: Cousins et al., 2009; Heinz and Schlagenhauf, 2010; Miyake et al., 2011). Because dopamine is an EM precursor, an increase in EM levels in CNS tissues would be expected. Using a mouse model of synaptic and hyperdopaminergic aspects of schizophrenia (Andrieux et al., 2002; Brun et al., 2005), we recently observed increased levels of brain EM in STOP mice compared to wild-type mice (Charlet et al., 2010). Interestingly, COMT, a potential factor in the methylation of morphine precursors, is more active in certain brain regions of schizophrenic patients. This increase in activity is primarily due to the presence of the Val105/158 Met polymorphism (reviewed in: (Sagud et al., 2010)).

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However, no data concerning the EM levels in schizophrenic patients are available. It is interesting to note that in some cases, opioid antagonists (e.g., naloxone, naltrexone) were previously used to treat schizophrenia associated with addiction, implicating MOR (McNicholas and Martin, 1984; Welch and Thompson, 1994; Wonodi et al., 2004). Thus, high levels of EM may be related to some of the symptoms observed in this pathology. Endogenous morphine, schizophrenia and nociception. Among the symptoms of schizophrenia, clinical observations reveal a lack of pain expression during normally painful situations in schizophrenic patients (burns, fractures, etc.). However, this phenomenon is poorly understood, and two hypotheses have been proposed: (i) a modification of endogenous pain controls and (ii) differing conscious interpretations of nociceptive signals (Blumensohn et al., 2002; Autie et al., 2009; Bonnot et al., 2009). The higher levels of EM potentially present in schizophrenic patients may explain functional changes in endogenous pain control. The A118G polymorphism of the OPRM1 gene occurs more frequently in patients with schizophrenia, suggesting the involvement of MOR and the opioid/opiate system in schizophrenic pain symptoms (Sery et al., 2010). Interestingly, patients with this polymorphism are described to be less sensitive to morphine (for review: Mague and Blendy, 2010). More investigations are needed to determine the precise role of EM in schizophrenia and may lead to the development of new treatments for schizophrenic symptoms. Parkinson’s disease Parkinson’s disease is primarily characterized by movement disorders, such as slowness (akinesia), rigid movements (hypertonia) and tremors, which are consequences of the degeneration of dopaminergic neurons in the substantia nigra pars compacta. Ultimately, these changes result in a depletion of dopamine in the CNS. We recently reported an increase in the levels of EM and its metabolites in the CNS of Parkinson’s disease patients and in different animal models (Charron et al., 2011). This increase may be the result of increased dopaminergic catabolism and higher amounts of the morphine precursor THP. Higher levels of endogenous opiates in the brain may have varying implications: (i) they may be responsible for some motor symptoms of Parkinson’s disease or (ii) they may be one of the many compensation mechanisms established by the CNS (Bezard and Gross, 1998; Bezard et al., 2003). Because EM and its metabolites are present in CNS areas involved in motor control, movement and posture, these alkaloids may be implicated in motor function. Thus, a change in the levels or ratios of endogenous opiates may result in an impairment of these functions (Laux et al., 2011, 2012). In addition to motor symptoms, Parkinson’s disease is also associated with pain symptoms at a prevalence of approximately 40% (for review: Chaudhuri and Odin, 2010; Ford, 2010; Meissner et al., 2011). Warm thermal

hyperalgesia is observed in some patients (Schestatsky et al., 2007), while thermal hypoalgesia to cold is observed in others (Brefel-Courbon et al., 2005). In both cases, an improvement in these symptoms is observed after L-DOPA treatment. We have similarly observed a return to normal EM levels following L-DOPA treatments in patients and in Parkinson’s disease models (Charron et al., 2011). Because EM and its derivatives may be involved in nociception control, increases in EM or specific EMM species may be contributing factors or the cause of pain symptoms observed in Parkinson’s disease patients. The correlation between L-DOPA treatments and pain relief as well as reduced EM further supports the hypothesis of EM involvement in these symptoms. At the present time, it is not possible to determine whether increased levels of endogenous opiates are a cause or consequence of parkinsonian symptoms. Further investigations are required to reveal whether these endogenous compounds represent therapeutic targets.

CONCLUSION Although no clear function has yet been attributed to EM and EMM in the CNS, their presence in specific neurons and astrocytes cannot be considered as fortuitous. Besides its potential implication in pain-regulatory processes, the presence of these endogenous compounds in brain regions that are not usually involved in pain modulation opens exciting perspectives to extend the role and function of this endogenous opiate far beyond analgesic functions. Acknowledgements—We are most grateful to Mrs. Elise Savier and Mr. Leny teyssier for their advice on the manuscript.

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(Accepted 7 December 2012) (Available online 21 December 2012)