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389
An. R. Acad. Nac. Farm., 2009, 75 (3): 389-418
REVISIONES
Central and peripheral endogenous morphine
Yannick Goumon*, Alexis Laux, Arnaud Muller, Dominique Aunis
*Inserm U575. Strasbourg, France.
Recibido el 28 de abril de 2009.
ABSTRACT
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 and
morphine-6-glucuronide has been shown to be present in the human
neuroblastoma 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 neuro-
transmission.
An increasing number of publications have demonstrated its pre-
sence 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 phy-
siological processes.
Key words: Morphine; Alkaloid; Morphine-6-Glucuronide; Anal-
gesia; μ Opioid Receptor.
YANNICK GOUMON Y COLS.AN. R. ACAD. NAC. FARM.
390
RESUMEN
Morfina endógena a nivel central y periférico
La morfina se identificó por primera vez en el opio procedente
de Papaver somniferum, y sigue siendo uno de los analgésicos más
potentes conocidos empleados en los hospitales. Desde comienzos de
la década de los 80s, la morfina endógena, con una estructura idén-
tica a la morfina aislada de las amapolas, se caracterizado en nume-
rosas células y tejidos de mamíferos. En mamíferos, la biosíntesis de
la morfina endógena está asociada a la dopamina, como se ha de-
mostrado en la línea celular neuronal humana productora de cate-
colaminas SH-SY5Y. Más recientemente, se ha demostrado la pre-
sencia 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 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 im-
portantes sobre la presencia e implicación en procesos fisiológicos
de la morfina endógena.
Palabras clave: Morfina; Alcaloide; Morfina-6-Glucorónido; Anal-
gesia; Receptor opioide μ.
1. EXOGENOUS MORPHINE
a) History
When the capsule of the poppy Papaver somniferum is incised, a
milky fluid exudes which, after harvesting and drying, yields a brown
gum-like substance known as opium. Mention is made of this
substance’s efficacy as a painkiller, against diarrhoea and as a
narcotic, in texts dating back as far as 4000 years BC. Morphine
is one of the forty alkaloids present in opium (1) and today, its
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analgesic activities —as well as its dangers addiction and overdose—
are common knowledge. At this time, morphine and its precursor
codeine remain the gold standard in pain relief (2, 3).
Morphine is still the most commonly used analgesic in hospitals,
mainly to relieve acute pain (especially following surgery) but
sometimes for chronic pain which is refractory to other active
compounds. After administration (orally, subcutaneous or
intravenous injection, or infusion), morphine has a half-life of about
three hours. There is no recommended maximum dose and the
amount administered can be progressively stepped up until pain
relief is obtained, as long as there are no side effects: a typical
daily dosage for chronic cancer pain in adults is 30 milligrams a
day (administered by infusion). The analgesic activity is due to the
binding of morphine to μ opiate receptors. Morphine often has
unwanted side effects, including constipation, drowsiness, nausea
and vomiting; other, less common side effects are confusion,
nightmares and, at excessive doses, respiratory depression (which
can cause apnoea and lead to death).
b) Mu (μμ
μμ
μ) opiate receptors
At the beginning of the 1970’s, the existence of specific morphine-
binding receptors was hypothesised on the basis of the drug’s
physiological effects, and soon such opiate receptors were indeed
discovered in the central nervous system (4). Most opiates (alkaloids)
and opioids (peptides) preferentially bind to Mu (μ), Delta (δ) and
Kappa (κ) receptors, all proteins with seven membrane-crossing
segments coupled to G proteins. Morphine and its derivative
morphine-6-glucuronide (M6G) preferentially bind a receptor
referred to as the Mu (μ) opiate receptor (MOR) or the Mu (μ) opioid
peptide receptor [MOP (5)]. Like morphine, the endogenous peptide
ligands, endomorphin-1 and endomorphin-2, have a high affinity for
μ receptors (6). The extracellular N-terminal portion of the receptor
molecule carries the ligand binding site while the intracellular
C-terminal portion is involved in signal transduction (7). These
receptors are found in both the central nervous system and the
periphery. In the brain, the highest densities of μ receptors are found
YANNICK GOUMON Y COLS.AN. R. ACAD. NAC. FARM.
392
in the thalamus, the putamen, the black substance, the cortex, the
ventral tegmental zone, the nucleus accumbens and the amygdala
(8, 9). In the periphery, µ receptors are expressed on endothelial
cells (10, 11) and cells of the immune system (12), among others.
Pharmacological experiments and ligand binding studies have
shown that there are several different isoforms of the μ receptor:
—μ1 is mainly expressed in the CNS and has a high affinity for
morphine -it is this form that mediates the drug’s analgesic
activity;
—μ2 is expressed in the CNS, the respiratory system and the gut,
and mediates most of the drug’s side effects (3);
—μ3 is found on human monocytes, granulocytes and endothe-
lial cells.
However, whether or not these three different isoforms really
exist is now controversial because all μ receptors are encoded by a
single gene, Opioid Receptor Mu 1 (OPRM1) in humans and Oprm1
in mice, and the different forms are generated by alternative splicing.
Currently, 28 different variants have been described in mice (13, 14),
and ten different forms have been characterised in humans (15).
This classification system is continually changing and it is more
than likely that other variants will be identified.
Signal transduction mechanisms of the
μ
receptors
The activation of Gi/Go proteins inhibits cellular activity by
means of three main mechanisms [reviewed in (5)]:
— by inhibiting adenylate cyclase activity leading to a reduction
in cAMP generation. However, it is important to remember
that repeated exposure to morphine will lead to enhanced
adenylate cyclase activity, a phenomenon which seems to
contribute to the addictiveness of opiates (16);
— by opening potassium channels which leads to increased K+
flux and hyperpolarisation of the cell (17);
— by blocking voltage-dependent calcium channels and decrea-
sing permeability to Ca2+ (17).
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Activating potassium channels at the same time as blocking
voltage-dependent calcium channels inhibits neurotransmitter
release so two typical effects of binding at μ receptors are, on the
one hand reduced GABA release by hippocampal interneurones (18),
and on the other reduced glutamate release by neurones in the
striatum (19).
In addition to these three mechanisms, it has been shown that
activation of μ receptors can affect other signalling pathways
specific to other cell-types (20, 21), e.g. in endothelial cells and some
types of immune cell (notably leukocytes). Thus, the stimulation
of μ receptors induces the generation and release of nitrogen oxide
(NO) via a PKC-dependent mechanism (1, 22, 23). Morphine’s
immunosuppressive activity seems to be dependent on µ receptor-
mediated induction of a cascade of MAP kinases in lymphocytes and
polymorphonuclear cells (24).
It has also been shown that GIRK (G-protein activated Inwardly
Rectifying K+ current) channels are involved in the analgesia induced
by the bolus injection of morphine, especially through its action at
the Periaqueductal gray matter (PAG): the drug inhibits GABAergic
interneurones thereby lifting the inhibition of PAG neurones which
are involved in descending control of nociception (25-27). Other
experiments have shown that opiate inhibition of neurones in the
dorsal layer of the spinal cord also involves GIRK channels. This
mechanism underlies the analgesic effect obtained by injecting μ
receptor agonists intrathecally (28, 29). Finally, recent work has
revealed the role played by GIRK channels in tolerance and addiction
when morphine is administered on a long-term basis (30, 31).
c) Morphine catabolism
Enzymes
Exogenous morphine is mainly inactivated (i.e. detoxified) in
the liver by a superfamily of enzymes referred to as the UDP-
glucuronosyltransferases [UGT (32)]. Different forms of UGT are
found in the gut and kidneys [reviewed in (33)] and recently, the
presence of UGT2B7 was found in the brain, suggesting that
YANNICK GOUMON Y COLS.AN. R. ACAD. NAC. FARM.
394
endogenous and exogenous morphine could be glucuronized inside
this organ (34, 35).
These various forms of UGT have distinct but overlapping
substrate specificities. To date, 28 different UGT genes have been
identified and these have been divided into two families and sub-
families (UGT1, UGT2A and UGT2B) on the basis of their homology
(36). Only certain of the known human UGTs appear to be catalytically
active, namely UGT1A1, 1A3, 1A4, 1A5, 1A6, 1A7, 1A8, 1A9, 1A10, 2A1,
2B4, 2B7, 2B15, 2B17 and 2B28 (37, 38). In the liver, morphine
is glucuronidated by a UGT at Carbon 3 or 6, to generate either
morphine-3-glucuronide (M3G) or morphine-6-glucuronide (M6G;
Figure 1), both highly hydrophilic compounds which are quickly
excreted in the urine.
Figure 1. UGT2B7-catalysed generation of M3G and M6G from morphine in the
liver.
M3G accounts for about 90% of the glucuronide products and has
no analgesic activity at all (Figure 1). In contrast, M6G (the other 10%
of morphine glucuronide products in the liver) is reported to be a
more potent analgesic than the parent molecule [reviewed in: (32)].
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In humans, most M6G and M3G formation is catalysed by
UGT2B7, although UGT1A1 and 1A8 have been described as making a
minor contribution to M6G (38), while UGT1A3, 1A6, 1A9 and 1A10
can only catalyse the formation of M3G (34). Most UGT enzymes
are mainly expressed in the liver but some are found elsewhere,
e.g. UGT1A6 and UGT2B7 are expressed in the brains of rats and
humans (39), UGT1A6 is also expressed by rat neurones and astrocytes
in primary tissue culture (40), and UGT1A6 has been detected by in
situ hybridisation in pyramidal hippocampal neurones and Purkinje
cells in the rat cerebellum (41).
In humans, M3G —the major glucuronide product of exogenous
morphine— has zero affinity for opiate receptors and no analgesic
activity (32). M6G —which accounts for 10% of the morphine
breakdown products in the liver— can bind the μ receptor and has
been reported as being 1-600 times more active as an analgesic than
morphine itself, the variability depending on the model studied
(notably species) and the route of administration [intraventricular,
intravenous, etc.; reviewed in (2, 32 42)]. As well as these two
glucuronides, smaller amounts of other metabolites are also formed
and found in the urine, including morphine 3,6-diglucuronide,
normorphine, normorphine-6-glucuronide and morphine-3-sulphate,
with no or little analgesic activity (43, 44).
Before morphine or M6G can have any effect in the central
nervous system, they have to cross the blood-brain barrier (BBB).
M6G has been reported as crossing this barrier 32-57 less efficiently
than morphine (45) and its analgesic effects are mainly obtained
by intrathecal and intraventricular injection. Studies on the efficacy
of M6G in humans have yielded inconsistent results. One study
suggested that intrathecal M6G is 4-5 times more effective against
postoperative pain (46) while a number of studies have indicated
that very high doses of intravenous M6G are required for effective
analgesia [reviewed in (3, 32)]. Because of its strongly hydrophilic
nature, M6G does not cross the BBB efficiently and very high
intravenous doses (of the order of 0.3 mg/kg) are necessary to reach
a high enough M6G concentration in the brain for effective analgesia.
In contrast to morphine, M6G analgesia seems to last longer [6-8
hours compared with just 2-4 hours (47)] and it induces fewer side
effects, notably less vomiting and respiratory depression [reviewed
YANNICK GOUMON Y COLS.AN. R. ACAD. NAC. FARM.
396
in (47)]. The longer-lasting action of M6G coupled with its lower
incidence of adverse reactions make it a very promising analgesic. A
number of clinical studies are currently underway to compare the
analgesic activities of M6G and morphine in the management of
postoperative pain (48).
2. ENDOGENOUS MORPHINE
a) History
In 1903, a French scientist, Dr. Mavrojannis, observed that, when
he injected morphine into rats, they presented symptoms similar to
those of a cataleptic attack (49). He hypothesised that an endogenous
substance similar to morphine was present in the rat brain. However,
it was necessary to wait for the improved detection methods of the
1970’s —notably radioimmunoassay (RIA)— before a compound
related to morphine could be detected in the brains of mice, rabbits
and cats (50). This substance was not sensitive to proteases and had
the same pharmacological receptor-binding profile as morphine. It
was first called Morphine-Like Compound (MLC) (50) and was
isolated from human cerebrospinal fluid (CSF), urine and brain
extracts [prepared from patients who had never been given morphine
(51)]. Subsequently, large quantities of MLC were purified from
rabbit, rat and toad (Bufo marinus) epidermal tissue (52, 53). In
1985, Goldstein et al. purified a morphine-related compound from
an extract of bovine hypothalamus and adrenal glands (54). Using
chromatography and nuclear magnetic resonance (NMR), they
showed that the substance was indeed morphine, with a structure
identical to that of the plant molecule (54).
Subsequent experiments showed that this alkaloid could be found
in the neural and immune cells of invertebrates (55). Endogenous
alkaloids also seem to be produced by certain parasites, morphine
having been detected in both Schistosoma mansoni (56) and Ascaris
suum (57). Some scientists remained sceptical and believed that this
morphine could have come from food: morphine had been detected
in many plants (including hay and lettuce) as well as in cow’s and
human breast milk, at concentrations of between 200 and 500 ng/L
(58). Experiments in livestock reared in strictly morphine-free
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conditions, and in tissue cultures similarly depleted of any trace of
morphine, showed that de novo endogenous morphine synthesis is a
real phenomenon.
b) Synthesis of endogenous morphine
Definitive proof of the existence of endogenous morphine
required demonstration of the de novo synthesis of the compound in
mammalian cells. To do this, various intermediates in the synthesis
of morphine were sought (Figure 2). Morphine and codeine were
the first to be characterised (by mass spectrometry) in bovine
hypothalamus, and then in rat brain extracts (59). Moreover, the
injection of salutaridine, thebaine and codeine (three known
intermediates in the plant synthetic pathway) led to rises in
morphine levels in the rat brain (60). The conversion of reticulin to
salutaridine by a cytochrome P-450 (an essential step in formation
of the morphine skeleton) was also detected in rat liver (61) and
then in microsome extracts from porcine liver tissue. It is interesting
to note that higher levels of morphine and codeine can be detected
in the urine of patients with Parkinson’s disease on L-DOPA (62);
in the brain, L-DOPA is decarboxlated to generate dopamine, an
intermediate in the morphine synthetic pathway (Figure 2).
Moreover, it was shown in vivo that mice whose tyrosine hydroxylase
gene had been knocked out (tyrosine hydroxylase being necessary
for dopamine formation) did not produce any endogenous morphine
(63): morphine synthesis appears to depend on the presence of
dopamine.
Incontrovertible proof that mammals synthesise morphine was
provided by Zenk et al. in 2004 and 2005: when the cell line
SH-SY5Y (derived from a human neuroblastoma) was cultured
in the presence of 18O2, the group were able to isolate a series of
radiolabelled intermediates in the morphine synthetic pathway, as
well as radiolabelled morphine [Figure 2 (64-66)]. Experiments with
different radiolabelled precursors made it possible to define the
entire pathway.
Most steps in the mammalian morphine synthetic pathway are
the same as those in plants. The pathway begins with the
YANNICK GOUMON Y COLS.AN. R. ACAD. NAC. FARM.
398
condensation of dopamine and 4-hydroxyphenylacetaldehyde
(DOPAL), both of which are derivatives of tyrosine (64). This reaction
proceeds spontaneously in an aqueous environment without
catalysis. It is worth noting that the enzyme CYP2D6 is important
because it is known to be involved in the formation of dopamine
from tyramine, and the conversion of codeine to morphine (67). At
this time, experimental results suggest that this enzyme is present in
the liver, kidneys and immune cells (human white blood cells) (68).
It seems that, as in plants, mammals have two ways of synthesising
morphine from thebaine (Figure 2) although only a few of the
enzymes actually involved in this pathway have been characterised
and much information has been extrapolated from data about
enzymes discovered in plants.
It is also worth noting that dopamine can be synthesised from
tyramine through the action of DOPA decarboxylase/L-aromatic
amino acid decarboxylase so the possibility that cells deficient in
tyrosine hydroxylase (TH) could generate dopamine and therefore
morphine cannot be ruled out. This is all the more likely given
that TH-deficient neurones expressing DOPA decarboxylase (and
therefore capable of producing morphine) have been described in
different parts of the brain (69-73).
Recently, Bianchi et al. conducted experiments on conditional
tyrosine hydroxylase knockout mice [i.e. in which expression of the
gene is only abolished in central dopaminergic neurons (63)]. In the
brains of conditional TH-/- mice, morphine was below the detection
limit of the assay. However, the detection limit was relatively high and
it cannot be excluded that some morphine was present; unfortunately,
no immunohistochemical analysis was performed to complement
these findings. In practice, non-dopaminergic/catecholaminergic cells
such as those of the DAN-G line (pancreas) and HepG2 (hepatocytes)
(66) are capable of producing morphine de novo, and it cannot
be ruled out that morphine may also be synthesised by other cells
in the central nervous system in a pathway dependent on DOPA
decarboxylase or on an unknown enzyme rather than TH. In addition,
it can also not be excluded that some particular cells are able to uptake
morphine precursors (i.e. dopamine) to form morphine.
By means of immunohistochemical analysis, high-performance
liquid chromatography (HPLC) and mass spectrometry, it has been
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possible to characterise the presence of endogenous morphine
unambiguously in various mammalian tissues, both central and
peripheral.
Figure 2. Intermediates on the morphine synthetic pathway in mammals:
enzymes that may be involved. This synthetic pathway can take place in particu-
lar cells, but might also involve uptake of intermediates (e.g. dopamine) by other
cells that will finish the morphine synthesis.
YANNICK GOUMON Y COLS.AN. R. ACAD. NAC. FARM.
400
c) Endogenous morphine: localisation in the central nervous
c) system and physiological functions
Maps of the brains of the dog and rat generated using various
methods (HPLC and RIA) have shown the presence of morphine
and/or its derivatives in neurones and nerve fibres (74, 75). In 1999,
Meijerink et al. detected and quantified morphine in the thalamus,
cortex, hypothalamus and cerebellum [Figure 3 (76)]. However, these
experiments were conducted after 24 hours of fasting and fasting
has been reported to increase the concentration of endogenous
morphine in the brain (77). More recently, our group (78) was able
to complete this work, characterizing morphine amounts present in
the normal mouse brain (Figure 3). Moreover, morphine has also
been detected in human cerebrospinal fluid (79). At the intracellular
level, morphine has been detected in the cell bodies, axons and
terminals of neurones in the putamen, hippocampus, hypothalamus,
brain stem, cerebellum and spinal cord (74). Bianchi et al. have also
shown that these neurones can accumulate tritiated morphine after
intra-cerebroventricular infusion (74, 80), suggesting that these
neurones have a system to uptake morphine.
Figure 3. Cerebral distribution of endogenous morphine. Amounts of morphi-
ne present in the mouse (78), and in the fasting rat and dog (76).
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Our recent studies have focused on the functional roles
of endogenous morphine in the central nervous system (78).
In experiments on the human neurone line SH-SY5Y [which is
used to study neuronal secretion (81)], immunohistochemical
analysis showed morphine/M6G/M3G (Figure 4) colocalizing with
chromogranin A (CGA, a granular marker) in vesicle-like organelles
with a dense core observed in certain neurons.
Figure 4. Confocal laser micrograph showing SH-SY5Y cells immunostained for
morphine. Colocalisation highlighted in yelow.
Morphine and M6G were purified from SH-SY5Y cells and
analysed by mass spectrometry. The identification of M6G pointed to
the presence of UGT2B7 which is the main enzyme known to be able
to convert morphine into this metabolite, and experiments based on
RT-PCR and Western blotting showed, for the first time, that UGT2B7
was being expressed in these cells. Our experiments showed that
morphine secretion by SH-SY5Y cells following nicotine-induced
depolarisation was Ca2+-dependent. Patch-clamping experiments
showed that morphine and M6G could both induce naloxone-
dependent electrophysiological responses at low concentrations (10-10-
10-9M).
In order to extend the observations made in the in vitro model,
we looked for endogenous morphine in various parts of the brain of
mice [which are reported not to produce M6G (82)] using microscopy
YANNICK GOUMON Y COLS.AN. R. ACAD. NAC. FARM.
402
and a morphine-specific ELISA. This showed the presence of
endogenous morphine in various places, including the hippocampus
and the cerebellum (Figure 3 and Figure 5).
Figure 5. Localization of endogenous morphine present in the mouse brain.
A) Immunodetection of endogenous morphine in the murine brain. The immu-
nostaining was done with an antibody that recognises morphine and its glucuro-
nidated derivatives. B) Localization of morphine label in the cerebellum using
mouse monoclonal anti-morphine antibody and an HRP-conjugated secondary
antibody. Morphine labelling was observed around Purkinje cells. ML, molecu-
lar layer; GL, granular layer. C) Electron micrograph (anti-morphine antibody) of
murine cerebellum showing nerve terminals on the cell body of a Purkinje cell. The
arrows indicate synaptic contacts with morphine-containing, presynaptic vesicles.
PC, Purkinje cell; N, nucleus.
Focussing on the cerebellum, we showed a morphine immuno-
histochemical signal in basket cells (78) and their terminals synaps-
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ing with the cell bodies of Purkinje cells (Figures 5B and C) which
are reported as expressing μ receptors (83, 84).
Morphine in the nervous system
Various studies have pointed to the involvement of endogenous
morphine in analgesia, memory, plasticity and development, as well
as in the installation of addiction. A number of studies have described
the effects of exogenous morphine in the CNS although, without an
understanding of effective concentrations inside the synaptic cleft, it
is difficult to speculate about the functions of endogenous morphine
by extrapolating from results obtained with exogenous morphine. It
is also likely that the level of endogenous morphine being produced
and secreted varies with physiological conditions (notably stress). It
is important to note that the concentrations used in most experiments
(therapeutic doses) are often greater than 1 μM. Nevertheless, on the
basis of the observed effects of exogenous morphine coupled with
an understanding of the distribution of endogenous morphine and
its receptors in the CNS, hypotheses can be formulated about the
physiological roles of endogenous morphine.
Endogenous morphine and nociception
An experiment conducted in the year 2000 showed that
endogenous morphine colocalises with μ receptors in various parts
of the brain stem. These include the locus coeruleus, the parabrachial
nucleus, the periaqueductal grey substance and the nucleus raphe,
all structures known to be involved in supraspinal nociception
modulation (1). On the other hand, experiments carried out by
Guarna et al. showed that the injection of antibodies directed against
morphine into murine CSF (a procedure which lowers the level
of endogenous morphine in the brain) induced hypersensitivity to
heat-associated pain (85). Moreover, knockout mice which do not
express μ receptors are abnormally sensitive to thermal stimuli but
not to mechanical stimuli (86). These findings seem to suggest that
endogenous morphine mainly modulates sensitivity to thermal pain.
YANNICK GOUMON Y COLS.AN. R. ACAD. NAC. FARM.
404
It should also be noted that morphine seems to have paradoxical
effects according to its concentration. One study showed that the
subcutaneous injection of a small quantity of morphine (1-10 μg/kg)
induced hyperalgesia whereas high doses (1000-7000 μg/kg) induced
analgesia (87). These results are consistent with those of Robuvitch et
al. who showed that DAMGO, a μ receptor agonist, stimulated cAMP
production in the SK-N-SH cell line (derived from a neuroblastoma)
at a concentration of 10 nM, but inhibited it at 0.1 μM (88). One of
this group’s hypotheses is that some nociceptive neurones could be
expressing a low level of G protein-coupled μ receptors with a high
affinity for morphine, and these could be mediating the hyperalgesia
in the presence of low morphine concentrations; these same neurones
would also be expressing classic μ receptors coupled with Gi/o
proteins, to mediate the analgesia observed at the higher morphine
concentration (89).
Endogenous morphine and memory
An experiment in which antibodies against morphine were injected
into murine CSF pointed to a link between endogenous morphine and
memorisation (90): after twelve hours of fasting, control mice showed
deficient memorisation whereas memorisation processes were
unaffected in the mice in which the level of endogenous morphine had
been artificially depressed (i.e. neutralized by the antibodies).
Endogenous morphine could therefore inhibit memorisation at times
of stress (e.g. during starvation).
Exogenous morphine has been observed to have various effects
on hippocampal function, the hippocampus being known to be a
key structure in memorisation. Morphine is known to be able to
modulate neurotransmission in the hippocampus by inhibiting its
GABAergic interneurones. Such inhibition would result in an
increase in the discharge amplitude of pyramidal neurones in the
CA1 zone (91) and modify the efficacy of glutamatergic synapses by
acting on the expression of proteins important for the post-synaptic
density [i.e. receptors (92)]. Morphine would therefore act to
consolidate memories by promoting long-term potentiation (LTP).
However, other experiments have shown that prenatal exposure to
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morphine impairs memorisation processes and reduces LTP at
hippocampal synapses (93). Moreover, another experiment in rats
showed that one-off exposure to high-dose morphine (10 mg/kg
administered intraperitoneally) impaired memorisation in a cross-
maze model. The effects of exogenous morphine on memorisation
are therefore as yet poorly understood and seem to depend on
both dosage and stage of development. In the light of all these
observations and because both endogenous morphine and μ receptors
are known to be present in the hippocampus, it could be that
endogenous morphine plays a role in controlling memorisation
functions.
Morphine, neurogenesis and the growth of nerve cells
In the brain of adult mammals, there are two sites of ongoing
neurogenesis, namely the sub-ventricular zone (SVZ) along the edge
of the lateral ventricle, and the sub-granular zone (SGZ) of the
hippocampal dentate gyrus. Many contradictory results have been
published on the effects of morphine on the multiplication of neuronal
progenitors in the SGZ, reflecting the complexity of the underlying
mechanisms [reviewed in (94)]. In rats and mice on long-term
morphine (10 mg/kg administered intraperitoneally), neurogenesis has
been observed to be decreased by a factor of over 30% in the SGZ (95,
96), whereas other experiments have shown that morphine stimulated
multiplication (97, 98). Moreover, in μ receptor knock-out mice, an
increase of over 50% was documented in the rate of survival of newly
formed neurones in the granular zone of the dentate gyrus four weeks
after the injection of bromodeoxyuridine [BrdU, a marker for
neurogenesis (99)]. Endogenous morphine in the hippocampus (78)
could therefore regulate the production and survival of neuronal
progenitors, thereby affecting synaptic plasticity in the hippocampus.
These data are supported by the results of clinical studies which point
to hippocampal plasticity problems in heroin addicts (100).
With respect to cell growth, in vitro studies have shown that
morphine has dose-dependent effects on the growth of neural
processes (axons and dendrites) in cell lines and primary neurones
in tissue culture: at high concentrations (10 mM-10 μM), morphine
YANNICK GOUMON Y COLS.AN. R. ACAD. NAC. FARM.
406
inhibited the growth of neuronal processes in primary cultures of
hippocampal and cerebellar granule neurones in a naloxone-
dependent fashion (101), whereas morphine concentrations of
between 1 nM and 10 fM enhanced process growth in spinal cord
and cortical neurones by a factor of over 20% (101), and a low
concentration of morphine (10 pM) stimulated process elongation
in PC-12 cells (102). These effects were not reversed by naloxone
suggesting that they are mediated by special, high-affinity receptors.
In addition, a recent study showed that low doses of morphine
had beneficial effects on synaptic regeneration and reconstruction at
the nerve terminals of non-myelinated afferent fibres of the second
lamina of the spinal cord following damage to the sciatic nerve (103).
Even more recent experiments described that low morphine
concentrations (1-100 nM, 2 hours) significantly stimulated migration
in rat microglial cells exposed to ATP (acting as a chemotactic agent)
(104). Significant effects were observed from the concentration of 1
nM with peak activity observed at 100 nM. CTAP, a specific antagonist
of µ receptors, inhibited this migration. These results suggest that
endogenous morphine in the brain could modulate immune responses
in the CNS.
Endogenous morphine and addiction
Experiments in two invertebrate models, Mytulis edulis and
Homarus americanus, showed that exposure to addictive substances
(ethanol, nicotine and cocaine) stimulates a two-fold increase in
the release of endogenous morphine by the nervous system (105). A
number of groups have postulated the existence of a link between
endogenous morphine and addiction (106) although no experiments
have yet been conducted in animals.
d) Localisation and physiological roles of endogenous
d) morphine in the periphery
In the periphery, morphine has been detected in the liver and in
a hepatocyte cell line (61, 66), and in the adrenal glands of a number
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of mammalian species. The adrenal gland is one of the major organs
involved in responses to stress and is composed of a cortical part
(where glucocorticoids are produced) and a medullary part [mainly
composed of chromaffin cells (107-109)]. Previously, morphine had
also been found in PC-12 cells which are transformed rat chromaffin
cells (66, 110), and in eel chromaffin cells (111). Chromaffin cells are
neuroendocrine cells derived from the neural crest which are full
of secretory granules containing catecholamines together with many
different proteins and peptides, including chromogranins (107). In
a stressful situation, chromaffin cell stimulation by the splanchnic
nerve induces membrane depolarisation and degranulation which
leads to emptying of catecholamines and the other granule contents
into the blood stream. It is worth noting that the catecholamines
(dopamine, adrenaline and noradrenaline) are all derived from
dopamine and tyrosine precursors, as is morphine.
In experiments carried out in 2006 in a bovine chromaffin cell
model, we detected M6G inside these cells’ secretory granules (Figure
6). M6G secretion was also observed when nicotine was used to
induce depolarisation in these cells in primary tissue culture.
Because these chromaffin cells degranulate in response to stress, it
is likely that M6G is secreted along with the catecholamines in such
responses. Once in the circulation, the M6G could bind μ receptors
on diverse cell types (immune cells, endothelial cells, etc.) and trigger
physiological responses. We also showed that the M6G in chromaffin
granules is stored as a complex with phosphatidylethanolamine-
Figure 6. Confocal laser micrograph showing primary chromaffin cells in
tissue cells labelled with antibodies against morphine and CGA (a granular
marker). Colocalisation highlighted in yellow.
YANNICK GOUMON Y COLS.AN. R. ACAD. NAC. FARM.
408
binding protein (PEBP) which would prevent it being cleared via the
kidneys. Our findings also showed that, in the chromaffin cell model,
M6G is the final product of the alkaloid synthesis pathway, and
could represent a neuroendocrine effector.
A number of studies conducted on invertebrate immune cells
have shown that morphine synthesis is stepped up at times of stress
(55). More recently, a number of articles have reported that immune
cells [notably polymorphonuclear cells and monocytes (68, 112)]
synthesise morphine, pointing to a role in immune responses.
The intensive use of morphine in hospitals has spurred many
studies focusing on the presence of morphine in the serum or plasma
after the i.v. administration of exogenous morphine. In contrast to
this body of work on exogenous morphine, there is little information
about endogenous morphine in the blood although some findings
suggest that endogenous morphine may play a role in stress and
immune responses. Since the beginning of the 1990’s, a number of
studies on invertebrate immune cells had demonstrated morphine
synthesis in response to stress (55). Three other studies conducted
on pigs and patients who had undergone invasive surgery (e.g.
coronary bypass surgery or laparotomy) showed elevated levels of
endogenous morphine in the blood. Tonnesen et al. showed that
coronary bypass surgery induced a rise in the amount of morphine
in human serum from 0.28 nM to 3.9 nM (113-115) and, in another
experiment, the same group measured a morphine concentration of
about 10 nM in the blood of piglets who had undergone coronary
bypass surgery while none was detectable in control animals (113).
Finally, a human study suggested that the concentration of morphine
in the blood was higher after open cholecystectomy than it was after
laparascopy (0.2 nM versus 0.018 nM) (115). Surgical procedures
such as bypass surgery or thoracotomy elicit massive inflammation,
e.g. the extracorporeal circulation associated with coronary bypass
surgery always causes inflammation. Taken together, these
observations point to a link between rises in blood morphine and
inflammation. It is interesting to note that estimated IC50’s for µ
receptors [of the order of 10 nM (116)] are consistent with the levels
observed in the circulation, suggesting that endogenous morphine
may have effects on different cell types, including immune cells and
endothelial cells.
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The injection of lipopolysaccharide (LPS, an integral component
of the cell walls of Gram-negative bacteria) into rats is used to study
sepsis. In this system, LPS is known to induce an increase in the
concentration of endogenous morphine in the adrenal glands (76) and
brain (77). Other types of stress (like starvation) also induce rises in
morphine levels in the spinal cord (76).
Morphine is known to be able to have immunosuppressant
activity via various distinct mechanisms (117):
(i) direct action on monocytes, macrophages and granulocytes:
by inducing nitrogen oxide [NO (22)] and thereby inhibiting
the production of pro-inflammatory cytokines as well as
chemotaxis and phagocytosis (116). On the other hand, the
binding of morphine to µ receptors on endothelial cells
also induces NO production and inhibits the expression of
certain adhesion molecules involved in the recruitment of
immune cells to sites of infection and inflammation;
(ii) activation of the hypothalamic-pituitary-adrenal axis (HPA)
and release of immunosuppressive glucocorticoids (119);
(iii) inhibition of the differentiation of stem cells into lympho-
cytes, and inhibition of T lymphocyte multiplication (120);
(iv) inhibition of the cytolytic activity of NK cells (119-121):
this has been observed after the direct injection of morphine
into the cerebrospinal fluid, suggesting that the effect is
mediated in the central nervous system.
The release of morphine in the blood could represent a physiolo-
gical mechanism designed to mitigate over-exuberant inflammatory
reactions. Morphine is also known to induce analgesia so it could, if
present at high enough levels in the blood (113, 114) or brain (77)
at times of stress, modulate the activity of peripheral nociceptors.
3. CONCLUSION
In summary, numerous studies have established a role for
morphine as an endogenous signalling molecule. Thus, endogenous
morphine appears to be both a neurotransmitter and endocrine
YANNICK GOUMON Y COLS.AN. R. ACAD. NAC. FARM.
410
mediator playing a role in physiological processes. Investigation
on endogenous morphine will continue to be an important axis of
research and efforts will be focus on the characterization of its
implication in physiology.
4. ACKNOWLEDGEMENT
This work was funded by Inserm, CNRS, the University Louis-
Pasteur at Strasbourg, the Fondation de France (to Y.G), the French
Ministère délégué à la Recherche et à l’Enseignement Supérieur
(Ph.D fellowship to A.M. and A.L.), the Fondation Transplantation
(to Y.G.) and the Fondation pour la Recherche Médicale (to Y.G.).
5. BIBLIOGRAPHY
1. Stefano, G. B., Goumon, Y., Casares, F., Cadet, P., Fricchione, G. L., Rialas,
C., et al. (2000) Endogenous morphine. Trends Neurosci. 23(9): 436-442.
2. Lotsch, J. (2005) Pharmacokinetic-pharmacodynamic modeling of opioids. J.
Pain Symptom. Manage. 29(5 Suppl): S90-103.
3. Lotsch, J. & Geisslinger, G. (2001) Morphine-6-glucuronide: an analgesic of
the future? Clin. Pharmacokinet. 40(7): 485-499.
4. Pert, C. B., Pasternak, G. & Snyder, S. H. (1973) Opiate agonists and
antagonists discriminated by receptor binding in brain. Science. 182(119):
1359-1361.
5. Trescot, A. M., Datta, S., Lee, M. & Hansen, H. (2008) Opioid pharmacology.
Pain Physician. 11(2 Suppl): S133-153.
6. Zadina, J. E., Hackler, L., Ge, L. J. & Kastin, A. J. (1997) A potent and
selective endogenous agonist for the mu-opiate receptor. Nature. 386(6624):
499-502.
7. Zhang, Y., Chen, Q. & Yu, L. C. (2008) Morphine: a protective or destructive
role in neurons? Neuroscientist. 14(6): 561-570.
8. Ding, Y. Q., Kaneko, T., Nomura, S. & Mizuno, N. (1996) Immunohisto-
chemical localization of mu-opioid receptors in the central nervous system
of the rat. J. Comp. Neurol. 367(3): 375-402.
9. Mansour, A., Fox, C. A., Burke, S., Akil, H. & Watson, S. J. (1995)
Immunohistochemical localization of the cloned mu opioid receptor in the
rat CNS. J. Chem. Neuroanat. 8(4): 283-305.
10. Bianchi, C., Sellke, F. W., Del Vecchio, R. L., Tonks, N. K. & Neel, B. G.
(1999) Receptor-type protein-tyrosine phosphatase mu is expressed in specific
vascular endothelial beds in vivo. Exp. Cell Res. 248(1): 329-338.
VOL. 75 (3), 389-418, 2009 CENTRAL AND PERIPHERAL ENDOGENOUS MORPHINE
411
11. Vidal, E. L., Patel, N. A., Wu, G., Fiala, M. & Chang, S. L. (1998) Interleukin-
1 induces the expression of mu opioid receptors in endothelial cells.
Immunopharmacology. 38(3): 261-266.
12. McCarthy, L., Wetzel, M., Sliker, J. K., Eisenstein, T. K. & Rogers, T. J.
(2001) Opioids, opioid receptors, and the immune response. Drug Alcohol
Depend. 62(2): 111-123.
13. Doyle, G. A., Sheng, X. R., Lin, S. S., Press, D. M., Grice, D. E., Buono, R.
J., et al. (2007) Identification of five mouse mu-opioid receptor (MOR) gene
(Oprm1) splice variants containing a newly identified alternatively spliced
exon. Gene. 395(1-2): 98-107.
14. Xu, J., Xu, M., Hurd, Y. L., Pasternak, G. W. & Pan, Y. X. (2009) Isolation
and characterization of new exon 11-associated N-terminal splice variants of
the human mu opioid receptor gene. J. Neurochem. 108(4): 962-972.
15. Pan, Y. X. (2005) Diversity and complexity of the mu opioid receptor gene:
alternative pre-mRNA splicing and promoters. DNA Cell Biol. 24(11): 736-
750.
16. Nevo, I., Avidor-Reiss, T., Levy, R., Bayewitch, M. & Vogel, Z. (2000) Acute
and chronic activation of the mu-opioid receptor with the endogenous ligand
endomorphin differentially regulates adenylyl cyclase isozymes. Neurophar-
macology. 39(3): 364-371.
17. Williams, J. T., Christie, M. J. & Manzoni, O. (2001) Cellular and synaptic
adaptations mediating opioid dependence. Physiol. Rev. 81(1): 299-343.
18. Cohen, G. A., Doze, V. A. & Madison, D. V. (1992) Opioid inhibition of GABA
release from presynaptic terminals of rat hippocampal interneurons. Neuron.
9(2): 325-335.
19. Jiang, Z. G. & North, R. A. (1991) Membrane properties and synaptic
responses of rat striatal neurones in vitro. J. Physiol. 443: 533-553.
20. Connor, M. & Henderson, G. (1996) delta- and mu-opioid receptor
mobilization of intracellular calcium in SH-SY5Y human neuroblastoma
cells. Br. J. Pharmacol. 117(2): 333-340.
21. Tang, Y., Chen, K. X., Jiang, H. L., Wang, Z. X., Ji, R. Y. & Chi, Z. Q.
(1996) Molecular modeling of mu opioid receptor and its interaction with
ohmefentanyl. Zhongguo Yao Li Xue Bao. 17(2): 156-160.
22. Pasternak, G. W. (2007) When it comes to opiates, just say NO. J. Clin.
Invest. 117(11): 3185-3187.
23. Stefano, G. B., Goumon, Y., Bilfinger, T. V., Welters, I. D. & Cadet, P.
(2000) Basal nitric oxide limits immune, nervous and cardiovascular
excitation: human endothelia express a mu opiate receptor. Prog. Neurobiol.
60(6): 513-530.
24. Chuang, L. F., Killam, K. F., Jr. & Chuang, R. Y. (1997) Induction and
activation of mitogen-activated protein kinases of human lymphocytes as
one of the signaling pathways of the immunomodulatory effects of morphine
sulfate. J. Biol. Chem. 272(43): 26815-26817.
25. Ikeda, K., Kobayashi, T., Kumanishi, T., Niki, H. & Yano, R. (2000)
Involvement of G-protein-activated inwardly rectifying K (GIRK) channels in
opioid-induced analgesia. Neurosci. Res. 38(1): 113-116.
YANNICK GOUMON Y COLS.AN. R. ACAD. NAC. FARM.
412
26. Marker, C. L., Stoffel, M. & Wickman, K. (2004) Spinal G-protein-gated
K+ channels formed by GIRK1 and GIRK2 subunits modulate thermal
nociception and contribute to morphine analgesia. J. Neurosci. 24(11): 2806-
2812.
27. Vaughan, C. W., Bagley, E. E., Drew, G. M., Schuller, A., Pintar, J. E., Hack,
S. P., et al. (2003) Cellular actions of opioids on periaqueductal grey neurons
from C57B16/J mice and mutant mice lacking MOR-1. Br. J. Pharmacol.
139(2): 362-367.
28. Marker, C. L., Lujan, R., Loh, H. H. & Wickman, K. (2005) Spinal G-protein-
gated potassium channels contribute in a dose-dependent manner to the
analgesic effect of mu- and delta- but not kappa-opioids. J. Neurosci. 25(14):
3551-3559.
29. Nakatsuka, T., Fujita, T., Inoue, K. & Kumamoto, E. (2008) Activation of
GIRK channels in substantia gelatinosa neurones of the adult rat spinal cord:
a possible involvement of somatostatin. J. Physiol. 586(10): 2511-2522.
30. Cruz, H. G., Berton, F., Sollini, M., Blanchet, C., Pravetoni, M., Wickman,
K., et al. (2008) Absence and rescue of morphine withdrawal in GIRK/Kir3
knock-out mice. J. Neurosci. 28(15): 4069-4077.
31. Torrecilla, M., Quillinan, N., Williams, J. T. & Wickman, K. (2008) Pre- and
postsynaptic regulation of locus coeruleus neurons after chronic morphine
treatment: a study of GIRK-knockout mice. Eur. J. Neurosci. 28(3): 618-624.
32. Coller, J. K., Christrup, L. L. & Somogyi, A. A. (2008) Role of active
metabolites in the use of opioids. Eur. J. Clin. Pharmacol.
33. Ohno, S. & Nakajin, S. (2009) Determination of mRNA expression of human
UDP-glucuronosyltransferases and application for localization in various
human tissues by real-time reverse transcriptase-polymerase chain reaction.
Drug Metab. Dispos. 37(1): 32-40.
34. Radominska-Pandya, A., Little, J. M. & Czernik, P. J. (2001) Human UDP-
glucuronosyltransferase 2B7. Curr. Drug Metab. 2(3): 283-298.
35. Yamada, H., Ishii, K., Ishii, Y., Ieiri, I., Nishio, S., Morioka, T., et al. (2003)
Formation of highly analgesic morphine-6-glucuronide following physiologic
concentration of morphine in human brain. J. Toxicol. Sci. 28(5): 395-401.
36. Mackenzie, P. I., Owens, I. S., Burchell, B., Bock, K. W., Bairoch, A.,
Belanger, A., et al. (1997) The UDP glycosyltransferase gene superfamily:
recommended nomenclature update based on evolutionary divergence.
Pharmacogenetics. 7(4): 255-269.
37. Mackenzie, P. I., Walter Bock, K., Burchell, B., Guillemette, C., Ikushiro,
S., Iyanagi, T., et al. (2005) Nomenclature update for the mammalian UDP
glycosyltransferase (UGT) gene superfamily. Pharmacogenet. Genomics. 15
(10): 677-685.
38. Ohno, S., Kawana, K. & Nakajin, S. (2008) Contribution of UDP-glucuro-
nosyltransferase 1A1 and 1A8 to morphine-6-glucuronidation and its kinetic
properties. Drug Metab. Dispos. 36(4): 688-694.
39. King, C. D., Rios, G. R., Assouline, J. A. & Tephly, T. R. (1999) Expression
of UDP-glucuronosyltransferases (UGTs) 2B7 and 1A6 in the human brain
VOL. 75 (3), 389-418, 2009 CENTRAL AND PERIPHERAL ENDOGENOUS MORPHINE
413
and identification of 5-hydroxytryptamine as a substrate. Arch. Biochem.
Biophys. 365(1): 156-162.
40. Suleman, F. G., Abid, A., Gradinaru, D., Daval, J. L., Magdalou, J. & Minn,
A. (1998) Identification of the uridine diphosphate glucuronosyltransferase
isoform UGT1A6 in rat brain and in primary cultures of neurons and astro-
cytes. Arch. Biochem. Biophys. 358(1): 63-67.
41. Brands, A., Munzel, P. A. & Bock, K. W. (2000) In situ hybridization studies
of UDP-glucuronosyltransferase UGT1A6 expression in rat testis and brain.
Biochem. Pharmacol. 59(11): 1441-1444.
42. Lotsch, J. (2005) Opioid metabolites. J. Pain Symptom. Manage. 29(5 Suppl):
S10-24.
43. Yeh, S. Y., Gorodetzky, C. W. & Krebs, H. A. (1977) Isolation and identi-
fication of morphine 3- and 6-glucuronides, morphine 3,6-diglucuronide,
morphine 3-ethereal sulfate, normorphine, and normorphine 6-glucuronide
as morphine metabolites in humans. J. Pharm. Sci. 66(9): 1288-1293.
44. Zheng, M., McErlane, K. M. & Ong, M. C. (1998) High-performance liquid
chromatography-mass spectrometry-mass spectrometry analysis of morphine
and morphine metabolites and its application to a pharmacokinetic study in
male Sprague-Dawley rats. J. Pharm. Biomed. Anal. 16(6): 971-980.
45. Wu, D., Kang, Y. S., Bickel, U. & Pardridge, W. M. (1997) Blood-brain barrier
permeability to morphine-6-glucuronide is markedly reduced compared with
morphine. Drug Metab Dispos. 25(6): 768-771.
46. Grace, D. & Fee, J. P. (1996) A comparison of intrathecal morphine-6-
glucuronide and intrathecal morphine sulfate as analgesics for total hip
replacement. Anesth. Analg. 83(5): 1055-1059.
47. Dahan, A., van Dorp, E., Smith, T. & Yassen, A. (2008) Morphine-6-
glucuronide (M6G) for postoperative pain relief. Eur. J. Pain. 12(4): 403-411.
48. van Dorp, E. L., Romberg, R., Sarton, E., Bovill, J. G. & Dahan, A. (2006).
Morphine-6-Glucuronide: Morphine’s Successor for Postoperative Pain
Relief? Anesth. Analg. 102(6): 1789-1797.
49. Mavrojannis, M. (1903) Action Cataleptique de la Morphine chez les Rats.
Contribution a la Theorie Toxique da la Catalepsie. Comptes rendues Soc.
Biol. 55: 1092-1094.
50. Gintzler, A. R., Mohacsi, E. & Spector, S. (1976) Radioimmunoassay for the
simultaneous determination of morphine and codeine. Eur. J. Pharmacol.
38(1): 149-156.
51. Shorr, J., Foley, K. & Spector, S. (1978) Presence of a non-peptide morphine-
like compound in human cerebrospinal fluid. Life Sci. 23(20): 2057-2062.
52. Oka, K., Kantrowitz, J. D. & Spector, S. (1985) Isolation of morphine from
toad skin. Proc. Natl. Acad. Sci. USA. 82(6): 1852-1854.
53. Spector, S., Kantrowitz, J. D. & Oka, K. (1985) Presence of endogenous
morphine in toad skin. Prog. Clin. Biol. Res. 192: 329-332.
54. Goldstein, A., Barrett, R. W., James, I. F., Lowney, L. I., Weitz, C. J.,
Knipmeyer, L. L., et al. (1985) Morphine and other opiates from beef brain
and adrenal. Proc. Natl. Acad. Sci. USA. 82(15): 5203-5207.
YANNICK GOUMON Y COLS.AN. R. ACAD. NAC. FARM.
414
55. Stefano, G. B., Digenis, A., Spector, S., Leung, M. K., Bilfinger, T. V.,
Makman, M. H., et al. (1993) Opiate-like substances in an invertebrate, an
opiate receptor on invertebrate and human immunocytes, and a role in
immunosuppression. Proc. Natl. Acad. Sci. USA. 90(23): 11099-11103.
56. Leung, M. K., Dissous, C., Capron, A., Woldegaber, H., Duvaux-Miret, O.,
Pryor, S., et al. (1995) Schistosoma mansoni: the presence and potential use
of opiate-like substances. Exp. Parasitol. 81(2): 208-215.
57. Goumon, Y., Casares, F., Pryor, S., Ferguson, L., Brownawell, B., Cadet,
P., et al. (2000) Ascaris suum, an intestinal parasite, produces morphine.
J. Immunol. 165(1): 339-343.
58. Hazum, E., Sabatka, J. J., Chang, K. J., Brent, D. A., Findlay, J. W. &
Cuatrecasas, P. (1981) Morphine in cow and human milk: could dietary
morphine constitute a ligand for specific morphine (mu) receptors? Science.
213(4511): 1010-1012.
59. Weitz, C. J., Lowney, L. I., Faull, K. F., Feistner, G. & Goldstein, A. (1986)
Morphine and codeine from mammalian brain. Proc. Natl. Acad. Sci. USA.
83(24): 9784-9788.
60. Donnerer, J., Oka, K., Brossi, A., Rice, K. C. & Spector, S. (1986) Presence
and formation of codeine and morphine in the rat. Proc. Natl. Acad. Sci. USA.
83(12): 4566-4567.
61. Weitz, C. J., Faull, K. F. & Goldstein, A. (1987) Synthesis of the skeleton of
the morphine molecule by mammalian liver. Nature. 330(6149): 674-677.
62. Matsubara, K., Fukushima, S., Akane, A., Kobayashi, S. & Shiono, H. (1992)
Increased urinary morphine, codeine and tetrahydropapaveroline in
parkinsonian patient undergoing L-3,4-dihydroxyphenylalanine therapy: a
possible biosynthetic pathway of morphine from L-3,4-dihydroxyphenyla-
lanine in humans. J. Pharmacol. Exp. Ther. 260(3): 974-978.
63. Neri, C., Ghelardini, C., Sotak, B., Palmiter, R. D., Guarna, M., Stefano, G.,
et al. (2008) Dopamine is necessary to endogenous morphine formation in
mammalian brain in vivo. J. Neurochem. 106(6): 2337-2344.
64. Boettcher, C., Fellermeier, M., Boettcher, C., Drager, B. & Zenk, M. H. (2005).
How human neuroblastoma cells make morphine. Proc. Natl. Acad. Sci. USA.
102(24): 8495-8500.
65. Poeaknapo, C. (2005) Mammalian morphine: de novo formation of morphine
in human cells. Med. Sci. Monit. 11(5): MS6-17.
66. Poeaknapo, C., Schmidt, J., Brandsch, M., Drager, B. & Zenk, M. H. (2004)
Endogenous formation of morphine in human cells. Proc. Natl. Acad. Sci.
USA. 101(39): 14091-14096.
67. Zhu, W., Mantione, K. J., Shen, L., Cadet, P., Esch, T., Goumon, Y., et al.
(2005) Tyrosine and tyramine increase endogenous ganglionic morphine
and dopamine levels in vitro and in vivo: cyp2d6 and tyrosine hydroxylase
modulation demonstrates a dopamine coupling. Med. Sci. Monit. 11(11):
BR397-404.
68. Zhu, W., Cadet, P., Baggerman, G., Mantione, K. J. & Stefano, G. B. (2005)
Human white blood cells synthesize morphine: CYP2D6 modulation. J.
Immunol. 175(11): 7357-7362.
VOL. 75 (3), 389-418, 2009 CENTRAL AND PERIPHERAL ENDOGENOUS MORPHINE
415
69. Baker, H., Abate, C., Szabo, A. & Joh, T. H. (1991) Species-specific
distribution of aromatic L-amino acid decarboxylase in the rodent adrenal
gland, cerebellum, and olfactory bulb. J. Comp. Neurol. 305(1): 119-129.
70. Ikemoto, K., Kitahama, K., Jouvet, A., Arai, R., Nishimura, A., Nishi, K.,
et al. (1997) Demonstration of L-dopa decarboxylating neurons specific to
human striatum. Neurosci. Lett. 232(2): 111-114.
71. Ikemoto, K., Kitahama, K., Nishimura, A., Jouvet, A., Nishi, K., Arai, R., et
al. (1999) Tyrosine hydroxylase and aromatic L-amino acid decarboxylase do
not coexist in neurons in the human anterior cingulate cortex. Neurosci. Lett.
269(1): 37-40.
72. Kitahama, K., Geffard, M., Araneda, S., Arai, R., Ogawa, K., Nagatsu, I.,
et al. (2007) Localization of L-DOPA uptake and decarboxylating neuronal
structures in the cat brain using dopamine immunohistochemistry. Brain
Res. 1167: 56-70.
73. Ugrumov, M. V., Melnikova, V. I., Lavrentyeva, A. V., Kudrin, V. S. &
Rayevsky, K. S. (2004) Dopamine synthesis by non-dopaminergic neurons
expressing individual complementary enzymes of the dopamine synthetic
pathway in the arcuate nucleus of fetal rats. Neuroscience. 124(3): 629-635.
74. Bianchi, E., Alessandrini, C., Guarna, M. & Tagliamonte, A. (1993)
Endogenous codeine and morphine are stored in specific brain neurons.
Brain Res. 627(2): 210-215.
75. Bianchi, E., Guarna, M. & Tagliamonte, A. (1994) Immunocytochemical
localization of endogenous codeine and morphine. Adv. Neuroimmunol. 4(2):
83-92.
76. Meijerink, W. J., Molina, P. E. & Abumrad, N. N. (1999) Mammalian opiate
alkaloid synthesis: lessons derived from plant biochemistry. Shock. 12(3):
165-173.
77. Goumon, Y., Bouret, S., Casares, F., Zhu, W., Beauvillain, J. C. & Stefano, G.
B. (2000) Lipopolysaccharide increases endogenous morphine levels in rat
brain. Neurosci. Lett. 293(2): 135-138.
78. Muller, A., Glattard, E., Taleb, O., Kemmel, V., Laux, A., Miehe, M., et al.
(2008) Endogenous Morphine in SH-SY5Y Cells and the Mouse Cerebellum.
PLoS One. 3(2): Epub www.plosone.org/doi/pone.0001641.
79. Cardinale, G. J., Donnerer, J., Finck, A. D., Kantrowitz, J. D., Oka, K. &
Spector, S. (1987) Morphine and codeine are endogenous components of
human cerebrospinal fluid. Life Sci. 40(3): 301-306.
80. Guarna, M., Neri, C., Petrioli, F. & Bianchi, E. (1998) Potassium-induced
release of endogenous morphine from rat brain slices. J. Neurochem. 70(1):
147-152.
81. Agis-Torres, A., Ball, S. G. & Vaughan, P. F. (2002) Chronic treatment with
nicotine or potassium attenuates depolarisation-evoked noradrenaline release
from the human neuroblastoma SH-SY5Y. Neurosci. Lett. 331(3): 167-170.
82. van Dorp, E. L., Morariu, A. & Dahan, A. (2008) Morphine-6-glucuronide:
potency and safety compared with morphine. Expert Opin. Pharmacother.
9(11): 1955-1961.
YANNICK GOUMON Y COLS.AN. R. ACAD. NAC. FARM.
416
83. Mrkusich, E. M., Kivell, B. M., Miller, J. H. & Day, D. J. (2004) Abundant
expression of mu and delta opioid receptor mRNA and protein in the
cerebellum of the fetal, neonatal, and adult rat. Brain Res. Dev. Brain Res.
148(2): 213-222.
84. Zhang, Y., Pan, Y. X., Kolesnikov, Y. & Pasternak, G. W. (2006)
Immunohistochemical labeling of the mu opioid receptor carboxy terminal
splice variant mMOR-1B4 in the mouse central nervous system. Brain Res.
1099(1): 33-43.
85. Guarna, M., Bianchi, E., Bartolini, A., Ghelardini, C., Galeotti, N., Bracci, L.,
et al. (2002) Endogenous morphine modulates acute thermonociception in
mice. J. Neurochem. 80(2): 271-277.
86. Kieffer, B. L. & Gaveriaux-Ruff, C. (2002) Exploring the opioid system by
gene knockout. Prog. Neurobiol. 66(5): 285-306.
87. Galeotti, N., Stefano, G. B., Guarna, M., Bianchi, E. & Ghelardini, C. (2006)
Signaling pathway of morphine induced acute thermal hyperalgesia in mice.
Pain. 123(3): 294-305.
88. Rubovitch, V., Gafni, M. & Sarne, Y. (2003) The mu opioid agonist DAMGO
stimulates cAMP production in SK-N-SH cells through a PLC-PKC-Ca++
pathway. Brain Res. Mol. Brain Res. 110(2): 261-266.
89. Crain, S. M. & Shen, K. F. (2001) Acute thermal hyperalgesia elicited by
low-dose morphine in normal mice is blocked by ultra-low-dose naltrexone,
unmasking potent opioid analgesia. Brain Res. 888(1): 75-82.
90. Guarna, M., Ghelardini, C., Galeotti, N., Bartolini, A., Noli, L., Neri, C., et al.
(2004) Effects of endogenous morphine deprivation on memory retention
of passive avoidance learning in mice. Int. J. Neuropsychopharmacol. 7(3):
311-319.
91. Miller, K. K. & Lupica, C. R. (1997) Neuropeptide FF inhibition of morphine
effects in the rat hippocampus. Brain Res. 750(1-2): 81-86.
92. Moron, J. A., Abul-Husn, N. S., Rozenfeld, R., Dolios, G., Wang, R. & Devi,
L. A. (2007) Morphine administration alters the profile of hippocampal
postsynaptic density-associated proteins: a proteomics study focusing on
endocytic proteins. Mol. Cell. Proteomics. 6(1): 29-42.
93. Niu, L., Cao, B., Zhu, H., Mei, B., Wang, M., Yang, Y., et al. (2008) Impaired
in vivo synaptic plasticity in dentate gyrus and spatial memory in juvenile
rats induced by prenatal morphine exposure. Hippocampus.
94. Sargeant, T. J., Day, D. J., Mrkusich, E. M., Foo, D. F. & Miller, J. H. (2007)
Mu opioid receptors are expressed on radial glia but not migrating neuro-
blasts in the late embryonic mouse brain. Brain Res. 1175: 28-38.
95. Eisch, A. J., Barrot, M., Schad, C. A., Self, D. W. & Nestler, E. J. (2000)
Opiates inhibit neurogenesis in the adult rat hippocampus. Proc. Natl. Acad.
Sci. USA. 97(13): 7579-7584.
96. Mandyam, C. D., Norris, R. D. & Eisch, A. J. (2004) Chronic morphine
induces premature mitosis of proliferating cells in the adult mouse sub-
granular zone. J. Neurosci. Res. 76(6), 783-794
VOL. 75 (3), 389-418, 2009 CENTRAL AND PERIPHERAL ENDOGENOUS MORPHINE
417
97. Persson, A. I., Thorlin, T., Bull, C. & Eriksson, P. S. (2003) Opioid-induced
proliferation through the MAPK pathway in cultures of adult hippocampal
progenitors. Mol. Cell. Neurosci. 23(3): 360-372.
98. Persson, A. I., Thorlin, T., Bull, C., Zarnegar, P., Ekman, R., Terenius, L., et
al. (2003) Mu- and delta-opioid receptor antagonists decrease proliferation
and increase neurogenesis in cultures of rat adult hippocampal progenitors.
Eur. J. Neurosci. 17(6): 1159-1172.
99. Harburg, G. C., Hall, F. S., Harrist, A. V., Sora, I., Uhl, G. R. & Eisch, A. J.
(2007) Knockout of the mu opioid receptor enhances the survival of adult-
generated hippocampal granule cell neurons. Neuroscience. 144(1): 77-87.
100. Weber, M., Modemann, S., Schipper, P., Trauer, H., Franke, H., Illes, P., et
al. (2006) Increased polysialic acid neural cell adhesion molecule expression
in human hippocampus of heroin addicts. Neuroscience. 138(4): 1215-1223.
101. Brailoiu, E., Hoard, J., Brailoiu, G. C., Chi, M., Godbolde, R. & Dun, N. J.
(2004) Ultra low concentrations of morphine increase neurite outgrowth in
cultured rat spinal cord and cerebral cortical neurons. Neurosci. Lett. 365(1):
10-13.
102. Tenconi, B., Lesma, E., DiGiulio, A. M. & Gorio, A. (1996) High opioid doses
inhibit whereas low doses enhance neuritogenesis in PC12 cells. Brain Res.
Dev. Brain Res. 94(2): 175-181.
103. Zeng, Y. S., Nie, J. H., Zhang, W., Chen, S. J. & Wu, W. (2007) Morphine
acts via mu-opioid receptors to enhance spinal regeneration and synaptic
reconstruction of primary afferent fibers injured by sciatic nerve crush. Brain
Res. 1130(1): 108-113.
104. Horvath, R. J. & DeLeo, J. A. (2009) Morphine enhances microglial migration
through modulation of P2X4 receptor signaling. J. Neurosci. 29(4): 998-1005.
105. Zhu, W., Mantione, K. J., Casares, F. M., Cadet, P., Kim, J. W., Bilfinger,
T. V., et al. (2006) Alcohol-, nicotine-, and cocaine-evoked release of morphine
from invertebrate ganglia: model system for screening drugs of abuse. Med.
Sci. Monit. 12(5): BR155-161.
106. Stefano, G. B., Bianchi, E., Guarna, M., Fricchione, G. L., Zhu, W., Cadet, P.,
et al. (2007) Nicotine, alcohol and cocaine coupling to reward processes via
endogenous morphine signaling: the dopamine-morphine hypothesis. Med.
Sci. Monit. 13(6): RA91-102.
107. Aunis, D. (1998) Exocytosis in chromaffin cells of the adrenal medulla. Int.
Rev. Cytol. 181: 213-320.
108. Aunis, D., Miras-Portugal, M. T. & Mandel, P. (1974) Bovine adrenal
medullary dopamine-beta-hydroxylase: studies on the structure. Biochim.
Biophys. Acta. 365: 259-273.
109. Aunis, D., Miras-Portugal, M. T. & Mandel, P. (1975) Bovine adrenal
medullary dopamine-beta-hydroxylase: studies on interaction with
concanavalin A. J. Neurochem. 24: 425-431.
110. Goumon, Y., Zhu, W., Weeks, B. S., Casares, F., Cadet, P., Bougaeva, M.,
et al. (2000) Identification of morphine in the adrenal medullary chromaffin
PC-12 cell line. Brain Res. Mol. Brain Res. 81(1-2): 177-180.
YANNICK GOUMON Y COLS.AN. R. ACAD. NAC. FARM.
418
111. Epple, A., Navarro, I., Horak, P. & Spector, S. (1993) Endogenous morphine
and codeine: release by the chromaffin cells of the eel. Life Sci. 52(16):
PL117-121.
112. Boettcher, C., Fischer, W. & Zenk, M. H. (2006) Comment on «Human White
Blood Cells Synthesize Morphine: CYP2D6 Modulation». J. Immunol. 176(10):
5703-5704.
113. Brix-Christensen, V., Goumon, Y., Tonnesen, E., Chew, M., Bilfinger, T.
& Stefano, G. B. (2000) Endogenous morphine is produced in response to
cardiopulmonary bypass in neonatal pigs. Acta Anaesthesiol. Scand. 44(10):
1204-1208.
114. Brix-Christensen, V., Tonnesen, E., Sánchez, R. G., Bilfinger, T. V. & Stefano,
G. B. (1997) Endogenous morphine levels increase following cardiac surgery
as part of the antiinflammatory response? Int. J. Cardiol. 62(3): 191-197.
115. Yoshida, S., Ohta, J., Yamasaki, K., Kamei, H., Harada, Y., Yahara, T., et al.
(2000) Effect of surgical stress on endogenous morphine and cytokine levels
in the plasma after laparoscopoic or open cholecystectomy. Surg. Endosc.
14(2): 137-140.
116. Pan, Y. X., Xu, J., Bolan, E., Moskowitz, H. S., Xu, M. & Pasternak, G. W.
(2005) Identification of four novel exon 5 splice variants of the mouse mu-
opioid receptor gene: functional consequences of C-terminal splicing. Mol.
Pharmacol. 68(3): 866-875.
117. Sharp, B. M., Roy, S. & Bidlack, J. M. (1998) Evidence for opioid receptors
on cells involved in host defense and the immune system. J. Neuroimmunol.
83(1-2): 45-56.
118. Dinda, A., Gitman, M. & Singhal, P. C. (2005) Immunomodulatory effect of
morphine: therapeutic implications. Expert. Opin. Drug. Saf. 4(4): 669-675.
119. Mellon, R. D. & Bayer, B. M. (1998) Evidence for central opioid receptors
in the immunomodulatory effects of morphine: review of potential mecha-
nism(s) of action. J. Neuroimmunol. 83(1-2): 19-28.
120. Mellon, R. D. & Bayer, B. M. (1998) Role of central opioid receptor subtypes
in morphine-induced alterations in peripheral lymphocyte activity. Brain Res.
789(1): 56-67.
121. Mellon, R. D. & Bayer, B. M. (1999) The effects of morphine, nicotine and
epibatidine on lymphocyte activity and hypothalamic-pituitary-adrenal axis
responses. J. Pharmacol. Exp. Ther. 288(2): 635-642.
* Address Correspondence:
Dr. Yannick Goumon.
Inserm U575; 5, Rue Blaise Pascal. F-67084-
Strasbourg Cedex, France.
Phone: (33) 3 88 45 67 18. Fax: (33) 3 88 60 08 06.
email: yannick.goumon@inserm.u-strasbg.fr
