Download III. μ-Opioid receptors in the enteric nervous system

Am J Physiol Gastrointest Liver Physiol
281: G8–G15, 2001.
themes
Receptors and Transmission in the Brain-Gut Axis:
Potential for Novel Therapies
III. ␮-Opioid receptors in the enteric nervous system
Sternini, Catia. Receptors and Transmission in the
Brain-Gut Axis: Potential for Novel Therapies. III. ␮-Opioid
receptors in the enteric nervous system. Am J Physiol Gastrointest Liver Physiol 281: G8–G15, 2001.—G protein-coupled receptors are cell surface signal-transducing proteins,
which elicit a variety of biological functions by the activation
of different intracellular effector systems. Many of these
receptors, including the ␮-opioid receptor (␮OR), have been
localized in the gastrointestinal tract. ␮OR is the target of
opioids and alkaloids, potent analgesic drugs with high potential for abuse. ␮OR is expressed by enteric neurons, and it
undergoes ligand-selective endocytosis. It is of clinical importance because it mediates tolerance and other major side
effects of opiate analgesics, including impairment of gastrointestinal propulsion. An important observation of ␮OR is its
differential trafficking and desensitization properties in response to individual agonists, which might have long-term
physiological consequences and be involved in the development of opiate side effects. Receptor activation by agonists is
the basis for signaling, and alterations of the mechanisms
controlling cellular responses of G protein-coupled receptors
to agonists might be the basis of several diseases, including
gastrointestinal diseases. Therefore, understanding these
basic cellular mechanisms is important for developing appropriate therapeutic agents.
G protein-coupled receptors; receptor endocytosis; motoneurons; receptor trafficking
A VARIETY OF CHEMICAL MESSENGERS,
including classic
transmitters and numerous peptides, modulate multiple digestive functions, including motility, secretion,
and absorption. These effects are mediated by the
activation of specific receptors, most of which belong to
the superfamily of seven-transmembrane G proteincoupled receptors. G protein-coupled receptors represent a large and versatile class of cell surface signalAddress for reprint requests and other correspondence: C. Sternini, CURE Digestive Diseases Res. Ctr. Bldg. 115, Rm. 224,
VAGLAHS, 11301 Wilshire Blvd., Los Angeles, CA 90073 (E-mail:
[email protected]).
G8
transducing proteins, which activate different effector
systems to induce a variety of biological functions (46).
Biological responses to activated G protein-coupled receptors are regulated by receptor desensitization and
resensitization, which are mediated by a cascade of
events induced by ligand-receptor interaction and
which govern cellular responsiveness to agonist stimulation (3). Desensitization, which refers to the diminution of agonist effect after stimulation, is a mechanism to prevent uncontrolled response of the cell to
stimuli, whereas resensitization represents the process
by which cells become responsive again to stimuli.
These regulatory mechanisms are of clinical importance because they control signaling, and defects in
their regulation may result in uncontrolled cellular
response and diseases. Among the many events induced by ligand-receptor interaction, receptor endocytosis is of particular interest because it contributes to
the regulation of receptor-mediated signal transduction by removing receptors from the cell surface and
provides a means to identify sites of ligand release and
neuronal circuits activated by a ligand.
G protein-coupled receptors comprise receptors for
hormones and transmitters, including peptides (46).
Peptides represent a major group of signaling molecules that exert many biological effects in the enteric
nervous system. They can act as primary transmitters
by directly exciting target cells, as cotransmitters by
contributing to the excitability of the effector, as modulators by influencing the excitability of the target cell,
or as growth factors (14). The molecular cloning of
peptide receptors and the availability of peptide receptor antibodies have allowed the definition of the specific cell targets of peptides, which is essential to the
elucidation of their sites and modes of action, but have
also provided a valuable tool to establish whether these
receptors are functional by visualizing their translocation from the cell surface to the cytoplasm after agonist
stimulation. Several studies have investigated the endocytotic and sorting pathway of G protein-coupled
0193-1857/01 $5.00 Copyright © 2001 the American Physiological Society
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CATIA STERNINI
CURE Digestive Diseases Research Center, Department of Veterans Affairs Greater Los Angeles
Healthcare System, Digestive Diseases Division, Departments of Medicine and Neurobiology,
University of California, Los Angeles, California 90095
␮-OPIOID RECEPTORS IN ENTERIC NERVOUS SYSTEM
OPIOID RECEPTORS AND THEIR LIGANDS
Opioid receptors constitute an important group of G
protein-coupled receptors that mediate the effects of
endogenous opioid peptides and of structurally distinct
alkaloid opiate drugs in the nervous system, including
the enteric nervous system (25, 35). Three major receptors mediate opioid and opiate effects, the ␦-, ␬-, and
␮-opioid receptors, which are distinguished by their
affinity for opioids and alkaloids (35). Opioid receptors
have specific pharmacological profiles and physiological functions, maintain a certain degree of selectivity
for various opioid ligands, and display unique patterns
of expression in the nervous system, even though there
is overlap in their binding affinity, distribution, and
function (34, 35). For instance, endorphins bind to ␮and ␦-receptors with similar affinity, whereas dynorphins display some selectivity for ␬-opioid receptor and
enkephalins are the preferred ligands for ␦- but also
have remarkable affinity for ␮-opioid receptors (9). By
contrast, the recently discovered opioid peptides, endomorphin-1 and -2, which have been isolated from the
brain (49), have the highest affinity and selectivity for
␮OR of any opioid peptides described to date. Endomorphins have several thousand-fold preference for
␮OR over ␦- and ␬-receptors. They have affinity for
␮OR similar to that of [D-Ala2,N-Me-Phe4,Glyol5]enkephalin (DAMGO), one of the most potent ␮ORselective enkephalin analogs, but far greater selectivity. Furthermore, these peptides have potent biological
effects that mimic those of other ␮OR agonists (49).
Endomorphin-1 induces analgesia in mice with a
higher potency than morphine, and, like morphine and
DAMGO, endomorphin-1 and endomorphin-2 inhibit
electrically induced contraction of the guinea pig ileum
(28, 44).
Unlike the well-established endogenous opioids,
most alkaloids used clinically preferentially activate
the ␮OR (34). Opiate alkaloids, which include morphine and fentanyl, are the most efficacious and potent
analgesics used in humans for pain treatment. They
are very important therapeutic drugs with high potential for abuse. They have been extensively studied
because of their profound effects on the nervous system. Alkaloids exert a multitude of biological actions
that include analgesia, respiratory depression, and inhibition of intestinal transit and secretion (24). Gene
targeting studies have provided direct evidence for
functional roles of the ␮OR in several biological activities of opiates including analgesia and inhibition of
gastrointestinal transit, which are altered in ␮OR
knockout mice (27, 36).
Three known families of opioid peptides that are
endogenous to mammals have been reported in the
gastrointestinal tract, where they are localized either
in the enteric nervous system or in chromaffin cells (6).
Specifically, in the enteric nervous system, the derivatives of proenkephalins are mostly confined to myenteric neurons projecting to the circular muscle and
submucosal plexus (16), whereas prodynorphin-derived peptides are localized to submucosal and myenteric neurons and to fibers originating from the celiac
ganglion (39). Opioids and opiates affect a variety of
functions within the digestive system, including motility, transit, secretion, and electrolyte and fluid transport. The involvement of opioids in the control of contraction and propulsion is supported by a large body of
anatomic and functional evidence (19, 20, 24). Opioid
peptides and alkaloids impair intestinal transit in humans and other mammalian species by changing the
coordinated reflex motor activity into a segmenting and
nonpropulsive motility pattern (24, 25). In the guinea
pig, which has been used extensively as a model for
functional studies to characterize the physiological and
pathophysiological effects of opioids, morphine-induced blockade of propulsion and transit is not associated with muscular spasm (24, 25). In the gastrointestinal tract, opioid receptor binding sites corresponding
to the three classes of opioid receptors have been associated with different structures including smooth muscle and neurons (6, 24). However, it appears that the
neural effects of opioids in intact tissues are more
relevant than their direct effects on muscle (6). To date,
morphological studies using specific antibodies for the
cloned opioid receptors have provided direct evidence
for the presence of ␮- and ␬-opioid receptors in the
enteric nervous system (1, 41). Furthermore, functional and pharmacological evidence indicates that the
effects of opioids on intestinal motility induced by the
activation of opioid receptors on neuronal structures
are predominantly mediated by ␮- and ␬-opioid receptors (21, 25).
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receptors in transfected cells, and an increasing number of studies have also investigated these processes in
highly differentiated cells including neurons. Many
peptide receptors have been identified in neuronal and
nonneuronal structures of the gastrointestinal tract.
For instance, the receptors for tachykinins have been
localized to enteric neurons as well as to smooth muscle cells and interstitial cells of Cajal (17, 43) and have
been shown to undergo endocytosis in vitro and in vivo
in response to exogenous ligands and to tachykinins
endogenously released in response to stimuli (18, 37,
38). The focus of this themes article is on ␮-opioid
receptor (␮OR) distribution, activation, and trafficking
in the enteric nervous system. ␮OR is of clinical importance because it is the main mediator of the opiateinduced impairment of gastrointestinal transit, one of
the many side effects of opiate drugs, and it mediates
the development of tolerance and drug addiction that
limit the usefulness of these therapeutic compounds
(26, 35). ␮OR is expressed by functionally distinct
enteric neurons, and it undergoes agonist-selective receptor endocytosis in enteric neurons in vivo and in
vitro (40, 41). Agonist selectivity plays an important
role in internalization, which in turn might influence
the biological actions of ␮OR ligands, including opiate
alkaloids, important therapeutic drugs commonly used
in humans for pain control.
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␮-OPIOID RECEPTORS IN ENTERIC NERVOUS SYSTEM
␮-OPIOID RECEPTOR DISTRIBUTION IN THE ENTERIC
NERVOUS SYSTEM
␮-OPIOID RECEPTOR ACTIVATION AND TRAFFICKING
␮OR is an inhibitory G protein-coupled receptor
functionally coupled to several effector pathways, including inhibition of adenylyl cyclase and of cAMP
formation, increase of potassium currents, inhibition of
calcium currents, modulation of inositol trisphosphate
turnover, and activation of mitogen-activated protein
kinase (11, 32, 35). Coupling of the ␮OR to these
effector systems attenuates neuronal activity by inhibiting neurotransmitter release and changing neuronal
excitability by pre- and postsynaptic mechanisms. After ligand-receptor interaction, ␮OR undergoes adap-
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␮OR has been reported in enteric neurons of the rat
and guinea pig gastrointestinal tract. In the rat, ␮OR
has been observed in neurons of both the submucosal
and myenteric plexus and in fibers distributed to the
muscle, vasculature, and mucosa, as well as in presumed interstitial cells of Cajal in the myenteric plexus
and deep muscular plexus (1, 42). By contrast, in the
guinea pig, ␮OR immunoreactivity is primarily localized to neurons of the myenteric plexus, which are
more abundant in the small intestine, particularly the
ileum, than in the stomach and colon, and to fibers
distributed to the interconnecting strands and smooth
muscle layer, where they form a dense network in the
deep muscular plexus. ␮OR enteric neurons have the
morphological characteristics of Dogiel type I myenteric neurons, with an oval cell body, many thick dendrites protruding from the soma, and a long axonal
process, which can be followed within the plexus and
between plexuses in the interconnecting strands (41).
Dogiel type I neurons comprise motoneurons that
transmit information to the muscle cells and control
smooth muscle activity and interneurons that transmit
information to other enteric neurons (15). ␮OR-immunoreactive neurons represent a large population of
myenteric neurons, roughly corresponding to 30% of
myenteric neurons in the guinea pig ileum. ␮OR immunoreactivity is predominantly localized at the cell
surface in nonstimulated conditions, and it translocates to endosomes after activation with selective ␮OR
ligands (Refs. 40 and 41; see below). The ␮OR immunoreactivity distribution closely matches the distribution of the opioid peptide enkephalin (16). Indeed,
fibers containing enkephalin immunoreactivity are in
some cases in close vicinity to myenteric neurons bearing ␮OR. In addition, ␮OR and enkephalin immunoreactivities colocalize in some myenteric neurons and
fibers distributed to the circular muscle and the deep
muscular plexus, where they often surround interstitial cells of Cajal (unpublished observations). Enkephalins are capable of activating ␮OR and of triggering ␮OR endocytosis in enteric neurons and are likely
to be the endogenous opioids that primarily activate
neuronal ␮OR in the gut, even though they are not
selective for this opioid receptor type (35). It is possible
that endomorphins also act as endogenous ligands for
the ␮OR in the enteric nervous system, because these
novel opioids activate and induce internalization of
␮OR in enteric neurons in vitro (28). However, there is
a lack of evidence for these peptides in the gastrointestinal tract.
␮OR myenteric neurons comprise functionally distinct types of neurons. These include cholinergic and
tachykinergic ascending, excitatory motoneurons to
the muscle, as demonstrated by the presence of substance P and choline acetyltransferase (ChAT), a
marker for cholinergic neurons, in a large proportion of
␮OR myenteric neurons (unpublished observations).
They also likely include ascending interneurons, which
contain the same combination of chemical messengers
as excitatory motoneurons (10). In addition, ␮OR enteric neurons comprise a large proportion of descending neurons, as indicated by the localization of nitric
oxide synthase, the enzyme that synthesizes nitric
oxide (NO), and vasoactive intestinal polypeptide
(VIP), the major markers of descending inhibitory motoneurons (10, 14), as well as cholinergic descending
interneurons, as indicated by their coexpression of
ChAT and VIP.
The presence of ␮OR on cholinergic ascending excitatory neurons that innervate the muscle is consistent
with the functional evidence that opioids and opiates
inhibit the electrically evoked release of acetylcholine
by acting on enteric neurons primarily via the ␮OR.
This in turn results in inhibition of muscle contraction,
which is responsible for the delayed gastrointestinal
transit and severe constipation induced by opiates (24).
Opioid receptors and ligands might also modulate acetylcholine’s effect on motor activity, as suggested by
the hypersensitivity of ascending reflexes to acetylcholine in morphine-tolerant animals (24). On the other
hand, the presence of ␮OR on a substantial population
of VIP/NO descending neurons is in agreement with a
modulatory effect of opioids on the release of VIP and
the production of NO. Reduced release of inhibitory
transmitters would account for the reported excitatory
effect of opioids on smooth muscle (2). This is also in
agreement with the opioid inhibitory effect on compliance of the intestinal wall during the preparatory
phase of peristalsis in the intact segment of the guinea
pig ileum (45). Indeed, because ␮OR agonists do not
appear to act directly on the muscle of the guinea pig
small intestine (21) and they inhibit enteric neurons
(33), ␮OR agonist-induced inhibition of compliance of
intestinal wall resistance could be attributed to direct
activation of inhibitory motoneurons to the circular
muscle and/or to a reduction of the excitability of interneurons located in the reflex pathway. Inhibition of
VIP release and NO production could also suppress
descending relaxation and consequently interfere with
intestinal propulsion (19, 20). Together, these findings
support the hypothesis that the opioid effects on intestinal transit mediated by the activation of ␮OR result
from the inhibition of selected enteric neurons and that
opioids act by modulating transmitter release from
neurons of the ascending and descending pathways.
␮-OPIOID RECEPTORS IN ENTERIC NERVOUS SYSTEM
ally desensitizing the ␮OR (22, 23, 28, 41, 47, 48, 50).
Unlike etorphine and opioid peptides that induce rapid
␮OR internalization and desensitization, morphine,
which is the prototype of opiate analgesics and which
activates the same intracellular pathways as other
opiates (35), fails to induce ␮OR internalization and
desensitization, even at saturating concentrations far
in excess of those inducing maximal inhibition of electrically induced muscle contraction (40) and inhibition
of cAMP formation (22). Morphine does not trigger
␮OR endocytosis in enteric neurons, but it partially
inhibits ␮OR endocytosis induced by the opiate alkaloid etorphine, providing evidence that it activates and
occupies ␮ORs (41). This agonist selectivity of ␮OR
endocytosis that we have observed in enteric neurons
(28, 40, 41) has been confirmed in neurons of the
central nervous system and in cell lines transfected
with ␮OR cDNA (22, 23), showing that it is a generalized phenomenon of this receptor. This agonist-selective ␮OR trafficking provides the basis for the hypothesis that individual agonists might have differential
abilities to regulate biological effects mediated by the
␮OR. This would be in agreement with the observation
that the ability to induce tolerance is inversely correlated with opiate effectiveness in inducing endocytosis,
i.e., opiates that trigger ␮OR endocytosis appear to be
less prone to induce tolerance compared with morphine, which does not induce receptor endocytosis under the same experimental conditions (12).
␮OR internalization in enteric neurons also occurs in
response to endogenously released opioids. Indeed,
electrical stimulation of longitudinal muscle-myenteric
plexus preparations with frequencies within the range
that has been shown to induce enkephalin release (8) is
a potent stimulus for evoking ␮OR internalization in
enteric neurons (40). There is a relationship between
stimulus intensity and level of internalization. Low
Fig. 1. ␮-Opiate receptors (␮OR) on enteric neurons in unstimulated (A) and stimulated (B and C) conditions. ␮OR
immunoreactivity is predominantly confined at the cell surface in unstimulated (A) neurons. Enteric neurons have
the morphology of Dogiel type I with a long axonal process (arrow) and many thick dendrites protruding from the
soma (arrowheads). B and C: ␮OR immunoreactivity in endosomes after stimulation with either DAMGO (B), an
enkephalin analog, or etorphine (C). Shown are confocal images of enteric neurons from organotypic cultures of the
intestine incubated with DAMGO (1 ␮M) for 60 min at 4°C to allow ligand-receptor binding, washed, and either
fixed immediately (A) or incubated at 37°C in ligand-free solution for 30 min to allow receptor endocytosis (B). C
shows ␮OR in endosomes after exposure to 100 nM etorphine.
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tations such as desensitization, downregulation, and
resensitization in response to agonist treatment. These
events regulate cellular responsiveness to receptor activation and result from receptor-mediated processes
including phosphorylation, receptor endocytosis, intracellular sorting, and recycling (3).
The presence of ␮OR at the cell surface is essential
for cellular activation, and agonist-induced receptor
internalization plays an important role in regulating
cellular responsiveness by depleting the cell surface of
receptors and by contributing to the process of resensitization (3). ␮OR undergoes rapid, ligand-induced
internalization in transfected cells as well as in neurons (22, 23, 41). In the enteric nervous system, ␮OR
internalization occurs in both the soma and neuronal
processes, it is prevented by the opioid receptor antagonist naloxone (41) and by the selective ␮OR antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2
(CTOP) (40), and it persists for 4–6 h. ␮OR internalization in enteric neurons is concentration dependent,
and it occurs predominantly via a clathrin-mediated
mechanism. After appropriate intracellular sorting requiring endosomal acidification, ␮OR recycles to the
cell surface at ⬃6 h (unpublished observations). Rapid
␮OR internalization in enteric neurons is triggered by
opioids, including enkephalins (unpublished observations) and endomorphins in vitro (40), and by opiates
such as etorphine and fentanyl in vitro and in vivo (41)
(Fig. 1). By contrast, morphine, a high-affinity ␮OR
agonist, does not induce ␮OR endocytosis in enteric
neurons under the same experimental conditions, suggesting that different mechanisms regulate cellular
responsiveness to opioid ligands. Indeed, an interesting and intriguing aspect of ␮OR trafficking and signaling is that opioid agonists with similar abilities to
activate ␮OR signaling have remarkably different abilities in inducing ␮OR internalization and in function-
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␮-OPIOID RECEPTORS IN ENTERIC NERVOUS SYSTEM
Fig. 2. Agonist-selective ␮OR endocytosis
and recycling in enteric neurons. Following interaction with appropriate ligands,
G protein coupling, and phosphorylation
by G protein receptor kinases, ␮ORs interact with ␤-arrestins that induce uncoupling from G protein and facilitate
dynamin-dependent endocytosis via clathrin-mediated pathway. After appropriate
sorting, dephosphorylation, and separation from ␤ arrestins, ␮ORs recycle to the
cell surface. The magnitude of spare receptor fraction influences neuronal responsiveness as indicated by the reduction
of the maximal response in conditions in
which spare receptors are inactivated by
an alkylating agent, such as ␤-chlornaltrexamine (␤-CNA), that irreversibly blocks the
receptors. All of these components are likely
to play an important role in the regulation
of biological functions mediated by the ␮OR.
Other intracellular proteins might be involved in ␮OR trafficking as shown for other
G protein-coupled receptors but are not illustrated here.
ceptors have been inactivated by pretreatment with an
alkylating agent, ␤-chlornaltrexamine (␤-CNA), at concentrations that irreversibly inactivate opioid receptor
reserve (40). However, the reduction of neurogenic response by ligand-induced ␮OR endocytosis was not observed in preparations in which the receptor spare fraction was not reduced. This indicates that the reduction of
␮-opioid spare receptors plays an important role in the
diminution of the nerve-mediated response to opioids
induced by ␮OR endocytosis. On the other hand, the
observation that concentrations of morphine that completely abolish electrically induced muscle contractions
but fail to induce receptor endocytosis do not affect the
subsequent muscle twitch response to increasing concentrations of morphine, even in conditions in which spare
receptors have been inactivated, provides strong support
for the proposal that receptor endocytosis is an important
element in the attenuation of cellular responsiveness to
agonist stimulation. These studies clearly indicate that
both receptor endocytosis and reduction of spare receptor
fraction contribute to the diminution of neuronal responsiveness to opioids.
Studies on cell lines have shown that etorphine and
opioid peptides induce ␮OR phosphorylation and
plasma membrane translocation of ␤-arrestins followed by dynamin-dependent receptor internalization
(50), whereas morphine’s failure to induce desensitization is accompanied by a lack of ␮OR phosphorylation
and translocation of ␤-arrestins with consequent lack
of receptor internalization. However, morphine induces ␮OR phosphorylation with ␤-arrestin translocation and receptor sequestration when G protein kinase
2 is overexpressed (50). This suggests that alterations
of the machinery involved in receptor trafficking might
play an important role in the regulation of cellular
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frequency induces low levels of internalization in a few
neurons, whereas high frequency results in high levels
of internalization in many neurons. These findings
clearly indicate that native ␮ORs endocytose after activation by endogenously released opioids. Thus ␮OR
endocytosis can serve as an indication of opioid release
and also can be used as a means to visualize neuronal
pathways activated by endogenous opioids.
An important component of ␮OR function and regulation in the gastrointestinal tract is the presence of spare
␮ORs. Spare ␮ORs are functional receptors that can be
activated by agonists. The existence of spare ␮ORs implies that an agonist can exert its full effect by activating
only a fraction of the total ␮OR population on the cell
surface (7). The greater the excess of functional ␮ORs in
a system, the lower is the concentration of agonist required for an effect to occur. The ␮OR reserve in the
guinea pig myenteric plexus is substantial, comprising
⬃90% of the total ␮OR population; therefore, only 10%
occupancy is required for ␮OR agonist effects to occur.
The level of opioid receptor reserve is important in the
regulation of cellular sensitivity to opioids and opiates
(7). The magnitude of ␮-opioid spare receptor fraction
appears to control the potency of individual opioid ligands, and alteration of this fraction has been proposed
as one of the putative mechanisms of opioid tolerance. We
have provided evidence that reduction of ␮-opioid spare
receptor reserves together with ligand-induced ␮OR endocytosis might serve as mechanisms to regulate ␮OR
responsiveness to stimulation. This proposal is based on
our observation that ligand-induced ␮OR endocytosis in
enteric neurons reduces the nerve-mediated response
(i.e., electrically induced muscle twitch contraction and
acetylcholine release) in vitro in neuromuscular preparations of the small intestine in which ␮-opioid spare re-
␮-OPIOID RECEPTORS IN ENTERIC NERVOUS SYSTEM
CONCLUSION AND FUTURE PROSPECTS
Since the first discovery, a few years ago, that individual opioid ligands that activate ␮OR through the
same signaling pathways differ in their ability to induce rapid endocytosis of ␮OR, there has been increasing interest in trying to understand the physiological
implications of this dissociation between receptor signaling and endocytosis. This, together with the obser-
vation that the ability of opioid agonists to induce
receptor internalization is inversely correlated with
their efficacy to induce tolerance (12), supports the
concept of ligand-specific effects on intracellular adaptations induced by alkaloids. In addition, it is becoming
evident that different ␮OR ligands form different receptor conformations, that there are differences within
the ␮OR binding domains among agonists, and that
ligand-specific effects on intracellular proteins involved in receptor trafficking may occur. Together,
these findings have forced a reevaluation of the mechanisms underlying ␮OR activation and regulation (47).
The high analgesic potency of opiate alkaloid drugs is
limited by the development of important side effects
including tolerance, dependence, respiratory depression, and profound impairment of gastrointestinal
transit often resulting in severe constipation. Therefore, an elucidation of the mechanisms responsible for
these side effects is of clinical importance for the development of therapeutic agents that preserve their
efficacy as analgesics but are less effective in inducing
tolerance and other side effects. A better knowledge of
the roles and functional implications of receptor-mediated processes will provide the basis for the development of novel therapies targeted to the specific processes that affect neuronal responsiveness. For
instance, ␮OR endocytosis might play a more complex
role than being involved in the regulation of signal
transduction and in the maintenance of cellular desensitization by controlling cell surface receptor expression and might also regulate physiological responses
and biological actions of agonists. This is particularly
important for its clinical implications, suggesting that
␮OR endocytosis might influence the therapeutic action of potent analgesics like the opiate alkaloids. The
concept that receptor endocytosis might regulate physiological and pathophysiological processes, as proposed
for the ␮OR, can also be applicable to other G proteincoupled receptors. It is reasonable to speculate that
general biological roles of receptor endocytosis will be
identified for other G protein-coupled receptors and
that ligand-induced receptor endocytosis is involved in
biological and adaptive responses mediated by activated G protein-coupled receptors.
The author thanks Dr. Nicholas C. Brecha for helpful discussions
and James Minnis for the preparation of the illustrations.
The author is supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grants DK-54155, DK-41301, and
DK-35740.
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2. Bauer AJ, Sarr MG, and Szurszewski JH. Opioids inhibit
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4. Bohn LM, Gainetdinov RR, Lin F-T, Lefkowitz RJ, and
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responsiveness to ␮OR agonists. Because activation
and internalization of native ␮OR are comparable to
that observed in cell lines, it is reasonable to assume
that these processes described in transfected cells also
occur in highly differentiated cells. Figure 2 schematically illustrates what occurs to ␮OR after activation
with ligands capable of triggering receptor endocytosis.
Activated ␮ORs are phosphorylated by G protein
receptor kinases, they bind ␤-arrestins, and they endocytose via a dynamin-dependent mechanism that involves clathrin-coated pits (47) as other protein-coupled receptors. In addition to phosphorylate-activated
receptors, G protein receptor kinases promote interaction with ␤-arrestins, which block agonist-mediated
signal transduction by uncoupling receptors from G
proteins, therefore inducing desensitization (13). ␤-Arrestins also act as adaptor proteins for dynamin-dependent clathrin-mediated endocytosis, linking the receptor to the endocytic machinery, and regulate the rate at
which endosomal receptors are dephosphorylated and
recycled, therefore contributing to the resensitization
process. Dynamin is a cytosolic GTPase that regulates
the formation and internalization of clathrin-coated
vesicles and mediates early endosome formation (29).
Dynamin is required for the internalization of many G
protein-coupled receptors, including the ␮OR (47, 50).
These intracellular proteins play an important role in
the regulation of ␮OR function. Indeed, overexpression
or disruption of ␤-arrestins in cell lines affects ␮OR
trafficking and signaling. Overexpression of ␤-arrestins as well as overexpression of G protein receptor
kinase 2 facilitate endocytosis of morphine-activated
␮OR (47, 50), and functional deletion of ␤-arrestin 2
gene induces remarkable potentiation and prolongation of morphine-induced analgesia and prevents the
occurrence of ␮OR desensitization and tolerance with
chronic morphine treatment (4, 5). Furthermore, overexpression and translocation of dynamin from intracellular pools to plasma membranes have been observed
after chronic treatment with morphine (31), suggesting
that dynamin upregulation may be an important component of the increased neuronal plasticity that has
been recognized at the basis of morphine addiction
(30). Alterations of intracellular regulatory proteins
involved in receptor trafficking are hypothesized to
play a role in the development of tolerance and perhaps
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