(Received for publication, June 28, 1996, and in revised form, October 14, 1996)
From the Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
µ opioid receptors mediate the analgesia induced by morphine. Prolonged use of morphine causes tolerance development and dependence. To investigate the molecular basis of tolerance and dependence, the cloned mouse µ opioid receptor with an amino-terminal epitope tag was stably expressed in human embryonic kidney (HEK) 293 cells, and the effects of prolonged opioid agonist treatment on receptor regulation were examined. In HEK 293 cells the expressed µ receptor showed high affinity, specific, saturable binding of radioligands and a pertussis toxin-sensitive inhibition of adenylyl cyclase. Pretreatment (1 h, 3 h, or overnight) of cells with 1 µM morphine or [D-Ala2MePhe4,Gly(ol)5]enkephalin (DAMGO) resulted in no apparent receptor desensitization, as assessed by opioid inhibition of forskolin-stimulated cAMP levels. In contrast, the morphine and DAMGO pretreatments (3 h) resulted in a 3-4-fold compensatory increase in forskolin-stimulated cAMP accumulation. The opioid agonists methadone and buprenorphine are used in the treatment of addiction because of a markedly lower abuse potential. Pretreatment of µ receptor-expressing HEK 293 cells with methadone or buprenorphine abolished the ability of opioids to inhibit adenylyl cyclase. No compensatory increase in forskolin-stimulated cAMP accumulation was found with methadone or buprenorphine; these opioids blocked the compensatory effects observed with morphine and DAMGO. Taken together, these results indicate that methadone and buprenorphine interact differently with the mouse µ receptor than either morphine or DAMGO. The ability of methadone and buprenorphine to desensitize the µ receptor and block the compensatory rise in forskolin-stimulated cAMP accumulation may be an underlying mechanism by which these agents are effective in the treatment of morphine addiction.
Opioid agonists are the therapeutic choice in the management of
severe chronic pain (1). Despite effective analgesic benefit, extended
periods of opioid treatment can result in a range of undesirable side
effects, not the least of which is the development of tolerance.
Opioids such as morphine demonstrate potent and stereoselective effects
that are reversed by selective antagonists, suggesting an interaction
with a specific membrane receptor (1). Selective, saturable, and high
affinity membrane binding sites were subsequently demonstrated by
radioligand binding assays on native tissue preparations (1). Martin
et al. (2) proposed the hypothesis of multiple opioid
classes and defined µ receptors as the site of morphine action. The
classification of opioid receptors into µ, , and
was
subsequently reinforced by the development of highly selective ligands
for each class (3). Recently, the genetic basis for the three opioid
classes was obtained, with the molecular cloning of the
,
, and µ opioid receptors (for review, see Ref. 4).
Of the three opioid receptor classes, the µ receptor is thought to be the principal site of analgesic interaction, since most of the clinically relevant opioids used in pain management bind to this receptor with high affinity (5, 6, 7). Studies on the µ receptor offer potential molecular insights into the cellular basis for tolerance and dependence, serious side effects of prolonged opioid usage which may result from effects on receptor regulation (8).
Whereas morphine induces dependence, methadone is used in the treatment of opioid addiction, despite being a full agonist at the µ receptor (9). The therapeutic efficacy of methadone has been linked to a lower abuse potential, although it is not established why methadone has lower addictive properties than morphine. Buprenorphine is the other opioid agent used in the treatment of addiction and has not been found to precipitate withdrawal in animal and human studies (9, 10). Buprenorphine has been reported to be a partial agonist or mixed agonist/antagonist at the µ receptor, and these properties have been implicated as the basis for addiction therapy (10).
Despite the clinical importance of tolerance development and dependence, relatively little is known about the molecular and cellular events induced by morphine (8). Also, the cellular events that accompany the therapeutic actions of methadone and buprenorphine in treating addiction are poorly understood.
The recent cloning and heterologous expression of the µ receptor have facilitated attempts to determine the biochemical mechanisms of clinically used opioids. In this study we investigated the molecular consequences of prolonged opioid agonist exposure on the cloned murine µ receptor stably expressed in HEK1 293 cells. Continuous treatment of the µ receptor with morphine or DAMGO does not appear to desensitize the receptor, but it does result in an adaptive cellular response that is manifested by an increase in forskolin-stimulated intracellular cAMP levels. In contrast, methadone and buprenorphine treatments result in a pronounced desensitization of the µ receptor while also blocking the compensatory increases in forskolin-stimulated cAMP levels observed with either morphine or DAMGO. These differential actions of opioid agonists at the µ receptor may explain the mechanism by which morphine induces dependence, whereas methadone and buprenorphine are effective therapeutic agents in the treatment of dependence.
HEK 293 cells were grown and maintained in
minimal essential medium with Earle's salts (Life Technologies, Inc.)
containing 10% fetal bovine serum, 1,000 units ml1
penicillin and streptomycin sulfate in 10% CO2 at
37 °C. The mouse µ opioid receptor gene modified with the FLAG
epitope (DYKDDDDK) at the amino terminus was a gift from Dr. Mark von
Zastrow, University of California at San Francisco. The modified
cDNA in the expression vector pcDNA3 (Invitrogen) was stably
transfected into HEK 293 cells by a modification of the calcium
phosphate protocol (11). Briefly, HEK 293 cell monolayers at
approximately 70% confluence in T 75-cm2 flasks were
transfected with 30 µg of plasmid. After an overnight incubation, the
medium was removed, and the cells were treated with 5 ml of
phosphate-buffered saline containing 10% glycerol for 10 min at room
temperature. Cells were then washed twice with phosphate-buffered
saline and incubated for 3 h at 37 °C with growth medium
containing 100 µg ml
1 chloroquine. Cells were then
washed twice and incubated for 48 h at 37 °C in growth medium.
Stable transformants were selected in growth medium containing 1 mg
ml
1 Geneticin (Life Technologies, Inc.) and maintained in
T 75-cm2 tissue culture flasks in 10% CO2 at
37 °C.
Receptor binding studies were
performed using membranes from stably transfected HEK 293 cells
expressing the µ-FLAG cDNA. Membranes were prepared and receptor
binding studies conducted as described (5) and as noted in the table
and figure legends. Briefly, cell monolayers were harvested in 6 ml of
buffer containing 50 mM Tris-HCl (pH 7.8) containing 1 mM EGTA, 5 mM MgCl2, 10 µg ml1 leupeptin, 10 µg ml
1 pepstatin, 200 µg ml
1 bacitracin, and 0.5 µg ml
1
aprotinin and placed on ice. A cell pellet was prepared by
centrifugation at 24,000 × g for 7 min at 4 °C and
was homogenized in the same buffer using a Polytron (Brinkmann
Instruments) at setting 2.5, 30 s. The cell homogenate was
centrifuged for at 48,000 × g for 20 min at 4 °C,
and the resulting cell pellet was homogenized and placed on ice for the
binding assays. Binding assays were carried out at 25 °C for 40 min
in a final volume of 200 µl in the presence or absence of competing
ligands.
For agonist pretreatment studies, a 10-fold concentrated stock of
agonist was diluted into growth medium and added to individual culture
flasks. The final concentration of all agonists used in regulation
studies was 1 µM. Cell monolayers were harvested at the
times indicated in the table and figure legends. Pertussis toxin
treatments were carried out either in tissue culture flasks or 12-well
plates overnight at 37 °C with 100 ng ml1 pertussis
toxin (List Biological Laboratories, Campbell, CA).
Stably transfected HEK 293 cells
were subcultured in 12-well culture plates and allowed to recover for
72 h prior to experiments. For agonist pretreatment and pertussis
toxin experiments, the growth medium was replaced for the times
indicated in the figure legends with medium containing either ligand or
pertussis toxin. After treatment, the medium was removed and replaced
with 1 ml of growth medium containing 0.5 mM
isobutylmethylxanthine, and the cells were incubated for 30 min at
37 °C. The culture medium was then removed and replaced with fresh
medium, with or without 10 µM forskolin and opioids, and
the cells were transferred to 37 °C. After 5 min the medium was
removed, 1.0 ml of 0.1 N HCl was added, and the monolayers
were frozen at 20 °C. For determination of the cAMP content of
each well, the monolayers were thawed, placed on ice, sonicated, and
the intracellular cAMP levels measured by radioimmunoassay (Amersham
plc, Buckinghamshire, UK). Data obtained from the dose-response curves
were analyzed by nonlinear regression analysis with GraphPad Prism 2.01 (GraphPad Software, Inc., San Diego, CA).
The monoclonal
antibody M2 against the FLAG epitope was purchased from Eastman Kodak.
Antibody radioiodination was performed by a chloramine-T procedure
(12). Briefly, 250 µg of M2 antibody was incubated in 200 mM NaPO4 buffer (pH 7.3) with 0.5 mCi of Na125I and the reaction initiated with 100 µl of
chloramine T (0.5 mg ml1 in NaPO4 buffer).
After 30 s at room temperature, the reaction was terminated by the
addition of 100 µl of sodium metabisulfite (1.25 mg ml
1
in NaPO4 buffer). The iodinated protein was separated from
free 125I by column chromatography with Sephadex G-25, and
aliquots from the collected fractions were counted in an LKB
scintillation counter and then stored at 4 °C.
After agonist treatment
of cell monolayers, approximately 200,000 cpm of 125I-M2
antibody was added to individual wells in 24-well plates in growth
medium containing 10% fetal calf serum. Cells were incubated for 30 min at 37 °C, the medium aspirated, and the monolayers washed twice
with growth medium. Bound radioactivity was determined after
solubilization of the cell monolayer with 0.5 ml of 0.5 N NaOH and counted in a scintillation counter.
Nonspecific radiolabeled antibody binding was determined in the
presence of 10 µM FLAG peptide (Eastman Kodak) and
accounted for 30% or less of the total binding.
Cell monolayers were pretreated
with the selective µ receptor agonists DAMGO, morphine, and methadone
and the nonselective opioids levorphanol, etorphine, and buprenorphine.
The chemical structures of these opioid ligands are shown in Fig.
1.
To investigate the prolonged effects of opioid agonist treatment,
we stably expressed the mouse µ receptor in HEK 293 cells. Pharmacological characterization of the stably transformed cells showed
saturable, high affinity binding for selective µ opioid ligands,
which was best defined as a single, noninteracting site (Table
I). Saturation radioligand binding with the nonselective opioid antagonist [3H]diprenorphine revealed a µ receptor density (Bmax) of 7.7 ± 0.5 pmol
mg1 membrane protein with a dissociation constant
(Kd) of 0.61 ± 0.14 nM
(n = 3). No specific radioligand binding was detected in untransfected HEK 293 cells (data not shown). Competition binding experiments with the selective µ peptide agonist
[3H]DAMGO showed that the cloned and expressed mouse µ receptor shared some pharmacological characteristics with the
previously characterized rat and human µ receptors (6, 7). As shown in Table I, selective µ agonists such as morphine, DAMGO, and methadone showed specific, high affinity membrane binding, as did the
nonselective opioid agonists levorphanol, etorphine, and buprenorphine.
The affinity for buprenorphine was approximately 3-fold higher than
reported previously by Raynor et al. (6). This observed
difference in affinity may reflect differences in the surrogate cell
lines used (COS-7 in Raynor et al. (6); HEK 293 cells in
this study); the presence of the amino-terminal epitope tag; or
differences inherent in the cloned and expressed human, rat, and mouse µ receptors. The rank order of potency for opioid agonists to
displace [3H]DAMGO membrane binding at the epitope-tagged
mouse µ receptor is etorphine > levorphanol > morphine = buprenorphine = DAMGO > methadone (Table
I).
|
Previous studies from this and other laboratories have shown that the
opioid receptors couple to the inhibition of adenylyl cyclase activity
in HEK 293 cells (13, 14, 15). Consistent with the competition binding
results, the opioid agonists were found to retain a similar rank order
of potency when tested for the ability to inhibit forskolin-stimulated
cAMP accumulation (Fig. 2A). The nonselective
opioids etorphine and levorphanol were found to be the most potent,
followed by the selective µ opioids DAMGO and morphine. Buprenorphine
and methadone were the least potent for inhibiting cAMP accumulation
(Fig. 2A). However, although buprenorphine appeared to be
slightly more potent than methadone in the inhibition of
forskolin-stimulated cAMP accumulation, the maximal inhibition was
significantly different from that of the other opioids examined in this
study (p < 0.05, n = 10). The inability of buprenorphine to inhibit cAMP accumulation maximally in
the HEK 293 cells is consistent with the partial agonist activity that
has been described by others (9, 10). As shown in Fig. 2B,
the opioid agonist inhibition of forskolin-stimulated cAMP was
sensitive to pertussis toxin pretreatment. Overnight treatment of cell
monolayers with 100 ng ml1 pertussis toxin abolished the
ability of morphine, levorphanol, or etorphine to inhibit cAMP
accumulation. This suggests that the µ receptor inhibition of cAMP
accumulation in HEK 293 cells couples to either the Gi or
Go class of G proteins. The effects of morphine on the
inhibition of forskolin-stimulated cAMP accumulation were blocked by
the nonselective opioid antagonist naloxone (Fig. 2C).
[3H]DAMGO binding to the µ receptor was reduced by
100 µM GTP
S or by overnight treatment with pertussis
toxin, in agreement with the effects observed on functional inhibition
of cAMP accumulation (Fig. 3).
Opioid agonist inhibition of
forskolin-stimulated cAMP accumulation in µ-FLAG-expressing HEK 293 cells. Panel A, HEK 293 cell monolayers were treated for 30 min at 37 °C with growth medium containing 0.5 mM
isobutylmethylxanthine. After treatment, the medium was replaced with
growth medium containing agonist over the concentration range of
1011 to 10
6 M and incubated at
37 °C for 5 min. The medium was then aspirated, and 1 ml of 0.1 N HCl was added; the cells were sonicated. Intracellular cAMP levels were measured using a commercially available cAMP radioimmunoassay kit (Amersham Corp.). The inhibition of
forskolin-stimulated cAMP accumulation is expressed as a percentage of
the forskolin control. Forskolin-stimulated cAMP levels were typically
5-20-fold higher than basal values. The data presented are the
means ± S.E. of three or more separate experiments, each
performed in duplicate.
, buprenorphine;
, morphine;
,
levorphanol;
, DAMGO;
, etorphine;
, methadone. Panel
B, pertussis toxin effects on morphine (
), levorphanol (
),
or etorphine (
) inhibition of forskolin-stimulated cAMP
accumulation. Cell monolayers were treated overnight with 100 ng
ml
1 of pertussis toxin (List Biologicals), and cAMP
accumulation was determined the following day. The data shown are the
means ± S.E. of three independent experiments, each performed in
duplicate. Panel C, naloxone effects on morphine inhibition
of forskolin-stimulated cAMP accumulation in HEK 293 cells. Cell
monolayers were treated as in panel A, and either morphine
(1 µM) or morphine plus naloxone (1 µM) was
added to the monolayers. After 5 min at 37 °C the medium was
aspirated, and the samples were prepared for cAMP radioimmunoassay. The results are
the mean ± S.E. of three separate experiments, each performed in
duplicate.
To determine the effects of agonist pretreatment on the mouse µ receptor, the reduction of [3H]DAMGO binding was used as a measure of receptor-G protein coupling. Using a 3-h agonist pretreatment protocol, the effects of the opioid agonists morphine, levorphanol, the synthetic peptide DAMGO, and the highly potent opioid etorphine were examined. In addition, two opioids currently used in the treatment of opioid addiction, methadone and buprenorphine, were also studied. Pretreatment with five of the six opioid agonists reduced high affinity [3H]DAMGO binding (Fig. 3). This suggests that pretreatment with these three opioids results in an apparent altered coupling of the µ receptor with G proteins in the HEK 293 cells. Since high affinity [3H]DAMGO binding is also reduced by overnight pertussis toxin treatment, the effects of the agonist pretreatment impair the coupling of the µ receptor to the Gi or Go class of G proteins in the HEK 293 cells. DAMGO pretreatment was found to have no effect on high affinity [3H]DAMGO binding (102 ± 18%, n = 3). Buprenorphine and etorphine pretreatments appeared to abolish [3H]DAMGO binding relative to control binding (99 ± 1% reduction, n = 4 for buprenorphine; 99 ± 0.3% reduction, n = 3 for etorphine); and methadone, levorphanol, and morphine pretreatments reduced radiolabeled agonist binding (reductions of 79 ± 13%, 73 ± 6% and 35 ± 8%, respectively, n = 3). The effects of buprenorphine were stereoselective, as (+) buprenorphine, the inactive stereoisomer, did not bind to the mouse µ receptor (data not shown).
To determine the effects of agonist pretreatment on µ-FLAG receptor
density, two independent studies were performed. The binding of
[3H]diprenorphine, a nonselective opioid antagonist, was
found to be decreased by four of the six agonist pretreatments
examined, in agreement with the [3H]DAMGO studies (Fig.
4A). Morphine and DAMGO pretreatments had negligible effects on [3H]diprenorphine binding
(reductions of 9 ± 11% and 19 ± 8%, respectively). And,
whereas levorphanol reduced antagonist binding by 28 ± 4%, methadone, buprenorphine, and etorphine pretreatments resulted in
marked reductions in [3H]diprenorphine binding (61 ± 12%, 99 ± 1%, and 68 ± 8%, respectively). Since
etorphine and buprenorphine have been proposed to be highly lipophilic
opioids (10), the dramatic reductions in
[3H]diprenorphine binding may result from an irreversible
component of the ligand binding that occurred during the 3-h
pretreatment, making direct radioligand measurements inaccurate. To
assess the effects of agonist pretreatment on receptor density
independently of radioligand binding, an iodinated monoclonal antibody
against the amino-terminal FLAG epitope was used. If the continued
presence of the pretreatment agonist (e.g. buprenorphine)
affected the affinity of [3H]diprenorphine binding, then
the use of the radiolabeled M2 antibody should provide an alternative
means to measure the cell surface receptor density. At present, the
amino terminus of the mouse µ receptor is predicted to be an
extracellular site not known to be directly involved in ligand binding
(3, 16, 17). Fig. 4B demonstrates that the binding of the
radiolabeled M2 antibody to intact cell monolayers is unaffected by
pretreatment with the agonists levorphanol (101 ± 7% of control,
n = 4), buprenorphine (109 ± 7% of control,
n = 8), or morphine binding (95 ± 3% of control,
n = 3), whereas DAMGO resulted in small decreases in antibody binding (83 ± 5% of control, n = 4). In
contrast, the 3-h pretreatment of cells with 1 µM
etorphine resulted in a reduction of M2 binding to 64 ± 6%
(n = 8) of the untreated control (Fig. 4B).
The antibody binding studies indicate that despite the ability of
levorphanol, etorphine, DAMGO, methadone, and buprenorphine pretreatments to reduce high affinity [3H]diprenorphine
binding, only DAMGO and etorphine pretreatments significantly reduced
M2 antibody binding to the µ-FLAG receptor in HEK 293 cells.
As shown in Fig. 5, the functional consequences of
agonist pretreatment were examined on the inhibition of
forskolin-stimulated cAMP accumulation. Fig. 5A shows that
the morphine inhibition of forskolin-stimulated adenylyl cyclase is
dose-dependent over the concentration range of
1011 to 10
6 M. In untreated
monolayers, a maximal inhibition of cAMP accumulation (75 ± 8%,
n = 8) was observed at 1 µM morphine.
Three-hour or overnight morphine pretreatment appeared to have little
additional effect on the EC50 of the morphine dose-response
curve (Fig. 5A). However, the pretreatments did appear to
enhance the maximal inhibitions observed at the 1 µM
morphine concentration (93 ± 5%, n = 7 and 92 ± 5%, n = 4, inhibition of
forskolin-stimulated cAMP accumulation for 3-h and overnight
pretreatments, respectively). Levorphanol also inhibited
forskolin-stimulated cAMP accumulation in a dose-dependent manner, with a maximal inhibition in control cells of 76 ± 6% (n = 6) at 1 µM. In contrast to the
effects of morphine pretreatment on morphine inhibition of cAMP
accumulation, pretreatment of the HEK 293 cells with levorphanol did
not appear to enhance the maximal cAMP inhibition (82 ± 8%,
n = 6, for 3-h pretreatment; 81 ± 7%, n = 4, for overnight pretreatment), while having little
effect on the observed agonist EC50 (Fig. 5B).
In agreement with the morphine and levorphanol results, DAMGO (Fig.
5C) inhibition was also dose-dependent, with a
maximal inhibition of 73 ± 5% at 1 µM
(n = 4). Unlike either morphine or levorphanol
pretreatment, DAMGO pretreatment appeared to shift the EC50
for cAMP inhibition to higher concentrations, with the control
EC50 = 3.3 ± 1.9 nM (n = 4) and DAMGO-treated EC50 = 8.4 ± 1.4 nM
(n = 7). DAMGO pretreatment did not appear to enhance
the maximal inhibition of cAMP accumulation by DAMGO (Fig.
5C). Levorphanol and DAMGO 3-h pretreatments resulted in a
rightward shift (Fig. 5D) in the EC50 values for
morphine inhibition of cAMP accumulation (10 ± 1.3 nM, n = 8, for DAMGO pretreatment and
37 ± 2 nM, n = 8, for levorphanol
pretreatment). Neither pretreatment appeared to enhance the maximal
inhibition of cAMP accumulation by morphine. Thus, although morphine
and levorphanol pretreatments can reduce high affinity agonist binding, as assessed with [3H]DAMGO, these agonists do not cause a
functional desensitization of the µ receptor.
Agonist pretreatment effects on opioid
inhibition of forskolin-stimulated intracellular cAMP levels.
Stably transfected HEK 293 cells were plated in 12-well dishes, and
intracellular cAMP levels were assayed as described under
"Experimental Procedures." Forskolin-stimulated cAMP values, in the
absence of opioid agonist, were defined as 100%, and the opioid
percentage of inhibition of cAMP was calculated from the forskolin
control value. Panel A, dose-dependent morphine
inhibition of forskolin-stimulated cAMP levels from control and 1 µM morphine-pretreated cell monolayers. The results
presented represent the mean ± S.E. of at least three separate
experiments, each performed in duplicate and assayed in duplicate.
, control;
, 3-h pretreatment;
, overnight pretreatment; *p < 0.05. Panel B, levorphanol
dose-dependent inhibition of forskolin-stimulated cAMP
levels from control and 1 µM levorphanol-pretreated cell monolayers. The results shown are the mean ± S.E. of three
independent experiments, each performed and assayed in duplicate.
Symbols are as in panel A. Panel C,
DAMGO concentration-dependent inhibition of
forskolin-stimulated cAMP levels from control and 1 µM
DAMGO-pretreated cells. The results shown are the means ± S.E. of
three independent experiments performed in duplicate.
, control;
, 3-h pretreatment. Panel D, levorphanol or DAMGO
pretreatment effects on morphine inhibition of forskolin-stimulated
cAMP accumulation. HEK 293 cells were pretreated with either 1 µM levorphanol (
) or DAMGO (
) for 3 h at
37 °C, and the concentration-dependent effects of
morphine on intracellular cAMP accumulation were determined. The
results are the mean ± S.E. of three independent experiments, each performed in duplicate.
The inability of morphine, DAMGO, and levorphanol treatments to
desensitize the response to forskolin-stimulated cAMP accumulation could be the result of the HEK 293 cells lacking critical cellular factors needed for opioid receptor desensitization. To test this possibility, HEK 293 cells were pretreated with etorphine, an extremely
potent opioid (18, 19, 20). Etorphine pretreatment desensitized the mouse µ receptor in HEK 293 cells (Fig. 6A). The
3-h etorphine exposure resulted in a rightward shift of the etorphine
dose-response curve (control EC50 = 0.044 ± 0.001 nM, n = 6; treated EC50 = 2.1 ± 1 nM, n = 7) as well as a
substantial reduction in the levels of maximal inhibition (control,
86 ± 3%; treated, 14 ± 12%). These results suggest that
the mouse µ receptor expressed in HEK 293 cells can undergo
desensitization when pretreated with the potent opioid etorphine.
Although pretreatment of µ-FLAG-expressing HEK 293 cells with the
clinically used analgesic morphine did not desensitize the µ receptor, a pronounced receptor desensitization was observed after
pretreatment with methadone and buprenorphine, opioids used in the
treatment of morphine and heroin dependence (Fig. 6, B and
C). A 3-h pretreatment of cell monolayers with methadone
resulted in a marked rightward shift in the EC50 for the
methadone inhibition of forskolin-stimulated cAMP accumulation (Fig.
6B; methadone control EC50 = 9.6 ± 1.5 nM, n = 5; 3-h treated EC50 = 230 ± 3.7 nM, n = 5). The 3-h
methadone pretreatment reduced the maximal inhibition of cAMP
accumulation (control, 84 ± 6%, n = 7; 3-h
pretreated, 55 ± 6%, n = 5). Buprenorphine
pretreatment (3 h) also resulted in receptor desensitization (Fig.
6C) at buprenorphine concentrations above 108
M, with the maximal inhibition of cAMP accumulation reduced
from 67 ± 3% (n = 14) for control to 2 ± 8% (n = 5) for treated cells. No effect on
forskolin-stimulated cAMP accumulation was found with (+) buprenorphine
(data not shown). The three opioid agonists etorphine, methadone, and
buprenorphine also appeared to desensitize the effects of morphine on
cAMP accumulation (Fig. 6D) by markedly reducing the ability
of maximal concentrations of morphine to inhibit forskolin-stimulated
cAMP accumulation. Methadone pretreatment appeared to have a pronounced
effect on the observed EC50 for morphine inhibition, with
an approximately 200-fold shift to higher agonist concentrations when
compared with control (Fig. 6D; 3-h treatment
EC50 = 467 ± 19 nM, n = 3, compared with Fig. 4A; control EC50 = 2.3 ± 1.5 nM). Thus, the opioids methadone and
buprenorphine are able to desensitize the mouse µ receptor to
morphine, with rightward shifts in the EC50 for inhibition
and a reduction in the maximal levels of inhibition after pretreatment.
The pretreatment effects of these three opioids differ markedly from
the effects observed on the mouse µ receptor after pretreatment with
morphine, levorphanol, or DAMGO.
Etorphine, methadone, or buprenorphine
desensitization of opioid agonist inhibition of forskolin-stimulated
cAMP accumulation. Panel A, etorphine desensitization of
etorphine effects on forskolin-stimulated cAMP accumulation. Monolayers
were pretreated for 3 h with 1 µM etorphine and
assayed for intracellular cAMP accumulation. The results are the
mean ± S.E. of at least three independent experiments.
Statistical significance (*p < 0.05) was determined by
a paired Student's t test. , control;
, 3-h pretreatment. Panel B, control or methadone pretreatment
effects on methadone inhibition of forskolin-stimulated cAMP
accumulation. Monolayers were pretreated with methadone for 3 h at 37 °C and then
prepared for cAMP accumulation experiments as described under "Experimental Procedures." The results shown are the means ± S.E. of at least three separate experiments, all performed in
duplicate. Symbols are as in panel A. Panel
C, control or buprenorphine pretreatment effects on buprenorphine
inhibition of forskolin-stimulated cAMP accumulation in µ-FLAG
expressing HEK 293 cells. Cells were either untreated or treated for
3 h with 1 µM buprenorphine and assayed for
intracellular cAMP accumulation, as described under "Experimental Procedures." The results are the means ± S.E. of three or more independent experiments, each performed in duplicate. Statistical significance was determined as in panel A.
Symbols are as in panel A. Panel D,
etorphine, methadone, and buprenorphine effects on morphine inhibition
of forskolin-stimulated cAMP accumulation. µ-FLAG-expressing HEK 293 cells were pretreated with 1 µM etorphine (
),
methadone (
), or buprenorphine (
), and the
dose-dependent inhibition of intracellular cAMP by morphine
was measured. Each point represents the mean ± S.E. of at least
three independent experiments, performed and assayed in
duplicate.
Adaptive sensitization or overshoot of adenylyl cyclase activity has
been implicated in the cellular mechanisms of opioid tolerance (21,
22), and a recent study on the cloned rat µ receptor suggested that
the physiological consequences of receptor sensitization, rather than
receptor desensitization, may be critical to tolerance development
(22). We examined whether a physiological adaptation phenomenon
occurred after opioid agonist pretreatment of HEK 293 cells by
determining the levels of forskolin-stimulated cAMP after agonist
pretreatment. As shown in Fig. 7, a 3-h pretreatment with 1 µM morphine or DAMGO leads to an approximately
3.4 ± 0.3-fold increase (ANOVA, n = 17, p < 0.001) and 2.9 ± 0.2-fold (n = 12, p < 0.001) in forskolin-stimulated intracellular
cAMP accumulation, respectively, for morphine and DAMGO, when compared
with untreated cells. This increase was not observed with levorphanol
(n = 8), methadone (n = 12), or
buprenorphine (n = 9) pretreatment, which was
significantly different from the effects of morphine or DAMGO (ANOVA,
p < 0.001). A combination of morphine and DAMGO
resulted in no additive increase in the adaptive cAMP response above
that seen with the individual agonists (data not shown), suggesting that morphine and DAMGO may use a common mechanism in causing the
increases in intracellular forskolin-stimulated cAMP. The effect of the
potent opioid etorphine (n = 9) was unique among the
opioid agonists examined in this study, since the pretreatment reduced
the intracellular cAMP levels by approximately 50% but was
statistically insignificant compared with the individual effects of
levorphanol, methadone, and buprenorphine (ANOVA, p > 0.05). These results indicate that the opioid agonists differ in their ability to cause compensatory increases in forskolin-stimulated cAMP
with the mouse µ receptor, as only morphine and DAMGO pretreatments resulted in an adaptive, or overshoot, response.
The compensatory effects of morphine and DAMGO on adenylyl cyclase were blocked by the addition of other opioid agonists. Fig. 7 shows that when cell monolayers were pretreated with morphine in combination with other opioid agonists such as levorphanol (n = 3, p < 0.001), methadone (n = 3, p < 0.001), etorphine (n = 2, p < 0.001), or buprenorphine (n = 3, p < 0.001), the forskolin-stimulated increase of adenylyl cyclase activity was reversed. In a similar manner, DAMGO pretreatment in combination with other opioid agonists also resulted in a reduction in the forskolin-stimulated levels of intracellular cAMP (Fig. 7). No statistically significant differences were observed for the effects of methadone when compared with the combinations of methadone and morphine (n = 3, p > 0.05) or methadone and DAMGO (n = 3, p > 0.05). These results demonstrate that the opioid agonists levorphanol, etorphine, methadone, and buprenorphine are capable of blocking the compensatory increases in intracellular cAMP observed after chronic treatment with morphine or DAMGO.
In the current study we have stably expressed an epitope-tagged
mouse µ receptor (23) in HEK 293 cells and examined the pharmacological and functional consequences of prolonged agonist exposure. The mouse receptor binds the selective µ agonists morphine, DAMGO, and methadone with high affinity, as well as the nonselective opioids levorphanol, etorphine, and buprenorphine. In agreement with
earlier studies on the heterologous expression of cloned opioid
receptors in HEK 293 cells (13, 14, 15), the high affinity agonist binding
of the expressed mouse µ receptor was sensitive to the effects of
GTPS and pertussis toxin pretreatments. The sensitivity to pertussis
toxin indicates that in the HEK 293 cells, the expressed mouse µ receptor couples to a member of the Gi or Go
family of G proteins. Such a result is consistent with immunoprecipitation studies on other surrogate cell lines that stably
express the cloned opioid receptors (24).
The most significant findings of the current study are the demonstrations that the opioid agonists differentially regulate the mouse µ receptor and that the two opioids currently used in the treatment of addiction, methadone and buprenorphine, desensitize the µ receptor. The agonist pretreatment studies presented here show that morphine, levorphanol, etorphine, methadone, and buprenorphine effectively reduce high affinity agonist binding, although not to the same extent. In contrast, pretreatment with DAMGO had no effect on high affinity agonist binding. Buprenorphine and etorphine were found to be the most potent opioids in that both decreased high affinity agonist and antagonist binding, but only etorphine clearly down-regulated the µ receptor in HEK 293 cells. Of considerable importance is the demonstration that the two opioids currently used in the treatment of addiction, methadone and buprenorphine, both potently desensitize the mouse µ receptor, whereas morphine and levorphanol, which are highly addictive, did not desensitize the µ receptor. This suggests that the therapeutic role of methadone and buprenorphine in treating addiction may somehow be related to the ability to desensitize the µ receptor. Further differences between the opioid agonists were noted on the pretreatment sensitization of adenylyl cyclase. Morphine and DAMGO pretreatments substantially increased forskolin-stimulated intracellular cAMP levels, and this process was blocked by methadone and buprenorphine, which themselves did not cause the response. These results suggest both opioid agonists and antagonists are capable of reversing the supersensitization events seen after morphine or DAMGO pretreatment, indicating that the cellular basis for withdrawal may be more complex than previously proposed (21, 22).
The interplay among tolerance, functional desensitization, receptor down-regulation, and the compensatory increases observed in intracellular second messengers is intricate. All of these phenomena have been proposed to play a role in opioid tolerance and dependence. The development of opioid tolerance has been reported to result from a loss of opioid responsiveness, or a desensitization of opioid action, rather than from a significant receptor down-regulation (25, 26). To develop a cellular paradigm for tolerance development, a number of studies have been conducted on the effects of prolonged opioid treatment with opioid receptor-expressing cell lines. In cells that endogenously express opioid receptors, the results of receptor down-regulation and desensitization studies have been complicated by the presence of one or more opioid receptor classes in the cell lines studied (27). Recently, studies on the cloned human and rat µ receptors in the COS-7 transient expression system have demonstrated that chronic µ agonist exposure results in neither receptor down-regulation nor desensitization (6). Arden et al. (13) were able to demonstrate down-regulation and phosphorylation of the rat µ receptor in stably transfected HEK 293 cells; however, it is unclear whether the down-regulation event was accompanied by receptor desensitization. Chakrabarti et al. (28) stably expressed the rat µ receptor in a murine neuroblastoma cell line and examined the effects of morphine and DAMGO pretreatment on opioid receptor binding and adenylyl cyclase inhibition. The results of this study (28) indicated that both morphine and DAMGO were able to reduce the maximum number of receptor binding sites and, following extended treatment, diminished the maximal levels of opioid-mediated inhibition of cAMP. The differences in µ receptor regulation observed in the studies of Raynor et al. (6) and Arden et al. (13) suggest that agonist regulation of the µ receptor may be a function of the surrogate cell lines or of the species isoforms of the µ receptor. None of these studies (6, 13, 28) addressed the actions of buprenorphine or methadone on µ receptor regulation.
The results presented here demonstrate that the desensitization of the µ receptor by buprenorphine is independent of down-regulation or internalization of the receptor, since cell surface labeling of the receptor with a radiolabeled monoclonal antibody revealed no changes in antibody binding after buprenorphine treatment. The loss of high affinity agonist and antagonist binding after buprenorphine pretreatment may be due to the continued presence of the opioid on the receptor, with the continuous activation of the receptor resulting in functional desensitization. Such a notion would be consistent with previous studies that have demonstrated a slow off-rate for buprenorphine binding to the µ opioid receptor (10).
However, activation of the receptor is not the sole requirement for desensitization since morphine, DAMGO, and levorphanol are more potent and effective than buprenorphine in inhibiting forskolin-stimulated cAMP accumulation. Clearly, buprenorphine must stimulate the µ receptor to induce a cascade of cellular events resulting in a desensitization of the receptor which does not occur with these other opioid agonists. This may be due to buprenorphine binding differently to the µ receptor. Preliminary mutagenesis studies suggest that buprenorphine has different requirements for binding to the µ receptor than morphine, DAMGO, or levorphanol, since point mutations at key amino acids which abolish morphine, DAMGO, or levorphanol binding do not affect buprenorphine binding (29).
In the current study etorphine induced cellular responses distinct from the other opioids examined, as a 3-h pretreatment resulted in the down-regulation and complete desensitization of the µ receptor while also lowering the compensatory increases observed for forskolin-stimulated cAMP accumulation. This unique agonist profile may be due to the extremely potent effects of etorphine and may also reflect the lipophilic nature of this opioid, as membrane binding studies point to a component of etorphine binding which is essentially irreversible (30). The in vivo actions of etorphine, as assessed in animal and human behavioral studies, also indicate that etorphine is profoundly more potent than morphine by a factor of as much as 500-10,000-fold, depending upon the species tested and behavioral paradigm employed (18, 20). It has also been noted that although etorphine has a short duration of action in man, it is capable of suppressing abstinence in morphine-dependent subjects, suggesting that etorphine may be effective in the short term curbing of opioid withdrawal (20). However, etorphine has been reported as being highly addictive in rodents and primates (19) and has been reported as having abuse potential in man (20). Studies on the ability of etorphine to induce tolerance and addiction indicate that tolerance can occur in rats and monkeys, but no evidence of tolerance has been observed in humans (19). Thus, the unique and complex biochemical actions of etorphine observed in the current study are also mirrored by the unusual effects reported for in vivo behavioral studies in a variety of species.
Methadone pretreatment also desensitized the µ receptor and prevented the adaptive sensitization of forskolin-stimulated cAMP accumulation by morphine. As observed for buprenorphine, the desensitization appeared to be independent of receptor down-regulation. Methadone, like morphine, is a full agonist at the mouse µ receptor (9). The ability of methadone to desensitize the receptor may also be due to its ability to bind differently to the µ receptor than morphine, DAMGO, or levorphanol. Consistent with this notion, recent studies have delineated distinct domains of the rat µ receptor involved in the binding of µ-selective alkaloids and peptides (17, 31).
The inability of morphine and levorphanol to desensitize the µ receptor suggests that uncoupling of the receptor from adenylyl cyclase is unrelated to the tolerance development associated with these agonists. Both agonists induce dependence in rodents (1), yet only morphine causes an adaptive sensitization of adenylyl cyclase (21, 22). These results suggest that the enhanced stimulation of forskolin-stimulated cAMP accumulation is unlikely to be the biochemical basis for morphine addiction. A recent study on adaptive sensitization in HEK 293 cells has shown that the type VI isoform of adenylyl cyclase may be involved, suggesting that in these cells the compensatory response occurs downstream of the receptor (32).
The ability of buprenorphine and methadone to desensitize the µ receptor functionally may be critical for the therapeutic efficacy of these opioids in the treatment of addiction. Morphine and heroin dependence may be a consequence of the continued activation of the µ receptor, with subsequent long term cellular changes in the nervous system. These prolonged neurochemical alterations may result from the inability of opioids such as morphine to desensitize the µ receptor. The therapeutic benefit derived from methadone or buprenorphine treatment may reflect the ability of these agents to interfere with the effects initiated by morphine, even in its continued presence, thereby interrupting a cascade of cellular events. However, methadone and buprenorphine are not opioid antagonists and do not precipitate withdrawal, thereby making them useful in the treatment of narcotic addiction. Furthermore, buprenorphine treatment does not appear to induce dependence (10) despite having agonist properties at the µ opioid receptor. Buprenorphine and morphine have been proposed to induce their in vivo analgesic action in animals through distinct cellular mechanisms (33). The therapeutic actions and long term consequences in man may also be due to distinct cellular actions.
Our findings show that chemically distinct opioid agonists can bind to the µ receptor and produce different cellular consequences. The studies presented here reveal that the addictive agents, such as morphine, and the opioids used in the treatment of addiction, buprenorphine and methadone, can be distinguished by different effects on µ receptor regulation. These studies provide a model system for understanding the molecular and cellular basis of dependence and tolerance, thereby facilitating studies on the design and development of new opioids that are potent analgesics but devoid of addictive properties.
We thank Dr. Mark von Zastrow, University of California at San Francisco, for the generous gift of the epitope tagged mouse µ opioid receptor and for a critical reading of the manuscript.