(Received for publication, September 13, 1995)
From the
Using CHO cells stably transfected with rat µ-opioid
receptor cDNA, we show that the µ-agonists morphine and
[D-Ala, N-methyl-Phe
,Gly-ol
]enkephalin
are negatively coupled to adenylylcyclase and inhibit
forskolin-stimulated cAMP accumulation. Chronic exposure of cells to
morphine leads to the rapid development of tolerance. Withdrawal of
morphine or [D-Ala
, N-methyl-Phe
,Gly-ol
]enkephalin
following chronic treatment (by wash or addition of the antagonist
naloxone) leads to an immediate increase in cyclase activity
(supersensitization or overshoot), which is gradually reversed upon
further incubation with naloxone. Phosphodiesterase inhibitors do not
affect the overshoot, indicating that it results from cyclase
stimulation rather than phosphodiesterase regulation. Morphine's
potency to inhibit cAMP accumulation is the same before and after
chronic treatment, suggesting that the apparent tolerance results from
cyclase activation, rather than from receptor desensitization. The
similar kinetics of induction of tolerance and overshoot support this
idea. Both the overshoot and acute opioid-induced cyclase inhibition
are blocked by naloxone and are pertussis toxin-sensitive, indicating
that both phenomena are mediated by the µ-receptor and
G
/G
proteins. The supersensitization is
cycloheximide-insensitive, indicating that it does not require newly
synthesized proteins. This is supported by the rapid development of
supersensitization. Taken together, these results show that
µ-transfected cells can serve as a model for investigating
molecular and cellular mechanisms underlying opiate drug addiction.
Pharmacological studies have defined three types of opioid
receptors, µ, , and
, that differ in their affinity for
various opioid ligands and in their distribution in the nervous system
(Herz, 1993). The three types of opioid receptors have recently been
cloned and are all members of the seven-transmembrane domain
GTP-binding protein (G protein)-coupled (
)receptor
superfamily. Activation of all three types of opioid receptors leads to
inhibition of adenylylcyclase (AC) activity, and this effect is
mediated through pertussis toxin (PTX)-sensitive G proteins (for review
see Reisine and Bell(1993); Uhl et al.(1994)). Much less is
currently known about the cellular and molecular mechanisms that
accompany prolonged opiate exposure leading to opiate tolerance and,
upon removal of the agonist, to opiate withdrawal.
Various
neuroblastoma cell lines have been used to study the regulation of AC
activity by prolonged opioid exposure. Sharma et al. (1975,
1977) showed that chronic exposure of NG108-15 neuroblastoma
glioma hybrid cells (a cell line that expresses mainly
-opioid receptors) to morphine leads to an increase in AC activity
(see also Hamprecht(1977)) and suggested that this phenomenon may
underlie the tolerant state. Withdrawal of the agonist (i.e. by adding the antagonist naloxone or by washing, which relieves
the inhibition of AC exerted by the agonist) revealed the phenomenon of
AC supersensitization or overshoot. Additional processes have been
suggested to underlie tolerance to opioids in this cell line. These
include receptor desensitization and down-regulation, as well as
uncoupling of the receptor from G proteins (for reviews see Loh et
al.(1988) and Way(1993)).
The effect of prolonged opioid
exposure on µ-opioid receptor signaling has been less well
characterized. In the human neuroblastoma SH-SY5Y cell line, which
expresses both µ- and -opioid receptors (Kazmi and Mishra,
1987), chronic activation of µ-opioid receptors by morphine was
shown to lead to partial desensitization (Yu et al., 1990; Yu
and Sadée, 1988) and, upon withdrawal of the
opiate agonist, to overshoot in the production of cAMP (Ammer and
Schulz, 1993a; Wang et al., 1994; Yu et al., 1990).
No receptor down-regulation or changes in guanyl nucleotide regulation
of agonist affinity were reported in this cell line after chronic
morphine treatment (Yu et al., 1990). On the other hand, in
the mouse 7315c cell line, which contains a homogeneous population of
µ-opioid receptors (Frey and Kebabian, 1984), chronic morphine
treatment induced a rapid loss of µ-opioid receptor-mediated
inhibition of AC, which was accompanied by a loss of guanyl nucleotide
regulation of agonist affinity (Puttfarcken et al., 1988).
Reduction in receptor number developed more slowly and required a
higher concentration of morphine (Puttfarcken and Cox, 1989;
Puttfarcken et al., 1988). In addition, no AC
supersensitization was observed in these cells following the chronic
opiate exposure (Puttfarcken and Cox, 1989).
The mechanism by which
chronic opioid treatment increases AC activity and cellular cAMP levels
is not known. According to some authors, AC supersensitization could be
the result of a secondary regulatory process during the chronic
exposure to opiates, the nature of which is not yet understood (Nestler et al., 1993; Sharma et al., 1975). According to
other authors, the increase in cAMP accumulation could be due to the
ability of opioid receptors to couple to G and to stimulate
AC directly, whereas the chronic opioid exposure would attenuate the
G
-mediated AC opioid inhibition (Crain and Shen, 1990; Shen
and Crain, 1992; Wang and Gintzler, 1994). Ammer and Schulz (1993a,
1993b) suggested that AC supersensitivity may be due to enhanced
coupling of the prostaglandin E
receptor to G
in NG108-15 cells and showed that it is correlated with
elevated levels of functionally intact G
in SH-SY5Y cells,
whereas Griffin et al.(1985) suggested that it may be due to
the loss of tonic G
-mediated inhibition of AC.
Chinese
hamster ovary (CHO) cells have been reported to be a suitable system
for expressing opioid receptors, with the expressed receptors showing
the expected ligand-binding selectivity (Fukuda et al., 1993;
Raynor et al., 1994). Moreover, expressed - and
-receptors were shown to inhibit AC activity through a
PTX-sensitive G protein (Avidor-Reiss et al., 1995; Law et
al., 1994). In this report, we show that this cell system is very
useful for the study of the effects of chronic exposure to opioids,
including the development of tolerance and dependence. Using CHO cells
transfected with the µ-opioid receptor, we show that (i)
µ-opioid agonists markedly inhibit forskolin (FS)-stimulated AC
activity; (ii) chronic morphine treatment leads to tolerance in the
ability of the drug to reduce the level of FS-induced cAMP; and (iii)
removal of the agonist following chronic treatment reveals a large
overshoot of AC activity. This overshoot does not require protein
synthesis and is responsible, at least in part, for the tolerance
observed. These opioid receptor-transfected cells are a good model for
studying the mechanisms of opiate drug addiction.
The application of
µ-opioid agonists to CHO-µ/1 cells led to a marked inhibition
of FS-stimulated cAMP accumulation in these cells. The µ-selective
opioid agonists DAMGE and morphine, as well as the nonselective opioid
agonist etorphine, were very effective in inhibiting FS-stimulated cAMP
accumulation, reaching levels of 85% inhibition with 100 nM of added opioid agonist. The
- and
-selective opioid
agonists U69593 and [D-penicillamine
, D-penicillamine
]enkephalin, respectively,
were not active (Fig. 1a). The nonselective opioid
antagonist naloxone (1 µM) did not inhibit FS-stimulated
cAMP accumulation and blocked the inhibitory effects of the
µ-agonists (Fig. 1b). In agreement with the lack of
specific opioid binding in CHO-K1 parental cells, morphine (1
µM) did not lead to any inhibition of cAMP accumulation in
these cells (data not shown). Altogether, these results demonstrate
that µ-agonists activate the µ-receptor in the transfected
cells, leading to inhibition of FS-stimulated cAMP accumulation.
Figure 1:
Effect of various opioid ligands on
FS-stimulated cAMP accumulation in CHO-µ cells. a,
inhibition of FS-stimulated cAMP accumulation by various opioid
agonists. The agonists, etorphine (etorph), morphine (morph), U69593, DAMGE, and
[D-penicillamine, D-penicillamine
]enkephalin (DPDPE),
were used at a concentration of 100 nM. The data are expressed
as the means ± S.E. of four experiments. 100% defines
FS-stimulated cAMP accumulation in cells that have not been treated
with opioids. b, blockade by naloxone of the opioid agonist
inhibition of FS stimulation. Morphine and DAMGE were used at
concentrations of 100 and 10 nM, respectively; naloxone (nalox) was used at 1 µM. The data show the means
± S.E. of a representative experiment. 100% defines
FS-stimulated cAMP accumulation in cells that have not been treated
with opioids and is equivalent to 1561 ± 42 cpm. cont,
control.
As
can be seen in Fig. 2, both morphine and DAMGE inhibit
FS-stimulated cAMP accumulation in a dose-dependent manner. However,
their ability to inhibit AC is different. Morphine is much less potent,
with an EC of 41 ± 7 nM, whereas DAMGE has
an EC
of 1.7 ± 0.8 nM. The maximal level
of inhibition (90-98% inhibition of FS-stimulated cAMP
accumulation) was obtained with 0.032-1 µM of DAMGE
and with 1 µM of morphine. Pretreatment of the cells with
PTX blocked the inhibitory effects of DAMGE on AC activity, indicating
the involvement of the G
/G
type of G proteins
in mediating this process.
Figure 2:
Effects of acute and chronic exposures to
DAMGE and morphine on cAMP accumulation in CHO-µ cells. a,
inhibition of FS-stimulated cAMP accumulation by various concentrations
of morphine () and DAMGE (
). b, induction of AC
supersensitization following chronic (4 h) exposure to DAMGE or
morphine and their withdrawal by the addition of naloxone prior to the
AC assay (see ``Experimental Procedures''). Where indicated
(
), cells were pretreated with PTX (100 ng/ml) for 24 h, and the
effect of DAMGE was studied. The data are expressed as the means
± S.E. of three or four experiments.
Figure 3: Effect of chronic treatment with naloxone on agonist-induced AC supersensitization. CHO-µ cells were treated for 4 h with 1 µM of DAMGE or morphine (morph) in the presence (shaded bars) or absence (white bars) of 1 µM naloxone (nalox). After this time, all cultures were washed once, and 1 µM naloxone was added just prior to the addition of FS. The data show the means ± S.E. of a representative experiment. Control (cont) represents cells that have not been pretreated with opioids.
Figure 6:
Effect of morphine pretreatment on
morphine inhibition of FS-stimulated cAMP accumulation. CHO-µ cells
were either pretreated with morphine (0.1 µM; ) for
4 h or served as controls (
). Withdrawal was achieved by four
washes as described under ``Experimental Procedures.''
Morphine was then readded at the indicated concentrations and cAMP
accumulation (a) and percent of inhibition (b) were
determined. The data are expressed as the means ± S.E. of a
representative experiment.
Fig. 2b shows
that both morphine and DAMGE evoke AC supersensitization in a
dose-dependent manner. The maximal level of overshoot was obtained when
the cells were exposed to 0.32-1 µM of DAMGE or to 1
µM of morphine. However, whereas DAMGE induced AC
supersensitization with an EC of 34 ± 6
nM, morphine induced AC supersensitization with an EC
of 102 ± 21 nM. Thus, as with the acute
inhibition of AC, DAMGE is more potent than morphine. Moreover, for
both ligands, the induction of AC supersensitization requires chronic
incubation with higher concentrations of agonists than those needed to
induce the acute opioid inhibition of FS-stimulated cAMP accumulation.
However, although DAMGE was 24-fold more potent than morphine in
inhibiting AC activity, it was only 3-fold more potent in evoking AC
supersensitization.
Pretreatment of the cells with PTX, which
prevents the opioid inhibition of AC (see Fig. 2a),
also blocked the naloxone-induced overshoot of cAMP accumulation in the
chronically opioid-treated cells (Fig. 2b). This
finding demonstrates that the overshoot phenomenon is linked to the
chronic activation of the opioid receptors, as well as to the
activation of the PTX-sensitive G/G
proteins
during the chronic opioid exposure.
To exclude the possibility of involvement of phosphodiesterase regulation in the cAMP overshoot phenomenon, we routinely included phosphodiesterase inhibitors in the assay. In Fig. 4, we show that the presence of phosphodiesterase inhibitors (1-methyl-3-isobutylxanthine alone or together with RO-20-1724) during the 10-min assay period increased the level of cAMP accumulation both in control and in chronic DAMGE-treated cells. However, the extent of the overshoot, in comparison with cells that were not pretreated with opioid agonist, was not affected by the phosphodiesterase inhibitors. This suggests that the AC supersensitization involves changes in AC activity rather than in phosphodiesterase activity.
Figure 4:
Effects of phosphodiesterase inhibitors on
FS-stimulated cAMP accumulation. FS-stimulated cAMP accumulation was
determined in control and in chronically (4 h, 1 µM DAMGE)
pretreated CHO-µ cells under withdrawal conditions. The
phosphodiesterase inhibitors 1-methyl-3-isobutylxanthine and
RO-20-1724 were present during the assay only where indicated.
Withdrawal was achieved by a rapid wash and the addition of naloxone to
all cultures. The data show the average values (in cpm of
[H]cAMP) of a representative experiment. The
ratio between the levels of cAMP in FS-stimulated chronic DAMGE-treated
and control cells is provided. PDE, phosphodiesterase; IBMX, 1-methyl-3-isobutylxanthine.
Fig. 5depicts the kinetics of
the effect of morphine on the activity of AC in the presence of the
drug and after its withdrawal. It shows that the cells have to be
exposed to morphine (0.32 µM) for at least 4 h in order to
reach the maximal level of AC supersensitization. This level is
maintained even after 35 h of exposure to the drug. The time needed to
achieve half-maximal supersensitization was equivalent to 2 h.
Chronic treatment with morphine also leads to the development of
tolerance in the µ-receptor-transfected CHO cells. As can be seen
from Fig. 5, the kinetics of the appearance of tolerance are
very similar to those of the overshoot. Namely, acute morphine (0.32
µM) inhibited FS-stimulated cAMP accumulation by 80%.
However, despite the continued presence of morphine, FS-stimulated cAMP
accumulation slowly recovered (after 4-10 h with morphine) to
approximately the level of untreated control cells. This tolerance to
morphine is not due to a loss of the capacity of morphine to inhibit
AC, because removal of morphine by adding naloxone (withdrawal
conditions) markedly increased the level of FS-stimulated cAMP
accumulation. Thus, morphine was still able to inhibit FS-stimulated
cAMP accumulation and to a similar extent (around 80%) as before the
chronic treatment.
Figure 5:
Time course of chronic morphine
pretreatment on FS-stimulated cAMP accumulation. CHO-µ cells were
incubated with 0.32 µM morphine for the periods indicated.
At the end of the preincubations, the morphine was removed (together
with the [H]adenine), and morphine (0.32
µM;
) or naloxone (1 µM;
) was
added to the cultures in assay medium, followed by the addition of FS
for the 10-min assay period. The data are expressed as the means
± S.E. of a representative experiment. 100% (1283 ± 55
cpm) represents the FS-stimulated cAMP value in cells not pretreated
with morphine.
To confirm the observation that morphine does not lead to receptor desensitization, we checked the ability of increasing concentrations of morphine to inhibit FS-stimulated cAMP accumulation both before and after chronic treatment with morphine (Fig. 6a). Four sequential washes were found to be effective in removing the bound agonist, as was deduced from the inability of added naloxone to induce a further increase in FS-stimulated cAMP accumulation (data not shown). Chronic treatment with morphine (0.1 µM) followed by these four sequential washes led to a 3.5-fold increase in the level of FS-stimulated cAMP accumulation. The readdition of morphine for the 10-min assay period inhibited FS-stimulated cAMP accumulation in both the control and the chronic morphine-treated cells in a dose-dependent manner. Moreover, as shown in Fig. 6b (by normalizing the data obtained in the experiment shown in Fig. 6a), it is clear that morphine is able to inhibit FS-stimulated cAMP accumulation following chronic morphine treatment to a similar extent as in control cells.
Although the addition of an antagonist during the assay after the
chronic treatment reveals the phenomenon of AC supersensitization (due
to blockade of the inhibition induced by the agonist), prolonged
exposure to the antagonist, after the chronic treatment, results in a
gradual reduction of FS-stimulated cAMP accumulation (Fig. 7).
The half-life of the disappearance of the AC supersensitization was
30 min, and after 4 h, the levels of cAMP in the cells had
returned to their original values (i.e. those obtained under
normal, nonopioid-treated conditions). It is therefore apparent that
the occurrence of AC supersensitization depends upon sustained
activation of the receptor and that the cells maintain the ability to
rapidly return to the original levels of cyclase activity and cAMP
concentration following the withdrawal of the opioid agonist.
Figure 7: Reversibility of AC supersensitization following long term treatment with naloxone. CHO-µ cells were pretreated for 18 h with 1 µM DAMGE. Naloxone (1 µM) was added for the indicated times prior to (and remained present during) the 10-min AC assay. Control (cont) represents cells that were not pretreated with DAMGE or naloxone and is defined as 100% (586 ± 32 cpm). The data are expressed as the means ± S.E. of a representative experiment.
In
order to clarify whether protein synthesis is involved in the induction
of AC supersensitization, we checked the effect of the protein
synthesis inhibitor cycloheximide on this phenomenon. We found that
incubation with 10 or 50 µM cycloheximide did not affect
the ability of the cells to induce AC supersensitization (Fig. 8). Under these conditions, cycloheximide was able to
inhibit >85% of the incorporation of
[H]-labeled amino acids (which were added to the
cells in parallel to the opioid agonist). Some inhibition of
FS-stimulated AC activity (
24%) was observed in chronic
opioid-treated cells exposed to 50 µM cycloheximide, but
the same level of inhibition was also observed in cells that had not
been treated with opioids. This result suggests that the opioid-induced
AC supersensitization does not require newly synthesized proteins.
Figure 8: Effect of cycloheximide on AC supersensitization. 10 or 50 µM cycloheximide (CHX) was added to the cultures 1 h before and was present during a 4-h chronic pretreatment with 1 µM DAMGE. FS-stimulated cAMP accumulation was determined in control and in DAMGE-pretreated CHO-µ cells under withdrawal conditions. The data are expressed as the means ± S.E. of a representative experiment. 100% is equivalent to the level of cAMP in FS-stimulated control cells not treated with cycloheximide.
In this study, we have used CHO cells expressing the rat µ-opioid receptor to gain information on µ-receptor signal transduction and its role in opiate addictive processes. We have demonstrated that the µ-transfected cells are able to interact with µ-agonists and that this interaction leads to several processes of AC regulation, including the inhibition of FS-stimulated cAMP accumulation, apparent tolerance, and the development of AC supersensitization.
Long term exposure of the transfected cells to opioids can offer a model system to study the molecular mechanisms of drug tolerance and withdrawal. In their review, Nestler et al.(1993) defined tolerance as ``a reduced effect upon repeated exposure to a constant drug dose, or the need for an increased dose to maintain the same effect.'' Using the µ-opioid receptor-transfected CHO cell line, we have observed that chronic morphine treatment led to an increase in the level of FS-stimulated cAMP accumulation, which could be overcome by higher levels of the drug. This result indicates the development of tolerance to the effect of morphine.
Withdrawal of opioid agonists after chronic treatment
(either by wash or by the addition of an antagonist) markedly enhances
FS-stimulated cAMP accumulation, indicating that AC was under
inhibition during the chronic treatment and that this treatment led to
AC supersensitization. Moreover, we have observed that the µ-opioid
receptors did not desensitize during the chronic morphine treatment and
that the ability of morphine to inhibit the FS-stimulated AC remained
the same both before and after the chronic exposure. These results
demonstrate that the tolerance observed is a result of the capacity of
the cells to undergo opioid-induced AC supersensitization and is not
due to changes in the receptor capacity to transduce the signal. These
results are consistent with the model suggested by Sharma et
al.(1975) for the role of AC regulation in the development of
morphine tolerance and dependence in NG108-15 cells. On the other
hand, these results differ from the observations reported by Frey and
Kebabian(1984), who detected full desensitization by morphine in mouse
7315c cells, and from those of Yu and Sadée(1988)
and Yu et al. (1990), who detected partial desensitization
(4-fold increase in the EC in SH-SY5Y cells).
The
degrees of both AC inhibition and supersensitization by DAMGE or
morphine are concentration-dependent. Although application of low
concentrations of agonist was sufficient to inhibit FS-stimulated cAMP
production, the supersensitization required pretreatment with higher
doses of the drugs. A similar observation was also obtained for the
-agonist U69593 applied to CHO cells transfected with the
-receptor (Avidor-Reiss et al., 1995). This difference in
concentrations needed for the two phenomena suggests that activation of
the overshoot requires the recruitment of threshold amounts of
signaling components, the nature of which remains to be clarified. It
may involve spare receptors, as suggested by Law et al.(1994),
or other mechanisms (see below). Nevertheless, this difference in
agonist concentration needed for the two phenomena is in agreement with
the clinical observation that the development of physical dependence
requires prolonged exposure to relatively high doses of opiates
(Bhargava, 1994).
It has been reported that various µ-ligands could differentially activate the µ-opioid receptor, leading to different effects on the AC signaling system. For example, although both morphine and DAMGE inhibited AC activity in SH-SY5Y cells (through the µ-receptors), it was shown that morphine but not DAMGE was able to induce AC supersensitization (Ammer and Schulz, 1993a). In contrast, in our µ-transfected CHO cells, both DAMGE and morphine were able to induce AC supersensitization. However, although DAMGE was 24-fold more potent than morphine in inhibiting AC activity, it was only 3-fold more potent in evoking AC supersensitization. This difference could in part be due to the ability of DAMGE but not of morphine to induce receptor desensitization (Carter and Medzihradsky, 1993). In this regard, it is worthwhile to note that Zimprich et al.(1995) reported a difference in the ability of DAMGE to induce desensitization of the inhibition of FS-stimulated cAMP accumulation with two isoforms of the rat µ-opioid receptor. The longer of the two forms of µ-opioid receptor, which is equivalent to the one used in our study, was more prone to desensitization than the shorter form. No information was provided to account for the mechanism of this desensitization phenomenon.
The increase in cAMP that occurs in intact NG108-15 and in several other cells following withdrawal after chronic treatment with substances that stimulate receptors negatively coupled to AC has been suggested to be due to the increased AC activity detected in membrane preparations from these cells (Sabol and Nirenberg, 1979; Sharma et al., 1975; Thomas and Hoffman, 1987). Moreover, in our experiments, the relative increase in FS-stimulated cAMP accumulation was the same in both the presence and absence of phosphodiesterase inhibitors. Thus, the increase in cAMP appears to result from an up-regulation of AC activity rather than from opioid-induced inhibition of phosphodiesterase. Nevertheless, it is worth noting that in intact NG108-15 cells but not in membrane preparations chronic treatments with carbachol or opiate agonists were shown to result in an attenuated degradation rate constant for cAMP, suggesting a contribution of phosphodiesterase regulation to the cAMP overshoot observed in these cells (Law and Loh, 1993; Thomas et al., 1990).
Several reports suggest that activation of opioid
receptors could either inhibit or stimulate AC activity, depending on
opioid agonist concentration. Opioid agonists at low concentrations
were shown to activate the G pathway and stimulate AC
activity, whereas high concentrations of opioid agonists activate
G
and inhibit AC activity in myenteric plexus (Wang and
Gintzler, 1994) and in dorsal root ganglion cells (Crain and Shen,
1990). Using µ-opioid receptor-transfected CHO cells, we found that
low concentrations of DAMGE or morphine (e.g. as low as 0.1
nM) did not stimulate AC activity. Moreover, PTX pretreatment
abolished the inhibitory opioid effect without revealing any opioid
activation (see Fig. 2a), suggesting that the acute
opioid effects in the CHO cells are mediated through
G
/G
and not through G
proteins.
The PTX sensitivity clearly demonstrates that chronic opioid
activation, like acute opioid inhibition, has to be mediated through
both opioid receptors and PTX-sensitive G proteins in order to produce
the elevated cyclase activity observed following removal of the
agonist. The PTX sensitivity is in agreement with the observation that
PTX prevents the development of opiate dependence when administered to
rats (Parolaro et al., 1990). Because µ-receptors
expressed in CHO-K1 cells were found to activate G,
G
and G
(Chakrabarti et
al., 1995), this PTX sensitivity could be attributable to these
G
and/or G
protein
subunits. Similarly,
in NG108-15 cells, where the
-receptor is coupled to
G
, G
, and G
proteins (McKenzie
and Milligan, 1990; Roerig et al., 1992), PTX abolished the AC
supersensitivity (Griffin et al., 1985).
Regarding the
mechanism of the AC supersensitization, it has been suggested, based on
results obtained using S49 mouse lymphoma cells chronically treated
with a somatostatin analog, that the mechanism involved in AC
supersensitization could be attributed to a decrease in the
concentration of intracellular cAMP, due to the inhibition of AC by
G, which would affect PKA activity in the cells (Thomas and
Hoffman, 1988). However, this possibility does not fit with our finding
that elevated intracellular cAMP levels, as obtained using cholera
toxin or dibutyryl cAMP, were unable to block the development of AC
supersensitivity (data not shown). It has recently been suggested that
chronic opiate exposure could up-regulate AC expression. For example,
Matsuoka et al.(1994) demonstrated increased mRNA of AC type
VIII in the amygdala and locus coeruleus of chronic morphine-treated
rats. However, we show here that cycloheximide at concentrations that
inhibit protein synthesis does not inhibit AC supersensitivity.
Moreover, the development of the overshoot is relatively rapid, with
half-maximal supersensitivity achieved within 2 h, kinetics that do not
seem to be consistent with induction of new protein synthesis. Ammer
and Schulz (1993b) suggested that AC supersensitivity in
morphine-treated NG108-15 cells may be due to enhanced coupling
of prostaglandin E
receptors to G
. However, as
described here for CHO cells and by Ammer and Schulz (1993a) for
SH-SY5Y cells, AC supersensitization is also being detected when AC is
activated directly by FS in a receptor-independent manner.
Another
possible mechanism that could be related to AC supersensitivity is the
activation of AC by the dimers released from
G
/G
proteins upon opioid receptor activation.
dimers have been shown to activate a variety of signal
transduction pathways (for review see Clapham and Neer(1993)),
including AC of type II (Federman et al., 1992) and of type IV
(Gao and Gilman, 1991). Additional experiments will have to be
performed to determine the involvement of the
dimers in AC
supersensitization. Nevertheless, the finding of a supersensitized
state up to 2 h after withdrawal from chronic treatment is consistent
with a model requiring a secondary regulatory process that could be
mediated by the
dimers (i.e. phosphorylation)
rather than a direct action on the AC of the various types of G protein
subunits upon receptor activation.
In summary, using CHO cells transfected with rat µ-opioid receptor, we have shown that acute µ-agonist exposure leads to inhibition of AC, whereas chronic exposure leads to the development of supersensitization of AC activity. This activation of AC is reversible and is gradually lost following removal of the agonist. Our results suggest that the primary mechanism for morphine tolerance in chronic morphine-treated CHO cells is the underlying increase in AC activity, resulting in the necessity to use higher concentrations of agonist to reach the same low levels of cAMP in the cell as before. These observations are in line with the phenomena of opiate action, tolerance, and withdrawal.