![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 54, Issue 2, 419-426, August 1998
Department of Neurobiology, The Weizmann Institute of Science, 76100 Rehovot, Israel (I.N., T.A.-R., R.L., M.B., Z.V.), and The Israel Institute for Biological Research, 74100 Ness Ziona, Israel (E.H.)
 |
Summary |
|---|
Top
Summary
Introduction
Procedures
Results
Discussion
References
|
|---|
Adenylyl cyclase superactivation, a phenomenon by which chronic activation of inhibitory Gi/o-coupled receptors leads to an increase in cAMP accumulation, is believed to play an important role as a compensatory response of the cAMP signaling system in the cell. However, to date, the mechanism by which adenylyl cyclase activity is regulated by chronic exposure to inhibitory agonists and the nature of the adenylyl cyclase isozymes participating in this process remain largely unknown. Here we show, using COS-7 cells transfected with the various AC isozymes, that acute activation of the D2 dopaminergic and m4 muscarinic receptors inhibited the activity of adenylyl cyclase isozymes I, V, VI, and VIII, whereas types II, IV, and VII were stimulated and type III was not affected. Conversely, chronic receptor activation led to superactivation of adenylyl cyclase types I, V, VI, and VIII and to a reduction in the activities of types II, IV, and VII. The activity of AC-III also was reduced. This pattern of inhibition/stimulation of the various adenylyl cyclase isozymes is similar to that we recently observed on acute and chronic activation of the µ-opioid receptor, suggesting that isozyme-specific adenylyl cyclase superactivation may represent a general means of cellular adaptation to the activation of inhibitory receptors and that the presence/absence and intensity of the adenylyl cyclase response in different brain areas (or cell types) could be explained by the expression of different adenylyl cyclase isozyme types in these areas.
| |
Introduction |
|---|
|
Top
Summary
Introduction
Procedures
Results
Discussion
References
|
|---|
The
stimulation of seven-transmembrane domain G protein-coupled inhibitory
receptors leads to inhibition of AC and a consequent reduction in
cellular cAMP levels (Birnbaumer et al., 1990
). We and
others have shown, however, that when certain of these inhibitory receptors (e.g., µ-, 
-, and 
-opioid;
2-adrenergic; somatostatin) are chronically
activated, there is an increase in cAMP accumulation, which is
particularly apparent on withdrawal of the inhibitory agonist (Sharma
et al., 1975, 1977; Parsons and Stiles, 1987; Thomas and
Hoffman, 1987, 1992; Avidor-Reiss et al., 1995a, 1995b, 1996, 1997; McDermott and Sharp, 1995). This phenomenon, referred to as
AC superactivation (also termed AC overshoot, supersensitivity, or
sensitization), is believed to represent a possible biochemical substratum for the development of opiate tolerance and dependence, commonly observed on prolonged exposure to opiate drugs (Sharma et al., 1975; Nestler et al., 1993). Moreover, it
has been suggested that such regulation of AC could be a general means
of cellular adaptation to the activation of inhibitory
Gi/o-coupled receptors (Thomas and Hoffman,
1987).
Despite the fact that this phenomenon has been studied for a long time,
the mechanism by which it is evoked remains largely unknown. AC
superactivation has been described in many different cell types (Sharma
et al., 1975; Parsons and Stiles, 1987; Thomas and Hoffman,
1992; Avidor-Reiss et al., 1995a, 1997; McDermott and Sharp,
1995) and, as mentioned above, on chronic treatment with agonists of
several different inhibitory receptors, yet under similar conditions,
other cell types do not display AC superactivation (McDermott and
Sharp, 1995; Puttfarcken and Cox, 1989) or do so only under certain
stimulation conditions (Sharma et al., 1977; Jones and
Bylund, 1988; Ammer and Schulz, 1993). An explanation for this may
reside in the fact that there are several isozymes of AC that differ in
their properties and that different cell types vary in their AC isozyme
populations. To date, mRNAs encoding nine distinct isozymes of AC have
been identified, and it has been shown that they differ in their
capacity to be inhibited or stimulated by G protein i, s, and
subunits; protein kinase C; and Ca2+ (Mons
and Cooper, 1995; Sunahara et al., 1996).
Using COS cells cotransfected with µ-opioid receptor and various AC
isozymes, we observed superactivation of AC-I, -V, -VI, and -VIII
(Avidor-Reiss et al., 1996, 1997). In addition, we have shown that AC-V transfected into COS-7 cells is susceptible to superactivation after chronic activation of the -opioid or m2 muscarinic receptors (Avidor-Reiss et al., 1996). In partial
agreement with those results, Thomas and Hoffman (1996) showed that
AC-VI transfected into HEK 293 cells is susceptible to superactivation after chronic activation of the inhibitory m2 muscarinic or
D2 dopaminergic receptors. However, contrary to
our results with the µ-opioid receptor, they did not observe
superactivation of AC-I after chronic m2 activation. It therefore
became of interest to determine the pattern of differential
stimulation/inhibition of the various AC isozymes on acute and chronic
activation by various inhibitory receptors. In the present study, we
examined the effect of acute and chronic activation of the
D2 dopaminergic and m4 muscarinic receptors on
the activities of AC isozyme types I-VIII.
| |
Experimental Procedures |
|---|
|
Top
Summary
Introduction
Procedures
Results
Discussion
References
|
|---|
Materials.
2-[3H]adenine (18.0 Ci/mmol) was purchased from American Radiolabeled Chemicals (St. Louis,
MO). Ionomycin and the phosphodiesterase inhibitors
1-methyl-3-isobutylxanthine and RO-20-1724 were from Calbiochem (La
Jolla, CA). FS, cAMP, TSH, and atropine were from Sigma Chemical (St.
Louis, MO). (
)-Quinpirole, ()-sulpiride, and McN-A-343 were from
Research Biochemicals (Natick, MA). Tissue culture reagents were from
Life Technologies (Gaithersburg, MD).
Plasmids.
AC-containing plasmids (pXMD1-AC-I, pXMD1-AC-II,
pXMD1-AC-III, pXMD1-AC-IV, pXMD1-AC-V, pCMV5-neo-AC-VI, pXMD1-AC-VII,
pCMV5-neo-AC-VIII), pXMD1-gal, and pSG5-TSH have been described
previously (Avidor-Reiss et al., 1997). Other plasmids used
were the rat D2L dopamine receptor (referred to
here as the D2 receptor) cDNA in pcDNAI Amp
(obtained from Dr. S. Fuchs, Weizmann Institute, Rehovot, Israel) and
human m4 muscarinic receptor cDNA in PCD (provided by Dr. T. Bonner, National Institute of Mental Health, National Institutes of Health, Bethesda, MD).
Transient cell transfection.
Twenty-four hours before
transfection, a confluent 10-cm plate of COS-7 cells in DMEM
supplemented with 5% fetal calf serum, 100 units/ml penicillin, and
100 µg/ml streptomycin in a humidified atmosphere consisting of 5%
CO2/95% air at 37° was trypsinized and split
into four 10-cm plates. Using the DEAE-dextran chloroquine method as
described previously (Avidor-Reiss et al., 1997), the cells
were transfected, unless otherwise indicated, with 2 µg/plate of
either one of the AC isozyme cDNAs or pXMD1-gal (for mock DNA transfection), and, where indicated, with 1 µg/plate of
D2 dopamine receptor, m4 muscarinic receptor, or
TSH receptor cDNA. Twenty-four hours later, the cells were trypsinized
and recultured in 24-well plates, and after an additional 24 hr, the
cells were assayed for cAMP content (as a measure of AC activity) as
described below. Transfection efficiencies were normally in the range
of 40-80%, as determined by staining for -galactosidase activity
(Lim and Chae, 1989).
cAMP accumulation.
The assay was performed in triplicate as
described previously (Avidor-Reiss et al., 1996, 1997). In
brief, cells cultured in 24-well plates were incubated for 2 hr with
0.25 ml/well of fresh growth medium containing 5 µCi/ml
[3H]adenine and then washed three times with
0.5 ml/well of DMEM containing 20 mM HEPES, pH 7.4, and 0.1 mg/ml bovine serum albumin. This medium was replaced with 0.5 ml/well
of DMEM containing 20 mM HEPES, pH 7.4, 0.1 mg/ml bovine
serum albumin, and the phosphodiesterase inhibitors
1-methyl-3-isobutylxanthine (0.5 mM) and RO-20-1724 (0.5 mM). AC activity was stimulated in the presence or absence of the examined m4 or D2 ligands by the addition
of either FS, TSH, or ionomycin. After a 10-min (FS or TSH) or 20-min
(ionomycin) incubation at room temperature, the medium was removed, and
the reaction was terminated by the addition of perchloric acid
containing 0.1 mM unlabeled cAMP, followed by
neutralization with KOH, and the amount of
[3H]cAMP was determined by a two-step column
separation procedure as described previously (Avidor-Reiss et
al., 1996). Unless otherwise indicated, chronic agonist treatment
was achieved by incubating the cells for 18 hr with 200 nM
quinpirole (for the D2 receptor) or 100 µM McN-A-343 (for the m4 receptor), followed by agonist withdrawal (achieved by quick removal of medium and the addition of new
medium containing antagonist: 10 µM ()-sulpiride for
the D2 receptor or 10 µM atropine
for the m4 receptor, concentrations that effectively remove the agonist
used) and the addition of the appropriate AC stimulator (see above) to
assay cAMP accumulation. The incubation with
[3H]adenine took place during the last 2 hr of
the chronic exposure. Uptake of [3H]adenine
into the cells was not affected by the chronic agonist treatments.
| |
Results |
|---|
|
Top
Summary
Introduction
Procedures
Results
Discussion
References
|
|---|
Effect of D2 agonist on AC activity endogenously
expressed in COS-7 cells.
In COS-7 cells transfected with
D2 dopaminergic receptor cDNA, cAMP accumulation
could be stimulated (by ~3-fold) on activation with 1 µM FS (Fig. 1), and this
stimulated cAMP accumulation could be inhibited (by ~35%) by the
application of 200 nM of the D2 agonist quinpirole. Although a high concentration (10 µM)
of the nonselective dopaminergic receptor antagonist ()-sulpiride did not in itself affect FS-stimulated cAMP accumulation, application of
quinpirole in the presence of the antagonist completely prevented the
inhibitory effect of the agonist. Chronic pretreatment of the cells
with quinpirole followed by withdrawal of the agonist led to an
increase (of ~50%) in FS-stimulated cAMP accumulation, in agreement
with the phenomenon of AC superactivation (Sharma et al.,
1975; Thomas and Hoffman, 1987; Avidor-Reiss et al., 1995a, 1995b, 1996). Readdition of the agonist after chronic pretreatment with
quinpirole reduced cAMP accumulation (by ~30%) to a level similar to
that observed on acute application of the agonist, thus demonstrating
that the D2 receptor remains functional after this long period of chronic exposure. The addition of ()-sulpiride together with the agonist after the chronic quinpirole treatment led to
an equivalent level of AC superactivation (~50%) as that observed
when the antagonist alone was added.
|
; Sunahara et al., 1996), only
very slightly stimulated the endogenous AC activity in these cells
(Fig. 2b), suggesting that COS cells contain little if any of these AC
isozymes.
|
). To determine the nature of the regulation of the various AC
isozymes by acute and chronic exposure to D2
receptor agonists, we transfected AC types I-VIII into COS-7 cells and
monitored the effect of acute and chronic agonist treatment on AC
activity. All of the exogenous ACs were found to be functionally
active, as determined by the increase in cAMP accumulation in the cells
after stimulation with the appropriate stimulant (ionomycin for AC-I
and -VIII or TSH for the other isozymes). Moreover, Western blotting
performed for AC isozymes I, II, IV, V, VI, and VIII showed that these
isozymes are expressed in the transfected cells (data not shown).
Regulation of AC-V by D2 receptor activation.
We
have previously shown that AC-V expressed in COS-7 cells after
cotransfection with µ-opioid receptor is acutely inhibited by
activation of µ receptors and undergoes superactivation on withdrawal
from chronic treatment with µ receptor agonists (Avidor-Reiss et al., 1996). Fig. 3 shows
that similar results were obtained with the D2
dopaminergic receptor. Acute application of quinpirole strongly
inhibited TSH-stimulated cAMP accumulation (Fig. 3a), but this could be
completely prevented by inclusion of ()-sulpiride during the acute
agonist exposure. Withdrawal from chronic quinpirole treatment led to
AC superactivation (Fig. 3b). The continued presence of the antagonist
prevented the development of AC superactivation by chronic agonist
treatment, whereas chronic treatment with the antagonist alone had no
effect on cAMP accumulation. Pertussis toxin, which is known for its
ability to catalyze the ADP-ribosylation of Gi/o
proteins at cysteine residues at the carboxyl terminus of the
G
subunit, thus preventing the activation of
these G proteins (Birnbaumer et al., 1990), had a small
inhibitory effect on the level of stimulation of AC-V by TSH (Fig. 3c)
but was found to abolish both the inhibition and superactivation of
this isozyme by acute and chronic quinpirole treatment, respectively.
It therefore follows that both the inhibition and superactivation of
AC-V after short and prolonged activation of the
D2 receptor are mediated by the
Gi/o family of G proteins.
|
Differential regulation of AC isozymes by D2 receptor
activation.
In cells transfected with AC-I or AC-VIII, stimulation
with ionomycin resulted in a large increase in cAMP accumulation
compared with the unstimulated basal level (Fig.
4a). The finding that AC-I- and
AC-VIII-transfected cells show strong activation by ionomycin is in
agreement with previous reports demonstrating that
Ca2+/calmodulin has a strong stimulatory effect
on these isozymes (Mons and Cooper, 1995; Sunahara et al.,
1996) and indicates that the transfected AC-I and AC-VIII are expressed
and functionally active because, as described earlier, the endogenous
AC of COS is only very weakly stimulated by ionomycin (Fig. 2b). Acute
activation of the D2 receptor by quinpirole led
to inhibition of the ionomycin-stimulated activity of these two
isozymes, whereas chronic D2 receptor activation followed by withdrawal of the agonist led to superactivation (Fig. 4a).
|
s-activating receptors or by a constitutively active
s mutant (Mons and Cooper, 1995; Sunahara et al., 1996).
Stimulation of these AC isozymes, cotransfected together with
D2 and TSH receptors, by 0.1 µM
TSH, led to a large increase in cAMP accumulation compared with the unstimulated basal level (Fig. 4, b-d). In cells transfected with the
AC-V or -VI isozymes, acute quinpirole significantly inhibited the
TSH-stimulated cAMP accumulation, whereas withdrawal of the agonist
after chronic treatment led to superactivation. Similar results were
observed when AC-V or -VI were stimulated with 1 µM FS
(data not shown).
Taken together, the above results demonstrate that AC-I, -V, -VI, and
-VIII exhibit inhibition and superactivation by acute and chronic
D2 agonist exposure, respectively, and that the
two functions are not dependent on the agent used to stimulate the particular AC activity [i.e., FS, Ca2+
(ionomycin), or s (TSH)].
In contrast to the above results, acute exposure to quinpirole of cells
transfected with AC-II, -IV, or -VII (which are known to be closely
related according to their sequences and regulatory patterns; Cooper
et al., 1995; Sunahara et al., 1996), induced an
increase in TSH-stimulated cAMP accumulation (Fig. 4c). Moreover, after
withdrawal from chronic agonist treatment, not only was no
superactivation of these isozymes apparent, but a reduction in their
activities actually was observed.
As depicted in Fig. 4d, the activity observed in AC-III-transfected
cells was only slightly reduced by acute exposure to the D2 agonist. However, when these cells were
chronically treated with quinpirole and the agonist was withdrawn, a
further reduction in AC-III activity was observed.
Taken together, the results show that based on criteria of
agonist-induced inhibition and superactivation, the AC isozymes can be
divided into three functional groups: (1) AC-I, -V, -VI, and -VIII
exhibit inhibition by acute, and superactivation by chronic,
D2 agonist exposure; (2) AC-II, -IV, and -VII
exhibit AC stimulation on acute D2 agonist
treatment and inhibition of AC after chronic exposure; and (3) AC-III
is slightly inhibited by acute D2 agonist
treatment and further inhibited after chronic exposure.
Dose-response curves for the effects of acute and chronic agonist
exposures for AC-I, -V, and -II are depicted in Fig.
5. Significant inhibition of AC-I (Fig.
5a) and V (Fig. 5c) activities were observed in cells acutely treated
with quinpirole, with ED50 concentrations of
12 ± 3.6 and 8.7 ± 2.9 nM, respectively. A
dose-dependent superactivation was observed for both of these isozymes
after chronic exposure (Fig. 5, b and d). Interestingly, and as shown previously for the µ- and -opioid receptors (Avidor-Reiss et al., 1995a, 1995b), the ED50 concentrations
obtained for the chronic superactivation (AC-I, 37 ± 7.7 nM; AC-V, 23 ± 5.9 nM) were higher than
those observed for acute inhibition. Contrary to AC-I and -V, AC-II
showed a dose-dependent increase in activity on acute exposure to
quinpirole (Fig. 5e) and a dose-dependent inhibition of its activity
after chronic treatment with the D2 agonist (Fig. 5f), with ED50 values of 65 ± 24 and
12 ± 2.8 nM, respectively.
|
), the chronic
D2 agonist-induced superactivation of this
isozyme is reversible and could be abolished by prolonged exposure to the antagonist. Pretreatment for 18 hr with quinpirole followed by its
withdrawal and incubation with ()-sulpiride resulted in a nearly
total abolishment of the superactivated state of AC-V and recovery of
the original level of AC-V activity (data not shown).
|
Differential regulation of AC isozymes by m4 muscarinic receptor activation. Cells transfected with the m4 muscarinic receptor and the various AC isozymes were also tested on acute and chronic exposure to the muscarinic agonist McN-A-343 (Fig. 7). As seen previously (Fig. 4), AC isozymes I and VIII were stimulated by ionomycin (Fig. 7a), whereas activation of the transfected TSH receptor activated the other AC isozymes (Fig. 7, b-d).
|
| |
Discussion |
|---|
|
Top
Summary
Introduction
Procedures
Results
Discussion
References
|
|---|
We and others have reported that the chronic application of
agonists of various inhibitory seven-transmembrane G protein-coupled receptors produces a time- and concentration-dependent increase (as
opposed to the decrease observed on acute activation of these receptors) in the activity of AC (Sharma et al., 1975;
Parsons and Stiles, 1987; Thomas and Hoffman, 1992; Avidor-Reiss
et al., 1995a, 1995b). This phenomenon, originally described
on chronic treatment of cells with opiates (Sharma et al.,
1975), is particularly evident on withdrawal of the agonist and has
been referred to as AC superactivation, or "overshoot". In the case
of opiates, this phenomenon has been hypothesized to represent, at
least in part, a biochemical basis of opiate drug addiction (Sharma
et al., 1975; Nestler et al., 1993).
However, the actual situation seems to be more complicated, as we now
know that there are several subtypes of AC that differ in their
activation and inhibition patterns (by s, i/o, , and so
on). To date, nine AC isozymes have been cloned (AC types I-IX); the
activities of these AC isozymes seem to be stimulated by
Gs, although to different extents. These ACs
can be categorized according to sequence and functional similarities
into five classes: (1) AC-I and -VIII are stimulated by
Ca2+/calmodulin; (2) AC-V and -VI are inhibited
by low levels of Ca2+; (3) AC-II, -IV, and -VII
are activated by G
subunits in the presence
of activated Gs and have been reported to be
affected by activation of protein kinase C; (4) AC-III has been
reported to be either stimulated or inhibited by
Ca2+/calmodulin in the presence of
Gs; and (5) AC-IX has thus far been found to
be stimulated only by Gs (Mons and Cooper, 1995; Sunahara et al., 1996; Zimmermann and Taussig, 1996).
It was recently shown that acute activation of the m2 receptor inhibits
the activity of AC types I and VI and stimulates that of type II,
whereas chronic activation of this receptor results in superactivation
of AC-VI, with no effect on the AC-I and -II isozymes (Thomas and
Hoffman, 1996). D2 receptor activation also was
shown to lead to AC-VI superactivation. However, the effects of acute
and chronic activation of inhibitory dopaminergic or muscarinic
receptors on other AC isozymes have been for the most part unknown.
Interestingly, as we show here for the D2 and m4 receptors, and similar to what was observed by Avidor-Reiss et al. (1997) for the µ-opioid receptor, most of the AC isozymes are affected differentially by acute or chronic agonist activation. AC
types I, V, VI, and VIII are inhibited by acute agonist application and
exhibit superactivation on chronic treatment, whereas the activities of
types II, IV, and VII are stimulated on acute exposure and inhibited on
chronic agonist treatment; AC-III was only slightly inhibited by acute
agonist application, whereas its activity was further reduced on
chronic agonist treatment. Because the AC isozymes differ in the
parameters that define their stimulation and inhibition characteristics, the fact that various tissues, brain areas, and cell
types are known to vary in their repertoire of AC isozyme populations
(Mons and Cooper, 1994, 1995) may offer an explanation to the different
regulatory effects on AC activity of Gi/o-coupled inhibitory receptor activation depending on the tissues examined. Indeed, whereas Gi/o-coupled receptor activation
normally inhibits AC activity, it has been shown that opioid (Olianas
and Onali, 1995) or inhibitory muscarinic (Olianas and Onali, 1996)
receptor activation can stimulate AC activity in the rat olfactory bulb and that cannabinoid receptor activation stimulates AC activity in the
globus pallidus and in heart (Hillard et al., 1990; Maneuf and Brotchie, 1997). This also may explain the observation of different
levels of AC superactivation in various cell types (Puttfarcken and
Cox, 1989; McDermott and Sharp, 1995). For example, the COS-7 cells
used in this study have been shown to contain at least the AC-VII
isozyme (Premont, 1994). However, because AC-VII was shown here not to
undergo superactivation, the fact that COS cells were found here to
exhibit endogenous AC superactivation implies that at least one of the
AC isozymes that does exhibit superactivation (AC-I, -V, -VI, or -VIII)
must also be endogenously present in COS cells. The AC-I and VIII
isozymes can be eliminated a priori because they are
activated by Ca2+, and ionomycin was not able to
activate AC in nontransfected COS cells (Fig. 2b). It therefore follows
that one or both of AC-V and AC-VI must be endogenously present in COS.
It would be interesting to examine whether various brain areas also
exhibit different patterns of AC superactivation and, if so, whether
this can be correlated to localization patterns of the AC isozymes in
the brain.
The data obtained here, taken together with previously published
results (Avidor-Reiss et al., 1997), indicate that the
specific pattern of AC modulation observed here (with transfected COS
cells) seems to be a general means of cellular adaptation to the
activation of inhibitory receptors. It is conceivable that other cell
systems/brain areas (which may differ in their G protein subunits and
various signal transduction components) also may exhibit AC
isozyme-specific mechanisms for adaptation to inhibitory receptor
activation, even though variations in the final result could be
expected, depending on the AC isozyme pattern and G protein subunit
composition.
In this regard, Thomas and Hoffman (1996) did not find superactivation
of AC-I by chronic m2 muscarinic agonist application. Because both m2
and m4 inhibit AC via Gi/o proteins, their
chronic effects on AC-I could be expected to be similar. Indeed, in
preliminary experiments, we observed AC-I superactivation on chronic m2
agonist application in COS cells transfected with the appropriate
receptors, although it was much weaker than that produced in
m4-transfected cells (data not shown). The difference between our
observation of slight AC-I superactivation as opposed to that of Thomas
and Hoffman that this isozyme is not superactivated by chronic m2 activation might be accounted for by the difference in the cell lines
used in the two studies (HEK 293 cells as opposed to COS in the current
study). For example, HEK 293 cells show a high level of background
superactivation (in the nontransfected cells) on stimulation with FS
(used as an AC stimulant by Thomas and Hoffman), making it more
difficult to observe a weak increase in AC activity. Another possible
explanation for the difference is the fact that these cell lines can
differ in their Gi protein subunit populations.
Indeed, G dimers are known to have a role
in AC superactivation (Avidor-Reiss et al., 1996; Thomas and
Hoffman, 1996), and we have recently shown that different subunits
display different profiles for activation of AC-II (Bayewitch et
al., 1998). Different i populations also can affect coupling to
various receptor types in these cell lines, as shown, for example,
regarding the differential coupling of m2 versus m4 (Migeon et
al., 1995).
It should be noted that the transfected m4 receptor is the only
muscarinic receptor subtype that will be activated by application of
the agonist McN-A-343, even though this ligand is not selective for the
m4 receptor, because no muscarinic receptors are endogenously present
in COS cells. This agonist was selected for these experiments due to
the fact that it does not lead to significant down-regulation of
muscarinic receptors (Heldman E and Vogel Z, unpublished
observations). On the other hand, although carbachol (the more
classically used nonspecific muscarinic agonist) also led to
superactivation of AC-VI via activation of m2 (Thomas and Hoffman,
1996), this ligand has been shown to down-regulate all types of
muscarinic receptors, including m4 (Maloteaux and Hermans, 1994).
The findings of the presence or absence of superactivation of the
various AC isozymes on chronic D2 receptor
activation are of interest from several perspectives. First, in the
brain, AC-V is known to be highly expressed in the nucleus accumbens
(Glatt and Snyder, 1993; Mons and Cooper, 1994). This region, which is rich in D2 receptors, is one of the key nuclei in
the "dopamine reward pathway" in the brain (Koob, 1996). According
to the dopamine reward theory, addiction phenomena would have as a
common biochemical denominator a release of dopamine in the nucleus
accumbens, which then would exert its effects predominantly via local
D2 receptors (White et al., 1991;
Ranaldi and Beninger, 1994). The positive reinforcement generated by
the feeling of well-being produced by this release of dopamine would
contribute to the development of dependence on the event that caused
the initial release, thus initiating the vicious cycle of the
development of addiction. If a drug that augments extracellular
dopamine levels (e.g., opiates, which enhance its release, or cocaine,
which inhibits its uptake) is present in the system for a long period
of time, it is possible to envisage that the biochemical mechanism
underlying the short and long term effects of this neurotransmitter
could involve modulation of the activity of AC, particularly of the
isozyme predominantly present in the nucleus accumbens: AC-V. This also
could explain why certain drugs, which are addictive when taken over
the long term, can in "naive" users, who are using the drug for the
first time, actually have effects that are diametrically opposed to those observed in chronic abusers (Gulati, 1995).
In this study, we found that chronic activation of the inhibitory m4
muscarinic receptor also led to a similar pattern of superactivation/inhibition of the various AC isozyme types. The finding
that this is a characteristic common to several (e.g., m4,
D2, µ-opioid), if not all, inhibitory G
protein-coupled receptors, is of important physiological relevance. For
instance, the basis of the action of many pharmaceutical drugs is the
activation of inhibitory receptors. To illustrate this point,
D2 dopaminergic agonists such as bromocriptine or
pergolide, alone or in conjunction with the dopamine precursor
L-DOPA, are routinely used as antiparkinsonian agents
(Lieberman and Goldstein, 1985; Gimenez-Roldan et al., 1997). The abrupt discontinuation (withdrawal) of these
antiparkinsonian drugs has been reported to lead to the emergence of
the neuroleptic malignant syndrome, usually associated with neuroleptic
(dopamine antagonist) medications and generally believed to be related
to blockage of dopamine receptors in the brain (Olmsted, 1988; Ebadi et al., 1990). It thus is conceivable that the chronic use
of drugs that activate inhibitory receptors, even those that are not
considered to have addictive properties, could lead to superactivation of AC or, more specifically, of particular AC isozymes. Indeed, one
study reports that unilateral lesions of the nigrostriatal dopamine
pathway in rats (a model of parkinsonism) enhanced the sensitivity of
striatal AC to dopamine stimulation and that this AC hypersensitivity
was further enhanced on prolonged L-DOPA administration (Groppetti et al., 1986).
Furthermore, among the downstream effects of the phenomenon of AC
superactivation, the augmented cAMP concentration resulting from the
increased level of AC activity will affect protein phosphorylation, thereby increasing phosphorylation of, among others, cAMP-responsive element binding protein, which could in turn affect transcription factor regulation (Nestler et al., 1993). It thus is evident
that the implications of the chronic use of inhibitory receptor
agonists as therapeutic treatments may be more far reaching than they
would seem at first glance and that possible indirect effects of such drugs arising from AC regulation should be examined more closely.
| |
Acknowledgments |
|---|
We are grateful to the following scientists for the kind donation of the indicated plasmids: Dr. Sara Fuchs, Weizmann Institute, Rehovot, Israel (rat D2L dopamine receptor); Dr. Shinji Kosugi, Kyoto University, Kyoto, Japan (rat TSH receptor); Dr. Tom Bonner, National Institute of Mental Health, National Institutes of Health, Bethesda, MD (human m4 muscarinic receptor); Dr. Alfred Gilman, University of Texas Southwestern Medical Center, Dallas, TX (AC-IV); Dr. Franz-Werner Kluxen, University of Dusseldorf, Dusseldorf, Germany (pXMD1 plasmid and pXMD1-gal); Dr. Thomas Pfeuffer, Heinrich-Heine University, Dusseldorf, Germany (AC-I, AC-II, and AC-V in pXMD1); Dr. Randy Reed, Johns Hopkins School of Medicine, Baltimore, MD (AC-III); and Dr. John Krupinski and Dr. Peter A. Watson, Geisinger Clinic, Danville, PA (AC-VI, AC-VII, and AC-VIII).
| |
Footnotes |
|---|
Received December 29, 1997; Accepted May 11, 1998
This work was supported by the National Institute of Drug Abuse (Grant DA06265), German-Israeli Foundation for Scientific Research and Development, Forschheimer Center for Molecular Genetics, and Israeli Ministries of Science and Arts and of Absorption (fellowship to I.N.).
Send reprint requests to: Dr. Zvi Vogel, Department of Neurobiology, Weizmann Institute of Science, Rehovot 76100, Israel. E-mail: bnvogel{at}weizmann.weizmann.ac.il
| |
Abbreviations |
|---|
AC, adenylyl cyclase; DMEM, Dulbecco's modified Eagle's medium; HEK, human embryonic kidney; FS, forskolin; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; TSH, thyroid-stimulating hormone.
| |
References |
|---|
|
Top
Summary
Introduction
Procedures
Results
Discussion
References
|
|---|
.
J Biol Chem
271:
21309-21315-Opioid receptor-transfected cell lines: modulation of adenylyl cyclase activity following acute and chronic opioid treatments.
FEBS Lett
361:
70-74[Medline]. subunits.
J Biol Chem
273:
2273-2276
9-tetrahydrocannabinol on adenylate cyclase activation in heart.
J Pharmacol Exp Ther
252:
1075-10822-adrenergic receptor-mediated sensitization of forskolin-stimulated cyclic AMP production in HT29 cells.
J Biol Chem
263:
14236-14244-cell RINm5F.
Cell Signal
7:
277-285[Medline]. and Go subunits.
J Biol Chem
270:
16070-16074 subunits in transducing enhanced activity of the type VI isoform.
Mol Pharmacol
49:
907-914[Abstract].This article has been cited by other articles:
|
|
 |
|
  C. M. Darnell, J. Thompson, D. Stromberg, L. Roy, and P. Sheeran Effect of Low-Dose Naloxone Infusion on Fentanyl Requirements in Critically Ill Children Pediatrics, May 1, 2008; 121(5): e1363 - e1371. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. B. Penn and J. L. Benovic Regulation of Heterotrimeric G Protein Signaling in Airway Smooth Muscle Proceedings of the ATS, January 1, 2008; 5(1): 47 - 57. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Roy, J. Wang, R. Charboneau, H. H. Loh, and R. A. Barke Morphine Induces CD4+ T Cell IL-4 Expression through an Adenylyl Cyclase Mechanism Independent of the Protein Kinase A Pathway J. Immunol., November 15, 2005; 175(10): 6361 - 6367. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H.-M. Zhang, D.-P. Li, S.-R. Chen, and H.-L. Pan M2, M3, and M4 Receptor Subtypes Contribute to Muscarinic Potentiation of GABAergic Inputs to Spinal Dorsal Horn Neurons J. Pharmacol. Exp. Ther., May 1, 2005; 313(2): 697 - 704. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. J. Watts Molecular Mechanisms for Heterologous Sensitization of Adenylate Cyclase J. Pharmacol. Exp. Ther., July 1, 2002; 302(1): 1 - 7. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. K. Sunahara and R. Taussig Isoforms of Mammalian Adenylyl Cyclase: Multiplicities of Signaling Mol. Interv., June 1, 2002; 2(3): 168 - 184. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Rubenzik, E. Varga, D. Stropova, W. R. Roeske, and H. I. Yamamura Expression of alpha -Transducin in Chinese Hamster Ovary Cells Stably Transfected with the Human delta -Opioid Receptor Attenuates Chronic Opioid Agonist-Induced Adenylyl Cyclase Superactivation Mol. Pharmacol., November 1, 2001; 60(5): 1076 - 1082. [Abstract] [Full Text]
|
|
|
|
|
|
M. G. Cumbay and V. J. Watts Heterologous Sensitization of Recombinant Adenylate Cyclases by Activation of D2 Dopamine Receptors J. Pharmacol. Exp. Ther., June 1, 2001; 297(3): 1201 - 1209. [Abstract] [Full Text]
|
|
) with 1 µM FS
(basal, unstimulated), in the presence or absence of 200 nM of the agonist quinpirole (quin) and/or
10 µM of the antagonist (
) with
200 nM quinpirole followed by a rapid wash and readdition
(at the beginning of the cAMP accumulation assay) of either 10 µM sulpiride (sulp; withdrawal conditions)
or 200 nM quinpirole (quin) or by the
readdition of 200 nM quinpirole together with 10 µM sulpiride (quin + sulp). Data represent
the mean ± standard error of triplicate determinations of a
representative experiment of three experiments that gave similar
results. **, p < 0.01 versus FS-stimulated
cAMP accumulation.
),
after stimulation in the presence of acute 200 nM
quinpirole (
), and after withdrawal (by rapid wash and addition of
(
