 |
 Previous Article | Next Article 
The Journal of Neuroscience, June 1, 2001, 21(11):3715-3720
Involvement of Spinal Protein Kinase C in the Attenuation of
Opioid µ-Receptor-Mediated G-Protein Activation after Chronic
Intrathecal Administration of
[D-Ala2,N-MePhe4,Gly-Ol5]Enkephalin
Minoru
Narita1, 2,
Hirokazu
Mizoguchi2,
Michiko
Narita2,
Hiroshi
Nagase2, 3,
Tsutomu
Suzuki1, and
Leon F.
Tseng2
1 Department of Toxicology, Hoshi University, School of
Pharmacy, Tokyo, 142-8501, Japan, 2 Department of
Anesthesiology, Medical College of Wisconsin, Milwaukee, Wisconsin
53226, and 3 Pharmaceutical Research Laboratories, Toray
Industries Incorporated, Kamakura 248-8555, Japan
 |
ABSTRACT |
The present study was designed to investigate the role of a protein
kinase C (PKC) isoform in the uncoupling of the µ-opioid receptor
from G-proteins after repeated intrathecal injection of a
selective µ-receptor agonist,
[D-Ala2,N-MePhe4,Gly-ol5]enkephalin
(DAMGO), in the spinal cord of mice. The activation of G-proteins by
opioids was measured by monitoring the
guanosine-5'-o-(3-[35S]thio)triphosphate
([35S]GTP S) binding. Mice were injected
intrathecally with saline or DAMGO once a day for 1-7 d. At 24 hr
after every injection the spinal cord membranes were prepared for the
assay. The enhanced [35S]GTPS binding by
µ-agonists DAMGO, endomorphin-1, or endomorphin-2 was attenuated
clearly in spinal cord membranes obtained from mice that were
treated intrathecally with DAMGO for 5 and 7 d, but not for 1 or
3 d. By contrast, no change in levels of
[35S]GTPS binding induced by the  -receptor
agonist SNC-80 or  -receptor agonist U-50,488H was noted in membranes
obtained from mice that were treated with DAMGO. Concomitant
intrathecal administration of a specific PKC inhibitor Ro-32-0432 with
DAMGO blocked the attenuation of DAMGO-induced G-protein activation
that was caused by chronic DAMGO treatment. Western blotting analysis
showed that chronic DAMGO treatment increased the levels of PKC, but
not PKC , PKC I, and PKCII isoforms, in spinal cord membranes.
Furthermore, mice lacking PKC failed to exhibit the desensitization
of the DAMGO-stimulated [35S]GTPS binding after
repeated DAMGO injection. These findings indicate that repeated
intrathecal administration of DAMGO may activate the PKC isoform and
in turn cause a desensitization of µ-receptor-mediated G-protein
activation in the mouse spinal cord.
Key words:
µ-opioid receptor; protein kinase C; phosphorylation; tolerance; G-protein; spinal cord
| |
INTRODUCTION |
The opioid agonists modulate a
number of physiological processes including pain, reward, stress, and
immune responses via the stimulation of various opioid receptors
(Mansour et al., 1988 ). One of the major opioid receptor types, the
µ-opioid receptor, was cloned in 1993 and classified as a
G-protein-coupled receptor (Chen et al., 1993). Opioids mainly inhibit
cyclic AMP formation, close voltage-sensitive
Ca2+ channels, and open
K+ channels via the stimulation of
Gi/o proteins (Childers, 1991). Over the past few
years opioids, including µ-opioids, also have been shown to activate
the phosphoinositide-signaling cascade in a variety of cells and neural
tissues (Chen and Huang, 1991; Mangoura and Dawson, 1993; Smart et al.,
1995; Ueda et al., 1995).
The receptor-coupled hydrolysis of membranal phosphoinositides,
particularly phosphatidyl inositol 4,5-bisphosphate
(PIP2), yields two intracellular second
messengers, diacylglycerol (DAG) and inositol triphosphate
(IP3). DAG activates protein kinase C (PKC), and
IP3 mobilizes Ca2+
after binding with cytoplasmic IP3
receptors (Berridge, 1987; Fisher et al., 1992, 1993). These processes
appear to be an important part of the signal transduction mechanism for
controlling the various cellular events in the CNS.
PKC is a key regulatory enzyme that modulates both presynaptic and
postsynaptic neuronal function, synthesis and release of neurotransmitters, and the regulation of receptors. PKC has expanded into a family of closely related protein, which can be subdivided and
classified on the basis of certain structural and biochemical similarities. Several PKC isoforms, especially conventional PKCs (cPKCs) including , I, II, and that are
Ca2+-dependent and activated by both
phosphatidylserine (PtdSer) and DAG, have been identified in neurons of
the spinal cord; in each case immunocytochemistry has shown that they
are concentrated in the superficial laminae of the dorsal horn
(Malmberg et al., 1997; Martin et al., 1999).
We previously reported that activation of PKC in the spinal cord is
implicated in the development of spinal antinociceptive tolerance to
µ-opioid receptor agonists in mice (Narita et al., 1995). The
µ-opioid receptor contains several potential phosphorylation sites
(Chen et al., 1993; Kaufman et al., 1995). It has been hypothesized that activated PKC directly phosphorylates the opioid receptor and
subsequently induces the uncoupling of opioid receptors from G-proteins
(Pei et al., 1995). However, there is little or no direct evidence to
support the contention that repeated stimulation of µ-opioid
receptors by opioid µ-agonists produces the PKC-dependent uncoupling
of µ-opioid receptors from G-proteins. In the present study we
therefore investigated whether repeated intrathecal injection of
a highly selective µ-opioid receptor agonist,
[D-Ala2,N-MePhe4,Gly-ol5]enkephalin
(DAMGO), causes any changes in the increase of µ-opioid receptor-stimulated
guanosine-5'-o-(3-[35S]thio)triphosphate
([35S]GTPS) binding and the levels of
membrane-located cPKC isoforms in the mouse spinal cord. We also
investigated whether repeated intrathecal injections of DAMGO could
affect the DAMGO-stimulated [35S]GTPS
binding in mice lacking the PKC isoform.
| |
MATERIALS AND METHODS |
Animals. Male CD-1 mice (Charles River Breeding
Laboratories, Wilmington, MA) and PKC knock-out mice (The Jackson
Laboratory, Bar Harbor, MA), which were maintained on C57BL/6 and 129Sv
mixed genetic backgrounds as described previously (Abeliovich et al., 1993), were used. Animals were housed five per cage in a room maintained at 22 ± 0.5°C with an alternating 12 hr light/dark cycle.
[35S]GTPS binding assay. The spinal
cord was homogenized in ice-cold Tris-Mg2+
buffer containing (in mM) 50 Tris-HCl, pH 7.4, 5 MgCl2, and 1 EGTA for the
[35S]GTPS binding assay. The
homogenate was centrifuged at 48,000 × g at 4°C for
10 min. The pellets were resuspended in
[35S]GTPS binding assay buffer
containing (in mM) 50 Tris-HCl, pH 7.4, 5 MgCl2, 1 EGTA, and 100 NaCl and recentrifuged at
48,000 × g at 4°C for 10 min. The final pellets were
resuspended in assay buffer as membranous fractions for the
[35S]GTPS binding. The reaction was
initiated by the addition of membrane suspension (3-8 µg of protein
for each assay as determined by the method of Bradford, 1976) into the
assay buffer with the opioid receptor agonists, 30 µM guanosine-5'-diphosphate (GDP), and 50 pM [35S]GTPS
(1000 Ci/mmol; Amersham, Arlington Heights, IL). The suspensions were
incubated at 25°C for 2 hr, and the reaction was terminated by
filtering through Whatman GF/B glass filters, which had been soaked
previously in a soaking buffer of 50 mM Tris-HCl,
pH 7.4, and 5 mM MgCl2 at
4°C for 2 hr, using a Brandel cell harvester (model M-24; Brandel,
Gaithersburg, MD). Then the filters were washed three times with 5 ml
of an ice-cold Tris-HCl buffer, pH 7.4, and transferred to
scintillation counting vials containing scintillation cocktail, 0.5 ml
of Soluene-350 (Packard Instrument, Meriden, CT), and 4 ml of Hionic
Fluor (Packard Instrument). The radioactivity in the samples was
determined with a liquid scintillation analyzer (model 1600CA, Packard
Instrument). Nonspecific binding was measured in the presence of 10 µM unlabeled GTPS.
Western blotting. The spinal cord was removed quickly after
decapitation of mice and homogenized in ice-cold buffer A containing (in mM) 20 Tris-HCl, pH 7.5, 2 EDTA, 0.5 EGTA, and 1 phenylmethylsulfonyl fluoride plus 25 µg/ml leupeptin, 0.1 mg/ml
aprotinin, and 0.32 M sucrose. The homogenate was
centrifuged at 1,000 × g for 10 min, and the
supernatant was ultracentrifuged at 100,000 × g for 30 min at 4°C. The resulting supernatant was retained as the cytosolic fraction. The pellets were washed with buffer B (buffer A without sucrose) and homogenated in buffer B with 1% Triton X-100. After incubation for 45 min, soluble fractions were obtained by
ultracentrifugation at 100,000 × g for 30 min and then
were retained as membranous fractions for Western blotting. An aliquot
of tissue sample was diluted with an equal volume of 2×
electrophoresis sample buffer (Protein Gel Loading Dye-2X, Amresco,
Solon, OH) containing 2% SDS and 10% glycerol with 0.2 M dithiothreitol. Proteins (5-20 µg/lane as
determined by the method of Bradford, 1976) were separated by size on
4-20% SDS-polyacrylamide gradient gel by using the buffer system of
Laemmli (1970) and were transferred to nitrocellulose membranes in
Tris-glycine buffer containing 25 mM Tris and 192 mM glycine. For immunoblot detection of PKC
isozymes the membranes were blocked in Tris-buffered saline (TBS)
containing 5% nonfat dried milk (Bio-Rad Laboratories, Hercules, CA)
for 1 hr more at room temperature with agitation. The membrane was
incubated with primary antibody diluted in TBS [PKC, PKCI,
PKCII, and PKC at ratios of 1:4000 (), 1:3000 (I and
II), 1:1000 (); Santa Cruz Biotechnology, Santa Cruz, CA]
containing 5% nonfat dried milk overnight at 4°C. Then the membrane
was washed twice for 5 min and twice for 10 min in Triton X-TBS (TTBS)
containing TBS and 0.05% Triton X-100, followed by 2 hr of incubation
at room temperature with horseradish peroxidase-conjugated goat
anti-rabbit IgG (Southern Biotechnology Associates, Birmingham, AL)
diluted 1:10,000 in TBS containing 5% nonfat dried milk. After this
incubation the membranes were washed twice for 5 min and then three
times for 10 min in TTBS. The antigen-antibody peroxidase complex was detected finally by enhanced chemiluminescence (Pierce, Rockford, IL)
according to the manufacturer's instructions and visualized by
exposure to Amersham Hyperfilm (Amersham Life Sciences, Arlington Heights, IL).
Intrathecal injection. Intrathecal administration was
performed by following the method described by Hylden and Wilcox
(1980), using a 25 µl Hamilton syringe with a 30 gauge needle.
Injection volumes were 5 µl for intrathecal injection.
Drugs. The drugs used were DAMGO (Bachem California,
Torrance, CA); endomorphin-1 (Tocris Cookson, Ballwin, MO);
endomorphin-2 (Tocris Cookson);
D-Phe-Cys-Tyr-D-Try-Orn-Thr-Pen-Thr-NH2
(CTOP; Bachem California);
[D-Pen2,5]enkephalin (DPDPE;
Bachem California);
[D-Ala2]deltorphin II
(Molecular Research Laboratories, Durham, NC); SNC-80 (Tocris Cookson);
naltrindole (NTI; Research Biochemicals, Natick, MA); U-50,488H
(Research Biochemicals); U-69,593 (Research Biochemicals); nor-BNI
(Research Biochemicals); Ro-32-0432 (Calbiochem-Novabiochem, San
Diego, CA); GTPS (Research Biochemicals); and GDP (Sigma, St. Louis, MO).
Statistical analysis. The data are expressed as the
mean ± SEM. The statistical significance of differences between
the groups was assessed with a one-way ANOVA, followed by Dunnett's
test (comparison with a control group) or Newman-Keuls test
(comparisons between multiple groups).
| |
RESULTS |
Effects of chronic intrathecal treatment with DAMGO on increases of
the [35S]GTPS binding induced by µ-, -,
and -opioid agonists in the spinal cord
Under these conditions groups of mice were injected intrathecally
with saline (5 µl/mouse) or DAMGO (50 ng/mouse) once a day for 1-7
d. At 24 hr after the last injection of each group the spinal cord
membranes were prepared for each assay. As shown in Table
1, the µ-opioid receptor agonists
DAMGO, endomorphin-1, and endomorphin-2, each at 10 µM,
produced marked increases in [35S]GTPS binding to spinal cord
membranes obtained from mice treated intrathecally with saline for
5 d. To determine whether these increases of the
[35S]GTPS binding were mediated by
the stimulation of µ-opioid receptors, we studied the effects
of the µ-opioid receptor antagonist CTOP on µ-agonist-stimulated
[35S]GTPS binding. The increase of
[35S]GTPS binding by DAMGO,
endomorphin-1, or endomorphin-2 was blocked completely by coincubation
with 10 µM CTOP (Table 1). The incubation of CTOP alone
had no effect on the basal [35S]GTPS
binding level (data not shown). Figure 1
shows the changes in the 10 µM DAMGO-induced increase in
[35S]GTPS binding to spinal cord
membranes after the daily intrathecal treatments with saline or DAMGO.
The increased [35S]GTPS binding
induced by DAMGO (10 µM) to spinal cord membranes was not
affected by intrathecal treatment with DAMGO (50 ng) for 1-3 d but was
attenuated significantly in mice treated for 5 and 7 d. The
concentration-response curve and also the maximal increase for the
DAMGO-induced increases in [35S]GTPS
binding to spinal cord membranes were attenuated significantly in mice
treated intrathecally with DAMGO for 5 d as compared with saline-treated mice (Fig. 2). Chronic
intrathecal treatment with DAMGO for 5 d also significantly
reduced the levels of [35S]GTPS
binding to spinal cord membranes induced by either endomorphin-1 or
endomorphin-2 (Fig. 3).
View larger version (14K):
[in this window]
[in a new window]
|
Figure 1.
Time course effect of repeated intrathecal
injection of DAMGO on the DAMGO-induced increase in
[35S]GTPS binding to spinal cord membranes.
Groups of mice were injected intrathecally with saline (5 µl/mouse)
or DAMGO (50 ng/mouse) once a day for 1-7 d. At 24 hr after every
injection the spinal cord membranes were prepared for each assay. The
assay was conducted with or without DAMGO (10 µM). Data
points represent the mean ± SEM for 4-17 independent samples.
The basal [35S]GTPS binding in mice treated
with saline for 1, 3, 5, and 7 d was 31.2 ± 3.6, 31.8 ± 1.1, 29.2 ± 1.2, and 30.3 ± 1.9 fmol/mg protein,
respectively. On the contrary, the basal
[35S]GTPS binding in mice treated with DAMGO
for 1, 3, 5, and 7 d was 33.2 ± 1.2, 33.2 ± 7.9, 28.6 ± 1.3, and 31.1 ± 1.5 fmol/mg protein, respectively.
The statistical significance of differences between the groups was
assessed with a one-way ANOVA, followed by Dunnett's test.
*p < 0.05 versus saline-treated group.
F values of one-way ANOVA in 5 and 7 d treatment
are F(1,29) = 27.659 and
F(1,6) = 11.381, respectively.
|
|
View larger version (17K):
[in this window]
[in a new window]
|
Figure 2.
Concentration-response curve for the
DAMGO-induced increase in [35S]GTPS binding to
spinal cord membranes obtained from chronically saline- or
DAMGO-treated mice. Groups of mice were injected intrathecally with
saline (5 µl/mouse) or DAMGO (50 ng/mouse) once a day for 5 d.
At 24 hr after the last injection the spinal cord membranes were
prepared for each assay. The assay was conducted with or without DAMGO
(0.001-10 µM). Data points represent the mean ± SEM for 3-17 independent samples. The basal
[35S]GTPS binding in mice treated with saline
and DAMGO for 5 d was 29.2 ± 1.2 and 28.6 ± 1.3 fmol/mg protein, respectively. The statistical significance of
differences between the groups was assessed with a one-way ANOVA,
followed by Dunnett's test. *p < 0.05 versus
saline-treated group. F values of one-way ANOVA in 0.1, 0.3, 1, 3, and 10 µM DAMGO are
F(1,25) = 32.158, F(1,13) = 17.020, F(1,25) = 25.531, F(1,13) = 11.379, and
F(1,29) = 27.659, respectively.
|
|
View larger version (18K):
[in this window]
[in a new window]
|
Figure 3.
Effect of repeated intrathecal injection of DAMGO
on the endomorphin-induced increase in
[35S]GTPS binding to spinal cord membranes.
Groups of mice were injected intrathecally with saline (5 µl/mouse)
or DAMGO (50 ng/mouse) once a day for 5 d. At 24 hr after the last
injection the spinal cord membranes were prepared for each assay. The
assay was conducted with or without endomorphin-1 and endomorphin-2
(0.1-10 µM). Data represent the mean ± SEM for
three to nine independent samples. The basal
[35S]GTPS binding in mice treated with saline
and DAMGO for 5 d was 29.2 ± 1.2 and 28.6 ± 1.3 fmol/mg protein, respectively. The statistical significance of
differences between the groups was assessed with a one-way ANOVA,
followed by Dunnett's test. *p < 0.05 versus
saline-treated group. F values of one-way ANOVA in 10 µM endomorphin-1, 0.1 µM endomorphin-2, and
10 µM endomorphin-2 are
F(1,13) = 9.135, F(1,4) = 9.351, and
F(1,10) = 14.037, respectively.
|
|
The -opioid receptor agonists DPDPE,
[D-Ala2]deltorphin II, and
SNC-80 produced robust stimulation of
[35S]GTPS binding at 10 µM in mice treated intrathecally with saline for 5 d
(Table 1), These effects were blocked completely by coincubation with
the specific -opioid receptor antagonist NTI (Table 1). Incubation
of the -opioid receptor agonist U-50,488 H or U-69,593 increased the
[35S]GTPS binding to the spinal cord
membranes obtained from mice treated intrathecally with saline for
5 d (Table 1). The increase of
[35S]GTPS binding induced by a
-agonist was abolished by the -opioid receptor antagonist nor-BNI
(Table 1). Under these conditions the increases of the
[35S]GTPS binding stimulated by -
and -opioid receptor agonists were not affected by chronic
intrathecal treatment with DAMGO (Fig.
4). The levels of
[35S]GTPS binding stimulated by
DPDPE,
[D-Ala2]deltorphin II,
SNC-80, U-50,488H, or U-69,593 at 10 µM in mice treated
with DAMGO for 5 d were similar to those found in mice treated
with saline for 5 d.
View larger version (15K):
[in this window]
[in a new window]
|
Figure 4.
Effect of repeated intrathecal injection of DAMGO
on the -opioid receptor agonists DPDPE-,
[D-Ala2]deltorphin II-, and SNC-80- or
-opioid receptor agonists U-50,488H- and U-69,593-induced increases
in [35S]GTPS binding to spinal cord membranes.
Groups of mice were injected intrathecally with saline (5 µl/mouse)
or DAMGO (50 ng/mouse) once a day for 5 d. At 24 hr after the last
injection the spinal cord membranes were prepared for each assay. The
assay was conducted with or without 10 µM DPDPE,
[D-Ala2]deltorphin II
(DELT), SNC-80, U-50,488H (U-50),
and U-69,593 (U-69). Data represent the mean ± SEM
for three to nine independent samples. The basal
[35S]GTPS binding in mice treated with saline
and DAMGO for 5 d was 29.2 ± 1.2 and 28.6 ± 1.3 fmol/mg protein, respectively. The statistical significance of
differences between the groups was assessed with a one-way ANOVA,
followed by Dunnett's test.
|
|
The role of PKC in the development of µ-opioid receptor
desensitization induced by chronic treatment with DAMGO
Then the role of PKC in the development of µ-opioid receptor
desensitization was investigated. Groups of mice were
treated intrathecally with DAMGO in the absence or presence of a
specific PKC inhibitor, Ro-32-0432 (250 ng/mouse). As shown in eFigure 5, treatment of Ro-32-0432 coadministered
with DAMGO completely blocked the decrease of the DAMGO-stimulated
[35S]GTPS binding induced by repeated
DAMGO injection for 5 d. Treatment with Ro-32-0432 alone had no
effect on the DAMGO-stimulated
[35S]GTPS binding (Fig. 5).
View larger version (21K):
[in this window]
[in a new window]
|
Figure 5.
Blockade of PKC inhibitor on the decreased level
of the DAMGO-stimulated [35S]GTPS binding
induced by repeated intrathecal injection of DAMGO. Groups of mice were
treated intrathecally with saline (5 µl/mouse) or DAMGO (50 ng/mouse)
in the absence or presence of the specific PKC inhibitor Ro-32-0432
(250 ng/mouse) once a day for 5 d. At 24 hr after the last
injection the spinal cord membranes were prepared for each assay. The
assay was conducted with or without DAMGO (0.1-10 µM).
Each column represents the mean ± SEM for 3-17
samples. The basal [35S]GTPS binding in mice
treated with saline alone, DAMGO alone, DAMGO combined with Ro-32-0432,
and Ro-32-0432 alone for 5 d was 29.2 ± 1.2, 28.6 ± 1.3, 29.8 ± 3.1, and 30.1 ± 1.8 fmol/mg protein,
respectively. The statistical significance of differences among the
groups was assessed with a one-way ANOVA, followed by Newman-Keuls
test. *p < 0.05 versus saline-treated group;
#p < 0.05 versus DAMGO-treated group.
F values of one-way ANOVA in 0.1, 1, and 10 µM DAMGO are F(3,35) = 11.096, F(3,35) = 8.486, and
F(3,39) = 16.199, respectively.
|
|
Identification of cPKC isoforms involved in µ-opioid receptor
desensitization induced by chronic treatment with DAMGO
The levels of cPKC isoforms in membranes of the spinal cord after
the repeated injection of DAMGO were analyzed quantitatively by Western
blot analysis. Groups of mice were treated intrathecally with DAMGO (50 ng/mouse) or saline for 5 d; on day 6 spinal cord membranes were
prepared for the assay. Immunoreactivity of the PKC isoform was
enhanced significantly by DAMGO treatment as compared with that of
saline treatment, whereas the other three cPKC isoforms, PKC,
PKCI, and PKCII, were not altered (Fig. 6A,B).
View larger version (20K):
[in this window]
[in a new window]
|
Figure 6.
The levels of cPKC isoforms in spinal cord
membranes after repeated intrathecal injections of DAMGO. Groups of
mice were injected intrathecally with saline (5 µl/mouse) or DAMGO
(50 ng/mouse) once a day for 5 d. At 24 hr after the last
injection the spinal cord membranes were prepared for immunoblotting.
A, Representative Western blot of PKC, PKCI,
PKCII, and PKC isoform proteins. B, Changes in the
membrane-located protein levels of cPKC isoforms in spinal cord
membranes after repeated intrathecal injections of DAMGO. Each
column represents the mean ± SEM for four samples.
The statistical significance of differences between the groups was
assessed with a one-way ANOVA, followed by Dunnett's test.
*p < 0.05 versus saline-treated group. The
F value of one-way ANOVA in the PKC isoform is
F(1,6) = 46.531.
|
|
Lack of µ-opioid receptor desensitization induced by chronic
treatment with DAMGO in PKC knock-out mice
To investigate further the role of the PKC isoform in the
process of chronic DAMGO-induced desensitization, we next investigated whether repeated intrathecal injections of DAMGO caused no effect on
the DAMGO-stimulated [35S]GTPS
binding in mice lacking the PKC isoform. Immunoblot analysis showed
that no PKC protein could be detected in both membranous and
cytosolic fractions of the spinal cord obtained from PKC knock-out
mice (Fig. 7A).
Immunoreactivities of the other three cPKC isoforms, PKC, PKCI,
and PKCII, in mice lacking the PKC isoform were detectable to the
same degree as the wild type (data not shown), confirming the
specificity of these mutant mice. As shown in Figure 7B,
repeated intrathecal treatment with DAMGO once a day for 7 d
consecutively produced a 20.0% reduction in the
[35S]GTPS binding stimulated by DAMGO
at 10 µM to spinal membranes of wild-type mice
(p < 0.05 vs saline pretreatment). However, the
same chronic treatment with DAMGO did not affect significantly the
increases of the DAMGO-stimulated
[35S]GTPS binding in mice lacking the
PKC isoform (Fig. 7C).
View larger version (17K):
[in this window]
[in a new window]
|
Figure 7.
A, Immunoblot analysis of protein
levels of membranous or cytosolic fractions of PKC in the spinal
cord obtained from wild-type and PKC knock-out (KO)
mice. B, Effect of repeated intrathecal injections of
DAMGO on the DAMGO-induced increase in
[35S]GTPS binding to spinal cord membranes from
wild-type mice. Groups of wild-type mice were injected intrathecally
with saline (5 µl/mouse) or DAMGO (50 ng/mouse) once a day for 7 d. At 24 hr after the last injection the spinal cord membranes were
prepared for each assay. The assay was conducted with or without DAMGO
(0.1-10 µM). Data represent the mean ± SEM for 11 independent samples. The basal [35S]GTPS
binding in wild-type mice treated with saline and DAMGO for 7 d
was 31.1 ± 2.8 and 27.5 ± 1.2 fmol/mg protein,
respectively. The statistical significance of differences between the
groups was assessed with a one-way ANOVA, followed by Dunnett's test.
*p < 0.05 versus saline-treated group.
F values of one-way ANOVA in 0.1, 1, and 10 µM DAMGO are F(1,20) = 5.954, F(1,20) = 10.640, and
F(1,20) = 8.244, respectively.
C, No development of the µ-opioid receptor-mediated
downregulation by repeated DAMGO injection in mice lacking the PKC
isoform. Groups of PKC knock-out mice were injected intrathecally
with saline (5 ml/mouse) or DAMGO (50 ng/mouse) once a day for 7 d. At 24 hr after the last injection the spinal cord membranes were
prepared for each assay. The assay was conducted with or without DAMGO
(0.1-10 µM). Data represent the mean ± SEM for 11 independent samples. The basal [35S]GTPS
binding in PKC knock-out mice treated with saline and DAMGO for
7 d was 26.7 ± 2.1 and 29.1 ± 3.2 fmol/mg protein,
respectively. The statistical significance of differences between the
groups was assessed with a one-way ANOVA, followed by Dunnett's
test.
|
|
| |
DISCUSSION |
Chronic daily intrathecal DAMGO treatment attenuates the increases
of [35S]GTPS bindings stimulated by µ-opioid,
but not - or -opioid, agonists in the mouse spinal cord
We found in the present study that mice repeatedly intrathecally
injected with a selective µ-opioid receptor agonist DAMGO showed the
time-dependent desensitization of a µ-opioidergic system to activate
G-proteins in the spinal cord. This desensitization was selective to
µ-, but not to - and -, opioidergic systems to activate
G-proteins. These results indicate that chronic intrathecal injection
of µ-opioids uncouples the spinal µ-opioid receptor from
G-proteins, resulting in the desensitization of a µ-opioidergic system in the mouse spinal cord.
PKC in the spinal cord is involved in µ-opioid receptor
desensitization induced by chronic treatment with DAMGO
Tolerance to opioid analgesia is a major drawback in clinical use
of these analgesics. It has been proposed that chronic adaptive molecular mechanisms in opioid tolerance involve some protein kinases,
including protein kinase A (PKA), PKC, and G-protein-coupled receptor
kinase. We have reported previously that intrathecal pretreatment with
a membrane-permeable PKC inhibitor, calphostin C, but not with a
specific PKA inhibitor, blocks the development of the antinociceptive
tolerance to intrathecally administered DAMGO in mice (Narita et al.,
1995). We also found that the activation of PKC by phorbol esters
attenuates either the opioid-induced antinociceptive effect or the
G-protein activation (Narita et al., 1996, 1997). These findings
indicate that activated PKC is involved in the process of the
development of opioid tolerance.
Treatment of a specific PKC inhibitor Ro-32-0432 coadministered with
DAMGO into the spinal cord completely blocked the desensitization of
spinal µ-opioidergic systems induced by chronic DAMGO treatment. Considering that the activation of PKC induces the phosphorylation of
several membrane proteins, the receptor itself seems to be one of the
good targets. Indeed, the opioid receptors can be phosphorylated by PKC
(Pei et al., 1995). It is therefore most likely that the activation of
PKC by chronic treatment with µ-opioids may lead to the
phosphorylation of membrane-bound µ-opioid receptors and in turn
causes the uncoupling of µ-opioid receptors from G-proteins after
chronic treatment with µ-opioids in the spinal cord.
Identification of the PKC isoform involved in µ-opioid
receptor desensitization induced by chronic treatment with DAMGO
Recent cloning studies revealed that the PKC family consists of at
least 12 isoforms possessing distinct differences in structure, substrate requirement, expression, and localization that therefore may
underlie diverse physiological functions (Way et al., 2000). In the
present study we thus examined whether repeated intrathecal administration of DAMGO for 5 d could alter the levels of
membrane-bound PKC isoforms, especially cPKCs, in the mouse spinal
cord. In spinal cord membranes obtained from mice injected repeatedly
with DAMGO for 5 d, the upregulation of the PKC isoform in the
spinal cord was clearly noted. Three other cPKC isoforms, PKC,
PKCI, and PKCII, were not altered significantly after repeated
administration of DAMGO. The results are consistent with the previous
finding that chronic spinal administration of morphine caused a
significant increase in levels of PKC in spinal cord membranes of
rats (Mao et al., 1995). These data raise the possibility that the
PKC isoform dominantly modulates an autoinhibition of spinal
µ-opioidergic systems.
The PKC isoform has been identified in neurons of the brain and
spinal cord. A recent study in mice lacking the PKC isoform has
shown that the PKC isoform may be critical for synaptic plasticity such as neuropathic pain (Malmberg et al., 1997). The neuropathic pain
after partial nerve ligation results in a somatotopically organized
upregulation of PKC in the superficial dorsal horn and its
translocation from the cytoplasm and nucleus toward the plasma membrane
of immunoreactive neurons (Mayer et al., 1995). The present study with
PKC knock-out mice revealed that repeated spinal administration of
µ-agonist in mice lacking the PKC isoform gene failed to cause any
desensitization of G-protein activation by the µ-agonist. Taking
together with the present results of the immunoblotting, the PKC
isoform is likely to be one of the most important factors to modulate
the homologous desensitization of spinal µ-opioidergic systems.
Recent studies with dual immunofluorescence labeling showed that PKC
was present in only 5% of cells expressing µ-opioid receptors in rat
spinal cord (Polgar et al., 1999). If this is also the case in the
mouse, one wonders how µ-opioid receptors in the spinal cord could be
phosphorylated directly by PKC. The results obtained from this study
would suggest the possibility that chronic treatment with DAMGO may
cause an increase of the expression of PKC in µ-opioid
receptor-containing cells.
Because it has been well recognized that there is no cross-tolerance to
opioid-induced analgesia/antinociception among opioid receptor types,
the lack of cross-talk between µ- and - or -opioidergic systems
on the desensitization of opioid-induced G-protein activation after
repeated treatment with µ-agonists was expected. However, it should
be pointed out that either - or -opioid receptors possess the
phosphorylation sites for PKC (Mollereau et al., 1994; Yasuda et al.,
1998). Although the exact mechanism is unclear at this time, it is
possible that the remaining PKC isoforms activated by chronic treatment
with a - or -opioid receptor agonist provide sufficient kinase
activity to regulate -and -opioidergic systems.
In conclusion, the present results indicate a potential role for PKC,
especially the PKC isoform, in the process of uncoupling of the
spinal µ-opioid receptor from G-proteins in mice chronically treated
intrathecally with a specific µ-opioid receptor agonist, DAMGO. We
propose that PKC may be activated after the repeated administration
of µ-opioids and may play an important role in the development of
µ-opioid receptor-mediated tolerance.
| |
FOOTNOTES |
Received Sept. 12, 2000; revised March 5, 2001; accepted March 13, 2001.
This study was supported by National Institutes of Health Grant DA
03811 (L.F.T.).
Correspondence should be addressed to Dr. Leon F. Tseng, Department of
Anesthesiology, MEB-462c, Medical College of Wisconsin, 8701 Watertown
Plank Road, Milwaukee, WI 53226. E-mail: ltseng{at}mcw.edu.
| |
REFERENCES |
-
Abeliovich A,
Chen C,
Goda Y,
Sliva AJ,
Stevens CF,
Tonegawa S
(1993)
Modified hippocampal long-term potentiation in PKC-mutant mice.
Cell
75:1253-1262[Web of Science][Medline].
-
Berridge MJ
(1987)
Inositol trisphosphate and diacylglycerol: two interacting second messengers.
Annu Rev Biochem
56:159-193[Web of Science][Medline].
-
Berridge MJ
(1993)
Inositol trisphosphate and calcium signaling.
Nature
361:315-325[Medline].
-
Bradford MM
(1976)
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding.
Anal Biochem
72:248-254[Web of Science][Medline].
-
Chen L,
Huang LY
(1991)
Sustained potentiation of NMDA receptor-mediated glutamate responses through activation of protein kinase C by a µ-opioid.
Neuron
7:319-326[Web of Science][Medline].
-
Chen Y,
Mestek A,
Liu J,
Hurley JA,
Yu L
(1993)
Molecular cloning and functional expression of a µ-opioid receptor from rat brain.
Mol Pharmacol
44:8-12[Abstract].
-
Childers SR
(1991)
Opioid receptor-coupled second messenger systems.
Life Sci
48:1991-2003[Web of Science][Medline].
-
Fisher SK,
Heacock AM,
Agranoff BW
(1992)
Inositol lipids and signal transduction in the nervous system: an update.
J Neurochem
58:18-38[Web of Science][Medline].
-
Hylden JL,
Wilcox GL
(1980)
Intrathecal morphine in mice: a new technique.
Eur J Pharmacol
67:313-316[Web of Science][Medline].
-
Kaufman DL,
Keith Jr DE,
Anton B,
Tian J,
Magendzo K,
Newman D,
Tran TH,
Lee DS,
Wen C,
Xia YR,
Lusis AJ,
Evans CJ
(1995)
Characterization of the murine µ-opioid receptor gene.
J Biol Chem
270:15877-15883[Abstract/Free Full Text].
-
Laemmli UK
(1970)
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:680-685[Medline].
-
Malmberg AB,
Chen C,
Tonegawa S,
Basbaum AI
(1997)
Preserved acute pain and reduced neuropathic pain in mice lacking PKC.
Science
278:279-283[Abstract/Free Full Text].
-
Mangoura D,
Dawson G
(1993)
Opioid peptides activate phospholipase D and protein kinase C
 chicken embryo neuron cultures.
Proc Natl Acad Sci USA
90:2915-2919[Abstract/Free Full Text]. -
Mansour A,
Khachaturian H,
Lewis ME,
Akil H,
Watson SJ
(1988)
Anatomy of CNS opioid receptors.
Trends Neurosci
11:308-314[Web of Science][Medline].
-
Mao J,
Price DD,
Phillips LL,
Lu J,
Mayer DJ
(1995)
Increases in protein kinase C immunoreactivity in the spinal cord of rats associated with tolerance to the analgesic effects of morphine.
Brain Res
677:257-267[Web of Science][Medline].
-
Martin WJ,
Liu H,
Wang H,
Malmberg AB,
Basbaum AI
(1999)
Inflammation-induced up-regulation of protein kinase C immunoreactivity in rat spinal cord correlates with enhanced nociceptive processing.
Neuroscience
88:1267-1274[Web of Science][Medline].
-
Mayer DJ,
Mao J,
Price DD
(1995)
The association of neuropathic pain, morphine tolerance and dependence, and the translocation of protein kinase C.
NIDA Res Monogr
147:269-298[Medline].
-
Mollereau C,
Parmentier M,
Mailleux P,
Butour JL,
Moisand C,
Chalon P,
Caput D,
Vassart G,
Meunier JC
(1994)
ORL1, a novel member of the opioid receptor family. Cloning, functional expression, and localization.
FEBS Lett
341:33-38[Web of Science][Medline].
-
Narita M,
Narita M,
Mizoguchi H,
Tseng LF
(1995)
Inhibition of protein kinase C, but not of protein kinase A, blocks the development of acute antinociceptive tolerance to an intrathecally administered µ-opioid receptor agonist in the mouse.
Eur J Pharmacol
280:R1-R3[Web of Science][Medline].
-
Narita M,
Mizoguchi H,
Tseng LF
(1996)
Phorbol ester blocks the increase of a high affinity GTPase activity induced by 2-opioid receptor agonist in the mouse spinal cord.
Eur J Pharmacol
310:R1-R3[Medline].
-
Narita M,
Ohsawa M,
Mizoguchi H,
Kamei J,
Tseng LF
(1997)
Pretreatment with protein kinase C activator phorbol 12,13-dibutyrate attenuates the antinociception induced by µ- but not -opioid receptor agonist in the mouse.
Neuroscience
76:291-298[Medline].
-
Pei G,
Kieffer BL,
Lefkowitz RJ,
Freedman NJ
(1995)
Agonist-dependent phosphorylation of the mouse -opioid receptor: involvement of G-protein-coupled receptor kinases but not protein kinase C.
Mol Pharmacol
48:173-177[Abstract].
-
Polgar E,
Fowler JH,
McGill MM,
Todd AJ
(1999)
The types of neuron which contain protein kinase C in rat spinal cord.
Brain Res
833:71-80[Web of Science][Medline].
-
Smart D,
Smith G,
Lambert DG
(1995)
µ-Opioids activate phospholipase C in SH-SY5Y human neuroblastoma cells via calcium channel opening.
Biochem J
305:577-581.
-
Ueda H,
Miyamae T,
Fukushima N,
Takeshima H,
Fukuda K,
Sasaki Y,
Misu Y
(1995)
Opioid µ- and -receptors mediate phospholipase C activation through Gi1 in Xenopus oocytes.
Brain Res Mol Brain Res
32:166-170[Medline].
-
Way KJ,
Chou E,
King GL
(2000)
Identification of PKC isoform-specific biological actions using pharmacological approaches.
Trends Pharmacol Sci
21:181-187[Medline].
-
Yasuda H,
Lindorfer MA,
Myung CS,
Garrison JC
(1998)
Phosphorylation of the G-protein 12 subunit regulates effector specificity.
J Biol Chem
273:21958-21965[Abstract/Free Full Text].
Copyright © 2001 Society for Neuroscience 0270-6474/01/21113715-06$05.00/0
This article has been cited by other articles:
|
|
|
|
 
J. Mao, B. Sung, R.-R. Ji, and G. Lim
Chronic Morphine Induces Downregulation of Spinal Glutamate Transporters: Implications in Morphine Tolerance and Abnormal Pain Sensitivity
J. Neurosci.,
September 15, 2002;
22(18):
8312 - 8323.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Mao, B. Sung, R.-R. Ji, and G. Lim
Neuronal Apoptosis Associated with Morphine Tolerance: Evidence for an Opioid-Induced Neurotoxic Mechanism
J. Neurosci.,
September 1, 2002;
22(17):
7650 - 7661.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. D. Mandyam, D. R. Thakker, J. L. Christensen, and K. M. Standifer
Orphanin FQ/Nociceptin-Mediated Desensitization of Opioid Receptor-Like 1 Receptor and {micro} Opioid Receptors Involves Protein Kinase C: A Molecular Mechanism for Heterologous Cross-Talk
J. Pharmacol. Exp. Ther.,
August 1, 2002;
302(2):
502 - 509.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
TOP
ABSTRACT
INTRODUCTION
Compound via MeSH
