From the Department of Toxicology, School of
Pharmacy, Hoshi University, Tokyo 142-8501, Japan,
§ Department of Anesthesiology, Medical College of
Wisconsin, Milwaukee, Wisconsin 53226, ¶ Pharmaceutical
Research Laboratories, Toray Industries, Inc., Kamakura 248-8555, Japan, and
Department of Pharmacology, James H. Quillen College of Medicine, East Tennessee State University, Johnson
City, Tennessee 37614
Received for publication, October 24, 2000, and in revised form, January 24, 2001
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ABSTRACT |
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The protein kinase C (PKC) The PKC1 family
of enzymes plays an important role in signal transduction in several
physiological processes (1). Molecular cloning has revealed a family of
closely related proteins that can be subdivided on the basis of certain
structural and biochemical similarities: the
Ca2+-dependent or conventional isoforms
(PKC PKC The µ-opioid receptor, which is G-protein-coupled (9-12), is one of
several opioid receptors, the activation of which modulates a number of
physiological processes including pain, reward, stress, immune
responses, neuroendocrine functions, and cardiovascular control (13).
At the spinal level, µ-opioid receptors are concentrated in the
superficial layers of the dorsal horn and mediate
analgesia/antinociception (14). We reported previously that PKC in the
spinal cord is implicated in the development of spinal antinociceptive
tolerance to µ-opioid receptor agonists in mice (15, 16).
Furthermore, activation of PKC inhibits the opioid-induced G-protein
activation in the mouse spinal cord (17). Chronic treatment with the
µ-opioid receptor agonist morphine causes an increase in PKC Animals--
The PKC Western Blotting--
Spinal cords were removed quickly after
decapitation and homogenized in ice-cold buffer A containing 20 mM Tris-HCl (pH 7.5), 2 mM EDTA, 0.5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 25 µg of leupeptin per ml, 0.1 mg of aprotinin per ml, 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 incubating for 45 min, soluble
fractions were obtained by ultracentrifugation at 100,000 × g for 30 min and then 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-2×; AMRESCO, Solon, OH) containing 2% SDS and 10% glycerol with
0.2 M dithiothreitol. Proteins (5-20 µg/lane) were
separated by size on 4-20% SDS-polyacrylamide gradient gel using the
buffer system of Laemmli (19) and transferred to nitrocellulose membranes in Tris/glycine buffer containing 25 mM Tris and
192 mM glycine. For immunoblot detection of PKC isozymes,
membranes were blocked in Tris-buffered saline (TBS) containing 5%
nonfat dried milk (Bio-Rad) for 1 h at room temperature with
agitation. The membrane was incubated with primary antibody diluted in
TBS (PKC Immunohistochemical Approach--
Mice were anesthetized with
urethane (1.0 g/kg, intraperitoneal) and perfused intracardially with
chilled 0.1 M phosphate-buffered saline followed by freshly
prepared 4% paraformaldehyde in phosphate-buffered saline (pH 7.4).
Spinal cords were removed, postfixed in the same fixative for 2 h,
and cryoprotected in 30% sucrose/phosphate-buffered saline overnight.
Transverse spinal sections of 40 µm were prepared with the use of a
Vibratome. The standard avidin-biotin complex method described
previously was used (20, 21). Tissues were treated first with 3%
H2O2 to quench endogenous peroxidase, washed several times, and blocked with 10% normal goat sera. Tissues were
incubated in primary antisera to either µ-opioid receptor (1:10,000-15,000 dilution with 0.4% Triton X-100 and 1% bovine serum
albumin) or PKC [35S]GTP Assessment of Antinociception--
Antinociception was
determined by the tail-flick test (22). For measurement of the latency
of the tail-flick response, mice were held gently by hand with their
tail positioned in an apparatus (Model TF6, EMDIE Instrument Co.,
Maidens, VA) for radiant heat stimulation on the dorsal surface of the
tail. The intensity of heat stimulus was adjusted so that the animal
flicked its tail after 3-5 s. The inhibition of the tail-flick
response was expressed as percentage of maximum possible effect, which
was calculated as ((T1 Intrathecal Injection--
Intrathecal (i.t.) administration was
performed following the method described by Hylden and Wilcox (23)
using a 25-µl Hamilton syringe with a 30-gauge needle. Injection
volume was 5 µl for i.t. injection.
RNA Preparation and Quantitative Analysis by Reverse
Transcription-Polymerase Chain Reaction (RT-PCR)--
Total RNA in the
spinal cord was extracted using SV Total RNA Isolation system (Promega,
Madison, WI) following the manufacturer's instructions. The purified
total RNA was quantified by a spectrophotometer at
A260. To prepare first strand cDNA, 1 µg
of RNA was incubated in 100 µl of buffer containing 10 mM
dithiothreitol, 2.5 mM MgCl2, dNTP mixture, 200 units of reverse transcriptase II (Life Technologies, Inc.), and 0.1 mM oligo (dT)12 µ-Opioid Receptor Binding Assay--
The µ-opioid receptor
binding assays were carried out with
[tyrosyl-3,5-3H]DAMGO
([3H]DAMGO, 67.0 Ci mmol Statistical Analysis--
The data are expressed as the
mean ± S.E. The statistical significance of differences between
the groups was assessed with the Newman-Keuls multiple comparison test.
Identification of PKC
In the wild-type mice, PKC
In wild-type mice, µ-opioid receptor-IR was distributed mainly in
laminae I and II of all segments of the spinal cord; the deeper laminae
contained much less µ-opioid receptor-IR (Fig. 2C). In
PKC Enhanced µ-Opioid Receptor-mediated G-protein Activation and
Antinociception in PKC
Additional evidence for increased sensitivity of the µ-opioid
receptor functions in PKC Protection of µ-Opioid Receptor Degradation in PKC
To evaluate whether the increased responses to µ opioids in PKC PKC is an integral part of the cell signaling machinery (1, 2).
Biochemical and molecular cloning analysis has revealed that similar to
most other signaling proteins, the enzyme comprises a large family with
multiple isoforms exhibiting individual characteristics and
distinct patterns of tissue distribution (1, 2).
In the present study, we confirm the findings by Mori et al.
(4) and Malmberg et al. (8) using both wild-type and PKC We have reported previously that i.t. pretreatment with the selective,
potent, and membrane-permeable PKC inhibitor calphostin C enhances the
antinociception induced by i.t. administration of opioid receptor
agonists (15). In contrast, activation of PKC by phorbol esters
attenuates either the antinociceptive effect (16) or the G-protein
activation induced by opioids (17). More recently, disruption of the
phospholipase C Here we report for the first time that µ-opioid receptor-mediated
activation of G-proteins in the spinal cord is enhanced in PKC The present observation of increased maximal binding sites for
µ-opioid receptor ligands in PKC The µ-opioid receptor contains several potential phosphorylation
sites (9, 12). Furthermore, activated PKC has been shown to
phosphorylate the opioid receptor directly and subsequently induce its
internalization (28). Taken together, our result raises the possibility
that the protection of functional µ-opioid receptors from
phosphorylation and/or internalization by the deletion of PKC In view of the cloning studies of those receptors possessing the
phosphorylation sites for PKC (29, 30), the apparent lack of effects in
the deletion of PKC In summary, our analysis of PKC isoform is a
major pool of the PKC family in the mammalian spinal cord. PKC
is
distributed strategically in the superficial layers of the dorsal horn
and, thus, may serve as an important biochemical substrate
in sensory signal processing including pain. Here we report that
µ-opioid receptor-mediated analgesia/antinociception and activation
of G-proteins in the spinal cord are enhanced in PKC
knockout mice.
In contrast,
- and
-opioidergic and ORL-1
receptor-mediated activation of G-proteins in PKC
knockout mice was
not altered significantly relative to the wild-type mice. Deletion
of PKC
had no significant effect on the mRNA product of spinal
µ-opioid receptors but caused an increase of maximal
binding of the µ-opioid receptor agonist
[3H][D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin
in spinal cord membranes obtained from PKC
knockout mice. These
findings suggest that deletion of PKC
genes protects the
functional µ-opioid receptors from degradation by phosphorylation. More importantly the present data provide direct evidence that PKC
constitutes an essential pathway through which phosphorylation of
µ-opioid receptors occurs.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, -
I, -
II, and -
), the Ca2+-independent or
novel isoforms (PKC
, -
, -
, and -
), the atypical isoforms
(PKC
and PKC
,), and finally PKCµ, which seems to take an
intermediate position between the novel and atypical PKC isoforms (2).
These isozymes show subtle differences in their regional distribution,
subcellular localization, and enzymological characteristics and,
therefore, may underlie diverse physiological functions (2).
is found to be the major form of the PKC family within the
mammalian spinal cord (3-5). A role of PKC
in spinal nociceptive processing has been proposed on the basis of the observation that persistent inflammation induces an up-regulation of PKC
in the rat
spinal cord associated with enhanced nociceptive responses (6).
Recently knockout mice with PKC
gene deletion have been developed
successfully by homologous recombination (7). These knockout mice allow
a direct assessment of PKC
-dependent physiological and
pharmacological responses. Malmberg et al. (8) have reported that mice that lack the PKC
isozyme display normal responses to
acute pain stimuli but show reduced signs of neuropathic pain after
partial ligation of the sciatic nerve, suggesting that PKC
isozyme
is involved in nociception processing in the spinal cord.
immunoreactivity in the spinal cord of rats associated with the
development of tolerance to the antinociceptive effects of
morphine (18), indicating that PKC
may modulate the
functionality of µ-opioid receptors. The purpose of this study was to
investigate whether mice with a deletion of the gene that encodes the
neuronal-specific PKC
isoform exhibit any changes in two spinal
µ-opioid receptor-mediated functions, namely antinociception and
activation of G-proteins.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
knockout mice and their wild-type mice
were used in the present study. PKC
knockout mice were maintained on
C57BL/6 and 129Sv mixed genetic backgrounds (The Jackson Laboratory,
Bar Harbor, ME). All experiments were approved by and conformed to the
guidelines of the Medical College of Wisconsin Animal Care Committee.
Animals were housed five per cage in a room maintained at 22 ± 0.5 °C with an alternating 12-h light-dark cycle.
, PKC
I, PKC
II, and PKC
, 1:4000 (
), 1:3000 (
I
and
II), 1:1000 (
); Santa Cruz Biotechnology, Santa Cruz, CA)
containing 5% nonfat dried milk overnight at 4 °C. The membrane was
washed twice for 5 min and twice for 10 min in Triton X-TBS containing TBS and 0.05% Triton X-100 followed by a 2-h incubation at room temperature with horseradish peroxidase-conjugated goat anti-rabbit IgG
(Southern Biotechnology Associates, Inc., Birmingham, AL) diluted
1:10,000 in TBS containing 5% nonfat dried milk. Thereafter, the
membranes were washed twice for 5 min and then 3 times for 10 min in
Triton X-TBS. The antigen-antibody peroxidase complex then was detected
by enhanced chemiluminescence (Pierce) according to the manufacturer's
instructions and visualized by exposure to Amersham Hyperfilm
(Amersham Pharmacia Biotech).
(1:20,000 dilution with 0.4% Triton X-100 and 1%
bovine serum albumin) for 24 h at 4 °C with gentle agitation. The µ-opioid receptor and PKC
antisera were polyclonal raised in
rabbits and purchased from Oncogene Research Products (Cambridge, MA)
and Santa Cruz Biotechnology, Inc. After thorough rinsing, sections
were incubated with biotinylated goat anti-rabbit IgG (1:200, Vector
Laboratories) for 2 h. Sections were rinsed with phosphate-buffered saline and incubated in avidin-biotin complex solution (1:100, Vector Laboratories) for 1 h. After several
rinses in TBS, sections were developed in
diaminobenzidine-H2O2 solution and washed for
at least 2 h with TBS. Sections were mounted on slides with 0.25%
gel alcohol, air-dried, dehydrated with absolute alcohol followed by
xylene, and coverslipped with Permount. Control experiments were
carried out in which spinal sections were processed without the primary antisera.
S Binding Assay--
The spinal cord
was homogenized in ice-cold Tris-Mg2+ buffer containing 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2,
and 1 mM EGTA. The homogenate was centrifuged at
48,000 × g at 4 °C for 10 min. The pellets were
resuspended in [35S]GTP
S binding assay buffer
containing 50 mM Tris-HCl (pH 7.4), 5 mM
MgCl2, 1 mM EGTA, and 100 mM 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]GTP
S binding. The reaction was
initiated by the addition of membrane suspension (3-8 µg of membrane
proteins/assay) into the assay buffer with the opioid receptor
agonists, 30 µM GDP, and 50 pM
[35S]GTP
S (1000 Ci mmol
1, Amersham
Pharmacia Biotech). The suspensions were incubated at 25 °C for
2 h, and the reaction was terminated by filtering through Whatman
GF/B glass filters using a Brandel cell harvester (Model M-24, Brandel,
Inc., Gaithersburg, MD). The filters then were washed and transferred
to scintillation counting vials containing scintillation mixture (0.5 ml of Soluene-350 and 4 ml of Hionic Fluor mixture, Packard Instrument
Co., Inc., Meriden, CT). The radioactivity in the samples was
determined with a liquid scintillation analyzer (Model 1600 CA, Packard
Instrument Co.). Nonspecific binding was measured in the presence of 10 µM unlabeled GTP
S.
T0)/(T2
T0)) × 100, where
T0 and T1 were the
tail-flick latencies before and after the injection of opioid agonist
and T2 was the cut-off time that was set at
10 s for the tests to avoid injury to the tail.
18 (Life Technologies, Inc.). The µ-opioid receptor gene was amplified in a 50-µl PCR solution containing 0.8 mM MgCl2, dNTP mix, and DNA
polymerase with synthesized primers: a sense primer of µ-opioid
receptor, which is at position 299-320 (5'-AGA CTG CCA CCA ACA TCT ACA
T-3') of the receptor, and an antisense primer at position 623 to 643 (5'-TGG ACC CCT GCC TGT ATT TTG-3'), which was designed according to
GenBankTM sequence accession number U26915. Samples were
heated to 95 °C for 2 min, 55 °C for 2 min, and 72 °C for 3 min and cycled 40 times through 95 °C for 1 min, 55 °C for 2 min,
and 72 °C for 3 min. The final incubation was 72 °C for 7 min.
The resulting 344-base pair PCR product amplified with the above
primers was subcloned into pGEM-T vector (Invitrogen Corp., San Diego,
CA) by the T-A cloning method. The DNA sequencing for the inserted region confirmed that the amplified nucleotides corresponded to those
of murine µ-opioid receptor cDNA (24). The mixture was run on 1%
agarose gel electrophoresis with the indicated markers and primers for
the internal standard
2-microglobulin. The agarose gel
was stained with ethidium bromide and photographed with UV transillumination. The intensity of the bands was analyzed and semiquantified by computer-assisted densitometry using the NIH image.
1; Amersham
Pharmacia Biotech) at 0.2-20 nM in a final volume
of 1.0 ml, which contained 50 mM Tris-HCl buffer (pH 7.4)
and 0.1 ml of the homogenated membrane fraction. The amount of membrane protein used in each assay was in the range of 90-140 µg as
determined by the method of Lowry et al. (25). The test
tubes were incubated for 2 h at 25 °C. The specific binding was
defined as the difference in binding observed in the absence and
presence of 10 µM unlabeled naloxone. The incubation was
terminated by collecting the membranes on Whatman GF/B filters using a
Brandel cell harvester. The filters then were washed three times with 5 ml of Tris-HCl buffer (pH 7.4) at 4 °C and transferred to
scintillation vials. Then 0.5 ml of Soluene-350 and 4 ml of Hionic
Fluor mixture were added to the vials. After a 12-h equilibration
period, the radioactivity in the samples was determined in the liquid
scintillation analyzer. Values for Scatchard analysis represent the
mean ± S.E. of three independent determinations.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Isoform in the Spinal Cord--
Western
blot analysis of total spinal cord proteins with a rabbit polyclonal
IgG specific to PKC
showed that the PKC
protein was not detected
in both membranous and cytosolic fractions of spinal cords of PKC
knockout mice (Fig. 1, A and
B). Immunoreactivity to the other three conventional PKC
isoforms, PKC
, PKC
I, and PKC
II, in PKC
knockout mice was
comparable with that observed in the wild-type (Fig. 1, A
and B), confirming the specific deletion of the PKC
genes. This is consistent with the report in the brain tissue obtained
from PKC
knockout mice (26). Additionally, we investigated the
existence of other novel PKC isoforms in spinal cord tissues obtained
from both wild-type and PKC
knockout mice. There was no significant
difference in the levels of PKC
, PKC
, and PKC
between the
wild-type and PKC
knockout mice, whereas PKC
, PKC
, PKCµ, and
PKC
isoforms were not detectable in spinal cord tissues from both
types of animals (data not shown).
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Fig. 1.
Immunoblot analysis of protein levels of
membranous (A) or cytosolic (B)
fractions of PKC isoforms ( ,
I,
II, and
) in the spinal cords from wild-type and
PKC
knockout (KO)
mice.
immunoreactivity (PKC
-IR) was
concentrated in the lamina II of all spinal segments; deeper laminae were nearly devoid of PKC
-IR (Fig.
2A). In addition to the lamina II, PKC
-IR was noted in the white matter dorsolateral to the central
canal (Fig. 2A). In PKC
knockout mice, immunoreactivity was completely absent in the spinal cord (Fig. 2B).
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Fig. 2.
Photomicrographs of sections through
wild-type and PKC knockout
(PKC
-KO) mouse spinal cords
labeled with PKC
and
µ-opioid receptor antisera. A, low
magnification of a lumbar spinal section from a wild-type mouse showing
a dense band of PKC
-IR mainly in the laminae II; a fairly dense
network of PKC
-IR cell processes is seen also in the white matter
dorsolateral to the central canal (cc). B, low
magnification showing a complete absence of PKC
-IR in a lumbar
spinal cord from a PKC
knockout mouse. C, low
magnification showing µ-opioid receptor IR is concentrated in the
laminae I and II of lumbar spinal cord from a wild-type mouse.
D, low magnification of a lumbar spinal section from a
PKC
knockout mouse in which the pattern of distribution of
µ-opioid receptor-IR is similar to that of the wild-type mouse.
(Scale bar, 500 µm for all panels).
knockout mice, the pattern of distribution of µ-opioid receptor-IR was similar to that observed in the spinal cord of wild-type mice (Fig. 2D).
Knockout Mice--
We investigated whether
ablation of PKC
-mediated pathways affected the G-protein
activation through stimulation of µ-opioid receptors. The ability of
µ-opioid receptor agonists to activate G-proteins in the spinal cord
of wild-type and PKC
knockout mice was examined by monitoring the
binding to membranes of [35S]GTP
S. The µ-opioid
receptor agonists, DAMGO, morphine, endomorphin-1, and endomorphin-2
(0.1-10 µM), produced
concentration-dependent increases in
[35S]GTP
S binding to spinal cord membranes
obtained from wild-type mice (Fig. 3,
A and B), which were abolished by coincubating
the membranes with the specific µ-opioid receptor antagonist
D-Phe-Cys-D-Tyr-Orn-Thr-Pen-Thr-NH2 (CTOP; data not shown). In PKC
knockout mice, the increase in [35S]GTP
S binding stimulated by either DAMGO,
morphine, endomorphin-1, or endomorphin-2 was significantly greater as
compared with that in wild-type mice (Fig. 3, A and
B). These effects were completely blocked by CTOP (data not
shown). The
1-opioid receptor agonist [D-Pen2,5]enkephalin, the
2-opioid receptor agonist
[D-Ala2]deltorphin II, the
1-opioid receptor agonist U-50,488H and the ORL-1
receptor agonist nociceptin produced a robust stimulation of
[35S]GTP
S binding in wild-type mice (Fig.
3C). The levels of [35S]GTP
S binding
stimulated by the latter group of opioid receptor agonists in PKC
knockout mice were similar to those found in wild-type mice (Fig.
3C).
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Fig. 3.
A and B, effects of several
µ-opioid receptor agonists DAMGO (A), morphine
(A), endomorphin-1 (B), and endomorphin-2
(B) on [35S]GTP S binding to spinal cord
membranes of wild-type and PKC
knockout
(PKC
-KO) mice. Membranes were incubated with
[35S]GTP
S (50 pM) and GDP (30 µM) with and without different concentrations of each
µ-opioid receptor agonist. The data are shown as the percentage of
basal [35S]GTP
S (50 pM) binding measured
in the presence of GDP (30 µM) and absence of agonist and
indicate the mean ± S.E. from at least three independent
experiments. *p < 0.05 versus wild-type
mice. C, there was no difference in the
[35S]GTP
S binding stimulated by the specific
-opioid receptor agonist (DPDPE,
[D-Pen2,5]enkephalin; DELT,
[D-Ala2]deltorphin II) or
-opioid receptor
agonist (U-50,488H) or the selective ORL-1 receptor agonist
nociceptin in spinal cord membranes obtained from wild-type and PKC
knockout (PKC
-KO) mice. Membranes were
incubated with [35S]GTP
S (50 pM) and GDP
(30 µM) with and without 1 or 10 µM of each
agonist.
knockout mice with respect to the antinociceptive response to µ-agonists was obtained in the tail-flick latency test. An i.t. administration of DAMGO produced marked antinociception in the wild-type mice (Fig.
4A). The DAMGO-induced antinociception was maximal at 10 min and had returned to control level
30 min after injection. The antinociceptive effect induced by DAMGO was
enhanced significantly in PKC
knockout mice as compared with
wild-type mice. The dose-response relationship for the DAMGO-induced antinociception in PKC
knockout mice was shifted to the left by
2.3-fold (Fig. 4B). The antinociceptive effect of DAMGO in PKC
knockout mice was inhibited completely by CTOP pretreatment (Fig. 4A).
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Fig. 4.
A, time-course changes in the
DAMGO-induced antinociception in wild-type ( ) and PKC
knockout (PKC
-KO) mice (
,
). Groups of
mice were administered i.t. injections of DAMGO (5.8 pmol) and CTOP
(47.1 pmol), and the tail-flick responses were measured at different
times after injection. Antinociception was expressed as a percentage of
maximal possible effect (% antinociception). The data
represent the mean ± S.E., *, p < 0.05 versus wild-type mice; #, p < 0.05 versus DAMGO alone. B, the dose-response curve
for the antinociceptive effect produced by i.t. injection of DAMGO in
wild-type (
) and PKC
knockout mice (
) is shown. The tail-flick
latencies were measured 10 min after the i.t. injection of DAMGO. The
data represent the mean ± S.E.
Knockout
Mice--
We next investigated whether the PKC
gene deletion could
influence the gene production of µ-opioid receptors directly. As shown in Fig. 5, the RT-PCR assay
revealed that the µ-opioid receptor production in the spinal cord did
not differ significantly between the two genotypes.
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Fig. 5.
No change in µ-opioid
receptor mRNA in PKC knockout
(PKC
-KO) mice. The levels of
µ-opioid receptor (MOR) mRNA in the spinal cord were
quantitated using RT-PCR. A, representative RT-PCR for the
µ-opioid receptor mRNA in the spinal cord obtained from PKC
knockout and wild-type mice is shown. The mixture was run on 1%
agarose gel electrophoresis with the indicated markers and primers of
the internal standard
2-microglobulin
(
2-MG). Three independent
experiments were performed in this study. B,
semiquantitation of the intensity of the bands was conducted by using
an NIH image. The value for PKC
knockout mice was expressed as a
percentage of the value in wild-type mice. Each column represents the
mean ± S.E. of three samples.
knockout mice could result from an increased population of µ-opioid
receptors in the spinal cord, we performed the saturation binding
analysis using [3H]DAMGO. Saturation binding studies with
[3H]DAMGO at different concentrations revealed a single
high affinity binding site that represents the µ-opioid receptor.
There was no difference between the wild-type and PKC
knockout mice
in the affinity (Kd value) of µ-opioid receptors
in the spinal cord (Table I). However,
the maximal binding of [3H]DAMGO in spinal cord membranes
prepared from PKC
knockout mice was increased significantly as
compared with that from the wild type (Table I).
Scatchard analyses of the µ-opioid receptors in the spinal cord
membrane obtained from wild-type and PKC knockout (PKC
-KO)
mice
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
knockout mice that PKC
-IR is localized predominantly in laminae II
of the dorsal horn of the spinal cord. A few PKC
-positive somata and
cell processes were detected in the lamina III, which is consistent
with the findings reported by Martin et al. (6) and Polgar
et al. (5). In contrast, µ-opioid receptor-IR was found to
be dense in the laminae I and II of the dorsal horn of the spinal cord.
It should be noted that the pattern of spinal distribution of
µ-opioid receptor-IR in PKC
knockout mice was qualitatively
similar to that observed in the wild-type mice. Furthermore, PKC
-IR
in the µ-opioid receptor knockout mice was detected clearly in the
same areas that contain PKC
-IR of the wild-type
mice.2 These data
suggest that there is no genetic cross-talk to produce individual
mRNA and protein between µ-opioid receptor and PKC
isoform in
the spinal cord. This contention is supported by the present RT-PCR
study in which mRNA production of µ-opioid receptors was
not changed significantly in PKC
knockout mice compared with the
wild-type mice.
3 gene in mice has been shown to enhance
sensitivity to behavioral and cellular responses to µ-opioids (27).
These findings indicate that activation of phospholipase
C
3 by µ-opioids may lead to a concomitant activation of PKC, subsequent phosphorylation, and internalization of µ-opioid receptors.
knockout mice. The greater efficacy of µ-opioid receptor-mediated signal transduction in PKC
knockout mice implies that this isoform may suppress the µ-opioid receptor-dependent actions in
the spinal cord. In conjunction with enhanced G-protein
activation, functional deletion of the PKC
gene resulted in a
marked potentiation of the spinal antinociceptive effect of µ opioids. Although the enhanced antinociception induced by
i.t.-administered µ agonist may involve a complex signaling pathway,
our biochemical evidence supports the interpretation that the enhanced µ agonist-induced antinociception in PKC
knockout mice results, at
least in part, from an up-regulation of the µ-opioid receptors.
knockout mice is of considerable interest in light of little or no change in mRNA levels of
µ-opioid receptors in PKC
knockout mice relative to the wild-type
mice. Viewed in this context, the increase in µ-ligand binding sites in spinal membranes from PKC
knockout mice may reflect an increase in functional membrane-bound µ-opioid receptors, which are
effectively coupled to G-proteins.
may
contribute to an increase in membrane-bound functional µ-opioid receptors.
gene with respect to the
- and
-opioidergic and ORL-1 systems was unexpected. These observations suggest that various G-protein-coupled receptors are affected differentially by the loss of the PKC
and that the remaining PKC
isoforms provide sufficient kinase activity to regulate
,
, and
ORL-1 receptors.
knockout mice supports the notion
that the loss of the PKC
gene may protect the functional µ-opioid
receptors from degradation in the spinal cord, leading to an
enhancement of the spinal µ-opioidergic system to activate G-protein
and produce antinociception. The present data provide direct evidence
for the critical role of PKC
in the negative modulatory pathway for
spinal µ-opioidergic systems.
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FOOTNOTES |
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* This study was supported by National Institutes of Health Grants DA 03811 (to L. F. T.) and NS18710 and NS39646 (to N. J. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: Medical College of Wisconsin, Dept. of Anesthesiology, Medical Education Bldg., Rm. M4308, 8701 Watertown Plank Rd., Milwaukee, WI 53226. Tel.: 414-456-5686; Fax: 414-456-6507; E-mail: ltseng@mcw.edu.
Published, JBC Papers in Press, January 29, 2001, DOI 10.1074/jbc.M009716200
2 M. Narita, H. Mizoguchi, M. Narita, N. J. Dun, I. Sora, F. S. Hall, G. R. Uhl, H. Nagase, T. Suzuki, and L. F. Tseng, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
PKC, protein kinase
C;
TBS, Tris-buffered saline;
i.t., intrathecal;
RT-PCR, reverse
transcription-polymerase chain reaction;
DAMGO, [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin;
IR, immunoreactivity;
CTOP, D-Phe-Cys-D-Tyr-Orn-Thr-Pen-Thr-NH2;
GTPS, guanosine-5'-O-(3-thio)triphosphate.
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