Enhanced µ-Opioid Responses in the Spinal Cord of Mice Lacking Protein Kinase Cgamma Isoform*

Minoru NaritaDagger §, Hirokazu Mizoguchi§, Tomohiko Suzuki, Michiko Narita§, Nae J. Dun||, Satoshi ImaiDagger , Yoshinori YajimaDagger , Hiroshi Nagase, Tsutomu SuzukiDagger , and Leon F. Tseng§**

From the Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The protein kinase C (PKC)gamma isoform is a major pool of the PKC family in the mammalian spinal cord. PKCgamma 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 PKCgamma knockout mice. In contrast, delta - and kappa -opioidergic and ORL-1 receptor-mediated activation of G-proteins in PKCgamma knockout mice was not altered significantly relative to the wild-type mice. Deletion of PKCgamma 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 PKCgamma knockout mice. These findings suggest that deletion of PKCgamma genes protects the functional µ-opioid receptors from degradation by phosphorylation. More importantly the present data provide direct evidence that PKCgamma constitutes an essential pathway through which phosphorylation of µ-opioid receptors occurs.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (PKCalpha , -beta I, -beta II, and -gamma ), the Ca2+-independent or novel isoforms (PKCdelta , -epsilon , -eta , and -theta ), the atypical isoforms (PKClambda and PKCzeta ,), 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).

PKCgamma is found to be the major form of the PKC family within the mammalian spinal cord (3-5). A role of PKCgamma in spinal nociceptive processing has been proposed on the basis of the observation that persistent inflammation induces an up-regulation of PKCgamma in the rat spinal cord associated with enhanced nociceptive responses (6). Recently knockout mice with PKCgamma gene deletion have been developed successfully by homologous recombination (7). These knockout mice allow a direct assessment of PKCgamma -dependent physiological and pharmacological responses. Malmberg et al. (8) have reported that mice that lack the PKCgamma isozyme display normal responses to acute pain stimuli but show reduced signs of neuropathic pain after partial ligation of the sciatic nerve, suggesting that PKCgamma isozyme is involved in nociception processing in the spinal cord.

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 PKCgamma immunoreactivity in the spinal cord of rats associated with the development of tolerance to the antinociceptive effects of morphine (18), indicating that PKCgamma 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 PKCgamma isoform exhibit any changes in two spinal µ-opioid receptor-mediated functions, namely antinociception and activation of G-proteins.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Animals-- The PKCgamma knockout mice and their wild-type mice were used in the present study. PKCgamma 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.

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 (PKCalpha , PKCbeta I, PKCbeta II, and PKCgamma , 1:4000 (alpha ), 1:3000 (beta I and beta II), 1:1000 (gamma ); 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).

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 PKCgamma (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 PKCgamma 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.

[35S]GTPgamma 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]GTPgamma 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]GTPgamma 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]GTPgamma 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 GTPgamma S.

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 - 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.

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)12-18 (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 beta 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.

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-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.

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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Identification of PKCgamma Isoform in the Spinal Cord-- Western blot analysis of total spinal cord proteins with a rabbit polyclonal IgG specific to PKCgamma showed that the PKCgamma protein was not detected in both membranous and cytosolic fractions of spinal cords of PKCgamma knockout mice (Fig. 1, A and B). Immunoreactivity to the other three conventional PKC isoforms, PKCalpha , PKCbeta I, and PKCbeta II, in PKCgamma knockout mice was comparable with that observed in the wild-type (Fig. 1, A and B), confirming the specific deletion of the PKCgamma genes. This is consistent with the report in the brain tissue obtained from PKCgamma knockout mice (26). Additionally, we investigated the existence of other novel PKC isoforms in spinal cord tissues obtained from both wild-type and PKCgamma knockout mice. There was no significant difference in the levels of PKCepsilon , PKCzeta , and PKCdelta between the wild-type and PKCgamma knockout mice, whereas PKClambda , PKCtheta , PKCµ, and PKCeta 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 (alpha , beta I, beta II, and gamma ) in the spinal cords from wild-type and PKCgamma knockout (KO) mice.

In the wild-type mice, PKCgamma immunoreactivity (PKCgamma -IR) was concentrated in the lamina II of all spinal segments; deeper laminae were nearly devoid of PKCgamma -IR (Fig. 2A). In addition to the lamina II, PKCgamma -IR was noted in the white matter dorsolateral to the central canal (Fig. 2A). In PKCgamma 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 PKCgamma knockout (PKCgamma -KO) mouse spinal cords labeled with PKCgamma and µ-opioid receptor antisera. A, low magnification of a lumbar spinal section from a wild-type mouse showing a dense band of PKCgamma -IR mainly in the laminae II; a fairly dense network of PKCgamma -IR cell processes is seen also in the white matter dorsolateral to the central canal (cc). B, low magnification showing a complete absence of PKCgamma -IR in a lumbar spinal cord from a PKCgamma 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 PKCgamma 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).

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 PKCgamma 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).

Enhanced µ-Opioid Receptor-mediated G-protein Activation and Antinociception in PKCgamma Knockout Mice-- We investigated whether ablation of PKCgamma -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 PKCgamma knockout mice was examined by monitoring the binding to membranes of [35S]GTPgamma S. The µ-opioid receptor agonists, DAMGO, morphine, endomorphin-1, and endomorphin-2 (0.1-10 µM), produced concentration-dependent increases in [35S]GTPgamma 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 PKCgamma knockout mice, the increase in [35S]GTPgamma 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 delta 1-opioid receptor agonist [D-Pen2,5]enkephalin, the delta 2-opioid receptor agonist [D-Ala2]deltorphin II, the kappa 1-opioid receptor agonist U-50,488H and the ORL-1 receptor agonist nociceptin produced a robust stimulation of [35S]GTPgamma S binding in wild-type mice (Fig. 3C). The levels of [35S]GTPgamma S binding stimulated by the latter group of opioid receptor agonists in PKCgamma 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]GTPgamma S binding to spinal cord membranes of wild-type and PKCgamma knockout (PKCgamma -KO) mice. Membranes were incubated with [35S]GTPgamma 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]GTPgamma 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]GTPgamma S binding stimulated by the specific delta -opioid receptor agonist (DPDPE, [D-Pen2,5]enkephalin; DELT, [D-Ala2]deltorphin II) or kappa -opioid receptor agonist (U-50,488H) or the selective ORL-1 receptor agonist nociceptin in spinal cord membranes obtained from wild-type and PKCgamma knockout (PKCgamma -KO) mice. Membranes were incubated with [35S]GTPgamma S (50 pM) and GDP (30 µM) with and without 1 or 10 µM of each agonist.

Additional evidence for increased sensitivity of the µ-opioid receptor functions in PKCgamma 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 PKCgamma knockout mice as compared with wild-type mice. The dose-response relationship for the DAMGO-induced antinociception in PKCgamma knockout mice was shifted to the left by 2.3-fold (Fig. 4B). The antinociceptive effect of DAMGO in PKCgamma 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 (open circle ) and PKCgamma knockout (PKCgamma -KO) mice (triangle ,black-triangle). 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 (open circle ) and PKCgamma 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.

Protection of µ-Opioid Receptor Degradation in PKCgamma Knockout Mice-- We next investigated whether the PKCgamma 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 PKCgamma knockout (PKCgamma -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 PKCgamma 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 beta 2-microglobulin (beta 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 PKCgamma knockout mice was expressed as a percentage of the value in wild-type mice. Each column represents the mean ± S.E. of three samples.

To evaluate whether the increased responses to µ opioids in PKCgamma 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 PKCgamma 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 PKCgamma knockout mice was increased significantly as compared with that from the wild type (Table I).

                              
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Table I
Scatchard analyses of the µ-opioid receptors in the spinal cord membrane obtained from wild-type and PKCgamma knockout (PKCgamma -KO) mice


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 PKCgamma knockout mice that PKCgamma -IR is localized predominantly in laminae II of the dorsal horn of the spinal cord. A few PKCgamma -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 PKCgamma knockout mice was qualitatively similar to that observed in the wild-type mice. Furthermore, PKCgamma -IR in the µ-opioid receptor knockout mice was detected clearly in the same areas that contain PKCgamma -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 PKCgamma 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 PKCgamma knockout mice compared with the wild-type mice.

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 Cbeta 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 Cbeta 3 by µ-opioids may lead to a concomitant activation of PKC, subsequent phosphorylation, and internalization of µ-opioid receptors.

Here we report for the first time that µ-opioid receptor-mediated activation of G-proteins in the spinal cord is enhanced in PKCgamma knockout mice. The greater efficacy of µ-opioid receptor-mediated signal transduction in PKCgamma 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 PKCgamma 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 PKCgamma knockout mice results, at least in part, from an up-regulation of the µ-opioid receptors.

The present observation of increased maximal binding sites for µ-opioid receptor ligands in PKCgamma knockout mice is of considerable interest in light of little or no change in mRNA levels of µ-opioid receptors in PKCgamma knockout mice relative to the wild-type mice. Viewed in this context, the increase in µ-ligand binding sites in spinal membranes from PKCgamma knockout mice may reflect an increase in functional membrane-bound µ-opioid receptors, which are effectively coupled to G-proteins.

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 PKCgamma may contribute to an increase in membrane-bound functional µ-opioid receptors.

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 PKCgamma gene with respect to the delta - and kappa -opioidergic and ORL-1 systems was unexpected. These observations suggest that various G-protein-coupled receptors are affected differentially by the loss of the PKCgamma and that the remaining PKC isoforms provide sufficient kinase activity to regulate delta , kappa , and ORL-1 receptors.

In summary, our analysis of PKCgamma knockout mice supports the notion that the loss of the PKCgamma 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 PKCgamma in the negative modulatory pathway for spinal µ-opioidergic systems.

    FOOTNOTES

* 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.

    ABBREVIATIONS

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; GTPgamma S, guanosine-5'-O-(3-thio)triphosphate.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Nishizuka, Y. (1995) FASEB J. 9, 484-496[Abstract/Free Full Text]
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