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

Abstract

The protein kinase C (PKC)γ isoform is a major pool of the PKC family in the mammalian spinal cord. PKCγ 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 PKCγ knockout mice. In contrast, δ- and κ-opioidergic and ORL-1 receptor-mediated activation of G-proteins in PKCγ knockout mice was not altered significantly relative to the wild-type mice. Deletion of PKCγ 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 PKCγ knockout mice. These findings suggest that deletion of PKCγ genes protects the functional μ-opioid receptors from degradation by phosphorylation. More importantly the present data provide direct evidence that PKCγ constitutes an essential pathway through which phosphorylation of μ-opioid receptors occurs. The protein kinase C (PKC)γ isoform is a major pool of the PKC family in the mammalian spinal cord. PKCγ 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 PKCγ knockout mice. In contrast, δ- and κ-opioidergic and ORL-1 receptor-mediated activation of G-proteins in PKCγ knockout mice was not altered significantly relative to the wild-type mice. Deletion of PKCγ 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 PKCγ knockout mice. These findings suggest that deletion of PKCγ genes protects the functional μ-opioid receptors from degradation by phosphorylation. More importantly the present data provide direct evidence that PKCγ constitutes an essential pathway through which phosphorylation of μ-opioid receptors occurs. protein kinase C Tris-buffered saline intrathecal reverse transcription-polymerase chain reaction [d-Ala2,N-Me-Phe4,Gly5-ol]enkephalin immunoreactivity d-Phe-Cys-d-Tyr-Orn-Thr-Pen-Thr-NH2 guanosine-5′-O-(3-thio)triphosphate The PKC1 family of enzymes plays an important role in signal transduction in several physiological processes (1Nishizuka Y. FASEB J. 1995; 9: 484-496Crossref PubMed Scopus (2352) Google Scholar). 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 (PKCα, -βI, -βII, and -γ), the Ca2+-independent or novel isoforms (PKCδ, -ε, -η, and -θ), the atypical isoforms (PKCλ and PKCζ,), and finally PKCμ, which seems to take an intermediate position between the novel and atypical PKC isoforms (2Way K.J. Chou E. King G.L. Trends Pharmacol. Sci. 2000; 21: 181-187Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar). These isozymes show subtle differences in their regional distribution, subcellular localization, and enzymological characteristics and, therefore, may underlie diverse physiological functions (2Way K.J. Chou E. King G.L. Trends Pharmacol. Sci. 2000; 21: 181-187Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar).PKCγ is found to be the major form of the PKC family within the mammalian spinal cord (3Mao J. Price D.D. Mayer D.J. Hayes R.L. Brain Res. 1992; 588: 144-149Crossref PubMed Scopus (119) Google Scholar, 4Mori M. Kose A. Tsujino T. Tanaka C. J. Comp. Neurol. 1990; 299: 167-177Crossref PubMed Scopus (120) Google Scholar, 5Polgar E. Fowler J.H. McGill M.M. Todd A.J. Brain Res. 1999; 833: 71-80Crossref PubMed Scopus (154) Google Scholar). A role of PKCγ in spinal nociceptive processing has been proposed on the basis of the observation that persistent inflammation induces an up-regulation of PKCγ in the rat spinal cord associated with enhanced nociceptive responses (6Martin W.J. Liu H. Wang H. Malmberg A.B. Basbaum A.I. Neuroscience. 1999; 88: 1267-1274Crossref PubMed Scopus (116) Google Scholar). Recently knockout mice with PKCγ gene deletion have been developed successfully by homologous recombination (7Abeliovich A. Chen C. Goda Y. Sliva A.J. Stevens C.F. Tonegawa S. Cell. 1993; 75: 1253-1262Abstract Full Text PDF PubMed Scopus (421) Google Scholar). These knockout mice allow a direct assessment of PKCγ-dependent physiological and pharmacological responses. Malmberg et al. (8Malmberg A.B. Chen C. Tonegawa S. Basbaum A.I. Science. 1997; 278: 279-283Crossref PubMed Scopus (582) Google Scholar) have reported that mice that lack the PKCγ isozyme display normal responses to acute pain stimuli but show reduced signs of neuropathic pain after partial ligation of the sciatic nerve, suggesting that PKCγ isozyme is involved in nociception processing in the spinal cord.The μ-opioid receptor, which is G-protein-coupled (9Chen Y. Mestek A. Liu J. Hurley J.A. Yu L. Mol. Pharmacol. 1993; 44: 8-12PubMed Google Scholar, 10Chen Y. Yu L. J. Biol. Chem. 1994; 269: 7839-7842Abstract Full Text PDF PubMed Google Scholar, 11Chen Y. Fan Y. Liu J. Mestek A. Tian M. Kozak C.A. Yu L. FEBS Lett. 1994; 347: 279-283Crossref PubMed Scopus (319) Google Scholar, 12Kaufman D.L. Keith Jr., D.E. Anton B. Tian J. Magendzo K. Newman D. Tran T.H. Lee D.S. Wen C. Xia Y.R. Lusis A.J. Evans C.J. J. Biol. Chem. 1995; 270: 15877-15883Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), 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 (13Mansour A. Khachaturian H. Lewis M.E. Akil H. Watson S.J. Trends Neurosci. 1988; 11: 308-314Abstract Full Text PDF PubMed Scopus (1074) Google Scholar). At the spinal level, μ-opioid receptors are concentrated in the superficial layers of the dorsal horn and mediate analgesia/antinociception (14Aicher S.A. Sharma S. Cheng P.Y. Liu-Chen L.Y. Pickel V.M. Synapse. 2000; 36: 12-20Crossref PubMed Scopus (40) Google Scholar). 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 (15Narita M. Narita M. Mizoguchi H. Tseng L.F. Eur. J. Pharmacol. 1995; 280: R1-R3Crossref PubMed Scopus (86) Google Scholar, 16Narita M. Ohsawa M. Mizoguchi H. Kamei J. Tseng L.F. Neuroscience. 1997; 76: 291-298Crossref PubMed Scopus (37) Google Scholar). Furthermore, activation of PKC inhibits the opioid-induced G-protein activation in the mouse spinal cord (17Narita M. Mizoguchi H. Tseng L.F. Eur. J. Pharmacol. 1996; 310: R1-R3Crossref PubMed Scopus (10) Google Scholar). Chronic treatment with the μ-opioid receptor agonist morphine causes an increase in PKCγ immunoreactivity in the spinal cord of rats associated with the development of tolerance to the antinociceptive effects of morphine (18Mao J. Price D.D. Phillips L.L. Lu J. Mayer D.J. Brain Res. 1995; 677: 257-267Crossref PubMed Scopus (124) Google Scholar), indicating that PKCγ 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 PKCγ isoform exhibit any changes in two spinal μ-opioid receptor-mediated functions, namely antinociception and activation of G-proteins.DISCUSSIONPKC is an integral part of the cell signaling machinery (1Nishizuka Y. FASEB J. 1995; 9: 484-496Crossref PubMed Scopus (2352) Google Scholar, 2Way K.J. Chou E. King G.L. Trends Pharmacol. Sci. 2000; 21: 181-187Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar). 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 (1Nishizuka Y. FASEB J. 1995; 9: 484-496Crossref PubMed Scopus (2352) Google Scholar, 2Way K.J. Chou E. King G.L. Trends Pharmacol. Sci. 2000; 21: 181-187Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar).In the present study, we confirm the findings by Mori et al.(4Mori M. Kose A. Tsujino T. Tanaka C. J. Comp. Neurol. 1990; 299: 167-177Crossref PubMed Scopus (120) Google Scholar) and Malmberg et al. (8Malmberg A.B. Chen C. Tonegawa S. Basbaum A.I. Science. 1997; 278: 279-283Crossref PubMed Scopus (582) Google Scholar) using both wild-type and PKCγ knockout mice that PKCγ-IR is localized predominantly in laminae II of the dorsal horn of the spinal cord. A few PKCγ-positive somata and cell processes were detected in the lamina III, which is consistent with the findings reported by Martin et al. (6Martin W.J. Liu H. Wang H. Malmberg A.B. Basbaum A.I. Neuroscience. 1999; 88: 1267-1274Crossref PubMed Scopus (116) Google Scholar) and Polgaret al. (5Polgar E. Fowler J.H. McGill M.M. Todd A.J. Brain Res. 1999; 833: 71-80Crossref PubMed Scopus (154) Google Scholar). 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 PKCγ knockout mice was qualitatively similar to that observed in the wild-type mice. Furthermore, PKCγ-IR in the μ-opioid receptor knockout mice was detected clearly in the same areas that contain PKCγ-IR of the wild-type mice. 2M. H. M. J. S. H. T. and L. These data suggest that is no to individual mRNA and protein between μ-opioid receptor and PKCγ isoform in the spinal cord. is by the present study in which mRNA of μ-opioid receptors was not significantly in PKCγ knockout mice with the wild-type have reported previously that with the and PKC C the antinociception by of opioid receptor agonists (15Narita M. Narita M. Mizoguchi H. Tseng L.F. Eur. J. Pharmacol. 1995; 280: R1-R3Crossref PubMed Scopus (86) Google Scholar). In contrast, activation of PKC by the antinociceptive effect M. Ohsawa M. Mizoguchi H. Kamei J. Tseng L.F. Neuroscience. 1997; 76: 291-298Crossref PubMed Scopus (37) Google Scholar) or the G-protein activation by (17Narita M. Mizoguchi H. Tseng L.F. Eur. J. Pharmacol. 1996; 310: R1-R3Crossref PubMed Scopus (10) Google Scholar). More of the gene in mice has been to to and responses to A. H. D. Sci. S. A. 1999; PubMed Scopus Google Scholar). These findings that activation of by may to a activation of and of μ-opioid we report the that μ-opioid receptor-mediated activation of G-proteins in the spinal cord is enhanced in PKCγ knockout mice. The of μ-opioid receptor-mediated signal transduction in PKCγ knockout mice that this isoform may the μ-opioid in the spinal cord. In with enhanced G-protein functional deletion of the PKCγ gene in a of the spinal antinociceptive effect of the enhanced antinociception by agonist may a signaling biochemical evidence the that the enhanced antinociception in PKCγ knockout mice in from an up-regulation of the μ-opioid present observation of maximal binding μ-opioid receptor in PKCγ knockout mice is of in of or no in mRNA of μ-opioid receptors in PKCγ knockout mice relative to the wild-type mice. in this the increase in binding in spinal membranes from PKCγ knockout mice may an increase in functional μ-opioid receptors, which are to μ-opioid receptor several phosphorylation (9Chen Y. Mestek A. Liu J. Hurley J.A. Yu L. Mol. Pharmacol. 1993; 44: 8-12PubMed Google Scholar, 12Kaufman D.L. Keith Jr., D.E. Anton B. Tian J. Magendzo K. Newman D. Tran T.H. Lee D.S. Wen C. Xia Y.R. Lusis A.J. Evans C.J. J. Biol. Chem. 1995; 270: 15877-15883Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Furthermore, PKC has been to the opioid receptor and Mol. Pharmacol. 1995; Google Scholar). the that the of functional μ-opioid receptors from phosphorylation by the deletion of PKCγ may to an increase in functional μ-opioid of the cloning of receptors the phosphorylation PKC H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, C. M. C. D. FEBS Lett. 1994; PubMed Scopus Google Scholar), the lack of effects in the deletion of PKCγ gene with to the δ- and κ-opioidergic and ORL-1 was These suggest that G-protein-coupled receptors are by the of the PKCγ and that the PKC isoforms provide kinase to and ORL-1 analysis of PKCγ knockout mice the that the of the PKCγ gene may the functional μ-opioid receptors from degradation in the spinal to an of the spinal to G-protein and The present data provide direct evidence the role of PKCγ in the pathway spinal The PKC1 family of enzymes plays an important role in signal transduction in several physiological processes (1Nishizuka Y. FASEB J. 1995; 9: 484-496Crossref PubMed Scopus (2352) Google Scholar). 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 (PKCα, -βI, -βII, and -γ), the Ca2+-independent or novel isoforms (PKCδ, -ε, -η, and -θ), the atypical isoforms (PKCλ and PKCζ,), and finally PKCμ, which seems to take an intermediate position between the novel and atypical PKC isoforms (2Way K.J. Chou E. King G.L. Trends Pharmacol. Sci. 2000; 21: 181-187Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar). These isozymes show subtle differences in their regional distribution, subcellular localization, and enzymological characteristics and, therefore, may underlie diverse physiological functions (2Way K.J. Chou E. King G.L. Trends Pharmacol. Sci. 2000; 21: 181-187Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar). PKCγ is found to be the major form of the PKC family within the mammalian spinal cord (3Mao J. Price D.D. Mayer D.J. Hayes R.L. Brain Res. 1992; 588: 144-149Crossref PubMed Scopus (119) Google Scholar, 4Mori M. Kose A. Tsujino T. Tanaka C. J. Comp. Neurol. 1990; 299: 167-177Crossref PubMed Scopus (120) Google Scholar, 5Polgar E. Fowler J.H. McGill M.M. Todd A.J. Brain Res. 1999; 833: 71-80Crossref PubMed Scopus (154) Google Scholar). A role of PKCγ in spinal nociceptive processing has been proposed on the basis of the observation that persistent inflammation induces an up-regulation of PKCγ in the rat spinal cord associated with enhanced nociceptive responses (6Martin W.J. Liu H. Wang H. Malmberg A.B. Basbaum A.I. Neuroscience. 1999; 88: 1267-1274Crossref PubMed Scopus (116) Google Scholar). Recently knockout mice with PKCγ gene deletion have been developed successfully by homologous recombination (7Abeliovich A. Chen C. Goda Y. Sliva A.J. Stevens C.F. Tonegawa S. Cell. 1993; 75: 1253-1262Abstract Full Text PDF PubMed Scopus (421) Google Scholar). These knockout mice allow a direct assessment of PKCγ-dependent physiological and pharmacological responses. Malmberg et al. (8Malmberg A.B. Chen C. Tonegawa S. Basbaum A.I. Science. 1997; 278: 279-283Crossref PubMed Scopus (582) Google Scholar) have reported that mice that lack the PKCγ isozyme display normal responses to acute pain stimuli but show reduced signs of neuropathic pain after partial ligation of the sciatic nerve, suggesting that PKCγ isozyme is involved in nociception processing in the spinal cord. The μ-opioid receptor, which is G-protein-coupled (9Chen Y. Mestek A. Liu J. Hurley J.A. Yu L. Mol. Pharmacol. 1993; 44: 8-12PubMed Google Scholar, 10Chen Y. Yu L. J. Biol. Chem. 1994; 269: 7839-7842Abstract Full Text PDF PubMed Google Scholar, 11Chen Y. Fan Y. Liu J. Mestek A. Tian M. Kozak C.A. Yu L. FEBS Lett. 1994; 347: 279-283Crossref PubMed Scopus (319) Google Scholar, 12Kaufman D.L. Keith Jr., D.E. Anton B. Tian J. Magendzo K. Newman D. Tran T.H. Lee D.S. Wen C. Xia Y.R. Lusis A.J. Evans C.J. J. Biol. Chem. 1995; 270: 15877-15883Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), 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 (13Mansour A. Khachaturian H. Lewis M.E. Akil H. Watson S.J. Trends Neurosci. 1988; 11: 308-314Abstract Full Text PDF PubMed Scopus (1074) Google Scholar). At the spinal level, μ-opioid receptors are concentrated in the superficial layers of the dorsal horn and mediate analgesia/antinociception (14Aicher S.A. Sharma S. Cheng P.Y. Liu-Chen L.Y. Pickel V.M. Synapse. 2000; 36: 12-20Crossref PubMed Scopus (40) Google Scholar). 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 (15Narita M. Narita M. Mizoguchi H. Tseng L.F. Eur. J. Pharmacol. 1995; 280: R1-R3Crossref PubMed Scopus (86) Google Scholar, 16Narita M. Ohsawa M. Mizoguchi H. Kamei J. Tseng L.F. Neuroscience. 1997; 76: 291-298Crossref PubMed Scopus (37) Google Scholar). Furthermore, activation of PKC inhibits the opioid-induced G-protein activation in the mouse spinal cord (17Narita M. Mizoguchi H. Tseng L.F. Eur. J. Pharmacol. 1996; 310: R1-R3Crossref PubMed Scopus (10) Google Scholar). Chronic treatment with the μ-opioid receptor agonist morphine causes an increase in PKCγ immunoreactivity in the spinal cord of rats associated with the development of tolerance to the antinociceptive effects of morphine (18Mao J. Price D.D. Phillips L.L. Lu J. Mayer D.J. Brain Res. 1995; 677: 257-267Crossref PubMed Scopus (124) Google Scholar), indicating that PKCγ 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 PKCγ isoform exhibit any changes in two spinal μ-opioid receptor-mediated functions, namely antinociception and activation of is an integral part of the cell signaling machinery (1Nishizuka Y. FASEB J. 1995; 9: 484-496Crossref PubMed Scopus (2352) Google Scholar, 2Way K.J. Chou E. King G.L. Trends Pharmacol. Sci. 2000; 21: 181-187Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar). 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 (1Nishizuka Y. FASEB J. 1995; 9: 484-496Crossref PubMed Scopus (2352) Google Scholar, 2Way K.J. Chou E. King G.L. Trends Pharmacol. Sci. 2000; 21: 181-187Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar).In the present study, we confirm the findings by Mori et al.(4Mori M. Kose A. Tsujino T. Tanaka C. J. Comp. Neurol. 1990; 299: 167-177Crossref PubMed Scopus (120) Google Scholar) and Malmberg et al. (8Malmberg A.B. Chen C. Tonegawa S. Basbaum A.I. Science. 1997; 278: 279-283Crossref PubMed Scopus (582) Google Scholar) using both wild-type and PKCγ knockout mice that PKCγ-IR is localized predominantly in laminae II of the dorsal horn of the spinal cord. A few PKCγ-positive somata and cell processes were detected in the lamina III, which is consistent with the findings reported by Martin et al. (6Martin W.J. Liu H. Wang H. Malmberg A.B. Basbaum A.I. Neuroscience. 1999; 88: 1267-1274Crossref PubMed Scopus (116) Google Scholar) and Polgaret al. (5Polgar E. Fowler J.H. McGill M.M. Todd A.J. Brain Res. 1999; 833: 71-80Crossref PubMed Scopus (154) Google Scholar). 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 PKCγ knockout mice was qualitatively similar to that observed in the wild-type mice. Furthermore, PKCγ-IR in the μ-opioid receptor knockout mice was detected clearly in the same areas that contain PKCγ-IR of the wild-type mice. 2M. H. M. J. S. H. T. and L. These data suggest that is no to individual mRNA and protein between μ-opioid receptor and PKCγ isoform in the spinal cord. is by the present study in which mRNA of μ-opioid receptors was not significantly in PKCγ knockout mice with the wild-type have reported previously that with the and PKC C the antinociception by of opioid receptor agonists (15Narita M. Narita M. Mizoguchi H. Tseng L.F. Eur. J. Pharmacol. 1995; 280: R1-R3Crossref PubMed Scopus (86) Google Scholar). In contrast, activation of PKC by the antinociceptive effect M. Ohsawa M. Mizoguchi H. Kamei J. Tseng L.F. Neuroscience. 1997; 76: 291-298Crossref PubMed Scopus (37) Google Scholar) or the G-protein activation by (17Narita M. Mizoguchi H. Tseng L.F. Eur. J. Pharmacol. 1996; 310: R1-R3Crossref PubMed Scopus (10) Google Scholar). More of the gene in mice has been to to and responses to A. H. D. Sci. S. A. 1999; PubMed Scopus Google Scholar). These findings that activation of by may to a activation of and of μ-opioid we report the that μ-opioid receptor-mediated activation of G-proteins in the spinal cord is enhanced in PKCγ knockout mice. The of μ-opioid receptor-mediated signal transduction in PKCγ knockout mice that this isoform may the μ-opioid in the spinal cord. In with enhanced G-protein functional deletion of the PKCγ gene in a of the spinal antinociceptive effect of the enhanced antinociception by agonist may a signaling biochemical evidence the that the enhanced antinociception in PKCγ knockout mice in from an up-regulation of the μ-opioid present observation of maximal binding μ-opioid receptor in PKCγ knockout mice is of in of or no in mRNA of μ-opioid receptors in PKCγ knockout mice relative to the wild-type mice. in this the increase in binding in spinal membranes from PKCγ knockout mice may an increase in functional μ-opioid receptors, which are to μ-opioid receptor several phosphorylation (9Chen Y. Mestek A. Liu J. Hurley J.A. Yu L. Mol. Pharmacol. 1993; 44: 8-12PubMed Google Scholar, 12Kaufman D.L. Keith Jr., D.E. Anton B. Tian J. Magendzo K. Newman D. Tran T.H. Lee D.S. Wen C. Xia Y.R. Lusis A.J. Evans C.J. J. Biol. Chem. 1995; 270: 15877-15883Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Furthermore, PKC has been to the opioid receptor and Mol. Pharmacol. 1995; Google Scholar). the that the of functional μ-opioid receptors from phosphorylation by the deletion of PKCγ may to an increase in functional μ-opioid of the cloning of receptors the phosphorylation PKC H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, C. M. C. D. FEBS Lett. 1994; PubMed Scopus Google Scholar), the lack of effects in the deletion of PKCγ gene with to the δ- and κ-opioidergic and ORL-1 was These suggest that G-protein-coupled receptors are by the of the PKCγ and that the PKC isoforms provide kinase to and ORL-1 analysis of PKCγ knockout mice the that the of the PKCγ gene may the functional μ-opioid receptors from degradation in the spinal to an of the spinal to G-protein and The present data provide direct evidence the role of PKCγ in the pathway spinal PKC is an integral part of the cell signaling machinery (1Nishizuka Y. FASEB J. 1995; 9: 484-496Crossref PubMed Scopus (2352) Google Scholar, 2Way K.J. Chou E. King G.L. Trends Pharmacol. Sci. 2000; 21: 181-187Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar). 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 (1Nishizuka Y. FASEB J. 1995; 9: 484-496Crossref PubMed Scopus (2352) Google Scholar, 2Way K.J. Chou E. King G.L. Trends Pharmacol. Sci. 2000; 21: 181-187Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar). In the present study, we confirm the findings by Mori et al.(4Mori M. Kose A. Tsujino T. Tanaka C. J. Comp. Neurol. 1990; 299: 167-177Crossref PubMed Scopus (120) Google Scholar) and Malmberg et al. (8Malmberg A.B. Chen C. Tonegawa S. Basbaum A.I. Science. 1997; 278: 279-283Crossref PubMed Scopus (582) Google Scholar) using both wild-type and PKCγ knockout mice that PKCγ-IR is localized predominantly in laminae II of the dorsal horn of the spinal cord. A few PKCγ-positive somata and cell processes were detected in the lamina III, which is consistent with the findings reported by Martin et al. (6Martin W.J. Liu H. Wang H. Malmberg A.B. Basbaum A.I. Neuroscience. 1999; 88: 1267-1274Crossref PubMed Scopus (116) Google Scholar) and Polgaret al. (5Polgar E. Fowler J.H. McGill M.M. Todd A.J. Brain Res. 1999; 833: 71-80Crossref PubMed Scopus (154) Google Scholar). 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 PKCγ knockout mice was qualitatively similar to that observed in the wild-type mice. Furthermore, PKCγ-IR in the μ-opioid receptor knockout mice was detected clearly in the same areas that contain PKCγ-IR of the wild-type mice. 2M. H. M. J. S. H. T. and L. These data suggest that is no to individual mRNA and protein between μ-opioid receptor and PKCγ isoform in the spinal cord. is by the present study in which mRNA of μ-opioid receptors was not significantly in PKCγ knockout mice with the wild-type mice. We have reported previously that with the and PKC C the antinociception by of opioid receptor agonists (15Narita M. Narita M. Mizoguchi H. Tseng L.F. Eur. J. Pharmacol. 1995; 280: R1-R3Crossref PubMed Scopus (86) Google Scholar). In contrast, activation of PKC by the antinociceptive effect M. Ohsawa M. Mizoguchi H. Kamei J. Tseng L.F. Neuroscience. 1997; 76: 291-298Crossref PubMed Scopus (37) Google Scholar) or the G-protein activation by (17Narita M. Mizoguchi H. Tseng L.F. Eur. J. Pharmacol. 1996; 310: R1-R3Crossref PubMed Scopus (10) Google Scholar). More of the gene in mice has been to to and responses to A. H. D. Sci. S. A. 1999; PubMed Scopus Google Scholar). These findings that activation of by may to a activation of and of μ-opioid receptors. Here we report the that μ-opioid receptor-mediated activation of G-proteins in the spinal cord is enhanced in PKCγ knockout mice. The of μ-opioid receptor-mediated signal transduction in PKCγ knockout mice that this isoform may the μ-opioid in the spinal cord. In with enhanced G-protein functional deletion of the PKCγ gene in a of the spinal antinociceptive effect of the enhanced antinociception by agonist may a signaling biochemical evidence the that the enhanced antinociception in PKCγ knockout mice in from an up-regulation of the μ-opioid receptors. The present observation of maximal binding μ-opioid receptor in PKCγ knockout mice is of in of or no in mRNA of μ-opioid receptors in PKCγ knockout mice relative to the wild-type mice. in this the increase in binding in spinal membranes from PKCγ knockout mice may an increase in functional μ-opioid receptors, which are to The μ-opioid receptor several phosphorylation (9Chen Y. Mestek A. Liu J. Hurley J.A. Yu L. Mol. Pharmacol. 1993; 44: 8-12PubMed Google Scholar, 12Kaufman D.L. Keith Jr., D.E. Anton B. Tian J. Magendzo K. Newman D. Tran T.H. Lee D.S. Wen C. Xia Y.R. Lusis A.J. Evans C.J. J. Biol. Chem. 1995; 270: 15877-15883Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Furthermore, PKC has been to the opioid receptor and Mol. Pharmacol. 1995; Google Scholar). the that the of functional μ-opioid receptors from phosphorylation by the deletion of PKCγ may to an increase in functional μ-opioid receptors. In of the cloning of receptors the phosphorylation PKC H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, C. M. C. D. FEBS Lett. 1994; PubMed Scopus Google Scholar), the lack of effects in the deletion of PKCγ gene with to the δ- and κ-opioidergic and ORL-1 was These suggest that G-protein-coupled receptors are by the of the PKCγ and that the PKC isoforms provide kinase to and ORL-1 receptors. In analysis of PKCγ knockout mice the that the of the PKCγ gene may the functional μ-opioid receptors from degradation in the spinal to an of the spinal to G-protein and The present data provide direct evidence the role of PKCγ in the pathway spinal