The Journal of Neuroscience, June 1, 2001, 21(11):3715–3720 Involvement of Spinal Protein Kinase C␥ in the Attenuation of Opioid ␮-Receptor-Mediated G-Protein Activation after Chronic 2 4 5 Intrathecal Administration of [D-Ala ,N-MePhe ,Gly-Ol ]Enkephalin Minoru Narita,1,2 Hirokazu Mizoguchi,2 Michiko Narita,2 Hiroshi Nagase,2,3 Tsutomu Suzuki,1 and Leon F. Tseng2 1Department of Toxicology, Hoshi University, School of Pharmacy, Tokyo, 142-8501, Japan, 2Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, and 3Pharmaceutical Research Laboratories, Toray Industries Incorporated, Kamakura 248-8555, Japan The present study was designed to investigate the role of a that were treated with DAMGO. Concomitant intrathecal admin- protein kinase C (PKC) isoform in the uncoupling of the istration of a specific PKC inhibitor Ro-32-0432 with DAMGO ␮-opioid receptor from G-proteins after repeated intrathecal blocked the attenuation of DAMGO-induced G-protein activa- 2 4 injection of a selective ␮-receptor agonist, [D-Ala ,N-MePhe ,Gly- tion that was caused by chronic DAMGO treatment. Western ol 5]enkephalin (DAMGO), in the spinal cord of mice. The activation blotting analysis showed that chronic DAMGO treatment in- of G-proteins by opioids was measured by monitoring the creased the levels of PKC␥, but not PKC␣, PKC␤I, and PKC␤II guanosine-5Ј-o-(3-[ 35S]thio)triphosphate ([ 35S]GTP␥S) binding. isoforms, in spinal cord membranes. Furthermore, mice lacking Mice were injected intrathecally with saline or DAMGO once a day PKC␥ failed to exhibit the desensitization of the DAMGO- for 1–7 d. At 24 hr after every injection the spinal cord membranes stimulated [ 35S]GTP␥S binding after repeated DAMGO injec- were prepared for the assay. The enhanced [ 35S]GTP␥S binding tion. These findings indicate that repeated intrathecal adminis- by ␮-agonists DAMGO, endomorphin-1, or endomorphin-2 was tration of DAMGO may activate the PKC␥ isoform and in turn attenuated clearly in spinal cord membranes obtained from mice cause a desensitization of ␮-receptor-mediated G-protein ac- that were treated intrathecally with DAMGO for 5 and 7 d, but not tivation in the mouse spinal cord. for 1 or 3 d. By contrast, no change in levels of [ 35S]GTP␥S binding induced by the ␦-receptor agonist SNC-80 or ␬-receptor Key words: ␮-opioid receptor; protein kinase C; phosphory- agonist U-50,488H was noted in membranes obtained from mice lation; tolerance; G-protein; spinal cord The opioid agonists modulate a number of physiological pro- PKC is a key regulatory enzyme that modulates both presyn- cesses including pain, reward, stress, and immune responses via aptic and postsynaptic neuronal function, synthesis and release of the stimulation of various opioid receptors (Mansour et al., 1988). neurotransmitters, and the regulation of receptors. PKC has ex- One of the major opioid receptor types, the ␮-opioid receptor, panded into a family of closely related protein, which can be was cloned in 1993 and classified as a G-protein-coupled receptor subdivided and classified on the basis of certain structural and (Chen et al., 1993). Opioids mainly inhibit cyclic AMP formation, biochemical similarities. Several PKC isoforms, especially con- ϩ ϩ close voltage-sensitive Ca 2 channels, and open K channels via ventional PKCs (cPKCs) including ␣, ␤I, ␤II, and ␥ that are 2ϩ the stimulation of Gi/o proteins (Childers, 1991). Over the past Ca -dependent and activated by both phosphatidylserine (Ptd- few years opioids, including ␮-opioids, also have been shown to Ser) and DAG, have been identified in neurons of the spinal cord; activate the phosphoinositide-signaling cascade in a variety of in each case immunocytochemistry has shown that they are con- cells and neural tissues (Chen and Huang, 1991; Mangoura and centrated in the superficial laminae of the dorsal horn (Malmberg Dawson, 1993; Smart et al., 1995; Ueda et al., 1995). et al., 1997; Martin et al., 1999). The receptor-coupled hydrolysis of membranal phosphoinositi- We previously reported that activation of PKC in the spinal des, particularly phosphatidyl inositol 4,5-bisphosphate (PIP2), cord is implicated in the development of spinal antinociceptive yields two intracellular second messengers, diacylglycerol (DAG) tolerance to ␮-opioid receptor agonists in mice (Narita et al., ␮ and inositol triphosphate (IP3). DAG activates protein kinase C 1995). The -opioid receptor contains several potential phos- 2ϩ (PKC), and IP3 mobilizes Ca after binding with cytoplasmic phorylation sites (Chen et al., 1993; Kaufman et al., 1995). It has IP3 receptors (Berridge, 1987; Fisher et al., 1992, 1993). These been hypothesized that activated PKC directly phosphorylates processes appear to be an important part of the signal transduction the opioid receptor and subsequently induces the uncoupling of mechanism for controlling the various cellular events in the CNS. opioid receptors from G-proteins (Pei et al., 1995). However, there is little or no direct evidence to support the contention that ␮ ␮ Received Sept. 12, 2000; revised March 5, 2001; accepted March 13, 2001. repeated stimulation of -opioid receptors by opioid -agonists ␮ This study was supported by National Institutes of Health Grant DA 03811 produces the PKC-dependent uncoupling of -opioid receptors (L.F.T.). from G-proteins. In the present study we therefore investigated Correspondence should be addressed to Dr. Leon F. Tseng, Department of Anesthesiology, MEB-462c, Medical College of Wisconsin, 8701 Watertown Plank whether repeated intrathecal injection of a highly selective 2 4 5 Road, Milwaukee, WI 53226. E-mail: [email protected]. ␮-opioid receptor agonist, [D-Ala ,N-MePhe ,Gly-ol ]enkephalin Copyright © 2001 Society for Neuroscience 0270-6474/01/213715-06$15.00/0 (DAMGO), causes any changes in the increase of ␮-opioid 3716 J. Neurosci., June 1, 2001, 21(11):3715–3720 Narita et al. • Tolerance of ␮-Receptor Function and PKC␥ Ј 35 receptor-stimulated guanosine-5 -o-(3-[ S]thio)triphosphate Table 1. Opioid-induced increase in [35S]GTP␥S binding to spinal cord ([ 35S]GTP␥S) binding and the levels of membrane-located membranes of mice cPKC isoforms in the mouse spinal cord. We also investigated whether repeated intrathecal injections of DAMGO could affect % Stimulation Treatment (% of control) the DAMGO-stimulated [ 35S]GTP␥S binding in mice lacking the PKC␥ isoform. 10 ␮M DAMGO (␮-agonist) 89.2 Ϯ 3.5 10 ␮M DAMGO ϩ 10 ␮M CTOP (␮-antagonist) Ϫ1.6 Ϯ 3.2* MATERIALS AND METHODS 10 ␮M Endomorphin-1 (␮-agonist) 64.8 Ϯ 3.6 Animals. Male CD-1 mice (Charles River Breeding Laboratories, Wil- 10 ␮M Endomorphin-1 ϩ 10 ␮M CTOP 5.7 Ϯ 1.9* ␥ mington, MA) and PKC knock-out mice (The Jackson Laboratory, Bar 10 ␮M Endomorphin-2 (␮-agonist) 68.9 Ϯ 3.4 Harbor, MA), which were maintained on C57BL/6 and 129Sv mixed ␮ ϩ ␮ Ϫ Ϯ genetic backgrounds as described previously (Abeliovich et al., 1993), 10 M Endomorphin-2 10 M CTOP 2.3 0.2* ␮ ␦ Ϯ were used. Animals were housed five per cage in a room maintained at 10 M DPDPE ( -agonist) 18.1 2.4 22 Ϯ 0.5°C with an alternating 12 hr light/dark cycle. 10 ␮M DPDPE ϩ 0.1 ␮M NTI (␦-antagonist) 0.4 Ϯ 1.2* 35 ␥ 2 [ S]GTP S binding assay. The spinal cord was homogenized in ice- 10 ␮M [D-Ala ]deltorphin II (␦-agonist) 38.0 Ϯ 6.0 2ϩ cold Tris-Mg buffer containing (in mM) 50 Tris-HCl, pH 7.4, 5 MgCl2, ␮ 2 ϩ ␮ Ϫ Ϯ 35 ␥ 10 M [D-Ala ]deltorphin II 1 M NTI 0.4 2.7* and 1 EGTA for the [ S]GTP S binding assay. The homogenate was ␮ ␦ Ϯ centrifuged at 48,000 ϫ g at 4°C for 10 min. The pellets were resus- 10 M SNC-80 ( -agonist) 30.4 4.3 35 ␮ ϩ ␮ Ϯ pended in [ S]GTP␥S binding assay buffer containing (in mM)50 10 M SNC-80 1 M NTI 1.5 4.5* Tris-HCl, pH 7.4, 5 MgCl2, 1 EGTA, and 100 NaCl and recentrifuged at 10 ␮M U-50,488H (␬-agonist) 15.3 Ϯ 1.2 ϫ 48,000 g at 4°C for 10 min. The final pellets were resuspended in assay 10 ␮M U-50,488H ϩ 0.1 ␮M nor-BNI (␬-antagonist) 3.5 Ϯ 2.7* 35 ␥ buffer as membranous fractions for the [ S]GTP S binding. The reac- ␮ ␬ Ϯ tion was initiated by the addition of membrane suspension (3–8 ␮gof 10 M U-69,593 ( -agonist) 16.1 2.0 ␮ ϩ ␮ Ϫ Ϯ protein for each assay as determined by the method of Bradford, 1976) 10 M U-69,593 0.1 M nor-BNI 0.6 3.5* into the assay buffer with the opioid receptor agonists, 30 ␮M guanosine- 35 The reaction was initiated by the addition of membrane suspension obtained from 5Ј-diphosphate (GDP), and 50 pM [ S]GTP␥S (1000 Ci/mmol; Amer- the mouse spinal cord into the assay buffer with 10 ␮M opioid, 30 ␮M GDP, and 50 sham, Arlington Heights, IL). The suspensions were incubated at 25°C 35 pM [ S]GTP␥S. Data represent the mean Ϯ SEM for 3–17 samples. The basal for 2 hr, and the reaction was terminated by filtering through Whatman [35S]GTP␥S binding in mice treated with saline for 5 d was 29.2 Ϯ 1.2 fmol/mg GF/B glass filters, which had been soaked previously in a soaking buffer protein. The statistical significance of differences between the groups was assessed Ͻ of 50 mM Tris-HCl, pH 7.4, and 5 mM MgCl2 at 4°C for 2 hr, using a with a one-way ANOVA, followed by Dunnett’s test.