Michiko Narita

Inhibition of protein kinase C, but not of protein kinase A, blocks the development of acute antinociceptive tolerance to an intrathecally administered μ-opioid receptor agonist in the mouse

A specific protein kinase C inhibitor, calphostin C, which injected alone had no effect on the antinociception induced by intrathecal (i.t.) administration of a selective mu-opioid receptor agonist, [D-Ala2,NMePhe4,Gly(ol)5]enkephalin (DAMGO), dose-dependently attenuated the development of acute tolerance to the i.t. DAMGO-induced antinociception in male ICR mice. On the other hand, a selective protein kinase A inhibitor, KT5720, did not have any effect on the development of acute tolerance to DAMGO antinociception. These findings suggest that protein kinase C, but not protein kinase A, plays an important role in the development of acute tolerance to the mu-opioid receptor agonist-induced antinociception.

Implications of the NR2B subunit-containing NMDA receptor localized in mouse limbic forebrain in ethanol dependence

The present study was designed to further investigate the direct involvement of the NR2B-containing NMDA receptor in ethanol dependence. Using the liquid diet method, mice were chronically treated with skimmed milk containing 5% ethanol for 5 days. After the discontinuation of ethanol, mice revealed tremor, handling-elicited convulsion and death. Treatment with a selective NR2B-containing NMDA receptor antagonist, ifenprodil, significantly suppressed the expression of ethanol withdrawal signs. The protein level of NR2B subunits in the limbic forebrain, but not the cerebral cortex, during chronic ethanol treatment was markedly increased with respect to the levels in control mice. The significant up-regulation of NR2B subunits lasted for at least 9 h after the discontinuation of ethanol and returned to the basal level by 48 h after the withdrawal. These findings suggest that the up-regulation of NR2B subunits during chronic ethanol exposure may be implicated in the initial development of physical dependence on ethanol.

The μ-opioid receptor gene-dose dependent reductions in G-protein activation in the pons/medulla and antinociception induced by endomorphins in μ-opioid receptor knockout mice

Abstract There appear to be different relationships between μ-opioid receptor densities and the acute and neuroadaptive μ-opioid agonist-induced responses of the multiple opioid neuronal systems, including important pons/medulla circuits. The recent success in creating μ-opioid receptor knockout mice allows studies of μ-opioid agonist-induced pharmacological and physiological effects in animals that express no, one or two copies of the μ-opioid receptor gene. We now report that the binding of μ-opioid receptor ligand, [3H][D-Ala2,NHPhe4,Gly-ol]enkephalin to membrane preparations of the pons/medulla was reduced by half in heterozygous μ-opioid receptor knockout mice and eliminated in homozygous μ-opioid receptor knockout mice. The endogenous μ-opioid agonist peptides endomorphin-1 and -2 activate G-proteins in the pons/medulla from wild-type mice in a concentration-dependent fashion, as assessed using [35S]guanosine-5′-o-(3-thio)triphosphate binding. This stimulation was reduced to half of the wild-type levels in heterozygous mice and eliminated in homozygous knockout mice. The intracerebroventricular injection of either endomorphin-1 or endomorphin-2 produced marked antinociception in the hot-plate and tail-flick tests in wild-type mice. These antinociceptive actions were significantly reduced in heterozygous μ-opioid receptor knockout mice, and virtually abolished in homozygous knockout mice. The μ-opioid receptors are the principal molecular targets for endomorphin-induced G-protein activation in the pons/medulla and the antinociception caused by the intracerebroventricular administration of μ-opioid agonists. These data support the notion that there are limited physiological μ-opioid receptor reserves for inducing G-protein activation in the pons/medulla and for the nociceptive modulation induced by the central administration of endomorphin-1 and -2.

Identification of the G-protein-coupled ORL1 receptor in the mouse spinal cord by [35S]-GTPγS binding and immunohistochemistry

Although the ORL1 receptor is clearly located within the spinal cord, the functional signalling mechanism of the ORL1 receptor in the spinal cord has not been clearly documented. The present study was then to investigate the guanine nucleotide binding protein (G‐protein) activation mediated through by the ORL1 receptor in the mouse spinal cord, measuring the modulation of guanosine‐5′‐o‐(3‐[35S]‐thio) triphosphate ([35S]‐GTPγS) binding by the putative endogenous ligand nociceptin, also referred as orphanin FQ. We also studied the anatomical distribution of nociceptin‐like immunoreactivity and nociceptin‐stimulated [35S]‐GTPγS autoradiography in the spinal cord. Immunohistochemical staining of mouse spinal cord sections revealed a dense plexus of nociceptin‐like immunoreactive fibres in the superficial layers of the dorsal horn throughout the entire length of the spinal cord. In addition, networks of fibres were seen projecting from the lateral border of the dorsal horn to the lateral grey matter and around the central canal. In vitro [35S]‐GTPγS autoradiography showed high levels of nociceptin‐stimulated [35S]‐GTPγS binding in the superficial layers of the mouse dorsal horn and around the central canal, corresponding to the areas where nociceptin‐like immunoreactive fibres were concentrated. In [35S]‐GTPγS membrane assay, nociceptin increased [35S]‐GTPγS binding of mouse spinal cord membranes in a concentration‐dependent and saturable manner, affording maximal stimulation of 64.1±2.4%. This effect was markedly inhibited by the specific ORL1 receptor antagonist [Phe1Ψ (CH2‐NH) Gly2] nociceptin (1–13) NH2. None of the μ‐, δ‐, and κ‐opioid and other G‐protein‐coupled receptor antagonists had a significant effect on basal or nociceptin‐stimulated [35S]‐GTPγS binding. These findings suggest that nociceptin‐containing fibres terminate in the superficial layers of the dorsal horn and the central canal and that nociceptin released in these areas may selectively stimulate the ORL1 receptor to activate G‐protein. Furthermore, the unique pattern of G‐protein activation in the present study provide additional evidence that nociceptin is distinct from the μ‐, δ‐ or κ‐opioid system.

Up-regulation of the TrkB receptor in mice injured by the partial ligation of the sciatic nerve.

Partial nerve injury induced by tying a tight ligature around the sciatic nerve induced a marked hyperalgesia, and this persistent painful state lasted for 14 days in mice. Under these conditions, the nerve injury induced a significant increase in protein level of protein kinase Cgamma isoform in plasma membranes in the spinal cord. We report here for the first time that protein level of TrkB receptor located in plasma membranes was clearly up-regulated in the spinal cord obtained from the nerve-injured mice. These findings suggest that the up-regulation of protein kinase Cgamma associated with activated TrkB receptors following partial sciatic nerve ligation may induce sensitization of synaptic transmission and may in turn cause the persistent pain in mice.

Absence of G-protein activation by μ-opioid receptor agonists in the spinal cord of μ-opioid receptor knockout mice

The ability of μ‐opioid receptor agonists to activate G‐proteins in the spinal cord of μ‐opioid receptor knockout mice was examined by monitoring the binding to membranes of the non‐hydrolyzable analogue of GTP, guanosine‐5′‐O‐(3‐[35S]thio)triphosphate ([35S]GTPγS). In the receptor binding study, Scatchard analysis of [3H][D‐Ala2,NHPhe4,Gly‐ol]enkephalin ([3H]DAMGO; μ‐opioid receptor ligand) binding revealed that the heterozygous μ‐knockout mice displayed approximately 40% reduction in the number of μ‐receptors as compared to the wild‐type mice. The homozygous μ‐knockout mice showed no detectable μ‐binding sites. The newly isolated μ‐opioid peptides endomorphin‐1 and ‐2, the synthetic selective μ‐opioid receptor agonist DAMGO and the prototype of μ‐opioid receptor agonist morphine each produced concentration‐dependent increases in [35S]GTPγS binding in wild‐type mice. This stimulation was reduced by 55–70% of the wild‐type level in heterozygous, and virtually eliminated in homozygous knockout mice. No differences in the [35S]GTPγS binding stimulated by specific δ1‐ ([D‐Pen2,5]enkephalin), δ2‐ ([D‐Ala2]deltorphin II) or κ1‐ (U50,488H) opioid receptor agonists were noted in mice of any of the three genotypes. The data clearly indicate that μ‐opioid receptor gene products play a key role in G‐protein activation by endomorphins, DAMGO and morphine in the mouse spinal cord. They support the idea that μ‐opioid receptor densities could be rate‐limiting steps in the G‐protein activation by μ‐opioid receptor agonists in the spinal cord. These thus indicate a limited physiological μ‐receptor reserve. Furthermore, little change in δ1‐, δ2‐ or κ1‐opioid receptor‐G‐protein complex appears to accompany μ‐opioid receptor gene deletions in this region.

Pretreatment with protein kinase C activator phorbol 12,13-dibutyrate attenuates the antinociception induced by μ- but not ϵ-opioid receptor agonist in the mouse

Abstract The effects of pretreatment with a protein kinase C activator, phorbol 12,13-dibutyrate, on antinociception induced by i.c.v.-administered μ-opioid receptor agonist [ d -Ala 2 , NMePhe 4 , Gly(ol) 5 ] enkephalin (DAMGO) or morphine and ϵ-opioid receptor agonist β-endorphin were studied in male ICR mice. The tail-flick responses were used for antinociceptive tests. I.c.v. pretreatment with phorbol 12,13-dibutyrate (50xa0pmol) for 30 or 60 but not 10xa0min attenuated antinociception induced by i.c.v. administered DAMGO. I.c.v. pretreatment with phorbol 12,13-dibutyrate (10 and 50xa0pmol) for 60xa0min caused a dose-dependent attenuation of DAMGO (19.5xa0pmol)- or morphine (6.0xa0nmol)-induced antinociception. The dose-response curve for DAMGO-induced antinociception was shifted to the right by 7.3-fold by i.c.v. pretreatment with phorbol 12,13-dibutyrate (50xa0pmol) for 60xa0min. However, the i.c.v.-administered β-endorphin-induced antinociception was not affected by the same pretreatment with phorbol 12,13-dibutyrate. The attenuation of i.c.v.-administered DAMGO- and morphine-induced antinociception by phorbol 12,13-dibutyrate was reversed by concomitant i.c.v. pretreatment with a selective protein kinase C inhibitor calphostin C. These results suggest that activation of protein kinase C by phorbol 12,13-dibutyrate leads to the desensitization of μ-, but not ϵ-opioid receptor-mediated antinociception. These findings also provide additional evidence for differential intracellular modulation on antinociceptive action of μ- and ϵ-opioid receptor agonists.

Role of the phosphatidylinositol-specific phospholipase C pathway in δ-opioid receptor-mediated antinociception in the mouse spinal cord

Stimulation of delta-opioid receptors has been shown to activate phospholipase C via the activation of G-proteins in vitro. The present study was designed to determine, with the tail-flick method, whether the stimulatory effect of delta-opioid receptor agonists on phospholipase C and inositol lipid turnover participates in the mechanisms of the delta-opioid receptor-mediated antinociception in the mouse spinal cord. Intrathecal pretreatment with the phospholipase C inhibitors neomycin and U73122, which produced no changes in the basal tail-flick latencies when they were injected alone, significantly attenuated the antinociception induced by intrathecal administration of the selective delta-opioid receptor agonist [D-Ala(2)]deltorphin II in mice. The selective phosphatidylinositol-specific phospholipase C inhibitor ET-18-OCH(3) inhibited the antinociception induced by intrathecal administration of [D-Ala(2)]deltorphin II in a dose-dependent manner. In mice undergoing treatment with LiCl, which impairs phosphatidylinositol synthesis, the antinociception induced by intrathecal administration of [D-Ala(2)]deltorphin II was significantly reduced. Co-administration of D-myo-inositol-1,4,5-trisphosphate restored the [D-Ala(2)]deltorphin II-induced antinociception in LiCl-pretreated mice. On the other hand, intrathecal pretreatment with the selective protein kinase C inhibitor calphostin C, but not the protein kinase A inhibitor KT5720, resulted in a dose-dependent enhancement of the [D-Ala(2)]deltorphin II-induced antinociception. These results indicate a potential role for the phospholipase C-inositol-1,4, 5-trisphosphate pathway in the expression of delta-opioid receptor-mediated antinociception in the mouse spinal cord. Furthermore, activation of protein kinase C by the stimulation of delta-opioid receptors may constitute a significant pathway involved in negative modulation of spinal delta-opioid receptor-mediated antinociception.

Differential involvement of μ1-opioid receptors in endomorphin- and β-endorphin-induced G-protein activation in the mouse pons/medulla

Several genetic mouse models of differential sensitivity to opioids have been used to investigate the mechanisms underlying individual variation in responses to opioids. The CXBK mice are inbred recombinant mice which have a lower level of mu(1)-opioid receptors than their parental strain. Endomorphin-1 and endomorphin-2 are endogenous opioid peptides that are highly selective for mu-opioid receptors, while beta-endorphin, which is also an endogenous opioid peptide, is non-selective for mu-, delta- and putative epsilon-opioid receptors. The present study was designed to investigate the effects of these endogenous opioid peptides on G-protein activation by monitoring guanosine-5-o-(3-[35S]thio)triphosphate binding to pons/medulla membranes of CXBK mice and their parental strain C57BL/6 ByJ mice. Endomorphin-1 (0.1-10 microM), endomorphin-2 (0.1-10 microM) and beta-endorphin (0.1-10 microM) increased guanosine-5-o-(3-[35S]thio)triphosphate binding to the pons/medulla membranes from C57BL/6 ByJ and CXBK mice in a concentration-dependent manner. However, the increases of guanosine-5-o-(3-[35S]thio)triphosphate binding induced by either endomorphin-1 or endomorphin-2 in CXBK mice were significantly much lower than those in C57BL/6ByJ mice. However, no significant difference was found in the increases of the guanosine-5-o-(3-[35S]thio)triphosphate binding induced by beta-endorphin in C57BL/6 ByJ and CXBK mice. Moreover, whereas the increase of guanosine-5-o-(3-[35S]thio)triphosphate binding induced by 10 microM endomorphin-1 or endomorphin-2 were almost completely blocked by a mu-opioid receptor antagonist beta-funaltrexamine (10 microM) in both strains, the increase of guanosine-5-o-(3-[35S]thio)triphosphate binding induced by 10 microM beta-endorphin was attenuated to approximately 70% of stimulation by co-incubation with 10 microM beta-funaltrexamine in both strains. The residual stimulation of [35S]guanosine-5-o-(3-thio)triphosphate binding by 10 microM beta-endorphin in the presence of 10 microM beta-funaltrexamine was further attenuated by the addition of putative epsilon-opioid receptor partial agonist beta-endorphin (1-27) (1 microM) in both strains. Like the endomorphins, the synthetic mu-opioid receptor agonist [D-Ala(2),N-MePhe(4), Gly-ol(5)]enkephalin at 10 microM showed lower increases of guanosine-5-o-(3-[35S]thio)triphosphate binding in CXBK mice than those in C57BL/6ByJ mice. However, there was no strain difference in the stimulation of guanosine-5-o-(3-[35S]thio)triphosphate binding induced by 10 microM of the selective delta(1)-opioid receptor agonist [D-Pen(2,5)]enkephalin, delta(2)-opioid receptor agonist [D-Ala(2)]deltorphin II or kappa-opioid receptor agonist U50,488H. The results indicate that the G-protein activation by endomorphin-1 and endomorphin-2 in the mouse pons/medulla is mediated by both mu(1)- and mu(2)-opioid receptors. Moreover, beta-endorphin-induced G-protein activation in the mouse pons/medulla is, in part, mediated by mu(2)- and putative epsilon-, but not by mu(1)-opioid receptors.

G protein activation by endomorphins in the mouse periaqueductal gray matter.

The midbrain periaqueductal gray matter (PAG) is an important brain region for the coordination of mu-opioid-induced pharmacological actions. The present study was designed to determine whether newly isolated mu-opioid peptide endomorphins can activate G proteins through mu-opioid receptors in the PAG by monitoring the binding to membranes of the non-hydrolyzable analog of GTP, guanosine-5-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS). An autoradiographic [(35)S]GTPgammaS binding study showed that both endomorphin-1 and -2 produced similar anatomical distributions of activated G proteins in the mouse midbrain region. In the mouse PAG, endomorphin-1 and -2 at concentrations from 0.001 to 10 microM increased [(35)S]GTPgammaS binding in a concentration-dependent manner and reached a maximal stimulation of 74.6+/-3.8 and 72.3+/-4.0%, respectively, at 10 microM. In contrast, the synthetic selective mu-opioid receptor agonist [D-Ala(2),NHPhe(4), Gly-ol]enkephalin (DAMGO) had a much greater efficacy and produced a 112.6+/-5.1% increase of the maximal stimulation. The receptor specificity of endomorphin-stimulated [(35)S]GTPgammaS binding was verified by coincubating membranes with endomorphins in the presence of specific mu-, delta- or kappa-opioid receptor antagonists. Coincubation with selective mu-opioid receptor antagonists beta-funaltrexamine or D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Phe-Thr-NH(2) (CTOP) blocked both endomorphin-1 and-2-stimulated [(35)S]GTPgammaS binding. In contrast, neither delta- nor kappa-opioid receptor antagonist had any effect on the [(35)S]GTPgammaS binding stimulated by either endomorphin-1 or -2. These findings indicate that both endomorphin-1 and -2 increase [(35)S]GTPgammaS binding by selectively stimulating mu-opioid receptors with intrinsic activity less than that of DAMGO and suggest that these new endogenous ligands might be partial agonists for mu-opioid receptors in the mouse PAG.