Shigehisa Kato

cDNA cloning and regional distribution of a novel member of the opioid receptor family

We have cloned a cDNA for a novel member of the opioid receptor family, designated as ROR‐C, from the rat cerebrum cDNA library using the probe derived from the δ‐opioid receptor subtype cDNA. The deduced amino acid sequence of ROR‐C shows high homology with those of ROR‐A (rat δ‐opioid receptor subtype), ROR‐B (rat μ‐subtype) and ROR‐D (rat k‐subtype). RNA blot hybridization and in situ hybridization analysis revealed that ROR‐C mRNA is expressed in discrete regions of the rat central nervous system.

Primary structures and expression from cDNAs of rat opioid receptor δ-and μ-subtypes

The complete amino acid sequences of rat opioid receptors (designated as ROR‐A and ROR‐B) have been deduced by cloning and sequencing the cDNAs. The ligand‐binding properties of ROR‐A and ROR‐B expressed from the cloned cDNAs in Chinese hamster ovary cells correspond most closely to those of the pharmacologically defined δ‐ and μ‐opioid receptor subtypes, respectively. RNA blot hybridization analysis revealed that cerebrum and brainstem contain both ROR‐A and ROR‐B mRNAs, whereas neither ROR‐A nor ROR‐B mRNAs can be detected in cerebellum.

Functional coupling of the δ-, μ-, and κ-opioid receptors to mitogen-activated protein kinase and arachidonate release in Chinese hamster ovary cells

Abstract: To examine whether the mitogen‐activated protein kinase (MAPK) cascade and phospholipase A2 (PLA2) are involved in the signal transduction mechanism of the opioid receptor, the δ‐, μ‐, and κ‐opioid receptors were stably expressed from cDNA in Chinese hamster ovary cells. Activation of the δ‐, μ‐, and κ‐receptors by agonists induced a rapid and transient increase in MAPK activity accompanied by reduced electrophoretic mobility of the 42‐kDa isoform of MAPK (p42), probably owing to phosphorylation. The opioid receptor‐mediated increase in MAPK activity was suppressed not only by pretreatment with genistein, a tyrosine protein kinase inhibitor, but also by prolonged exposure to phorbol 12‐myristate 13‐acetate and pretreatment with GF 109203X, a selective protein kinase C (PKC) inhibitor, suggesting the involvement of PKC as well as tyrosine protein kinase. Furthermore, stimulation of the δ‐, μ‐, and κ‐receptors with opioid agonists in the presence of A23187, a calcium ionophore, resulted in an increase in arachidonate release, suggesting that PLA2 is activated by the opioid receptors when the intracellular Ca2+ concentration is elevated. Both MAPK activation and increase in arachidonate release mediated by the opioid receptors were abolished by pretreatment with pertussis toxin, suggesting that these responses are mediated by Gi or Go types of GTP‐binding regulatory proteins.

cDNA cloning and pharmacological characterization of an opioid receptor with high affinities for κ‐subtype‐selective ligands

The amino acid sequence of a rat opioid receptor, designated as ROR‐D, has been deduced by cloning and sequencing the cDNA. The ROR‐D expressed from the cDNA exhibits high affinities for ligands selective for the opioid receptor κ‐subtype and low affinities for ligands selective for the δ‐ and μ‐subtypes. RNA blot hybridization analysis indicated that ROR‐D mRNA is distributed in cerebrum and brainstem but not in cerebellum.

Activation of mitogen‐activated protein kinase by the nociceptin receptor expressed in Chinese hamster ovary cells

Activation of the nociceptin receptor stably expressed in Chinese hamster ovary cells induced a transient mitogen‐activated protein kinase (MAPK) activation, via pertussis toxin‐sensitive G‐proteins. The nociceptin receptor‐mediated MAPK activation was partially blocked by down‐regulation or inhibition of protein kinase C, and suppressed by pretreatment with a phosphatidylcholine‐specific phospholipase C inhibitor, D609. Furthermore, a tyrosine protein kinase inhibitor, genistein, and phosphatidylinositol 3‐kinase inhibitors, wortmannin and LY294002, affected the nociceptin‐induced MAPK activity. The nociceptin‐induced MAPK activation may lead to activation of phospholipase A2 and induce changes in gene expression.

Nociceptin receptor-mediated Ca2+ channel inhibition and its desensitization in NG108-15 cells

It has been shown that the membrane of hybrid NG108-15 neuroblastoma x glioma cells contains a high-affinity binding site for nociceptin. In the present study, we first demonstrated the expression of nociceptin receptor mRNA in NG108-15 cells. Application of nociceptin to NG108-15 cells produced a concentration-dependent (EC50 = 29 nM) inhibition of Ca2+ channel currents in a pertussis toxin-sensitive fashion. This nociceptin-induced inhibition of Ca2+ channel currents was prevented in the presence of omega-conotoxin GVIA, a blocker of the N-type Ca2+ channel, and had both voltage-dependent and -independent components. Prolonged application of nociceptin elicited homologous desensitization of the inhibition with a time constant of 5.3 min. These results indicate that the nociceptin receptor is coupled to the N-type Ca2+ channel via pertussis toxin-sensitive G proteins in NG108-15 cells and that this coupling is associated with rapid and homologous desensitization.

Coupling of the cloned μ-opioid receptor with the ω-conotoxin-sensitive Ca2+ current in NG108-15 cells

Abstract: Voltage‐dependent Ca2+ currents were measured in NG108‐15 neuroblastoma × glioma hybrid cells transformed to express the rat μ‐opioid receptor by the whole‐cell configuration of the patch‐clamp technique with Ba2+ as charge carrier. A μ‐opioid receptor‐selective agonist, [d‐Ala2, N‐Me‐Phe4,Gly5‐ol]enkephalin caused significant inhibition of voltage‐dependent Ca2+ currents in μ‐receptor‐transformed NG108‐15 cells but not in nontransfected or vector‐transformed control cells. On the other hand, a δ‐opioid receptor‐selective agonist, [d‐penicillamine2,d‐penicillamine5]enkephalin, induced inhibition of voltage‐dependent Ca2+ currents in both control and μ‐receptor‐transformed cells, which is mediated by the δ‐opioid receptor expressed endogenously in NG108‐15 cells. The inhibition of voltage‐dependent Ca2+ currents induced by [d‐Ala2,N‐Me‐Phe4,Gly5‐ol]enkephalin and [d‐penicillamine2,d‐penicillamine5]enkephalin was reduced by pretreatment of the cells with pertussis toxin or ω‐conotoxin GVIA. These results indicate that the μ‐opioid receptor expressed from cDNA functionally couples with ω‐conotoxin‐sensitive N‐type Ca2+ channels through the action of pertussis toxin‐sensitive G proteins in NG108‐15 cells.

Desensitization and resensitization of δ-opioid receptor-mediated Ca2+ channel inhibition in NG108-15 cells

1 To approach the mechanisms underlying desensitization of the opioid receptor‐mediated Ca2+ channel inhibition, the effects of prolonged application of [D‐Ala2, D‐Leu5]enkephalin (DADLE) on Ba2+ currents (IBa) through Ca2+ channels were analysed in NG108‐15 neuroblastoma × glioma hybrid cells. 2 Inhibition of IBa by 100 nM DADLE desensitized by 57% with a time constant of 4.4 min. 3 Maximal desensitization of the δ‐opioid receptor‐Ca2+ channel coupling was attained by 1 μM DADLE. The EC50 value for desensitization was estimated to be 78 nM. 4 RNA blot hybridization analysis and immunoblot analysis revealed the expression of β‐adrenoceptor kinase‐1 (βARK1) in NG108‐15 cells. 5 Heparin, an inhibitor of βARK, significantly reduced the magnitude and rate of desensitization, whereas Rp‐cyclic AMPS and PKI (14‐24)amide, inhibitors of cyclic AMP‐dependent protein kinase (PKA), or long‐term treatment with phorbol 12‐myristate 13‐acetate to induce down‐regulation of protein kinase C (PKC) had no significant effect. 6 Recovery from desensitization (resensitization) proceeded with a time constant of 6.7 min. Okadaic acid, an inhibitor of serine/threonine phosphatases 1 and 2A, significantly attenuated the degree of resensitization. 7 In summary, we have characterized the time course and concentration‐dependence of the desensitization of DADLE‐induced IBa inhibition in NG108‐15 cells. This desensitization was reversible after removal of DADLE. It is suggested that βARK, but neither PKA nor PKC, is involved in desensitization, while serine/threonine phosphatases mediate resensitization.

Adaptations to chronic agonist exposure of μ-opioid receptor-expressing Chinese hamster ovary cells

To investigate cellular adaptation responses induced by chronic agonist treatment of the mu-opioid receptor, Chinese hamster ovary (CHO) cells were stably transfected with the rat mu-opioid receptor cDNA. Chronic treatment with agonists selective for the mu-opioid receptor, [D-Ala2, N-MePhe4, Gy-ol5]enkephalin (DAMGO), morphine and fentanyl, time- and dose-dependently induced down-regulation of the mu-opioid receptor. The down-regulation was not significantly affected by pretreatment with pertussis toxin, but was completely blocked by treatment with hypertonic sucrose, suggesting that receptor internalization mediated by clathrin-coated vesicles is an essential step in the mu-opioid receptor down-regulation. On the other hand, forskolin-stimulated cyclic AMP formation was increased by chronic DAMGO treatment, which was inhibited by pertussis toxin pretreatment. These results indicate that two adaptation responses induced by chronic agonist treatment of the mu-opioid receptor-expressing CHO cells, down-regulation of the mu-opioid receptor and supersensitization of adenylate cyclase, are mediated by distinct mechanisms.

Partial Agonistic Activity of Naloxone on the Opioid Receptors Expressed from Complementary Deoxyribonucleic Acids in Chinese Hamster Ovary Cells

Naloxone is a widely used opioid antagonist.To analyze the cellular responses induced by naloxone in the absence of opioid agonists, Chinese hamster ovary (CHO) cells, which do not endogenously express the opioid receptors, have been permanently transfected with the cloned complementary DNAs to produce the [micro sign]-, delta-, and kappa-opioid receptors. Naloxone dose-dependently reduced forskolin-stimulated cyclic adenosine monophosphate (cAMP) formation in the cells expressing the [micro sign]- and kappa-opioid receptors, although the effect was less than that of opioid agonists [D-Ala2, N-Me-Phe4, Gly-ol5]enkephalin and U50,488, respectively. The naloxone-induced cAMP reduction was abolished by pretreatment of the cells with pertussis toxin, which suggests that pertussis toxin-sensitive G proteins (Gi and/or Go) are involved in the response. Cellular guanosine triphosphatase activity was significantly increased by naloxone in the cells expressing the [micro sign]- and kappa-opioid receptors, which suggests that the application of naloxone to these receptors induces activation of the G proteins. We conclude that naloxone possesses partial agonistic activity on the [micro sign]- and kappa-opioid receptors expressed from complementary DNAs in CHO cells. Implications: In this study, we examined whether naloxone has agonistic activity on the opioid receptors by using cultured cells transfected with delta-, [micro sign]-, and kappa-opioid receptor complementary DNAs. Our data indicate that naloxone is a partial agonist on the [micro sign]- and kappa-opioid receptors. (Anesth Analg 1998;87:450-5)