Takehiro Shoda

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.

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.

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.

Activation of μ-opioid receptor induces expression of c-fos and junB via mitogen-activated protein kinase cascade

BackgroundOpioid-induced long-term functional alterations of the nervous system, such as tolerance, addiction, and dependence, conceivably involve changes in gene expression. The authors have previously reported that opioid receptors are functionally coupled to extracellular signal–regulated kinase, a class of the mitogen-activated protein kinase. To address whether activation of the opioid receptor induces changes in gene expression through the activation of extracellular signal–regulated kinase, the authors examined &mgr;-opioid receptor (MOR)–induced immediate early gene expression. MethodsChinese hamster ovary cells stably expressing MOR were used. Cells were stimulated by MOR agonists after 24-h serum starvation. Expression of c-fos and junB genes was analyzed by RNA blot hybridization. To explore the mechanism of MOR-mediated c-fos and junB expression, activity of a transcription factor, Elk-1, was assessed by reporter assay. Furthermore, to investigate the functional consequences of c-fos and junB induction, MOR-mediated formation of the functional transcription factor complex AP-1 was examined by reporter assay and electrophoretic mobility shift assay. Results&mgr;-Opioid receptor activation induced c-fos and junB messenger RNAs, which were inhibited by pretreatment of the cells with pertussis toxin and PD98059, an inhibitor of extracellular signal–regulated kinase cascade. MOR stimulation elevated Elk-1-mediated transcriptional activity by about 10-fold. AP-1–mediated transcriptional activity was stimulated by MOR agonists by about twofold. Electrophoretic mobility shift assay revealed that AP-1 binding activity in the nuclear extract was elevated by MOR activation and further showed that products of c-fos and junB genes are involved in formation of AP-1 complex. Conclusions&mgr;-Opioid receptor activation induces c-fos and junB expression and elevates AP-1–mediated transcriptional activities via the mitogen-activated protein kinase cascade.

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)

Activation of Phospholipase A2 by the Nociceptin Receptor Expressed in Chinese Hamster Ovary Cells

Abstract: To gain insight into the molecular mechanism for nociceptin function, functional coupling of the nociceptin receptor expressed in Chinese hamster ovary (CHO) cells with phospholipase A2 (PLA2) was examined. In the presence of A23187, a calcium ionophore, activation of the nociceptin receptor induced time‐ and dose‐dependent release of arachidonate, which was abolished by pretreatment of the cells with pertussis toxin (PTX). Immunoblot analysis using anti‐Ca2+‐dependent cytosolic PLA2 (cPLA2) monoclonal antibody demonstrates that activation of the nociceptin receptor induces a time‐ and dose‐dependent electrophoretic mobility shift of cPLA2, suggesting that phosphorylation of cPLA2 is induced by the nociceptin receptor. Pretreatment of the cells with PD98059, a specific mitogen‐activated protein kinase/extracellular signal‐regulated kinase kinase 1 inhibitor, or staurosporine, a potent inhibitor of serine/threonine protein kinases and tyrosine protein kinases, partially inhibited the nociceptin‐induced cPLA2 phosphorylation and arachidonate release. These results indicate that the nociceptin receptor expressed in CHO cells couples with cPLA2 through the action of PTX‐sensitive G proteins and suggest that cPLA2 is activated by phosphorylation induced by the nociceptin receptor via mechanisms partially dependent on p44 and p42 mitogen‐activated protein kinases.

OPIOID POTENTIATION OF N-TYPE CA2+ CHANNEL CURRENTS VIA PERTUSSIS-TOXIN-SENSITIVE G PROTEINS IN NG108-15 CELLS

Abstract Opioids have both inhibitory and stimulatory effects on neurotransmitter release. While the inhibitory effect has been ascribed to presynaptic inhibition of Ca2+ channels, the cellular mechanism underlying the stimulatory effect is not clear. In order to address this issue, we analyzed the effects of [d-Ala2, d-Leu5]-enkephalin (DADLE) on whole-cell Ba2+ currents (IBa) through voltage-gated Ca2+ channels in NG108–15 neuroblastoma × glioma hybrid cells. Application of DADLE inhibited and washout of DADLE transiently potentiated IBa. Furthermore, potentiation of IBa was elicited even in the presence of DADLE, when inhibition was relieved by a large depolarizing prepulse. DADLE-induced potentiation, as well as inhibition, had both voltage-sensitive and -insensitive components and was abolished by treatment with ICI174864, a δ-opioid antagonist, pertussis toxin (PTX) and ω-conotoxin GVIA. Potentiation developed over @3 min and took 5–20 min to recover, whereas inhibition was complete within 30 s and recovered within 1 min. Although this potentiation should contribute to DADLE-induced desensitization of Ca2+ channel inhibition, it was not the sole mechanism for desensitization. We conclude that the δ-opioid receptor exerts a dual action on N-type Ca2+ channels via PTX-sensitive G proteins, i.e., rapid inhibition followed by slowly developing potentiation.

Morphine induces DNA damage and P53 activation in CD3+ T cells.

BACKGROUND Morphine has been shown to affect the function of immune system, but the precise mechanism remains to be elucidated. The present study was aimed to clarify the mechanism for the morphine-induced immune suppression by analyzing the direct effect of morphine on human CD3+ T cells. METHODS To identify genes up-regulated by action of morphine on the opioid receptor expressed in CD3+ T cells, PCR-select cDNA subtraction was performed by the use of total RNA from human CD3+ T cells treated with morphine in the presence and absence of naloxone. RESULTS We show that p53 and damage-specific DNA binding protein 2 (ddb2) genes are up-regulated by morphine in a naloxone-sensitive manner. Furthermore, the results indicate that DNA damage, quantified by apurinic-apyrimidinic site counting assay and phosphorylation of Ser-15 in P53 protein, is induced in CD3+ T cells by morphine in a naloxone-sensitive manner. GENERAL SIGNIFICANCE Because it was shown that only the kappa opioid receptor gene is expressed in CD3+ T cells in the opioid receptor family, the present study suggests that morphine induces DNA damage through the action on the kappa opioid receptor, which leads to immune suppression by activation of P53-mediated signal transduction.