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

BACKGROUND: Opioid-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 mu-opioid receptor (MOR)-induced immediate early gene expression. METHODS: Chinese 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: Mu-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: Mu-opioid receptor activation induces c-fos and junB expression and elevates AP-1-mediated transcriptional activities via the mitogen-activated protein kinase cascade.

Opioid potentiation of N-type Ca2+ channel currents via pertussis-toxin-sensitive G proteins in NG108-15 cells.

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 x 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 delta-opioid antagonist, pertussis toxin (PTX) and omega-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 delta-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.

The ultrastructural characteristics of eccrine sweat glands in a Fabry disease patient with hypohidrosis.

As an investigation of the pathogenetic mechanism of diminished sweating in Fabry disease, an electron microscopy ultrastructural study was conducted on specimens of eccrine sweat glands from a typical patient with Fabry disease who had hypohidrosis, a low skin moisture content, and diminished thermoregulation ability. Numerous characteristic cytoplasmic inclusions were observed in the eccrine sweat glands, the lamellar pattern of which was considerably variable in various types of gland cells. Large vacuolar inclusions predominated in clear cells of secretory coil; lesser vacuoles were also seen in the coiled duct, and the basal cells of the straight duct toward the coiled duct displayed mulberry-like figures. There were some clear cells showing cell damage and necrosis in the secretory coil. Lamellated inclusions were noted in the unmyelinated axons innervating the eccrine sweat glands. The small blood vessels around the eccrine glands were narrowed by swollen endothelial cells with heavy inclusions. These intracytoplasmic deposits may be responsible for the decreased sweating ability in Fabry disease. The factors related to hypohidrosis are also discussed.

Activation of phospholipase A2 by the nociceptin receptor expressed in Chinese hamster ovary cells.

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.

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.

Partial agonistic activity of naloxone on the opioid receptors expressed from complementary deoxyribonucleic acids in Chinese hamster ovary cells.

UNLABELLED: 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 mu-, delta-, and kappa-opioid receptors. Naloxone dose-dependently reduced forskolin-stimulated cyclic adenosine monophosphate (cAMP) formation in the cells expressing the mu- 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 mu- 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 mu- 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-, mu-, and kappa-opioid receptor complementary DNAs. Our data indicate that naloxone is a partial agonist on the mu- and kappa-opioid receptors.

Tyrosine kinase inhibitors suppress N-type and T-type Ca2+ channel currents in NG108-15 cells.

Modulation of Ca2+ channel activity by protein kinases constitutes one of the major mechanisms regulating neuronal functions. Here, we explored the possible modulation of neuronal Ca2+ channels by protein tyrosine kinases (PTKs). To this end, the effects of PTK inhibitors on whole-cell Ba2+ currents (IBa) through voltage-gated Ca2+ channels were analysed in differentiated NG108-15 neuroblastoma x glioma hybrid cells. Genistein suppressed IBa in a concentration-dependent fashion (IC50 = 22 microM). Although daidzein, an analogue of genistein that is devoid of PTK inhibitory activity, also suppressed IBa, we estimated that specific PTK inhibition by genistein reduced IBa amplitude by 30%. In addition, lavendustin A (20 microM) and herbimycin A (20 microM), two other distinct PTK inhibitors, depressed IBa by 22% and 20%, respectively. Genistein suppressed N-type and T-type currents, sparing L-type current, and its effect was independent of G protein activation. The results suggest that the activity of neuronal Ca2+ channels can be modulated by PTKs, opening the possibility that some of the functions of PTKs in the nervous system are mediated by Ca2+ channel modulation.

Adaptations to chronic agonist exposure of mu-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.

Desensitization and resensitization of delta-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 (I(Ba)) through Ca2+ channels were analysed in NG108-15 neuroblastoma x glioma hybrid cells. 2. Inhibition of I(Ba) by 100 nM DADLE desensitized by 57% with a time constant of 4.4 min. 3. Maximal desensitization of the delta-opioid receptor-Ca2+ channel coupling was attained by 1 microM DADLE. The EC50 value for desensitization was estimated to be 78 nM. 4. RNA blot hybridization analysis and immunoblot analysis revealed the expression of beta-adrenoceptor kinase-1 (betaARK1) in NG108-15 cells. 5. Heparin, an inhibitor of betaARK, 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 I(Ba) inhibition in NG108-15 cells. This desensitization was reversible after removal of DADLE. It is suggested that betaARK, but neither PKA nor PKC, is involved in desensitization, while serine/threonine phosphatases mediate resensitization.

Ca2+ channel inhibition by endomorphins via the cloned mu-opioid receptor expressed in NG108-15 cells.

Endomorphin-1 and -2, recently isolated endogenous peptides specific for the mu-opioid receptor, inhibited Ca2+ channel currents with EC50 of 6 and 9 nM, respectively, in NG108-15 cells transformed to express the cloned rat mu-opioid receptor. On the other hand, they elicited no response in nontransfected NG108-15 cells. It is concluded that endomorphin-1 and -2 induce Ca2+ channel inhibition by selectively activating the mu-opioid receptor.

Acquired laryngomalacia as a cause of airway obstruction immediately after unilateral mouth floor surgery.

We report a case of acquired laryngomalacia in which airway obstruction due to prolapse of the epiglottis during inspiration was observed immediately after a unilateral mouth floor resection. Suggested causes are resection of unilateral elevator muscles of the hyoid bone, epiglottic oedema and transient loss of pharyngeal motor control due to surgical intervention and high-dose radiation.

[A case report of prostate cancer resistant to endocrine therapy successfully treated with intra-arterial chemotherapy].

A 73-year-old male with low abdominal pain on urination and frequent urination was diagnosed as poorly differentiated adenocarcinoma of prostate. He received endocrine therapy with DESD and bilateral orchiectomy. This treatment was not effective, so he was given intra-arterial infusion chemotherapy with MTX, ADM and CDDP using the reservoir system. After 2 courses of this chemotherapy the regression rate was 75%, and the pathological examination after the chemotherapy revealed no cancer cells. There is no established chemotherapy for prostate cancer at present. Thus this case is very suggestive for the treatment of prostate cancer.

Oxygen embolism due to hydrogen peroxide irrigation during cervical spinal surgery.

Hydrogen peroxide is used to cleanse and irrigate wounds. As it decomposes immediately into water and oxygen on contact with organic tissue, it is usually regarded as a safe agent. We report a case of oxygen embolism associated with hydrogen peroxide irrigation of the surgical field during anterior fusion of the cervical vertebrae. It was accompanied by precipitous hypotension and decrease in pulse oximetry oxygen saturation and end-tidal CO2 tension. Semi-closed spaces formed under the apatite dowel and between the apatite dowel and vertebral bodies may have precipitated the absorption of oxygen bubbles into the vasculature. Although this case was associated with a rapid recovery and uneventful sequelae, it discourages the use of hydrogen peroxide in this procedure because of the potential hazards including cardiovascular collapse.

Vaginal absorption of a potent luteinizing hormone-releasing hormone analogue (leuprolide) in rats. IV: Evaluation of the vaginal absorption and gonadotropin responses by radioimmunoassay.

The vaginal absorption of leuprolide (a potent luteinizing hormone-releasing hormone analogue), which has the potential for producing regression of hormone-dependent tumors as well as high gonadotropin-releasing and ovulation-inducing activities, was evaluated in rats by radioimmunoassay. Gonadotropin (luteinizing hormone and follicle-stimulating hormone) release was concomitantly determined. Although leuprolide disappeared rapidly from the serum after intravenous administration (the biological half-lives were 8.4 min in the alpha-phase and 33.2 min in the beta-phase), long-lasting serum levels were observed when the analogue was administered vaginally. The vaginal absorption was enhanced by adding citric acid to the test solution. The absolute bioavailability, estimated by the AUC of serum leuprolide levels, was 25.8% over 6 h and 38.0% over 12 h in the 5% citric acid solution (pH 3.5). The sustained release of gonadotropin was also obtained after vaginal administration of the analogue. A linear dose absorption correlation of leuprolide was obtained in the range of 10-1000 micrograms/kg in an aqueous solution or methylcellulose jelly. The release of gonadotropin showed a plateau level at greater than 10 micrograms/kg, which corresponds to an effective dose for antitumor activity. The vaginal absorption of leuprolide varied with the estrous cycle, but this effect was eliminated by prior subcutaneous pretreatment with the analogue.

Desensitization of gonadotropin-releasing response following vaginal consecutive administration of leuprolide in rats.

The vaginal absorption of a potent luteinizing hormone-releasing hormone analog (leuprolide), and the gonadotropins (luteinizing hormone and follicule-stimulating hormone)-releasing response after continuous vaginal and subcutaneous administration of the analog to rats were determined by the radioimmunoassay. A marked suppressive effect on the gonadotropin-releasing response along with long-lasting serum levels of leuprolide were paradoxically observed after consecutive 3-d vaginal administration of the analog at doses of 1 microgram/kg or greater. Pituitary function recovered progressively with cessation of the treatment but was not complete 4 d after cessation. Continuous vaginal and subcutaneous infusion resulted in an almost complete inhibition of both gonadotropins response. It is suggested that effective desensitization of the pituitary gonadotropin response is elicited by continuous administration of leuprolide and, therefore, the vaginal administration resulting in prolonged serum levels of the analog could be preferable as a self-administration method for medical treatments such as anti-tumor therapy and birth control.

Regression of rat mammary tumors by a potent luteinizing hormone-releasing hormone analogue (leuprolide) administered vaginally.

Potent luteinizing hormone-releasing hormone analogues are known to cause regression of hormone-dependent mammary tumors. We have observed that high and long-lasting serum levels of a potent luteinizing hormone-releasing hormone analogue [desglycyl10-(D-leucyl6) luteinizing hormone-releasing hormone ethylamide, leuprolide] resulted from vaginal administration which effectively caused down regulation in the pituitary by chronic treatment. Regression of 7,12-dimethylbenz(a)-anthracene-induced mammary tumors in Sprague-Dawley rats by consecutive daily vaginal administration of leuprolide was investigated. In untreated rats, 71% of tumors were growing at 8 weeks, whereas after i.p. injection of leuprolide (500 micrograms/kg) all tumors were regressing 2 weeks after commencement of treatment and 86.7% of tumors disappeared by 8 weeks. Vaginal administration of 100 micrograms/kg for 8 weeks produced regression in 80% of tumors and disappearance in 35%. The vaginal administration of a higher dose (500 to 5000 micrograms/kg) produced highly significant antitumor effects [regression in 82.2 +/- 4.0% (S.E.) and disappearance in 52.9 +/- 2.1%]. These results are consistent with the effects produced by ovariectomy. Whereas 13 and 7 new tumors appeared in untreated rats and those treated vaginally with leuprolide (100 micrograms/kg), respectively, only one or two tumors appeared in i.p. and vaginally (above 500 micrograms/kg) treated rats during treatment. Histological classification of the mammary tumors after treatment indicated therapeutic effects similar to those shown by tumor size determination. Thus, it was concluded that vaginal application of leuprolide at doses above 500 micrograms/kg might be a potentially useful method for antitumor therapy.

Vaginal absorption of a potent luteinizing hormone-releasing hormone analogue (leuprolide) in rats III: Effect of estrous cycle on vaginal absorption of hydrophilic model compounds.

The effect of estrous cycle stages on vaginal absorption was determined by the use of insulin, phenolsulfonphthalein, and salicylic acid as hydrophilic model compounds. Absorption of these compounds was markedly affected by the stage, possibly due to the change of transport rate through the pore-like pathways. The absorption of phenolsulfonphthalein during proestrus and estrus is roughly one-tenth of that during metestrus and diestrus. An increase of the nonionized form of salicylic acid, produced by a lowered pH, resulted in an enhancement of absorption during proestrus and diestrus; higher contribution of the transport through the cell membrane possibly reduced an effect of the estrous cycle. However, consecutive daily administration of leuprolide halted the cycle at diestrus and reduced the cycle effect on the vaginal absorption of phenolsulfonphthalein; when the treatment was started at any of the four stages of the cycle, vaginal absorption was enhanced approximately 20%, with less variance than that observed in normal diestrous rats.