naloxone and Glioma

In our earlier studies we have demonstrated that recombinant human interferon-alpha 2A (rHu-IFN-alpha 2A) inhibits hypothalamo-pituitary-adrenocortical (HPA) secretion following both peripheral and central administration. Furthermore, this effect is antagonized by mu-opioid receptor antagonists, suggesting transduction by this subtype of opioid receptors. In the present studies, we demonstrate that this effect is also observed with the hybrid recombinant preparation, rHu-IFN-alpha A/D, and a leucocyte-derived rat IFN-alpha preparation. The inhibitory effects on HPA activity were observed after intraperitoneal (i.p.) injections of rHu-IFN-alpha 2A (10(3) U), rHu-IFN-alpha A/D (10(4) U), and of Rat-IFN-alpha (1 and 10 U). Similar effects were observed with intracerebroventricular (i.c.v.) administration of all three IFN-alpha preparations. No increases in plasma concentrations of corticosterone were observed with doses of rHu-IFN-alpha A/D up to 10(6) U (i.p.) or 7 x 10(5) U (i.c.v.), but increases were found following i.c.v. administration of high doses of Rat-IFN-alpha (10(3) and 5 x 10(3) U). The inhibitory effects of all of the IFN-alpha preparations tested were antagonized by naloxone, but the stimulatory effects of 5 x 10(3) U Rat-IFN-alpha were not. Injections of rHu-IFN-alpha 2A (10(4) U i.p.) to urethane-anesthetized rats decreased the electrical activity of the majority of hypothalamic paraventricular nucleus neurons tested, including putative corticotropin-releasing factor-secreting neurons antidromically identified as projecting to the median eminence. These electrophysiological data suggest that the decreases in HPA activity evoked by IFN-alpha are mediated by a rapid inhibitory effect at the level of the corticotropin-releasing factor-secreting neurons. The sensitivity of many central nervous system effects of IFN-alpha to mu-receptor antagonists strongly suggests that the cytokine serves as an endogenous opioid agonist arising from the immune system. In support of this hypothesis we have shown that SH-SY5Y human neuroblastoma cells, differentiated with retinoic acid treatment to express predominantly mu-receptors, are sensitive to rHu-IFN-alpha 2A in vitro. This sensitivity took the form of a dose-dependent inhibition of forskolin-stimulated adenylyl cyclase activity. The data yielded an IC50 (95% confidence intervals) value of 7.93 (5.70-11.04) nM for this effect. Neither undifferentiated SH-SY5Y cells nor NG108-15 mouse neuroblastoma x rat

Topics: Animals; Corticosterone; Depression, Chemical; Electrophysiology; Glioma; Humans; Hybrid Cells; Hypothalamo-Hypophyseal System; Interferon Type I; Interferon-alpha; Male; Mice; Naloxone; Neuroblastoma; Neurons; Paraventricular Hypothalamic Nucleus; Pituitary-Adrenal System; Rats; Rats, Sprague-Dawley; Recombinant Fusion Proteins; Recombinant Proteins; Species Specificity; Tumor Cells, Cultured

Topics: Animals; Bacterial Toxins; Carbachol; Cell Membrane; Cyclic AMP; Drug Tolerance; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Glioma; Hybrid Cells; Mice; Naloxone; Narcotics; Neuroblastoma; Norepinephrine; Prostaglandins E; Rats; Receptors, Opioid; Virulence Factors, Bordetella

This study demonstrates that activation of delta-opioid receptors (DORs) in neuroblastomaxglioma (NG108-15) hybrid cells by [D-Ala2, D-Leu5]enkephalin (DADLE) and etorphine significantly enhances cell adhesion to fibronectin-coated wells. This effect is blocked by both naloxone and integrin binding RGDT peptides. In addition, cell adhesion turned out to be a prerequisite for DOR-stimulated transactivation of Tropomyosin-related kinase A (TrkA) and extracellular signal-regulated kinases 1/2 (ERK1/2). Because inhibition of TrkA activation by AG879 completely blocked DOR- and integrin-mediated ERK1/2 signaling, the present results indicate that in NG108-15 cells DOR-stimulated ERK1/2 activation is mediated by integrin-induced transactivation of TrkA.

Topics: Cell Adhesion; Enkephalin, Leucine-2-Alanine; Etorphine; Glioma; Humans; Hybrid Cells; Integrins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Naloxone; Neuroblastoma; Neurons; Oligopeptides; Protein Kinase Inhibitors; Receptor, trkA; Receptors, Opioid, delta; Transcriptional Activation; Tyrphostins

Acute mu opioid application has been shown to activate extracellular signal-related kinases (ERKs) in various non-neural cell lines. However, ERK activation in neuronal cells following acute morphine treatment is more questionable. Moreover, the ERK activation phenomenon observed in vivo after withdrawal of chronic opioids has never been demonstrated in vitro. The goal of this study was to determine if mu agonist treatment induced ERK activation acutely or after withdrawal of chronic opioids in one glial and three neuronal cell lines. We found that acute application of opioids was not able to activate ERK in neuronal cell lines but was able to activate ERK in a glial cell line. In another set of experiments, cells were chronically treated with escalating doses of a mu opioid agonist. After 8 days, the agonist was removed from the media and naloxone applied. Acute ERK activation was not seen in any tested cell line after agonist removal. These findings suggest that opioids may acutely activate ERK in non-neuronal cells, and that the acute ERK activation observed in some brain regions during opioid withdrawal in vivo might be mediated by indirect effects on neuronal cells.

Topics: Animals; Blotting, Western; Cell Line, Tumor; Diprenorphine; Drug Administration Schedule; Enzyme Activation; Fentanyl; Glioma; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Ligands; Mitogen-Activated Protein Kinases; Naloxone; Narcotic Antagonists; Narcotics; Neuroblastoma; Neuroglia; Neurons; Radioligand Assay; Rats; Time Factors

To investigate the effect of morphine on catabolism and anabolism of purine nucleotide in c6 glioma cells.. C6 glioma cells were cultured and divided into 3 groups: 1) morphine group: morphine (10 micro g/ml culture) was added for 6 h, 12 h, 24 h, 48 h, and 72 h; 2) morphine + naloxone group: naloxone (1 micro mol/L) was added for 1 hour and then morphine (10 micro g/ml) was added for 24 hours; and 3) control group: normal saline was used for 6, 12, 24, 48, and 72 hours. The C6 glioma cells were centrifuged. RT-PCR was used to examine the gene transcripts of key enzymes of purine salvage way, hypoxanthine-guanine-phosphoribosyl transferase (HGPRT) and adenylate kinase (AK). RT-PCR-Southern blotting was used to examine the gene transcripts of key enzymes of purine salvage way, xanthine dehydrogenase (XD)/xanthine oxidase (XO) mRNA.. Compared with that in the control group, the transcript of AK mRNA was significantly lower in the C6 cells treated with morphine for 24 hours, and began to re-increase 48 hours after morphine treatment. The transcript of AK mRNA remained at a low level after treatment of naloxone for 1 hour and treatment of morphine for 24 hours. The levels of transcript of HGPRT mRNA were lower in the morphine group than in the control group at all time points after treatment. However, the level of transcript of HGPRT mRNA 72 hours after treatment was higher in the morphine group than in the control group. The level of transcript of HGPRT mRNA 24 hours after exposure to morphine in the naloxon2 + morphine group was still lower than in the control group. The levels of transcripts of XD/XO mRNA were significantly higher after exposure to morphine in comparison with those in the control group at all time points after treatment. However, the levels of XD/XO mRNA 24 hours after exposure to morphine in the naloxone + morphine group recovered to the normal level.. The downregulation effect of morphine on the gene expression of AK and HGPRT may not be mediated by mu receptor. The upregulation effect of morphine on the gene expression of XD/XO may be mediated by mu receptor. Naloxone reverses the effect of morphine on enhancement of XD/XO gene expression and cannot reverse the inhibitory effect of morphine on HGPRT and AK gene expression.

Topics: Adenosine Kinase; Analgesics, Opioid; Animals; Drug Interactions; Gene Expression; Glioma; Hypoxanthine Phosphoribosyltransferase; Morphine; Naloxone; Narcotic Antagonists; Purine Nucleotides; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured

To investigate the dual effects by the delta opioid receptor agonists DPDPE on the delayed rectified potassium channels in NG108-15 cells.. A series of outward currents were evoked in NG108-15 cells by depolarizing voltage from -50 mV to +80 mV at holding potential of -90 mV. These currents were delayed rectified potassium currents. Relatively selected delta opioid receptor agonists DPDPE of higher and lower concentrations were used to modulate the delayed rectified K+ current in NG108-15 cells. Opioid receptor antagonist Naloxone (NAL) and relatively selected delta opioid receptor antagonist Naltrindole (NTI) were used in the present experiments for the characterization of the actions of opioid receptors.. The relatively higher concentrations of delta opioid receptor agonist DPDPE (> or = 10(-6) mol/L) significantly increased the amplitude of the delayed rectified K+ current. On the contrary, the relatively lower concentrations of DPDPE (< or = 10(-12) mol/L) decreased the amplitude of the delayed rectified K+ current (P < 0.05). Furthermore both the increase and decrease were time-dependent.. delta opioid receptor agonist has dual regulatory effects on the delayed rectified potassium channels in NG108-15 cells.

Topics: Animals; Cell Membrane; Enkephalin, D-Penicillamine (2,5)-; Glioma; Hybrid Cells; Mice; Naloxone; Naltrexone; Neuroblastoma; Patch-Clamp Techniques; Potassium Channels, Inwardly Rectifying; Rats; Receptors, Opioid, delta; Tumor Cells, Cultured

The guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding assay for the determination of relative opioid efficacy has been adapted to measure G protein activation in digitonin-permeabilized C6 rat glioma cells expressing a cloned mu-opioid receptor. The mu-agonist [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) caused a 3-fold increase in [35S]GTPgammaS binding over basal in a naloxone-sensitive manner. Relative mu-agonist efficacy was DAMGO > fentanyl > or = morphine > buprenorphine. Nalbuphine showed no efficacy. G protein activation by receptors has been predicted to occur by random encounter. In this model a reduction in the number of receptors will decrease the rate of G protein activation but not the maximum number of G proteins activated. To test this model C6 mu cells were treated with the irreversible mu-antagonist beta-funaltrexamine (10 nM) prior to permeabilization. This reduced the number of mu-opioid receptors determined with [3H]diprenorphine to 23 +/- 3% of control with no change in affinity. A commensurate reduction (to 29 +/- 10% of control) in the level of [35S]GTPgammaS binding stimulated by DAMGO was observed, but the t(1/2) for [35S]GTPgammaS binding remained unchanged. Thus, random encounters of receptor and G protein failed to occur in this permeabilized cell preparation. A model that assumes an organized association of G proteins with receptors better describes the activation of G proteins by opioid mu-receptors.

Topics: Analgesics, Opioid; Animals; Digitonin; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Glioma; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Indicators and Reagents; Naloxone; Naltrexone; Narcotic Antagonists; Rats; Receptors, Opioid, mu; Tumor Cells, Cultured

A C6 glioma cell line stably transfected with the human kappa opioid receptor (kappaOR) was used to characterize receptor binding and G protein activation via the kappaOR by a comprehensive series of opioid ligands. The ligand-binding affinity for [3H]5alpha,7alpha, 8beta(-)-N-methyl-N-(7-Cl-pyrrolidinyl)-1-oxaspiro(4, 5)dec-8-yl)benzene acetamide (U69593) was similar to that observed in monkey brain membranes and was 10-fold lower in the presence of sodium and GDP. Both peptide and nonpeptide agonists maximally stimulated [35S]GTPgammaS binding. The stimulation of [35S]GTPgammaS binding was blocked by pretreatment of cells with pertussis toxin. Partial stimulation of [35S]GTPgammaS binding via the kappaOR was observed for several ligands that are antagonists at the mu opioid receptor, suggesting an additional mechanism of drug action. The ability of isomers of tifluadom and levallorphan to stimulate [35S]GTPgammaS binding indicates that the chiral carbon of levallorphan, a benzomorphan derivative, imparts a greater degree of stereoselectivity than does the chiral carbon in the benzodiazepine derivative tifluadom. In addition, (-)tifluadom, the less potent isomer of tifluadom, which is also a gamma-aminobutyric acidA receptor agonist, stimulated [35S]GTPgammaS binding. In contrast, d-pentazocine, (+)SKF10047, (+)cyclazocine, and d-ethylketocyclazocine displayed no agonist activity. kappaOR-selective antagonist norbinaltorphimine competitively inhibited the stimulation of [35S]GTPgammaS binding by the active isomers of ethylketocyclazocine, cyclazocine, and nalorphine to the same degree, indicating that all three ligands are eliciting an effect via the kappaOR. The results suggest that these cells express a homogeneous population of kappaOR, and that their [35S]GTPgammaS-binding properties make them an excellent means to assess kappaOR efficacy.

Topics: Analgesics, Opioid; Animals; Benzeneacetamides; Glioma; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Ligands; Naloxone; Naltrexone; Narcotic Antagonists; Pyrrolidines; Rats; Receptors, Opioid, kappa; Stereoisomerism; Transfection; Tritium; Tumor Cells, Cultured

To compare activation of G proteins by opioid receptors, opioid agonist-stimulated guanosine 5'-O-(3-[35S]thiotriphosphate) ([35S]GTP gamma S) binding in the presence of excess GDP was assayed in membranes from NG108-15 (delta) and SK-N-SH (primarily mu) cells. Basal [35S]GTP gamma S binding consisted of a single class of low-affinity sites (KD 400-500 nM). Addition of agonists produced a high-affinity site 100-300-fold higher in affinity than the basal site. The receptor/transducer amplification factor (ratio of activated G protein Bmax to opioid receptor Bmax) was 10-fold higher for SK-N-SH mu receptors than for NG108-15 delta receptors. Chronic delta agonist ([D-Ser2]-Leu-enkephalin-Thr; DSLET) treatment of NG108-15 cells resulted in an 80% loss of DSLET-stimulated [35S]-GTP gamma S binding within 1 h. Morphine treatment of SK-N-SH cells decreased mu agonist ([D-Ala2, N-Me-Phe4,Gly5-ol]-enkephalin; DAMGO)-stimulated [35S]GTP gamma S binding by 45% after 16 h, with no effect after 1 h. Loss of agonist response was due to a decrease in the Bmax of activated G proteins with no change in the KD. These results provide a quantitative description of G protein activation occurring on acute and chronic exposure to opioid agonists.

Topics: Analgesics; Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalins; Glioma; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Hybrid Cells; Membrane Proteins; Mice; Naloxone; Naltrexone; Narcotic Antagonists; Narcotics; Neuroblastoma; Protein Binding; Rats; Receptors, Opioid, delta; Receptors, Opioid, mu; Signal Transduction; Sulfur Radioisotopes; Time Factors

Prolonged exposure of neuroblastoma x glioma (NG 108-15) hybrid cells to inhibitory acting drugs results in sensitization of adenylate cyclase. We now report that chronic activation (3 days) of either inhibitory delta-opioid receptors, alpha 2B-adrenoceptors, or muscarinic M4 receptors significantly decreases the number of stimulatory, adenylate cyclase-coupled prostaglandin E1 receptors. Pharmacological characterization further revealed that the loss of [3H]prostaglandin E1-binding sites most likely corresponds to a reduction of the number of high-affinity, G protein-coupled prostaglandin E1 receptors. The decline in functionally active prostaglandin E1 receptors developed in a time- and dose-dependent manner and could be prevented by pretreatment of the cells with pertussis toxin. Heterologous prostaglandin E1 receptor regulation was blocked by concomitant exposure of the cells to antagonists for inhibitory receptors and was rapidly reversed (t 1/2 < 30 min) upon termination of chronic inhibitory drug treatment. The decrease in high-affinity prostaglandin E1 receptors developed regardless of whether full or partial agonists were used for pretreatment. In addition, the concentrations of inhibitory drugs required to maximally affect prostaglandin E1 receptor number closely resembled those mediating maximal adenylate cyclase inhibition. The data demonstrate that chronic inhibitory drug treatment of NG 108-15 hybrid cells reduces the number of functionally active, excitatory prostaglandin E1 receptors. Thus, it is proposed that adaptations at the level of stimulatory receptor systems contribute to the regulatory mechanisms associated with drug dependence.

Topics: Adenylate Cyclase Toxin; Adenylyl Cyclases; Adrenergic alpha-Agonists; Alprostadil; Animals; Binding, Competitive; Cattle; Clonidine; Enkephalin, Leucine-2-Alanine; Glioma; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Hybrid Cells; Muscarinic Agonists; Naloxone; Narcotic Antagonists; Neuroblastoma; Oxotremorine; Pertussis Toxin; Receptors, Opioid, delta; Receptors, Prostaglandin E; Virulence Factors, Bordetella

gamma-Hydroxybutyrate (GHB) and morphine induce a number of similar effects. Moreover, the effects they elicit can be reversed by the opiate antagonist naloxone (NX), suggesting that GHB may produce at least some of its central effects by acting as an opiate agonist. The present study considered this possibility by examining the effect of GHB on mu, delta, and kappa-opioid receptor binding in concentrations of 1 nM-0.1 mM. GHB was inactive in each instance, at every dose examined. GHB is consequently not a direct opiate receptor agonist. It is also unlikely to be an indirect (enkephalin or dynorphin release-stimulating) agonist. The mechanism of action involved whereby NX can reverse the effects of GHB must therefore not involve opioid mechanisms; at least not directly.

Topics: Adjuvants, Anesthesia; Analgesics; Animals; Diprenorphine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Etorphine; Glioma; Guinea Pigs; Naloxone; Narcotic Antagonists; Narcotics; Neuroblastoma; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Sodium Oxybate; Tritium; Tumor Cells, Cultured

Opioids elicit an increase in the intracellular free Ca2+ concentration ([Ca2+]i) in neuroblastoma x glioma hybrid NG108-15 cells, which, depending upon growth conditions, results from either Ca2+ influx in differentiated cells or Ca2+ release from internal stores in undifferentiated cells (Jin et al., 1992). In this report we describe fura-2-based digital imaging studies that demonstrate that opioid-evoked Ca2+ release in these cells results from the activation of phospholipase C (PLC) and subsequent mobilization of the inositol 1,4,5-trisphosphate (IP3)-sensitive store. D-Ala2-D-Leu5-enkephalin (DA-DLE) evoked concentration-dependent increases in [Ca2+]i (EC50 approximately equal to 4 nM). The response was blocked by naloxone (1 microM). In single cells, sequential application of selective opioid agonists (10 nM) evoked responses of the rank order DADLE = D-Pen2, D-Pen5-enkephalin (DPDPE) > trans-(+/-) 3,4-dichloro-N-methyl-N-(2-[1- pyrrolidinyl]cyclohexyl) benzeneacetamide (U50488) > D-ala2, N-Me-Phe4, Gly5-ol-enkephalin (DAMGO), consistent with activation of a delta-opioid receptor. Forty percent (n = 198) of the cells responded to 100 nM DADLE with a net [Ca2+]i increase of 483 +/- 40 nM. Bradykinin (100 nM) elicited a response in 91% of the cells with a mean net amplitude of 707 +/- 36 nM. The DADLE-evoked responses were not blocked by removal of extracellular Ca2+; instead, they were abolished by treatment with 10 nM thapsigargin, an agent that depletes and prevents refilling of IP3-sensitive Ca2+ stores. A 1 microM concentration of U73122, an aminosteroid inhibitor of PLC, completely blocked the DADLE-evoked [Ca2+]i increase, while an inactive analog, U73433, was without effect. To explore the possible role of G-proteins in mediating opioid-induced [Ca2+]i increases in NG108-15 cells, we pretreated cells with pertussis or cholera toxin; pertussis toxin blocked the opioid-induced response while cholera toxin was without effect, consistent with a Gi- or Go-mediated effect. Activation of the opioid inhibitory pathway previously described for these cells appears to stimulate the phosphoinositide (PI) cascade as well. Including the PI cascade among the multiple second messenger systems modulated by opioids may be key to understanding the biochemical events that underlie acute and chronic opioid action.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics; Animals; Calcium; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine-2-Alanine; Enkephalins; Fluorescent Dyes; Fura-2; Glioma; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Kinetics; Mice; Microscopy, Fluorescence; Naloxone; Narcotics; Neuroblastoma; Pyrrolidines; Rats; Tumor Cells, Cultured

In an NG 108-15 neuroblastoma x glioma hybrid cell suspension, extracellular ATP (via P2-purinergic receptors) and bradykinin stimulated Ins(1,4,5)P3 formation, which was accompanied by an increase in the cytosolic Ca2+ concentration ([Ca2+]i). Leucine enkephalin (EK) also slightly increased [Ca2+]i in the absence, but not in the presence, of apyrase, which hydrolyses extracellular ATP and ADP to AMP. When the cells were stimulated by P2-agonists or bradykinin prior to the application of EK, EK induces a remarkable rise in [Ca2+]i. This P2-agonist- or bradykinin-assisted EK action was also observed in single cells on a coverslip. A decrease in the extracellular Ca2+ concentration only slightly lowered the EK-induced rise in [Ca2+]i, but treatment of the cells with thapsigargin, an agent which depletes Ca2+ in the Ins(1,4,5)P3-sensitive pool, almost completely abolished EK action. The observed permissive stimulation by EK of Ins(1,4,5)P3 formation induced by a P2-agonist or bradykinin may be a primary event for the EK-induced [Ca2+]i rise. These actions of EK were antagonized by naloxone and completely reversed by prior treatment of the cells with pertussis toxin, whereas the toxin hardly affected the actions of P2-agonists and bradykinin themselves. Thus EK can induce phospholipase C activation and subsequent Ca2+ mobilization, provided that the cells have been previously or are simultaneously stimulated by endogenous adenine nucleotides or by externally applied P2-agonists or bradykinin. In this cross-talk mechanism between opioid receptors and these Ca(2+)-mobilizing agonist receptors, pertussis toxin-sensitive G-proteins play a permissive role.

Topics: Adenosine Triphosphate; Bradykinin; Calcium; Enkephalin, Leucine; Enkephalins; Enzyme Activation; Glioma; GTP-Binding Proteins; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Naloxone; Neuroblastoma; Pertussis Toxin; Receptors, Bradykinin; Receptors, Neurotransmitter; Receptors, Opioid; Receptors, Purinergic; Terpenes; Thapsigargin; Tumor Cells, Cultured; Type C Phospholipases; Virulence Factors, Bordetella

We previously reported that transfection of antisense OBCAM (opioid-binding cell adhesion molecule) cDNA into NG108-15 neuroblastoma x glioma hybrid cells, which contain delta-opioid receptors, results in greatly reduced opioid binding (Ann, D. K., Hasegawa, J., Ko, J. L., Chen, S. T., Lee, N. M., and Loh, H. H. (1992) J. Biol. Chem. 267, 7921-7926. Here we report that these cells show altered coupling between opioid receptors and G-proteins. G-proteins were identified using cholera toxin (CTX)-induced ADP-ribosylation and antisera selective for Gi2 and Go alpha subunits. In the presence of delta-opioid agonists, CTX induced the incorporation of [32P]ADP-ribose into a 39-41-kDa protein with the same electrophoretic mobility as Gi2 and Go alpha subunits. This band, which was also a pertussis toxin (PTX) substrate, exhibited decreased CTX-induced ADP-ribosylation in membranes of cells treated chronically with D-Ala2-D-Leu5-enkephalin (DADLE). In cells transfected with antisense cDNA for OBCAM, labeling of this band was also decreased, compared with either sense-transfected or untransfected cells. DADLE inhibition of adenylyl cyclase and DADLE stimulation of GTPase were also greatly impaired in antisense cells, as well as GTP and GppNHp inhibition of basal and forskolin-stimulated adenylyl cyclase. These results provide further evidence for a role of OBCAM in opioid receptor function.

Topics: Adenosine Diphosphate Ribose; Adenylate Cyclase Toxin; Adenylyl Cyclases; Animals; Carrier Proteins; Cell Adhesion Molecules; Cell Membrane; Cholera Toxin; DNA, Antisense; Electrophoresis, Polyacrylamide Gel; Enkephalin, Leucine-2-Alanine; Glioma; GPI-Linked Proteins; GTP Phosphohydrolases; GTP-Binding Proteins; Hybrid Cells; Kinetics; Membrane Proteins; Mice; Molecular Weight; NAD; Naloxone; Neuroblastoma; Pertussis Toxin; Rats; Receptors, Opioid, delta; Transfection; Tumor Cells, Cultured; Virulence Factors, Bordetella

According to current concepts, agonists can effect the down-regulation of cell surface receptors, whereas antagonists can cause their up-regulation. We have discovered that the opioid antagonists naltrexone, naloxone, and ICI174864 induce a transient down-regulation of delta-opioid receptors before up-regulation, in NG108-15 cells. The possibility of an apparent loss of sites due to blockade by residual antagonist was ruled out by several lines of evidence. The reduction in delta receptors was time, temperature, and antagonist concentration dependent. This down-regulation could not be induced by either the highly mu-selective opioid antagonist cyclic D-Phe-Cys-Try-D-Trp-Arg-Thr-Pen-Thr-amide or the muscarinic antagonist atropine. In the same neurohybrid cells, the opioid agonist [D-Ala2,D-Leu5]enkephalin (0.1 microM, 60 min) effected a greater down-regulation of delta-opioid receptors. Similar qualitative changes in opioid binding of subcellular fractions were elicited with [D-Ala2,D-Leu5]enkephalin and naltrexone. However, the agonist was 2-fold more effective in reducing the heavy membrane population of receptors and 4-fold more potent in increasing the light membrane sites. Because heavy membranes are enriched in plasma membrane, whereas light membranes contain intracellular sites, these findings indicate that internalization occurs in both instances. Naltrexone and the delta-specific antagonists ICI174864 and naltrindole also diminished specific activities of two lysosomal enzymes, whereas opioid agonist-induced down-regulation was accompanied by an increase in their specific activities. Pretreatment of cell cultures with concanavalin A blocked both down-regulation and alterations in the lysosomal enzyme activities elicited by agonists and antagonists, suggesting that the latter is an opioid receptor-mediated process. The up-regulation of delta-opioid receptors by antagonists appears, then, to entail down-regulation that differs from that of agonists.

Topics: Amino Acid Sequence; Binding Sites; Binding, Competitive; Concanavalin A; Down-Regulation; Enkephalin, Leucine-2-Alanine; Glioma; Kinetics; Lysosomes; Molecular Sequence Data; Naloxone; Naltrexone; Narcotic Antagonists; Neuroblastoma; Receptors, Opioid; Receptors, Opioid, delta; Subcellular Fractions; Tritium; Tumor Cells, Cultured

Effect of dalargin, an opioid peptide (a synthetic analogue of Leu-enkephalin), on proliferation and intensity of DNA synthesis of C6 glioma cells was studied. Specific conditions of cultivation were selected, with a low control value of proliferation, which permitted to assess growth-stimulating effect of the peptide. Growth curves were plotted to assess peptide activity, which demonstrated that reaction was a many-phase process: a significant increase in cell number under peptide effect was observed only at the beginning of the logarithm phase and at the beginning of the prestationary phase of the growth curve. Cell number increased on average by 25-27% in the presence of dalargin as compared to control. Reaction of glioma DNA synthesis to dalargin also demonstrates complexity of the process: the peptide changes DNA synthesis, but as a rule, this process has a three-phase character and is not directly associated with the duration of cultivation in the presence of dalargin. Effect of naloxone, an opiate receptor blocker, was analysed to assess the receptor mechanism. It was found that reaction for naloxone and for combined effect of naloxone and dalargin was not the same.

Topics: Animals; Brain Neoplasms; Cell Division; Cell Line; DNA, Neoplasm; Drug Interactions; Enkephalin, Leucine-2-Alanine; Glioma; Naloxone; Rats; Thymidine; Time Factors; Tritium; Tumor Cells, Cultured

In membranes of neuroblastoma x glioma (NG108-15) hybrid cells, the photoreactive GTP analog, [alpha-32P] GTP azidoanilide, was incorporated into 39-41-kDa proteins comigrating in urea-containing sodium dodecyl sulfate-polyacrylamide gels with immunologically identified G-protein alpha-subunits, i.e. a 39-kDa Go alpha-subunit, a 40-kDa Gi2 alpha-subunit, and a 41-kDa Gi alpha-subunit of an unknown subtype. The synthetic opioid, D-Ala2,D-Leu5-enkephalin (DADLE), stimulated photolabeling of the 39-41-kDa proteins. In the presence of GDP, which increased the ratio of agonist-stimulated to basal photolabeling, DADLE at a maximally effective concentration stimulated photolabeling of the 39- and the 40-kDa protein 2-3-fold. Somatostatin, adrenaline, and bradykinin were less potent than DADLE and, to varying degrees, stimulated photolabeling of the 40-kDa protein more than that of the 39-kDa protein. Prostaglandin E1 was inactive. The present data represent direct evidence for an activation of endogenous Go and Gi2 via opioid receptors and other receptors in the native membrane milieu.

Topics: Alprostadil; Bradykinin; Carrier Proteins; Cell Adhesion Molecules; Electrophoresis, Polyacrylamide Gel; Enkephalin, Leucine-2-Alanine; Epinephrine; Glioma; GTP-Binding Proteins; Hybrid Cells; Membrane Glycoproteins; Naloxone; Neuroblastoma; Somatostatin; Tumor Cells, Cultured

In synaptosomal membranes from rat brain cortex, the mu selective agonist [3H]dihydromorphine in the absence of sodium, and the nonselective antagonist [3H]naltrexone in the presence of sodium, bound to two populations of opioid receptor sites with Kd values of 0.69 and 8.7 nM for dihydromorphine, and 0.34 and 5.5 nM for naltrexone. The addition of 5 microM guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]) strongly reduced high-affinity agonist but not antagonist binding. Exposure of the membranes to high pH reduced the number of GTP[gamma-35S] binding sites by 90% and low Km, opioid-sensitive GTPase activity by 95%. In these membranes, high-affinity agonist binding was abolished and modulation of residual binding by GTP[gamma S] was diminished. High-affinity (Kd, 0.72 nM), guanine nucleotide-sensitive agonist binding was reconstituted by polyethylene glycol-induced fusion of the alkali-treated membranes with (opioid receptor devoid) C6 glioma cell membranes. Also restored was opioid agonist-stimulated, naltrexone-inhibited GTPase activity. In contrast, antagonist binding in the fused membranes was unaltered. Alkali treatment of the glioma cell membranes prior to fusion inhibited most of the low Km GTPase activity and prevented the reconstitution of agonist binding. The results show that high-affinity opioid agonist binding reflects the ligand-occupied receptor-guanine nucleotide binding protein complex.

Topics: Animals; Binding, Competitive; Cell Line; Cell Membrane; Cells, Cultured; Cerebral Cortex; Dihydromorphine; Glioma; GTP Phosphohydrolases; Guanosine 5'-O-(3-Thiotriphosphate); Kinetics; Male; Membrane Fusion; Naloxone; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, mu; Synaptosomes

The effects of morphine on the basal cAMP level in the neuroblastoma X glioma NG108-15 hybrid cell line have been studied. Morphine (10 microM) added to the incubation media at hr 0 caused a rapid and significant decrease in the cAMP level up to hr 1; the level then slowly returned to the control at hr 6, and gradually increased to its peak at hr 36, returning to the control at hr 60. These results provide the first evidence for a delayed rebound increase of cAMP during morphine treatment. Naloxone (10 microM) added at hr 0 concomitantly with morphine blocked the morphine-induced decrease in cAMP level at hr 1 and attenuated its increase at hr 36. However, when naloxone was added at hr 5.5, the cAMP level significantly increased at hr 6, and at hr 36 the cAMP level increase was the same as in the case of morphine alone. Furthermore when naloxone was added 0.5 hr prior to harvesting the cells at hr 6, 12, 24 and 36, the cAMP level showed an immediate increase at each time point up to about the same level as observed with morphine alone at hr 36. Chloramphenicol, a protein synthesis inhibitor (100 microM) itself caused little or no change in the cAMP level. Added 30 min before morphine, chloramphenicol decreased the morphine-induced rebound increase at hr 36 in a concentration-dependent manner without any significant effect on cAMP decrease at hr 1. However when chloramphenicol was added at hr 5.5, the morphine-induced rebound increase at hr 36 was also attenuated, thereby suggesting an involvement of macromolecular synthesis in the rebound increase of cAMP which may be used as a model for the development of morphine dependence.

Topics: Chloramphenicol; Cyclic AMP; Glioma; Hybrid Cells; Morphine; Naloxone; Neuroblastoma; Osmolar Concentration; Tumor Cells, Cultured

The neuroblastoma-glioma NG108 cell line has been shown to contain both a delta-opiate receptor and enkephalin peptides. In this paper, the presence of authentic proenkephalin mRNA and proenkephalin-derived peptides are demonstrated. Growth of the cells in the presence of etorphine for 5-7 days resulted in a 3-fold increase of proenkephalin mRNA, which was accompanied by comparable increases in proenkephalin peptides and free enkephalin. The effect was mimicked by either morphine or [D-Ala2,D-Met5]enkephalinamide, and was blocked by naloxone. The EC50 for the effect of etorphine was 10(-9) M. The cyclic AMP content of cells grown for 5 days in the presence of etorphine was the same as that of control cells. Forskolin treatment also increased the proenkephalin mRNA content of the cells: the effect was not additive with that of etorphine, suggesting that the effect of opiate agonists was not occurring through their inhibition of adenylate cyclase. The results suggest that proenkephalin synthesis in NG108 cells can be regulated by two different mechanisms, one involving cyclic AMP while the other, regulated by the opiate receptor, is yet to be determined.

Topics: Animals; Cell Line; Colforsin; Enkephalin, Methionine; Enkephalins; Etorphine; Genes; Glioma; Hybrid Cells; Morphinans; Morphine; Naloxone; Neuroblastoma; Protein Precursors; RNA, Messenger; Transcription, Genetic

Opioids' modulation of beta-receptors' density and function has been investigated in a cultured cell line system. Rat C6 glioma cells do not have opioid receptors or, at least, the number of these receptors is very low, but cell exposure to desmethylimipramine (DMI) causes expression of functional opioid receptors as indicated by the increased [3H]DHM binding and by the acquired ability of opioids to inhibit ISO-stimulated cAMP accumulation. Cell exposure to DMI also causes beta-receptors' down-regulation as indicated by the decline in [3H]DHA binding coupled to a reduced ability of isoproterenol (ISO) to stimulate cAMP accumulation in intact cells. In the present paper we show that cell exposure to opioid agonists during DMI treatment counteracted DMI-induced beta-receptor loss. Similarly, opioid agonists added at the beginning of ISO exposure in DMI-pretreated cells, inhibited ISO-induced beta-receptor tachyphylaxis. These results suggest that opioids may exert a protective effect on beta-receptor function and this appears to be a common mechanism which is operant when overstimulation of beta-receptors takes place.

Topics: Animals; Cell Line; Cell Membrane; Cyclic AMP; Desipramine; Glioma; Isoproterenol; Kinetics; Morphine; Naloxone; Rats; Receptors, Adrenergic, beta; Receptors, Opioid

The putative delta-opiate receptor complex has been identified by a new approach which employed an antibody that is directed against a peptide which binds gamma-endorphin and is specified by RNA that is complementary to that of gamma-endorphin mRNA. This antibody competes with beta-endorphin and naloxone for binding sites on the surface of neuroblastoma X glioma NG108-15 hybrid cells. The opiate receptor complex has an apparent molecular weight of 210,000 and is composed of four noncovalently associated subunits with apparent molecular weights of 68,000, 58,000, 45,000 and 30,000.

Topics: Antibodies; beta-Endorphin; Binding, Competitive; Cell Line; Endorphins; gamma-Endorphin; Glioma; Hybrid Cells; Immunologic Techniques; Molecular Weight; Naloxone; Neuroblastoma; Peptides; Receptors, Opioid; Receptors, Opioid, delta; RNA, Messenger

Leucine-enkephalin, methionine-enkephalin, and morphine caused a reversible block of Ca2+ channel currents in neuroblastoma-glioma hybrid cells (NG108-15). The long-lasting (type 2) component of the Ca2+ channel current was blocked by leucine-enkephalin, while the transient (type 1) component was not affected. The enkephalin-induced blocking action was antagonized by naloxone and appears to be mediated by delta-opiate receptors. Two different aspects of the blocking effect were detected, a resting block and a recovery from block during prolonged depolarizing pulses. Recovery from block was more complete, and its time course was more rapid, with depolarization to more positive potentials. The dose dependence of the type 2 channel block at rest indicated a one-to-one binding stoichiometry, with an apparent dissociation constant of 8.8 nM. Somatostatin exerted a similar selective blocking action on the type 2 Ca2+ channel. The time- and voltage-dependent block of type 2 Ca2+ channels may provide a mechanism underlying the enkephalinergic presynaptic inhibition of transmitter release and the somatostatin block of pituitary growth hormone release.

Topics: Animals; Calcium; Calcium Channel Blockers; Cell Line; Dose-Response Relationship, Drug; Electric Conductivity; Enkephalin, Leucine; Glioma; Ion Channels; Naloxone; Neuroblastoma; Rats; Somatostatin

Ethanol inhibits opioid peptide binding to the delta-opioid receptor. When neuroblastoma x glioma NG108-15 hybrid cells are grown with 25-200 mM ethanol, opioid receptor density increases up to 2-fold without a change in receptor affinity. Since changes in neurotransmitter receptor density may be important in neuronal adaptations to ethanol, we investigated the underlying mechanisms and functional consequences of this phenomenon. The opiate antagonist, naloxone, also increased opioid receptor number, but produced a smaller effect than ethanol with greater fractional inhibition of binding; long term enhancement of binding by ethanol is therefore not a simple function of acute receptor inhibition. Ethanol did not inhibit receptor down-regulation by etorphine, an opiate agonist, and therefore is not likely to increase receptor expression through interference with tonic down-regulation by endogenous opioid peptides. Ethanol increased opioid receptor expression in NG108-15 cells treated with actinomycin D, but not cycloheximide; hence, normal protein synthesis, but not DNA transcription, may be required for this response. The opioid receptors induced in ethanol-treated cells were subject to normal up-regulation by naloxone, down-regulation by etorphine, and acute inhibition of agonist binding by Na+. Etorphine maximally inhibits cyclic AMP accumulation in NG108-15 cells with only fractional occupancy of opioid receptors. Chronic ethanol exposure increased the receptor reserve for this response, resulting in a 3.5-fold increase in the potency of etorphine for inhibiting phenylisopropyladenosine-stimulated cyclic AMP accumulation. Neuronal adaptation to ethanol may involve changes in the density of receptors that regulate cellular levels of cyclic AMP.

Topics: Cell Line; Cyclic AMP; Cycloheximide; Dactinomycin; Diprenorphine; DNA; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Ethanol; Etorphine; Glioma; Hybridomas; Naloxone; Neuroblastoma; Phenylisopropyladenosine; Receptors, Opioid; Receptors, Opioid, delta; Sodium; Transcription, Genetic

The effects of varying the sodium concentration (at constant ionic strength) on opioid binding at mu- and delta-opioid receptors in 7315c and NG108-15 cells has been examined. The binding of [3H]etorphine to mu-receptors on 7315c cells was increased by replacing the sodium in the incubation medium with potassium or N-methyl-D-glucamine. This effect was shown to be attributable to an increase in affinity, with no change in the maximum number of binding sites, both in cell membrane suspensions and in intact 7315c cells. Replacement of sodium with potassium or N-methyl-D-glucamine in NG108-15 membrane or intact cell suspensions also resulted in an increase in [3H]etorphine binding, but in these cells the effect was associated with an increase in the number of binding sites measurable under these experimental conditions. The effects of sodium on opioid inhibition of adenylate cyclase in membrane preparations from 7315c and NG108-15 cells also differed. Sodium reduced apparent agonist affinity in 7315c membranes. In NG108-15 cell membranes, sodium was essential for the demonstration of opioid inhibition of cyclase activity. Increasing the sodium concentration above 0.5 mM resulted in an increase in the fraction of total enzyme activity inhibited by opioid, but the opioid IC50 did not change. In the companion paper, it is shown that the effects of sodium removal on mu- and delta-receptor binding in guinea pig brain neural membranes were similar to those observed in the cell preparations. An increase in intracellular sodium concentration without change in extracellular concentration was effected by incubation of 7315c and NG108-15 cells with the sodium-selective ionophore, monensin. When sodium was present in the extracellular medium, monensin reduced [3H]etorphine binding by 50% or more, both at mu-receptors in 7315c cells and at delta-receptors in NG108-15 cells. In the absence of sodium, however, monensin treatment produced only a small inhibition of binding. These results suggest that sodium acts at an intracellular site to regulate opioid agonist binding at both mu- and delta-receptors, but that the mode of regulation is not identical at each site. Since a reduction in intracellular sodium concentration by removal of extracellular sodium increases agonist binding, and an increase in intracellular sodium following monensin treatment reduces agonist binding, it is probable that the intracellular sodium concentration is a critical regulator of opioid agonist b

Topics: Adenylyl Cyclase Inhibitors; Animals; Cell Line; Cell Membrane; Cyclazocine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Ethylketocyclazocine; Etorphine; Glioma; Guinea Pigs; Monensin; Naloxone; Neuroblastoma; Pituitary Neoplasms; Rats; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Sodium

The adenylate cyclase (AC) of the neuroblastoma-glioma hybrid cells (NG108-15), is generally considered to be a model for the study of the biochemical correlates of opiate tolerance and dependence. However, the naloxone-induced rebound response of adenylate cyclase, described in some recent reports, is much smaller than that originally described by Sharma, Klee and Nirenberg (1975). Possible explanations for these discrepancies are: (1) a marked down-regulation of opioid receptors and tolerance produced by the use of delta agonists or (2) the use of etorphine, a relatively hydrophobic drug which has slower dissociation rates than morphine. To test these possibilities, neuroblastoma-glioma hybrid cells were treated cells with morphine, etorphine, [D-Ala2,D-Leu5]enkephalin (DADLE), [D-Ala2]Leu-enkephalinamide (DALAMID) or vehicle. In addition, some of the cells treated with etorphine were washed with DADLE to replace the etorphine without producing the rebound response of adenylate cyclase prior to the addition of naloxone. The cells treated with morphine, DADLE and DALAMID, and incubated with prostaglandin E1 (PGE1) and naloxone showed a significant rebound of adenylate cyclase when compared with control groups and opiate-treated cells, incubated only with PGE1. In contrast, naloxone did not induce any significant rebound response in cells treated with etorphine unless they were previously washed with DADLE. These results demonstrate that the lack of a rebound response in cells treated with etorphine was due to the slow dissociation rates of the opiate and not to tolerance or to down-regulation of opioid receptors produced by agonists of high intrinsic activity.

Topics: Adenylyl Cyclases; Alprostadil; Cell Line; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalin, Methionine; Etorphine; Glioma; Morphinans; Morphine; Naloxone; Neuroblastoma; Receptors, Opioid

Opioid receptor binding and opioid-mediated inhibition of cAMP accumulation were studied simultaneously in intact NG108-15 cells. The dose-response curves for the biological response were suggestive of positive cooperativity and systematically occurred at lower ligand concentrations than those for the binding of [3H] [D-Ala2, D-Leu5]enkephalin (DADLE), which were instead shallow and suggestive of a site heterogeneity or of a cooperative phenomenon. Computer modeling of the binding isotherms revealed that the data are best described assuming two binding sites with different affinities for the agonist; the mean ratio between the DADLE concentrations yielding half-maximal occupancy of the high affinity site and half-maximal response was 1.5, but it was 36 when the fractional occupancy of the sum of the two sites was considered. On examining several opioids, no direct correlation was found between high affinity site and biological response; however, several agonists displayed different affinities for the two sites, while the antagonist naloxone and the partial agonist diprenorphine bound to them with identical affinities. Furthermore, naloxone exhibited a good agreement between half-maximal receptor occupancy and Ki in blocking the agonist response. Thus, the binding heterogeneity detectable in intact cells is agonist-specific, and suggests rather that the sites are states of an identical receptor population. When [3H]diprenorphine was used to label the opioid receptors, the competition curves for DADLE were consistent with the existence of an additional, very low affinity state undetectable by direct binding with labeled agonist and, again, not discriminated by naloxone. Multiple affinity states of the opioid receptor in intact cells may reflect its interaction with the effector system in the plasma membrane.

Topics: Adenylyl Cyclases; Animals; Cell Line; Cyclic AMP; Diprenorphine; Dose-Response Relationship, Drug; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Etorphine; Glioma; Hybrid Cells; Kinetics; Ligands; Morphine; Naloxone; Neuroblastoma; Neurons; Receptors, Opioid; Receptors, Opioid, delta

Neuroblastoma-glioma NG108-15 cells that were cultured for 48 h with the opiate antagonist, naloxone, respond to the guanosine 5'-triphosphate (GTP) analogue guanosine 5'-[beta, gamma-imido]-triphosphate (GMP-PNP) in the binding assay as the control, non-treated, cells. This was observed when the guanyl nucleotide was tested in the presence or absence of sodium chloride and also after subcellular fractionation of the membranes on a sucrose gradient which separated between two receptor-containing fractions. The findings suggest that the increase in delta type enkephalin receptors in naloxone-treated NG108-15 cells does not reflect an alteration in the interaction between the receptor and the adenylate cyclase-GTP-binding protein system.

Topics: Animals; Cells, Cultured; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Glioma; GTP-Binding Proteins; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Naloxone; Neuroblastoma; Rats; Receptors, Opioid; Receptors, Opioid, delta

Topics: Chromatography, Affinity; Diprenorphine; Glioma; Hybrid Cells; Naloxone; Neuroblastoma; Neurons; Receptors, Opioid

Topics: Adaptation, Physiological; Adenylyl Cyclases; Animals; Cell Line; Clone Cells; Cytosol; Endorphins; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Etorphine; Glioma; Guanylyl Imidodiphosphate; Lanthanum; Mice; Naloxone; Narcotics; Neuroblastoma; Rats; Receptors, Opioid; Sodium; Time Factors

Previous studies with membranes from rat heart (Mol. Pharmacol. 21: 570-580, 1982) and human platelets (J. Biol. Chem. 257: 2829-2833, 1982) have suggested that inhibition of adenylate cyclase by occupation of hormone receptors is blocked by pretreatment of membranes with relatively low concentrations of N-ethylmaleimide (NEM). Using membranes derived from NG108-15 neuroblastoma X glioma cells as a model system, we have examined the effect of NEM on the interaction of three inhibitory receptors with adenylate cyclase. Pretreatment of membranes with 100 to 216 microM NEM resulted in a loss of the capacity of agonists to inhibit adenylate cyclase through muscarinic cholinergic and opiate receptors and a loss of GTP-sensitive high-affinity binding of agonists to both of these receptors. Under the same conditions, stimulation of adenylate cyclase by prostaglandin E1 was unchanged. In contrast to the total loss of capacity to inhibit adenylate cyclase by muscarinic and opiate receptor activation, the inhibition of adenylate cyclase by activation of alpha-2 adrenergic receptors was only partially blocked by maximally effective concentrations of NEM. Similarly, GTP-sensitive high-affinity binding of epinephrine to alpha-2 receptors still occurred in NEM (316 microM)-treated membranes. Whereas only a decrease in the efficacy of muscarinic and opiate receptor agonists for inhibition of adenylate cyclase occurred as a result of NEM treatment, pretreatment of membranes with 316 microM NEM resulted in a 30-fold decrease in the potency of epinephrine for inhibition of adenylate cyclase.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenylyl Cyclase Inhibitors; Animals; Cell Line; Epinephrine; Ethylmaleimide; Glioma; GTP-Binding Proteins; Morphine; Naloxone; Neuroblastoma; Oxotremorine; Quinuclidinyl Benzilate; Receptors, Adrenergic, alpha; Receptors, Cell Surface; Receptors, Cholinergic; Receptors, Muscarinic; Receptors, Neurotransmitter; Receptors, Opioid; Yohimbine

Two different -SH groups associated with the opiate receptors of the mouse neuroblastoma X rat glioma hybrid NG108-15 have been identified. Modification of these by N-ethylmaleimide (NEM) (presumed to be via alkylation) or by para-chloromercuribenzoic acid (presumed to be via formation of mercury adducts) decreases the binding of both opiate agonists and antagonists to these receptors. Agonist binding is more sensitive than antagonist binding to modification by NEM. Losses in antagonist binding are accounted for totally by decreases in the number of binding sites; there are no corresponding losses in antagonist affinity. Losses of antagonist binding exhibit a pseudo-first order rate constant; the modification of only one such group completely destroys the binding site. Both agonists and antagonists protect against modification of this group by NEM. Sodium and lithium, but not GTP, also protect this group, indicating that the action of these monovalent cations is directly on the receptor moiety. Losses in agonist binding stem not only from decreases in receptor number but also from selective losses in affinity. This -SH group appears to be different from the one at the binding site as sodium, GTP, and antagonist ligands do not protect against losses in agonist affinity. Agonist high affinity also is lost in a pseudo-first order fashion indicating that an alteration of only one -SH group per receptor complex is sufficient to produce this effect. The possible roles of two sulfhydryls in opiate receptor function are discussed.

Topics: Animals; Cell Line; Dithiothreitol; Enkephalin, Methionine; Ethylmaleimide; Glioma; Hybrid Cells; Kinetics; Mice; Naloxone; Naltrexone; Neuroblastoma; Rats; Receptors, Opioid; Sulfhydryl Reagents

It has been repeatedly demonstrated that the neuroblastoma-glioma (NG 108-15) cell line has opiate receptors that inhibit adenylate cyclase and it has been proposed that this inhibition is mediated by a naloxone reversible stimulation of a low Km GTPase (Koski and Klee, Proc. Natl. Acad. Sci. 78:4185, 1981). The guanine nucleotides of NG cells were labeled with [3H]guanine followed by incubation with 10(-6)M guanine. Etorphine (10(-6)M) or vehicle were added and the incubations continued for 1-4 min. The reaction was stopped with 5 percent TCA containing nucleotides as carriers and markers for the HPLC. Marker nucleotides were detected at 254 nm and the labeled nucleotides by liquid scintillation spectrometry. In several experiments, etorphine failed to produce any measurable change in the labeled nucleotides or in the GTP/GDP ratios. To verify that the opiate receptors were functional we measured its capacity to inhibit the formation of cAMP induced by PGE1. We also studied the effects of naloxone and PGE1 on the formation of cAMP in opiate tolerant cells. Tolerant cells responded to naloxone with a 50 percent increase in cAMP, indicating again that the opiate receptors were functional. Our results are consistent with the idea that in intact NG108-15 cells the opiate-mediated hydrolysis of GTP observed in cell membrane preparations is of very small magnitude.

Topics: Alprostadil; Animals; Cell Line; Cyclic AMP; Etorphine; Glioma; Guanine Nucleotides; Guanosine Triphosphate; Mice; Naloxone; Neuroblastoma; Prostaglandins E; Tritium

BALB/c mice were immunized with an opioid receptor complex over the period of 1 year. Spleen cells from the mouse, whose serum inhibited opiate binding to rat neural membranes to the greatest extent, were fused with P3-X63-Ag8. 653.3 myeloma cells. By radioimmunoassay (RIA), 32 cell lines have been detected that secrete an antibody to a component of the isolated receptor complex. Antibodies from 2 of the cell lines have an effect on opiate binding to rat neural membranes. One antibody, OR-689.2.4 is an IgM cryoglobulin. This antibody partially inhibited the binding of 3H-dihydromorphine (3H-DHM), 3H-naloxone, 3H-ethylketocyclazocine (3H-EKC), and 3H-D-Ala2, D-Leu5 enkephalin (3H-DADLE) to rat neural membranes. The other antibody, OR-465.3, inhibited the binding of 3H-DHM and 3H-naloxone to rat neural membranes by a maximum of 70%. This antibody also inhibited the binding of 3H-DADLE to neural membranes but, did not affect the binding of this peptide to membranes from the neuroblastoma-glioma hybrid cell line, NG108-15. Work is ongoing to generate monoclonal antibodies specific for each subclass of opioid receptor.

Topics: Animals; Antibodies, Monoclonal; Cell Fusion; Cell Membrane; Glioma; Hybridomas; Mice; Mice, Inbred BALB C; Naloxone; Neuroblastoma; Neurons; Rats; Receptors, Opioid

"Undifferentiated" neuroblastoma glioma hybrid NG 108-15 cells have not previously been characterized at a light and ultrastructural level despite their use in opiate receptor studies and their possession of one or more opioids. Three cell types rather than one were found. Type A is the classic round hybridoma cell. Type B is a neuron like cell and Type C is a giant cell similar to Type A cells but larger and with dense cytoplasm. Dense core vesicles, 800-1200 Ao in diameter were found as were virus particles in most Type A cells. Multivesicular bodies with clear vesicles which are interpreted as glutaraldehyde artefacts were found but no clear neurosecretory vesicles were found in any A cells--Etorphine at 10(-6) M for 48 hours and naloxone 10(-4) M were employed to product dependence and precipitated abstinence but no clear cut morphologic differences were noted between control, opiate dependent and precipitated abstinent cells.

Topics: Animals; Cell Line; Etorphine; Glioma; Hybrid Cells; Mice; Naloxone; Neuroblastoma; Rats

Partially purified extracts from neuroblastoma X glioma hybrid cells 108CC15 inhibit, like opioids, the prostaglandin E1-evoked formation of cyclic AMP in a dose-dependent manner in the same hybrid cells. The inhibition is prevented by the opioid antagonist naloxone. In addition, the same extract competes with [3H]naloxone and [3H]Leu-enkephalin for binding to opioid receptors of hybrid cell membranes and to a specific antiserum, respectively. The opioid activity in the extracts is destroyed by carboxypeptidase A and leucine aminopeptidase, but not by trypsin. Further purification of the extracts by HPLC, TLC, or high-voltage paper electrophoresis reveals in each case two active fractions which behave like Met- and Leu-enkephalin. The Met-enkephalin-like, but not the Leu-enkephalin-like, fraction is inactivated by treatment with BrCN. Dimethylaminonaphtylsulfonyl (dansyl) derivatives of Met- and Leu-enkephalin correspond to [3H]dansyl derivatives of Met-like substances from hybrid cells. Three to four times as much Met-enkephalin-like as Leu-enkephalin-like material is present in the extract. The overall concentration of opioid peptides in the hybrid cells varies between 0.03 and 1.0 pmol Leu-enkephalin equivalents per mg protein. The amount of opioids in the hybrid cells is strongly dependent on the cell density. The findings suggest that neuroblastoma X glioma hybrid cells contain opioid peptides that are very similar, if not identical, to Met- and Leu-enkephalin. Opioid activity can also be detected in other neuronal cell lines and even in glioma cells.

Topics: Alprostadil; Animals; Cell Line; Cyclic AMP; Endorphins; Enkephalin, Leucine; Enkephalin, Methionine; Enkephalins; Glioma; Hybrid Cells; Mice; Naloxone; Neuroblastoma; Oxidation-Reduction; Prostaglandins E; Rats

The effect of cerebroside sulfate, phosphatidylserine, and other phospholipids on opiate receptor function in neuroblastoma N18TG2 cells was studied by incorporation of lipids into the membrane bilayer of viable cells. A concentration- and time-dependent incorporation of sulfatide by N18TG2 cells was observed. The incorporated lipid was not metabolized during the incubation period of up to 48 hr at 37 degrees. Optimal conditions for lipid incorporation were determined to be 4 days after the cell seeding and in 1% fetal calf serum. The incorporated lipid was established to be associated with the plasma membrane fraction of the crude cell homogenate. Furthermore, increases in Vmax but not Km values of the adenylate cyclase for Mg2+, ATP, and prostaglandin E1 were observed in neuroblastoma N18TG2 cells exposed to cerebroside sulfate for 4--6 hr. The incorporation of cerebroside sulfate or phosphatidylserine by N18TG2 cells did not increase the number of opiate binding sites in this cell line as determined by [3H]naloxone, [3H]etorphine, or 3H-labeled D-Ala2-Met5-enkephalinamide binding. Although there was an increase in the affinity of [3H]naloxone binding, linear correlation between the amount of cerebroside sulfate incorporated and the quantity of binding increase was not observed. However, augmentation of both the potencies and the efficacies (maximal inhibitory level) of morphine and enkephalin to regulate adenylate cyclase activity was observed after sulfatide incorporation. At the maximal concentration of cerebroside sulfate used (67 microM) the opiate receptor activity in N18TG2 cells approached that of NG108-15 cells. Identical treatment of N18TG2 cells with cerebroside or psychosine sulfate did not produce any potentiation of the opiate inhibition of adenylate cyclase. Of all of the phospholipids tested--phosphatidylserine, phosphatidylinositol, and phosphatidylcholine--only phosphatidylcholine produced a potentiation of the opiate effect. Both synthetic dipalmitoyl phosphatidylcholine or brain phosphatidylcholine could elicit the potentiation.

Topics: Adenylyl Cyclases; Animals; Cell Line; Glioma; Kinetics; Lipid Metabolism; Mice; Naloxone; Narcotics; Neoplasms, Experimental; Neuroblastoma; Rats; Receptors, Opioid; Subcellular Fractions; Sulfoglycosphingolipids

Topics: Alprostadil; Animals; Chromatography, High Pressure Liquid; Cyclic AMP; Dexamethasone; Endorphins; Enkephalin, Leucine; Enkephalin, Methionine; Enkephalins; Glioma; Glucocorticoids; Hybrid Cells; Naloxone; Neuroblastoma; Prostaglandins E

Topics: Animals; Cyclic AMP; Enkephalin, Methionine; Enkephalins; Glioma; Hybrid Cells; Mice; Naloxone; Naltrexone; Neoplasms, Experimental; Neuroblastoma; Nitrogen Mustard Compounds; Prostaglandins E, Synthetic; Rats; Receptors, Opioid

Topics: Animals; Cell Line; Endorphins; Enkephalins; Galactosamine; Galactose; Gangliosides; Glioma; Glucosamine; Glycosphingolipids; Hybrid Cells; Kinetics; Mice; Morphine; Naloxone; Narcotics; Neuroblastoma

Topics: Cell Line; Endorphins; Glioma; Hybrid Cells; Naloxone; Neoplasms, Experimental; Neuroblastoma; Prostaglandins E

Topics: Animals; Cells, Cultured; Cerebral Cortex; Glioma; Guinea Pigs; Humans; Hybrid Cells; Mice; Myenteric Plexus; Naloxone; Narcotics; Neuroblastoma; Neurons; Opioid-Related Disorders; Rats; Receptors, Opioid; Synaptic Transmission

Topics: Cell Membrane; Cells, Cultured; Enkephalins; Etorphine; Glioma; Guanylyl Imidodiphosphate; Hybrid Cells; Magnesium; Manganese; Naloxone; Neuroblastoma; Nucleotides; Receptors, Opioid; Sodium

Topics: beta-Lipotropin; Cyclic AMP; Dose-Response Relationship, Drug; Glioma; Hybrid Cells; Leucine; Methionine; Naloxone; Narcotics; Neuroblastoma; Oligopeptides; Receptors, Opioid; Structure-Activity Relationship

Topics: Cyclic AMP; Drug Interactions; Endorphins; Glioma; Hybrid Cells; Naloxone; Neuroblastoma; Peptides; Prostaglandins E

The unnatural (+) enantiomer of morphine had minimal activity in three opiate assays in vitro: the rat brain homogenate binding assay, the electrically stimulated guinea pig ileum assay, and the inhibition of adenylate cyclase in neuroblastoma X glioma hybrid cell homogenates. When (+)-morphine was microinfected into the periaqueductal gray (a site known to mediate morphine analgesia) of drug-naive rats, there was only minimal analgesia, but the hyperresponsivity usually observed after microinfection of (-)-morphine occurred. Also, when (+)-morphine was microinfected into the midbrain reticular formation of drug-naive rats, rotation similar to that following microinjection of (-)-morphine occurred. These behaviors were not blocked by naloxone. Significantly, they typically occur in precipitated abstinence in morphine-dependent rats. These observations suggest that there are at least two classes of receptors, one stereospecific and blocked by naloxone and the other only weakly stereospecific and not blocked by naloxone, and that precipitated abstinence may be due, in part, to a selective blockade of receptors of the former class but not of the latter.

Topics: Adenylyl Cyclase Inhibitors; Animals; Behavior, Animal; Binding Sites; Binding, Competitive; Brain; Glioma; Guinea Pigs; In Vitro Techniques; Molecular Conformation; Morphine; Morphine Derivatives; Muscle Contraction; Muscle, Smooth; Naloxone; Neuroblastoma; Rats; Receptors, Opioid; Stereoisomerism; Structure-Activity Relationship

Topics: Animals; Binding, Competitive; Cyclic AMP; Enkephalins; Etorphine; Glioma; Hybrid Cells; Naloxone; Neuroblastoma; Neurons; Prostaglandins E; Rats; Receptors, Cell Surface; Receptors, Opioid; Somatostatin

Reactions mediated by the opiate receptors that inhibit adenylate cyclase (EC 4.6.1.1) are closely coupled to subsequent reactions that gradually increase adenylate cyclase activity of neuroblastoma X glioma NG108-15 hybrid cells. Opiate-treated cells have higher basal-, prostaglandin E1-, and 2-chloroadenosine-stimulated activities than do control cells. However, NaF or guanosine 5'-(beta, gamma-imido)triphosphate abolishes most of the differences in adenylate cyclase activity observed with homogenates from control and opiate-treated cells. Cycloheximide blocked some, but not all, of the opiate-dependent increase in adenylate cyclase activity. These results suggest that the opiate-dependent increase in adenylate cyclase is due to conversion of adenylate cyclase to a form with altered activity. Protein synthesis also is required for part of the opiate effect. We propose that activity of adenylate cyclase determines the rate of conversion of the enzyme from one form to the other and that opiates, by inhibiting adenylate cyclase, alter the relative abundance of low- and high-activity forms of the enzyme.

Topics: Adenosine; Adenylyl Cyclases; Cell Line; Cycloheximide; Enzyme Activation; Etorphine; Fluorides; Glioma; Guanylyl Imidodiphosphate; Hybrid Cells; Kinetics; Morphinans; Morphine; Naloxone; Neuroblastoma; Prostaglandins E

Topics: Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; beta-Lipotropin; Cell Line; Enzyme Activation; Glioma; Hybrid Cells; Kinetics; Ligands; Melanocyte-Stimulating Hormones; Morphine; Naloxone; Nerve Tissue Proteins; Neuroblastoma; Oligopeptides; Prostaglandins E; Receptors, Opioid

Incubation of neuroblastoma X glioma hybrid cells for 12-97 hr with methionine-enkephalin results in an increase in adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] that is mediated by the opiate receptor. The results show that cells become tolerant to, and dependent upon, enkephalin.

Topics: Adenylyl Cyclases; Cells, Cultured; Drug Tolerance; Enzyme Activation; Etorphine; Glioma; Humans; Hybrid Cells; Ligands; Naloxone; Neuroblastoma; Oligopeptides; Prostaglandins E; Substance-Related Disorders

Topics: Animals; Cell Line; Cyclic AMP; Drug Synergism; Glioma; Hybrid Cells; Mice; Morphine; Naloxone; Neuroblastoma; Neuroglia; Neurons; Prostaglandin Antagonists; Prostaglandins; Rats; Stimulation, Chemical

Topics: Animals; Cyclic AMP; Cyclic GMP; Dextrorphan; Dose-Response Relationship, Drug; Glioma; Hybrid Cells; Levorphanol; Mice; Morphine; Naloxone; Neuroblastoma; Neuroglia; Neurons; Rats; Receptors, Drug

Morphine inhibits adenylate cyclase (EC 4.6.1.1) activity of neuroblastoma times glioma hybrid cells. The inhibition is stereospecific and is reversed by the antagonist, naloxone. The relative affinities of narcotics for the opiate receptor agree well with their effectiveness as inhibitors of adenylate cyclase. Morphine-sensitive and -insensitive cell lines were found, and the degree of sensitivity was shown to be dependent upon the abundance of narcotic receptors. Thus, morphine receptors are functionally coupled to adenylate cyclase. A molecular mechanism for narcotic addiction and tolerance is proposed.

Topics: Adenylyl Cyclase Inhibitors; Animals; Cell Line; Glioma; Hybrid Cells; Mice; Morphine; Naloxone; Neuroblastoma; Prostaglandins; Rats; Receptors, Drug

A neuroblastoma x glioma hybrid cell line with well-developed neural properties was found that has high-affinity morphine receptors. The average cell contains approximately 3 x 10(6) receptors. In contrast, parent cells and other neuroblastoma or hybrid cell lines tested had few or no morphine receptors.

Topics: Animals; Cell Line; Glioma; Hybrid Cells; Mice; Morphine; Morphine Derivatives; Naloxone; Nerve Tissue Proteins; Neuroblastoma; Rats; Receptors, Drug; Tritium