enkephalin--leucine-2-alanine and Glioma

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

The delta opioid peptide [D-Ala2,D-Leu5]enkephalin (DADLE) has been shown to be a neuroprotective agent via mechanisms that are not totally understood. We previously demonstrated that the i.p. injection of DADLE in mice causes an increase of nerve growth factor (NGF) in the brain. To further clarify the NGF-increasing action of DADLE, we examined here the NGF-increasing effect of DADLE in vitro, using cultured NG-108 cells. DADLE dose-dependently increases the immunoreactive level of NGF in NG-108 cells in a bell-shape manner, with the effective DADLE concentrations in the picomolar range (0.01-100 pM). Also, DADLE at 1 pM selectively increases c-Jun and c-Fos, but not c-Rel. These results indicate that DADLE is one of the most potent agents in increasing the NGF in the biological system and that this action of DADLE involves selective increases of c-Jun and c-Fos, transcription factors that promote the NGF expression.

Topics: Animals; Blotting, Western; Cell Line, Tumor; Dose-Response Relationship, Drug; Enkephalin, Leucine-2-Alanine; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Glioma; Mice; Nerve Growth Factor; Neuroblastoma; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Rats

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.

Topics: Barium; Calcium Channels; Electric Conductivity; Enkephalin, Leucine-2-Alanine; Glioma; GTP-Binding Proteins; Hybrid Cells; Kinetics; Neuroblastoma; Pertussis Toxin; Receptors, Opioid, delta; Tumor Cells, Cultured; Virulence Factors, Bordetella

GM1 ganglioside was previously shown to function as a specific regulator of excitatory opioid activity in dorsal root ganglion neurons and F11 hybrid cells, as seen in its facilitation of opioid-induced activation of adenylyl cyclase and its ability to dramatically reduce the threshold opioid concentration required to prolong the action potential duration. The elevated levels of GM1 resulting from chronic opioid exposure of F11 cells were postulated to cause the ensuing opioid excitatory supersensitivity. We now show that GM1 promotes opioid (DADLE)-induced activation of adenylyl cyclase in NG108-15 cells which possess the delta-type of receptor. In keeping with previous studies of other systems, this can be envisioned as conformational interaction of GM1 with the receptor that results in uncoupling of the receptor from Gi and facilitated coupling to Gs. This would also account for the observation that DADLE-induced attenuation of forskolin-stimulated adenylyl cyclase was reversed by GM1, provided the cells were not pretreated with pertussis toxin. When the cells were so pretreated, GM1 evoked an unexpected attenuation of forskolin-stimulated adenylyl cyclase attributed to GM1-promoted influx of calcium which was postulated to inhibit a calcium-sensitive form of adenylyl cyclase. This is concordant with several studies showing GM1 to be a potent modulator of calcium flux. Pertussis toxin in these experiments exerted dual effects, one being to promote interaction of the delta-opioid receptor with Gs through inactivation of Gi, and the other to enhance the GM1-promoted influx of calcium by inactivation of Go; the latter is postulated to function as constitutive inhibitor of the relevant calcium channel. NG108-15 cells thus provide an interesting example of competitive interaction between two GM1-regulated systems involving enhancement of both opioid receptor excitatory activity and calcium influx.

Topics: Adenylate Cyclase Toxin; Adenylyl Cyclases; Aminopterin; Binding, Competitive; Calcium Channels; Cholera Toxin; Cyclic AMP; Enkephalin, Leucine-2-Alanine; Fluorescent Dyes; G(M1) Ganglioside; Glioma; Hybrid Cells; Hypoxanthine; Neuroblastoma; Pertussis Toxin; Receptors, Opioid; Thymidine; Tumor Cells, Cultured; Virulence Factors, Bordetella

Opioid-receptor adaptation may lead to changes in transcriptional regulation by sequence-specific DNA-binding proteins. Gel-shift assays of nuclear extracts from NG108-15 cells revealed that an increase of AP-1 DNA-binding activity ensues under conditions previously established to induce down- or up-regulation of delta-opioid receptors.

Topics: Animals; Base Sequence; Binding Sites; Cell Nucleus; Consensus Sequence; Down-Regulation; Enkephalin, Leucine-2-Alanine; Glioma; Hybrid Cells; Mice; Naltrexone; Neuroblastoma; Oligodeoxyribonucleotides; Rats; Receptors, Opioid, delta; Transcription Factor AP-1; Tumor Cells, Cultured; Up-Regulation

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

Leucine-enkephalin (Leu-EK) dose-dependently elicited an increase in cytosolic Ca2+ concentration ([Ca2+]i) with an EC50 of 1.2 microM via the phosphoinositide cascade in NG108-15 cells. Chronic treatment of cells with [D-Ala2,D-Leu5]enkephalin caused time-dependent homologous desensitization. In the presence of extracellular Ca2+, ATP as well as bradykinin stimulated significantly higher increases in inositol 1,4,5-trisphosphate (IP3) generation than did Leu-EK; however, the magnitude of intracellular Ca2+ pools increased after ATP stimulation, whereas bradykinin depleted intracellular pools. Hence, cells lost their [Ca2+]i response to Leu-EK if bradykinin was first added to induce a [Ca2+]i increase, whereas the response was unchanged if Leu-EK was added after addition of ATP. When Leu-EK was added simultaneously with bradykinin or ATP, an additive response was observed in IP3 generation; however, the rise in [Ca2+]i reached the same level as that induced by bradykinin or ATP alone. In the absence of extracellular Ca2+ in which the replenishment of intracellular pools was not possible, ATP displayed an inhibitory effect similar to that of bradykinin on the Leu-EK-induced [Ca2+]i increase. Prior treatment of cells with Leu-EK slightly heterologously desensitized the action of bradykinin, but had no effect on the ATP response. Our results suggest that a shared intracellular Ca2+ pool is sensitive to the opioid, bradykinin and P2-purinoceptor agonists; however, a defined pool of phosphatidylinositol 4,5-bisphosphate or a specific phospholipase C is responsible for each receptor.

Topics: Adenosine Triphosphate; Bradykinin; Calcium; Cyclic AMP; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Glioma; Inositol 1,4,5-Trisphosphate; Neuroblastoma; Receptors, Opioid, delta; Tumor Cells, Cultured

Cross-regulation from the stimulatory phospholipase C to the adenylyl cyclase pathways was explored in neuroblastoma-glioma NG-108-15 cells in culture. Activation of protein kinase C by phorbol myristic acid resulted in a markedly attenuated activation of the inhibitory adenylyl cyclase response to delta-opiate agonists and epinephrine but not to the muscarinic agonist carbachol. The ability of okadaic acid to mimic the effects of phorbol myristic acid on the inhibitory response suggested a role for protein phosphorylation. Adenylyl cyclase activity from cells in which protein kinase C had been activated demonstrated a loss in the inhibitory adenylyl cyclase response at the level of the G-protein. Activation of protein kinase C prompted a 2-4-fold increase in phosphorylation of G1 alpha 2 in cells metabolically labeled with [32P]orthophosphate. The phosphate content of Gi alpha 2 was determined to be approximately 0.5 mol/mol subunit in the unstimulated cells and approximately 1.5 mol/mol subunit for cells in which protein kinase C was activated. The effects of okadaic acid, 4-alpha-phorbol, and calphostin C on inhibition of adenylyl cyclase in cells treated with phorbol myristic acid correlate with the effects of these agents on phosphorylation of Gi alpha 2. The time courses for attenuation of inhibitory adenylyl cyclase and that for phosphorylation of Gi alpha 2 were similar in cells challenged with phorbol myristic acid. These data argue for cross-regulation from the stimulatory protein kinase C to inhibitory adenylyl cyclase pathways at the level of Gi alpha 2 via protein phosphorylation.

Topics: Adenylyl Cyclases; Enkephalin, Leucine-2-Alanine; Enzyme Activation; Ethers, Cyclic; Glioma; GTP-Binding Proteins; Hybrid Cells; Naphthalenes; Neuroblastoma; Okadaic Acid; Phorbols; Phosphorylation; Polycyclic Compounds; Protein Kinase C; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

A study was made of the effect of opioid peptides, (Leu)-enkephalin and its synthetic analog dalargin, on the maturation speed (differentiation) and proliferation activity of glioma C-6 cells. This effect on phenotypes of glioma C-6 cells was determined using some biochemical parameters: changes in the activity of glutamine synthetase (astrocytic marker) and cyclic nucleotide phosphorohydrolase (oligodendrocytic marker) in the culture of glioma C-6 cells in the early and late passages. The biochemical analysis was made at the Laboratory in Denver, USA, and we thank Prof. A. Vernadakis for the possibility to carry out a part of this work that helped us to find the growth activity of opioid peptides on the C-6 cells. Proliferation was examined in cultivation conditions approximately conforming the conditions of cultivation for the primary glial cell cultures. The control proliferation level was high in this case. It is demonstrated that opioid peptides accelerate (or strengthen) the expression of phenotypic signs in C-6 glioma cells in early and late passages changing specific activity of the marker enzymes, i.e. operating as a growth factor. Opioid peptides show glial growth factor characteristics on the glioma C-6 glial cell model as well, for glioma C-6 is known to be a perfect model to analyse the action of different substances on the glia.

Topics: 2',3'-Cyclic-Nucleotide Phosphodiesterases; Animals; Biomarkers, Tumor; Cell Division; Cell Transformation, Neoplastic; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Glioma; Glutamate-Ammonia Ligase; Phenotype; Rats; Time Factors; Tumor Cells, Cultured

We have investigated the effects of the C-terminal amyloid precursor protein fragment His 657-Lys 676 upon calcium currents in NG108-15 neuroblastoma x glioma hybrid cells. The amyloid precursor protein fragment His 657-Lys 676 (1-10 microM) did not affect calcium currents per se, but clearly blocked the calcium current suppression mediated by both adrenergic alpha 2B- and opioid delta receptors in a concentration-dependent manner. The reverse amyloid precursor protein fragment Lys 676-His 657 and the shorter amyloid precursor protein fragment Gly 659-Lys 676 did not affect calcium current suppression by adrenergic alpha 2B- and opioid delta receptors. The similar interaction of C-terminal amyloid precursor protein with adrenergic alpha 2B- and opioid delta receptors suggest that the effect occurs downstream of the receptor, possibly via the GTP binding protein Go.

Topics: Amyloid; Animals; Calcium; Calcium Channels; Enkephalin, Leucine-2-Alanine; Glioma; GTP-Binding Proteins; Hybrid Cells; Neuroblastoma; Norepinephrine; Peptide Fragments; Prions; Protein Precursors; Receptors, Adrenergic, alpha-2; Receptors, Opioid, delta; Tumor Cells, Cultured

Mouse N18TG2 neuroblastoma and rat C6 glioma cell lines were injected into male nude mice, and the tumors were passaged serially. At each generation, tumors were analyzed for delta opioid binding using [3H][D-Ala2,D-Leu5]enkephalin and for sigma 1 and sigma 2 binding with 1,3-[3H]di-o-tolylguanidine in the presence and absence of 1 microM pentazocine. Receptor density (Bmax) and affinity (KD) were estimated by homologous competition binding assays. Opioid and sigma Bmax values in the solid tumors were significantly lower than their original levels in vitro. KD values for opioid/sigma ligands were similar in vitro and in vivo. With successive passages in the murine host, delta opioid and sigma 1 binding of the neuroblastoma-derived solid tumors became undetectable. In contrast, sigma 2 receptor Bmax values were unchanged with successive passages of the neuroblastoma-derived tumors and doubled in the nude mouse-borne gliomas. When neuroblastoma-derived solid tumors that were devoid of delta opioid binding were returned to culture, opioid receptors appeared to be up-regulated as compared with their original in vitro levels. Serial passaging of these recultured cells in vivo again resulted in a rapid decline in opioid receptor content. The opioid data are consistent with our prior findings on opioid binding diminution in human brain tumors. The pattern of change for sigma binding was more complex, with the sigma 2 response in late passages of the glioma being reminiscent of the formerly observed increase in number of sigma sites in transformed human meninges, kidney, and colon tissue.

Topics: Animals; Binding, Competitive; Cell Line; Cell Membrane; Convulsants; Enkephalin, Leucine-2-Alanine; Glioma; Guanidines; Kinetics; Male; Mice; Mice, Nude; Neuroblastoma; Pentazocine; Rats; Receptors, Opioid; Receptors, sigma; Transplantation, Heterologous; 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

Continuous elevation of intracellular cyclic AMP (cAMP) by culturing neuroblastoma x glioma NG108-15 hybrid cells in the presence of forskolin and isobutylmethylxanthine (IBMX) in a chemically defined medium resulted in differentiation of the hybrid cells, as indicated by extension of neurite-like structures and induction of a subclass of G-protein, Go, as monitored by Western analysis. This cellular differentiation also resulted in an initial 25 to 30% increase in [3H]diprenorphine binding 3 hr after forskolin and IBMX treatment, followed by a decrease in opioid receptor density to the maximal level of 35% of control 4 days later. However, the potencies and maximal inhibitory levels of various opioid agonists to inhibit adenylate cyclase activity was not altered during differentiation. When the differentiated hybrid cells were treated with DADLE chronically, an apparent decrease in the ability of the agonist to desensitize and to down-regulate the delta-opioid receptor was observed. It is unlikely that this observed attenuation was due to activation of cAMP-dependent protein kinase A, because (1) attenuation of DADLE desensitization was time-dependent, reaching maximal effects 48 hr after the initiation of treatment; and (2) pretreatment of NG108-15 cells with forskolin and IBMX resulted in attenuation of forskolin's ability to stimulate adenylate cyclase activity and parallel decrease in the ability of forskolin to activate the cAMP-dependent protein kinases in these cells was also observed. Thus, it is unlikely that the activation of protein kinase A by forskolin and IBMX is the cause for the attenuation of DADLE-induced delta-opioid receptor desensitization in differentiated NG108-15 cells.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Cell Differentiation; Colforsin; Cyclic AMP; Enkephalin, Leucine-2-Alanine; Glioma; Hybrid Cells; Neuroblastoma; Protein Kinases; Receptors, Opioid, delta; Tumor Cells, Cultured

Functional delta opioid receptors were expressed in Xenopus oocytes following injection of poly(A+)RNA isolated from the mouse neuroblastoma X rat glioma hybrid cell line, NG108-15. Oocytes coinjected with in vitro transcribed beta 2-adrenergic receptor mRNA and NG108-15 cell mRNA had 3-fold elevated cAMP levels following isoproterenol treatment. Application of delta opioids to these oocytes caused dose-dependent inhibition of isoproterenol-induced increase of cAMP, which was antagonized by naltrexone. This is the first demonstration of the expression of opioid receptors in Xenopus oocytes. The ability to assay expression of delta opioid receptors functionally coupled to the inhibition of adenylate cyclase in Xenopus oocytes following injection of exogenous mRNA will provide a system to investigate the relationship of molecular structure to function of opioid receptors.

Topics: Adenylyl Cyclases; Analgesics; Analysis of Variance; Animals; Cyclic AMP; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine-2-Alanine; Enkephalins; Female; Glioma; Hybrid Cells; Isoproterenol; Mice; Naltrexone; Neuroblastoma; Oocytes; Poly A; Rats; Receptors, Opioid, delta; RNA; RNA, Messenger; Xenopus laevis

In this study we used as glial cell models, early and late passage C-6 glial cells, 2B clone, and advanced passages of glial cells derived from aged mouse cerebral hemispheres (MACH) to examine responsiveness to opioids. We have previously reported that early passage C-6 glial cells, 2B clone, are bipotential and can be geared toward oligodendrocyte or astrocytic expression, whereas late passage C-6 glial cells are astrocytic. In addition, MACH cultures have been previously characterized and consist of astrocytes type 1 and 2, some oligodendrocytes, and few glial precursors. In this study, early passage (17-20) and late passage (106-108) C-6 glial cells or MACH cells of passages 16-19 were grown from plating time until harvesting, day 7 or 8, in DMEM + 10% FBS in the presence or absence of opioid peptides, Leu-enkephalin (10(-8) to 10(-10) M) or its synthetic analog, dalargin (Tyr-D-Ala-Gly-Phe-Leu-Arg; 10(-8) to 10(-10) M). We examined for the activities of glutamine synthetase (GS) and cyclic nucleotide phosphohydrolase (CNP), enzyme markers for astrocytes and oligodendrocytes, respectively. We found that CNP activity was markedly increased in the early passage following opioid treatment, indicative of a shift to oligodendrocytic expression. In the late passage cells, already committed to astrocytic expression, opioid treatment enhanced GS activity suggesting that astrocytes respond to opioids. GS activity was markedly increased in MACH cultures grown in the presence of opioids with no changes in CNP. Thus, type 1 astrocytes, the predominant glial type in MACH cultures, responded to opioids.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 2',3'-Cyclic-Nucleotide Phosphodiesterases; Amino Acid Sequence; Animals; Astrocytes; Cellular Senescence; Endorphins; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Glioma; Glutamate-Ammonia Ligase; Mice; Mice, Inbred C3H; Molecular Sequence Data; Nerve Tissue Proteins; Neuroglia; Neuronal Plasticity; Oligodendroglia; Tumor Cells, Cultured

This study investigates the functional state of the stimulatory GTP-binding protein GS in neuroblastoma x glioma NG108-15 hybrid cells chronically exposed to an opioid. For this purpose, a novel in situ reconstitution protocol was established using membranes selectively depleted of GS function by transient exposure to low pH and then reconstituted with purified exogenous stimulatory GTP-binding proteins. With prostaglandin E1 (PGE1) receptor-stimulated adenylate cyclase activity as an indicator, reconstituted membranes of cells previously rendered tolerant to the delta-opioid [D-Ala2,D-Leu5]enkephalin (DADLE) exhibited approximately 3-fold elevated cAMP generation upon stimulation with PGE1, compared with nontolerant reconstituted cell membranes. This effect developed dose-dependently with respect to the opioid concentration used for pretreatment of the cells and was blocked by concomitant exposure to naloxone. In contrast, receptor-independent activation of GS by the stable GTP analogue guanosine-5'-O-(3-thio)triphosphate did not reveal any difference in adenylate cyclase activity between reconstituted membranes of control and chronically DADLE-pretreated cells. Furthermore, the functional activity of endogenous GS displayed no difference between control and DADLE-tolerant cells, as assessed in S49 cyc- reconstitution assays using sodium cholate extracts derived from NG108-15 membranes. The data presented suggest that the increase in PGE1 receptor-mediated adenylate cyclase activity in opioid-tolerant/dependent NG108-15 hybrid cells most likely relates to enhanced coupling efficiency between the PGE1 binding site (receptor) and GS. Moreover, our results support the concept that supersensitivity to excitatory drugs reflects an adaptive mechanism of cells chronically exposed to an opioid.

Topics: Adenylyl Cyclases; Amino Acid Sequence; Animals; Enkephalin, Leucine-2-Alanine; Glioma; GTP-Binding Proteins; Hybrid Cells; Hydrogen-Ion Concentration; Mice; Molecular Sequence Data; Neuroblastoma; Rabbits; Receptors, Prostaglandin; Receptors, Prostaglandin E

In neuroblastoma x glioma NG108-15 hybrid cells, opioid agonists inhibited both basal and prostaglandin E1-stimulated adenylate cyclase activities assayed in the presence of the phosphodiesterase (PDE) inhibitors isobutylmethylxanthine and ZK62711 (rolipram). However, when intracellular [3H]cAMP was measured in the absence of the PDE inhibitors the maximal inhibitory level was increased, using the opioid agonist D-Ala2,D-Leu5-enkephalin. This increase in opioid activity was due to agonist stimulation of cAMP degradation, because when the degradation rate of [3H] cAMP was measured in intact hybrid cells it was observed to increase from the control value of 0.495 +/- 0.003 min-1 to 0.760 +/- 0.003 min-1 in the presence of 1 microM D-Ala2,D-Leu5-enkephalin; this was reversed by naloxone. Dose-dependent studies with various opioid agonists, partial agonists, and antagonists revealed that there was a direct correlation between the abilities of these opioid ligands to inhibit adenylate cyclase activity and to stimulate PDE activity, with enkephalin and its analogs being the most potent agonists. Chronic agonist treatment also resulted in a reduction of the opioid agonist stimulation of cAMP degradation, with an apparent decrease in the PDE activity upon addition of naloxone after chronic treatment. However, treatment of the hybrid cells with pertussis toxin, which attenuated the agonist inhibition of adenylate cyclase activity, did not abolish this opioid response. When selective inhibitors for various types of PDE were used, the type I PDE inhibitor W-7 attenuated the opioid effect, whereas the type II PDE inhibitor trequinsin (HL725), the type III PDE inhibitor indolidan, and the type IV PDE inhibitor rolipram had no effect on opioid-stimulated cAMP degradation. The stimulation of type I PDE activity by delta-opioid receptors was independent of extracellular Ca2+ and was not observed with membrane preparations. Therefore, in NG108-15 cells delta-opioid receptors regulate intracellular cAMP levels by coupling to a pertussis toxin-insensitive guanine nucleotide-binding protein, resulting in an increase in intracellular Ca2+ and in Ca2+/calmodulin-dependent PDE activity.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Adenylate Cyclase Toxin; Calcium; Cyclic AMP; Enkephalin, Leucine-2-Alanine; Enzyme Activation; Glioma; Hybrid Cells; Neuroblastoma; Neurons; Pertussis Toxin; Phosphodiesterase Inhibitors; Receptors, Opioid, delta; Tumor Cells, Cultured; 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

Depolarization-induced increases in intracellular free calcium concentration ([Ca2+]i) were measured in single NG108-15 cells using indo-1 based microfluorimetry. In cells differentiated for 6 days in serum-free forskolin (5 microM) supplemented media, application of micromolar concentrations of [D-Ala2-D-Leu5]enkephalin (DADLE) inhibited Ca2+ influx mediated by voltage-gated Ca2+ channels. Inhibition of 50 mM K(+)-induced Ca2+ influx by DADLE was concentration-dependent over the range of 0.1 to 10 microM and blocked by 100 microM naloxone. Differentiation increased the amplitude of depolarization-induced [Ca2+]i transients from 78 +/- 21 nM in undifferentiated cells to 1,282 +/- 318 nM after 6 days. One microM nitrendipine inhibited Ca2+ influx by at least 65% at all stages of differentiation, while sensitivity to omega-conotoxin GVIa (omega-CgTx) did not appear until day 3. omega-CgTx inhibited a dihydropyridine-sensitive Ca2+ channel. DADLE inhibition of Ca2+ channels did not appear until 3 days of differentiation. Thus, opioid inhibition of depolarization-induced Ca2+ influx paralleled the expression of omega-CgTx sensitive voltage-gated Ca2+ channels.

Topics: Calcium Channel Blockers; Cell Differentiation; Colforsin; Culture Media; Cytophotometry; Endorphins; Enkephalin, Leucine-2-Alanine; Glioma; Ion Channel Gating; Nervous System Neoplasms; Neuroblastoma; omega-Conotoxin GVIA; Peptides, Cyclic; Tumor Cells, Cultured

Five separate guanine nucleotide-binding proteins (G proteins) were immunologically identified in membranes from neuroblastoma x glioma NG108-15 hybrid cells. These alpha subunit proteins were Gi2 alpha, two isoforms of Gi3 alpha, and two isoforms of Go alpha. The G proteins that interacted with delta-opioid receptors in these membranes were identified using cholera toxin (CTX)-induced ADP-ribosylation and antisera selective for various G protein alpha subunits. In the presence of delta-opioid agonists, CTX induced the incorporation of [32P]ADP-ribose into three pertussis toxin substrates. Using antisera generated against peptide sequences from G alpha subunits, these three pertussis toxin substrates were identified as Gi2 alpha, Go2 alpha, and one isoform of Gi3 alpha, which has yet to be identified. This CTX-induced labeling was demonstrated to be mediated via the delta-opioid receptor in these hybrid cells by the observation that delta agonists D-Ala2-D-Leu5-enkephalin (DA-DLE) and D-Pen2-D-Pen5-enkephalin, as well as the nonselective agonists etorphine and bremazocine, were active, but the mu agonist PL017 and the kappa agonist U-50-488H did not show this activity. This incorporation into all three substrates induced by DADLE was dose dependent, with EC50 (95% confidence interval) values ranging from 12 (3-52) to 183 (65-520) nM, which compared with the Kd value of 10 +/- 1.5 nM for this agonist, a dose that produces maximal inhibition of adenylate cyclase activity. Furthermore, pretreatment of the cells with pertussis toxin or treatment of the membranes with the antagonist naloxone blocked the incorporation induced by DADLE. Incorporation of [32P]ADP-ribose into all three substrates decreased 35-83% in membranes in which the receptors had been down-regulated by chronic treatment of the cells with DADLE. Thus, a single opioid receptor type can interact with three separate G proteins.

Topics: Adenosine Diphosphate Ribose; Adenylate Cyclase Toxin; Analgesics; Animals; Autoradiography; Cell Line; Cell Membrane; Cholera Toxin; Electrophoresis, Polyacrylamide Gel; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine-2-Alanine; Enkephalins; Glioma; GTP-Binding Proteins; Hybrid Cells; Kinetics; Mice; Molecular Weight; Neuroblastoma; Pertussis Toxin; Phosphorus Radioisotopes; Rats; Receptors, Opioid; Receptors, Opioid, delta; 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

The intracellular free calcium concentration ([Ca2+]i) was measured in single NG108-15 cells using indo-1-based microfluorimetry. In cells differentiated for 6-14 days in serum-free, forskolin (5 microM)-supplemented medium, application of micromolar concentrations of [D-Ala2,D-Leu5]-enkephalin (DADLE) inhibited Ca2+ influx mediated by voltage-gated Ca2+ channels. DADLE, at concentrations ranging from 1 nM to 1 microM, also produced rapid transient increases in [Ca2+]i (EC50 = 10 nM). The [Ca2+]i increases elicited by DADLE did not correlate with the inhibitory effects of the peptide. DADLE-induced [Ca2+]i increases were blocked by naloxone. In single cells, sequential application of selective opioid agonists (30 nM) evoked responses of the rank order DADLE = [D-Pen2,D-Pen5]-enkephalin > (trans)-(+-)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]cyclohexyl) benzeneacetamide > [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin, consistent with activation of a delta-opioid receptor. The response was completely blocked by removal of extracellular Ca2+ or application of 1 microM nitrendipine, indicating that the increase in [Ca2+]i results from Ca2+ influx via dihydropyridine-sensitive, voltage-gated Ca2+ channels. Substitution of N-methyl-D-glucamine for extracellular Na+ or application of 1 microM tetrodotoxin greatly reduced, and in some cases blocked, the DADLE-induced [Ca2+]i increase, consistent with amplification of the response by voltage-gated Na+ channels. The [Ca2+]i increase was mimicked by both dibutyryl-cAMP and phorbol 12,13-dibutyrate. These findings are consistent with a delta-opioid-induced depolarization, possibly mediated by a second messenger, that subsequently recruits voltage-sensitive Ca2+ channels. In contrast to differentiated cells, undifferentiated cells responded to DADLE with a modest [Ca2+]i increase that was not sensitive to nitrendipine. In these cells, activation of the same second messenger system may elevate [Ca2+]i by mobilization from intracellular stores rather than influx. In addition to previously described inhibitory coupling to adenylyl cyclase and Ca2+ channels in NG108-15 cells, these results suggest that a novel, excitatory, effector system may also couple to opioid receptors.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Bucladesine; Calcium; Calcium Channels; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine-2-Alanine; Enkephalins; Glioma; Hybrid Cells; Ion Channel Gating; Narcotics; Neuroblastoma; Phorbol 12,13-Dibutyrate; Pyrrolidines; Receptors, Opioid, delta; 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

Mouse neuroblastoma x rat glioma hybrid cells (N x G, 108CC15) were used to study the inhibitory effects of the synthetic opioid D-Ala2-D-Leu5-enkephalin (DADLE), somatostatin, adrenaline-alpha 2 and angiotensin II on voltage-dependent Ca(2+)-currents (ICa) using the patch-clamp technique in the whole-cell configuration mode. The inhibitory effects could be abolished by pretreatment of N x G cells with pertussis toxin or intracellular infusion of GDP beta S indicating an involvement of a pertussis toxin sensitive GTP-binding protein (G-protein), presumably Go. The effect of DADLE, the strongest inhibitor of ICa, was studied during dibutyryl cyclic AMP (dBcAMP) induced differentiation. Using omega-conotoxin GVIA (omega-CTX) and methoxyverapamil (D600) as specific Ca(2+)-channel blockers of the N- and L-type Ca(2+)-channels, it was found that in N x G cells DADLE predominantly induces inhibition of T- and N-type Ca(2+)-channels.

Topics: Animals; Calcium Channels; Cell Differentiation; Enkephalin, Leucine-2-Alanine; Gallopamil; Glioma; GTP-Binding Proteins; Hybrid Cells; Mice; Neuroblastoma; omega-Conotoxin GVIA; Peptides, Cyclic; Protein Kinase C; Rats; 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

Cholera toxin treatment (up to 1 microgram/ml, 16 h) of neuroblastoma x glioma hybrid NG108-15 cells produced a decrease of some 35% in both delta opioid receptor-mediated stimulation of high-affinity GTPase activity and inhibition of forskolin-amplified adenylate cyclase. Coincident with these decreases was a down-regulation of some 35% in the delta opioid receptor population. A similar pattern of a decrease in signalling capacity was noted for the alpha 2B-adrenergic receptor in these cells after cholera toxin treatment. Half-maximal effects of cholera toxin on all of the parameters assayed were noted at concentrations between 2 and 5 ng/ml. Neither levels of Gi2, as assessed by immunoblotting with specific antisera, nor the intrinsic activity of the alpha subunit of the guanine-nucleotide-binding protein which acts as the inhibitory G-protein of the adenylate cyclase in these cells, as assessed by guanosine 5'-[beta gamma-imido]triphosphate (Gpp[NH]p)-mediated inhibition of adenylate cyclase, was lowered by cholera toxin treatment. Furthermore, levels of another pertussis toxin-sensitive G-protein (Go) expressed by these cells was also not lowered by cholera toxin treatment. However, as previously noted in other cells [Milligan, Unson & Wakelam (1989) Biochem. J. 262, 643-649], marked down-regulation of the alpha subunit of the stimulatory G-protein (Gs) of the adenylate cyclase cascade was observed in response to cholera toxin treatment. Previous studies [Klee, Milligan, Simonds & Tocque (1985) Mol. Aspects Cell Regul. 4, 117-129] have shown that cholera toxin treatment can result in a decrease in the maximal effectiveness of agonists which function to inhibit adenylate cyclase. These data have been used as evidence to suggest a functional interaction between Gs and 'Gi'. The results provided herein demonstrate that such effects of the toxin can be explained adequately by a decrease in the number of receptors that function to produce inhibition of adenylate cyclase.

Topics: Adenylate Cyclase Toxin; Adenylyl Cyclase Inhibitors; Adrenergic alpha-Agonists; Brimonidine Tartrate; Cholera Toxin; Colforsin; Enkephalin, Leucine-2-Alanine; Glioma; GTP Phosphohydrolases; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Hybrid Cells; Immunoblotting; Neuroblastoma; Pertussis Toxin; Quinoxalines; Receptors, Opioid; Receptors, Opioid, delta; Signal Transduction; Tumor Cells, Cultured; Virulence Factors, Bordetella

Chronic treatment of neuroblastoma x glioma NG108-15 hybrid cells with the opioid agonist D-Ala,2 D-Leu5-enkephalin (DADLE) induces a homologous desensitization of the delta opioid receptors present in these cells. Since the Kd value of the delta opioid receptor's high-affinity state reflects the potency of the agonist, we examined the effect of receptor desensitization in NG108-15 cells on the percentage of receptor in the high-affinity state. When NG108-15 hybrid cells were treated with 10 or 100 nM DADLE for 4 hr at 24 degrees C, loss of DADLE's ability to inhibit adenylate cyclase was observed. However, when competition binding experiments were carried out with P2P3 membranes isolated from the delta opioid-desensitized hybrid cells, it was determined that 41.7 +/- 3.4% of the total binding sites remained in the high-affinity state, with no apparent alteration in the Kd value of either high- or low-affinity states. Similarly, when NG108-15 cells were treated with 100 ng/ml of pertussis toxin for 3 hr at 37 degrees C, 39.9 +/- 3.6% of the binding sites remained in the high-affinity state. This reduction in the percentage of receptor in high-affinity state was agonist specific, for chronic treatment of hybrid cells with levorphanol, a partial agonist, or the antagonist naloxone did not alter the percentage of opioid receptors in the high-affinity state. Furthermore, the delta opioid receptors remaining in the high-affinity state after chronic DADLE treatment were still sensitive to both Na+ and guanyldylimidodiphosphate, indicating that opioid ligand binding remained coupled to the G-proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenylate Cyclase Toxin; Binding, Competitive; Enkephalin, Leucine-2-Alanine; Glioma; GTP-Binding Proteins; Guanosine Diphosphate; Guanylyl Imidodiphosphate; Humans; Hybrid Cells; Magnesium; Neuroblastoma; Pertussis Toxin; Receptors, Opioid; Receptors, Opioid, delta; Sodium; Virulence Factors, Bordetella

Mouse neuroblastoma x rat glioma hybrid cells (NG108-15) express an opioid receptor of the delta subclass which both stimulates high-affinity GTPase activity and inhibits adenylate cyclase by interacting with a pertussis-toxin-sensitive guanine-nucleotide-binding protein(s) (G-protein). Four such G-proteins have now been identified without photoreceptor-containing tissues. We have generated anti-peptide antisera against synthetic peptides which correspond to the C-terminal decapeptides of the alpha-subunit of each of these G-proteins and also to the stimulatory G-protein of the adenylate cyclase cascade (Gs). Using these antisera, we demonstrate the expression of three pertussis-toxin-sensitive G-proteins in these cells, which correspond to the products of the Gi2, Gi3 and Go genes, as well as Gs. Gi1, however, is not expressed in detectable amounts. IgG fractions from each of these antisera and from normal rabbit serum were used to attempt to interfere with the interaction of the opioid receptor with the G-protein system by assessing ligand stimulation of high-affinity GTPase activity, inhibition of adenylate cyclase activity and conversion of the receptor to a state which displays reduced affinity for agonists. The IgG fraction from the antiserum (AS7) which specifically identifies Gi2 in these cells attenuated the effects of the opioid receptor. This effect was complete and was not mimicked by any of the other antisera. We conclude that the delta-opioid receptor of these cells interacts directly and specifically with Gi2 to cause inhibition of adenylate cyclase, and that Gi2 represents the true Gi of the adenylate cyclase cascade. The ability to measure alterations in agonist affinity for receptors following the use of specific antisera against a range of G-proteins implies that such techniques should be applicable to investigations of the molecular identity of the G-protein(s) which interacts with any receptor.

Topics: Adenylate Cyclase Toxin; Adenylyl Cyclase Inhibitors; Amino Acid Sequence; Animals; Base Sequence; Cell Line; DNA, Neoplasm; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Glioma; GTP Phosphohydrolases; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Hybrid Cells; Immune Sera; Kinetics; Mice; Molecular Sequence Data; NAD; Neuroblastoma; Oligonucleotide Probes; Pertussis Toxin; Rats; Receptors, Opioid; Receptors, Opioid, delta; RNA, Neoplasm; Signal Transduction; Virulence Factors, Bordetella

We have characterized the pertussis toxin substrate in NG 108-15 cell membranes using site-specific antisera and ADP-ribosylation. Cell membranes contain two pertussis toxin-sensitive guanine nucleotide-binding protein alpha-subunits (G alpha) whose Rf values in gel electrophoresis coincide with those of G alpha o and G alpha i2. The total quantity of Gi and Go immunoreactivity amounted to 24.3 +/- 2.8 pmol/mg, whereas only 1.5 +/- 0.2 pmol/mg are capable of undergoing ADP-ribosylation catalyzed by pertussis toxin. Pretreatment of cells with the agonist [D-Ala2,D-Leu2]-enkephalin (DADLE) for 24 h and DADLE or morphine for 72 h did not alter the incorporation of ADP-ribose or the immunoreactive amount of Gi and Go subunits. However, pretreatment for 72 h with naloxone increased the incorporation of ADP-ribose without an apparent change in affinity or in the immunochemically determined protein levels of Gi and Go. This indicates that the process of down-regulation and desensitization of the delta-opioid receptor neither requires quantitative alterations in the levels of Gi and Go nor changes in the degree of coupling among their subunits. In contrast, chronic exposure to antagonists seems to alter the degree of precoupling between alpha- and beta-subunits of Gi and/or Go.

Topics: Adenosine Diphosphate Ribose; Electrophoresis, Polyacrylamide Gel; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Glioma; GTP-Binding Proteins; Hybrid Cells; Immunoblotting; Morphine; NAD; Neuroblastoma; Pertussis Toxin; Time Factors; Tumor Cells, Cultured; Virulence Factors, Bordetella

Recently we reported the synthesis of the first enantiomeric pair of irreversible opioid ligands [(3S,4R)-(-)- and (3R,4S)-(+)-cis-4, SUPERFIT] and specific interaction of the latter with the delta receptor. Here we report another enantiomeric pair of irreversible opioid ligands, (+)-trans- and (-)-trans-3-methylfentanyl isothiocyanates [(3S,4S)-(+)-trans- and (3R,4R)-(-)-trans-4]. A single-crystal X-ray analysis of the 2,4,6-trinitrobenzenesulfonic acid salt of (+)-trans-3-methyl-N-phenyl-4-piperidinamine [(+)-trans-8] revealed it (and, therefore, 4) to have the trans configuration and the absolute configuration of (+)-trans-8 to be 3S,4S. The (+)-trans enantiomer of 4 was shown to be highly potent and about 10-fold more selective as an acylating agent than (-)-trans-4 for the higher affinity [3H]DADL (delta) binding site in rat brain membranes. In that assay, (+)-trans-4 and (+)-cis-4 were essentially equipotent as affinity ligands, and the levo enantiomers were considerably less potent. (+)-trans-4 was, thus, a potent, subtype-selective acylating agent for the delta opioid receptor in vitro. With membranes from NG108-15 neuroblastoma x glioma hybrid cells, containing only delta receptors, (+)-cis-4 was found to be a little more potent than (+)-trans-4. Similarly, (+)-cis-4 is the most effective inhibitor of adenylate cyclase in these membranes, (+)-trans-4 has weak activity, and the levo enantiomers are inactive. Only (+)-cis-4 was found to have antinociceptive activity in vivo.

Topics: Acylation; Adenylyl Cyclase Inhibitors; Analgesia; Animals; Brain; Cell Membrane; Chemical Phenomena; Chemistry; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Fentanyl; Glioma; Hybrid Cells; Indicators and Reagents; Ligands; Male; Molecular Structure; Neuroblastoma; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, delta; Stereoisomerism; Structure-Activity Relationship; Tumor Cells, Cultured

According to classical models of drug-receptor interactions, competitive antagonists share with agonists the ability to bind to a common site on the receptor molecule. However, they are different from agonists, as they cannot trigger the "stimulus" that leads to biological responses--i.e., they lack intrinsic activity. For those receptors whose signals are transduced to effector systems by GTP-binding regulatory proteins (G proteins), a mechanistic equivalent of such a stimulus is an increased ability of agonist-bound receptor to accelerate nucleotide exchange and thus GTPase activity on the G-protein molecule. Here we show that for a member of this family of receptors (delta opioid receptors in membranes of NG108-15 neuroblastoma-glioma cells), two types of competitive antagonists can be distinguished. One type has no intrinsic activity, since it neither stimulates nor inhibits the GTPase activity of G proteins and its apparent affinity for the receptor is not altered by pertussis toxin-mediated uncoupling of receptor and G protein. The second type, however, can inhibit GTPase and thus exhibits negative intrinsic activity; its affinity for receptors is increased following uncoupling from G proteins. The existence of antagonists with negative intrinsic activity may be a general feature of several classes of neurotransmitters or hormone receptors and calls for a reevaluation of biological effects produced by competitive antagonists.

Topics: Animals; Benzomorphans; Cell Line; Cell Membrane; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Ethylmaleimide; Glioma; GTP Phosphohydrolases; GTP-Binding Proteins; Kinetics; Narcotic Antagonists; Neuroblastoma; Pertussis Toxin; Receptors, Opioid; Receptors, Opioid, delta; Virulence Factors, Bordetella

Cholera toxin catalyzes the ADP-ribosylation of 40 kDa pertussis toxin substrates in membranes from NG108-15 cells, which is increased in the presence of the opioid agonist DADLE. The basal ADP-ribosylation can be abolished by the opioid antagonist ICI 174864, suggesting that unoccupied opioid receptors interact spontaneously with the pertussis toxin substrates Gi/Go in the membrane. Treatment of NG108-15 cells with the opioid agonist DADLE leads to a reduction of agonist-stimulated and basal ADP-ribosylation of 40 kDa substrates catalyzed by cholera toxin. This indicates that the spontaneous interaction between opioid receptors and G-proteins is decreased in membranes of cells in which the receptor was desensitized by prolonged exposure to the agonist.

Topics: Adenosine Diphosphate Ribose; Animals; Cell Line; Cell Membrane; Cholera Toxin; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Glioma; GTP Phosphohydrolases; GTP-Binding Proteins; Hybrid Cells; NAD; Neuroblastoma; Pertussis Toxin; Receptors, Opioid; Virulence Factors, Bordetella

Topics: Animals; Calcium; Cell Line; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Glioma; GTP-Binding Proteins; Homeostasis; Hybrid Cells; Ion Channels; Neuroblastoma; Neurons

The properties of [125I]beta h-endorphin-binding sites from rat brain membranes and membranes from the NG108-15 cell line were compared using a monoclonal antibody directed against the opioid receptor and opioid peptides as probes. The binding of [125I]beta h-endorphin to both rat brain and NG108-15 membranes yielded linear Scatchard plots with Kd values of 1.2 nM and 1.5 nM, respectively, and Bmax values of 865 fmol/mg rat brain membrane protein and 1077 fmol/mg NG108-15 membrane protein. A monoclonal antibody, OR-689.2.4, capable of inhibiting mu and delta binding but not kappa binding to rat brain membranes, noncompetitively inhibited the binding of 1 nM [125I]beta h-endorphin to rat brain and NG108-15 membranes with an IC50 value of 405 nM for rat brain membranes and 543 nM for NG108-15 membranes. The monoclonal antibody also inhibited the binding of 3 nM [3H] [D-penicillamine2, D-penicillamine5] enkephalin to NG108-15 membranes with an IC50 value of 370 nM. In addition to blocking the binding of [125I]beta h-endorphin to brain membranes, the antibody also displaced [125I]beta h-endorphin from membranes. Site-specific opioid peptides had large variations in their IC50 values depending on whether they were inhibiting [125I]beta h-endorphin binding to rat brain or the NG108-15 membranes. When the peptides were tested with the monoclonal antibody for their combined ability to inhibit [125I]beta h-endorphin binding to both membrane preparations, the peptides and antibody blocked binding as though they were acting at allosterically coupled sites, not two totally independent sites. These studies suggest that mu-, delta-, and beta-endorphin-binding sites share some sequence homology with the 35,000-dalton protein that the antibody is directed against.

Topics: Animals; Antibodies, Monoclonal; beta-Endorphin; Brain; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Glioma; Immunoglobulin M; In Vitro Techniques; Iodine Radioisotopes; Male; Molecular Weight; Neuroblastoma; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Tumor Cells, Cultured

Opioid receptor activity in neuroblastoma x glioma NG108-15 hybrid cell membranes was attenuated by acid phosphatase purified by high performance liquid chromatography and devoid of protease activity. Treatment of membranes with this phosphatase decreased opioid inhibition of adenylate cyclase and this effect was potentiated by the presence of the opioid agonist during the phosphatase treatment. Phosphatase treatment did not affect the number of opioid receptors but it did alter the distribution of receptors among affinity states, by increasing the percentage of receptors in the low affinity state. The similarities between these effects and desensitization of the opioid receptor, during chronic opioid treatment, are discussed.

Topics: Acid Phosphatase; Animals; Chromatography, High Pressure Liquid; Cyclic AMP; Diprenorphine; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Etorphine; Glioma; Guanfacine; Guanidines; Hybrid Cells; Neuroblastoma; Phenylacetates; Receptors, Opioid

Incubation of the neuroblastoma x glioma hybrid cell line NG108-15 in tissue culture with dibutyryl cyclic AMP (1 mM) for up to 8 days produced a morphological differentiation of the cells, during which they extended neurite-like processes. Pertussis-toxin-catalysed ADP-ribosylation indicated that amounts of guanine-nucleotide-binding proteins (G-proteins), which are substrates for this toxin, were approximately doubled in membranes from the 'differentiated' cells in comparison with the control cells. Immunoblotting of membranes derived from either untreated or dibutyryl cyclic AMP-treated cells with anti-peptide antisera specific for the alpha subunits of the pertussis-toxin-sensitive G-proteins Gi and Go demonstrated that amounts of these G-proteins were reciprocally modulated during the differentiation process. In comparison with the untreated cells, the amount of Gi in the 'differentiated' cells was decreased, whereas the amount of Go was substantially increased. Stimulation of high-affinity GTPase activity in response to opioid peptides, which in this cell line interact with an opioid receptor of the delta subclass, was much decreased, and inhibition of adenylate cyclase activity was almost entirely attenuated in the 'differentiated'-cell membranes in comparison with membranes of untreated cells. Opioid receptor number was also decreased in membranes of the dibutyryl cyclic AMP-treated cells in comparison with the control cells. These data demonstrate that relatively small changes in the observed pattern of pertussis-toxin-catalysed ADP-ribosylation of membranes can mask more dramatic alterations in amounts of the individual pertussis-toxin-sensitive G-proteins, and further demonstrate the importance of methodologies able to discriminate between the different gene products.

Topics: Bucladesine; Cell Differentiation; Cell Membrane; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalin, Methionine; Glioma; GTP-Binding Proteins; Hybrid Cells; Neuroblastoma; Time Factors

Opioid-receptor binding and the opioid-mediated stimulation of low Km GTPase and inhibition of adenylate cyclase were studied in membranes derived from NG 108-15 cells pretreated with either the opioid peptide [D-Ala2, D-Leu5]enkephalin (DADLE) or morphine. Pretreatment with DADLE resulted in a concentration-dependent loss of responsiveness of GTPase to the peptide; this effect was entirely accounted for by a reduction in the maximal stimulation produced acutely by DADLE, without changes in the EC50 of the peptide, indicating a non-competitive type of desensitization. The degree of desensitization of GTPase was similar after one and 24 hr of pretreatment with DADLE, indicating that the process occurs rapidly. In contrast, morphine, which was 70-80% as potent as DADLE in stimulating GTPase and inhibiting adenylate cyclase in acute conditions, induced only a minimal desensitization of the opioid-GTPase system and, in contrast to DADLE, did not desensitize adenylate cyclase. Pretreatment with DADLE for one hour led to a decrease in opioid receptor density which was quantitatively similar to the degree of desensitization of GTPase: both these effects of DADLE were antagonized to a similar extent when morphine was also present in the pretreatment. Thus, desensitization of the opioid-stimulated GTPase appears to be correlated with down-regulation of the opioid receptor. Moreover, these findings suggest that partial agonists cannot induce this process.

Topics: Adenylyl Cyclase Inhibitors; Cell Line; Cell Membrane; Drug Tolerance; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Glioma; GTP Phosphohydrolases; Hybrid Cells; Morphine; Neuroblastoma; Phosphoric Monoester Hydrolases; Receptors, Opioid

Treatment of NG108-15 cells in culture with the opiate peptide [D-Ala2,D-Leu5]enkephalin produces maximal inhibition of cyclic AMP synthesis in less than 15 min. The activity of [GM3]:N-acetylgalactosaminyltransferase is similarly inhibited, but maximal inhibition is not observed for at least 30 min following the addition of [D-Ala2,D-Leu5]enkephalin. Conversely, the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine rapidly potentiates the intracellular accumulation of cyclic AMP and, in a more gradual fashion, increases [GM3]:N-acetylgalactosaminyltransferase activity. The reductions in the activity of [GM3]:N-acetylgalactosaminyltransferase that occur following treatment of NG108-15 cells with indomethacin argues for a direct role of cyclic AMP in the observed changed in [GM3]:N-acetylgalactosaminyltransferase activity. By adding low concentrations of cyclic AMP (but not cyclic GMP) to microsomes derived from neonatal rat brain, we were able to demonstrate a dose-dependent phosphorylation of membrane protein and subsequent doubling of [GM3]:N-acetylgalactosaminyltransferase activity.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Brain; Cell Line; Cyclic AMP; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enzyme Activation; Galactosyltransferases; Glioma; Kinetics; Mice; Microsomes; N-Acetylgalactosaminyltransferases; Neuroblastoma; Phosphoproteins; Phosphorylation; Polypeptide N-acetylgalactosaminyltransferase; Protein Kinases; Rats

In membranes of neuroblastoma x glioma hybrid (NG108-15) cells, bradykinin (EC50 approximately equal to 5 nM) stimulates GTP hydrolysis by a high-affinity GTPase (Km approximately equal to 0.2 microM). The octapeptide, des-Arg9-bradykinin, was inactive. Stimulation of GTP hydrolysis by bradykinin and an opioid agonist was partially additive. Treatment of NG108-15 cells with pertussis toxin, which inactivates Ni, eliminated GTPase stimulation by the opioid agonist but not by bradykinin. The data suggest that bradykinin activates in NG108-15 membranes a guanine nucleotide-binding protein which is not sensitive to pertussis toxin and which may be involved in bradykinin-induced stimulation of phosphoinositide metabolism in these cells.

Topics: Bradykinin; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Glioma; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine Triphosphate; Hybrid Cells; Hydrolysis; Membranes; Neuroblastoma; Pertussis Toxin; Phosphatidylinositols; Phosphoric Monoester Hydrolases; Virulence Factors, Bordetella

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

A subclone of NG108-15 neuroblastoma-glioma hybrid cells was used to study the intracellular distribution of opioid receptors. Subcellular organelles were separated on self-generating Percoll-sucrose gradients and the enzymes beta-glucuronidase, galactosyltransferase, 5'-nucleotidase, and glucose-6-phosphatase were used as markers to localize the various structures. Analysis of the receptor distribution from untreated cells shows that the plasma membranes contained the highest receptor density, but a significant portion of the opioid binding sites was unevenly distributed between the lysosomes, microsomes, and Golgi elements. The enzyme markers indicated that appearance of opioid receptors in these intracellular structures does not result merely from contamination with plasma membranes. About 11% of the receptors appeared in a fraction lighter than plasma membranes. The antilysosomal agent chloroquine altered the intracellular compartmentation of the receptors, possibly by blocking their translocation in the cells. Leu-enkephalin induced time-dependent loss of receptors from all four intracellular compartments examined, but a kinetic analysis showed that the rate of receptor loss in these fractions was not identical. Thus, the percent of receptors appearing in the lysosomal fraction that could still bind [3H]D-Ala2-D-Leu5-enkephalin in vitro was increased on treatment with Leu-enkephalin. As an additional approach to follow the intracellular fate of the receptors, cells were labeled with [3H]diprenorphine, chased with various unlabeled opiates, and the distribution of 3H-ligand-receptors in the cells was monitored. Leu-enkephalin and etorphine altered the distribution of receptor-bound [3H]diprenorphine between the plasma membranes, lysosomes, and Golgi elements, whereas morphine had no such effect. The study sheds light on the role of intracellular structures in the metabolism of opioid receptors in untreated and opioid-treated cells.

Topics: Animals; Cell Line; Cell Membrane; Centrifugation, Density Gradient; Chloroquine; Diprenorphine; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Etorphine; Glioma; Golgi Apparatus; Kinetics; Lysosomes; Microsomes; Neuroblastoma; Receptors, Opioid

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

The existence of multiple affinity states for the opiate receptor in neuroblastoma x glioma NG108-15 hybrid cells has been demonstrated by competition binding studies with tritiated diprenorphine and [D-Ala2, D-Leu5]enkephalin (DADLE). In the presence of 10 mM Mg2+, all receptors exist in a high affinity state with Kd = 1.88 +/- 0.16 nM. Addition of 10 microM guanyl-5'-yl imidodiphosphate (Gpp(NH)p) decreased the affinity of DADLE to Kd = 8.08 +/- 0.93 nM. However, in the presence of 100 mM Na+, which is required for opiate inhibition of adenylate cyclase activity, analysis of competition binding data revealed three sites: the first, consisting of 17.5% of total receptor population has a Kd = 0.38 +/- 0.18 nM; the second, 50.6% of the population, has a Kd = 6.8 +/- 2.2 nM; and the third, 31.9% of the population, has a Kd of 410 +/- 110 nM. Thus, in the presence of sodium, a high affinity complex between receptor (R), GTP binding component (Ni), and ligand (L) was formed which was different from that formed in the absence of sodium. These multiple affinity states of receptor in the hybrid cells are agonist-specific, and the percentage of total opiate receptor in high affinity state is relatively constant in various concentrations of Na+. Multiple affinity states of opiate receptor can be demonstrated further by Scatchard analysis of saturation binding studies with [3H]DADLE. In the presence of Mg2+, or Gpp(NH)p, analysis of [3H]DADLE binding demonstrates that opiate receptor can exist in a single affinity state, with apparent Kd values of [3H]DADLE in 10 mM Mg2+ = 1.75 +/- 0.28 nM and in 10 microM Gpp(NH)p = 0.85 +/- 0.12 nM. There is a reduction of Bmax value from 0.19 +/- 0.02 nM in the presence of Mg2+ to 0.14 +/- 0.03 nM in the presence of Gpp(NH)p. In the presence of 100 mM Na+, Scatchard analysis of saturation binding of [3H]DADLE reveals nonlinear plots; two-site analysis of the curves yields Kd = 0.43 +/- 0.09 and 7.9 +/- 3.2 nM. These Kd values are analogous to that obtained with competition binding studies. Again, this conversion of single site binding Scatchard plots to multiple sites binding plots in the presence of Na+ is restricted to 3H-agonist binding only.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Animals; Binding, Competitive; Cell Line; Diprenorphine; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Glioma; Guanylyl Imidodiphosphate; Hybrid Cells; Kinetics; Magnesium; Neuroblastoma; Receptors, Opioid; Sodium

Specific binding properties of the tritium-labeled delta opiate receptor agonist [3H][2-D-penicillamine, 5-D-penicillamine]enkephalin [( 3H][D-Pen2, D-Pen5]enkephalin) were characterized in the rat brain and in a mouse neuroblastoma-rat glioma hybrid cell line (NG 108-15). Saturation isotherms of [3H][D-Pen2, D-Pen5]enkephalin binding to rat brain and NG 108-15 membranes gave apparent Kd values of 1-6 nM. These values are in good agreement with the Kd value obtained from the kinetic studies. The Bmax value in NG 108-15 membranes was 235.3 fmol/mg of protein. An apparent regional distribution of [3H][D-Pen2, D-Pen5]enkephalin binding was observed in the rat brain. A number of enkephalin analogues inhibited [3H][D-Pen2, D-Pen5]enkephalin binding with high affinity (IC50 values of 0.5-5.0 nM) in both NG 108-15 and rat brain membranes. However, putative mu receptor-selective ligands such as morphine, [D-Ala2, MePhe4, Gly5-ol]enkephalin, [MePhe3, D-Pro4]morphiceptin, and naloxone were less effective inhibitors of [3H][D-Pen2, D-Pen5]enkephalin binding in both systems tested. These data suggest that [3H][D-Pen2, D-Pen5]enkephalin is a potent and selective ligand for the delta opioid receptor.

Topics: Animals; Brain; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Glioma; Hybrid Cells; Kinetics; Membranes; Mice; Neuroblastoma; Rats; Receptors, Opioid; Receptors, Opioid, delta

Binding activity of the enkephalin dimer [D-Ala2, Leu5-NH-CH2-]2 (DPE2) to NG108-15 hybrid cells was compared to that of the monomer [D-Ala2, Leu5]enkephalin amide (DALEA). At 25 degrees C, the values of the apparent affinity constant for DPE2, measured to intact and lysed cells and membranes, was 5.0 (+/- 0.09) X 10(9) M-1 for n = 28 experiments, as compared to 0.9 (+/- 0.08) X 10(9) M-1 (n = 16) for DALEA. At 4 degrees C, the binding affinity of DPE2 decreased by 43% and that of DALEA by 33%. An important difference between the binding of DPE2 and DALEA was that, after necessary corrections for difference in maximal "bindability" of the respective tritiated enkephalins, the molar binding capacity for DALEA was twofold higher than for DPE2, although mutual cross-displacement studies indicated that binding occurred to one class of noninteracting homogeneous receptors. The binding capacity for intact and lysed cells and membranes was 20 (+/- 2) X 10(-11) M for DPE2 and 43 (+/- 2) X 10(-11) M for DALEA. The enkephalin monomers [D-Ala2, D-Leu5]enkephalin (DADLE) and [D-Ala2, Met5]enkephalin amide (DAMEA) showed binding characteristics similar to those of DALEA.

Topics: Animals; Cell Line; Cell Membrane; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalin, Methionine; Glioma; Neuroblastoma; Receptors, Opioid; Tritium

The equilibrium binding and dissociation kinetics of the enkephalin dimer bis-(D-Ala2-D-Leu5-enkephalin)-ethylenediamide (designated DPE2) to neuroblastoma glioma NG108-15 cells were investigated and compared with the monomers D-Ala2-D-Leu5-enkephalin (DADL) and D-Ala2-Leu5-enkephalinamide (DALEA). Binding was studied after exposure of the membrane to increasing concentrations of the irreversible delta receptor selective ligand FIT in order to decrease the density of binding sites on the cell membrane. The increased affinity of DPE2 did not revert to that of the monomer DADL by this reduction of binding sites. Similarly, the dissociation of DPE2 did not approach that of the monomer DALEA in the presence of 1 microM DALEA. These data strongly suggest that crosslinking does not occur, and fail to confirm the hypothesis that dimers with short spanning chain length aid the clustering of receptors. We postulate: 1) If the dimer binds to a bivalent binding site, the monovalent binding state of our bivalent ligand may not exist to an appreciable extent, and 2) the bivalent ligand cannot bind when the binding site is irreversibly blocked by a monovalent ligand.

Topics: Animals; Cell Line; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Glioma; Kinetics; Models, Neurological; Neuroblastoma; Receptors, Opioid; Receptors, Opioid, delta

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

Pretreatment of intact NG108-15 cells with pertussis toxin suppresses opioid inhibition of cyclic AMP accumulation mediated by the inhibitory guanine nucleotide-binding regulatory protein, Ni, which apparently also mediates the inhibitory nucleotide effects on opioid against binding. The toxin treatment had no effect on opioid agonist binding measured in NG108-15 cell membranes without sodium present. However, the toxin potentiated the inhibitory effect of sodium on agonist binding, leading to an agonist-specific reduction of opioid receptor affinity in the presence of sodium in the binding reaction. The potency of the stable GTP analog, GTP gamma S, to reduce agonist binding in the presence of sodium was little changed in membranes prepared from pertussis toxin-treated cells compared to control membranes, whereas the potency of the stable GDP analog, GDP beta S, was magnified. The data indicate that ADP-ribosylation of Ni by pertussis toxin potentiates sodium regulation of opioid agonist binding and that the communication between Ni and opioid receptors is not lost by the covalent modification of Ni.

Topics: Animals; Bacterial Toxins; Cell Line; Diprenorphine; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Glioma; Guanine Nucleotides; Hybrid Cells; Mice; Morphinans; Neuroblastoma; Pertussis Toxin; Rats; Receptors, Opioid; Sodium; Virulence Factors, Bordetella

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: 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

The effect of exogenous substances on the expression of opiate receptors on 108CC15 neuroblastoma X glioma hybrid cells has been studied. Cell differentiation by culture in the presence of N6-O2-dibutyryl adenosine 3',5'-cyclic monophosphate induced a three fold increase in opiate receptor density. When the cells were grown in the presence of 10(-5) M morphine hydrochloride for up to 23 days, opiate receptor densities were reduced by only 30% when compared with matched controls. Culture in the presence of 10(-7) M D-Ala2-D-Leu5-enkephalin produced opiate receptor down regulation of 73% compared to controls after only 4 h of treatment. The down regulation process could be inhibited by continued exposure to D-Ala2 D-Leu5-enkephalin at concentrations greater than 4 nM; below this concentration down regulation was rapid and irreversible. A model to explain these observations is described.

Topics: Animals; Bucladesine; Cell Differentiation; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalin, Methionine; Glioma; Hybrid Cells; Mice; Morphine; Neuroblastoma; Rats; Receptors, Opioid; Receptors, Opioid, delta

Opiate receptor down-regulation in neuroblastoma X glioma NG108-15 hybrid cells possibly involved the internalization of ligand-receptor complexes during chronic treatment. However, receptor internalization was not supported by the observed decrease in [3H] enkephalin(D-Ala2,D-Leu5) ( [3H]DADLE) associated with the hybrid cells during prolonged incubation with 10 nM [3H]DADLE at 37 degrees C. This decrease in [3H]DADLE bound was determined to be due to degradation of the ligand-receptor complexes, for a time-dependent increase in [3H]DADLE bound was observed when the incubations were carried out in the presence of 0.1 mM chloroquine. The increase did not exceed the amount of down-regulated receptor, could be blocked by naloxone, and was not observed at 24 degrees C. The [3H]DADLE bound in the presence of chloroquine was not sensitive to trypsin or to 20 microM diprenorphine. The accumulated [3H]DADLE was demonstrated to be intracellularly located by the fractionation of the homogenates in self-generating Percoll gradients. In the presence of chloroquine, a time-dependent translocation of [3H]DADLE from the plasma membrane-enriched fractions to the lysosome-enriched fractions was observed. The translocation was not observed at 24 degrees C in the presence of chloroquine or at 37 degrees C in the absence of chloroquine. The [3H]DADLE in the lysosome-enriched fractions was not sensitive to trypsin and remained bound in the presence of chloroquine. With the removal of chloroquine, an increase in the release of [3H]DADLE into the medium was observed. Sephadex G-50 column chromatography of the sodium deoxycholate extracts of the lysosome-enriched fractions suggested that the [3H]DADLE was bound to macromolecules intracellularly. Thus, chronic [3H]DADLE treatment of the hybrid cells resulted in an internalization of ligand-receptor complexes which were degraded in the lysosomes. Subsequently, the [3H]DADLE was regurgitated by the hybrid cells.

Topics: Animals; Cell Line; Chloroquine; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Glioma; Hybrid Cells; Kinetics; Lysosomes; Mice; Neuroblastoma; Rats; Receptors, Opioid

Rat glioma X mouse neuroblastoma hybrid neurotumor cells (NG108-15), synchronized by amino acid deprivation, showed a cell-cycle-dependent peak of activity of a ganglioside N-acetylgalactosaminyl transferase 14-24 h following release from the cell cycle block (S/G2 phase). Maximal expression of two typical lysosomal hydrolases, N-acetyl-beta-hexosaminidase and beta-galactosidase, occurred between 18 and 21 h following release (S phase), declining to G1 phase levels during the peak of N-acetylgalactosamine (GalNAc) transferase activity. In addition, glycosyltransferase activity in G2 phase cells showed an increase in apparent Vmax (suggesting the presence of more enzyme/mg of cell protein) and apparent binding affinity for uridine diphosphate N-acetylgalactosamine (UDP-GalNAc) (32 versus 14 microM) when compared to transferase activity in the G1 phase. However, the opioid peptide enkephalin [D-Ala2, D-Leu5], which inhibits ganglioside GalNAc transferase activity in unsynchronized NG108-15 cultures, was much more inhibitory in whole cells 8 h after release from the cell cycle block (G1 phase) than in cells 20 h after release (G2 phase), with 50% inhibition occurring at 2 X 10(-9) M and 2 X 10(-7) M, respectively. These results suggest that the GalNAc transferase activity is regulated in more than one way during the cell cycle, since both Vmax and Km changes are observed, and that the cyclic AMP-dependent mechanism by which opiates reduce transferase activity is receptor mediated and cell cycle dependent.

Topics: Animals; Cell Cycle; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; G(M2) Ganglioside; G(M3) Ganglioside; Galactosyltransferases; Gangliosides; Glioma; Hybrid Cells; Kinetics; Mice; N-Acetylgalactosaminyltransferases; Neuroblastoma; Rats

This study reports on the molecular mechanism of delta-opiate receptor down regulation on 108CC15 neuroblastoma X glioma hybrid cells. The down regulation induced by culture in the presence of 10(-5) M 2-D-Ala, 5-D-Leu-enkephalin (DADLE) can be prevented by continued exposure to ligand concentrations greater than 4 nM, the Kd of the binding site. Below this concentration, down regulation is a rapid and irreversible process. It is deduced that the internalization process in this cell line is initiated when unoccupied receptor dimers are present. These results have important implications for down regulation studies using cultured cell lines and studies of receptor regulation in vivo after chronic treatment with neuroactive drugs.

Topics: Animals; Cell Line; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalin, Methionine; Glioma; Hybrid Cells; Kinetics; Mice; Neuroblastoma; Rats; Receptors, Opioid; Receptors, Opioid, delta