Glioma Hybrid NG108-15 Cells Research Articles

Cyanide sensitive and insensitive bioenergetics in a clonal neuroblastoma x glioma hybrid cell line

The primary mechanism of cyanide (CN) intoxication is the inhibition of metabolism in the central nervous system. We determined the effects of CN on several biochemical processes in neuroblastoma x glioma hybrid NG108-15 cells, which possess numerous neuronal properties. These cells were not sensitive to a high concentration (1 mM) of NaCN, but became sensitive in the presence of the anaerobic glycolysis inhibitors sodium iodoacetate (IA) and 2-deoxyglucose (2-DG):cellular metabolic processes (e.g., DNA, RNA and protein synthesis) decreased to about 40% of control due to treatment with 0.5 mM NaCN + 0.05 mM IA and 0.1 mM NaCN + 20 mM 2-DG. ATP in cells exposed to 0.01 or 0.1 mM NaCN + 20 mM 2-DG was reduced 75% and 100% respectively within one min. Pretreatment of cells with the CN antidote cobalt (II) chloride (CoCl2) (0.06-0.18 mM) for 5 min prevented the depression of both [3H]leucine incorporation and ATP synthesis due to 1 mM NaCN + 20 mM 2-DG in a concentration-dependent manner. A proposed CN antidote alpha-ketoglutaric acid (disodium salt) also prevented the depression of cellular metabolism due to NaCN plus 2-DG. These results indicate that blocking anaerobic glycolysis makes NG108-15 cells sensitive to a low concentration of CN. Thus NG108-15 cells should be useful to study the mechanisms of neurotoxicity of CN and to test antidotes.

Cholera toxin impairment of opioid-mediated inhibition of adenylate cyclase in neuroblastoma x glioma hybrid cells is due to a toxin-induced decrease in opioid receptor levels.

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.

Entry and replication of Japanese encephalitis virus in cultured neurogenic cells

The entry mode and growth pattern of Japanese encephalitis (JE) virus in mouse neuroblastoma N18TG2 cells and mouse neuroblastoma x rat glioma NG108-15 hybrid cells were studied by electron microscopy. At two minutes after inoculation, JE virions adsorbed onto and directly penetrated through the plasma membrane of the hybrid cells, whereas virions did not adsorb nor entered the neuroblastoma cells. Correspondingly, the hybrid cells showed assembling progeny JE virions in the cistemae of rough endoplasmic reticulum (RER) 1 day postinoculation (p.i.) although virions were rarely found on the following days during the experiment. On the other hand, progeny virions did not assemble in the RER cisternae of the neuroblastoma cells throughout the experiment. The morphologic observations, therefore, suggest that (a) the hybrid cells express JE-virus receptors which facilitate the viral attachment onto and entry into the cells, while the neuroblastoma cells do not and (b) JE virus replicates very poorly after the entry into the hybrid cells while it does not replicate at all in the neuroblastoma cells. The virus titrations of the media of the neuroblastoma and hybrid cell cultures showed only titers indicative of residual virus of the inoculum that progressively decreased during the experiment. The present results show therefore that of the two neurogenic cell culture lines studied only the hybrid cell line can be used for the study of viral entry and replication, although it is not suited for virus production. Possible reasons for the poor replication of JE virus in the hybrid cells are discussed.

The α 2 B adrenergic receptor of undifferentiated neuroblastoma × glioma hybrid NG108-15 cells, interacts directly with the guanine nucleotide binding protein, G i2

In membranes of undifferentiated neuroblastoma × glioma hybrid cell line NG108-15, the apparent specific binding of [ 3H]yohimbine measured in the presence of 1 μM noradrenaline, was increased substantially by the presence of the poorly hydrolysed analogue of QTP, guanylyl-imidodiphosphate (Gpp[NH]p) or by preincubation of membranes with antibodies against the C-terminal decapeptide of the α subunit of the G-protein G i2. Such an effect was not produced by antibodies against the equivalent region of G oα G i3α or G sα or from non-immune serum. By contrast, total specific binding of [ 3H]yohimbine was not modified by co-incubation with Gpp[NH]p or by preincubation with the antibodies from any of the anti-G protein antisera. These results demonstrate a direct interaction of the α 2B adrenergic receptor of NG108-15 cells with G i2.

Bradykinin-evoked acetylcholine release via inositol trisphosphate-dependent elevation in free calcium in neuroblastoma x glioma hybrid NG108-15 cells.

The mechanism underlying the bradykinin (BK)-induced increase of acetylcholine (ACh) release was studied in neuroblastoma x glioma hybrid NG108-15 cells and their synapses formed onto mouse muscle cells. External application of BK or iontophoretic injection of extrinsic inositol 1,4,5-trisphosphate (InsP3) into the cytoplasm of NG108-15 cells produced membrane hyperpolarization in the hybrid cells and an increase in the frequency of miniature end-plate potentials (MEPPs) in paired myotubes. Ba2+ blocked the hyperpolarization in response to BK, but facilitation of MEPPs was still observed. InsP3-dependent facilitation of MEPPs was also observed in cells where the InsP3 injections produced no detectable hyperpolarization or even depolarization. Real-time quantitative monitoring of intracellular free Ca2+ concentration [( Ca2+]i) with fura-2 in single NG108-15 cells showed that BK application or InsP3 injection induced an elevation of [Ca2+]i which coincided in time with membrane hyperpolarization recorded from the same cell. The [Ca2+]i rise produced by InsP3 injection started from the single site of injection and that produced by BK began from a deep compartment of the cytoplasm of the NG108-15 cells. The BK- and InsP3-evoked facilitation of MEPPs and the [Ca2+]i rise were relatively independent of extracellular Ca2+. These findings suggest that the BK-induced ACh release results not from membrane potential changes but from a transient InsP3-dependent elevation of [Ca2+]i.

Bradykinin induces inositol 1,4,5‐trisphosphate‐dependent hyperpolarization in K+ M‐current‐deficient hybrid NL308 cells: Comparison with NG108–15 neuroblastoma × glioma hybrid cells

External application of bradykinin (BK) to mouse neuroblastoma X mouse fibroblast hybrid NL308 cells and mouse neuroblastoma X rat glioma hybrid NG108-15 cells produced a transient outward (hyperpolarizing) current. In NG108-15 cells, BK also induced an inward (depolarizing) current associated with a decrease in input membrane conductance, which results from the inhibition of a voltage-sensitive potassium current, the M-current. However, in NL308 cells, either no depolarization was elicited by BK or, even if the BK-induced depolarization was evoked, it was associated with an increased conductance. To explain the above difference, the intracellular second messenger system of NL308 cells was examined in detail. BK induced the rapid accumulation (three- to fivefold higher than the control level) of inositol 1,4,5-trisphosphate (InsP3) in NL308 cells. The cytosolic Ca2+ concentration was also elevated to 540 nM from 180 nM at a basal level. This seems to be enough to activate a voltage-independent and Ca2(+)-sensitive K+ current, resulting in the hyperpolarization. Intracellular injection of InsP3 replicated the hyperpolarization. NL308 cells possess protein kinase C (C-kinase), with specific activities of C-kinase in cytosolic and membrane fractions being 233 and 24 pmol/min/mg protein, respectively. The activity associated with particulates became higher after phorbol dibutyrate (PDBu) treatment. But NL308 cells did not show the characteristic inward relaxation by step hyperpolarizations and the outward rectification in the current-voltage relationship, indicating that the M current is deficient in NL308 cells. Therefore, application of PDBu failed to mimic the inward current. The results suggest the role of InsP3 and C-kinase in controlling two K+ currents.

Cholinergic synapse formation between NG108-15 and muscle cells and modulation of transmission

Mouse neuroblastoma x rat glioma hybrid NG108-15 cells form cholinergic synapses with rat or mouse muscle cells in culture. The rate of synapse formation is greatly dependent on intracellular cyclic AMP concentrations. The synapse formation is lower in the presence of glia maturation factor, a partially purified brain extract. Once the synapse between NG108-15 cells and myotubes has been formed, this synapse is stable for days. Extracellular application of serotonin, PGF2 alpha, PGD2, neurotensin and bradykinin on NG108-15 cells increases synaptic transmission. Since bradykinin increases the level of intracellular inositol 1,4,5-trisphosphate (InsP3), bradykinin-induced facilitation is due to InsP3-dependent elevation of intracellular Ca concentrations.

Evidence for a GTP-dependent increase in membrane permeability for calcium in NG108-15 microsomes

The effect of GTP on Ca 2+ uptake and release was studied in a microsomal fraction isolated from neuroblastoma x glioma hybrid NG108-15 cells. GTP did not alter the ATP-dependent initial uptake of Ca 2+ but markedly enhanced the efflux of Ca 2+ from microsomes. GTP-dependent Ca 2+ release requires the presence of millimolar concentration of Mg 2+. The effect of GTP was not mimicked by other nucleotides and was competitively blocked by the thiophosphate analogue of GTP, GTPγS but not by the non-hydrolyzable nucleotide GMP-PNP. Addition of an inhibiting concentration of GTPγS after completion of GTP-induced calcium release did not result in a re-uptake of Ca 2+, showing the irreversibility of the releasing effect of GTP. Our data are consistent with the hypothesis of Ca 2+-dependent GTP-induced opening of a channel responsible for vectorial transport of Ca 2+ ions from one intracellular compartment to another. A model is proposed suggesting that the GTP-binding protein is a GTP-specific diacylglycerol kinase.

The GTP‐Binding Proteins, Go and Gi2, of Neural Cloned Cells and Their Changes During Differentiation

The levels of the alpha-subunits of two GTP-binding proteins, Go and Gi2, were determined in neural and nonneural cloned cells by immunoassays. Go alpha was detected in all neural cells and some of nonneural cells, but not in HL-60 leukemic cells and PYS-2 teratocarcinoma-derived cells. The level of Go alpha was highest in the PC12 pheochromocytoma cells. Gi2 alpha was present in all types of cells tested, and its level was highest in the HL-60 cells and relatively high in glioma cells. Treatment of PC12 cells and neuroblastoma x glioma hybrid NG108-15 cells with nerve growth factor and forskolin, respectively, caused the extension of neuronal-like processes and increase in the level of Go alpha by 60-80%, but small changes in the levels of Gi2 alpha.

Modification of the indolamine content in neuroblastoma x glioma hybrid NG108-15 cells upon induced differentiation.

1. The neuroblastoma x glioma hybrid NG108-15 cell line has been widely studied as a neuronal model for its serotonergic, cholinergic, and peptidergic properties. 2. The catecholamine and serotonin content and that of their major metabolites have been determined by high-performance liquid chromatography with electrochemical detection (HPLC-EC) in NG108-15 cells under differentiated and undifferentiated conditions. 3. Cellular contents of L-DOPA, norepinephrine, (NE), L-epinephrine (EPI), and dopamine (DA) in differentiated cells, induced by 1 mM dibutyryl cyclic AMP (dBcAMP), are 149, 40, 129, and 124%, respectively, higher than those in undifferentiated cells. 4. 3,4-Dihydroxyphenethylacetic acid (DOPAC), the major metabolite of DA, is detectable only in differentiated cells. Similarly, DOPAC is present only in culture medium from differentiated cells, and not that of undifferentiated cells. 5. Serotonin (5-HT) is detectable only in undifferentiated cells; and the level of 5-hydroxyindoleacetic acid (5-HIAA), the major metabolite of 5-HT, is also 12.7% higher is undifferentiated cells. 6. Comparative analyses of differentiated and undifferentiated cells in monolayer cultures and undifferentiated cells cultured in the presence of 1 mM dBcAMP under suspension conditions suggest that change in the indolamine content is due to cellular changes upon morphological differentiation. 7. The clonal NG108-15 cell line is also catecholaminergic, in addition to cholinergic and serotonergic; and a shift of neurotransmitter pattern from serotonin to dopamine production occurs during morphological differentiation.

Characterization of muscarinic acetylcholine receptors on intact neuroblastoma � glioma NG108-15 cell upon induced differentiation

Studies have established that major increases in muscarinic acetylcholine receptor (mAchR) binding in the brain appear to coincide with synaptogenesis. The neuroblastoma x glioma hybrid NG108-15 cell line has been demonstrated to possess numerous functional characteristics of intact neurons, including synapse formation with myotubes. The present study examines and characterizes the mAchR on the hybrid NG108-15 cells during differentiation, induced by 1 mM dBcAMP. Specific binding of [3H]-QNB for differentiated cells increases gradually to a final level of 130% (P less than 0.05) over the control undifferentiated cells during the first 24 hr of incubation. Further, this increase of receptor sites appears to correlate proportionately to the degree of neurite extension of the differentiating cells. The dissociation rate constant at equilibrium (Kd) and maximum binding capacity (Bmax) have been determined to be 5.6 nM and 920 fmol/10(6) cells, respectively, for differentiated cells, and 4.4 nM and 400 fmol/10(6) cells, respectively, for undifferentiated cells. Computer analyses of the data obtained from saturation experiments reveal a single class of binding sites for [3H]-QNB on both differentiated and undifferentiated cells. The Hill plot analysis of the QNB-binding indicates a Hill coefficient (nH) of 1.0 and 0.91 for differentiated and undifferentiated cells, respectively, suggesting the unity of receptor sites with no co-operativity. Our results depict that increases of mAchRs on intact cells correlate with the degree of cellular differentiation.

Mechanism of Agonist‐Induced Down‐Regulation and Subsequent Recovery of Muscarinic Acetylcholine Receptors in a Clonal Neuroblastoma X Glioma Hybrid Cell Line

The mechanisms of carbachol-induced muscarinic acetylcholine receptor (mAChR) down-regulation, and recovery following carbachol withdrawal, were studied in the neuroblastoma x glioma hybrid NG108-15 cell line by specific ligand binding assays. N-[3H]Methylscopolamine ([3H]NMS) and [3H]quinuclidinyl benzilate ([3H]QNB) were used as the ligands for the cell surface and total cellular mAChRs, respectively. Exposure of cells to 1 mM carbachol for 16 h decreased the specific binding of [3H]NMS and [3H]QNB by approximately 80%. Bacitracin (1-4 mg/ml) and methylamine (1-15 mM), inhibitors of transglutaminase and of endocytosis, prevented agonist-induced loss of surface mAChRs. Pretreatment of cells with the antimicrotubular agents nocodazole (0.1-10 microM) and colchicine (1-10 microM) prevented carbachol-induced loss of [3H]QNB binding, but not that of [3H]NMS binding. These results indicate that agonist-induced mAChR down-regulation occurs by endocytosis, followed by microtubular transport of receptors to their intracellular degradation sites. When carbachol was withdrawn from the culture medium following treatment of cells for 16 h, receptors recovered and were incorporated to the surface membrane. This recovery process was antagonized by monovalent ionophores monensin (0.1 microM) and nigericin (40 nM), which interfere with Golgi complex function. Receptor recovery was also prevented by the antimicrotubular agent nocodazole. Thus, recovery of receptors appears to be mediated via Golgi complex and microtubular transport to the surface membrane.

Rebound increase of basal cAMP level in NG108-15 cells during chronic morphine treatment: Effects of naloxone and chloramphenicol

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

Modification of opioid receptor activity by acid phosphatase in neuroblastoma X glioma NG108-15 hybrid cells

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.

A novel enhancement of SV40 enhancer activity by treatment of mouse neuroblastoma N18TG2 with protein kinase inhibitor H-7.

To determine the role of protein phosphorylation in transcription regulation, we have treated mouse neuroblastoma N18TG2 cells with the protein kinase inhibitor H-7 and tested its effect on transcription. After the preculture and transfection in the presence of H-7, the cell preparation was divided in half and cultured with and without H-7. The level of CAT expression of pSV2-CAT was found to be higher in the cells cultured in the absence of H-7 than in those cultured in the presence of H-7. This difference was observed only after pretreatment of the cells with H-7, suggesting that withdrawal of H-7 from the culture medium after preculture with H-7 gave an enhancing effect on CAT expression. This phenomenon was also observed with transformants that expressed the CAT gene of pSV2-CAT stably. The 72 base-pair (bp) repeat of SV40 DNA was responsible for this difference in CAT expression. A similar effect of H-7 on the SV40 enhancer activity was observed in mouse neuroblastoma x rat glioma hybrid NG108-15 cells, but not in rat glioma C6-BU-1 cells.

Role of a protein regulating guanine nucleotide binding in phosphoinositide breakdown and calcium mobilization by bradykinin in neuroblastoma × glioma hybrid NG108-15 cells: Effects of pertussis toxin and cholera toxin on receptor-mediated signal transduction

The addition of bradykinin to NG108-15 cells resulted in an increase in the intracellular Ca 2+ concentration ([Ca 2+] i) and the formation of inositol monophosphate, inositol biphosphate, and inositol trisphosphate in these cells. The bradykinin-stimulated formation of inositol polyphosphates in plasma membrane preparations was dependent on the presence of GTP or guanosine-5′-O-thiotriphosphate (GTPγS) but not of GDP. GTPγS, unlike GTP, increased the basal formation of inositol polyphosphate in NG108-15 membranes. Iontophoretic injection of GTPγS into single cells induced increases in [Ca 2+] i. These effects of bradykinin and GTPγS on [Ca 2+] i and the formation of inositol phosphates in the intact cells and membranes were not affected by treatment of the cells with pertussis toxin or cholera toxin. Data on binding of bradykinin to membrane preparations indicated the presence of two classes of binding sites with K d values of 0.80 ± 0.26 and 9.63 ± 0.13 nM. Approximately 74% of the receptors were in the high affinity state. In the presence of guanyl-5′-yl-imidodiphosphate [Gpp(NH)p], the high affinity sites in the membrane preparations were converted to low affinity sites with no change in the total receptor number. These toxin treatments had no effect on binding of bradykinin to its receptors. Thus. these results indicate that a guanine nucleotide regulatory protein, which is not a substrate of pertussis toxin or cholera toxin, is involved in mediating the effects of bradykinin on membrane-bound phosphoinositide-specific phospholipase C to induce the increase of cytosolic calcium.

Endogenous benzodiazepine-like molecules in the human, rat and bovine brains studied with a monoclonal antibody to benzodiazepines

The anti-benzodiazepine monoclonal antibody 21-7F9 has been used for the identification and study of endogenous benzodiazepine-like molecules in the human, rat and bovine brains. A sandwich radioimmunoassay has been designed for the quantification of the membrane-bound endogenous benzodiazepine-like molecules. The localization of these molecules is not restricted to the brain tissue. They are also present in kidney, liver and spleen as well as in the neuroblastoma × glioma NG108-15 hybrid cells line. Immunoblots show benzodiazepine-like immunoreactivity in the membrane proteins of all these tissues. The membrane-bound benzodiazepine-like molecules are resistant to limited proteolysis of the membranes. Moreover, this treatment increases the binding of the monoclonal antibody 21-7F9 to the membranes, probably by exposing sites that normally are not accessible to the antibody. Immunocyto experiments show that benzodiazepine-like molecules are also present in samples of human cerebella that have been stored in paraffin since 1940, 15 years before the first chemical synthesis of benzodiazepines. The results indicate that the cerebellar benzodiazepine-like molecules recognized by the antibody are the product of biological (not chemical) synthesis. Benzodiazepine-like immunoreactivity has also been detected in NG108-15 cells that have been cultured for 3 months ib serum-free medium. These results suggest that the cells could biosynthesize benzodiazepine-like molecules.

Membrane current responses to intracellular injections of inositol 1,3,4,5-tetrakisphosphate and inositol 1,3,4-trisphosphate in NG108-15 hybrid cells

Iontophoretic injections of inositol 1,4,5-trisphosphate inside neuroblastoma × glioma NG108-15 hybrid cells evoked an outward K + current across the outer cell membrane, probably activated by the release of intracellular Ca 2+. No such current was produced by equivalent intracellular injections of inositol 1,3,4-trisphosphate or inositol 1,3,4,5-tetrakisphosphate. Instead, these compounds evoked an inward current with a reversal potential of about −20 mV, and which may therefore be due to a non-specific cation conductance. This suggests that these derivatives are unable to release sufficient Ca 2+ to activate the Ca 2+-dependent K + current in these cells.

Effect of phospholipases on chronic opiate action in neuroblastoma X glioma NG108-15 hybrid cells.

Chronic treatment of neuroblastoma X glioma NG108-15 hybrid cells with opiate agonist resulted in loss of the acute opiate inhibition of adenylate cyclase activity with a concomitant increase in the enzymatic activity observable on addition of the antagonist naloxone. The role of membrane lipids in the cellular expression of these chronic opiate effects was investigated by the hydrolysis of phospholipids with various lipases. Treatment with phospholipase C from Clostridium welchii produced an enzyme concentration-dependent decrease of prostaglandin E1-stimulated adenylate cyclase activity in control or etorphine-treated (1 microM for 4 h) hybrid cells. In addition, incubation of hybrid cells with phospholipase C concentrations of greater than or equal to 0.5 U/ml completely abolished the compensatory increase in adenylate cyclase activity after chronic opiate treatment. This attenuation of the increase in adenylate cyclase activity by phospholipase C could be prevented by inclusion of phosphatidylcholine but not of phosphatidic acid during the enzymatic incubations. The specificity of the phospholipids involved in expression of the chronic opiate effect could be demonstrated further by the absence of effect exhibited by phospholipase C from Bacillus cereus and phospholipase D. Hydrolysis of the acyl side chains of phospholipids with phospholipase A2 did not alter the chronic opiate effect after removal of lysophosphatides with bovine serum albumin. Because the guanylylimidodiphosphate- and NaF-sensitive adenylate cyclase activities were not affected by these phospholipase treatments, the expression of the compensatory increase in adenylate cyclase activity is mediated via an increase in the coupling between hormonal receptor and adenylate cyclase with the participation of the polar head groups of the phospholipids and not the hydrophobic side chains.

An antibody to a peptide specified by an RNA that is complementary to γ-endorphin mRNA recognizes an opiate receptor

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