Glioma Hybrid NG108-15 Cells Research Articles

Cell-free desensitization of opioid inhibition of adenylate cyclase in neuroblastoma X glioma NG108-15 hybrid cell membranes.

When membranes from neuroblastoma X glioma NG108-15 hybrid cells were incubated in a cell-free system with opioid agonists, a time-, temperature-, and dose-dependent desensitization to opioid inhibition of adenylate cyclase activity was observed. The composition of the system during the incubation was manipulated to elucidate the biochemical mechanisms of desensitization. Receptor coupling appeared to be a prerequisite for desensitization, because both magnesium and sodium, which are necessary for coupling, were required for desensitization. Removal of ATP and addition of cyclic AMP or cyclic GMP had no effect on desensitization.

1-oleoyl-2-acetyl-glycerol and phorbol diester stimulate Ca 2+ influx through Ca 2+ channels in neuroblastoma × glioma hybrid NG108-15 cells

The effect of 1-oleoyl-2-acetyl-glycerol (OAG) and the phorbol diester 12-O-tetradecanoyl-phorbol-acetate (TPA) on the intracellular Ca 2+ concentration ([Ca 2+]i) in NG108-15 cells was studied using a Ca 2+ indicator, quin 2. OAG and TPA induced an increase in [Ca 2+]i from 100 ± 19 to 187 ± 24 nM and 192 ± 15 nM, respectively, within 15 min. The increase in [Ca 2+]i induced by activators of protein kinase C was dependent on the extracellular Ca 2+ concentration ([Ca 2+]o) and was inhibited by the Ca 2+ blockers, verapamil and nifedipine. These results indicate that the OAG- and TPA-induced [Ca 2+]i increase is mediated by the influx of extracellular Ca 2+ through voltage-sensitive Ca 2+ channels.

Bradykinin-induced intracellular Ca 2+ elevation in neuroblastoma × glioma hybrid NG108-15 cells; Relationship to the action of inositol phospholipids metabolites

The effect of bradykinin on the intracellular Ca 2+ concentration ([Ca 2+] i) in NG108-15 cells was studied using a Ca 2+ indicator quin 2. Bradykinin induced two phases of change in [Ca 2+] i. Bradykinin induced a spike phase of [Ca 2+] i increase which was detectable within 15 s and decayed to near-basal concentration in 3 min and then a prolonged plateau phase of [Ca 2+] i increase which continued for 15 min. The bradykinin-induced spike phase was not diminished by decreasing extracellular Ca 2+ concentration ([Ca 2+] o) to 1 μM. On the contrary, the plateau phase was dependent on [Ca 2+] o and inhibited by Ca 2+ blockers, verapamil (50 μM), nifedipine (1 μM). The iontophoretic injection of inositol-trisphosphate (IP3) into the single cell induced the increase of [Ca 2+] i, which was independent of [Ca 2+] o. These results indicate that the bradykinin-induced spike phase is mediated by the release of intracellular Ca 2+ stores induced by IP3, while the plateau phase is mediated by influx of extracellular Ca 2+ probably through voltage-sensitive Ca 2+ channels.

Ethanol increases the expression of functional delta-opioid receptors in neuroblastoma x glioma NG108-15 hybrid cells.

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.

Neuroblastoma X glioma NG108-15 hybrid cells cultured in a serum-free chemically defined medium: effects on acute and chronic opiate regulation of adenylate cyclase activity

Neuroblastoma X glioma NG108-15 hybrid cells cultured in a chemically defined medium within 3 cell passages, exhibited viability, growth rate and morphology similar to those of cells grown in medium supplemented with 5% fetal calf serum. Hybrid cells cultured in the chemically defined medium within these periods of time also did not exhibit a difference in basal adenylate cyclase activity nor in the enzymatic activities stimulated by adenosine, forskolin, NaF, GppNHp or Mn 2+. Furthermore, opiate receptor density in chemically defined medium cultured cells remained identical to that in cells cultured in 5% fetal calf serum. The acute and chronic effects of opiates on adenylate cyclase were similar for cells grown under either set of conditions.

Effect of pertussis toxin treatment on the down-regulation of opiate receptors in neuroblastoma X glioma NG108-15 hybrid cells.

Chronic treatment of neuroblastoma X glioma NG108-15 hybrid cells with 10 nM [D-Ala2,D-Leu5] enkephalin (DADLE) results in a reduction of cell-surface opiate delta receptors. Whether opiate receptor internalization requires the activation of the guanine nucleotide-binding protein (Ni) is unclear. Hence, activation of Ni was attenuated by treating hybrid cells with 100 ng/ml pertussis toxin (PT) for 3 h, which resulted in a decrease in DADLE's ability to inhibit adenylate cyclase activity. Despite this prior treatment with PT, chronic exposure of these cells to 10 nM DADLE resulted in a time-dependent decrease in both [3H]diprenorphine and [3H]DADLE binding. This reduction in 3H-ligand binding in cells previously treated with PT represented internalization of the receptors because translocation was observed of bound [3H]DADLE from plasma membrane fractions to the lysosomal fractions in the Percoll gradients. Thus, opiate receptors internalize without activation of Ni. The internalization of opiate receptors was not accompanied by Ni. By measuring the amount of the 41-kDa alpha subunit being labeled by PT with [32P]NAD+, it was determined that plasma membrane preparations, of both the control cells and cells treated with 10 nM of DADLE for 4 h, contained equal concentrations of Ni, 2 pmol of Ni/mg of protein. Additionally, there was no measurable alteration in the amount of PT substrate in the lysosomal fractions of the DADLE-treated cells as compared to that of control cells. Chronic DADLE treatment resulted in a decrease in Km value of NAD+ in the ADP-ribosylation of 41-kDa subunit of Ni. In summary, opiate receptors internalize as agonist-receptor complexes without the guanine nucleotide-binding component.

Dual pathways of receptor-mediated cyclic GMP generation in NG108-15 cells as differentiated by susceptibility to islet-activating protein, pertussis toxin

The cellular cGMP content increased in response to a variety of receptor agonists, which activate [e.g., prostaglandin (PG) E 1, E 2, and F 2α or inhibit (e.g., α-adrenergic, muscarinic, and opiate agonists) adenylate cyclase in neuroblastoma X glioma hybrid NG108-15 cells. The responses were additive when PGF 2α and enkephalin were mixed. The inhibitory guanine nucleotide regulatory protein (N i) is involved in adenylate cyclase inhibition; this function of N i is lost when it is ADP-ribosylated by islet-activating protein (IAP), pertussis toxin [ H. Kurose, T. Katada, T. Amano, and M. Ui (1983) J. Biol. Chem. 258, 4870–4875 ]. The cGMP rise induced by stimulation of the receptors linked to adenylate cyclase inhibition was also diminished by IAP; the time course and dose response for the IAP-induced diminution were the same between adenylate cyclase inhibition and cGMP generation. N i thus appears to mediate guanylate cyclase activation as well as adenylate cyclase inhibition initiated via the same receptors. Melittin also increased cGMP. No additivity was shown when enkephalin and melittin were combined, suggesting that phospholipase A 2 might play a role in N i-mediated guanylate cyclase activation. On the other hand, the PGF 2α-induced cGMP rise was associated with increased incorporation of 32 P i into phosphatidylinositol; was not affected by cholera toxin, IAP or forskolin; and showed no additivity when combined with A23187, which increased cGMP by itself. PGs would occupy receptors linked to phosphatidylinositol breakdown, thereby increasing the availability of intracellular Ca 2+, which is responsible for guanylate cyclase activation. Thus, dual pathways are proposed for a receptor-mediated cGMP rise in NG108-15 cells.

Evidence for a Ca 2+-independent hydrolysis of phosphatidylinositol 4,5-bisphosphate in neuron-like cell line NG108-15 cells

The addition of bradykinin to 32P i-labeled neuroblastoma × glioma hybrid NG108-15 cells caused a substantial loss of radioactivity from phosphatidylinositol 4,5-bisphosphate (PI-4,5-P 2). The bradykinin-induced hydrolysis of PI-4,5-P 2 was almost equally observed even when extracellular Ca 2+ was depleted with EGTA (100μM). On the other hand, high K + depolarization of the cells, which allows Ca 2+ influx through voltage-dependent Ca 2+ channels, failed to induce any significant decrease in the radioactivity of PI-4,5-P 2. These data indicate that the bradykinin-stimulated PI-4,5-P 2 hydrolysis in NG108-15 cells is independent of extracellular Ca 2+ and also that PI-4,5-P 2 hydrolysis is not stimulated by an elevation of intracellular Ca 2+ concentration.

Multiple affinity states of opiate receptor in neuroblastoma x glioma NG108-15 hybrid cells. Opiate agonist association rate is a function of receptor occupancy.

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)

Bradykinin-induced transient accumulation of inositol trisphosphate in neuron-like cell line NG 108-15 cells

Studies were undertaken to further elucidate the mechanism(s) by which bradykinin-dependent phosphoinositide metabolism takes place in neuroblastoma × glioma hybrid NG108-15 cells [(1984) J. Biol. Chem. 259, 10201–10207] using [ 3H]inositol-labelled cells. Bradykinin produced net increases in the level of [ 3H]inositol phosphates, especially of [ 3H]inositol trisphosphate which is formed transiently and most rapidly. The results indicate that bradykinin activates a phosphodiesterase to break down phosphatidylinositol 4,5-bisphosphate, generating two recently recognized intracellular messengers, 1,2-diacylglycerol and inositol trisphosphate.

Effects of cycloheximide and tunicamycin on opiate receptor activities in neuroblastoma X glioma NG108-15 hybrid cells

The molecular mechanism of opiate receptor down-regulation and desensitization was investigated by studying the effects of cycloheximide and tunicamycin on opiate receptor activities in neuroblastoma X glioma NG108-15 hybrid cells. Cycloheximide inhibited [ 35S]methionine and [ 3H]-glucosamine incorporation by hybrid cells, while tunicamycin inhibited [ 3H]glucosamine incorporation only. Exposing hybrid cells to these two agents did not alter the viability of the cell. Treatment of NG108-15 cells with cycloheximide or tunicamycin produced a decrease in [ 3H]diprenorphine binding dependent on both time and concentrations of inhibitors, with no measurable modification in the ability of etorphine to regulate intracellular cyclic AMP production. Cycloheximide attenuated [ 3H]-diprenorphine binding by decreasing both the number of sites, B max, and the affinity of the receptor, K d . Tunicamycin treatment produced a decrease in B max with no apparent alteration in K d values. Cycloheximide and tunicamycin did not potentiate the rate or magnitude of etorphine-induced down-regulation or desensitization of opiate receptor in NG108-15 cells. Furthermore, there was an apparent antagonism in cycloheximide action on receptor down-regulation. The reappearance of opiate binding sites after agonist removal was affected by these two inhibitors. Cycloheximide and tunicamycin eliminated the increase in [ 3H]diprenorphine binding in the chronic etorphine-treated cells after agonist removal. These two inhibitors did not alter the resensitization of hybrid cells to etorphine. Thus, the site of opiate agonist action to induce receptor down-regulation and desensitization is not at the site of protein synthesis or protein glycosylation. These data substantiate previously reported observations that receptor down-regulation and receptor desensitization are two different cellular adaptation processes.

Bradykinin-induced rapid breakdown of phosphatidylinositol 4,5-bisphosphate in neuroblastoma X glioma hybrid NG108-15 cells.

External application of bradykinin to neuroblastoma X glioma hybrid NG108-15 cells produced a sustained depolarization preceded by a transient hyperpolarization. Bradykinin also increased the frequency of miniature end-plate potentials recorded from cultured striated muscle cells which had been innervated by NG108-15 cells. Parallelism between facilitative phases of miniature end-plate potentials and depolarization indicates that bradykinin caused an enhanced synaptic transmission from NG108-15 cells due to depolarization. Effects of bradykinin on phospholipid metabolism in the hybrid cells were then examined to shed light upon the mechanism by which bradykinin-receptor interaction leads to facilitation of synaptic transmission. Bradykinin induced specific incorporation of 32Pi into phosphatidic acid and phosphatidylinositol without affecting [3H]glycerol incorporation into these phospholipids by 10 min after its addition. The addition of bradykinin to hybrid cells prelabeled with 32Pi caused a transient decrease (maximal effect seen at 10-30 s) in the radioactivity from phosphatidylinositol 4,5-bisphosphate (PI-4,5-P2) which was followed by the accumulation of radioactivity in phosphatidic acid and phosphatidylinositol. A Ca2+ ionophore, A23187, failed to induce the initial degradation of PI-4,5-P2. The data show that the magnitudes of bradykinin-induced PI-4,5-P2 degradation and membrane potential changes in NG108-15 cells are both dependent on the concentration of bradykinin and that the degradation of PI-4,5-P2 precedes the electrophysiological responses. Taken together with the finding that bradykinin induced a transient increase in Ca2+ influx (at 10-20 s), it appears that a rapid and transient degradation of PI-4,5-P2 might be related to the initiation of the NG108-15 cell activities through mobilization of extracellular Ca2+ into the cells.

β-Endorphin: Evidence for the existence of opioid and non-opioid binding components for the tritiated human hormone in NG108-15 cells

Human β-endorphin (β h-EP) binding on neuroblastoma x glioma hybrid NG108-15 cells using tritiated human beta endorphin ( 3H-β h-EP) as a primary ligand was found to have a component which was not displacable with [D-Ser 2]-Leu-enkephalin-Thr 6 (DSLET). The β h-EP binding on these cells after saturation of the δ opiate sites with 200 nM DSLET was further characterized with synthetic β h-EP analogs. the non-opioid binding site appears to recognize β h-EP-(6–31), β h-EP-(21–31) and β h-EP-(28–31). Under these conditions, these COOH-terminal segments fully displace the tritiated β h-EP. However, β h-EP-(1–27) does not further displace 3H-β h-EP in the presence of DSLET. The fact that a combination of DSLET and β h-EP-(6–31) results in a full displacement of 3H-β h-EP provides direct evidence for the existence of two binding sites for β h-EP in NG108-15 cells, one recognizing the NH 2-terminal enkephalin sequence and the other the non-opioid COOH-terminal segment.

A single class of neurotensin receptors with high affinity in neuroblastoma X glioma NG108-15 hybrid cells that mediate facilitation of synaptic transmission

Receptor binding of [3H]neurotensin was examined on membrane preparations derived from neuroblastoma X glioma NG108-15 hybrid cells. The specific binding was saturable and reversible, and a dissociation constant (Kd) was calculated to be about 0.24 nM from the rate constants. Scatchard analysis of neurotensin binding at equilibrium revealed a single class of binding sites with a Kd of 0.86 nM and a maximal binding capacity (Bmax) of 250 fmol/mg of protein (7700 receptor sites/cell). [D-Arg9]-Neurotensin had a high affinity (IC50 = 0.5 nM) for the neurotensin receptors, but [D-Phe11]-neurotensin had a lower affinity (IC50 = 280 nM), while angiotensin II and bradykinin had almost no affinity for [3H]neurotensin-binding sites. Under similar conditions [3H]neurotensin binding to mouse and rat brain synaptosomal fractions showed two binding sites with high (0.86 and 0.44 nM) and low (13 and 19 nM) affinities. We have examined several possible physiological consequences of neurotensin receptor binding. Neurotensin (10 microM) exhibited no influence on adenylate cyclase activity, 45Ca uptake, or 32Pi incorporation into phosphatidylinositol fractions of NG108-15 cells. Electrophysiological study of isolated NG108-15 cells revealed neurotensin-induced transient hyperpolarization followed by sustained depolarization with enhanced membrane excitability. Application of neurotensin to NG108-15 cells that had formed synapses with cultured striated muscle cells caused a considerable increase in frequency of miniature endplate potentials from the muscle cells. These data show that NG108-15 cells possess a single class of neurotensin receptors similar to a high affinity site of synaptosomal membranes from the murine brains.(ABSTRACT TRUNCATED AT 250 WORDS)

Down-regulation of opiate receptor in neuroblastoma x glioma NG108-15 hybrid cells. Chloroquine promotes accumulation of tritiated enkephalin in the lysosomes.

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.

Facilitation of synaptic transmission by prostaglandin D 2 at synapses between NG108-15 hybrid and muscle cells

The effect of prostaglandin (PG) D 2 on neuronal functions was investigated in neuroblastoma × glioma NG108-15 hybrid cells. PGD 2 caused a sustained increase in miniature end-plate potentials (MEPPs) recorded from cultured striated muscle cells which had formed junctions with NG108-15 cells. PGD 2 initially hyperpolarized and then depolarized NG108-15 cells. The time course of depolarization fitted well to the facilitative phase of MEPPs. The same action on synaptic transmission and membrane potentials was detected with PGF 2α but not with PGE 1. PGD 2 (10 −4M) produced a 3-fold increase of adenylate cyclase activity in NG108-15 cell homogenates through its receptors that are distinct from those of PGE 1 and PGI 2. These results show that PGD 2 facilitates MEPP frequency from NG108-15 cells due to depolarization, and suggest that PGD 2 may act as a physiological neuromodulator for synaptic transmission in vivo.

Modulation of adenylate cyclase activity by a cytosolic factor following chronic opiate exposure in neuroblastoma x glioma NG108-15 hybrid cells

A soluble cytosolic factor from neuroblastoma x glioma NG108-15 hybrid cells stimulates adenylate cyclase activity in isolated membrane preparations. This cytosolic component is heat stable, pronase insensitive, has a molecular weight less than 350 daltons and an absorbance peak at 260 nm. The stimulation is immediate, independent of Ca ++ and exhibits a sigmoidal concentration dependency curve. The cytosolic factor stimulated adenylate cyclase activity in etorphine treated cells (100 nM etorphine, 16 hrs) to a greater extent than in control cells. In addition, cytosolic factor derived from etorphine treated cells, as compared to control cells, displayed an increased capacity to stimulate adenylate cyclase. It is suggested that the observed cytosolic factor may be adenosine and that cells chronically treated with an opiate exhibit an increase in both concentration and sensitivity to this agent.

Attenuation of enkephalin activity in neuroblastoma X glioma NG108-15 hybrid cells by phospholipases.

The role of membrane phospholipids in enkephalin receptor-mediated inhibition of adenylate cyclase (EC 4.6.1.1) activity in neuroblastoma X glioma NG108-15 hybrids was studied by selective hydrolysis of lipids with phospholipases. When NG108-15 cells were treated with phospholipase C from Clostridium welchii at 37 degrees C, an enzyme concentration--dependent decrease in adenylate cyclase activity was observed. The basal and prostaglandin E1 (PGE1)-stimulated adenylate cyclase activities were more sensitive to phospholipase C (EC 3.1.4.3) treatment than were the NaF-5'-guanylylimidodiphosphate (Gpp(NH)p)-sensitive adenylate cyclase activities. Further, Leu5-enkephalin inhibition of basal or PGE1-stimulated adenylate cyclase activity was attenuated by phospholipase C treatment, characterized by a decrease of enkephalin potency and of maximal inhibitory level. [3H]D-Ala2-Met5-enkephalinamide binding revealed a decrease in receptor affinity with no measurable reduction in number of binding sites after phospholipase C treatment. Although opiate receptor was still under the regulation of guanine nucleotide after phospholipase C treatment, adenylate cyclase activity was more sensitive to the stimulation of Gpp(NH)p. Thus, the reduction of opiate agonist affinity was not due to the uncoupling of opiate receptor from N-component. Further, treatment of NG108-15 hybrid cell membrane with phospholipase C at 24 degrees C produced analogous attenuation of enkephalin potency and efficacy without alteration in receptor binding. The reduction in enkephalin potency could be reversed by treating NG108-15 membrane with phosphatidylcholine, but not with phosphatidylserine, phosphatidylinositol, or cerebroside sulfate. The enkephalin activity in NG108-15 cells was not altered by treating the cells with phospholipase A2 o phospholipase C from Bacillus cereus. Hence, apparently, there was a specific lipid dependency in enkephalin inhibition of adenylate cyclase activity.

Serotonin in neuroblastoma × glioma NG108-15 hybrid cells

Mouse neuroblastoma × rat glioma NG108-15 hybrid cells contain a considerable amount of serotonin, and possess small but significant tryptophan hydroxylase activity. The results suggest that NG108-15 hybrid cells are serotonergic, in addition to the known cholinergic property.

Identification of a Mr 58 000 glycoprotein subunit of the opiate receptor

A M r 58 000 subunit of the opiate receptor has been identified using tritiated fentanyl isothiocyanate, a potent opiate alkylating reagent with specificity for the δ-opiate receptor subclass. The subunit is alkylated in the presence of dextrorphan but not levorphanol. The specifically labelled protein was retained on columns of immobilized wheat germ agglutinin and is therefore presumably a glycoprotein. Partial purification of the M r 58 000 opiate receptor subunit from neuroblastoma × glioma NG108-15 hybrid cell membranes is described.