Effect of Phospholipases on Chronic Opiate Action in Neuroblastoma × 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.     

Inhibition of Bradykinin-Induced Calcium Increase by Phosphatase Inhibitors in Neuroblastoma × Glioma Hybrid NG108-15 Cells

Abstract: Prior treatment of NG108-15 cells with phosphatase inhibitors including okadaic acid and calyculin A inhibited the elevation of cytosolic Ca²⁺ concentration ([Ca²⁺]_i) induced by bradykinin by ∼63%. This inhibition was dependent on the concentration of okadaic acid with an IC₅₀ of 0.15 n M . Okadaic acid treatment only lowered the maximal response of [Ca²⁺]_i increase and had no effect on the EC₅₀ value for bradykinin regardless of the presence of extracellular Ca²⁺. Neither the capacity of ⁴⁵Ca²⁺ accumulation within intracellular nonmitochondrial Ca²⁺ stores nor the magnitude of [Ca²⁺]_i increase induced by thapsigargin was reduced by the treatment of okadaic acid. In contrast, the same phosphatase inhibitor treatment inhibited the bradykinin-evoked inositol 1,4,5-trisphosphate (IP₃) generation, the Mn²⁺ influx, and the capacity of mitochondrial Ca²⁺ accumulation. Furthermore, the sensitivity of IP₃ in the Ca²⁺ release was suppressed by okadaic acid pretreatment. Our results suggest that the reduction of bradykinin-induced [Ca²⁺]_i rise by the promotion of protein phosphorylation was attributed to the reduced activity of phospholipase C, the decreased sensitivity to IP₃, and the slowed rate of Ca²⁺ influx. Thus, phosphorylation plays a role in bradykinin-sensitive Ca²⁺ signaling cascade in NG108-15 cells.     

Possible Existence of Two Subsets of Platelet-Activating Factor Receptor to Mediate Polyphosphoinositide Breakdown and Calcium Influx in Neuroblastoma × Glioma Hybrid NG 108–15 Cells

Platelet-activating factor (PAF) initiated polyphosphoinositide (polyPI) breakdown and a rise of intracellular calcium concentration ([Ca2+]i) in neuroblastoma x glioma hybrid NG 108-15 cells. The accumulation of [3H]inositol trisphosphate and [3H]inositol bisphosphate was evident within 15 s after PAF stimulation, peaked at 1 min, and then gradually decayed. The increase in [3H]inositol monophosphate level was observed at 30 s, plateaued in 5 min, and was sustained up to 10 min in the presence of 10 mM LiCl. On the other hand, the rise of [Ca2+]i evoked by PAF reached a peak within 8-12 s and returned to basal levels within 1 min as measured in fura 2-loaded cells. When cells were suspended in Ca(2+)-depleted medium, the PAF-induced [Ca2+]i rise was reduced by 80%, indicating that the increase of [Ca2+]i was predominantly due to the Ca2+ influx from an extracellular source. Both PAF-induced accumulation of 3H-labeled inositol phosphates and [Ca2+]i elevation were concentration dependent with EC50 values of approximately 1 x 10(-10) and 5 x 10(-8) M, respectively. The PAF analogs 1-O-hexadecyl-2-hydroxy-sn-glycero-3-phosphocholine and 1-O-hexadecyl-2-O-methyl-rac-glycerol-3-phosphocholine were much poorer agonists at eliciting the same responses in these cells. Pretreatment of cells with pertussis toxin caused a substantial inhibition of PAF-induced accumulation of 3H-inositol phosphates. In contrast, the rise in [Ca2+]i was not significantly affected by toxin treatment at the same concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

G Protein Modulation of ω-Conotoxin Binding Sites in Neuroblastoma X Glioma NG 108-15 Hybrid Cells

Electrophysiological evidence shows that voltage-dependent calcium channel (VDCC) activity can be regulated by a large number of neurotransmitters. In particular, guanine nucleotide binding regulatory protein (G protein)-mediated inhibitory modulation of the channel activity has been deduced from evidence that GTP analogues and purified G proteins are able to mimic this effect. The G proteins involved are pertussis toxin (PTx) sensitive. The purpose of the present study was to investigate, using biochemical techniques, whether G protein activation modulates the recognition site for omega-conotoxin GVIA (CgTx), a peptide neurotoxin that selectively labels a population of high-threshold VDCC. Undifferentiated and differentiated (1 mM dibutyryl cyclic AMP, 4 days) NG 108-15 cells were used. In both crude cellular extracts specific binding of 125I-CgTx was characterized. Differentiation induced a sixfold increase in the number of binding sites and doubled the KD value. The in vitro addition of guanylylimidodiphosphate (GMP-PNP; a nonhydrolyzable analogue of GTP) to extracts prepared from differentiated cells reduced the 125I-CgTx binding by 48%. This effect, observed in undifferentiated cells as well, was also caused by other triphosphate guanine nucleotides, such as GTP, but not by guanosine 5'-O-(2-thiodiphosphate) or adenine nucleotides. Treatment of the cells with PTx prevented the GMP-PNP effect. Moreover, the results obtained after preincubation with specific antisera raised against the alpha subunits of Gi1-2 and Go suggest that Go is the G protein responsible for the observed effect.     

L-type calcium channel gating is modulated by bradykinin with a PKC-dependent mechanism in NG108-15 cells

Bradykinin (BK) excites dorsal root ganglion cells, leading to the sensation of pain. The actions of BK are thought to be mediated by heterotrimeric G protein-regulated pathways. Indeed there is strong evidence that in different cell types BK is involved in phosphoinositide breakdown following activation of G_q/11. In the present study we show that the Ca²⁺ current flowing through L-type voltage-gated Ca²⁺ channels in NG108-15 cells (differentiated in vitro to acquire a neuronal phenotype), measured using the whole-cell patch clamp configuration, is reversibly inhibited by BK in a voltage-independent fashion, suggesting a cascade process where a second messenger system is involved. This inhibitory action of BK is mimicked by the application of 1,2-oleoyl-acetyl glycerol (OAG), an analog of diacylglycerol that activates PKC. Interestingly, OAG occluded the effects of BK and both effects were blocked by selective PKC inhibitors. The down modulation of single L-type Ca²⁺ channels by BK and OAG was also investigated in cell-attached patches. Our results indicate that the inhibitory action of BK involves activation of PKC and mainly shows up in a significant reduction of the probability of channel opening, caused by an increase and clustering of null sweeps in response to BK.     

Intracellular Calcium Pools in Neuroblastoma × Glioma Hybrid NG108–15 Cells

The intracellular nonmitochondrial calcium pools of saponin-permeabilized NG108-15 cells were characterized using inositol 1,4,5-trisphosphate (IP3) and GTP. IP3 or GTP alone induced release of 47 and 68%, respectively, of the calcium that was releasable by A23187. GTP induced release of a further 24% of the calcium after IP3 treatment, whereas IP3 induced release of a further 11% of the calcium after GTP treatment. Guanosine 5'-O-(3-thio)triphosphate had little effect on IP3-induced calcium release but completely inhibited GTP-induced calcium release. In contrast, heparin inhibited the action of IP3 but not that of GTP. The results imply the existence of at least three nonmitochondrial pools: (a) 31% is releasable by IP3 and GTP, (b) 11% is releasable by IP3 alone, and (c) 24% is releasable by GTP alone. GTP enhanced calcium uptake in the presence of oxalate with an EC50 of 0.6 microM and stimulated calcium release in the absence of oxalate with an EC50 of 0.32 microM. The similar EC50 values for these dual effects of GTP on calcium movement suggest that GTP exerts its dual action by the same mechanism.     

ATP-Activated Nonselective Cation Current in NG108-15 Cells

Abstract: ATP (1 mM) induced a biphasic increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), i.e., an initial transient increase decayed to a level of sustained increase, in NG108-15 cells. The transient increase was inhibited by a phospholipase C inhibitor, 1-[6-[[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122), whereas the sustained increase was abolished by removal of external Ca²⁺. We examined the mechanism of the ATP-elicited sustained [Ca²⁺]_i increase using the fura-2 fluorescent method and the whole-cell patch clamp technique. ATP (1 mM) induced a membrane current with the reversal potential of 12.5 ± 0.8 mV (n = 10) in Tyrode external solution. The EC₅₀ of ATP was ～0.75 mM. The permeability ratio of various cations carrying this current was Na⁺ (defined as 1) > Li⁺ (0.92 ± 0.01; n = 5) > K⁺ (0.89 ± 0.03; n = 6) > Rb⁺ (0.55 ± 0.02; n = 6) > Cs⁺ (0.51 ± 0.01; n = 5) > Ca²⁺ (0.22 ± 0.03; n = 3) > N-methyl-d -glucamine (0.13 ± 0.01; n = 5), suggesting that ATP activated a nonselective cation current. The ATP-induced current was larger at lower concentrations of external Mg²⁺. ATP analogues that induced the current were 2-methylthio-ATP (2MeSATP), benzoylbenzoic-ATP, adenosine 5′-thiotriphosphate (ATPγS), and adenosine 5′-O-(2-thiodiphosphate), but not adenosine, ADP, α,β-methylene-ATP (AMPCPP), β,γ-methylene-ATP (AMPPCP), or UTP. Concomitant with the current data, 2MeSATP and ATPγS, but not AMPCPP or AMPPCP, increased the sustained [Ca²⁺]_i increase. We conclude that ATP activates a class of Ca²⁺-permeable nonselective cation channels via the P_2z receptor in NG108-15 cells.     

Involvement of Both Inhibitory and Stimulatory Guanine Nucleotide Binding Proteins in the Expression of Chronic Opiate Regulation of Adenylate Cyclase Activity in NG108-15 Cells

Abstract: Chronic etorphine treatment of neuroblastoma × glioma NG108-15 cells results in both an increase in adenylate cyclase activity (upon addition of the opiate antagonist naloxone) as well as an homologous desensitization of the opiate receptor. The continued ability of opiate agonists to regulate adenylate cyclase activity following opiate receptor desensitization can be understood by proposing that the catalytic subunit of adenylate cyclase in NG108-15 cells is under tonic regulation by both guanine nucleotide regulatory (N_i) and stimulatory (N_s) components. Inactivation of N_i by pertussis toxin (PT) treatment resulted in elevated adenylate cyclase activities comparable to those observed in control cells following chronic opiate treatment. This increased enzymatic activity could not be further induced by PT treatment of cells exposed to opiate previously. In addition, procedures that prevented receptor-mediated activation of N_s, i.e., treatment with NaF or desensitization of the stimulatory receptors (prostaglandin E₁, adenosine) eliminated the increase in adenylate cyclase activity induced by naloxone following chronic opiate exposure. Hence, the increase in enzymatic activity observed following chronic opiate treatment may be due to a loss in tonic inhibitory regulation of adenylate cyclase mediated through N_i resulting in the unimpeded expression of N_s activity. This tonic inhibition of adenylate cyclase activity is one of the multiple mechanisms by which N_i regulates adenylate cyclase in this cell line.     

Choline Uptake by the Neuroblastoma X Glioma Hybrid, NG108-15

The neuroblastoma X glioma hybrid clone NG108-15 is able to release acetylcholine upon depolarization and form cholinergic neuromuscular synapses in culture. Normal functioning of cholinergic synapses is thought to be dependent on the ability of a neuron to take up extracellular choline, since neurons are unable to synthesize choline de novo. For these two reasons it became important to characterize the choline uptake system of NG108-15 cells. The uptake system appears to bear little if any resemblance to the Na⁺-dependent high-affinity choline uptake system normally associated with cholinergic neurons. Although the cells appear to possess both high- and low- affinity choline uptake systems, neither system is dependent on Na⁺ and uptake actually is increased about 60% by the substitution of sucrose for NaCl. Acetylcholine synthesis also is not dependent on Na⁺, since sucrose, substituted for NaCl, also stimulates acetylcholine synthesis. Changes in the concentrations of the other ions in the uptake medium have little effect on uptake, with the exception that elevated Ca²⁺ or Mg²⁺ reverses the stimulation of choline uptake produced by substitution of sucrose for NaCl. Choline uptake is inhibited by hemicholinium-3, but only at high concentrations of the drug (IC₅₀= 30–80 μm ). The metabolic poisons cyanide and iodoacetate inhibit uptake by only 30-40%. Growth of the cells in N⁶,O^2′ dibutyryladenosine-3′,5′-cyclic monoposphate, which promotes functional and morphological differentiation of the cells, decreased slightly the total amount of choline taken up but had no additional effect on the uptake system. Thus, it appears that NG108-15 cells are capable of forming functional cholinergic synapses with muscle cells even though the neuroblastoma does not possess the high-affinity choline uptake system normally associated with cholinergic neurons.     

Inhibition of neuronal nitric-oxide synthase by phosphorylation at Threonine1296 in NG108-15 neuronal cells

We demonstrate that neuronal nitric-oxide synthase (nNOS) is directly inhibited through the phosphorylation of Thr(1296) in NG108-15 neuronal cells. Treatment of NG108-15 cells expressing nNOS with calyculin A, an inhibitor of protein phosphatase 1 and 2A, revealed a dose-dependent inhibition of nNOS enzyme activity with concomitant phosphorylation of Thr(1296) residue. Cells expressing a phosphorylation-deficient mutant in which Thr(1296) was changed to Ala proved resistant to phosphorylation and suppression of NOS activity. Mimicking phosphorylation mutant of nNOS in which Thr(1296) is changed to Asp showed a significant decrease in nNOS enzyme activity, being competitive with NADPH, relative to the wild-type enzyme. These data suggest that phosphorylation of nNOS at Thr(1296) may involve the attenuation of nitric oxide production in neuronal cells through the decrease of NADPH-binding to the enzyme.     

Prevention of Monensin-Induced Hyperpolarization in NG108-15 Cells

The NG108-15 (neuroblastoma X glioma hybrid) cell line was used as an in vitro neuronal model to evaluate potential antagonists of the Na⁺-selective carboxylic ionophore monensin. Changes in membrane electrical characteristics induced by monensin with and without the simultaneous administration of antagonists were measured using intracellular microelectrode techniques. Bath application of monensin (3 M) produced a hyperpolarization of 35 mV. Monensin also altered the generation of action potentials in response to electrical stimulation in 14 of 24 (58%) exposed cells, as evident in a partial or complete loss of action potentials or in an alteration of action potential waveform. The antagonists used were Na⁺-K⁺ pump inhibitor ouabain (1–3 M), the Ca²⁺-dependent K⁺ channel blocker quinine (3–30 M) or drugs known to influence Ca²⁺ signaling in cells, i.e., trifluoperazine (3–10 M), verapamil (1–10 M) or chlorpromazine (3–30 M). On a molar basis, ouabain was the most and trifluoperazine the least effective of the antagonists. Quinine, verapamil and chlorpromazine all prevented the development of the hyperpolarization in an approximate concentration-dependent manner. However, none of these drugs was able to block the effects of monensin on action potentials. Indeed, high concentrations of the antagonists that were most effective in preventing the hyperpolarization accentuated impairments in action potential generation and also reduced input resistance in many cells. Thus, none of these antagonists appears suitable for transition to in vivo antidotal protection studies.     

Extracellular ATP Stimulates Calcium Influx in Neuroblastoma Glioma Hybrid NG108–15 Cells

Abstract— ATP-induced changes in the intracellular Ca²⁺concentration ([Ca²⁺]_i) in neuroblastoma glioma hybrid NG108–15 cells were studied. Using the fluorescent Ca²⁺indicator fura-2, we have shown that the [Ca²⁺]_i increased in response to ATP. ATP at 3 mM caused the greatest increase in [Ca^z+]_i, whereas at higher concentrations of ATP the response became smaller. Two nonhydrolyzable ATP analogues, adenosine 5′-thiotriphosphate and 5′-adenylyl-β, γ-imidodiphosphate, could not trigger significant [Ca²⁺]_i change, but they could block the ATP effect. Other adenine nucleotides, including ADP, AMP, α,β-methylene-ATP, β,γ-methylene-ATP, and 2-methylthio-ATP, as well as UTP and adenosine, all had no effect on [Ca²⁺]_i at 3 mM. In the absence of extracellular Ca²⁺, the effect of ATP was inhibited totally, but could be restored by the addition of Ca²⁺ to the cells. Upon removal of Mg²⁺, the maximum increase in [Ca²⁺]_i induced by ATP was enhanced by about 42%. Ca²⁺-channel blockers partially inhibited the ATP-induced [Ca²⁺]_i rise. The ATP-induced [Ca²⁺]_i rise was not affected by thapsigargin pretreatment, though such pretreatment blocked bradykinin-induced [Ca²⁺]_i rise completely. No heterologous desensitization of [Ca²⁺]_i rise was observed between ATP and bradykinin. The magnitude of the [Ca²⁺]_i rise induced by ATP increased between 1.5 and 3.1 times when external Na⁺was replaced with Tris, N-methyl-d -glucamine, choline, or Li⁺. The addition of EGTA or verapamil to cells after their maximum response to ATP immediately lowered the [Ca²⁺]_i to the basal level in Na⁺-containing or Na⁺-free Tris solution. Our results suggest that ATP stimulates Ca²⁺influx via at least two pathways: ion channels that are permeable to Ca²⁺ and Na⁺, and pores formed by ATP^4-.     

Effects of Protonophores on Membrane Electrical Characteristics in NG108-15 Cells

Studies were conducted to determine the effects of bath application of the protonophores carbonyl cyanide m-chlorophenylhydrazone (CCCP) and carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone (FCCP) on membrane electrical characteristics of differentiated NG108-15 (neuroblastoma X glioma hybrid) cells. Membrane resting potential (V_m), input resistance (R_in) and electrically induced action potential generation were measured using intracellular micro-electrode techniques. Both compounds produced concentration-dependent depolarization rather than the hyperpolarization commonly found with other central mammalian neurons. CCCP and FCCP also reduced R_in and disrupted the generation of action potentials in a concentration-dependent manner. The contribution of the observed alterations to the in vivo toxicity of these compounds remains to be established.     

Stimulation of Phospholipase D Activity in Human Neuroblastoma (LA-N-2) Cells by Activation of Muscarinic Acetylcholine Receptors or by Phorbol Esters: Relationship to Phosphoinositide Turnover

We have investigated the coupling of muscarinic acetylcholine receptors (mAChR) to phospholipid hydrolysis in a human neuroblastoma cell line, LA-N-2, by measuring the formation of 3H-inositol phosphates (3H-IP) and of [3H]phosphatidylethanol ([3H]PEt) in cells prelabeled with [3H]inositol and [3H]oleic acid. The muscarinic agonist carbachol (CCh) stimulated the phospholipase C (PLC)-mediated formation of 3H-IP in a time- and dose-dependent manner (EC50 = 40-55 microM). In addition, in the presence of ethanol (170-300 mM), CCh elevated levels of [3H]PEt [which is regarded as a specific indicator of phospholipase D (PLD) activity] by three- to sixfold. The effect of CCh on PEt formation also was dose dependent (EC50 = 50 microM). Both effects of CCh were antagonized by atropine, indicating that they were mediated by mAChR. Incubation of LA-N-2 cells with the phorbol ester phorbol 12-myristate 13-acetate (PMA, 0.1 microM; 10 min) increased [3H]PEt levels by up to 10-fold. This effect was inhibited by the protein kinase C (PKC) inhibitor staurosporine (1 microM) or by pretreatment for 24 h with 0.1 microM PMA, by 74% and 65%, respectively. In contrast, the effect of CCh on PEt accumulation was attenuated by only 28% in the presence of staurosporine (1 microM). In summary, these results suggest that, in LA-N-2 neuroblastoma cells, mAChR are coupled both to phosphoinositide-specific PLC and to PLD. PKC is capable of stimulating PLD activity in these cells; however, it is not required for stimulation of the enzyme by mAChR activation.     

Elevated Phosphatidyl-CMP Is Not the Source of Diacylglycerol Accumulation Induced by Lithium in NG108-15 Cells

Abstract: Previous studies have shown that in the neuroblastoma X glioma hybrid cell line NG108-15 lithium is able to induce an increase in diacylglycerol levels. This effect was shown to be enhanced by the presence of bradykinin. Another striking effect of lithium was a marked gain in the level of the liponucleotide phosphatidyl-CMP. Increased phosphatidyl-CMP levels were detected in the presence of lithium alone but were considerably more pronounced in the presence of both lithium and bradykinin. These results are consistent with the inhibitory action of lithium on key enzymes of the degradation pathway of inositol phosphates, resulting in a decrease in cellular inositol content and in an elevation in levels of phosphorylated inositols. Comparison of the mass of the inositol phosphates and diacylglycerol showed that the lithium-induced diacylglycerol levels were substantially greater than would be expected from phosphoinositide hydrolysis alone. One possible reason for the increase in the level of diacylglycerol through the action of lithium is the reversal of the reaction for the formation of phosphatidyl-CMP. The resulting phosphatidic acid would then need to be further dephosphorylated to diacylglycerol. The lithium-induced elevation of phosphatidyl-CMP was prevented by addition of myo -inositol (10–30 m M ), suggesting that the increase in liponucleotide level was due to depletion of cellular inositol. Under the same conditions the elevated diacylglycerol concentration remained unchanged. Consequently, phosphatidyl-CMP is not its source, and diacylglycerol may arise through an effect of lithium on the degradation of phospholipids other than phosphoinositides. The action of phospholipase C or D on phosphatidylcholine is the most likely mechanism.     

δ-Opioids Stimulate Inositol 1,4,5-Trisphosphate Formation, and so Mobilize Ca2+ from Intracellular Stores, in Undifferentiated NG108-15 Cells

Abstract: δ-Opioids mobilize Ca²⁺ from intracellular stores in undifferentiated NG108-15 cells, but the mechanism involved remains unclear. Therefore, we examined the effect of [d -Pen^2,5]enkephalin on inositol 1,4,5-trisphosphate formation in these cells. [d -Pen^2,5]enkephalin caused a dose-dependent (EC₅₀ = 3.1 nM) increase in inositol 1,4,5-trisphosphate formation (measured using a specific radioreceptor mass assay), which peaked (25.7 ± 1.2 pmol/mg of protein with 1 µM, n = 9) at 30 s and returned to basal levels (10.6 ± 0.9 pmol/mg of protein, n = 9) within 4–5 min. This response was fully naloxone (1 µM) reversible and pertussis toxin (100 ng/ml for 24 h) sensitive. Preincubation with Ni²⁺ (2.5 mM) or nifedipine (1 µM) had no effect on the [d -Pen^2,5]enkephalin (1 µM)-induced inositol 1,4,5-trisphosphate response, and K⁺ (80 mM) was unable to stimulate inositol 1,4,5-trisphosphate formation, indicating Ca²⁺ influx-induced activation of phospholipase C is not involved. Preincubation with the protein kinase C inhibitor Ro 31-8220 (1 µM) enhanced, whereas acute exposure to phorbol 12,13-dibutyrate (1 µM) abolished, the [d -Pen^2,5]enkephalin (0.1 µM)-induced inositol 1,4,5-trisphosphate response, suggesting protein kinase C exerts an autoinhibitory feedback action. [d -Pen^2,5]Enkephalin also dose-dependently (EC₅₀ = 2.8 nM) increased the intracellular [Ca²⁺], which was maximal (24 nM increase with 1 µM, n = 5) at 30 s. This close temporal and dose-response relationship strongly suggests that δ-opioid receptor-mediated increases in intracellular [Ca²⁺] results from inositol 1,4,5-trisphosphate-induced Ca²⁺ release from intracellular stores, in undifferentiated NG108-15 cells.     

Involvement of Growth-Associated Protein-43 with Irreversible Neurite Outgrowth by Dibutyryl Cyclic AMP and Phorbol Ester in NG108–15 Cells

Simultaneous treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) and dibutyryl cyclic AMP (diBu-cAMP) for 72 h induced neurites in NG108-15 cells significantly longer than treatment with each alone. Treatment for 72 h with both drugs induced irreversible neurite extension and a decline in protein kinase C activity, although neurites extended by diBu-cAMP alone disappeared after the withdrawal of the drug. The expression of growth-associated protein-43 (GAP-43) mRNA was also observed by a combined application of TPA and diBu-cAMP. The increased level of GAP-43 mRNA induced by treatment with both drugs for 72 h was maintained at least 24 h after withdrawal of the drugs. In cells transfected with GAP-43 cDNA, neurites induced by treatment with diBu-cAMP alone for 72 h were maintained at least 48 h after removal of the drugs. These results suggest that GAP-43 could be involved in the maintenance of elongated neurites and that a decline in protein kinase C activity may be involved in the accumulation of GAP-43.     

Characteristics for Enhanced Response of Serotonin-Evoked Ion Dynamics in Differentiated NG108-15 Cells

Characteristics for the up-regulated response in the concentration of intracellular calcium ion ([Ca²⁺] _i ) and in the sodium ion (Na⁺) current by serotonin (5-HT) were investigated in differentiated neuroblastoma × glioma hybrid NG108-15 (NG) cells. The results for the changes in [Ca²⁺] _i by 5-HT were as follows, (1) The 5-HT-induced Ca²⁺ response was inhibited by 3 × 10⁻⁹ M tropisetron (a 5-HT₃ receptor blocker), but not by other types of 5-HT receptor blockers; (2) The 5-HT-induced Ca²⁺ response was mainly inhibited by calciseptine (a L-type Ca²⁺ blocker), but not by other types of Ca²⁺ channel blockers or 10⁻⁷ M TTX (a voltage-sensitive Na⁺ channel blocker); (3) When the extracellular Na⁺ was removed by exchange with choline chloride or N-methyl-d-glucamine, the 5-HT-induced Ca²⁺ response was extremely inhibited. The results for the 5-HT-induced Na⁺ current by the whole cell patch-clamp technique were as follows, (1) The 5-HT-induced Na⁺ current in differentiated cells was significantly larger than that in undifferentiated cells; (2) The ED₅₀ value for 5-HT-induced Na⁺ current in undifferentiated and differentiated cells was almost the same, about 4 × 10⁻⁶ M each other; (3) The 5-HT-induced Na⁺ current was completely blocked by 3 × 10⁻⁹ M tropisetron, but not by other 5-HT receptor antagonists and 10⁻⁷ M TTX. These results suggested that 5-HT-induced Ca²⁺ response in differentiated NG cells was mainly due to L-type voltage-gated Ca²⁺ channels allowing extracellular Na⁺ to enter via 5-HT₃ receptors, but not through voltage-gated Na⁺ channels.     

Chronic Activation of Inhibitory δ-Opioid Receptors Cross-Regulates the Stimulatory Adenylate Cyclase-Coupled Prostaglandin E1 Receptor System in Neuroblastoma × Glioma (NG108-15) Hybrid Cells

Abstract: The present article investigates chronic opioid regulation of the stimulatory adenylate cyclase-coupled prostaglandin E₁ (PGE₁) receptor system in neuroblastoma × glioma (NG108-15) hybrid cells. Persistent activation of δ-opioid receptors by morphine (10 µmol/L; 3 days) substantially down-regulates the number of PGE₁ binding sites by ～30%, without affecting their affinity. Radioligand binding studies performed in the presence of GTPγS (100 µmol/L) further revealed that the remaining PGE₁ binding sites are still capable of interacting functionally with their associated stimulatory G proteins, G_s. On the postreceptor level, neither changes in the abundance nor in the intrinsic activity of the α subunit of G_s (G_sα) were found during the state of opioid dependence, as has been verified by western blot and S49 cyc^? reconstitution experiments, respectively. Evaluation of the functional interaction between PGE₁ receptors and G_s by means of receptor-stimulated, cholera toxin-catalyzed ADP-ribosylation of G_sα revealed a significant increase in the ability of PGE₁ receptors to activate G_sα (3.3-fold increase in EC₅₀; p < 0.05) in cells chronically exposed to morphine. This effect was completely blocked by coincubation of the cells together with the opiate antagonist naloxone (100 µmol/L; 3 days), whereas precipitation of morphine withdrawal by naloxone (100 µmol/L) had no further effect on sensitization in PGE₁ receptor/G_s coupling. These findings provide evidence that the stimulatory adenylate cyclase-coupled PGE₁ receptor system represents a potential target of chronic δ-opioid receptor activation in NG108-15 hybrid cells. They further suggest that sensitization in stimulatory signal transduction plays a critical role in the generation of opioid dependence.     

Ethanol potentiates the uptake of [14C]Serine into phosphatidylserine by base-exchange reaction in NG 108-15 cells

Phospholipid base-exchange enzymes catalyze the incorporation of nitrogenous bases into phosphoglycerides by a calcium-dependent mechanism. In this study, we describe the effect of ethanol on the incorporation of radioactive serine, choline and ethanolamine into their respective phospholipids in a neuroblastoma x glioma hybrid cell line (NG 108-15). Long term ethanol exposure induced a potentiation of the incorporation of [¹⁴C]serine into phosphatidylserine. Moreover, the phosphorus content of PS was found to be increased after long-term ethanol exposure. No concomitant changes in the phosphorus content of other phospholipids were observed. The results indicate that in NG 108-15 cells, the incorporation of radiolabelled serine into PS is potentiated during chronic ethanol exposure.