Diacylglycerol kinase inhibitor R59022-induced autophagy and apoptosis in the neuronal cell line NG108-15

Phosphatidic acid (PA) is a lipid second messenger and is believed to be involved in cell proliferation and survival. PA is mainly produced by phospholipase D (PLD) and diacylglycerol kinase (DGK). Elevated PLD activity is believed to suppress apoptosis via activation of the mammalian target of rapamycin (mTOR). On the other hand, DGK inhibition has been demonstrated to induce apoptosis, but it is unclear whether DGK can regulate mTOR. Here, we investigated whether DGK inhibition can induce apoptosis and autophagy in neuronal cells, since mTOR is a key mediator of autophagy and the simultaneous activation of apoptosis and autophagy has been detected. A DGK inhibitor, R59022 induced autophagy and apoptosis without serum in NG108-15 cells. Autophagy preceded apoptosis, and apoptosis inhibition did not affect R59022-induced autophagy. R59022-induced autophagy was inhibited by exogenous PA, and protein kinase C activation and increases in intracellular Ca²⁺ levels, which are assumed to be caused by diacylglycerol accumulation, did not appear to be involved in R59022-induced autophagy. We also investigated the effects of R59022 on mTOR signaling pathway, and found that the pathway was not inhibited by R59022. These results imply that DGK plays an important role in cell survival via mTOR-independent mechanism.     

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.     

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.     

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.     

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.     

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.     

The Slow Cyclic GMP Increase Caused by Serotonin in NG108-15 Cells Is Not Inhibited by Antagonists of Known Serotonin Receptors: Possible Existence of a New Receptor Subtype Coupled with Membrane-Bound Guanylate Cyclase

Characterization of the serotonin (5-HT)-induced cyclic GMP (cGMP) elevation was investigated in comparison with bradykinin- and ANP-induced elevations in NG108-15 cells. At 20 s, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM, 100 microM), a membrane-permeabilized Ca2+ chelator, or N-monomethyl-L-arginine (NMMA, 300 microM), an inhibitor of L-arginine-derived nitric oxide (NO) synthesis, inhibited 5-HT-induced elevation by approximately 40%, and completely inhibited bradykinin-induced response. Neither 5-HT- nor ANP-induced cGMP elevation at 10 min was affected by BAPTA-AM or NMMA. The cGMP elevated by 5-HT as well as by ANP was effluxed to the extracellular medium. These results and our previous report suggest that 5-HT stimulates two subtypes of 5-HT receptors in NG108-15: first, 5-HT3 subtype stimulating Ca(2+)-sensitive cytosolic guanylate cyclase through NO derived from L-arginine and second, a probably novel 5-HT receptor subtype involved in activation of membrane-bound guanylate cyclase.     

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.     

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.     

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.     

Induction of choline acetyltransferase in the neuroblastoma x glioma cell line NG108-15

The mechanism of the induction of choline acetyltransferase activity in the hybrid cell line NG108-15 was studied. Induction by cyclic AMP analogs, forskolin, and prostaglandin E₁ + theophylline was found to be rapid with an increase in choline acetyltransferase specific activity detectable within 8 hrs and maximal after 24 hrs. Immunoblot analysis was used to demonstrate that the increase in choline acetyltransferase specific activity induced by prostaglandin E₁ + theophylline was due to an increase in enzyme protein. Cycloheximide effectively blocked the induction of choline acetyltransferase by prostaglandin E₁ + theophylline. These results demonstrate that the induction of choline acetyltransferase activity involves the synthesis of new enzyme protein. Attempts to measure choline acetyltransferase turnover by blocking its synthesis with cycloheximide indicated that this enzyme is a relatively stable protein with a half-life of greater than 24 hrs.     

Spontaneous firing of NG108-15 cells induced by transient exposure to ammonium chloride

Summary 1. We report that NG108-15 (neuroblastoma × glioma) cells differentiated in defined serum-free media are capable of exhibiting stable automaticity (the spontaneous occurrence of regenerative action potentials) following exposure to extracellular perfusates containing NH₄Cl. 2. Membrane depolarization (4–5 mV) concomitant with an increased pH_i during NH₄Cl exposure are followed by hyperpolarization (5–7 mV), subthreshold oscillations, and spontaneous firing after the removal of NH₄Cl. 3. Cells cultured in 10% serum did not exhibit automaticity. Cells cultured in serum-free media are twice as likely to show automaticity as those cultured in reduced (1.5%) serum media. 4. We have examined factors that contribute to the events following NH₄Cl exposure, namely, membrane depolarization and hyperpolarization, subthreshold oscillations, and automaticity. The inward currents activated at more negative potentials and the ionic currents associated with pronounced afterhyperpolarization in NG108-15 cells cultured in serum-free media provide a basis for the repetitive activity in general and automaticity in particular.     

Increase in Neurite Formation and Acetylene line Release by Transfection of Growth-Associated Protein-43 cDNA into NG108–15 Cells

Abstract: We previously reported that growth-associated protein-43 (GAP-43) could be involved in the maintenance of elongated neurites and that a decline in protein kinase C activity may be involved in accumulation of GAP-43. In the present study, to clarify the functional significance of GAP-43 for neurite maintenance and acetylcholine (ACh) release, we prepared NG-G11 cells by transfection of GAP-43 cDNA into NG108-15 cells. NG-G11 cells expressed GAP-43 mRNA at levels approximately twice that in nontransfected or vector-transfected cells under control conditions and after treatment with dibutyryl cyclic AMP (diBu-cAMP) or 12-O-tetradecanoylphorbol 13-acetate (TPA) plus diBu-cAMP. Neurite outgrowth after addition of diBu-cAMP was greater in NG-G11 than in control cells. In NG-G11 cells, neurites elongated by treatment with diBu-cAMP for 72 h were maintained after removal of the drug. Treatment with TPA plus diBu-cAMP for 24 h induced neurite outgrowth in NG-G11 cells, although control cells required 72 h. Depolarization by 50 m M KCI induced ACh release in both NG-G11 and control cells treated with diBu-cAMP or TPA/diBu-cAMP. Although removal of the drugs following diBu-cAMP treatment reversed ACh release to nontreated levels in control cells, a high-K⁺-induced level of ACh release remained in NG-G11 cells after removal of diBu-cAMP. ACh release induced by TPA plus diBu-cAMP for 24 h was further enhanced after removal of the drugs in NG-G11 cells, but it was not seen in control cells. These results suggest that levels of GAP-43 mRNA are correlated with neurite maintenance and the level of ACh release. Thus, GAP-43 may be involved in neuronal differentiation in NG108-15 cells.     

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.     

Heterogeneity of Nucleotide Receptors in NG108-15 Neuroblastoma and C6 Glioma Cells for Mediating Phosphoinositide Turnover

Abstract: We have compared the characteristics of receptors for nucleotide analogues and the involvement of phospholipase C (PLC) in the effector mechanism in NG108-15 neuroblastoma and C6 glioma cells. The relative potency of these analogues to stimulate inositol phosphate (IP) formation is UTP > UDP ? 2-methylthio-ATP (2-MeSATP), GTP > ATP, CTP > ADP > UMP in NG108-15 cells and ATP > UTP > ADP > GTP > UDP ? 2Me-SATP, CTP, UMP in C6 glioma cells. α,β-Methylene-ATP, β,γ-methylene-ATP, AMP, and adenosine had little or no effect in both types of cells. The EC₅₀ values were 3 and 106 µM for UTP in NG108-15 and C6 glioma cells, respectively. The EC₅₀ value for ATP in C6 glioma cells was 43 µM. 2-MeSATP was threefold more potent than ATP in NG108-15 cells but had little effect in C6 glioma cells at 1 mM. In NCB-20 cells, a similar rank order of potency to that found in NG108-15 cells, i.e., UTP ? GTP > ATP > CTP, was observed. In both NG108-15 and C6 glioma cells, preincubation with ATP or UTP caused a pronounced cross-desensitization of subsequent nucleotide-stimulated IP production. ATP and UTP displayed no additivity in terms of IP formation at maximally effective concentrations. In contrast, endothelin-1, bradykinin, and NaF interacted in an additive manner with either nucleotide in stimulating PI hydrolysis. Pretreatment with pertussis toxin did not affect ATP-, UTP-, and GTP-stimulated IP generation in these cells, indicating that nucleotide receptors coupled to PLC by a pertussis toxin-resistant G protein in both cell types. Short-term treatment of the cells with protein kinase C (PKC) activators [phorbol 12-myristate 13-acetate (PMA) and octylindolactam V] produced a dose-dependent inhibition of ATP- and UTP-induced IP formation with a greater extent and higher susceptibility in C6 glioma cells than in NG108-15 cells. Furthermore, a 24-h exposure of the cells to PMA resulted in an obvious attenuation of nucleotide-induced IP formation in C6 glioma cells but failed to change the response in NG108-15 cells. These results suggest that distinct nucleotide receptors that respond to ATP and UTP with different selectivity exist in NG108-15 and C6 glioma cells. These heterogeneous nucleotide receptors coupled to PLC undergo discriminative modulation by PKC. NG108-15 and NCB-20 neuroblastoma are two cell lines that showed the highest specificity to extracellular UTP rather than ATP among the nucleotide receptors so far studied in various cells, suggesting the presence of a pyrimidine receptor in these cells.     

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.     

Serotonin Stimulates Both Cytosolic and Membrane-Bound Guanylate Cyclase in NG108–15 Cells

The cyclic GMP (cGMP) content was rapidly (greater than 30 s) increased by serotonin [5-hydroxytryptamine (5-HT)] (EC50 = 10 microM), and the increase lasted for greater than 10 min in NG108-15 cells. The 5-HT-induced elevation of cGMP level (EC50 = 10 microM) at 20 s ("fast" elevation) was inhibited by ICS 205-930 or MDL 72,222 and by Ca2+ deficiency in the reaction medium but not by organic Ca2+ antagonists. The 5-HT effect at 10 min ("slow" elevation) was not inhibited by several antagonists for 5-HT receptors of the 1A, 1B, 1C, 1D, 2, and 3 subtypes and was independent from external Ca2+ concentration. The fast and slow effects of 5-HT were similar to the effects of bradykinin and atrial natriuretic peptide (ANP), respectively, in aspects of both Ca2+ dependency and time course of the effects. Bradykinin transiently stimulated formation of inositol phosphates as well as accumulation of cGMP, a finding suggesting that intracellular Ca2+ is involved in bradykinin-induced cGMP accumulation as shown in the fast response to 5-HT. ANP, an activator of membrane-associated guanylate cyclase (mGC), slowly (approximately 60 s) increased the cGMP content (EC50 = 10 nM), a result lasting for greater than 10 min, and the effects were independent from external Ca2+, as shown in the slow response to 5-HT. 5-HT and ANP did not induce formation of inositol phosphates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chronic Exposure of NG 108-15 Cells to Opiate Agonists Does Not Alter the Amount of the Guanine Nucleotide-Binding Proteins Gi and GO

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.     

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.     

Differential subcellular localization of two dopamine D2 receptor isoforms in transfected NG108-15 cells

The dopamine D2 receptor (D2R) is target for antipsychotic drugs and associated with several neuropsychiatric disorders. D2R has a long third cytoplasmic loop and a short carboxyl-terminal cytoplasmic tail. It exists as two alternatively spliced isoforms, termed D2LR and D2SR, which differ in the presence and absence, respectively, of a 29 amino acid insert in the third cytoplasmic loop. To evaluate the differential roles of the two D2R isoforms, we transfected both isoforms into NG108-15 cells and observed their subcellular localization by a confocal laser scanning light microscope. D2SR was predominantly localized at the plasma membrane, whereas D2LR was mostly retained in the perinuclear region around the Golgi apparatus. Using a yeast two hybrid system with a mouse brain cDNA library and coimmunoprecipitation assay, we found that heart-type fatty acid binding protein (H-FABP) interacts with D2LR but not with D2SR. H-FABP is a cytosolic protein involved in binding and transport of fatty acids. Overexpressed H-FABP and endogenous H-FABP were colocalized with the intracellular D2LR in NG108-15 cells. Furthermore, in the rat striatum, H-FABP was detected in the D2R-expressing neurons. From these results, H-FABP is associated with D2LR, and may thereby modulate the subcellular localization and function of D2LR.