星形孢菌素诱导NG108—15细胞凋亡

目的：研究星形孢菌素是否能引起NG108—15细胞的凋亡及它对数种与凋亡相关基因蛋白表达水平的影响．方法：用相差显微镜、荧光显微镜和透射电镜观察形态学变化;琼脂糖凝胶电泳检测DNA梯带;免疫印迹法检测凋亡相关基因蛋白的表达水平．结果：经星形孢菌素0．1μmol／L处理后NG108—15细胞呈典型的凋亡形态学变化．星形孢菌素处理后6h即出现DNA凋亡梯带,可持续至24h．Bax蛋白表达在星形孢菌素处理后6h开始上升,12h至最高峰,24h后下降．Bcl-2蛋白表达在星形孢菌素处理后3h明显上升,随后逐渐下降．星形孢菌素处理后6h可见caspase-3切割产物出现,但Cdk5蛋白的表达水平未见明显变化．p53蛋白表达在星形孢菌素处理12h后下降．结论：星形孢菌素诱导了NG108—15细胞的凋亡,可能与Bax蛋白表达的增加及caspase-3切割有关,而与p53和Cdk5无明显相关性．     

Isolation of RP-HPLC pure clonidine-displacing substance from NG108-15 cells

A crude extract of clonidine-displacing substance (CDS) has previously been extracted from the NG108-15 cell line. This study aimed to purify CDS extracted from this cell line further, by the technique of reverse phase-HPLC (RP-HPLC), and subsequently determine whether this refined CDS bears any similarity to CDS's extracted from other tissues. Crude CDS was extracted from NG108-cells and fractionated by RP-HPLC eluting with a linear gradient of methanol (5-65%; 1 ml min(-1) flow rate) over 50 min., and collected at 1 min. intervals. The pharmacological activities of the CDS fractions were determined by their abilities to displace bound [3H]clonidine to alpha(2)-adrenoceptors in rat brain membranes. RP-HPLC analysis of CDS revealed a pharmacologically active fraction distinct from agmatine, eluting at 24 min, corresponding to an absorbance peak observed at this time. Collectively, these results confirmed that CDS was present in the NG108-15 cell line. However, the RP-HPLC analysis showed the pharmacological activity to elute at a more hydrophobic gradient than previously observed with CDS's extracted from bovine tissues. These results support the notion of the existence of several CDS's.     

Antagonist-induced transient down-regulation of delta-opioid receptors in NG108-15 cells.

According to current concepts, agonists can effect the down-regulation of cell surface receptors, whereas antagonists can cause their up-regulation. We have discovered that the opioid antagonists naltrexone, naloxone, and ICI174864 induce a transient down-regulation of delta-opioid receptors before up-regulation, in NG108-15 cells. The possibility of an apparent loss of sites due to blockade by residual antagonist was ruled out by several lines of evidence. The reduction in delta receptors was time, temperature, and antagonist concentration dependent. This down-regulation could not be induced by either the highly mu-selective opioid antagonist cyclic D-Phe-Cys-Try-D-Trp-Arg-Thr-Pen-Thr-amide or the muscarinic antagonist atropine. In the same neurohybrid cells, the opioid agonist [D-Ala2,D-Leu5]enkephalin (0.1 microM, 60 min) effected a greater down-regulation of delta-opioid receptors. Similar qualitative changes in opioid binding of subcellular fractions were elicited with [D-Ala2,D-Leu5]enkephalin and naltrexone. However, the agonist was 2-fold more effective in reducing the heavy membrane population of receptors and 4-fold more potent in increasing the light membrane sites. Because heavy membranes are enriched in plasma membrane, whereas light membranes contain intracellular sites, these findings indicate that internalization occurs in both instances. Naltrexone and the delta-specific antagonists ICI174864 and naltrindole also diminished specific activities of two lysosomal enzymes, whereas opioid agonist-induced down-regulation was accompanied by an increase in their specific activities. Pretreatment of cell cultures with concanavalin A blocked both down-regulation and alterations in the lysosomal enzyme activities elicited by agonists and antagonists, suggesting that the latter is an opioid receptor-mediated process. The up-regulation of delta-opioid receptors by antagonists appears, then, to entail down-regulation that differs from that of agonists.     

Enhancement of veratridine-induced sodium dynamics in NG108-15 cells during differentiation

Developmental changes in dynamics of Na+ were studied in neuroblastomaxglioma hybrid NG108-15 cells during differentiation which was induced by dibutyryl cAMP (Bt2cAMP). Ratiometric Na+ imaging with a Na+-sensitive fluorescent dye SBFI (sodium-binding benzofuran isophthalate) revealed that the intracellular Na+ concentration ([Na+]i) was not affected by the application of high K+ (60 mM) solution to either control or differentiated cells. When cells were exposed to 50 microM veratridine (Vtd), an agonist of voltage-sensitive sodium channels (VSSCs), a significant increase in [Na+]i was observed in differentiated but not in undifferentiated cells. Calculated mean [Na+]i value increased from the basal 10.4 to 44.1 mM in response to 50 microM Vtd. This Vtd response was reversibly inhibited by tetrodotoxin (TTX), a specific blocker for VSSCs, in a dose-dependent manner (IC50 = 1 nM). It is suggested that VSSCs in NG108-15 cells are sensitive to TTX and Vtd and that the number of VSSCs increases during differentiation.     

Stereospecificity of opiate receptors in a synchronized culture of NG108-15 cells probed with fluorescent anti-idiotypic antibodies.

The stereospecificity of the binding of an anti-opiate receptor, anti-idiotypic antibody to receptors on a synchronized NG108-15 neuroblastoma x glioma cell culture has been examined by fluorescence labeling of the antibodies. We had previously found that unsynchronized NG108-15 cultures showed high specificity (reversibility by receptor-binding ligands) of antibody labeling. However, only a subpopulation additionally showed stereospecific labeling (nonreversibility by non-receptor-binding opiate enantiomers). In the present work we find that the percentage of cells that display stereospecific fluorescence is highly dependent upon position in the cell cycle. Furthermore, the properties of the bound fluorescence, with respect to sensitivity to photobleaching, also depend upon position in the cell cycle. The fluorescence behavior of labeled synchronized cell populations after treatment to inhibit de novo glycosylation is also reported. The results reported here may have implications concerning the structure of the opiate receptor complex and efforts to solubilize the binding protein in its native form.     

GTPase and adenylate cyclase desensitize at different rates in NG108-15 cells

The time course of opioid receptor binding disappearance and loss of responsiveness of the opioid-controlled GTPase and adenylate cyclase were compared in membranes derived from NG108-15 cells pretreated with the opioid peptide agonist [D-Ala2,D-Leu5]enkephalin (DADLE). Upon pretreatment with DADLE, a rapid desensitization of the opioid-stimulated GTPase occurred with a time course distinguishable as two exponential components having respective half-lives of 5-9 and 60-80 min. Opioid receptor binding activity, as assessed using [3H]diprenorphine, also decayed as two exponential components whose half-lives were similar to those for GTPase desensitization (7 and 120 min). However, when [3H]diprenorphine binding was measured in the presence of sodium and GTP, only the second, slow component was apparent. In contrast, desensitization of the opioid-controlled adenylate cyclase occurred as only one exponential decaying process, displaying a half-life of 57 min. Whereas the loss of responsiveness of GTPase to DADLE was entirely accounted for by a reduction in the maximal stimulation produced acutely by DADLE, desensitization of adenylate cyclase was characterized by both a decrease in maximal inhibition and a shift to the right of the EC50 of the agonist in inhibiting acutely the enzyme. In addition, after 1 hr of pretreatment with DADLE, the opioid-stimulated GTPase was desensitized by 65%, whereas 80% of maximal inhibition of adenylate cyclase could still be achieved. We suggest that: the rapid loss of responsiveness of the opioid-GTPase system results from an uncoupling between the receptor and the nucleotide-binding regulatory protein (N); the fast decaying GTPase activity appears to be not directly related to the opioid-mediated inhibition of adenylate cyclase; and the slow decaying GTPase activity, as well as the desensitization of the opioid-adenylate cyclase, is most likely accounted for by down-regulation of the opioid receptor. These findings may indicate that part of the opioid-stimulated GTPase in the membrane is not involved in inhibition of the cyclase and could reflect the activity of a regulatory protein which couples opioid receptors to another membrane effector. Alternatively, they might be interpreted on the basis of a model which involves a tight coupling between receptor activation and N protein and a large amplification mechanism between N protein and adenylate cyclase.     

Effects of neutral ionophores on membrane electrical characteristics of NG108-15 cells

The effects of several K(+)-selective neutral ionophores on membrane electrical characteristics of differentiated NG108-15 (neuroblastoma X glioma hybrid) cells were examined. Specifically, alterations in membrane resting potential (V(m)), input resistance (R(in)) and electrically-induced action potential generation were determined upon bath application of enniatin (0.1-10 microg/ml), nonactin (0. 1-10 microM) and valinomycin (0.1-10 microM). Although some cells exhibited a slight hyperpolarization and/or reduced R(in), i.e. membrane electrical correlates of enhanced K(+) loss, neither V(m) nor R(in) were significantly altered by any of the ionophores. However, valinomycin and especially nonactin affected action potentials induced by electrical stimulation. This was apparent in the ablation of action potentials in some cells and in the occurrence of degenerative changes in action potential shape in others. The simultaneous administration of the neutral ionophores and the protonophore CCCP or the superfusion of enniatin, nonactin or valinomycin in high (50 mM) glucose-containing physiological solution did not yield more extensive alterations in V(m) or R(in). These data suggest that the neutral ionophores are unable to materially enhance K(+) flux above the relatively high resting level in NG108-15 cells. Thus, alterations in action potentials appear to be unrelated to K(+) transport activity.     

Transformation of dopamine and alpha-methyldopamine by NG108-15 cells: formation of thiol adducts

The catecholamines, alpha-methyldopamine (alpha-MeDA) and dopamine (DA), have been implicated in 3,4-(methylenedioxy)amphetamine (MDA) toxicity. The toxicity and metabolic fate of alpha-MeDA, a metabolite of MDA, and DA, a neurotransmitter released by MDA administration, were examined in NG108-15 cells. Both catechols were found to accumulate intracellularly into NG108-15 cells. alpha-MeDA was about 4 times more toxic than DA in the cells. The depletion of glutathione (GSH) by buthionine sulfoximine (BSO) resulted in a drastic increase (10 times) in the alpha-MeDA mediated toxicity while the toxicity of DA was enhanced by 2 times. DA was largely metabolized to dihydroxyphenylacetic acid (DOPAC) and, to a smaller extent, formed an adduct with GSH. alpha-MeDA was primarily metabolized to a GSH adduct. alpha-MeDA was also metabolized to a product which was identified as the cysteinyl adduct. These adducts were identified by HPLC coelution with authentic standards. The GSH and cysteinyl adducts are presumably formed through conjugation of the thiols with intermediary quinone oxidation products of DA and alpha-MeDA. Previous studies indicate that alpha-MeDA is significantly more toxic than DA, especially under conditions of GSH depletion. The results of this study suggest that alpha-MeDA toxicity may occur through cytoplasmic accumulation and oxidation to a reactive quinone species followed by reaction with vital thiol functions or generation of reactive oxygen species. Cytoplasmic DA levels, on the other hand, appear to be significantly lower due to MAO metabolism and vesicular storage, and therefore, DA appears less likely to form conjugates with thiol groups or participate in possible redox cycling.     

Characterization of bradykinin receptors solubilized from rat uterus and NG108-15 cells

In this paper we report the solubilization of bradykinin B2 binding sites from membranes prepared from NG 108-15 tumours and rat uterus with retention of binding activity. Digitonin was found to solubilize the receptors from both tissues, and the addition of CHAPS increased the yield of soluble receptor from rat uterus only. The affinity of a range of bradykinin analogues has been shown to have the same rank order for both the soluble and membrane receptors from both tissues, and in corresponding functional assays. In addition, the binding of bradykinin ligands to the soluble and membrane receptors is similarly modulated by the presence of sodium ions. We conclude that the soluble binding sites correspond to the physiological bradykinin B2 receptor present in these tissues.     

Inhibition of ATP-induced cAMP formation by 5’-p-fluorosulfonylbenzoyladenosine in NG108-15 cells

ATP is known to increase intracellular cAMP levels in NG108-15 cells via a novel purinoceptor and this response is inhibited by the P₁ purinoceptor antagonist methylxanthine. In the present study, we examined the effects of 5’-p-fluorosulfonylbenzoyladenosine (FSBA), an affinity ligand for ATP-binding proteins, on cAMP formation mediated by activation of adenylate cyclase (AC)-linked purinoceptors in NG108-15 cells. cAMP levels were determined by RIA using an anti-succinyl-cAMP antiserum. FSBA (100 μM) increased intracellular cAMP about 2.6-fold. However, FSBA-induced cAMP formation was abolished by pretreatment with adenosine deaminase, suggesting that adenosine, a breakdown product of FSBA, is involved in FSBA-induced cAMP formation. In contrast, pretreatment of cells with FSBA in the presence of adenosine deaminase inhibited cAMP formation induced by ATP and β,γ-methylene-ATP (β,γ-MeATP), without affecting the prostaglandin E₁ (PGE₁)-induced response. The inhibitory effect of FSBA on ATP-induced cAMP formation was concentration-dependent with a concentration required for half-maximal inhibition (IC₅₀) of around 3 μM. The inhibitory effect of FSBA was not affected by pertussis toxin (PTX)-treatment. Pretreatment with FSBA (10 μM) depressed the maximal response to β,γ-MeATP by 60%, but did not affect the response to 5’-N-ethylcarboxamidoadenosine. The inhibitory effect of FSBA (100 μM) increased time-dependently during pretreatment and partly resisted wash-out. The inhibition by FSBA was protected by simultaneous addition of β,γ-MeATP during the FSBA pretreatment, indicating that both FSBA and the ATP analogue interacted with the same receptor site. The pretreatment with FSBA did not affect the increase in [Ca²⁺]_i induced by ATP, UTP or benzoylbenzoic ATP. These results suggest that FSBA inhibits cAMP accumulation induced in NG108-15 cells by ATP or related agonists by selective modification of an AC-linked purinoceptor. Received: 28 July 1997 / Accepted: 14 April 1998     

Delta-opioid-induced liberation of Gbetagamma mobilizes Ca2+ stores in NG108-15 cells.

Activation of delta-opioid receptors in NG108-15 cells releases Ca2+ from an intracellular store through activation of a pertussis toxin-sensitive G protein. We tested the hypothesis that activation of delta-opioid receptors mobilizes inositol 1,4,5-trisphosphate (IP(3))-sensitive Ca2+ stores via liberation of Gbetagamma. Fura-2-based digital imaging was used to study the mechanism of opioid-induced increases in [Ca2+](i) in NG108-15 cells. Exposure to D-Ala(2)-D-Leu(5) enkephalin (100 nM) for 90 s induced increases in [Ca2+](i) that were blocked by microinjection of the IP(3) receptor antagonist heparin (pipette concentration = 100 mg/ml) but not by sham injection. Microinjection of a peptide that binds Gbetagamma (QEHA, 1 mM) decreased the D-Ala(2)-D-Leu(5) enkephalin-evoked response. Microinjection of an inactive peptide (SKEE, 1 mM) that does not bind to Gbetagamma failed to inhibit the opioid-induced increase in [Ca2+](i). Microinjection of a peptide (QLKK, 15 mM) that binds to free Galpha(q) blocked the increase evoked by 3 nM bradykinin, but microinjection of an inactive peptide (ADRK, 15 mM) did not. Microinjection of QLKK did not significantly affect the opioid-induced increase in [Ca2+](i). Collectively, these data demonstrate that activation of delta-opioid receptors induces the release of Ca2+ from IP(3)-sensitive stores in NG108-15 cells through activation of the betagamma subunits of inhibitory G proteins.     

Slowing of ERG current deactivation in NG108-15 cells by the histidine-specific reagent diethylpyrocarbonate

The aim of this study was to explore and characterize the effect of the histidine-specific reagent diethylpyrocarbonate (DEPC) on the ERG (ether-à-go-go related gene) channels of whole-cell voltage-clamped NG108-15 neuroblastoma x glioma hybrid cells. The channels were fully activated by long depolarizing prepulses. Hyperpolarizing pulses elicited K+ inward currents which deactivated after reaching a peak. DEPC (0.26-2.1 mM, externally applied for 5-12 min) irreversibly decreased tau(-1), the rate constant of deactivation. At a pulse potential of -120 mV x tau(-1) decreased on average by 60%. The effect can be described as a -25 mV shift of the tau(-1)(V) curve. The activation curve and the curve relating steady-state current to pulse potential were shifted by similar amounts. The decrease of tau(-1) was the same at 0.26 and at 2.1 mM, but developed faster at the higher concentration. The slowing of deactivation was only seen when the cells were held at a potential of -20 mV. At this potential it developed with a time constant of 47 s. At more negative holding potentials (-40 or -70 mV) only a slight reduction of the peak occurred. The observations suggest preferential binding of DEPC to the open and inactivated channel states. The DEPC effect can possibly be explained by the reaction of DEPC with histidine residues or other amino acids in the external loops of the channel. However, dichloro-(2,2':6',2"-terpyridine)-platinum (II) dihydrate (DTPD), another histidine-specific reagent, markedly decreased the peak current without affecting tau(-1). Therefore, the possibility that (some of) the effects of DEPC and DTPD are unrelated to their property as histidine-specific reagents cannot be excluded.     

Effects of anions on ATP-induced [Ca2+], increase in NG108-15 cells

We investigated the effects of anions on different P2 receptors by measuring ATP-induced increase in intracellular Ca2+ concentration ([Ca2+]i) in fura-2-loaded NG108-15 and PC12 cells. In NG108-15 cells, ATP at 100 microM and 1 mM induced a transient and a sustained [Ca2+]i increase, respectively. The former, but not the latter, was inhibited by U-73122, indicating that the former was via the P2Y2 receptor and the latter via the P2X7 receptor. When external Cl- was replaced by other anions, the [Ca2+]i increase mediated by the P2Y2 receptor was not changed, but that mediated by the P2X7 receptor varied in the order of aspartate- > methanesulfonate > Cl- > Br > or = I-. In PC12 cells, transient [Ca2+]i increases mediated by the P2Y2 and P2X2 receptors were not affected by various anions. These results suggest that modulation by anions is unique to the P2X7 receptor and does not occur in P2Y2 and P2X2 receptors. This may be because the mechanism of ATP binding to the P2X7 receptor may be different than that to other P2 receptors.     

Chronic ethanol promotes the neuronal differentiation of NG108-15 cells independently of toxin-sensitive G-proteins

The ability of ethanol to promote neuronal differentiation of NG108-15 mouse neuroblastoma x rat glioma hybrid cells was investigated using morphological, biochemical and electrophysiological techniques. Ethanol concentration—(10–200 mM) and time—(1 h–3 days) dependently reduced cell proliferation, but increased acetylcholinesterase (AChE) activity and cell protein content. Chronic ethanol (200 mM) also time-dependently increased voltage-sensitive Ca²⁺ currents in the cells. Similar effects were obtained with chronic treatment of the cells with the standard differentiating agents sodium butyrate or forskolin. Chronic treatment of NG108-15 cells with primary alcohols (0.1–200 mM) of varying chain length all reduced cell proliferation and increased cell protein content and AChE activity with the potency order butanol>propanol>ethanol>methanol. Chronic treatment of NG108-15 cells with cholera toxin (50 ng ml⁻¹) or pertussis toxin (50 ng ml⁻¹) did not induce differentiation of the cells, nor did it modify the effects of 50 or 200 mM ethanol on cell proliferation, AChE activity or cellular protein content. Chronic cholera toxin did however abolish agonist-stimulated adenylyl cyclase activity in the cells, whereas pertussis toxin abolished receptor-mediated inhibition of adenylyl cyclase activity. Furthermore, inhibitors of protein kinase C (GF 109203X, 5 μM), protein kinase A (H-89, 10 μM) or Ca²⁺/calmodulin-dependent protein kinase II (KN-62, 3 μM) all failed to modify the effects of 200 mM ethanol on cell proliferation, AChE activity and cellular protein content. These experiments indicate that chronic ethanol is able to promote neuronal differentiation of NG108-15 cells independently of toxin-sensitive G-proteins and some protein kinases.