calcimycin and Neuroblastoma

Ca(2+) dysregulation is an important factor implicated in Alzheimer's disease pathogenesis. The mechanisms mediating the reciprocal regulation of Ca(2+) homeostasis and amyloid precursor protein (APP) metabolism, function, and protein interactions are not well known. We have previously shown that APP interacts with Homer proteins, which inhibit APP processing toward amyloid-β. In this study, we investigated the effect of Ca(2+) homeostasis alterations on APP/Homer3 interaction. Influx of extracellular Ca(2+) upon treatment of HEK293 cells with the ionophore A23187 or addition of extracellular Ca(2+) in cells starved of calcium specifically reduced APP/Homer3 but not APP/X11a interaction. Endoplasmic reticulum Ca(2+) store depletion by thapsigargin followed by store-operated calcium entry also decreased the interaction. Interestingly, application of a phospholipase C stimulator, which causes inositol 1,4,5-trisphosphate-induced endoplasmic reticulum Ca(2+) release, caused dissociation of APP/Homer3 complex. In human neuroblastoma cells, membrane depolarization also disrupted the interaction. This is the first study showing that changes in Ca(2+) homeostasis affect APP protein interactions. Our results suggest that Ca(2+) and Homers play a significant role in the development of Alzheimer's disease pathology.

Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Calcimycin; Calcium; Calcium Ionophores; Carrier Proteins; Endoplasmic Reticulum; HEK293 Cells; Homer Scaffolding Proteins; Humans; Membrane Potentials; Neuroblastoma; Protein Binding; Thapsigargin; Tumor Cells, Cultured

Immortalized neuronal cell lines can be induced to differentiate into more mature neurons by adding specific compounds or growth factors to the culture medium. This property makes neuronal cell lines attractive as in vitro cell models to study neuronal functions and neurotoxicity. The clonal human neuroblastoma BE(2)-M17 cell line is known to differentiate into a more prominent neuronal cell type by treatment with trans-retinoic acid. However, there is a lack of information on the morphological and functional aspects of these differentiated cells.. We studied the effects of trans-retinoic acid treatment on (a) some differentiation marker proteins, (b) types of voltage-gated calcium (Ca2+) channels and (c) Ca2+-dependent neurotransmitter ([3H] glycine) release in cultured BE(2)-M17 cells. Cells treated with 10 μM trans-retinoic acid (RA) for 72 hrs exhibited marked changes in morphology to include neurite extensions; presence of P/Q, N and T-type voltage-gated Ca2+ channels; and expression of neuron specific enolase (NSE), synaptosomal-associated protein 25 (SNAP-25), nicotinic acetylcholine receptor α7 (nAChR-α7) and other neuronal markers. Moreover, retinoic acid treated cells had a significant increase in evoked Ca2+-dependent neurotransmitter release capacity. In toxicity studies of the toxic gas, phosgene (CG), that differentiation of M17 cells with RA was required to see the changes in intracellular free Ca2+ concentrations following exposure to CG.. Taken together, retinoic acid treated cells had improved morphological features as well as neuronal characteristics and functions; thus, these retinoic acid differentiated BE(2)-M17 cells may serve as a better neuronal model to study neurobiology and/or neurotoxicity.

Topics: Antineoplastic Agents; Calcimycin; Calcium; Calcium Channel Blockers; Calcium Channels, N-Type; Calcium Ionophores; Cell Differentiation; Cell Line, Tumor; Cell Size; Chemical Warfare Agents; Choline O-Acetyltransferase; Gene Expression Regulation; Glycine; Humans; Nerve Tissue Proteins; Neuroblastoma; Neurotransmitter Agents; Phosgene; Potassium Chloride; Receptors, Cholinergic; Synapses; Synapsins; Tretinoin; Tritium; Tubulin

Mitochondrial oxidative stress plays important roles in aging and age-related degenerative disorders. The newly identified mitochondrial thioredoxin (mtTrx; Trx2) is a key component of the mitochondrial antioxidant system which is responsible for the clearance of reactive intermediates and repairs proteins with oxidative damage. Here, we show that in cultured SH-SY5Y human neuroblastoma 1cells, overexpression of mtTrx inhibited apoptosis and loss of mitochondrial membrane potential induced by a chemical oxidant, tert-butylhydroperoxide (tBH). The effects of calcium ionophore (Br-A23187) were not affected by mtTrx, suggesting the protection was specific against oxidative injury. The mitochondrial glutathione pool was oxidized by tBH, and this oxidation was not inhibited by increased mtTrx. Consequently, the antioxidant function of mtTrx is not redundant, but rather in addition, to that of GSH. Mutations of Cys90 and Cys93 to serines rendered mtTrx ineffective in protection against tBH-induced cytoxicity. These data indicate that mtTrx controls the mitochondrial redox status independently of GSH and is a key component of the defensive mechanism against oxidative stress in cultured neuronal cells.

Topics: Anti-Bacterial Agents; Apoptosis; Calcimycin; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Drug Antagonism; Glutathione; Humans; Mitochondrial Proteins; Neuroblastoma; Neurons; Oxidants; Oxidation-Reduction; tert-Butylhydroperoxide; Thioredoxins

Each skeletal muscle of the body contains a unique composition of "fast" and "slow" muscle fibers, each of which is specialized for certain challenges. This composition is not static, and the muscle fibers are capable of adapting their molecular composition by altered gene expression (i.e., fiber type conversion). Whereas changes in the expression of contractile proteins and metabolic enzymes in the course of fiber type conversion are well described, little is known about possible adaptations in the electrophysiological properties of skeletal muscle cells. Such adaptations may involve changes in the expression and/or function of ion channels. In this study, we investigated the effects of fast-to-slow fiber type conversion on currents via voltage-gated Na+ channels in the C(2)C(12) murine skeletal muscle cell line. Prolonged treatment of cells with 25 nM of the Ca2+ ionophore A-23187 caused a significant shift in myosin heavy chain isoform expression from the fast toward the slow isoform, indicating fast-to-slow fiber type conversion. Moreover, Na+ current inactivation was significantly altered. Slow inactivation less strongly inhibited the Na+ currents of fast-to-slow fiber type-converted cells. Compared with control cells, the Na+ currents of converted cells were more resistant to block by tetrodotoxin, suggesting enhanced relative expression of the cardiac Na+ channel isoform Na(v)1.5 compared with the skeletal muscle isoform Na(v)1.4. These results imply that fast-to-slow fiber type conversion of skeletal muscle cells involves functional adaptation of their electrophysiological properties.

Topics: Animals; Calcimycin; Calcium; Cell Line, Tumor; Electric Stimulation; Ion Channel Gating; Ionophores; Membrane Potentials; Mice; Mice, Inbred C3H; Muscle Fibers, Fast-Twitch; Muscle Fibers, Slow-Twitch; Muscle, Skeletal; Myosin Heavy Chains; Neuroblastoma; Patch-Clamp Techniques; Sodium Channels

Inhibition of protein translation plays an important role in apoptosis. While double-stranded RNA-dependent protein kinase (PKR) is named as it is activated by double-stranded RNA produced by virus, its activation induces an inhibition of protein translation and apoptosis via the phosphorylation of the eukaryotic initiation factor 2alpha (eIF2alpha). PKR is also a stress kinase and its levels increase during ageing. Here we show that PKR activation and eIF2alpha phosphorylation play a significant role in apoptosis of neuroblastoma cells and primary neuronal cultures induced by the beta-amyloid (Abeta) peptides, the calcium ionophore A23187 and flavonoids. The phosphorylation of eIF2alpha and the number of apoptotic cells were enhanced in over-expressed wild-type PKR neuroblastoma cells exposed to Abeta peptide, while dominant-negative PKR reduced eIF2alpha phosphorylation and apoptosis induced by Abeta peptide. Primary cultured neurons from PKR knockout mice were also less sensitive to Abeta peptide toxicity. Activation of PKR and eIF2alpha pathway by Abeta peptide are triggered by an increase in intracellular calcium because the intracellular calcium chelator BAPTA-AM significantly reduced PKR phosphorylation. Taken together, these results reveal that PKR and eIF2alpha phosphorylation could be involved in the molecular signalling events leading to neuronal apoptosis and death and could be a new target in neuroprotection.

Topics: Amyloid beta-Peptides; Animals; Apoptosis; Calcimycin; Calcium; Cell Count; eIF-2 Kinase; Eukaryotic Initiation Factor-2; Genistein; Humans; Ionophores; Mice; Mice, Inbred C57BL; Neuroblastoma; Neurons; Peptide Fragments; Phosphorylation; Quercetin; Rats; Rats, Sprague-Dawley

Intracellular calcium ([Ca2+](i)), cell volume, membrane potential and currents were measured in neuroblastomaxglioma hybrid cells to gain insight into how [Ca2+](i) controls cell volume. [Ca2+](i) was increased by fluid shear stress, mechanical stimulation of the cells, the Ca2+ ionophore A23187, caffeine and thapsigargin. The increase in [Ca2+](i) induced by mechanical stimulation was decreased by about 50% by caffeine and abolished after incubation of the cells in a Ca2+-free solution. Mechanical stimulation by stirring the cell suspension induced cell shrinkage that was abolished by caffeine, but induced cell swelling in Ca2+-free solution. In the presence of caffeine, A23187 induced cell shrinkage whereas thapsigargin induced cell swelling. Both cell volume changes were inhibited by the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid. The cells were hyperpolarized by fluid shear stress and A23187 and depolarized by caffeine, thapsigargin and intracellular EGTA. Under all these conditions, the membrane input resistance was decreased. Voltage-clamp experiments suggested that, in addition to an increased anionic current, fluid shear stress and A23187 increased a K+ current, whereas caffeine and intracellular Ca2+ chelation increased a non-selective cation current and thapsigargin increased both a K+ and a non-selective cation current. Taken together, these results suggest that, if cell volume is closely dependent on [Ca2+](i) and the activity of Cl- channels, its relative value is dependent on the ionic selectivity of co-activated channels and the membrane potential.

Topics: Brain Neoplasms; Caffeine; Calcimycin; Calcium; Cell Size; Chloride Channels; Electric Stimulation; Electrophysiology; Glioma; Humans; Hybrid Cells; Ion Channels; Ionophores; Membrane Potentials; Neuroblastoma; Nitrobenzoates; Patch-Clamp Techniques; Phosphodiesterase Inhibitors; Physical Stimulation; Thapsigargin; Tumor Cells, Cultured

The E693G (Arctic) mutation of the amyloid precursor protein was recently found to lead to early-onset Alzheimer's disease in a Swedish family. In the present study, we report that the Arctic mutation decreases cell viability in human neuroblastoma cells. The cell viability, as measured by the MTT assay and propidium iodide staining, was further compromised following exposure to calcium ionophore A23187, microtubule-binding colchicine or oxidative stress inducer hydrogen peroxide. The manner of cell death was found to be apoptotic. During apoptosis, cells with the Arctic mutation also decreased their secretion of beta-secretase cleaved amyloid precursor protein. The enhanced sensitivity to toxic stress in cells with the Arctic mutation most likely contributes to the pathogenic pathway leading to Alzheimer's disease.

Topics: Alkaloids; Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Apoptosis; Blotting, Western; Calcimycin; Colchicine; Humans; Hydrogen Peroxide; Ionophores; Mutation; Neuroblastoma; Tumor Cells, Cultured

Cyclooxygenases-1 and -2 are both expressed in neuronal cells in vivo. In the neuroblastoma cell lines NG108 and N2a, however, only cyclooxygenase-1 was detectable. Differentiation of the cells with retinoic acid increased cyclooxygenase-1 mRNA and protein expression within 24 and 48 h, respectively. A further increase was observed when the cells were concomitantly treated with the glucocorticoid dexamethasone (a 2-3-fold increase compared with retinoic acid alone). In the absence of retinoic acid, dexamethasone only slightly up-regulated cyclooxygenase-1 expression. The inhibitor of protein synthesis cycloheximide abrogated the effect of dexamethasone, indicating the involvement of newly synthesised proteins. Retinoic acid increased the transcription of cyclooxygenase-1 mRNA, determined with a luciferase-coupled promoter construct. Dexamethasone only slightly augmented cyclooxygenase-1-promoter activity but increased cyclooxygenase-1 mRNA stability. Other corticosteroids, hydrocortisone and aldosterone, also up-regulated cyclooxygenase-1 whereas neurosteroids or oestrogen were ineffective. Up-regulation was mediated primarily by the glucocorticoid receptor, because the receptor antagonist RU486 strongly reduced the effects of all corticosteroids. This indicated that in NG108 cells, the mineralocorticoid aldosterone may bind to the glucocorticoid receptor. Treatment of NG108 or N2a cells with corticosteroids did not alter the morphological phenotype obtained during differentiation. We thus show that corticosteroids, which down-regulate cyclooxygenase expression in most cell types, up-regulate cyclooxygenase-1 during neuronal differentiation.

Topics: Adrenal Cortex Hormones; Aldosterone; Animals; Benzimidazoles; Bucladesine; Calcimycin; Cell Differentiation; Cycloheximide; Cyclooxygenase 1; Cyclooxygenase 2; Dehydroepiandrosterone; Dehydroepiandrosterone Sulfate; Dexamethasone; Dinoprostone; Drug Synergism; Enzyme Induction; Estradiol; Gene Expression Regulation, Neoplastic; Genes, Reporter; Glioma; Hybrid Cells; Hydrocortisone; Ionophores; Isoenzymes; Luciferases; Membrane Proteins; Mice; Mifepristone; Neoplasm Proteins; Nerve Tissue Proteins; Neuroblastoma; Promoter Regions, Genetic; Prostaglandin-Endoperoxide Synthases; Protein Synthesis Inhibitors; Receptors, Glucocorticoid; Recombinant Fusion Proteins; RNA, Messenger; RNA, Neoplasm; Tetradecanoylphorbol Acetate; Transfection; Tretinoin; Tumor Cells, Cultured

Presenilins (PSs) are mutated in a majority of familial Alzheimer disease (FAD) cases. Mutated PSs may cause FAD by a number of pro-apoptotic mechanisms, or by regulating gamma-secretase activity, a protease involved in beta-amyloid precursor protein processing to the neurotoxic beta-amyloid peptide. Besides their normal endoproteolytic processing, PSs are substrates for caspases, being cleaved to alternative N-terminal and C-terminal fragments. So far little is known about the role of PSs cleavage in the apoptotic machinery. Here, we used SH-SY5Y neuroblastoma cells stably transfected with wild-type or exon 9 deleted presenilin 1 (PS1) in a time-course study after the exposure to the calcium ionophore A23187. During and after exposure to A 23187, intracellular calcium levels were higher in exon 9 deleted PS1 cells as compared with non-transfected and wild-type PS1 transfected cells. Cell death and the enrichment of apoptotic cells after A23187 exposure were increased by overexpression of exon 9 deleted PS1 as compared with the control cell lines. Wild-type PS1 cells were compared with exon 9 deleted PS1 cells and the temporal relationship between PS1 and other caspase substrates cleavages was analyzed. Exon 9 deleted PS1 cells exhibited a higher caspase-3 activation and a greater cleavage of PS1 and poly(ADP-ribose) polymerase (PARP) compared with wild-type PS1 cells. Exon 9 deleted PS1 cleavage occurred earlier than other caspase substrate cleavages (i.e., PARP and gelsolin), simultaneous with minimum detectable caspase-3 activation. Therefore, alternative cleavage of PS1 may play an important role for the regulation of the proteolytic cascade activated during apoptosis.

Topics: Apoptosis; Buffers; Calcimycin; Calcium; Caspases; Cell Adhesion; Exons; Gene Deletion; Gene Expression; Homeostasis; Humans; Ionophores; Membrane Proteins; Neuroblastoma; Neurons; Presenilin-1; Transfection; Tumor Cells, Cultured

Comparative analyses were conducted to determine the effects of Na(+) (monensin, MON), K(+) (nigericin, NIG) and Ca(2+) (A23187) selective carboxylic ionophores on differentiated NG108-15 (neuroblastoma X glioma hybrid) cells. Alterations in membrane potential (V(m)), input resistance (Rin) and electrically induced action potential generation were measured using intracellular microelectrode techniques in cells treated with 0.1-30 microM MON and NIG and 0.1-10 microM A23187. Responses to the ionophores were similar in that membrane hyperpolarization and unchanged R(in) predominated with all three compounds. However, significant differences between the ionophores were also detected. MON- and A23187-induced hyperpolarization was generally maintained throughout the 24-min superfusion whereas that produced by NIG diminished with time or was replaced by depolarization. In addition, action potential generation was blocked by NIG, whereas MON had no effect and action potential alterations were evident only with the highest A23187 concentration (10 microM). This study represents the initial comprehensive analysis of the effects of carboxylic ionophores on membrane electrical characteristics of an intact cell system and forms the basis for subsequent work using NG108-15 cells as a model system to evaluate potential therapeutic treatments against the carboxylic ionophores.

Topics: Calcimycin; Dose-Response Relationship, Drug; Glioma; Ionophores; Membrane Potentials; Monensin; Neuroblastoma; Neurons; Nigericin; Tumor Cells, Cultured

The immunophilin FKBP12 associates with intracellular Ca2+ channels and this interaction can be disrupted by the immunosuppressant FK506. We have investigated the effect of FK506 on Ca2+ release and Ca2+ uptake in permeabilized cell types. Changes in medium free [Ca2+] were detected by the fluorescent Ca2+ indicator fluo-3 in digitonin-permeabilized SH-SY5Y human neuroblastoma cells, DT40 and R23-11 (i.e. triple inositol 1,4,5-trisphosphate (IP3) receptor knockout cells) chicken B lymphocytes and differentiated and undifferentiated BC3H1 skeletal muscle cells. 45Ca2+ fluxes were studied in saponin-permeabilized A7r5 rat smooth muscle cells. Addition of FK506 to permeabilized SH-SY5Y cells led to a sustained elevation of the medium [Ca2+] corresponding to approximately 30 % of the Ca2+ ionophore A23187-induced [Ca2+] rise. This rise in [Ca2+] was not dependent on mitochondrial activity. This FK506-induced [Ca2+] rise was related to the inhibition of the sarcoplasmic/endoplasmic reticulum Ca2+-Mg2+-ATPase (SERCA) Ca2+ pump. Oxalate-facilitated 45Ca2+ uptake in SH-SY5Y microsomes was inhibited by FK506 with an IC50 of 19 microM. The inhibition of the SERCA Ca2+ pump was not specific since several macrocyclic lactone compounds (ivermectin > FK506, ascomycin and rapamycin) were able to inhibit Ca2+ uptake activity. FK506 (10 microM) did not affect IP3-induced Ca2+ release in permeabilized SH-SY5Y and A7r5 cells, but enhanced caffeine-induced Ca2+ release via the ryanodine receptor (RyR) in differentiated BC3H1 cells. In conclusion, FK506 inhibited active Ca2+ uptake by the SERCA Ca2+ pump; in addition, FK506 enhanced intracellular Ca2+ release through the RyR, but it had no direct effect on IP3-induced Ca2+ release.

Topics: Animals; Antiprotozoal Agents; Aorta; B-Lymphocytes; Biological Transport; Caffeine; Calcimycin; Calcium; Calcium Channels; Calcium Signaling; Calcium-Transporting ATPases; Chickens; Enzyme Inhibitors; Humans; Immunosuppressive Agents; Inositol 1,4,5-Trisphosphate Receptors; Ionophores; Ivermectin; Mice; Microsomes; Muscle, Smooth, Vascular; Neuroblastoma; Oxalates; Phosphodiesterase Inhibitors; Rats; Receptors, Cytoplasmic and Nuclear; Sirolimus; Spermine; Tacrolimus; Thapsigargin; Tumor Cells, Cultured

Large-conductance anion channel characteristics were investigated in neuroblastoma cells (N2A) by using different configurations of the patch-clamp technique. In excised patches, the channel was induced by depolarising potentials in 90% of experiments, had a conductance of 340 pS in symmetrical 135 mmol/l NaCl and exhibited the typical bell-shape activity. Neither the channel induction nor the channel activity was affected by rising the Ca2+ concentration on the cytopasmic side of membranes. In cell-attached configuration the maximal channel activity was shifted towards more positive potentials in comparison to that of excised patches and an increase in intracellular Ca2+, obtained by extracellular application of the Ca2+-ionophore A23187 in the presence of 0.2 micromol/l Ca2+, induced single-channel currents in 80% of patches compared to 31% of cell-attached experiments showing channel activity in normal conditions. In turn, application of 2 micromol/l Ca2+ induced channel activity in 100% of patches. The reversal potential of the channel in cell-attached patches was around -10 mV as the resting potential of cells eliciting channel activity. For cells where channel activity was not detected in cell-attached mode, the resting potential was around -45 mV. Channel activity could be restored in most whole-cell recordings in the presence of 2 micromol/l or more intracellular Ca2+ concentrations. The Ca2+-induction and the relation between channel activity and cell resting potential seem to suggest a role of the large-conductance anion channel in resting potential modulation during some basic functions of the neuroblastoma cell proliferation.

Topics: Animals; Anions; Calcimycin; Calcium Channels; Cell Division; Ionophores; Kinetics; Neuroblastoma; Patch-Clamp Techniques; Rats; Sodium Chloride; Time Factors; Tumor Cells, Cultured

Our previous studies have shown that noradrenaline release is enhanced by activation of protein kinase Calpha in SH-SY5Y cells. In the present study, we report that activation of protein kinase Calpha leads to (a) partial redistribution of the F-actin cytoskeleton and (b) a 2.5-fold increase in the number of large dense-cored vesicles within 100 nm of the plasma membrane. This redistribution can be prevented by down-regulation of protein kinase Calpha by up to 48 h exposure to phorbol dibutyrate. Treatment with the secretagogues 100 mM KCl, the Ca2+ ionophore A23187 (20 microM) and 1 mM carbachol also leads to a partial disassembly of the F-actin cytoskeleton. This is accompanied by an increase in the number of large dense cored vesicles at the plasma membrane following exposure to KCl and A23187 but not following exposure to carbachol. These results are discussed in relation to the hypothesis that a key step in the enhancement of noradrenaline release following activation of protein kinase Calpha and elevation of intracellular calcium is the movement of large dense cored vesicles to the plasma membrane following partial disassembly of the F-actin cytoskeleton.

Topics: Actins; Calcimycin; Carbachol; Cell Membrane; Down-Regulation; Enzyme Activation; Humans; Ionophores; Isoenzymes; Liposomes; Neuroblastoma; Potassium; Protein Kinase C; Protein Kinase C-alpha; Secretory Rate; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

To determine if neurochemical function might be impaired in cell models with altered cholesterol balance, we studied the effects of U18666A (3-beta-[(2-diethyl-amino)ethoxy]androst-5-en-17-one) on intracellular cholesterol metabolism in three human neuroblastoma cell lines (SK-N-SH, SK-N-MC, and SH-SY5Y). U18666A (< or =0.2 microg/ml) completely inhibited low density lipoprotein (LDL)-stimulated cholesterol esterification in SK-N-SH cells, while cholesterol esterification stimulated by 25-hydroxycholesterol or bacterial sphingomyelinase was unaffected or partially inhibited, respectively. U18666A also blocked LDL-stimulated downregulation of LDL receptor and caused lysosomal accumulation of cholesterol as measured by filipin staining. U18666A treatment for 18 h resulted in 70% inhibition of K+-evoked norepinephrine release in phorbol ester-differentiated SH-SY5Y cells, while release stimulated by the calcium ionophore A23187 was only slightly affected. These results suggest that U 18666A may preferentially block a voltage-regulated Ca2+ channel involved in norepinephrine release and that alterations in neurotransmitter secretion might be a feature of disorders such as Niemann-Pick Type C, in which intracellular cholesterol transport and distribution are impaired.

Topics: Androstenes; Anticholesteremic Agents; Biological Transport; Calcimycin; Calcium Channels; Cholesterol; Culture Media; Down-Regulation; Humans; Hydroxycholesterols; Kinetics; Lysosomes; Neuroblastoma; Norepinephrine; Potassium; Receptors, LDL; Sphingomyelin Phosphodiesterase; Staphylococcus aureus; Tumor Cells, Cultured

Missense mutations in the human presenilin-1 (PS1) gene, which is found on chromosome 14, cause early-onset familial Alzheimer's disease (FAD). FAD-linked PS1 variants alter proteolytic processing of the amyloid precursor protein and cause an increase in vulnerability to apoptosis induced by various cell stresses. However, the mechanisms responsible for these phenomena are not clear. Here we report that mutations in PS1 affect the unfolded-protein response (UPR), which responds to the increased amount of unfolded proteins that accumulate in the endoplasmic reticulum (ER) under conditions that cause ER stress. PS1 mutations also lead to decreased expression of GRP78/Bip, a molecular chaperone, present in the ER, that can enable protein folding. Interestingly, GRP78 levels are reduced in the brains of Alzheimer's disease patients. The downregulation of UPR signalling by PS1 mutations is caused by disturbed function of IRE1, which is the proximal sensor of conditions in the ER lumen. Overexpression of GRP78 in neuroblastoma cells bearing PS1 mutants almost completely restores resistance to ER stress to the level of cells expressing wild-type PS1. These results show that mutations in PS1 may increase vulnerability to ER stress by altering the UPR signalling pathway.

Topics: Alzheimer Disease; Animals; Brain; Calcimycin; Carrier Proteins; Cell Death; Cell Line; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Endoribonucleases; Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Humans; Intracellular Membranes; Membrane Proteins; Mice; Mice, Transgenic; Molecular Chaperones; Mutation; Neuroblastoma; Neurons; Phosphorylation; Presenilin-1; Protein Binding; Protein Denaturation; Protein Folding; Protein Serine-Threonine Kinases; RNA, Messenger; Signal Transduction; Transfection; Tunicamycin

Extensive necrotic death of MSN neuroblastoma cells could be induced after incubation with the calcium ionophore, A23187. The reaction was concentration-dependent and time course-dependent. Levels of the 66 kd/alpha-internexin neurofilament protein (NF-66) and the cognate heat shock protein 70 (Hsc 70) decreased during the Ca2+-activated cell death. Addition of the calcium chelator, ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) restored the normal level of NF-66 and partially that of the Hsc 70. Use of either calpain I or calpain II inhibitor could alleviate the reduction of 66 kd protein during the ionophore treatment whereas only calpain I inhibitor treatment was effective in restoring the normal level of the Hsc 70. Neither of these calpain inhibitors could block the ionophore triggered cell death. EGTA was toxic to cells in a wide range of concentration suggesting a calcium-independent activation of cell death mechanism.

Topics: Calcimycin; Calpain; Carrier Proteins; Cell Death; HSC70 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Humans; Intermediate Filament Proteins; Leupeptins; Nerve Tissue Proteins; Neuroblastoma; Neurofibrils; Oligopeptides; Tumor Cells, Cultured

The vasoactive intestinal peptide cytokine response element (VIP CyRE) is responsible for mediating the transcriptional induction of the VIP gene to the neuropoietic cytokines leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF). In investigating the sequence and function of the CyRE, we found a region of DNA with homology to the distal NFAT site in the IL-2 promoter. In this paper we characterize this sequence and show that the VIP NFAT site recognizes T cell NFAT with similar affinity to the previously characterized IL-2 NFAT site. However, despite its location in the middle of the CyRE, we find no CNTF/LIF induced binding to it. Instead we show that in NBFL neuroblastoma cells, the calcium ionophore A23187 induces a protein to bind to the VIP NFAT site. This A23187-mediated induction of nuclear protein binding to an NFAT oligonucleotide is dependent on extracellular calcium but not dependent on de novo protein synthesis. Thus, this protein has the characteristics of an NFAT-like protein and is recognized by an NFAT3-specific antiserum suggesting that it is indeed an NFAT protein. The location of the NFAT site in the VIP CyRE suggests that this may be one mechanism through which different signaling pathways engage in cross talk to alter VIP gene transcription.

Topics: Animals; Base Sequence; Binding Sites; Calcimycin; Ciliary Neurotrophic Factor; Cytokines; DNA; DNA Primers; DNA-Binding Proteins; Growth Inhibitors; Humans; Interleukin-6; Leukemia Inhibitory Factor; Lymphokines; Mutagenesis, Site-Directed; Nerve Growth Factors; Nerve Tissue Proteins; Neuroblastoma; NFATC Transcription Factors; Nuclear Proteins; Recombinant Proteins; Regulatory Sequences, Nucleic Acid; Signal Transduction; Transcription Factors; Transcription, Genetic; Transfection; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Regulation of the microM-calcium-requiring form of calpain (mu calpain) was studied in SH-SY-5Y human neuroblastoma cells. Immunoblot analysis demonstrated that the vast majority of mu calpain is localized within cytosolic pools. Calpain activation was monitored as a function of autolysis within intact cells following calcium influx from the culture medium by calcium ionophores A23187 or ionomycin, or following release of calcium from intracellular stores by thapsigargin. Within intact neuronal cells, following an influx of calcium into the cytosolic from either extracellular or intracellular sources, mu calpain is preferentially activated at the plasma membrane as evidenced by autolytic generation of faster-migrating isoforms. By contrast, similar autolytic profiles for mu calpain in membrane or cytosolic fractions following addition of calcium were observed under cell-free conditions and within cells following death due to extended ionophore-mediated calcium influx. These differential activation profiles for cytosolic mu calpain within living cells and following cellular fractionation/cell death indicate the presence of a regulatory system within neuronal cells. As in previous studies in other systems, we demonstrate the presence of a calpain activator protein. Cycloheximide treatment depleted the autolytic capacity of membrane-associated mu calpain within 4-6 hr without a corresponding decline in total mu calpain protein levels, indicating that the activator protein undergoes rapid turnover in comparison to calpain; pulse-chase radiolabeling confirmed the half-life of mu calpain to exceed 24 hr. Our data suggest that this labile protein represents a major rate-limiting step for in situ calpain activation within neuronal cells, and that, given the tremendous latent mu calpain activity within the cytosol, the interplay of the activator protein and the endogenous inhibitor calpastatin are crucial for maintaining neuronal homeostasis.

Topics: Calcimycin; Calcium; Calpain; Cell Membrane; Cycloheximide; Cytosol; Enzyme Activation; Enzyme Inhibitors; Enzyme Precursors; Half-Life; Humans; Ionomycin; Ionophores; Isoenzymes; Membrane Proteins; Neoplasm Proteins; Nerve Tissue Proteins; Neuroblastoma; Neurons; Protein Synthesis Inhibitors; Thapsigargin; Tumor Cells, Cultured

SH-SY-5Y human neuroblastoma cells were treated with 22 microM of a synthetic peptide corresponding to amino acid residues 25-35 of beta-amyloid (betaA) or 3 microM calcium ionophore A23187 in culture medium containing 1.8 mM extracellular calcium. Both agents increased tau immunoreactivity towards antibodies (PHF-1, ALZ-50) that recognize epitopes common with paired helical filaments (PHFs) and towards an antibody (5E2) that recognized a phosphate-independent tau epitope. However, only ionophore increased immunoreactivity with an additional phosphate-dependent antibody (AT-8) that recognized an epitope of tau when phosphorylated, and induced a corresponding decrease in immunoreactivity towards an additional antibody (Tau-1) that recognizes the same site when that site is not phosphorylated. Moreover, the ionophore-mediated increase in PHF-1 was blocked by EGTA, by the calpain inhibitor calpeptin and by the PKC inhibitor H7, while that evoked by betaA treatment was not inhibited by any of these treatments. Since ionophore-mediated calpain activation induces proteolytic PKC activation, we further examined the influence of PKC inhibition on betaA and ionophore-mediated PHF-1 induction. Antisense oligonucleotide-mediated downregulation of PKCepsilon in a stable transfectant SH-SY-5Y subclone diminished the ionophore-mediated, but not the betaA-mediated, increase in PHF-1 immunoreactivity. These data indicate specific differences in the intracellular cascade of events invoked by betaA and ionophore A23187. Moreover, although betaA invoked calcium influx in these cells, our findings further suggest that the induction of tau hyperphosphorylation by betaA may not be due to calcium influx.

Topics: Amyloid beta-Peptides; Antibodies, Monoclonal; Antibody Specificity; Biomarkers; Calcimycin; Calcium; Calpain; Epitopes; Humans; Ionophores; Neuroblastoma; Phosphoproteins; Phosphorylation; Protein Kinase C; tau Proteins; Tumor Cells, Cultured

We report the pharmacological characterization and cytoprotective effect of DY-9760e, 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-( 4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate, a novel antagonist of calmodulin. DY-9760e inhibited calmodulin-dependent enzymes, including calmodulin-dependent protein kinase II and IV, calcineurin, [corrected] calmodulin-dependent phosphodiesterase and myosin light chain kinase with Ki values of 1.4, 12, 2.0, 3.8 and 133 microM, respectively. These antagonistic effects of DY-9760e were more potent than those of W-7, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, another calmodulin antagonist. This compound showed little or no effect on calmodulin-independent enzymes, such as protein kinase A and C and calpain I and II. Analysis of the hydrophobic interaction of DY-9760e with calmodulin by using 2-p-toluidinylnaphthalene-6-sulfonate and 9-anthroylcholine revealed that, like W-7, DY-9760e bound to the hydrophobic regions of calmodulin. The [14C]DY-9760e binding assay indicated that DY-9760e bound to calmodulin at one class of binding site. Finally, DY-9760e substantially protected N1E-115 neuroblastoma cells from cytotoxicity induced by the Ca2+ ionophore, A23187. These results indicate that DY-9760e, a novel calmodulin antagonist, possesses a cytoprotective action and suggest that calmodulin plays a critical role in mediating some of the biochemical events leading to cell death following Ca2+ overload.

Topics: Calcimycin; Calcineurin Inhibitors; Calcium; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Cell Death; Indazoles; Ionophores; Neuroblastoma; Sulfonamides; Tumor Cells, Cultured

We expressed the gamma-subspecies of protein kinase C (gamma-PKC) fused with green fluorescent protein (GFP) in various cell lines and observed the movement of this fusion protein in living cells under a confocal laser scanning fluorescent microscope. gamma-PKC-GFP fusion protein had enzymological properties very similar to that of native gamma-PKC. The fluorescence of gamma-PKC- GFP was observed throughout the cytoplasm in transiently transfected COS-7 cells. Stimulation by an active phorbol ester (12-O-tetradecanoylphorbol 13-acetate [TPA]) but not by an inactive phorbol ester (4alpha-phorbol 12, 13-didecanoate) induced a significant translocation of gamma-PKC-GFP from cytoplasm to the plasma membrane. A23187, a Ca2+ ionophore, induced a more rapid translocation of gamma-PKC-GFP than TPA. The A23187-induced translocation was abolished by elimination of extracellular and intracellular Ca2+. TPA- induced translocation of gamma-PKC-GFP was unidirected, while Ca2+ ionophore-induced translocation was reversible; that is, gamma-PKC-GFP translocated to the membrane returned to the cytosol and finally accumulated as patchy dots on the plasma membrane. To investigate the significance of C1 and C2 domains of gamma-PKC in translocation, we expressed mutant gamma-PKC-GFP fusion protein in which the two cysteine rich regions in the C1 region were disrupted (designated as BS 238) or the C2 region was deleted (BS 239). BS 238 mutant was translocated by Ca2+ ionophore but not by TPA. In contrast, BS 239 mutant was translocated by TPA but not by Ca2+ ionophore. To examine the translocation of gamma-PKC-GFP under physiological conditions, we expressed it in NG-108 cells, N-methyl-D-aspartate (NMDA) receptor-transfected COS-7 cells, or CHO cells expressing metabotropic glutamate receptor 1 (CHO/mGluR1 cells). In NG-108 cells , K+ depolarization induced rapid translocation of gamma-PKC-GFP. In NMDA receptor-transfected COS-7 cells, application of NMDA plus glycine also translocated gamma-PKC-GFP. Furthermore, rapid translocation and sequential retranslocation of gamma-PKC-GFP were observed in CHO/ mGluR1 cells on stimulation with the receptor. Neither cytochalasin D nor colchicine affected the translocation of gamma-PKC-GFP, indicating that translocation of gamma-PKC was independent of actin and microtubule. gamma-PKC-GFP fusion protein is a useful tool for investigating the molecular mechanism of gamma-PKC translocation and the role of gamma-PKC in the central nervous sys

Topics: 3T3 Cells; Amino Acid Sequence; Animals; Calcimycin; Calcium; CHO Cells; COS Cells; Cricetinae; Cytochalasin D; Glioma; Green Fluorescent Proteins; Hybrid Cells; Isoenzymes; Kinetics; Luminescent Proteins; Mice; Microscopy, Confocal; Molecular Sequence Data; Neuroblastoma; Protein Kinase C; Receptors, N-Methyl-D-Aspartate; Recombinant Fusion Proteins; Tetradecanoylphorbol Acetate; Transfection

Calcium influx into SH-SY5Y human neuroblastoma cells after ionophore treatment or transient permeabilization in calcium-containing medium increased ALZ-50 immunoreactivity markedly. This increase was prevented by inhibitors active against calpain or against protein kinase C (PKC), suggesting that both of these enzymes were required to mediate the effect of calcium influx on ALZ-50 immunoreactivity. Treatment with PKC activator TPA increased ALZ-50 immunoreactivity in the absence of calcium influx or after intracellular delivery of the specific calpain inhibitor calpastatin, indicating that the influence of PKC was downstream from that of calpain. Calcium influx also resulted in mu-calpain autolysis (one index of calpain activation) and the transient appearance of PKM (i.e., free PKC catalytic subunits, generated by calpain-mediated cleavage of the regulatory and catalytic PKC domains). Inhibition of calpain within intact cells resulted in a dramatic increase in steady-state levels of total tau (migrating at 46-52 kDa) but resulted in a relatively minor increase in 68-kDa ALZ-50-immunoreactive tau isoforms. Although calcium influx into intact cells resulted in accumulation of ALZ-50 immunoreactivity, total tau levels were, by contrast, rapidly depleted. Incubation of isolated fractions with calpain in the presence of calcium indicated that ALZ-50-immunoreactive tau isoforms were more resistant to calpain-mediated proteolysis than were non-ALZ-50 reactive tau isoforms. These data therefore indicate that calpain may regulate tau levels directly via proteolysis and indirectly through PKC activation. A consequence of the latter action is altered tau phosphorylation, perhaps involving one or more kinase cascades, and the preferential accumulation of ALZ-50-immunoreactive tau isoforms due to their relative resistance to degradation. These findings provide a basis for the possibility that disregulation of calcium homeostasis may contribute to the pathological levels of conversion of tau to A68 by hyperactivation of the calpain/PKC system.

Topics: Antigens; Calcimycin; Calcium; Calpain; Endopeptidases; Humans; Hydrolysis; Immunoblotting; Isomerism; Nerve Tissue Proteins; Neuroblastoma; Phosphorylation; Protein Kinase C; tau Proteins; Tumor Cells, Cultured

The human neuroblastoma SH-SY5Y was found to express annexins I, II, IV, V, and VI by western blot analysis. Calcium-dependent membrane-binding proteins were isolated from SH-SY5Y and analysed by 2-dimensional gel electrophoresis. Proteins with Mr and Pi values similar to those of annexins I, II, III, IV, V, and VI were observed. The identity of annexins II and V was confirmed by western blotting. The membrane association of annexins II and V was studied in cells that had been stimulated to release noradrenaline by K+ depolarisation or by treatment with the ionophore A23187. Annexins II and V were both found to associate with membranes in a manner that was resistant to elution with EGTA and required Triton X-100 for their solubilisation. Homogenisation of cells in calcium-containing buffers also resulted in the formation of EGTA-resistant membrane-associated annexins II and V. The results demonstrate calcium-dependent relocation of annexins II and V to membranes in intact cells and suggest that these annexins bind in a calcium-dependent manner to non-phospholipid components of SH-SY5Y membranes. Examination of cells by immunofluorescence microscopy demonstrated that annexin II was homogeneously associated with the plasma membrane before treatment with ionophore and relocated to discrete patches of staining after treatment. Annexin V was found by immunofluorescence to be present in the cytoplasm and in the nucleus, Stimulation of the cells produced no change in the cytoplasmic staining pattern but resulted in a partial relocation of nuclear annexin V to the periphery of the nucleus. The results argue for a general role for both annexins in calcium signalling at discrete intracellular locations. The results are not consistent with the specific involvement proposed previously for annexin II in membrane fusion at sites of vesicle exocytosis.

Topics: Annexin A2; Annexin A5; Calcimycin; Calcium; Cell Compartmentation; Electrophoresis, Gel, Two-Dimensional; Humans; Membrane Potentials; Neuroblastoma

Growth of cultured N1E-115 neuroblastoma cells in 1 microM A23187 for 2 days to elevate internal Ca reduced both membrane Na current and the transient, but not steady state, component of outward K current. Na channel mRNA abundance was reduced by an average value of 45% without effect on Kv3.1. Increases in internal Ca may therefore control excitability by independent regulation of Na and K channel mRNA abundance in neurons.

Topics: Base Sequence; Calcimycin; Down-Regulation; Molecular Sequence Data; Neuroblastoma; Neurons; Polymerase Chain Reaction; RNA, Messenger; Sodium Channels; Tumor Cells, Cultured

The senile plaque in Alzheimer's disease (AD) consists mainly of the amyloid beta-peptide (A beta) derived from a family of large integral membrane glycoproteins, beta-amyloid precursor proteins (beta APP). Soluble derivatives of beta APP generated by the proteolytic processing of full-length beta APP are normally secreted into the conditioned medium of cultured cells. Here we have investigated the possibility that the processing of beta APP can be regulated by the cholinesterase inhibitors physostigmine and tacrine. Both drugs mildly improve cognitive functions in some patients with AD. We analyzed the level of beta APP in glial, neuroblastoma, and pheochromocytoma cells by immunoblotting cell lysates and conditioned media using a monoclonal antibody, MAb22C11. The levels of soluble beta APP derivatives normally present in conditioned media were severely inhibited by treating cells with tacrine but not with physostigmine. Whereas the treatment of cells with tacrine resulted in a small decrease in the intracellular levels of beta APP, treating cells with physostigmine resulted in a slight increase in the intracellular levels of beta APP compared to untreated cells. The effect of tacrine on the secretion of beta APP was not affected by cotreating cells with muscarinic agents, staurosporine, or the calcium ionophore. Our results suggest that a decrease in the secretion of beta APP by tacrine did not depend on its anticholinesterase activity and that tacrine operates via a noncholinergic mechanism.

Topics: Amyloid beta-Protein Precursor; Animals; Arecoline; Astrocytes; Atropine; Calcimycin; Carbachol; Cholinesterase Inhibitors; Enzyme Inhibitors; HeLa Cells; Humans; Ionophores; Muscarinic Agonists; Muscarinic Antagonists; Neuroblastoma; PC12 Cells; Physostigmine; Rats; Staurosporine; Tacrine

A significant fraction of the beta-amyloid precursor protein is proteolytically processed to yield large secreted forms (sAPP). These proteins have pleiotropic effects which potentially involve control of gene expression. We have investigated the influence of sAPP on the class of transcription factors which bind kappa B enhancer sequences. Transcription dependent on a kappa B element was enhanced by sAPP in several cell lines, as measured by expression of a transfected chloramphenicol acetyltransferase reporter gene. Secreted APP also induced an increase in kappa B DNA-binding activity in hippocampal neurons treated with sAPP. Both effects were mimicked by an analog of cyclic GMP and inhibited by an antagonist of cyclic GMP-dependent protein kinase. Such activation of kappa B-dependent transcription was correlated in two ways with the ability of sAPP to protect neuronal cells against calcium-mediated damage: (1) tumor necrosis factor beta also protected against calcium-mediated insults and induced kappa B-dependent transcription; (2) antisense oligonucleotide-mediated reduction of an endogenous inhibitor of NF-kappa B activated kappa B-binding activity and attenuated calcium-mediated toxicity in both a neuronal cell line and in primary neurons. These findings suggest that a kappa B-binding transcription factor can act as a coordinator of neuroprotective gene expression in response to cytokines.

Topics: Amyloid beta-Protein Precursor; Base Sequence; Calcimycin; Calcium; Cell Line; Cell Survival; Chloramphenicol O-Acetyltransferase; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; DNA-Binding Proteins; Enhancer Elements, Genetic; Glioma; Humans; I-kappa B Proteins; Kidney; Kinetics; Neuroblastoma; Neurons; NF-kappa B; NF-KappaB Inhibitor alpha; Oligonucleotides, Antisense; Recombinant Fusion Proteins; Thionucleotides; Transcription, Genetic; Transfection; Tumor Cells, Cultured

Topics: Calcimycin; Carbachol; Down-Regulation; Enzyme Inhibitors; Humans; Indoles; Maleimides; Neuroblastoma; Norepinephrine; Phorbol 12,13-Dibutyrate; Potassium Chloride; Protein Kinase C; Time Factors; Tumor Cells, Cultured

SH-SY-5Y human neuroblastoma cells were treated with combinations of the kinase inhibitors HA-1004, W-7 and H-7 and calcium ionophore A23187. Microdensitometric analyses revealed that, in the absence of ionophore-mediated calcium influx, PHF-1 levels were reduced by approximately half in cultures treated with HA-1004 or W-7, but were not reduced by H-7. By contrast, the doubling in PHF-1 immunoreactivity that resulted following ionophore treatment was prevented by all three inhibitors. These analyses demonstrate the recruitment of an additional kinase or kinases in tau phosphorylation following calcium influx, and underscore the possibility that de novo hyperactivation of calcium-dependent kinases may be involved in the early events that propagate PHF formation.

Topics: Antibodies, Monoclonal; Brain Neoplasms; Calcimycin; Calcium; Densitometry; Humans; Immunohistochemistry; Ionophores; Neuroblastoma; Phosphorylation; Protein Kinase Inhibitors; Protein Kinases; tau Proteins; Tumor Cells, Cultured

beta-Amyloid peptide (A beta), a proteolytic fragment of the beta-amyloid precursor protein, is a major component of senile plaques in the brain of Alzheimer's disease patients. This neuropathological feature is accompanied by increased neuronal cell loss in the brain and there is evidence that A beta is directly neurotoxic. In the present study reduced cell viability in four different neuroblastoma cell types was observed after treatment with human A beta 1-42 for 1 day. Of the cell types tested rat PC12 and human IMR32 cells were most susceptible to A beta toxicity. Chromosomal condensation and fragmentation of nuclei were seen in PC12, NB2a, and B104 cells but not in IMR32 cells irrespective of their high sensitivity to A beta. Electrophoretic analysis of cellular DNA confirmed internucleosomal DNA fragmentation typical for apoptosis in all cell types except IMR32. These findings suggest that the form of A beta-induced cell death (necrosis or apoptosis) may depend on the cell type.

Topics: Amyloid beta-Protein Precursor; Animals; Apoptosis; Calcimycin; Cell Nucleus; DNA; DNA Damage; Humans; Mice; Microscopy, Fluorescence; Neuroblastoma; Neurotoxins; Peptide Fragments; Rats; Tumor Cells, Cultured

Intercellular adhesion molecule-1 (ICAM-1) is an important cell surface adhesion receptor of the immune system. Its cell surface expression on a wide variety of cells, including cancer cells, is regulated by various proinflammatory cytokines. In the present study, we investigated the role of calcium (Ca2+) and calmodulin (CaM) in the retinoic acid and gamma-interferon (IFN-gamma) signaling in the human neuroblastoma cell line SK-N-SH for up-regulating ICAM-1 expression. A 24-h incubation in the presence of Ca(2+)-mobilizing agents (A23187 and thapsigargin) resulted in the induction of ICAM-1 expression. Both Ca(2+)-mobilizing agents stimulated ICAM-1 expression additively to IFN-gamma but not to retinoic acid, suggesting that IFN-gamma does not use Ca2+ to stimulate ICAM-1, whereas retinoic acid might use it in part. As a second messenger, Ca2+ can be coupled with calmodulin. Using calmodulin inhibitors (W7 and calmidazolium), we found that retinoic acid-stimulated, A23187-stimulated, and thapsigargin-stimulated but not FIN-gamma-stimulated ICAM-1 were inhibited. Calmodulin signaling elicited by retinoic acid was an early event occurring within the first h of retinoic acid treatment, providing evidence that they may both be coupled to regulate gene expression. Using a novel CaM kinase II inhibitor, KN-62, we demonstrated that retinoic acid stimulated ICAM-1 expression in a CaM kinase II-dependent fashion. The mechanisms whereby CaM kinase II mediates retinoic acid activity on ICAM-1 expression remain to be elucidated.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Calcimycin; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Cell Adhesion Molecules; Enzyme Activation; Humans; Imidazoles; Intercellular Adhesion Molecule-1; Interferon-gamma; Isoquinolines; Neuroblastoma; Piperazines; Protein Kinase C; Sulfonamides; Terpenes; Thapsigargin; Tretinoin; Tumor Cells, Cultured; Up-Regulation

We have examined the effect of cytosolic calcium concentration ([Ca2+]i) on neurite outgrowth in two neuronal cells, cerebellar granule cells and N1E-115 neuroblastoma cells. The set point [Ca2+]i in unstimulated cells bathed in normal extracellular medium was 37 nM and 108 nM, respectively. When we altered extracellular calcium concentration to cause small excursions of [Ca2+]i either above or below the set point, neurite outgrowth from granule cells declined. Thus granule cells show the bell-shaped dependence of neurite outgrowth on [Ca2+]i characteristic of sensory and other neurones [Dev. Brain Res., 70 (1992) 287-290; The Axon, Oxford University Press, New York, 1994]. In contrast, neurite outgrowth from N1E-115 cells increased monotonically as [Ca2+]i was reduced. This result, which is consistent with results obtained by studying individual growth cones [J. Neurosci., 9 (1989) 4007-4020], implies that these transformed cells are aberrant in having no bell-shaped dependence of neurite outgrowth on [Ca2+]i. In both cell types an increase of [Ca2+]i above the set point reduced neurite outgrowth. However, this decline did not persist as [Ca2+]i was set to increasingly high levels by increasing extracellular calcium. Rather, in both cell types, an increase of extracellular calcium above 6.9 mM produced a second, increasing phase of neurite outgrowth.

Topics: Animals; Brain Neoplasms; Calcimycin; Calcium; Cerebellum; Cytosol; Fura-2; Mice; Neurites; Neuroblastoma; Tumor Cells, Cultured

A procedure is described to fill up cells in culture with ACh and study its calcium dependent release, by-passing the synthesis steps. Whether differentiated or not with dbc-AMP, the NG108-15 cells efficiently released ACh when stimulated with calcium and ionophore A23187. The release was also studied in the parent C6-BU-1 and N18TG2 cells. It was found that C6-BU-1 released ACh much better that N18TG2 in spite of their glial origin. The internalization by NG108-15 cells of an antisense oligonucleotide probe hybridizing the 16 kDa proteolipid messenger common to mediatophore and to the V-ATPase reduced ACh release indicated a role of this proteolipid in ACh translocation. This characteristic protein was found in the membrane extract of NG108-15 cells and also in the C6-BU-1 cells, but its amount was strongly reduced in the N18TG2 cell line and in the NG108-15 cells having internalized the antisense probe.

Topics: Acetylcholine; Adenosine Triphosphatases; Animals; Bucladesine; Calcimycin; Calcium; Cell Differentiation; Glioma; Hybrid Cells; Membrane Proteins; Mice; Molecular Weight; Neuroblastoma; Oligonucleotide Probes; Oligonucleotides, Antisense; Rats; Transfection; Tumor Cells, Cultured

Second messenger regulation of gene expression provides a mechanism by which neurons can transduce environmental stimuli into long-term changes in the expression of molecules involved in neuronal signaling. We have investigated calcium-dependent induction of vasoactive intestinal polypeptide (VIP) mRNA and compared it with induction of VIP mRNA by cyclic AMP. Depolarization with 60 mM KCl or exposure to the calcium ionophore A23187 increases VIP mRNA levels in SH-SY5Y cells. The increase in VIP mRNA content in response to Ca2+ mobilization is slow, independent of adenylate cyclase activation, and requires de novo protein synthesis. The increase in VIP mRNA content in response to elevation of cyclic AMP levels by forskolin/isobutylmethylxanthine is independent of Ca2+ influx and does not require new protein synthesis. The mRNA for the transcription factors ATF-3, c-fos, c-jun, junB, and zif/268 is induced by A23187. Of these, ATF-3 showed the greatest relative induction by A23187 compared with induction by forskolin/isobutylmethylxanthine.

Topics: 1-Methyl-3-isobutylxanthine; Calcimycin; Calcium; Colforsin; Cyclic AMP; Gene Expression Regulation, Neoplastic; Humans; Neuroblastoma; Potassium; RNA, Messenger; Signal Transduction; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Murine neuroblastoma clone N1E-115 cells possess neurotensin (NT) receptors, which are coupled to signal transduction systems resulting in polyphosphoinositide (Pl) hydrolysis and cyclic GMP synthesis. Previously, we have demonstrated that the process of down-regulation of NT receptors in N1E-115 cells involves intracellular sequestration of recyclable receptors followed by receptor degradation, causing true down-regulation. In this study, agonist-induced sequestration of NT receptors in N1E-115 cells was inhibited by an aminosteroid, 1-(6-([17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino)hexyl)-1H-pyrrole- 2,5-diane (U-73122). Pl hydrolysis elicited by NT or sodium fluoride, which stimulates GTP binding proteins, was also inhibited by U-73122, whereas Pl hydrolysis elicited by calcium ionophores, ionomycin or A23187, was not apparently affected. These data suggest that U-73122 affects a process that is distal to the cell surface receptor but not involving the sites just proximal to Pl hydrolysis or cyclic GMP synthesis. It is suggested that U-73122 may affect the coupling of GTP binding proteins and the NT receptor. We conclude that GTP binding proteins play an important role in the mechanism of agonist-induced down-regulation of NT receptors in N1E-115 cells. These results may indicate that GTP binding proteins also play a role in the mechanism of internalization of this receptor in the central nervous system in vivo.

Topics: Animals; Calcimycin; Calcium; Cell Line; Cyclic GMP; Estrenes; In Vitro Techniques; Ionomycin; Mice; Neuroblastoma; Nitroprusside; Phosphatidylinositols; Pyrrolidinones; Receptors, Neurotensin; Second Messenger Systems; Signal Transduction; Sodium Fluoride

Neurotransmitter receptors alter membrane excitability and synaptic efficacy by generating intracellular signals that ultimately change the properties of ion channels. Through expression studies in Xenopus oocytes and mammalian cells, we found that the G protein-coupled m1 muscarinic acetylcholine receptor potently suppresses a cloned delayed rectifier K+ channel through a pathway involving phospholipase C activation and direct tyrosine phosphorylation of the K+ channel. Furthermore, analysis of neuroblastoma cells revealed that a similar tyrosine kinase-dependent pathway links endogenous G protein-coupled receptors to suppression of the native RAK channel. These results suggest a novel mechanism by which neurotransmitters and hormones may regulate a specific type of K+ channel that is widely expressed in the mammalian brain and heart.

Topics: Amino Acid Sequence; Animals; Brain; Calcimycin; Calcium; Cell Line; Embryo, Mammalian; Embryo, Nonmammalian; Female; Genistein; GTP-Binding Proteins; Humans; Isoflavones; Kidney; Kinetics; Membrane Potentials; Molecular Sequence Data; Mutagenesis, Site-Directed; Myocardium; Neuroblastoma; Oocytes; Potassium Channel Blockers; Potassium Channels; Protein Kinase C; Protein-Tyrosine Kinases; Receptors, Muscarinic; Signal Transduction; Tetradecanoylphorbol Acetate; Transfection; Tumor Cells, Cultured; Xenopus

Treatment of Neuro2a cells with drugs known to affect the integrity of microfilaments and microtubules, as well as with a calcium ionophore produced damage to the cellular membrane that was quantifiable by measuring the release of LDH into the culture medium. Concurrent exposure of the cells to ORG 2766 was found to modulate the release of LDH in a dose- and time-dependent fashion. ORG 2766 treatment was also able to reduce the basal release of LDH into the culture medium. [table: see text] The ORG 2766-induced reduction in LDH release was not due to down-regulation of protein synthesis. The peptide produced significant increases in protein synthesis relative to control conditions at concentrations of 10(-11) to 10(-6) M with 10(-8) M being an optimal dose. SDS-PAGE and 2-D PAGE analysis showed that de novo synthesis of most polypeptides was increased by about 40%. Additionally, a family of polypeptides tentatively identified as actins appear to undergo ORG 2766-dependent post translational charge modifications. These data are consistent with the hypothesis that regulation of transcription and/or translation are mechanisms important to the neurotrophic actions of ORG 2766.

Topics: Adrenocorticotropic Hormone; Animals; Anticonvulsants; Biomarkers; Calcimycin; Colchicine; Cytochalasin D; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; L-Lactate Dehydrogenase; Methionine; Mice; Neoplasm Proteins; Neuroblastoma; Neurotoxins; Peptide Fragments; Sulfur Radioisotopes; Tumor Cells, Cultured; Vincristine

Nordihydroguaiaretic acid (NDGA; a lipoxygenase inhibitor), LY-270766 (an inhibitor of 5-lipoxygenase), and the diacylglycerol lipase inhibitor RG 80267 completely eliminated potassium-evoked release of [3H]-noradrenaline ([3H]NA) from the human neuroblastoma clone SH-SY5Y with IC50 values of 10, 15, and 30 microM, respectively. In contrast, these inhibitors only partially inhibited carbachol-evoked release and had little effect on the calcium ionophore A23187-evoked release of NA in this cell line. Arachidonic acid partially inhibited potassium- and A23187-evoked release but did not reverse the inhibition of potassium-evoked release observed in the presence of RG 80267. These studies suggest that arachidonic acid (or its lipoxygenase products) are not important intermediates in the regulation of exocytosis in SH-SY5Y. This conclusion is strengthened by our studies in which SH-SY5Y cells were grown in medium supplemented with bovine serum albumin-linoleic acid (50 microM). Under these conditions there was a selective increase in content of membrane polyunsaturated fatty acids of the omega 6 series, including arachidonic acid; however, these changes did not effect potassium-, veratridine-, carbachol-, or calcium ionophore-evoked release of [3H]NA.

Topics: Arachidonic Acid; Calcimycin; Carbachol; Cyclohexanones; Eicosanoids; Exocytosis; Humans; Linoleic Acid; Linoleic Acids; Lipase; Lipoxygenase Inhibitors; Masoprocol; Neuroblastoma; Norepinephrine; Organic Chemicals; Potassium; Tumor Cells, Cultured

The mechanism by which thrombin induces neurite retraction was studied in NB2a mouse neuroblastoma cells. The rapid effect of thrombin (completed within minutes) appears to involve an interaction between its anion-binding exosite and the thrombin receptor. Structural alterations of this site increase the EC50 for thrombin-mediated retraction, and a hirudin C-terminal peptide that blocks this site inhibits the response. The thrombin effect was mimicked by a 14 amino acid peptide starting with Ser-42, at the proposed cleavage site of the human thrombin receptor. The protein kinase inhibitors staurosporine and H-7 blocked thrombin-induced retraction. It is therefore proposed that thrombin-mediated neurite retraction is caused by cleavage-induced activation of the thrombin receptor and involves stimulation of a protein kinase(s).

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Alkaloids; Animals; Calcimycin; Cyclic AMP; Egtazic Acid; Isoquinolines; Mice; Neurites; Neuroblastoma; Piperazines; Protein Kinase Inhibitors; Protein Kinases; Receptors, Cell Surface; Receptors, Thrombin; Staurosporine; Thrombin; Transcription, Genetic; Tumor Cells, Cultured

Topics: Animals; Calcimycin; Endothelins; Enzyme Activation; Indoles; Kinetics; Neuroblastoma; Phospholipase D; Protein Kinase C; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Gangliosides are known to assert both neuritogenic and neuroprotective effects when applied to a variety of neuroblastoma and primary neuronal cultures. We have developed a model employing Neuro-2a neuroblastoma cells with Ca2+ ionophore A23187 as neurotoxic agent causing neurite retraction and eventual cell death. Gangliosides attenuated the toxicity of this substance, increasing both cell survival and neurite stability. In one series of experiments, cells were exposed to A23187 for 24 hr and then incubated in fresh medium (washout) for 18 hr; gangliosides were present at varying times. The paradigm in which cells were only preincubated (2 hr) with ganglioside provided no benefit, nor did incubation of the cells in both ionophore and ganglioside during the 24-hr exposure period. Significant protection was achieved by exposing the cells to ganglioside after washout of A23187, or continuously throughout the whole period. Bovine brain ganglioside mixture and the four major components (GM1, GD1a, GD1b, GT1b) applied individually were all effective. By contrast, GM3 and GM1-alcohol, a neutral derivative of GM1, provided little or no protection. Dichlorobenzamil, an inhibitor of the Na(+)-Ca2+ exchanger, tended to block the neurite stabilizing effect of gangliosides, suggesting that the mechanism might involve potentiation of this antiporter.

Topics: Animals; Brain Chemistry; Calcimycin; Calcium; Cell Survival; Cells, Cultured; Gangliosides; Ion Exchange; Mice; Neurites; Neuroblastoma; Neurons; Tumor Cells, Cultured

This study characterizes free fatty acid release in a neuroblastoma cell line (Neuro-2A), a potential model system for the study of factors that control phospholipase A2 in neurons. Two compounds, bicuculline (an antagonist at gamma-aminobutyric acid receptors), and A23187 (a Ca2+ ionophore), were examined. The release of endogenous fatty acids and the turnover of radiolabeled arachidonic and docosahexaenoic acids were measured. The cells actively incorporated radiolabeled fatty acids into various glycerolipid pools. Both endogenous fatty acids and radiolabeled fatty acids were released from glycerolipids in a time-dependent manner. Phosphatidylcholine was a major source of released fatty acids. Release of free fatty acids was markedly stimulated by both bicuculline and A23187. We conclude that the Neuro-2A cells contain phospholipase activity that is sensitive to Ca2+ ionophore and bicuculline, and may provide a good system for further studies on the regulation of phospholipase A2 in neurons.

Topics: Arachidonic Acid; Bicuculline; Calcimycin; Docosahexaenoic Acids; Fatty Acids, Nonesterified; Neuroblastoma; Neurons; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Triglycerides; Tumor Cells, Cultured

The human neuroblastoma clone SH-SY5Y expresses potassium-, carbachol-, and calcium ionophore A23187-evoked, calcium-dependent release of [3H]noradrenaline. Release in response to carbachol and potassium was greater than additive. Atropine (Ki = 0.33 nM), hexahydrosiladifenidol (Ki = 18 nM), and pirenzepine (Ki = 1,183 nM) completely inhibited the carbachol-evoked noradrenaline release, an order of potency suggesting that an M3 receptor was linked to release. In contrast, noradrenaline release was only partially inhibited by the M2-selective antagonists methoctramine (10(-4) M) and AFDX-116 (10(-4) M), by approximately 14 and 46%, respectively. The nicotinic antagonist d-tubocurarine (10(-4) M) resulted in a partial inhibition of release, a finding suggesting that a nicotinic receptor may also be involved. SH-SY5Y provides a suitable cell line in which to study the biochemical mechanisms underlying the cholinergic receptor regulation of noradrenaline release.

Topics: Calcimycin; Calcium Chloride; Carbachol; Egtazic Acid; Humans; Muscarine; Neuroblastoma; Norepinephrine; Parasympathomimetics; Potassium; Tritium; Tumor Cells, Cultured

Cell hybrids (BIM) were produced between human neuroblastoma cells (IMR-32) and thymidine auxotrophs (B3T) of rat nerve-like cells (B103) in order to obtain cell lines undergoing stable neuronal differentiation. BIM cells exhibited the growth properties of partial transformation: 1) When the cell growth reached a plateau, BIM cells ceased to proliferate and expressed a differentiated phenotype. The shape of the cells changed from flat to round and they extended neurites. 2) When cultured in methylcellulose, BIM cells formed colonies, indicating that BIM cells have the ability of anchorage-independent growth. By Southern blot analysis, BIM cells had both human and rat types of N-myc genes. The human N-myc genes were amplified, but the extent of the amplification was lower in BIM cells than that in the parental cell line IMR-32. The rat N-myc gene was detected at a similar level in BIM, B3T, B103, and rat fibroblastic cells 3Y1. Therefore, the decrease in amplification of human N-myc genes may be involved in the properties of partial reverse-transformation in BIM cells. When treated with various drugs such as db-cAMP, forskolin, and cAMP with isobutyl-methylxanthine, BIM cells expressed a nerve-like phenotype. These findings indicate that cell hybridization yielded partial normalization of transformed nerve-like cells.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Axons; Bucladesine; Calcimycin; Cell Cycle; Cell Transformation, Neoplastic; Colforsin; Cyclic AMP; Fibroblasts; Humans; Hybrid Cells; Methylcellulose; Neuroblastoma; Neurons; Phenotype; Rats; Thymidine; Tretinoin; Tumor Cells, Cultured

Cultures of mouse Neuro-2a neuroblastoma cells treated with 3-6 mM extracellular Ca2+ exhibited enhanced neurite extension characterized by increased neurite numbers and lengths. The ganglioside GM1 potentiated the effect of extracellular Ca2+ by increasing further the number and length of the neurites formed in response to exogenous Ca2+. Maximal neuritic numbers were achieved with 4 mM Ca2+ while the longest neurites were observed in medium containing 4-6 mM Ca2+. Stimulation of the Ca2+ influx with the ionophore A23187 or the amino acid taurine also enhanced neurite formation and GM1 potentiated these actions. Transmission electron microscopy revealed numerous microtubules and neurofilaments in neurites and microfilaments with the spine-like processes along fine neuritic branches and in the filopodia of growth cones. Neuritic varicosities and growth cones contained a variety of vesicles. All of these structures were increased in the presence of GM1 and were increased further by extracellular Ca2+ or A23187. The ability of GM1 to enhance neuritogenesis was diminished by EGTA or Ruthenium red. Similarly, the effect of GM1 was diminished or abolished by Ca2+ channel blockers such as CdCl2 or LaCl3. X-ray microprobe analysis revealed that GM1 alone enhanced intracellular levels of total ionic and membrane bound Ca2+, perhaps accounting for the increased neuritogenesis observed under conditions in which Ca2+ was manipulated. The present study suggest that the neuritogenic action of GM1 is Ca2+ dependent.

Topics: Animals; Axons; Cadmium; Cadmium Chloride; Calcimycin; Calcium; Cell Differentiation; Cell Line; Egtazic Acid; G(M1) Ganglioside; Intermediate Filaments; Lanthanum; Mice; Microtubules; Neuroblastoma; Neurons; Ruthenium Red; Taurine

Topics: Arginine; Calcimycin; Calcium; Carbachol; Cells, Cultured; Cyclic GMP; Enzyme Activation; Guanylate Cyclase; Ionophores; Neuroblastoma; Nitric Oxide; omega-N-Methylarginine; Tumor Cells, Cultured

Topics: Calcimycin; Carbachol; Clone Cells; Drug Synergism; Humans; Neuroblastoma; Neurons; Norepinephrine; Potassium; Tritium; Tumor Cells, Cultured

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.

Topics: Adenosine Triphosphate; Animals; Biological Transport, Active; Calcimycin; Calcium; Calcium-Transporting ATPases; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Line; Cell Membrane Permeability; Glioma; Guanosine Triphosphate; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Kinetics; Mice; Neuroblastoma; Oligomycins; Rats

Preincubation with receptor agonists or phorbol esters desensitized muscarinic-receptor-mediated [3H]cyclic GMP responses in mouse neuroblastoma N1E-115 cells. However, desensitization mediated by phorbol esters was heterologous, whereas that effected by receptor agonist was specific towards the muscarinic receptors. In addition, there was no loss of cell surface muscarinic receptors, as measured by the binding of the hydrophilic ligand [3H]N-methylscopolamine, when cells were treated with phorbol esters, but receptor-agonist-induced desensitization was accompanied by a decrease in cell surface receptor density. We examined the role of protein kinase C (PKC) in the desensitization of muscarinic receptors by employing a kinase inhibitor and by down-regulation of PKC by long-term incubation of cells with phorbol esters. Whereas these manoeuvres had marked effects on phorbol-ester-induced desensitization of muscarinic responses, they did not block agonist-induced down-regulation and desensitization of muscarinic receptors. In addition, when phosphoinositide hydrolysis was suppressed, the muscarinic agonist was still capable of mediating receptor sequestration and desensitization. These results suggest that the mechanisms for regulating muscarinic receptor sensitivity could be both PKC-dependent and PKC-independent, being mediated by phorbol esters and receptor agonists respectively.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Calcimycin; Carbachol; Cyclic GMP; Down-Regulation; Isoquinolines; Mice; N-Methylscopolamine; Neuroblastoma; Parasympathomimetics; Phorbol 12,13-Dibutyrate; Phorbol Esters; Piperazines; Protein Kinase C; Receptors, Muscarinic; Scopolamine Derivatives; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Ionophore (A23187)-mediated calcium influx induced rapid neurite outgrowth in NB2a/d1 cells. This outgrowth was prevented by colchicine but not by cycloheximide, demonstrating a requirement for microtubule assembly but not de novo synthesis. Cytochalasin B induced rapid, colchicine-sensitive outgrowth, indicating that depolymerization of the submembrane actin network may be sufficient to allow neurite outgrowth under conditions which permitted microtubule assembly. Neurites induced by serum-deprivation or calcium influx were rapidly retracted by colchicine unless cytochalasin B was first added, indicating that the actin network may provide the retractile force which mediates neurite retraction following microtubule depolymerization. We conclude that neurite outgrowth can be initiated in NB2a/d1 cells by calcium influx, and may involve alterations in actin and microtubule dynamics.

Topics: Actins; Animals; Axons; Calcimycin; Calcium; Cell Differentiation; Colchicine; Cycloheximide; Cytochalasin B; Cytoskeleton; Mice; Microtubules; Neuroblastoma; Tubulin; Tumor Cells, Cultured

The effect of GTP on Ca2+ uptake and release was studied in a microsomal fraction isolated from neuroblastoma x glioma hybrid NG108-15 cells. GTP did not alter the ATP-dependent initial uptake of Ca2+ but markedly enhanced the efflux of Ca2+ from microsomes. GTP-dependent Ca2+ release requires the presence of millimolar concentration of Mg2+. The effect of GTP was not mimicked by other nucleotides and was competitively blocked by the thiophosphate analogue of GTP, GTP gamma S but not by the non-hydrolyzable nucleotide GMP-PNP. Addition of an inhibiting concentration of GTP gamma S after completion of GTP-induced calcium release did not result in a re-uptake of Ca2+, showing the irreversibility of the releasing effect of GTP. Our data are consistent with the hypothesis of Ca2+-dependent GTP-induced opening of a channel responsible for vectorial transport of Ca2+ ions from one intracellular compartment to another. A model is proposed suggesting that the GTP-binding protein is a GTP-specific diacylglycerol kinase.

Topics: Animals; Calcimycin; Calcium; Cell Line; Glioma; Guanosine Triphosphate; Hybrid Cells; Intracellular Membranes; Kinetics; Magnesium; Mice; Microsomes; Neuroblastoma; Oxalates; Rats

The hypothesis that Na+-dependent calcium extrusion is important in protecting against neuronal excitotoxicity was tested. In cocultures of embryonic rat hippocampal neurons and mouse neuroblastoma hybrid (NCB-20) cells, calcium ionophore A23187 (1 microM) or high levels of extracellular K+ killed hippocampal neurons selectively, leaving NCB-20 cells unscathed. Hippocampal neurons showed large, sustained rises in intracellular calcium in response to A23187 or K+, whereas NCB-20 cells showed only transient calcium responses. The ability of NCB-20 cells to reduce the calcium load and to survive exposure to A23187 or K+ were dependent on extracellular Na+, suggesting that an active Na+/Ca2+ exchange mechanism was important in protecting against cell death. Finally, removal of extracellular Na+ reduced the threshold for glutamate neurotoxicity in hippocampal neurons, demonstrating the importance of Na+/Ca2+ exchange in protecting against excitotoxicity. Taken together, these findings suggest that differences in cell calcium-regulating systems may determine whether a neuron lives or degenerates in the face of an excitatory challenge.

Topics: Animals; Calcimycin; Calcium; Cell Survival; Cricetinae; Glutamates; Glutamic Acid; Hippocampus; Mice; Neuroblastoma; Neurons; Potassium; Rats; Sodium; Tumor Cells, Cultured

Mouse neuroblastoma X embryonic Chinese hamster brain explant hybrid cell line (NCB-20) forms functional synapses when intracellular cyclic AMP levels are elevated for a prolonged period of time. NCB-20 cells were labeled with [32P]orthophosphate under conditions where 2-chloroadenosine gave maximum increases of 32P incorporation into tyrosine hydroxylase in nerve growth factor dibutyryl cyclic AMP-differentiated PC12 (pheochromocytoma) cells. When NCB-20 cells were exposed to activators [5-hydroxytryptamine (5-HT), prostaglandin E1, or forskolin], resulting in activation of cyclic AMP-dependent protein kinase, increased 32P incorporation into two major proteins [130 kilodaltons (kDa) and 90 kDa] occurred. 5-HT (in the presence of phosphodiesterase inhibitor, isobutylmethylxanthine) gave a three- to fourfold increase, and forskolin a four- to sevenfold increase in 32P incorporation into the 90-kDa protein. [D-Ala2,D-Leu5]-enkephalin, which decreased cyclic AMP levels and reversed the 2-chloroadenosine-stimulated phosphorylation of tyrosine hydroxylase in differentiated PC12 cells, also reversed the stimulation of phosphorylation of the 90-kDa protein in NCB-20 cells. Pretreatment of NCB-20 cells with a calcium ionophore, A23187, gave increased phosphorylation of the 90- and 130-kDa proteins, but phorbol esters such as 12-O-tetradecanoylphorbol 13-acetate (tumor promoting agent), cell depolarization with high K+, or pretreatment with dibutyryl cyclic GMP had no effect on phosphorylation of these proteins. In contrast, phosphorylation of an 80-kDa protein was decreased by forskolin, but increased following activation of the calcium/phospholipid-dependent kinase with tumor promoting agent. Neither the 90-kDa nor the 80-kDa protein showed any immunological cross-reactivity with synapsin, a major synaptic protein known to be phosphorylated by cyclic AMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase, but not calcium/phospholipid-dependent protein kinase. This suggests that in NCB-20 cells, several unique proteins can be phosphorylated by cyclic AMP-dependent protein kinase in response to hormonal elevation of cyclic AMP levels. In contrast, an 80-kDa protein is the primary substrate for calcium/phospholipid-dependent protein kinase, and its phosphorylation is inhibited by agents that elevate cyclic AMP levels and thereby activate cyclic AMP-dependent protein kinase.

Topics: 1-Methyl-3-isobutylxanthine; 2-Chloroadenosine; Adenosine; Alprostadil; Animals; Brain; Calcimycin; Colforsin; Cricetinae; Cyclic AMP; Electrophoresis, Polyacrylamide Gel; Embryo, Mammalian; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Hybrid Cells; Immunoassay; Mice; Nerve Tissue Proteins; Neuroblastoma; Neurotransmitter Agents; Phosphoproteins; Phosphorylation; Protein Kinases; Serotonin; Tumor Cells, Cultured; Tyrosine 3-Monooxygenase

Intracellular calcium homeostasis and its modulation by different agents was studied in control and differentiated IMR32 human neuroblastoma cells by using the Ca2+-sensitive fluorescent dye quin2. The results obtained demonstrate the existence in IMR32 cells of (a) voltage-dependent, verapamil sensitive, Ca2+ channels, which are expressed before differentiation; (b) muscarinic receptors whose activation triggers both Ca2+ influx and Ca2+ redistribution from intracellular stores, whereas nicotinic receptors and alpha-bungarotoxin binding sites do not; and (c) receptors for alpha-latrotoxin (the major toxin of the black widow spider venom), which are well-known markers of the neuronal presynaptic membrane. Up to now, no cell lines of human origin sensitive to this toxin have been identified. These results confirm that IMR32 cells are very convenient model cells for studying specific aspects of the neurochemistry and neurobiology of the human neuron at the molecular and cellular levels.

Topics: Aminoquinolines; Arthropod Venoms; Atropine; Calcimycin; Calcium; Carbachol; Cytoplasm; Egtazic Acid; Fluorescent Dyes; Homeostasis; Humans; Ion Channels; Membrane Potentials; Neuroblastoma; Pirenzepine; Potassium Chloride; Receptors, Cholinergic; Receptors, Muscarinic; Receptors, Nicotinic; Spectrometry, Fluorescence; Spider Venoms; Tumor Cells, Cultured; Verapamil

Because previous studies in whole-animal models have reported inconsistent results regarding the effect of chronic lithium on the regulation of the neuronal muscarinic acetylcholine receptor (mAChR) number, we examined the effect of chronic lithium on the regulation of mAChR in cell cultures of N1E-115, a mouse neuroblastoma clone. Li+ induced a concentration- and time-dependent increase in the mAChR number, with a 30% increase in specific [3H]quinuclidinyl benzilate binding in membrane homogenates induced by a 5-day incubation with 10 mM Li+. Agonist-induced down-regulation of the mAChR number was also inhibited by lithium: chronic treatment with 10 mM Li+ caused a 25-35% reduction in the magnitude of carbachol. In contrast, the decrease in the mAChR number induced by the synergistic action of A23187 (300 nM) and phorbol myristate acetate (300 nM) was unaffected by Li+. These results demonstrate that chronic treatment with Li+ increases the basal mAChR number and dampens the decrease in receptor number induced by a muscarinic agonist in neuroblastoma cells. The implications of these results in understanding the functional regulation of neuronal mAChR number are discussed.

Topics: Animals; Calcimycin; Carbachol; Cell Line; Cell Membrane; Chlorides; Kinetics; Lithium; Lithium Chloride; Mice; Neuroblastoma; Neurons; Receptors, Muscarinic; Tetradecanoylphorbol Acetate

The effects of phorbol 12-myristate 13-acetate (PMA) on carbamylcholine (CBC)-induced [3H]cyclic GMP formation in mouse neuroblastoma cells (clone N1E-115) were studied. PMA, but not 4 alpha-phorbol, suppressed muscarinic receptor-mediated cyclic GMP responses in a time-dependent and a concentration-dependent fashion with an IC50 of 68.8 +/- 20.2 nM. The inhibitory effects of PMA on CBC-induced cyclic GMP formation were of a mixed competitive and noncompetitive type, being characterized by a depression of maximal cyclic GMP response to CBC and a significant increase in its EC50. PMA also significantly reduced [3H]cyclic GMP formation induced by histamine, without affecting the responses elicited either by sodium azide or the calcium ionophore A23187. Although the inhibitory effects of PMA on CBC-induced cyclic GMP formation were not reversed by washing, these effects were significantly attenuated by H-7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine], a protein kinase C inhibitor. PMA had no effect on binding of an antagonist ligand to muscarinic receptors, or on the binding characteristics of CBC to these receptors in intact cells. On the other hand, PMA competed for the specific binding of a labeled phorbol ester in intact cells with a potency similar to that of PMA in inhibiting muscarinic receptor-mediated [3H]cyclic GMP responses.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Calcimycin; Carbachol; Cell Line; Cyclic GMP; Dose-Response Relationship, Drug; Histamine; Isoquinolines; Mice; Neuroblastoma; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phorbols; Piperazines; Receptors, Muscarinic; Tetradecanoylphorbol Acetate

Adenosine 3',5'-cyclic monophosphate (cAMP) content of neurons is determined not only by the rate of synthesis but also by the rate of hydrolysis by cyclic nucleotide phosphodiesterases. Multiple forms of cyclic nucleotide phosphodiesterase exist in brain and other tissues, and these may be regulated by various hormones and neuromodulators. The present study examines this regulation in a cloned line of neuroblastoma cells (N18TG2). A biphasic Lineweaver-Burk plot of cAMP hydrolysis revealed two Kms approximating 5 and 25 microM. Lineweaver-Burk plots of cGMP hydrolysis were linear over a range of 1 microM to 1 mM and exhibited a Km of 37 microM. Neither cAMP nor cGMP competed for hydrolysis of the alternative cyclic nucleotide. No evidence for an allosteric activation of cAMP phosphodiesterase by cGMP was found. Calcium regulation of phosphodiesterase was not found in spite of preparation of the cell extract with several protease inhibitors, and addition of exogenous calmodulin. No effect of calmodulin antagonists (calmidazolium, W7, or trifluoperazine) was observed in vitro or in situ. Growth of the cells in the presence of 200 nM 3,5,3'-triiodothyronine (T3) resulted in an increased hydrolysis of cAMP but of cGMP. This increase was attributed to an increase in Vmax with no change in either high or low Km. This response was blocked by cycloheximide, suggesting that the thyroid hormone effect requires protein synthesis. The thyroid hormone response in neuroblastoma cells is compared with the results of other studies of thyroid hormone effects on phosphodiesterase in other tissues in vivo.

Topics: 2',3'-Cyclic-Nucleotide Phosphodiesterases; Animals; Calcimycin; Calcium; Calmodulin; Cell Line; Clone Cells; Isoenzymes; Mice; Neuroblastoma; Phosphodiesterase Inhibitors; Triiodothyronine

Calcium may act as a second messenger in normal cellular signal transduction systems. However, an excessive influx of calcium into the cytoplasm is well known to be a final common pathway causing cell death under various pathological conditions. The purpose of this study was to investigate the effect of a transient treatment with the calcium ionophore A23187 on the recovery process of cell viability, energy metabolism, amino acid incorporation and calcium uptake in a neuroblastoma cell line. When neuroblastoma cells were treated with 20 microM of the calcium ionophore A23187 in combination with extracellular calcium, rapid energy failure and marked inhibition of amino acid incorporation by the cells occurred together with a massive influx of calcium, and finally resulted in cell death. Recovery from this calcium-induced damage with regards to energy metabolism and prognosis of cell viability was better after a 10-min treatment than after a 30-min treatment with A23187. After a 10-min treatment, the viability was higher in calcium-free medium than in calcium-containing medium in contrast with the cases after treatment for 30 min. The above difference in viability after treatment for 10 min had a very significant correlation with the degree of exclusion of excessive calcium and the recovery of CTP, indicating that the recovery of CTP and the rate of calcium exclusion may be final markers of the recovery of cells from calcium-induced damage rather than the recovery of ATP or amino acid incorporation. Amino acid incorporation was restricted to a level lower than that of the control long after the recovery of GTP and the GTP/GDP ratio.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Calcimycin; Calcium; Calcium Radioisotopes; Cell Line; Cell Survival; Energy Metabolism; Humans; Kinetics; Leucine; Neoplasm Proteins; Neuroblastoma

Atriopeptin III and related atrial natriuretic peptide hormones strongly elevate the level of cyclic GMP in three neural tumor cell lines. At peptide concentrations of 1 microM clear-cut plateaus of the dose-response curves are not yet reached. Atriopeptin III increases the intracellular concentration of cyclic GMP to a maximum in the course of 30-40 min. The effect of atriopeptin III on the cellular cyclic GMP level is independent of the concentration of extracellular Ca2+ and is not affected by the Ca2+ ionophore A23187. These results suggest (1) that atrial natriuretic hormones may play an important role in the nervous system, and (2) that cultured neural cells may be useful tools in the elucidation of the mechanisms of action of these hormones.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Atrial Natriuretic Factor; Calcimycin; Calcium; Cell Line; Cyclic GMP; Glioma; Hybrid Cells; Kinetics; Neuroblastoma; Rats

The rat CNS neuroblastoma B50 cell line is known to differentiate on addition of 1 mM dibutyryl cyclic AMP or on withdrawal of serum. In this report it is shown that high levels of extracellular calcium (10-25 mM) cause neurite extension, an important component of morphological differentiation. Stimulation of calcium influx with the ionophore A 23187 or blockade of calcium efflux with lanthanum are less efficient than extracellular calcium in stimulating neurite extension. These data suggest that intracellular calcium is not sufficient to cause full expression of a calcium-dependent differentiated state. Furthermore, phosphatidylinositol turnover is sharply altered as early as 1 h after addition of calcium to the medium while cyclic nucleotide levels remain unaffected. This suggests that activation of the phosphatidylinositol second-messenger system by calcium at the level of the cell membrane is the initial step in the cascade of events leading to neurite extension. Later events include a decrease in DNA synthesis (6-10 h after addition of calcium), and increase in intracellular calcium levels (12-24 h after calcium addition) concurrent with neurite extension. The intracellular increase in calcium levels is facilitated by synergistic action of 1 mM dibutyryl cyclic AMP with high external calcium (10-25 mM). This combined treatment results in a more complex pattern of neurite formation characterized by many synaptic-like junctions; this pattern is not obtained when either dibutyryl cyclic AMP or calcium is used as sole inducer.

Topics: Animals; Bucladesine; Calcimycin; Calcium; Cell Differentiation; Cell Line; Cyclic AMP; DNA; Kinetics; Neuroblastoma; Phosphatidylinositols; Rats

The Ca2+ accumulating properties of a nonmitochondrial intracellular organelle within cultured N1E-115 neuroblastoma cells containing an (ATP + Mg2+)-dependent Ca2+ pump were recently described in detail (Gill, D. L., and Chueh, S. H. (1985) J. Biol. Chem. 260, 9289-9297). Using both saponin-permeabilized N1E-115 cells and microsomal membranes from cells, this report describes the effectiveness of both inositol 1,4,5-trisphosphate (IP3) and guanine nucleotides in mediating Ca2+ release from this internal organelle, believed to be endoplasmic reticulum. Using permeabilized N1E-115 cells, 2 microM IP3 effects rapid release (t1/2 less than 20 s) of approximately 40% of accumulated Ca2+ releasable with 5 microM A23187. Half-maximal Ca2+ release occurs with 0.5 microM IP3, and maximal release with 3 microM IP3. Using a frozen microsomal membrane fraction isolated from lysed cells, 2 microM IP3 rapidly releases (t1/2 less than 30 s) 10-20% of A23187-releasable Ca2+ accumulated within nonmitochondrial Ca2+-pumping vesicles, although only in the presence of 3% polyethylene glycol (PEG). 10 microM GTP, but not guanosine 5'-(beta, gamma-imido)triphosphate (GMPPNP), increases the extent of release in the presence of IP3. Importantly, however, GTP alone induces a substantial release of Ca2+ (up to 40% of releasable Ca2+) with a t1/2 value (60-90 s) slightly longer than that for IP3. The effects of IP3 and GTP are approximately additive, and both effects require 3% PEG. Half-maximal Ca2+ release occurs with 1 microM GTP, with maximal release at 3-5 microM GTP; 20 microM GMPPNP has no effect on release and only slightly inhibits 5 microM GTP; 20 microM GDP promotes full release, but only after a 90-s lag, and initially inhibits the action of 5 microM GTP. Using permeabilized N1E-115 cells, 5 microM GTP with 3% PEG releases greater than 50% of releasable Ca2+; without PEG, GTP still mediates approximately 30% release of Ca2+ from cells. Neither IP3, GTP, or both together (with or without PEG) effects release of Ca2+ accumulated within synaptic plasma membrane vesicles. The profound effectiveness of GTP on Ca2+ release has important implications for intracellular Ca2+ regulation and is probably related to Ca2+ release mediated by IP3.

Topics: Adenosine Triphosphate; Animals; Calcimycin; Calcium; Cell Line; Cell Membrane Permeability; Guanine Nucleotides; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Mice; Microsomes; Neuroblastoma; Neurons; Polyethylene Glycols; Saponins; Sugar Phosphates

The tumor-promoting phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA), which activates protein kinase C, acted synergistically with A23187 to decrease muscarinic acetylcholine receptor (mAChR) number in neuroblastoma cells (clone N1E-115) as determined by a filter binding assay using [3H]quinuclidinyl benzilate in membrane homogenates. After a 6-h incubation, 10(-7) M PMA and 3 X 10(-7) M A23187 reduced mAChR number 30-40%, compared to the 40-50% reduction observed after treatment with 10(-3) M carbachol, a muscarinic agonist. Incubation with 3 X 10(-7) M A23187 and 10(-7) M 4 alpha-phorbol 12,13-didecanoate, an inactive phorbol ester, did not alter mAChR number. The addition of PMA and A23187 to cultures incubated with 10(-3) M carbachol caused only a modest 6% further reduction in mAChR number as compared to incubation with carbachol alone. The kinetics of the decrease in mAChR number produced by PMA/A23187 were similar to those seen after carbachol treatment. Recovery of mAChR number after treatment with either carbachol or PMA/A23187 was blocked by treatment with the protein synthesis inhibitor cycloheximide. Intact cell binding studies employing [3H]N-methylscopolamine showed that treatment with either PMA/A23187 or carbachol caused a rapid (within 15 min) loss of receptors from the cell surface prior to the decrease in total mAChR number. PMA (10(-7) M), but not 4 alpha-phorbol 12,13-didecanoate, promoted the translocation of protein kinase C activity from the cytosol to the membrane. Incubation with carbachol increased membrane-associated protein kinase C activity within 5 min with an EC50 of 3 X 10(-6) M. This increase persisted for at least 60 min in the continued presence of carbachol and was blocked by simultaneous incubation with atropine. These results suggest that activation of protein kinase C may be involved in the regulation of mAChR number in response to agonist.

Topics: Animals; Calcimycin; Calcium; Carbachol; Cell Line; Diglycerides; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Activation; Kinetics; Mice; Neuroblastoma; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phosphatidylserines; Protein Kinase C; Quinuclidinyl Benzilate; Receptors, Cholinergic; Tetradecanoylphorbol Acetate; Time Factors

Human neuroblastoma cells (clone SHSY-5Y) induced to differentiate by 12-O-tetradecanoylphorbol-13-acetate (TPA) are shown to possess properties characteristic of mature ganglion cells. Elevation of the external K+ concentration, exposure to Ca2+ ionophore A23187, and acetylcholine all stimulate the release of preloaded 3H-noradrenaline in the presence but not in the absence of added Ca2+. Acetylcholine causes a fall in the 86Rb+ or 14C-TPMP equilibrium potential across the plasma membrane and stimulates 86Rb+ efflux. These responses are prevented by atropine. Acetylcholine and muscarine but not nicotine stimulate an increase in 45Ca2+ influx, an effect blocked by atropine. None of these responses have been observed in nondifferentiating cells. Muscarinic receptors, however, as measured by the binding of tritiated quinuclidinyl benzilate (3H-QNB), were present to a similar extent in control and differentiated cells. Both cell types also exhibit an accelerated release of Ca2+ in response to acetylcholine, but the control cells were at least 1 order of magnitude more sensitive to the agonist.

Topics: Acetylcholine; Atropine; Calcimycin; Calcium; Cell Differentiation; Clone Cells; Ganglia, Sympathetic; Humans; Membrane Potentials; Muscarine; Neuroblastoma; Norepinephrine; Phenotype; Potassium; Receptors, Muscarinic; Rubidium; Tetradecanoylphorbol Acetate

The cellular cGMP content increased in response to a variety of receptor agonists, which activate [e.g., prostaglandin (PG) E1, E2, and F2 alpha] or inhibit (e.g., alpha-adrenergic, muscarinic, and opiate agonists) adenylate cyclase in neuroblastoma X glioma hybrid NG108-15 cells. The responses were additive when PGF2 alpha and enkephalin were mixed. The inhibitory guanine nucleotide regulatory protein (Ni) is involved in adenylate cyclase inhibition; this function of Ni 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. Ni 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 A2 might play a role in Ni-mediated guanylate cyclase activation. On the other hand, the PGF2 alpha-induced cGMP rise was associated with increased incorporation of 32Pi 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 Ca2+, which is responsible for guanylate cyclase activation. Thus, dual pathways are proposed for a receptor-mediated cGMP rise in NG108-15 cells.

Topics: Adenylate Cyclase Toxin; Animals; Bacterial Toxins; Calcimycin; Cyclic AMP; Cyclic GMP; Dinoprost; Enkephalins; Glioma; Hybrid Cells; Melitten; Mice; Neuroblastoma; Pertussis Toxin; Phospholipids; Prostaglandins F; Rats; Receptors, Cell Surface; Virulence Factors, Bordetella

We have studied the induction of an amiloride-sensitive sodium influx into C6 glioma, NIE, and NB2A neuroblastoma cell lines. In late log phase, cells grown continuously in the presence of 10% fetal calf serum showed Na+ influxes of approximately 25-30 nmol/mg protein min; less than 5% of this flux was inhibited by amiloride. Removal of serum for 24 h caused a decrease in the total Na+ influx to 15-20 nmol/mg protein/min. Upon readdition of serum to the incubation medium, there was an increase in total Na+ influx, depending on the cell type, of 20-400% within 2 min. This increment in Na+ influx represented an increase in amiloride-sensitive Na+ transport with an apparent K'1 of 0.4 mM. By adding serum back at various times after serum deprivation, it was determined that 4 h was required to observe a detectable increase in the amiloride-sensitive Na+ flux. Thus, serum removal results in the induction of the amiloride transport system which, however, remains latent until the reintroduction of serum to the medium. Addition of 5 micrograms/ml of cycloheximide blocked the increase in Na+ transport, indicating that de novo protein synthesis mediated this serum deprivation-induced increase in Na+ transport. Moreover, inhibition of de novo lipid synthesis by 0.1 mM fenfluramine also blocked the induction of this transport activity, suggesting that a coordinated synthesis of lipid and protein is required for the expression of this sodium transport site. We have also found that this serum stimulated Na+ influx did not saturate with Na+ concentration, up to 140 mM. Also, among commonly used inhibitors of passive Na+ entry into epithelial tissues, only amiloride was capable of inhibiting this transport system in these neural cell lines.

Topics: Amiloride; Animals; Biological Transport; Blood; Calcimycin; Cell Division; Cell Line; Cycloheximide; Dose-Response Relationship, Drug; Fenfluramine; Glioma; Kinetics; Mice; Neuroblastoma; Neurons; Pyrazines; Rats; Sodium

Light and high voltage electron microscopy (HVEM) procedures have been employed to examine the processes regulating saltatory motion in neurons. Light microscope studies demonstrate that organelle transport occurs by rapid bidirectional saltations along linear pathways in cultured neuroblastoma cells. HVEM stereo images of axons reveal that microtubules (Mts) and organelles are suspended in a continuous latticework of fine microtrabecular filaments and that the Mts and lattice constitute a basic cytoskeletal structure mediating the motion of particles along axons. We propose that particle transport depends on dynamic properties of nonstatic microtrabecular lattice components. EXperiments were initiated to determine the effects of changes in divalent cation concentrations (Ca2+ and Mg2+) on: (a)the continuation of transport and (b) the corresponding structural properties of the microtrabecular lattice. We discovered that transport continues or is stimulated to a limited extent in cells exposed to small amounts of exogenously supplied Ca2+ and Mg2+ ions (less than 0.1 mM). Exposure of neurons to increased dosages of Ca2+ and Mg2+ (0.2-1.0 mM) stimulates transport for 2-4 min at 37 degrees C, but after a 5- to 20-min exposure the saltatory movements of organelles are observed gradually to become shorter in duration and rate particle motion ceases to occur. HVEM observations demonstrated that Ca2+ - and with the cessation of motion. Ca2+-containing solutions produced contractions of the microtrabecular filaments, whereas Mg2+-containing solutions had the opposing effect of stimulating an elongation and assembly (expansion) of microtrabeculae. On the basis of these observations we hypothesize that cycles of Ca2+/Mg2+-coupled contractions and expansions of the microtrabecular lattice probably regulate organelle motion in nerve cells.

Topics: Animals; Axonal Transport; Calcimycin; Calcium; Cell Line; Edetic Acid; Egtazic Acid; Magnesium; Microscopy, Electron; Microtubules; Models, Biological; Neuroblastoma; Neurons; Organoids; Rats

Topics: Animals; Calcimycin; Clone Cells; Methylation; Mice; Neoplasms, Experimental; Neuroblastoma; Neurons; Phospholipids; Quinacrine; S-Adenosylmethionine

Topics: Anti-Bacterial Agents; Axons; Calcimycin; Calcium; Cell Differentiation; Cell Line; Cell Membrane; Humans; Neuroblastoma

Topics: Animals; Azides; Calcimycin; Calcium; Carbachol; Cyclic GMP; Egtazic Acid; Methylnitronitrosoguanidine; Neuroblastoma; Ouabain; Prostaglandins E

The uptake of [45Ca] has been studied in clonal glial and neuronal cells. It was somewhat more efficient in the neuroblastoma clone M1 compared to glial clones. In all cases [45Ca] uptake was shown to depend on the phosphate concentration in the incubation medium. It was decreased by the ionophore A 23187 at 200 microM concentration in both neuronal and glial clones. The influence of amino acids some of which are putative neurotransmitters was investigated; the interactions between [45Ca] uptake and these amino acids were related to their concentration and the type of cells used (neuronal or glial). L-aspartate and taurine for example had two opposite effects on [45Ca] uptake by the glial clone NN at two different concentrations; they could therefore play a role in the control of calcium level in the synaptic cleft.

Topics: Amino Acids; Animals; Aspartic Acid; Brain Neoplasms; Calcimycin; Calcium; Clone Cells; Cricetinae; Neuroblastoma; Neuroglia; Phosphates; Synaptic Transmission; Taurine

The increase in intracellular cyclic GMP concentrations in response to muscarinic-receptor activation in N1E-115 neuroblastoma cells is dependent on extracellular Ca2+ ion. The calcium ionophore A23187 can also evoke an increase in cyclic GMP in the presence of Ca2+ ion. Most (about 85%) of the guanylate cyclase activity of broken-cell preparations is found in the soluble fraction. The soluble enzyme can utilize MnGTP (Km = 55 micrometer), MgGTP (Km = 310 micrometer) and CaGTP (Km greater than 500 micrometer) as substrates. Free GTP is a strong competitive inhibitor (Ki approximately 20 micrometer). The enzyme possesses an allosteric binding site for free metal ions (Ca2+, Mg2+ and Mn2+). The membrane-bound guanylate cyclase is qualitatively similar to the soluble form, but has lower affinity for the metal-GTP substrates. Entry of Ca2+ into cells may increase cyclic GMP concentration by activating guanylate cyclase through an indirect mechanism.

Topics: Calcimycin; Calcium; Carbachol; Cell Line; Cyclic GMP; Enzyme Activation; Guanylate Cyclase; Kinetics; Neuroblastoma; Subcellular Fractions