sq-23377 and Neuroblastoma

Reactive oxygen species induce neuronal cell death. However, the detailed mechanisms of cell death have not yet been elucidated. Previously, we reported neurite degeneration before the induction of cell death. Here, we attempted to elucidate the mechanisms of neurite degeneration before the induction of cell death using the neuroblastoma N1E-115 cell line and a time-lapse live cell imaging system. Treatment with the calcium ionophore ionomycin induced cell death and neurite degeneration in a concentration- and time-dependent manner. Treatment with a low concentration of ionomycin immediately produced a significant calcium influx into the intracellular region in N1E-115 cells. After 1-h incubation with ionomycin, the fluorescence emission of MitoSOX

Topics: Animals; Calcium; Cell Death; Ionomycin; Mice; Mitochondria; Neurites; Neuroblastoma; Oxidative Stress; Reactive Oxygen Species

Botulinum neurotoxin A (BoNT/A) cleaves SNAP-25 and inhibits acetylcholine (ACh) release at the neuromuscular junctions (NMJ) to cause neuroparalysis. Previous reports indicate a dyssynchrony between the inhibitory effect of BoNT/A on ACh release and SNAP-25 cleavage.. We tested the in vitro (acute; 90 min) and in vivo (chronic; 12 h) effects of BoNT/A on stimulus-evoked ACh release (SEAR), twitch tension, and SNAP-25 cleavage in isolated extensor digitorum longus (EDL) nerve-muscle preparations (NMP).. In vitro or in vivo BoNT/A poisoning inhibited SEAR and twitch tension. Conversely, SNAP-25 cleavage and inhibition of spontaneous release frequency were observed only in NMP poisoned with BoNT/A in vivo. Moreover, chronic treatment of BoNT/A inhibited ionomycin stimulated Ca(2+) signals in Neuro 2a cells.. These results demonstrate that the inhibition of SEAR precedes SNAP-25 cleavage and suggest involvement of a more complex mechanism for the inhibitory effect of BoNT/A at the NMJ.

Topics: 4-Aminopyridine; Acetylcholine; Acetylcholinesterase; Amifampridine; Animals; Botulinum Toxins, Type A; Calcium Ionophores; Cell Line, Tumor; Electric Stimulation; Electromyography; Evoked Potentials, Motor; In Vitro Techniques; Ionomycin; Mice; Mice, Inbred C57BL; Neuroblastoma; Neuromuscular Agents; Neuromuscular Junction; Potassium Channel Blockers; Reflex; Time Factors

Calpains are involved in calcium-induced neuronal cell toxicity, which is associated with the pathophysiology of Alzheimer's disease (AD). The activity of calpains is regulated by the inhibitor calpastatin, and increased activity of calpains and decreased calpastastin are often found in AD. Histone deacetylase (HDAC) inhibitors are implicated in AD treatment through the improvement of learning and memory but the underlying mechanism is yet to be understood. Here, using SH-SY5Y neuroblastoma cells and a calcium ionophore ionomycin, we examined whether and how HDAC inhibitor trichostatin A (TSA) inhibits calcium-induced neuronal cell death. TSA increased both the mRNA and protein levels of calpastatin, with no alterations in those of calpain 1 and calpain 2. Furthermore, TSA-stimulated increase of calpastatin was accompanied by a significant attenuation of ionomycin-induced autolysis of calpain 1, but not of calpain 2, and calpain-dependent 150 kDa αII spectrin cleavage. Under these conditions, however, caspase activity was unaltered. Moreover, ectopic expression of small interfering RNA of calpastatin reversed the inhibitory effect of TSA on ionomycin-induced calpain 1 autolysis and αII spectrin cleavage. Chromatin immunoprecipitation assay revealed the increased levels of acetylation at lysine 5 of histone H4 (H4K5-Ac), H3K9-Ac and H3K14-Ac within the calpastatin promoter region in TSA-treated cells relative to control cells. Finally, TSA significantly decreased ionomycin-induced cell toxicity. This study demonstrates that TSA attenuates calcium-induced neuronal cell death by the inhibition of calpain activity which is mediated in part by increased calpastatin expression via histone hyperacetylation within the calpastatin promoter region. Our study provides a novel mechanism for the neuroprotective effect of HDAC inhibitors on AD.

Topics: Acetylation; Apoptosis; Blotting, Western; Calcium; Calcium-Binding Proteins; Calpain; Cell Proliferation; Chromatin Immunoprecipitation; Epigenomics; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Ionomycin; Luciferases; Neuroblastoma; Promoter Regions, Genetic; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Tumor Cells, Cultured

Previously, we reported that purinergic ionotropic P2X7 receptors negatively regulate neurite formation in Neuro-2a (N2a) mouse neuroblastoma cells through a Ca(2+)/calmodulin-dependent kinase II-related mechanism. In the present study we used this cell line to investigate a parallel though faster P2X7 receptor-mediated signaling pathway, namely Ca(2+)-regulated exocytosis. Selective activation of P2X7 receptors evoked exocytosis as assayed by high resolution membrane capacitance measurements. Using dual-wavelength total internal reflection microscopy, we have observed both the increase in near-membrane Ca(2+) concentration and the exocytosis of fluorescently labeled vesicles in response to P2X7 receptor stimulation. Moreover, activation of P2X7 receptors also affects vesicle motion in the vertical and horizontal directions, thus, involving this receptor type in the control of early steps (docking and priming) of the secretory pathway. Immunocytochemical and RT-PCR experiments evidenced that N2a cells express the three neuronal SNAREs as well as vesicular nucleotide and monoamine (VMAT-1 and VMAT-2) transporters. Biochemical measurements indicated that ionomycin induced a significant release of ATP from N2a cells. Finally, P2X7 receptor stimulation and ionomycin increased the incidence of small transient inward currents, reminiscent of postsynaptic quantal events observed at synapses. Small transient inward currents were dependent on extracellular Ca(2+) and were abolished by Brilliant Blue G, suggesting they were mediated by P2X7 receptors. Altogether, these results suggest the existence of a positive feedback mechanism mediated by P2X7 receptor-stimulated exocytotic release of ATP that would act on P2X7 receptors on the same or neighbor cells to further stimulate its own release and negatively control N2a cell differentiation.

Topics: Adenosine Triphosphate; Animals; Autocrine Communication; Calcium; Calcium Signaling; Cell Differentiation; Cell Line, Tumor; Exocytosis; Ionomycin; Ionophores; Mice; Neoplasm Proteins; Neuroblastoma; Paracrine Communication; Receptors, Purinergic P2X7; Secretory Vesicles; SNARE Proteins; Vesicular Monoamine Transport Proteins

The formation of intraneuronal inclusions is a common feature of neurodegenerative polyglutamine disorders, including Spinocerebellar ataxia type 3. The mechanism that triggers inclusion formation in these typically late onset diseases has remained elusive. However, there is increasing evidence that proteolytic fragments containing the expanded polyglutamine segment are critically required to initiate the aggregation process. We analyzed ataxin-3 proteolysis in neuroblastoma cells and in vitro and show that calcium-dependent calpain proteases generate aggregation-competent ataxin-3 fragments. Co-expression of the highly specific cellular calpain inhibitor calpastatin abrogated fragmentation and the formation of inclusions in cells expressing pathological ataxin-3. These findings suggest a critical role of calpains in the pathogenesis of Spinocerebellar ataxia type 3.

Topics: Animals; Antibodies; Ataxin-3; Calpain; Cell Line, Tumor; Enzyme Inhibitors; Humans; Ionomycin; Ionophores; Kidney; Machado-Joseph Disease; Mice; Nerve Tissue Proteins; Neuroblastoma; Nuclear Proteins; Peptides; Rabbits; Rats; Repressor Proteins; Transcription Factors

The mechanisms of TDI (2,4-toluene diisocyanate)-induced occupational asthma are not fully established. Previous studies have indicated that TDI induces non-specific bronchial hyperreactivity to methacholine and induces contraction of smooth muscle tissue by activating 'capsaicin-sensitive' nerves resulting asthma. Cytosolic-free calcium ion concentrations ([Ca(2+)](c)) are elevated when either capsaicin acts at vanilloid receptors, or methacholine at muscarinic receptors. This study therefore investigated the effects of TDI on Ca(2+) mobilization in human neuroblastoma SH-SY5Y cells. TDI was found to elevate [Ca(2+)](c) by releasing Ca(2+) from the intracellular stores and extracellular Ca(2+) influx. 500 microM TDI induced a net [Ca(2+)](c) increase of 112+/-8 and 78+/-6 nM in the presence and absence of extracellular Ca(2+), respectively. In Ca(2+)-free buffer, TDI induced Ca(2+) release from internal stores to reduce their Ca(2+) content and this reduction was evidenced by a suppression occurring on the [Ca(2+)](c) rise induced by thapsigargin, ionomycin, and methacholine after TDI incubation. In the presence of extracellular Ca(2+), simultaneous exposure to TDI and methacholine led a higher level of [Ca(2+)](c) compared to single methacholine stimulation, that might explain that TDI induces bronchial hyperreactivity to methacholine. We conclude that TDI is capable of interfering the [Ca(2+)](c) homeostasis including releasing Ca(2+) from internal stores and inducing extracellular Ca(2+) influx. The interaction of this novel character and bronchial hyperreactivity need further investigation.

Topics: Atropine; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Calcium Signaling; Cell Line, Tumor; Cytosol; Dose-Response Relationship, Drug; Drug Synergism; Egtazic Acid; Endoplasmic Reticulum; Hexamethonium; Homeostasis; Humans; Intracellular Fluid; Ionomycin; Methacholine Chloride; Neuroblastoma; Potassium; Pyridines; Receptor, Muscarinic M3; Thapsigargin; Toluene 2,4-Diisocyanate; Verapamil

The neuronal form of nitric oxide synthase (nNOS) was generally assumed to be constitutively expressed at a constant level. However, it is now becoming recognized that its expression can be modulated by a number of physiological and pathophysiological conditions. Previously, we reported that nNOS expression is up-regulated after prolonged muscarinic M(1) receptor stimulation. In this work, we report that muscarinic receptor activation signals the up-regulation of nNOS via multiple pathways in N1E-115 mouse neuroblastoma cells. These include protein kinase C (PKC) activation, cytosolic calcium mobilization and NO production. Further characterization showed that the half-life of nNOS is slightly, but significantly, increased in agonist-pretreated cells compared with vehicle-treated control cells. Based on these data, it appears that the level of nNOS expression is modulated in a complex manner by a number of mechanisms that include, but might not be limited to, those described here.

Topics: Analysis of Variance; Animals; Atropine; Blotting, Western; Carbachol; Cell Line, Tumor; Chelating Agents; Cholinergic Agonists; Drug Interactions; Egtazic Acid; Enzyme Inhibitors; Ionomycin; Ionophores; Mice; Muscarinic Antagonists; Nerve Tissue Proteins; Neuroblastoma; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Phorbol 12,13-Dibutyrate; Phorbol Esters; Receptor, Muscarinic M1; Time Factors; Up-Regulation

Glutamate toxicity has been implicated in cell death in neurodegenerative diseases and injuries. Glutamate-induced Ca2+ influx may mediate activation of calpain, a Ca2+-dependent cysteine protease, which in turn may degrade key cytoskeletal proteins. We investigated glutamate-mediated apoptosis of VSC4.1 motoneurons and functional neuroprotection by calpain inhibition. Exposure of VSC4.1 cells to 10 microM glutamate for 24 hr caused significant increases in intracellular free [Ca2+], as determined by fura-2 assay. Pretreatment of cells with 10 or 25 microM calpeptin (a cell-permeable calpain-specific inhibitor) for 1 hr prevented glutamate-induced Ca2+ influx. Western blot analyses showed an increase in Bax:Bcl-2 ratio, release of cytochrome c from mitochondria, and calpain and caspase-3 activities during apoptosis. Cell morphology, as evaluated by Wright staining, indicated predominantly apoptotic features following glutamate exposure. ApopTag assay further substantiated apoptotic features morphologically as well as biochemically. Our data showed that calpeptin mainly prevented calpain-mediated proteolysis and apoptosis and maintained whole-cell membrane potential, indicating functional neuroprotection. The results imply that calpeptin may serve as a therapeutic agent for preventing motoneuron degeneration, which occurs in amyotrophic lateral sclerosis and spinal cord injury. In this investigation, we also examined glutamate receptor subtypes involved in the initiation of apoptosis in VSC4.1 cells following exposure to glutamate. Our results indicated that the N-methyl-D-aspartate (NMDA) receptors contributed more than alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptors to glutamate-mediated Ca2+ influx and cell death mechanism. Inhibition of the activities of both NMDA and AMPA receptors protected VSC4.1 cells from glutamate toxicity and preserved whole-cell membrane potential.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Calcium; Calpain; Caspase 3; Caspases; Cell Fusion; Cysteine Proteinase Inhibitors; Cytochromes c; Dipeptides; Glutamic Acid; Humans; Ionomycin; Ionophores; Mice; Mitochondria; Motor Neurons; Neuroblastoma; Neuroprotective Agents; Patch-Clamp Techniques; Proto-Oncogene Proteins c-bcl-2; Rats; Spectrin; Spinal Cord

Together with its adaptor protein, the adaptor protein of 12 kDa also known as KARAP and TYROBP (DAP12), triggering receptor expressed in myeloid cells 2 (TREM2) is a stimulatory membrane receptor of the immunoglobulin/lectin-like superfamily, well known in myeloid cells. In humans, however, loss-of-function mutations of TREM2/DAP12 leave myeloid cells unaffected but induce an autosomal recessive disease characterized, together with bone cysts, by a spectrum of pathological lesions in the cortex, thalamus and basal ganglia with clinical symptoms of progressive dementia (polycystic lipomembraneous osteodysplasia with sclerosing leukoencephalopathy). Nothing was known about the role of TREM2/DAP12 in brain cell biology and physiology. By confocal immunocytochemistry we demonstrate that, in both human and mouse cerebral cortex, TREM2/DAP12, strongly expressed by microglia, is also present in a fraction of neurons but not in astrocytes and oligodendrocytes. In contrast, in the hippocampal cortex TREM2-expressing neurons are rare. Both in neurons and microglia the receptor appears to be located mostly intracellularly in a discrete compartment(s) partially coinciding with (or adjacent to) the Golgi complex/trans-Golgi network. Four nerve cell lines were identified as expressing the intracellular receptor system. In living human microglia CHME-5 and glioblastoma T98G cells, activation of TREM2 by its specific antibody induced [Ca2+]i responses, documenting its surface expression and functioning. Surface expression of TREM2, low in resting CHME-5 and T98G cells, increases significantly and transiently (60 min) when cells are stimulated by ionomycin, as revealed by both surface biotinylation and surface immunolabeling. Our results provide the first information about the expression, distribution (mostly intracellular) and functioning of TREM2/DAP12 system in nerve cells, a necessary step in the understanding of the cellular mechanisms affected in polycystic lipomembraneous osteodysplasia with sclerosing leukoencephalopathy.

Topics: Adaptor Proteins, Signal Transducing; Animals; Antibodies; Brain; Calcium; Cell Line, Tumor; Cerebral Cortex; Dementia; Drug Interactions; Epilepsy; Flow Cytometry; Glial Fibrillary Acidic Protein; Glioblastoma; Golgi Apparatus; Golgi Matrix Proteins; Humans; Immunohistochemistry; Immunoprecipitation; Ionomycin; Ionophores; Membrane Glycoproteins; Membrane Proteins; Mice; Mice, Inbred BALB C; Microglia; Microscopy, Confocal; Microscopy, Immunoelectron; Myeloid Cells; Neuroblastoma; Neurons; Phosphopyruvate Hydratase; Receptors, Immunologic; Reverse Transcriptase Polymerase Chain Reaction; Subacute Sclerosing Panencephalitis; Time Factors; Triggering Receptor Expressed on Myeloid Cells-1

Stanniocalcin is a polypeptide hormone that was first reported in fish as a regulator of mineral metabolism. Its recent identification in mammals has opened a new area of investigation in basic and clinical endocrinology. In the present study, regulation of the stanniocalcin (STC) and stanniocalcin related protein (STCrP) genes were investigated in mouse neuroblastoma cells (Neuro-2A) in relation to neuronal cell differentiation. Neuro-2A is an undifferentiated cell line that contains measurable levels of STCrP mRNA, but undetectable levels of STC mRNA. Treatment of the cells with either dbcAMP (1-4 mM) or 50 microM euxanthone (PW1) resulted in extensive differentiation and neurite outgrowth. However, only neurites of dbcAMP-treated cells developed varicosities, a phenotypic marker of axon formation. Furthermore, following differentiation induced by dbcAMP, there was an upregulation of STC and downregulation of STCrP mRNA levels. In the first 24 and 48 h of treatments, there was a maximum twofold induction and 1.5-fold reduction in STC and STCrP mRNAs respectively. Following 96 h of treatment, an additional 14-fold STC induction and 1.2-fold STCrP reduction were observed. The increase in STC mRNA levels was accompanied by a concomitant increase in axon-specific low molecular form microtubule-associated protein (MAP-2c) mRNA and varicosities on the neurites, suggesting a possible role for STC in axonogenesis. There was no induction of STC mRNA levels when PW1 was added into the culture media, whereas ionomycin (1-10 microM) had no observable effects on cell differentiation or STC/STCrP mRNA. Immunocytochemical staining of dbcAMP-treated cells revealed abundant levels of immunoreactive STC, particularly in the varicosities, with only weak staining in control, untreated cells. Antisense oligodeoxynucleotides transfection studies indicated that the expression of STC was a cause of varicosity formation and a consequence of cell differentiation. Our findings lend further support to the notion that STC is involved in the process of neural differentiation.

Topics: Animals; Bucladesine; Cell Differentiation; Dose-Response Relationship, Drug; Gene Expression Regulation; Glycoproteins; Hormones; Ionomycin; Ionophores; Mice; Neurites; Neuroblastoma; Neurons; Oligonucleotides, Antisense; RNA, Messenger; Stimulation, Chemical; Time Factors; Tumor Cells, Cultured; Xanthenes; Xanthones

In fura 2-loaded N1E-115 cells, regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) following a Ca(2+) load induced by 1 microM thapsigargin and 10 microM carbonylcyanide p-trifluoromethyoxyphenylhydrazone (FCCP) was Na(+) dependent and inhibited by 5 mM Ni(2+). In cells with normal intracellular Na(+) concentration ([Na(+)](i)), removal of bath Na(+), which should result in reversal of Na(+)/Ca(2+) exchange, did not increase [Ca(2+)](i) unless cell Ca(2+) buffer capacity was reduced. When N1E-115 cells were Na(+) loaded using 100 microM veratridine and 4 microg/ml scorpion venom, the rate of the reverse mode of the Na(+)/Ca(2+) exchanger was apparently enhanced, since an approximately 4- to 6-fold increase in [Ca(2+)](i) occurred despite normal cell Ca(2+) buffering. In SBFI-loaded cells, we were able to demonstrate forward operation of the Na(+)/Ca(2+) exchanger (net efflux of Ca(2+)) by observing increases (approximately 6 mM) in [Na(+)](i). These Ni(2+) (5 mM)-inhibited increases in [Na(+)](i) could only be observed when a continuous ionomycin-induced influx of Ca(2+) occurred. The voltage-sensitive dye bis-(1,3-diethylthiobarbituric acid) trimethine oxonol was used to measure changes in membrane potential. Ionomycin (1 microM) depolarized N1E-115 cells (approximately 25 mV). This depolarization was Na(+) dependent and blocked by 5 mM Ni(2+) and 250-500 microM benzamil. These data provide evidence for the presence of an electrogenic Na(+)/Ca(2+) exchanger that is capable of regulating [Ca(2+)](i) after release of Ca(2+) from cell stores.

Topics: Animals; Brain Neoplasms; Calcium; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Enzyme Inhibitors; Ionomycin; Ionophores; Membrane Potentials; Mice; Neuroblastoma; Nickel; Ouabain; Scorpion Venoms; Sodium; Sodium-Calcium Exchanger; Thapsigargin; Tumor Cells, Cultured; Veratridine

The plasma and nuclear membranes of neural cells have been shown to express gangliosides to a limited extent before, and at increasing levels during, differentiation. Recent studies employing qualitative cytochemistry have shown that GM1 expression in particular is significantly elevated in both membranes by specific neuritogenic agents. The present study provides a more complete description of ganglioside patterns of the 2 membranes of NG108-15 cells and a mutated form of the latter lacking gangliotetraose gangliosides. Nuclei of wild type NG108-15 cells were found to contain predominantly GM1 and GD1a, whereas whole cells had those in addition to substantial amounts of GM2 and GM3. GM1 and GD1a levels increased 2--3.5-fold in both whole cells and nuclei following axonogenic stimulation, but changed little in response to dendritogenic agents. GM2 expression, limited to the plasma membrane, showed little if any change with axonogenic stimuli but a 1.5--2-fold increase following treatment with dendritogenic agents. GM3 resembled GM2 in being virtually absent from the nuclear membrane, while its presence in the plasma membrane showed only modest change at most with any of the stimuli. The gangliotetraose ganglioside-deficient mutant cell line, NG-CR72, had significantly higher basal levels of GM2 in the plasma membrane compared to wild type NG108-15 cells, and this level increased significantly on treatment with dendritogenic agents. Basal GM3 levels were greatly reduced in the mutant cells and changed little with any of the stimuli. As expected, nuclei of NG-CR72 cells were virtually devoid of gangliosides. These mutant cells were previously shown to extend well defined dendritic neurites but were incapable of forming stable axonal processes. This study thus demonstrates major differences in the ganglioside content of wild type and mutated NG108-15 cells and their nuclei, and in their response to different neuritogenic stimuli.

Topics: Animals; Axons; Bucladesine; Cell Differentiation; Cell Membrane; Cell Nucleus; Dendrites; Gangliosides; Ionomycin; Mice; Mutation; Neurites; Neuroblastoma; Nuclear Envelope; Oligosaccharides; Potassium Chloride; Rats; Tretinoin; Tumor Cells, Cultured

Volumes of neuroblastoma x glioma hybrid NG 108-15 cells were electronically measured in order to characterize the mechanisms involved in volume regulation in isosmotic and anisosmotic conditions. The cells behave as perfect osmometers when tonicity was changed at constant chloride concentration by adding sucrose or replacing NaCl with CaCl2 or MgCl2. In contrast, the cell volume was poorly dependent on tonicity when the Cl- concentration was changed by adding NaCl or H2O. Cell shrinkage was induced by cell stirring or after a hypotonicity-induced swelling. These volume decreases were abolished by caffeine but not by ryanodine or EGTA. Shrinkage was also induced by the Ca2+ ionophore ionomycin. The ionomycin-induced volume decrease was abolished by EGTA. Cell swelling induced an outwardly rectifying Cl- current which was blocked by 5-nitro-2-(3-phenylpropylamino)benzoic acid, 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid and dihydroindenyloxy-alkanoic acid. When the tonicity was reduced at constant Cl- concentration by replacing NaCl with CaCl2 or MgCl2, the volume increased and then slowly decreased towards its control value. This regulatory volume decrease was blocked by 5-nitro-2-(3-phenylpropylamino)benzoic acid, 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid and dihydroindenyl-oxy-alkanoic acid. Long-term (hours-days) cell shrinkage was induced by a reduction of the culture medium osmolarity. Long-term cell swelling was induced by an increase of the culture medium osmolarity. These volume changes were abolished by the protein translation inhibitor cycloheximide. The results suggest that NG 108-15 cell volume is regulated by at least four interacting mechanisms controlled, respectively, by intracellular Ca2+, extracellular NaCl, cell volume and intracellular ionic strength. The speculative nature of ionic systems responsible for these volume regulating mechanisms is discussed.

Topics: Animals; Caffeine; Cell Size; Chloride Channels; Culture Media; Egtazic Acid; Glioma; Hybrid Cells; Hypotonic Solutions; Ionomycin; Membrane Potentials; Neuroblastoma; Osmolar Concentration; Ryanodine; Water-Electrolyte Balance

In mouse neuroblastoma N18TG2 cells prelabeled with [3H]arachidonic acid ([3H]AA) the biosynthesis of 2-arachidonoylglycerol (2-AG) is induced by ionomycin in a fashion sensitive to an inhibitor of diacylglycerol (DAG) lipase, RHC 80267, but not to four different phospholipase C (PLC) blockers. Pulse experiments with [3H]AA showed that ionomycin stimulation leads to the sequential formation of [3H]phosphatidic acid ([3H]PA), [3H]DAG, and [3H]2-AG. [3H]2-AG biosynthesis in N18TG2 cells prelabeled with [3H]AA was counteracted by propranolol and N-ethylmaleimide, two inhibitors of the Mg2+/Ca2(+)-dependent brain PA phosphohydrolase. Pretreatment of cells with exogenous phospholipase D (PLD) led to a strong potentiation of ionomycin-induced [3H]2-AG formation. These data indicate that DAG precursors for 2-AG in intact N18TG2 cells are obtained from the hydrolysis of PA and not through the activation of PLC. The presence of 2% ethanol during ionomycin stimulation failed to elicit the synthesis of [3H]phosphatidylethanol and did not counteract the formation of [3H]PA, thus arguing against the activation of PLD by the Ca2+ ionophore. Selective inhibitors of secretory phospholipase A2 and the acyl-CoA acylase inhibitor thimerosal significantly reduced [3H]2-AG biosynthesis. The implications of these latter findings, and of the PA-dependent pathways of 2-AG formation described here, are discussed.

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Cyclohexanones; Diglycerides; Endocannabinoids; Enzyme Inhibitors; Glycerides; Hydrolysis; Ionomycin; Ionophores; Lipoprotein Lipase; Mice; Neuroblastoma; Phosphatidic Acids; Phospholipase D; Phosphoric Monoester Hydrolases; Prodrugs; Protease Inhibitors; Tumor Cells, Cultured; Type C Phospholipases

Near-field optics (NFO) overcomes the diffraction limit of light microscopes and permits visualization of single molecules. However, despite numerous applications of NFO in the physical sciences, there is still a paucity of applications in the neurosciences. In this work, the authors have developed NFO probes to image intracellular dynamic processes in living cells. This is the first time a NFO probe has been inserted inside a living cell to deliver light to a spatially controlled region for optical measurements and to record cellular responses to external stimuli. Two different optical detection systems (CCD camera and avalanche photon detection) were developed to monitor cellular responses to drug administration in two different cell types. NG108-15 neuroblastoma cells and vascular smooth muscle cells (VSMC) were penetrated with NFO probes. Intracellular Ca2+ increases post drug stimulation were detected by NFO probes. The cells were loaded with either fura-2/AM or fluo-3/AM calcium dyes. VSMC were stimulated with angiotensin II, resulting in a precise area of intracellular Ca2+ increase. Different response profiles of Ca2+ increases were observed after ionomycin and bradykinin administration in NG108-15 cells. Responsive heterogeneities due to ionomycin among different cells of the same type were recorded. The results show that NFO probes make possible real-time visualization of intracellular events. With refinement, intracellular NFO probes offer the potential of probing cell function with fast temporal and excellent spatial resolutions.

Topics: Angiotensin II; Animals; Aorta, Thoracic; Bradykinin; Calcium; Cells, Cultured; Fluorescent Dyes; Glioma; Hybrid Cells; Ionomycin; Mice; Microscopy, Fluorescence; Muscle, Smooth, Vascular; Neuroblastoma; Optics and Photonics; Photography; Photons; Rats; Rats, Sprague-Dawley

Extracellular application of lysophosphatidic acid (LPA) elevated intracellular Ca(2+) concentration ([Ca(2+)](i)) in human SH-SY5Y neuroblastoma cells. The maximal response to LPA occurred between 0. 1 and 1 microM, at which point [Ca(2+)](i) was increased by approx. 500 nM. This increase was of similar magnitude to that caused by the muscarinic acetylcholine receptor agonist methacholine (MCh), although the initial rate of release by LPA was slower. Both LPA and MCh released Ca(2+) from intracellular stores, as assessed by inhibition of their effects by thapsigargin, a blocker of endoplasmic reticular Ca(2+) uptake, and by the persistence of their action in nominally Ca(2+)-free extracellular medium. Similarly, both agonists appeared to stimulate store-refilling Ca(2+) entry. MCh produced a marked elevation in cellular Ins(1,4,5)P(3) and stimulated [(3)H]InsP accumulation in the presence of Li(+). In contrast, LPA failed to stimulate detectable phosphoinositide turnover. Chronic down-regulation of Ins(1,4,5)P(3) receptor (InsP(3)R) proteins with MCh did not affect Ca(2+) responses to LPA. In addition, heparin, a competitive antagonist of InsP(3)Rs, blocked Ca(2+)-mobilization in permeabilized SH-SY5Y cells in response to MCh or exogenously added Ins(1,4,5)P(3), but failed to inhibit Ca(2+)-release induced by LPA. Elevation of [Ca(2+)](i) elicited by LPA was blocked by guanosine 5'-[beta-thio]-diphosphate, indicating that this agonist acts via a G-protein-coupled receptor. However, pertussis toxin was without effect on LPA-evoked [Ca(2+)](i) responses, suggesting that G(i/o)-proteins were not involved. In the absence of extracellular Ca(2+), N,N-dimethylsphingosine (DMS, 30 microM), a competitive inhibitor of sphingosine kinase, blocked LPA-induced Ca(2+) responses by almost 90%. In addition, MCh-induced Ca(2+) responses were also diminished by the addition of DMS, although to a lesser extent than with LPA. We conclude that LPA mobilizes intracellular Ca(2+)-stores in SH-SY5Y cells independently of the generation and action of Ins(1,4,5)P(3). Furthermore, the Ca(2+)-response to LPA appears to be dependent on sphingosine kinase activation and the potential generation of the putative second messenger sphingosine 1-phosphate.

Topics: Calcium; Down-Regulation; Enzyme Activation; Guanosine Diphosphate; Heparin; Humans; Inositol 1,4,5-Trisphosphate; Ionomycin; Lysophospholipids; Methacholine Chloride; Neuroblastoma; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine; Tumor Cells, Cultured

Lithium, carbamazepine and sodium valproate are mood stabilizers used in the treatment of bipolar disorder, and although their mechanisms of action remain unknown, signal transduction systems and the associated modulation of gene expression may constitute significant actions. We examined if acute or chronic treatments with these agents modulated the activation of the AP-1 transcription factor or the increased intracellular calcium levels in human neuroblastoma SH-SY5Y cells caused by stimulation with carbachol. AP-1 activation stimulated by carbachol was reduced by pretreatment for 1 h, 24 h or 7 days with 1 mM lithium by 15%, 37%, and 60%, respectively, and with 0.05 mM carbamazepine by 3%, 21%, and 46%, respectively, but not by pretreatment with 0.5 mM sodium valproate. AP-1 DNA binding activity stimulated by carbachol or by phorbol ester-induced activation of protein kinase C was inhibited by the protein kinase C inhibitor Ro31-8220, but phorbol ester-stimulated AP-1 activation was unaltered by 7-day pretreatments with lithium or carbamazepine. Activation of AP-1 by carbachol was dependent on calcium, as it was inhibited by treatment with the extracellular calcium chelator EGTA, the intracellular calcium chelator BAPTA-AM, and the calcium/calmodulin kinase II inhibitor KN62. Pretreatment for 7 days with lithium or carbamazepine had no significant effect on carbachol-stimulated increases in intracellular calcium levels, but reduced the stimulation of AP-1 by the calcium ionophore ionomycin by 30% to 40%. Thus, chronic treatment with the antibipolar agents lithium and carbamazepine attenuates carbachol-stimulated AP-1 DNA binding activity, and these agents preferentially inhibit signaling cascades activated by the calcium rather than the protein kinase C arm of the phosphoinositide signaling pathway.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Antipsychotic Agents; Bipolar Disorder; Calcium; Calcium Signaling; Carbachol; Carbamazepine; Chelating Agents; DNA; Dose-Response Relationship, Drug; Edetic Acid; Egtazic Acid; Gene Expression Regulation; Humans; Indoles; Ionomycin; Ionophores; Lithium; Muscarinic Agonists; Nerve Tissue Proteins; Neuroblastoma; Neurons; Phosphatidylinositols; Protein Binding; Protein Kinase C; Signal Transduction; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Tumor Cells, Cultured; Valproic Acid

Oxidative stress appears to contribute to neuronal dysfunction associated with Alzheimer's disease and other CNS neurodegenerative disorders. This investigation examined if oxidative stress might contribute to impairments in cholinergic receptor-linked signaling systems and if intracellular glutathione levels modulated responses to oxidative stress. To do this the activation of the AP-1 and NF-kappaB transcription factors and of the phosphoinositide second-messenger system was measured in human neuroblastoma SH-SY5Y cells after exposure to the oxidants H2O2 or diamide, with or without prior depletion of cellular glutathione. H2O2 concentration-dependently inhibited carbachol-stimulated AP-1 activation and this inhibition was potentiated in glutathione-depleted cells. Carbachol-stimulated NF-kappaB activation was unaffected by H2O2 unless glutathione was depleted, in which case there was a H2O2 concentration-dependent inhibition. Glutathione depletion also potentiated the inhibition by H2O2 of carbachol- or G-protein (NaF)-stimulated phosphoinositide hydrolysis, whereas phospholipase C activated by the calcium ionophore ionomycin was not inhibited. The thiol-oxidizing agent diamide also inhibited phosphoinositide hydrolysis stimulated by carbachol or NaF, and glutathione depletion potentiated the diamide concentration-dependent inhibition. Unlike H2O2, diamide also inhibited ionomycin-stimulated phosphoinositide hydrolysis. Activation of both AP-1 and NF-kappaB stimulated by carbachol was inhibited by diamide, and glutathione depletion potentiated the inhibitory effects of diamide. Thus, diamide inhibited a wider range of signaling processes than did H2O2, but glutathione depletion increased the susceptibility of phosphoinositide hydrolysis and of transcription factor activation to inhibition by both H2O2 and diamide. These results demonstrate that the vulnerability of signaling systems to oxidative stress is influenced by intracellular glutathione levels, indicating that cell-selective susceptibility to inhibition of signal transduction systems by oxidative stress can arise from cellular variations in antioxidant capacity.

Topics: Analysis of Variance; Base Sequence; Binding Sites; Buthionine Sulfoximine; Carbachol; Consensus Sequence; Diamide; Glutathione; GTP-Binding Proteins; Humans; Hydrogen Peroxide; Ionomycin; Neuroblastoma; NF-kappa B; Oligodeoxyribonucleotides; Oxidative Stress; Phosphatidylinositols; Second Messenger Systems; Sodium Fluoride; Transcription Factor AP-1; Tumor Cells, Cultured; Vanadates

Neuroblastoma cells are widely utilized models for the study of the neuritic outgrowth phase of neuronal differentiation, but relatively few such studies have attempted to identify the nature of the process outgrowths. This identification will be necessary in developing strategies for utilizing these models to distinguish the underlying mechanisms involved in axonogenesis vs dendritogenesis. In an effort to identify procedures for inducing specific types of neurite outgrowth, and for distinguishing axon- from dendrite-like processes, we have subjected two neuroblastoma cell lines to a variety of stimuli previously shown to induce neurite outgrowth in these cells. These include neuraminidase, ionomycin, KCl+dibutyryl cAMP, cholera toxin B subunit, retinoic acid, dibutyryl cAMP (alone), GM1 ganglioside, and low serum. The first four of these (group 1) gave rise to neurites with axon-like characteristics, including immunostaining that was positive for phosphorylated high molecular weight neurofilament protein (NF-H) and synaptic vesicle protein-2 (SV2), but negative for microtubule-associated protein-2 (MAP2). The next three treatments (group 2) resulted in dendrite-like processes, as evidenced in immunostaining that was positive for MAP2 and negative for NF-H and SV2. Neurites produced by low serum had mixed properties. These cytoskeletal differences were supported by immunoblot analysis with antisera to the above cytoskeletal proteins. Striking morphological differences were also noted, group 2-induced neurites being significantly shorter with more branch points than those generated by group 1 stimulants. Time of exposure to stimulatory agent was crucial in determining expression of the neuritic phenotype. Correlation with previous studies suggests that axon-like neurites result from stimulants which elevate intracellular Ca2+, a dependence not previously reported to our knowledge. Dendrite-like process outgrowth, on the other hand, does not appear to depend on altered intracellular Ca2+.

Topics: Animals; Axons; Bucladesine; Cholera Toxin; Dendrites; G(M1) Ganglioside; Ionomycin; Membrane Glycoproteins; Microtubule-Associated Proteins; Nerve Tissue Proteins; Neuraminidase; Neuroblastoma; Neurofilament Proteins; Neurotransmitter Agents; Potassium Chloride; Rats; Tretinoin; Tumor Cells, Cultured

Peroxynitrite may contribute to oxidative stress involving neurodegeneration in several disorders, including Alzheimer's disease. As with other reactive oxygen species, peroxynitrite might affect neuronal signalling systems, actions that could contribute to adaptive or deleterious cellular outcomes, but such effects have not previously been studied. To address this issue directly, peroxynitrite (50-500 microM) was administered to human neuroblastoma SH-SY5Y cells to assess its effects on protein tyrosine nitration, phosphoinositide signalling and protein tyrosine phosphorylation. Peroxynitrite rapidly increased the nitrotyrosine immunoreactivity of numerous proteins, primarily in the cytosol. Peroxynitrite inhibited, in a concentration-dependent manner, phosphoinositide hydrolysis stimulated by activation of muscarinic receptors with carbachol and the inhibition was greater after the depletion of cellular glutathione. In comparison, muscarinic receptor-stimulated phosphoinositide hydrolysis in human astrocytoma 1321N1 cells was less vulnerable to inhibition by peroxynitrite either without or with prior depletion of glutathione. There was a large, rapid and reversible increase in the tyrosine phosphorylation of the p120 Src substrate in peroxynitrite-treated SH-SY5Y cells, a response that was potentiated by glutathione depletion; in contrast, peroxynitrite decreased the tyrosine phosphorylation of focal adhesion kinase and paxillin. Tyrosine phosphorylation of p120 in 1321N1 astrocytoma cells was less sensitive to modulation by peroxynitrite. Thus alterations in phosphoinositide signalling and protein tyrosine phosphorylation were greater in neuroblastoma than astrocytoma cells, and modulation of these signalling processes probably contributes to neuronal mechanisms of the response to peroxynitrite.

Topics: Astrocytoma; Cell Membrane; Cytosol; Humans; Hydrolysis; Ionomycin; Neuroblastoma; Nitrates; Phosphatidylinositols; Phosphoproteins; Phosphorylation; Phosphotyrosine; Signal Transduction; Sodium Fluoride; Tumor Cells, Cultured

It is generally believed that the neuronal form of nitric oxide synthase (nNOS) is constitutively expressed and that regulation of this enzyme's activity is mediated solely by changes in cytosolic calcium concentration. Serendipitously, however, we observed that pretreatment of Chinese hamster ovary (CHO) cells, which coexpress muscarinic M1 receptors and nNOS, with 3.3 microM or 1 mM carbachol (CCh) for 48 h resulted in marked enhancement of maximal muscarinic receptor-stimulated nNOS activity as determined by L-[3H]citrulline and cyclic [3H]GMP production. This was accompanied by a decrease in the potency of CCh. Muscarinic receptor density was reduced in the agonist-pretreated cells, as determined by specific [N-methyl-3H]scopolamine methyl chloride binding, whereas competition binding studies revealed no changes in agonist affinity. Both receptor-stimulated inositol phosphate formation and elevation of intracellular calcium concentrations were found to be desensitized in agonist-pretreated cells in a manner dependent on CCh pretreatment concentration. It is interesting that ionomycin-stimulated nNOS activity was greater in CCh-pretreated cells. Also, western analysis revealed increased nNOS immunoreactivity in pretreated cells. A similar increase in nNOS immunoreactivity following agonist treatment was demonstrated in N1E-115 neuroblastoma cells, which endogenously express nNOS and muscarinic M1 receptors. Thus, the enhancement of maximal receptor-stimulated nNOS activity following agonist pretreatment can be attributed to up-regulation of nNOS. It is interesting that this augmentation of the response takes place in spite of receptor down-regulation and desensitization of multiple steps involved in nNOS activation.

Topics: Animals; Arginine; Binding, Competitive; Calcium; Carbachol; CHO Cells; Citrulline; Cricetinae; Dose-Response Relationship, Drug; Humans; Ionomycin; Ionophores; Muscarinic Agonists; Nerve Tissue Proteins; Neuroblastoma; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Receptor, Muscarinic M1; Receptors, Muscarinic; Tritium; Tumor Cells, Cultured

Changes in the concentration of cytosolic free calcium ([Ca++]i) play fundamental roles in the initiation and regulation of many neuronal processes. Altered regulation of [Ca++]i has been implicated in the action of some anesthetics. We investigated the effects of nitrous oxide (N2O) on Ca++ mobilization and membrane potential in the human neuroblastoma cell line SK-N-SH. [Ca++]i was monitored by fluorescence spectrophotometry of cells loaded with fura-2 or fluo-3. N2O reversibly suppressed carbachol-stimulated increases in [Ca++]i. N2O also inhibited increases in [Ca++]i induced by calcium ionophore or depolarization suggesting a mechanism involving enhanced efflux or sequestration of cytosolic Ca++. The inhibitory effect of N2O was attenuated when the transmembrane Na+ gradient was altered either by suspending cells in nominally Na(+)-free buffer or by pretreating cells with ouabain. The inhibitory effect of N2O was also attenuated by the Na+/Ca++ exchange inhibitor 3,4-dichlorobenzamil. The effects of N2O on membrane potential were measured fluorimetrically using bis(1,3-dibutylthiobarbituric acid)-trimethine oxonol. In the presence of N2O, resting membrane potential was hyperpolarized, a condition that would favor Ca++ efflux mediated by the electrogenic Na+/Ca++ exchanger. Taken together, these findings indicate that N2O suppresses carbachol-stimulated increases in [Ca++]i by enhancing Na+/Ca++ exchange activity. Enhancement of neuronal Na+/Ca++ exchange may contribute to the anesthetic action of N2O.

Topics: Amiloride; Aniline Compounds; Calcium; Carrier Proteins; Cell Line; Fluorescent Dyes; Fura-2; Humans; Ionomycin; Kinetics; Membrane Potentials; Neuroblastoma; Nitrous Oxide; Ouabain; Sodium; Sodium-Calcium Exchanger; Spectrometry, Fluorescence; Tumor Cells, Cultured; Xanthenes

Stimulation of SK-N-BE(2) cells with 1 mM carbachol (Cch) elicited phosphoinositide (PPI) hydrolysis and a rapid elevation of cytosolic Ca2+ concentration ([Ca2+]i) from 115 nM to about 500 nM, followed by a plateau around 200 nM. In myo [3H]inositol-labelled cells, Cch-evoked accumulation of [3H]inositol phosphate (IPs) was not affected when [Ca2+]i was clamped at resting by cell loading with 10 microM BAPTA/AM; under these conditions, maximal 1,4,5-inositol trisphosphate accumulation was not reduced either. When [Ca2+]i was clamped around 700 nM by cell treatment with 600 nM ionomycin, Cch-evoked [3H]IPs accumulation was enhanced by less than 20%, but it was impaired by a 30% and a 55% after [Ca2+]i reduction to about 70 nM and 35-50 nM, by cell loading with 20 microM or 40 microM BAPTA/AM, respectively. These results show that, in SK-N-BE(2) cells, Cch-activated PPI-specific phospholipase C is sensitive to [Ca2+]i but it already operates under suboptimal conditions at resting [Ca2+]i.

Topics: Calcium; Calcium Channel Agonists; Carbachol; Chelating Agents; Egtazic Acid; Electrophysiology; Humans; Hydrolysis; Ionomycin; Ionophores; Miotics; Neuroblastoma; Phosphatidylinositols; Tumor Cells, Cultured

The monoacylglycerol 2-arachidonoylglycerol (2-AG) has been recently suggested as a possible endogenous agonist at cannabinoid receptors both in brain and peripheral tissues. Here we report that a widely used model for neuronal cells, mouse N18TG2 neuroblastoma cells, which contain the CB1 cannabinoid receptor, also biosynthesize, release and degrade 2-AG. Stimulation with ionomycin (1-5 microM) of intact cells prelabelled with [3H]arachidonic acid ([3H]AA) led to the formation of high levels of a radioactive component with the same chromatographic behaviour as synthetic standards of 2-AG in TLC and HPLC analyses. The amounts of this metabolite were negligible in unstimulated cells, and greatly decreased in cells stimulated in the presence of the Ca2+-chelating agent EGTA. The purified component was further characterized as 2-AG by: (1) digestion with Rhizopus arrhizus lipase, which yielded radiolabelled AA; (2) gas chromatographic-MS analyses; and (3) TLC analyses on borate-impregnated plates. Approx. 20% of the 2-AG produced by stimulated cells was found to be released into the incubation medium when this contained 0.1% BSA. Subcellular fractions of N18TG2 cells were shown to contain enzymic activity or activities catalysing the hydrolysis of synthetic [3H]2-AG to [3H]AA. Cell homogenates were also found to convert synthetic [3H]sn-1-acyl-2-arachidonoylglycerols (AcAGs) into [3H]2-AG, suggesting that 2-AG might be derived from AcAG hydrolysis. When compared with ionomycin stimulation, treatment of cells with exogenous phospholipase C, but not with phospholipase D or A2, led to a much higher formation of 2-AG and AcAGs. However, treatment of cells with phospholipase A2 10 min before ionomycin stimulation caused a 2.5-3-fold potentiation of 2-AG and AcAG levels with respect to ionomycin alone, whereas preincubation with the phospholipase C inhibitor neomycin sulphate did not inhibit the effect of ionomycin on 2-AG and AcAG levels. These results suggest that the Ca2+-induced formation of 2-AG proceeds through the intermediacy of AcAGs but not necessarily through phospholipase C activation. By showing for the first time the existence of molecular mechanisms for the inactivation and the Ca2+-dependent biosynthesis and release of 2-AG in neuronal cells, the present paper supports the hypothesis that this cannabimimetic monoacylglycerol might be a physiological neuromodulator.

Topics: Animals; Arachidonic Acids; Calcium; Calcium Channel Blockers; Cannabinoids; Endocannabinoids; Enzyme Inhibitors; Glycerides; Hydrolysis; Ionomycin; Ionophores; Mice; Neuroblastoma; Neurons; Polyunsaturated Alkamides; Receptors, Cannabinoid; Receptors, Drug; Subcellular Fractions; Tumor Cells, Cultured

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

The effect of the membrane-permeant calcium chelator 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA/AM) on ionomycin-induced cellular calcium overload was studied in single differentiated NH15-CA2 neuroblastoma x glioma hybrid cells. To monitor [Ca2+]i we used the fluorescent indicator Fura-2. Preincubation of the cells with 3 microM BAPTA/AM reduced the number of cells showing deregulation of [Ca2+]i during ionomycin-induced calcium influx. The calcium transients elicited by application of KCl were also severely affected by the chelator. These transients, although varying from cell to cell in shape, amplitude and duration, are well reproducible in individual cells. After incubation of cells for 1 h with 0.3-30 microM BAPTA/AM the time course of these cellular transients was markedly slowed. At 1 microM BAPTA/AM, the time constant of decline of [Ca2+]i was increased by a factor of 4.1 +/- 2.4 (n = 14) and the amplitude was reduced to about 50%. With 30 microM BAPTA/AM, the K(+)-induced calcium transients were almost completely inhibited. We conclude that intracellularly loaded calcium chelators may be used for the prevention of [Ca2+]i-induced cell damage, however, at the expense of a disturbed calcium signalling.

Topics: Animals; Calcium; Chelating Agents; Cytoplasm; Egtazic Acid; Electrophysiology; Glioma; Hybrid Cells; Ionomycin; Ionophores; Mice; Nerve Tissue Proteins; Neuroblastoma; Neurons; Phenotype; Potassium Chloride; Rats

To verify the effect of cell culture state on frequency dependent increase in proliferation as well as Ca2+ flux across the plasma membrane, tumorigenic bone (TE-85) and neuroblastoma x glioma (NG108-15) cells cultured in the presence of fetal bovine serum (FBS) were exposed to capacitively coupled electric (CCEF) fields in the extremely low frequency (ELF) range of 10 to 18 Hz. [3H]Thymidine incorporation and 45Ca2+ uptake were used as endpoints. TE-85 cells cultured in the presence of 10% FBS did not exhibit a frequency dependent increase in proliferation in contrast to previous studies under growth arrested culture conditions, in which the cells were deprived of FBS. However, both TE-85 and NG108-15 cells had an increase in 45Ca2+ uptake in response to a 16 Hz 18.3 mV/cm CCEF. Fura-2 digital imaging microscopy was used to verify addition of 0.5 mM La3+ and 0.5 mM ionomycin as negative and positive controls, respectively. Imaging microscopy data was combined with 45Ca2+ incorporation results to quantify free intracellular calcium ([Ca2+]i) increase in response to CCEF exposure. TE-85 [Ca2+]i increased from 140 to 189-210 nM where as NG108-15 [Ca2+]i increased from 67 to 189-210 nM. These results suggested that serum deprivation may be a requirement for a frequency dependent increase in proliferation in TE-85 cells but is not necessary for the electric field induced increase in 45Ca2+ uptake in both TE-85 and NG108 cells. The present study also represents the first demonstration of increased 45Ca2+ uptake by neuroblastoma and/or glioma cells in response to an electric field exposure.

Topics: Animals; Biological Transport; Bone Neoplasms; Calcium; Cattle; Cell Division; DNA, Neoplasm; Electricity; Glioma; Humans; Hybrid Cells; Ionomycin; Kinetics; Lanthanum; Neuroblastoma; Oscillometry; Osteosarcoma; Thymidine; Tumor Cells, Cultured

Topics: Animals; Arachidonic Acids; Endocannabinoids; Glycerides; Ionomycin; Mice; Models, Biological; Neomycin; Neuroblastoma; Neurotransmitter Agents; Phospholipases; Tumor Cells, Cultured

In this study, the effects of three related peptides, pituitary adenylate cyclase-activating polypeptide 38 (PACAP38), PACAP27, and vasoactive intestinal peptide (VIP), on cyclic AMP (cAMP) accumulation and intracellular Ca2+ concentration ([Ca2+]i) were compared in N1E-115 cells. PACAP38 and PACAP27 stimulated cAMP accumulation up to 60-fold with EC50 values of 0.54 and 0.067 nM, respectively. The effect of VIP on cAMP accumulation was less potent. The binding of 125I-PACAP27 to intact cells was inhibited by PACAP38 and PACAP27 (IC50 values of 0.44 and 0.55 nM, respectively) but not by VIP. In fura-2-loaded cells, both PACAP38 and PACAP27 increased [Ca2+]i with EC50 values around 10 nM. The interactions of these three peptides with ionomycin, a Ca2+ ionophore, and 4 beta-phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, were also determined. Ionomycin increased the cAMP accumulation caused by all three peptides. With low concentrations of PACAP38 or PACAP27, the effect of PMA was inhibitory, whereas at higher concentrations of PACAP (> 1 nM), the effect of PMA was stimulatory. Similar to other agents that elevate cAMP, PACAP38 was an effective stimulator of neurite outgrowth. These results show that (a) PACAP27 and PACAP38 stimulate cAMP accumulation and increase [Ca2+]i through the type I PACAP receptors in N1E-115 cells, (b) ionomycin enhances cAMP accumulation by all three peptides, and (c) activation of protein kinase C has a dose-dependent stimulatory or inhibitory effect on the PACAP38- or PACAP27-stimulated cAMP accumulation.

Topics: Calcium; Carcinogens; Cyclic AMP; Iodine Radioisotopes; Ionomycin; Ionophores; Neurites; Neuroblastoma; Neuropeptides; Neurotransmitter Agents; Pituitary Adenylate Cyclase-Activating Polypeptide; Protein Kinase C; Signal Transduction; Tetradecanoylphorbol Acetate; Time Factors; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

In adherent SH-SY5Y human neuroblastoma cells cultured for 14 days to promote uptake and release of [3H]-noradrenaline, ionomycin induced a biphasic elevation of the intracellular [Ca2+] ([Ca2+]i). This consisted of a rapid transient elevation followed by a marked, persistent secondary elevation. Further study indicated that the peak [Ca2+]i elevation was dependent upon intracellular Ca2+ whilst the secondary elevation was dependent upon extracellular Ca2+. This profile of response and dependence upon intracellular and extracellular sources of Ca2+ was similar to that evoked by the muscarinic agonist, methacholine but was independent of inositol 1,4,5-trisphosphate generation. Ionomycin also stimulated the release of [3H]-noradrenaline from preloaded cells. Both intracellular and extracellular sources of Ca2+ were needed for the full response and synergised to effect release. Thus, in adherent SH-SY5Y cells, ionomycin elevates [Ca2+]i in a complex way in a manner partly analogous to the elevation of [Ca2+]i by agonists of phosphoinositidase C-linked receptors. Furthermore the effects of [Ca2+]i elevation on [3H]-noradrenaline release by these two processes are similar. Such functional consequences may, however, differ under circumstances where the profile and source of Ca2+ for ionomycin-mediated changes differs to that of receptor agonists.

Topics: Calcium; Extracellular Space; Humans; Inositol 1,4,5-Trisphosphate; Ionomycin; Ionophores; Neuroblastoma; Norepinephrine; Tritium; Tumor Cells, Cultured

We have proposed recently that a pertussistoxin-insensitive Ca2+ influx stimulated by Y2-type receptor activation in CHP-234 human neuroblastoma cells underlies increases in intracellular free Ca2+ concentration ([Ca2+]i) induced by neuropeptide Y (NPY), which were strictly dependent on extracellular Ca2+ and independent of internal Ca2+ stores. We describe here the actions of NPY in these same cells, using the activity of Ca(2+)-activated K+ channels as an indicator of [Ca2+]i. The elementary slope conductance of these channels was 110 +/- 3 pS (with an asymmetrical K+ gradient), their activity was greatly increased by application of ionomycin, and they were reversibly blocked by 1 mM tetraethylammonium (TEA) and 100 nM charybdotoxin. Application of 100 nM NPY, in the presence but not in the absence of extracellular Ca2+, increased the channel open probability. ATP applied in the absence of external Ca2+ caused rises both in channel open probability and [Ca2+]i. Inositol trisphosphate production was stimulated by ATP but not by NPY. In outside-out patches, NPY increased channel open probability, indicating that NPY-associated Ca2+ influx does not require all the intracellular machinery present in intact cells. Channel activation by NPY was unaffected by the replacement of guanosine 5'-triphosphate (GTP) by (guanosine 5'-O-(2-thiodiphosphate) (GDP[ beta S]), a non-hydrolysable GDP analogue, in the pipette internal solution, consistent with the lack of involvement of G-proteins in the coupling of Y2-type receptors to Ca2+ influx in CHP-234 cells.

Topics: Adenosine Triphosphate; Brain Neoplasms; Calcium; Electrophysiology; GTP-Binding Proteins; Guanosine Diphosphate; Humans; Inosine Triphosphate; Inositol 1,4,5-Trisphosphate; Ionomycin; Ionophores; Neuroblastoma; Patch-Clamp Techniques; Potassium Channels; Receptors, Neuropeptide Y; Thionucleotides; Tumor Cells, Cultured

The mechanism by which cyclic GMP synthesis is activated through a nucleotide receptor was studied in mouse neuroblastoma x rat glioma hybrid cells [108CC15 (NG 108-15)]. The transient increase in cyclic GMP level induced by ATP reached its maximum at 20 s and lasted for approximately 1 min. The maximal rise in cyclic GMP level achieved was highest for ATP and decreased in the following order: ATP = adenosine 5'(gamma-thio)triphosphate > UTP = 2-methylthio-ATP > ADP much greater than CTP, AMP, alpha,beta-methylene-ATP, 2'- and 3'-O-(4-benzoylbenzoyl)ATP. The EC50 of 1 +/- 0.2 microM for UTP was significantly lower than that for ATP (14 +/- 8 microM) and for all the other nucleotides tested. The rank order of potency is consistent with the pharmacology of a P2u receptor. At submaximal concentrations of the nucleotides ATP and UTP, the rise in cyclic GMP level was inhibited by suramin (IC50 = 40-60 microM) or the pyridoxal phosphate analogue pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (IC50 = 20-30 microM). Pretreatment of cells with the Ca2+ ionophore ionomycin or with 2,5-di(tert-butyl)-1,4-benzohydroquinone, an inhibitor of Ca(2+)-ATPase in the endoplasmic reticulum, a maneuver to deplete internal Ca2+ stores, suppressed the ATP- or UTP-induced stimulation of cyclic GMP synthesis. Similarly, loading of the cells with the Ca2+ chelator 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid inhibited cyclic GMP formation by ATP. Preincubation with forskolin to raise the cyclic AMP level potentiated the ATP-induced rise in cyclic GMP level by 60%.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine Triphosphate; Aminoquinolines; Animals; Bradykinin; Calcium; Colforsin; Cyclic GMP; Glioma; Hybrid Cells; Ionomycin; Mice; Neuroblastoma; Neurons; Nitric Oxide; Radioimmunoassay; Rats; Receptors, Purinergic P2; Signal Transduction; Suramin; Tritium; Tumor Cells, Cultured

In this study, we investigated the vasoactive intestinal polypeptide (VIP)-stimulated cAMP production and its interaction with protein kinase C activation and elevation of intracellular Ca2+ in N1E-115 neuroblastoma cells. VIP treatment caused a 55-fold increase in cAMP accumulation. Addition of 4 beta-phorbol 12-myristate 13-acetate reduced VIP- but not forskolin-stimulated cAMP response. In comparison, ionomycin potentiated both VIP- and forskolin-induced cAMP accumulation. Our results indicate that VIP stimulates cAMP accumulation in N1E-115 cells, and that although activation of protein kinase C inhibits the VIP-stimulated cAMP response, elevation of intracellular Ca2+ potentiates this signaling pathway.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; 1-Methyl-3-isobutylxanthine; Animals; Calcium; Carcinogens; Colforsin; Cyclic AMP; Dose-Response Relationship, Drug; Drug Interactions; Drug Synergism; Enzyme Activation; Ionomycin; Isoquinolines; Kinetics; Mice; Neuroblastoma; Phorbol Esters; Piperazines; Protein Kinase C; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Topics: Bradykinin; Butanols; Carbachol; Cell Line; Humans; Ionomycin; Kinetics; Methacholine Chloride; Neuroblastoma; Norepinephrine; Phosphates; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipase D; Phospholipids; Potassium; Tetradecanoylphorbol Acetate; Tritium; Tumor Cells, Cultured

Topics: Adenylyl Cyclases; Animals; Brain; Calcium; Calmodulin; Cations; Cell Line; Cell Membrane; Egtazic Acid; Hormones; Ionomycin; Kinetics; Neuroblastoma

Modes of Ca2+ activation by bradykinin, serotonin, and ATP and the possible receptor cross-talk were investigated in mouse neuroblastoma x rat glioma hybrid cells (108CC15) by monitoring fura-2 fluorescence in single cells. A transient rise of cytosolic Ca2+ activity was induced by short pulses of the hormones. Brief exposure of cells to ionomycin, which depletes intracellular Ca2+ stores, reduced the size of subsequent responses to bradykinin or ATP, but not to serotonin. Superfusion of the cells with Ca(2+)-free medium abolished the Ca2+ response to serotonin, whereas the responses to bradykinin and to ATP were only slightly reduced. This indicates that ATP, like bradykinin, induces the release of Ca2+ from intracellular stores. Serotonin, in contrast, activates Ca2+ entry from the extracellular space. To investigate whether ATP releases Ca2+ from the same stores as bradykinin, we examined the interaction of the hormones by applying them consecutively. When ATP was applied after bradykinin, the nucleotide did not evoke any response, irrespective of the presence or absence of extracellular Ca2+. The application of ATP before that of bradykinin reduced the size of a following bradykinin-induced Ca2+ response in Ca(2+)-free medium, but not in Ca(2+)-containing medium. This suggests that bradykinin may interact with the ATP-activated mechanism by cross-desensitization. Possibly, bradykinin receptors are coupled to additional Ca2+ stores not accessible to ATP that are refilled by extracellular Ca2+. Cyclic AMP and cyclic GMP apparently do not affect the Ca2+ responses to bradykinin and serotonin, as shown by the lack of influence of preincubation of the cells with forskolin or sodium nitroprusside.

Topics: Animals; Bradykinin; Calcium; Extracellular Space; Glioma; Hybrid Cells; Intracellular Membranes; Ionomycin; Mice; Neuroblastoma; Neurotransmitter Agents; Nucleotides, Cyclic; Rats; Receptors, Bradykinin; Receptors, Purinergic P2; Receptors, Serotonin; Serotonin; Tumor Cells, Cultured

We report a novel phenomenon in which the cytosolic Ca2+ concentration ([Ca2+]i) rise induced in neuroblastoma x glioma hybrid NG108-15 cells by bradykinin is synergistically enhanced by elevated extracellular K+ concentrations. Presence of extracellular Ca2+ during high-K+ treatment, but not after high-K+ treatment, was required for the synergism. In addition, when thapsigargin was added concurrently with high K+, bradykinin still induced a significantly higher [Ca2+]i rise than in cells treated with thapsigargin only. Both bradykinin-induced inositol 1,4,5-trisphosphate (IP3) generation and the size of the internal Ca2+ pool were increased by high-K+ treatment. Our data suggest that changes in membrane potential itself induced by high K+ probably do not cause the synergistic effect. The synergistic effect is apparently due to the stimulatory effects of high K+ on [Ca2+]i, which in turn modulates IP3 generation and increases the size of intracellular Ca2+ pools. If bradykinin is added following high K+, the synergism can be accounted for by increases both in IP3 production and in the size of the internal Ca2+ pools. If bradykinin is added simultaneously with high K+, enhanced Ca2+ release triggered by enhanced IP3 production is the major cause of the synergistic effects.

Topics: Bradykinin; Calcium; Drug Synergism; Egtazic Acid; Extracellular Space; Glioma; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Ionomycin; Neuroblastoma; Osmolar Concentration; Potassium; Signal Transduction; Tumor Cells, Cultured

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

Murine neuroblastoma clone N1E-115 cells possess neurotensin receptors that are coupled to polyphosphoinositide hydrolysis and cyclic guanosine 3',5'-monophosphate (cGMP) formation. These responses rapidly desensitize and these receptors rapidly down-regulate nearly completely in about 15 min. Although neurotensin is rapidly degraded by peptidases, in this study we show that at 37 degrees neurotensin (100 nM) in the absence of peptidase inhibitors caused this rapid desensitization and down-regulation (32 +/- 5 and 24 +/- 2% of control, respectively) of neurotensin receptors in N1E-115 cells. In addition, we demonstrated that this desensitization, resensitization, down-regulation and recovery of binding sites were temperature dependent. These data suggest that a certain degree of phospholipid fluidity or activity of some enzymes is required for these processes to occur. After addition of sodium nitroprusside or ionomycin to cells, cGMP increased in desensitized cells to the same degree as in control cells. Additionally, desensitization and down-regulation occurred in the absence of a change in the affinity of neurotensin for the remaining sites. These data suggest that desensitization is not caused by changes in nitric oxide synthesis, guanylyl cyclase activity or receptor affinity, but predominantly by a decrease in receptor number.

Topics: Animals; Binding Sites; Calcium; Clone Cells; Cyclic GMP; Down-Regulation; Intracellular Fluid; Ionomycin; Kinetics; Mice; Neuroblastoma; Neurotensin; Nitric Oxide; Nitroprusside; Receptors, Neurotensin; Receptors, Neurotransmitter; Stimulation, Chemical; Tumor Cells, Cultured

Under whole-cell recording, bradykinin (BK) produced an initial outward membrane current followed by an inward current in voltage-clamped NG108-15 cells. The initial outward current was associated with a rise in intracellular Ca2+ and was accompanied by the opening of Ca(2+)-dependent K(+)-channels recorded with a cell-attached patch electrode. This current was inhibited by intracellular Mg2+. The inward current was associated with inhibition of the voltage-dependent K(+)-current IK(M). These effects accord with those previously observed in microelectrode-impaled cells, with the difference that BK produced much more pronounced and long-lasting desensitization in the patch-clamped cells.

Topics: Acetylcholine; Animals; Apamin; Bradykinin; Calcium; Calcium Channels; Charybdotoxin; Fluorescent Dyes; Glioma; Hybrid Cells; Indoles; Inositol 1,4,5-Trisphosphate; Ionomycin; Membrane Potentials; Mice; Neuroblastoma; Neurotoxins; Norepinephrine; Potassium Channels; Rats; Scorpion Venoms; Virulence Factors, Bordetella

Application of bradykinin (Bk) to neuroblastoma x dorsal root ganglion (DRG) neurone hybrid cells (ND7/23) evoked an inward (depolarizing) current associated with an increase in membrane conductance. This response was antagonized by D-Arg0,Hyp3,Thi5,8,D-Phe7-Bk, but was not mimicked by des-Arg9-Bk, indicating the involvement of B2-receptors. The response was unaltered by replacement of extracellular Na+ by N-methylglucamine. Replacement of extracellular Cl by gluconate shifted the estimate reversal potential to a more positive value, while the use of potassium acetate filled recording electrodes shifted the reversal potential to a more negative value, and reduced the response amplitude, indicating the importance of Cl- in the response. This response to Bk was mimicked by the calcium ionophore ionomycin. Bk stimulated the formation of inositol 1,4,5-trisphosphate (IP3), and increased the release of arachidonic acid. In addition, Bk produced an increase in [Ca2+]i, as determined by microspectrofluorimetry. This was due to the release of Ca2+ from intracellular stores, since the response was unaltered when the cells were bathed in Ca(2+)-free solution. In summary, Bk depolarizes ND7/23 cells, probably through the activation of a chloride conductance. It seems likely that this is secondary to the rise in cytosolic Ca2+ concentration, due to the release of Ca2+ from internal stores by IP3. This Ca(2+)-activated chloride response is present in some sensory neurones, although its role in the activation of sensory neurones by Bk is at present unclear.

Topics: Alkaloids; Animals; Arachidonic Acid; Arachidonic Acids; Bradykinin; Calcium; Cell Membrane; Cells, Cultured; Cytosol; Evoked Potentials; Ganglia, Spinal; Hybrid Cells; Inositol Phosphates; Ionomycin; Membrane Potentials; Neuroblastoma; Neurons; Phorbol 12,13-Dibutyrate; Protein Kinase C; Rats; Receptors, Bradykinin; Receptors, Neurotransmitter; Second Messenger Systems; Staurosporine

This study evaluates the role of intracellular levels of Ca2+ [Ca2+]i in cyclic GMP formation mediated by muscarinic and histamine receptors in the mouse neuroblastoma clone N1E-115. Muscarinic agonists activated the turnover of phosphoinositides with a relative maximal response similar to that observed previously for cyclic GMP formation. Carbamylcholine induced a transient increase in inositol trisphosphate with a time course similar to that of cyclic GMP formation. In cells loaded with the fluorescent Ca2+ probe fura-2/acetoxymethyl ester, carbamylcholine as well as histamine induced a rapid and transient rise in [Ca2+]i. The time course of the changes in [Ca2+]i induced by agonists as well as by ionomycin closely paralleled that of cyclic GMP formation. Chelation of [Ca2+]i by loading of N1E-115 cells with quin 2/acetoxymethyl ester inhibited cyclic GMP formation induced by agonists in a dose-dependent manner. When cyclic GMP formation induced by agonists was assayed after the cells were exposed to 3 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) for 2 min, the formation of cyclic GMP was not inhibited significantly; however, it was completely abolished after 30-min exposure to EGTA. Treatment of cells with phospholipase A2 had no effect on resting [Ca2+]i and only slightly increased cyclic GMP formation, in spite of the induction of a marked release of [3H]arachidonate. Moreover, the formation of cyclic GMP induced by ionomycin was inhibited by the addition of phospholipase A2. Melittin contaminated with phospholipase A2 activity induced a rapid and sustained increase in cyclic GMP formation, as well as unesterified [3H]arachidonate release. However, after inactivation of the phospholipase A2 activity of melittin, its ability to stimulate cyclic GMP formation was enhanced. Our data indicate that receptor agonists stimulate cyclic GMP formation in N1E-115 cells by activating the formation of inositol trisphosphate, which is followed by the release of Ca2+ from intracellular stores. The evidence obtained does not support a major role for arachidonate release in receptor-mediated activation of guanylate cyclase. Conversely, it is consistent with an inhibitory role for arachidonic acid or its metabolites in this process.

Topics: Animals; Arachidonic Acids; Calcium; Cyclic GMP; Enzyme Activation; Guanylate Cyclase; Hydrolysis; Ionomycin; Mice; Models, Biological; Neuroblastoma; Phosphatidylinositols; Phospholipases A; Phospholipases A2; Receptors, Histamine; Receptors, Muscarinic; Tumor Cells, Cultured

The phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), which causes differentiation of SH-SY5Y neuroblastoma cells, reduces carbachol binding and carbachol-stimulated Ca2+ mobilization in these cells. The decrease in responsiveness to carbachol is due partially to a reduction in the amount of Ca2+ released by the cells and partially to a decrease in the sensitivity of the cells to carbachol. These effects probably can be attributed to a reduction in muscarinic receptor number and a decrease in receptor affinity, respectively. Forskolin, an alkaloid known to cause an increase in cellular cyclic AMP, enhances Ca2+ influx into the cells without affecting the cytosolic free Ca2+ concentration. The alkaloid causes an apparent restoration of the reduced Ca2+ release, caused by TPA, but does not affect the sensitivity of the cells to carbachol. Forskolin increases the decay of carbachol-induced increase in cytosolic Ca2+. The effects of TPA appear to be linked directly to receptor function, whereas those of forskolin are due to the effect of cyclic AMP on cellular Ca2+ metabolism.

Topics: Benzofurans; Binding, Competitive; Biomechanical Phenomena; Calcium; Carbachol; Colforsin; Cytosol; Fluorescent Dyes; Fura-2; Ionomycin; Membrane Potentials; N-Methylscopolamine; Neuroblastoma; Osmolar Concentration; Receptors, Muscarinic; Scopolamine Derivatives; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

This study reports increased intracellular Ca2+ and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in response to muscarinic-cholinergic stimulation of human neuroblastoma (SH-SY5Y) cells. Carbachol stimulation leads to a rapid increase in intracellular Ca2+ and Ins(1,4,5)P3 mass, both reaching a peak at around 10 s and then declining to a new maintained phase significantly above basal. Dose-response analysis of peak and plateau phases of intracellular Ca2+ shows different agonist potencies for both phases, carbachol being more potent for the plateau phase. The plateau-phase intracellular Ca2+ was dependent on extracellular Ca2+, which is admitted to the cell through a non-voltage-sensitive Ni2(+)-blockable Ca2+ channel. Using a Mn2+ quench protocol, we have shown that Ca2+ entry occurs early during the discharge of the internal stores. The plateau phase (Ca2(+)-channel opening) is dependent on the continued presence of agonist, since addition of atropine closes the Ca2+ channel and intracellular Ca2+ declines rapidly back to basal. We also failed to detect a refilling transient when we added back Ca2+ after intracellular Ca2+ had reached a peak and then declined in Ca2(+)-free conditions. These data strongly suggest that muscarinic stimulation of SH-SY5Y cells leads to a rapid release of Ca2+ from an Ins(1,4,5)P3-sensitive internal store and a parallel early entry of Ca2+ across the plasma membrane.

Topics: Arecoline; Atropine; Benzofurans; Calcium; Carbachol; Cell Line; Fluorescent Dyes; Fura-2; Humans; Inositol 1,4,5-Trisphosphate; Ionomycin; Kinetics; Manganese; Muscarine; Neuroblastoma; Receptors, Muscarinic; Tumor Cells, Cultured

The role of muscarinic receptor-mediated polyphosphoinositide hydrolysis and subsequent calcium signals in altering the subcellular localization of calmodulin (CaM) was examined in SK-N-SH human neuroblastoma cells. Upon incubation of the cells with the full agonist carbachol, a 4.5- to 5-fold increase in CaM in the cytosol was observed, from 126 ng of CaM to 629 ng of CaM. There was an accompanying 68% decrease in membrane-bound CaM. The increase in the cytosol was maximal by 15 min, as was a corresponding decrease in membrane-associated CaM. The redistribution of CaM was maintained for at least 2 hr, before returning toward control levels by 4 hr. The EC50 values for carbachol in eliciting the translocation were 3.7 microM for the increase in cytosol and 1.3 microM for the decrease in membranes. The maximal changes in CaM concentration in both membranes and cytosol occurred with 10 microM carbachol. Incubation of the SK-N-SH cells with the partial muscarinic agonists bethanechol and arecoline resulted in 27 and 26% decreases in membrane-associated CaM, respectively, and 28 and 35% increases in cytosolic CaM, respectively. Thus, the partial agonists were less efficacious than carbachol in eliciting changes in CaM localization. Atropine completely blocked the carbachol-stimulated translocation, whereas the nicotinic agonist 1,1-dimethyl 4-phenylpiperazinium had no effect on the localization of CaM. Activation of receptors coupled to adenylate cyclase did not affect distribution of CaM. CaM content in membranes and cytosol of cells incubated with prostaglandin E1 or the alpha 2-adrenergic agonist UK 14,304 was not different from control values. The ionophore ionomycin (10 microM) and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) (50 nM) were both able to elicit changes in CaM distribution. Ionomycin caused a 64% increase in CaM in the cytosol, with no significant change in membrane CaM. TPA elicited a decrease in membrane-associated CaM, with a corresponding increase in CaM in the cytosol. When TPA and ionomycin were incubated together, the translocation was equal in magnitude to that observed with carbachol alone. The protein kinase C inhibitor H-7 blocked the TPA-stimulated response and partially blocked the carbachol-stimulated response. The CaM-binding protein neuromodulin, which demonstrates a decreased affinity for CaM in the presence of Ca2+ and when phosphorylated by protein kinase C, was present in both membranes and cytosol of SK-N

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenylyl Cyclases; Biological Transport; Calcium; Calmodulin; Carbachol; Humans; Ionomycin; Isoquinolines; Kinetics; Neuroblastoma; Piperazines; Receptors, Muscarinic; Tetradecanoylphorbol Acetate

IMR-32 human neuroblastoma cells are unable to release [3H]dopamine in response to secretagogues. However, they express a normal complement of membrane receptors and ion channels which are efficiently coupled to second messenger production. In the present study we took advantage of the ability of this cell line to differentiate in vitro in the presence of either dibutyrryl-cAMP or 5-bromodeoxyuridine, to analyze any developmentally regulated changes in its secretory properties. Uptake, storage, and release of [3H]dopamine were studied biochemically and by autoradiography. The calcium ionophore ionomycin, phorbol 12-myristate 13-acetate and the presynaptic acting neurotoxin alpha-latrotoxin were used in both control and differentiated cells as secretagogue agents. The presence of secretory organelles was investigated by electron microscopy; the expression of secretory organelle markers, such as chromogranin/secretogranin proteins (secretory proteins) and synaptophysin (membrane protein), was detected by Western blotting and immunofluorescence. The results obtained indicate that IMR-32 cells acquire regulated secretory properties after in vitro drug-induced differentiation: (a) they assemble "de novo" secretory organelles, as revealed by electron microscopy and detection of secretory organelle markers, and (b) they are able to store [3H]dopamine and to release the neurotransmitter in response to secretagogue stimuli. Furthermore, secretagogue sensitivity was found to be different, depending on the differentiating agent. In fact, dibutyrryl-cAMP treated cells release [3H]dopamine in response to alpha-latrotoxin, but not in response to ionomycin, whereas 5-bromodeoxyuridine treated cells release the neurotransmitter in response to both secretagogues. All together these results suggest that IMR-32 cells represent an adequate model for studying the development of the secretory apparatus in cultured human neurons.

Topics: Biological Transport; Bromodeoxyuridine; Bucladesine; Calcium; Cell Compartmentation; Cell Differentiation; Cell Survival; Chromaffin Granules; Dopamine; Ethers; Humans; Ionomycin; Kinetics; Neuroblastoma; Protein Kinase C; Spider Venoms; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

The role of intracellular free Ca2+ in muscarinic-receptor linked depolarization of SH-SY5Y neuroblastoma cells has been determined by using the bisoxonol membrane potential probe DiBaC4-(3) and intracellular Ca2+ indicator fura-2 respectively. Carbachol and the Ca2+ ionophore, ionomycin, at concentrations which caused similar rises in intracellular Ca2+ increased the bisoxonol fluorescence (depolarization) to the same extent. The membrane potential responses, but not the changes in intracellular Ca2+, were dependent on extracellular Na+. Ionomycin depletion of intracellular Ca2+ with EGTA and ionomycin or loading the cells with a Ca2+ buffer, BAPTA, reduced the carbachol-induced depolarization. The results suggest that a rise in intracellular Ca2+ may cause depolarization through an increase in the Na+ permeability.

Topics: Calcium; Carbachol; Cytosol; Ethers; Humans; Ionomycin; Membrane Potentials; Neuroblastoma; Receptors, Muscarinic; Sodium; Tumor Cells, Cultured

Addition of bradykinin to mouse N1E-115 neuroblastoma cells evokes a rapid but transient rise in cytoplasmic free Ca2+ concentration ([Ca2+]i). The [Ca2+]i rise is accompanied by a transient membrane hyperpolarization, due to a several-fold increase in K+ conductance, followed by a prolonged depolarizing phase. Pretreatment of the cells with a Ca2+-ionophore abolishes the hormone-induced hyperpolarization but leaves the depolarizing phase intact. The transient hyperpolarization can be mimicked by iontophoretic injection of IP3(1,4,5) or Ca2+, but not by injection of IP3(1,3,4), IP4(1,3,4,5) or Mg2+ into the cells. Instead, IP3(1,3,4) evokes a small but significant membrane depolarization in about 50% of the cells tested. Microinjected IP4(1,3,4,5) has no detectable effect, nor has treatment of the cells with phorbol esters. These results suggest that, while IP3(1,4,5) triggers the release of stored Ca2+ to hyperpolarize the membrane, IP3(1,3,4) may initiate a membrane depolarization.

Topics: Animals; Bradykinin; Calcium; Cell Line; Electrophysiology; Ethers; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Ionomycin; Membrane Potentials; Microinjections; Neuroblastoma; Sugar Phosphates