thapsigargin and Glioma

Alzheimer's disease (AD) is a progressive neurodegenerative disorder leading to slow neuronal loss in several brain regions. It is characterised by the presence of cerebral senile plaques comprised of aggregated amyloid-β peptides. Transcriptional regulation of the γ-secretase complex, which cleaves the β-amyloid precursor protein to produce Aβ-peptides, could modulate the pathological phenotype of AD patients. This study investigates whether rosuvastatin, an HMG-CoA reductase inhibitor, modulates the expression of genes involved in the function of the γ-secretase complex, in a human cellular model for Aβ peptide accumulation. In particular, we analysed the effect of the statin combined with apoptotic induction. Experimental apoptosis was induced by thapsigargin treatment, a drug that depletes intracellular calcium stores via inhibition of the calcium ATPase pump. Notably, systemic calcium dysregulation accompanies almost all of the brain pathology processes observed in AD. We found differential transcriptional regulation of some γ-secretase cofactors relative to rosuvastatin treatment, in cells expressing Swedish mutant APP. Interestingly, this statin down-regulated the transcription of some enzyme cofactors, similar to treatment with thapsigargin. However, rosuvastatin neither affected the basal Aβ levels nor counteracted APP processing or Aβ over-production triggered by the thapsigargin. Our results provide evidence that rosuvastatin alters gene expression of the γ-secretase complex without affecting enzyme activity.

Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Cell Line, Tumor; Enzyme Inhibitors; Fluorobenzenes; Gene Expression; Glioma; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Pyrimidines; Rosuvastatin Calcium; Sulfonamides; Thapsigargin

In this study, the function of nitric oxide (NO) in endoplasmic reticulum (ER)-related cell death in human glioma cells was investigated. Treatment of human CRT-MG cells with the NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) and thapsigargin, an ER stress inducer, increased cytosolic Ca(2+) and caused apoptosis in a dose-dependent manner. Expression of the ER-associated molecules inositol-requiring enzyme 1 (IRE1)-alpha, p-eIF, and Ero1-alpha were also elevated in thapsigargin- or NO donor-treated cells. Furthermore, thapsigargin and SNAP treatment increased IRE1-alpha nuclease activity, induced IRE1-alpha/TRAF2 complex formation, and increased p-JNK1/2 levels, suggesting that NO activates the IRE1-alpha/TRAF2/JNK pathway in the ER. Expression of IRE1-alpha increased concomitantly with cAMP responsive element binding protein (CREB) phosphorylation. siRNA knock down of IRE1-alpha reduced phospho-CREB levels and abolished its nuclear translocation. The levels of phospho-CREB and IRE1-alpha increased with NO donor concentration, which resulted in cell death. IRE1-alpha and phospho-CREB levels in glioblastoma U87MG cells were higher than those in normal astrocytes in response to NO. In addition, treatment with the intracellular cytokine interleukin-1beta induced cell death associated with NO and increased IRE1-alpha and p-CREB levels. These data reveal that intracellular NO affects IRE1-alpha-dependent CREB phosphorylation in human glioma cells. Therefore, an IRE1-alpha-dependent phospho-CREB signaling pathway responsive to NO/Ca(2+) may play an important role in regulating ER-related cell death in glioma.

Topics: Apoptosis; Calcium; Cations, Divalent; Cell Line, Tumor; Cell Survival; Cyclic AMP Response Element-Binding Protein; Dose-Response Relationship, Drug; Endoplasmic Reticulum; Endoribonucleases; Fluorescent Antibody Technique; Glioma; Humans; JNK Mitogen-Activated Protein Kinases; Nitric Oxide; Phosphorylation; Protein Serine-Threonine Kinases; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; S-Nitroso-N-Acetylpenicillamine; Thapsigargin

Alterations in intracellular Ca(2+) concentration are amongst the most rapid responses to a variety of stimuli in mammalian cells. In the nervous system in particular, responses occur within nanoseconds. A major challenge in intracellular Ca(2+) analysis is to achieve measurements within this very fast time frame. To date, the dynamic intracellular Ca(2+) concentration has been monitored by confocal microscopy, plate-based assays, spectrofluorometry, and flow cytometry, although there are issues with the number of cells analyzed or gaps in recording due to the addition of compounds, with significant loss of detail of a rapid Ca(2+) response. The new generation of flow cytometers (such as Accuri C6) resolves this problem by allowing the addition of test compounds with continuous monitoring of thousands of cells, providing a method for dynamic Ca(2+) measurements. This system was tested with commonly used Ca(2+) modulating agents in C6 glioma cells. Thapsigargin (TG), a blocker of Ca(2+) uptake into the endoplasmic reticulum (ER), causes a significant increase in the intracellular calcium concentration via ER emptying followed by Ca(2+) entry via store-operated Ca(2+) channels (SOCC). This well-established pathway can be partially inhibited by 2-aminoethoxydiphenyl borate (2-APB), a blocker of SOCC. Both the increase with TG alone and the partial increase when coincubated with 2-APB were observed with continuous recording along with calibration curves using an Accuri C6 flow cytometer. With these new cytometers, dynamic Ca(2+) concentration measurement becomes extremely accessible and accurate, while also providing extensive and valuable data regarding population health and responsiveness.

Topics: Animals; Boron Compounds; Brain Neoplasms; Calcium; Calcium Channel Blockers; Calcium Channels; Calcium Signaling; Cell Line, Tumor; Enzyme Inhibitors; Flow Cytometry; Glioma; Rats; Thapsigargin

This study investigated how modulation of intracellular calcium alters the functional activity of the EAAC1 glutamate transporter in C6 glioma cells. Pre-incubation of C6 glioma cells with the endoplasmic reticulum Ca2+ ATP pump inhibitor, thapsigargin (10 microM) produced a time-dependent increase in the Vmax for D-[3H]aspartate transport that reached a maximum at 15 min (143% of control; P<0.001) that was accompanied by increased plasma membrane expression of EAAC1 and was blocked by inhibition of protein kinase C. Pre-incubation of C6 glioma cells with phorbol myristate-3-acetate (100 nM for 20 min) also caused a significant increase in the Vmax of sodium-dependent D-[3H]aspartate transport (190% of control; P<0.01). In contrast, in the absence of extracellular calcium, thapsigargin caused a significant inhibition in D-[3H]aspartate transport that was not mediated by protein kinase C. Blockade of store-operated calcium channels with 2-aminoethoxydiphenyl borate (50 microM) or SKF 96365 (10 microM) caused a net inhibition of D-[3H]aspartate uptake. Co-incubation of C6 glioma cells with both thapsigargin and 2-aminoethoxydiphenyl borate (but not SKF 96365) prevented the increase in D-[3H]aspartate transport that was observed in the presence of thapsigargin alone. Furthermore, 2-aminoethoxydiphenyl borate, but not SKF 96365, reduced the increase in intracellular calcium that occurred following pre-incubation of the cells with thapsigargin. It is concluded that, in C6 glioma cells, stimulation of EAAC1-mediated glutamate transport by thapsigargin is dependent on entry of calcium via the NSCC-1 subtype of store operated calcium channel and is mediated by protein kinase C. In contrast, in the absence of store operated calcium entry, thapsigargin inhibits transport.

Topics: Animals; Aspartic Acid; Biological Transport; Calcium; Cell Line, Tumor; Excitatory Amino Acid Transporter 3; Extracellular Space; Glioma; Mice; Protein Kinase C; Protein Kinase Inhibitors; Tetradecanoylphorbol Acetate; Thapsigargin

Photofrin photodynamic therapy (PDT) caused a dose-dependent decrease of enzymatic cell detachment by trypsin/ethylenediamine tetra-acetic acid (EDTA) in human glioma U251n and U87 cells. This happened coincidently with the increase of intracellular free calcium ([Ca(2+)](i)). Thapsigargin, which increased [Ca(2+)](i), induced further decrease in enzymatic cell detachment and increased cytotoxicity. Opposite effects were observed when 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetra-acetic acid tetrakis, an intracellular Ca(2+) chelator, was used. PDT-induced changes in [Ca(2+)](i) and cell detachment were not blocked by calcium channel antagonists nickel (Ni(2+)) or nimodipine, nor were they altered when cells were irradiated in a buffer free from Ca(2+) and magnesium (Mg(2+)), suggesting that [Ca(2+)](i) is derived from the internal calcium stores. Decreased cell migration was observed after PDT, as assessed by chemotactic and wound-healing assays. Our findings indicated that internal calcium store-derived [Ca(2+)](i) plays an important role in PDT-induced enzymatic cell detachment decrease and cytotoxicity. Cell migration may be affected by these changes.

Topics: Calcium; Cell Adhesion; Cell Line, Tumor; Cell Movement; Dihematoporphyrin Ether; Egtazic Acid; Glioma; Humans; Photochemotherapy; Photosensitizing Agents; Thapsigargin

Chondroitin sulfate (CS), which is known to be a neurite-preventing molecule, is a major component of the extracellular matrix (ECM) in the central nervous system (CNS). The CS expression is upregulated around damaged areas. Endoplasmic reticulum (ER) stress causes neuronal cell death in numerous neurodegenerative diseases. However, the effects of ER stress on glial cells remain to be clarified. The present study examined whether direct ER stress to glial cells can upregulate CS expression in C6 glioma cells and primary cultured mouse astrocytes, and also whether the expression of CS prevents neurite extension. ER stressors tunicamycin (TM) and thapsigargin (TG) significantly increased CS expression in both C6 cells and primary cultured astrocytes, while NO donor sodium nitroprusside (SNP) did not significantly alter the CS expression. The dosage of TM and TG treatment used in this study did not significantly induce cell death but upregulated the ER chaperone molecule Grp78 in C6 glioma cells and primary astrocytes. The ECM of glial cells exposed to ER stress prevented neurite extension in primary cultured mouse cortical neurons, and chondroitinase ABC (ChABC) treatment diminished the inhibitory effect on neurite extension. These findings suggest that direct ER stress to glial cells increases the CS expression, which thus prevents neurite extension.

Topics: Animals; Astrocytes; Cell Line, Tumor; Cell Survival; Cells, Cultured; Chondroitin Sulfates; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Glioma; Mice; Mice, Inbred C57BL; Neurites; Neurons; Nitric Oxide Donors; Nitroprusside; Rats; Thapsigargin; Tunicamycin

Despite much evidence that lithium and valproate, two commonly used mood stabilizers, exhibit neuroprotective properties against an array of insults, the pharmacological relevance of such effects is not clear because most of these studies examined the acute effect of these drugs in supratherapeutic doses against insults which were of limited disease relevance to bipolar disorder. In the present study, we investigated whether lithium and valproate, at clinically relevant doses, protects human neuroblastoma (SH-SY5Y) and glioma (SVG and U87) cells against oxidative stress and endoplasmic reticulum stress in a time-dependent manner. Pretreatment of SH-SY5Y cells for 7 days, but not 1 day, with 1 mM of lithium or 0.6 mM of valproate significantly reduced rotenone and H2O2-induced cytotoxicity, cytochrome c release and caspase-3 activation, and increased Bcl-2 levels. Conversely, neither acute nor chronic treatment of SH-SY5Y cells with lithium or valproate elicited cytoprotective responses against thapsigargin-evoked cell death and caspase-3 activation. Moreover, inhibitors of glycogen synthase kinase-3 (GSK-3), kenpaullone and SB216763, abrogated rotenone-induced, but not H2O2-induced, cytotoxicity. Thus the cytoprotective effects of lithium and valproate against H2O2-induced cell death is likely independent of GSK-3 inhibition. On the other hand, chronic lithium or valproate treatment did not ameliorate cytotoxicity induced by rotenone, H2O2, and thapsigargin in SVG astroglial and U87 MG glioma cell lines. Our results suggest that lithium and valproate may decrease vulnerability of human neural, but not glial, cells to cellular injury evoked by oxidative stress possibly arising from putative mitochondrial disturbances implicated in bipolar disorder.

Topics: Antimanic Agents; Caspase 3; Caspases; Cell Death; Cell Line, Tumor; Cytochromes c; Cytoprotection; Electron Transport Complex I; Endoplasmic Reticulum; Enzyme Activation; Enzyme Inhibitors; Glioma; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Hydrogen Peroxide; Lithium Compounds; Neuroblastoma; Neuroprotective Agents; Oxidative Stress; Proto-Oncogene Proteins c-bcl-2; Rotenone; Thapsigargin; Up-Regulation; Valproic Acid

There are several reports describing participation of small heat shock proteins (sHsps) in cellular protein quality control. In this study, we estimated the endoplasmic reticulum (ER) stress-induced response of Hsp27 and alphaB-crystallin in mammalian cells. Treatment targeting the ER with tunicamycin or thapsigargin induced the phosphorylation of Hsp27 but not of alphaB-crystallin in U373 MG cells, increase being observed after 2-10 h and decline at 24 h. Similar phosphorylation of Hsp27 by ER stress was also observed with U251 MG and HeLa but not in COS cells and could be blocked using SB203580, an inhibitor of p38 MAP kinase. Other protein kinase inhibitors, like Gö6983, PD98059, and SP600125, inhibitors of protein kinase C (PKC), p44/42 MAP kinase, and JNK, respectively, were without major influence. Prolonged treatment with tunicamycin but not thapsigargin for 48 h caused the second induction of the phosphorylation of Hsp27 in U251 MG cells. Under these conditions, the intense perinuclear staining of Hsp27, with some features of aggresomes, was observed in 10%-20% of the cells.

Topics: alpha-Crystallin B Chain; Animals; Antiviral Agents; Blotting, Western; Cell Nucleus; Chlorocebus aethiops; COS Cells; Endoplasmic Reticulum; Enzyme Inhibitors; Flow Cytometry; Fluorescent Antibody Technique; Glioma; Heat-Shock Proteins; HeLa Cells; HSP27 Heat-Shock Proteins; Humans; JNK Mitogen-Activated Protein Kinases; Mitogen-Activated Protein Kinase 3; Molecular Chaperones; Neoplasm Proteins; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Kinase C; Stress, Physiological; Thapsigargin; Tunicamycin

Parkinson's disease (PD) is one of the most prevalent neurodegenerative diseases but its etiology is unclear. Alpha-synuclein (alpha-SN) is a major component of Lewy bodies and Lewy neurites, and its missense mutations, A30P and A53T, cause familial PD. In PD, alpha-SN-positive glial inclusions are distributed mainly in the dorso-medial region of the substantia nigra, which contains most of the surviving dopaminergic neurons, suggesting that alpha-SN expression might have a neuroprotective function in glial cells. To investigate this hypothesis, we established alpha-SN transfected C6 glioma cell line clones and evaluated the expression of neurotrophins using semi-quantitative reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay. Brain-derived neurotrophic factor (BDNF) was induced by overexpression of wild-type alpha-SN but not by that of A30P and A53T. These data suggest that the pathogenic alpha-SN mutations, A30P or A53T, are linked to the loss of BDNF production in glial cells.

Topics: alpha-Synuclein; Amino Acid Substitution; Animals; Brain-Derived Neurotrophic Factor; Calcium; Cell Line, Tumor; Chelating Agents; Clone Cells; Dactinomycin; Egtazic Acid; Glioma; Humans; Immunoblotting; Immunohistochemistry; Lac Operon; Nerve Growth Factor; Nerve Tissue Proteins; Protein Synthesis Inhibitors; Rats; Recombinant Proteins; RNA, Messenger; Synucleins; Thapsigargin; Transfection

Ca2+-activated K+ channels were studied in C6-glioma cells in an attempt to correlate changes in expression with cell proliferation and differentiation. In this study, we treated C6-glioma cells with thapsigargin for 48 h. Cell proliferation was markedly inhibited, and cell morphology changed from round to a spindle differentiated shape. Furthermore, intracellular calcium concentration was initially increased during acute treatment with thapsigargin. The internal [Ca2+]i pool was eventually depleted after a 48-h thapsigargin treatment. We have characterized Ca2+-activated K+ currents in less differentiated C6 cells. After differentiation of C6 cells induced by thapsigargin, Ca2+-activated K+ currents were selectively suppressed. These data lend further support to the notion that the expression of Ca2+-activated K+ channels is intimately associated with the proliferation of C6-glioma cells, and the suppression of Ca2+-activated K+ channels coincides with the inhibition of proliferation and subsequent induction of cell differentiation.

Topics: Animals; Cell Differentiation; Cell Division; Cell Line, Tumor; Glioma; Membrane Potentials; Potassium Channels, Calcium-Activated; Thapsigargin

Capacitative calcium entry, usually evoked by receptor-ligand binding, may be also studied in the model system of calcium release after SERCA pump inhibition. We have previously found that disorganization of actin cytoskeleton has no effect on calcium influx into glioma C6 cells after thapsigargin administration [Biochem. Biophys. Res. Commun. 296 (2002) 484]. In the present work we show that the effect of other SERCA pump inhibitors depends on the endoplasmic reticulum distribution in a cell. Changing this distribution leads to changes in calcium release from ER stores. Intensity of calcium influx in the capacitative phase of cell answer does not depend on actin cytoskeleton state; however, administration of cytochalasin D significantly slows down signal build-up. While cyclopiazonic acid acts very similarly to thapsigargin, cytoskeleton disorganization leads to rise of calcium signal after administration of 2,5-di-(t-butyl)-1,4-benzohydroquinone. This effect may be caused by specific binding of this inhibitor to SERCA3 isoform of pump protein only.

Topics: Actin Cytoskeleton; Actins; Animals; Calcium; Calcium-Transporting ATPases; Cell Line, Tumor; Cytochalasin D; Endoplasmic Reticulum; Glioma; Rats; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin

Astrocytes express transient receptor potential channels (TRPCs), which have been implicated in Ca 2+ influx triggered by intracellular Ca 2+ stores depletion, a phenomenon known as capacitative Ca 2+ entry. We studied the properties of capacitative Ca 2+ entry in astrocytes by means of single-cell Ca 2+ imaging with the aim of understanding the involvement of TRPCs in this function. We found that, in astrocytes, capacitative Ca 2+ entry is not attributable to TRPC opening because the TRPC-permeable ions Sr2+ and Ba2+ do not enter astrocytes during capacitative Ca 2+ entry. Instead, natively expressed oleyl-acetyl-glycerol (OAG) (a structural analog of DAG) -sensitive TRPCs, when activated, initiate oscillations of cytosolic Ca 2+ concentration ([Ca 2+]i) pharmacologically and molecularly consistent with TRPC3 activation. OAG-induced [Ca 2+]i oscillations are not affected by inhibition of inositol trisphosphate (InsP3) production or blockade of the InsP3 receptor, therefore representing a novel form of [Ca 2+]i signaling. Instead, high [Ca 2+]i inhibited oscillations, by closing the OAG-sensitive channel. Also, treatment of astrocytes with antisense against TRPC3 caused a consistent decrease of the cells responding to OAG. Exogenous OAG but not endogenous DAG seems to activate TRPC3. In conclusion, in glial cells, natively expressed TRPC3s mediates a novel form of Ca 2+ signaling, distinct from capacitative Ca 2+ entry, which suggests a specific signaling function for this channel in glial cells.

Topics: Adenosine Triphosphate; Animals; Astrocytes; Biological Clocks; Calcium; Calcium Channels; Calcium Signaling; Cells, Cultured; Diglycerides; Enzyme Inhibitors; Fluorescent Dyes; Glioma; Immunohistochemistry; Intracellular Fluid; Ion Channels; Neuroglia; Oligonucleotides, Antisense; Protein Isoforms; Rats; Rats, Wistar; Strontium; Thapsigargin; TRPC Cation Channels

The effects of actin cytoskeleton disruption by cytochalasin D and latrunculin A on Ca2+ signals evoked by ADP, UTP or thapsigargin were investigated in glioma C6 cells. Despite the profound alterations of the actin cytoskeleton architecture and cell morphology, ADP and UTP still produced cytosolic calcium elevation in this cell line. However, calcium mobilization from internal stores and Ca2+ influx through store-operated Ca2+ channels induced by ADP and UTP were strongly reduced. Cytochalasin D and latrunculin A also diminished extracellular Ca2+ influx in unstimulated glioma C6 cells previously incubated in Ca2+ free buffer. In contrast, the disruption of the actin cytoskeleton had no effect on thapsigargin-induced Ca2+ influx in this cell line. Both agonist- and thapsigargin-generated Ca2+ entry was significantly decreased by the blocker of store-operated Ca2+ channels, 2-aminoethoxydiphenylborate. The data reveal that two agonists and thapsigargin activate store-operated Ca2+ channels but the mechanism of activation seems to be different. While the agonists evoke a store-mediated Ca2+ entry that is dependent on the actin cytoskeleton, thapsigargin apparently activates an additional mechanism, which is independent of the disruption of the cytoskeleton.

Topics: Actins; Adenosine Diphosphate; Animals; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Calcium Signaling; Chelating Agents; Cytochalasin D; Cytoskeleton; Enzyme Inhibitors; Fluorescent Dyes; Fura-2; Glioma; Microscopy, Confocal; Nucleic Acid Synthesis Inhibitors; Rats; Thapsigargin; Thiazoles; Thiazolidines; Tumor Cells, Cultured; Uridine Triphosphate

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

Riluzole is an effective neuroprotective drug. Its effect on intracellular free Ca2+ levels ([Ca2+]i) has not been explored. This study examined the effect of riluzole on [Ca2+]i in IMR32 neuroblastoma cells using fura-2 as a Ca2+ probe. Riluzole 0.1-1 mM increased [Ca2+]i in a concentration-dependent manner. Removal of extracellular Ca2+ inhibited the response by 52 +/- 5%. The [Ca2+]i increase induced by 0.2 mM riluzole was unaltered by 0.1 mM La3+ or 10 microM verapamil, but was inhibited by 51 +/- 4% by 10 microM nifedipine. In Ca2+-free medium, pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) reduced the 0.2 mM riluzole-induced Ca2+ release by 44 +/- 3%; this reduction was augmented to 66 +/- 5% by additionally depleting the Ca2+ stores in the Golgi complex with 50 microM brefeldin A. Inhibition of inositol 1,4,5-trisphosphate formation by 2 microM U73122, a phospholipase C inhibitor, did not affect Ca2+ release induced by 0.2 microM riluzole. It was concluded that the neuroprotective agent riluzole increased [Ca2+]i in IMR32 neuroblastoma cells concentration-dependently by releasing Ca2+ from multiple stores in an inositol 1,4,5-trisphosphate-independent manner and also by inducing nifedipine-sensitive Ca2+ influx.

Topics: Animals; Brefeldin A; Calcium; Calcium Channel Blockers; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Line; Dogs; Enzyme Inhibitors; Fura-2; Glioma; Humans; Ionophores; Kidney; Liver Neoplasms; Neuroblastoma; Neurons; Neuroprotective Agents; Protein Synthesis Inhibitors; Riluzole; Thapsigargin; Tumor Cells, Cultured

We examined the effects of dissolved nitric oxide (NO) gas on cytoplasmic calcium levels ([Ca(2+)](i)) in C6 glioma cells under anoxic conditions. The maximum elevation (27 +/- 3 nM) of [Ca(2+)](i) was reached at 10 microM NO. A second application of NO was ineffective if the first was >0.5 microM. The NO donor diethylamine/NO mimicked the effects of NO. Acute exposure of the cells to low calcium levels was without effect on the NO-evoked response. Thapsigargin (TG) increased [Ca(2+)](i) and was less effective if cells were pretreated with NO. Hemoglobin inhibited the effects of NO at a molar ratio of 10:1. 8-Bromo-cGMP was without effect on the NO-evoked response. If cells were pretreated with TG or exposed chronically to nominal amounts of calcium, NO decreased [Ca(2+)](i). The results suggest that C6 glioma cells have two receptors for NO. One receptor (NO(A)) elevates [Ca(2+)](i) and resides on the endoplasmic reticulum (ER). The other receptor (NO(B)) decreases [Ca(2+)](i) and resides on the plasmalemma or the ER. The latter receptor dominates when the level of calcium within intracellular stores is diminished.

Topics: Aerobiosis; Animals; Calcium; Cell Hypoxia; Cytoplasm; Cytosol; Digitonin; Egtazic Acid; Glioma; Hemoglobins; Homeostasis; Hydrazines; Kinetics; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides; Thapsigargin; Tumor Cells, Cultured

The effects of 2,5-di(tert-butyl)-1,4-benzohydroquinone (tBHQ), a synthetic phenolic antioxidant and a blocker of the sarco-endoplasmic ATPase, were evaluated on low and high voltage-activated Ca(2+) currents (ICas) with rodent dorsal root ganglion, hippocampal, and motor neurons. In all cell types tested, tBHQ (IC(50) = 35 microM) blocked ICa at concentrations used to inhibit sarco-endoplasmic ATPase. This effect was specific to tBHQ because the other sarco-endoplasmic reticulum calcium ATPase pump inhibitors (thapsigargin and cyclopiazonic acid) had no effect. Selective blockade of the N-type current with omega-conotoxin GVIA and of P- (motoneuron) or Q-type currents (hippocampal neuron) with omega-agatoxin IVA indicated that tBHQ inhibited N, P, and Q types of ICa. tBHQ had no effect on nitrendipine-sensitive (L-type) and residual drug-resistant (R-type) ICa, nor on the low voltage-activated T-type ICa. Contrary to neuronal cells, the L-type ICa was inhibited by tBHQ in a differentiated mouse neuroblastoma and rat glioma hybrid cell line. Injection of cDNAs encoding the alpha1A, alpha1B, alpha1C, and alpha1E subunits into oocytes showed that tBHQ blocked ICas at the level of the pore-forming protein. This effect of tBHQ on ICa should be considered when interpreting results obtained with tBHQ used on neuronal preparations. It also may be useful for developing new strategies for the generation of more potent intracellular calcium transient inhibitors.

Topics: Animals; Calcium; Calcium Channel Blockers; Calcium Channels; Calcium Channels, L-Type; Calcium Channels, N-Type; Calcium Channels, P-Type; Calcium Channels, Q-Type; Calcium Channels, R-Type; Calcium Channels, T-Type; Calcium-Transporting ATPases; Cells, Cultured; DNA, Complementary; Endoplasmic Reticulum; Enzyme Inhibitors; Ganglia, Spinal; Glioma; Hippocampus; Hybrid Cells; Hydroquinones; Mice; Motor Neurons; Neuroblastoma; Rats; Rats, Sprague-Dawley; Thapsigargin

Sodium butyrate is well known in stimulating growth and differentiation of cancer cells. In the present study, butyrate treatment caused decreases in thymidine incorporation in the early passages (45-60) of C6 glioma cells. In addition, butyrate also caused decreases in inositol incorporation and transient ATP-stimulated Ca2+ mobilization suggesting that butyrate altered general mechanisms of Ca2+ signaling in these cells. To gain direct insight into the crosstalk between sodium butyrate and Ca2+ signaling in transcriptional regulation, we investigated the induction of the Ca2+-sensitive immediate early genes (IEGs), c-fos, nur77 and c-myc. Sodium butyrate per se enhanced the expression of c-fos mRNA, and the enhanced levels were maintained for 24 h, but over the same time period, the initially increased levels of nur77 expression tailed off, while c-myc expression was slightly reduced. Increasing intracellular Ca2+ concentration ([Ca2+]i) by thapsgargin and A23187 induced the expression of both c-fos and nur77 mRNA expression, and synergistic effects were observed when cells were incubated with sodium butyrate plus thapsgargin and A23187. However, removal of both extracellular Ca2+ by EGTA, or intracellular free Ca2+ with BAPTA did not affect the sodium butyrate-induced c-fos and nur77 mRNA. These results suggest that although sodium butyrate altered Ca2+ signaling which is an important regulatory mechanism for c-fos and nur77 expression, nevertheless the sodium butyrate-induced c-fos and nur77 expression may be not in fact mediated through Ca2+ signaling.

Topics: Adenosine Triphosphate; Animals; Butyrates; Calcium; Calcium Signaling; Cell Differentiation; Chelating Agents; DNA-Binding Proteins; Egtazic Acid; Enzyme Inhibitors; Gene Expression; Glioma; Inositol; Nuclear Receptor Subfamily 4, Group A, Member 1; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-myc; Receptors, Cytoplasmic and Nuclear; Receptors, Steroid; RNA, Messenger; Thapsigargin; Thymidine; Transcription Factors; Tritium; Tumor Cells, Cultured

The effect of sphingosine and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on ATP-evoked Ca(2+) mobilization in glioma C6 cells was studied with the Fura-2 video-imaging technique. Treatment of the cells with TPA, an activator of protein kinase C, reduced the ATP-evoked release of Ca(2+) from the intracellular stores, whereas sphingosine, known from in vitro studies as a protein kinase C inhibitor, potentiated Ca(2+) release synergistically with ATP. ATP-induced Ca(2+) mobilization was also enhanced by a specific protein kinase C inhibitor, GF 109203X. Pretreatment of the cells with GF 109203X prevented TPA action, whereas TPA diminished the stimulatory effect of sphingosine. However, this sphingosine effect was only observed after a short (1 min) treatment, whereas a longer treatment (5 min) reduced ATP-evoked Ca(2+) release. It is therefore concluded that sphingosine has two apparent actions: it inhibits protein kinase C providing a positive feedback regulation of receptor signals and it releases Ca(2+) from intracellular stores by an unknown mechanism, possibly independent of protein kinase C.

Topics: Adenosine Triphosphate; Animals; Calcium; Calcium Signaling; Endoplasmic Reticulum; Enzyme Activation; Glioma; Indoles; Maleimides; Protein Kinase C; Rats; Sphingosine; Tetradecanoylphorbol Acetate; Thapsigargin; Time Factors; Tumor Cells, Cultured; Type C Phospholipases

PDGF-BB induces a rapid, sustained increase in intracellular calcium levels in U-1242 MG cells. We used several calcium channel blockers to identify the types of channels involved. L channel blockers (verapamil, nimodipine, nicardipine, nitrendipine and taicatoxin) had no effect on PDGF-BB induced alterations in intracellular calcium. Blockers of P, Q and N channels (omega-agatoxin-IVA, omega-conotoxin MVIIC and omega-conotoxin GVIA) also had no effect. This indicates that these channels play an insignificant role in supplying the Ca2+ necessary for PDGF stimulated events in U-1242 MG cells. However, a T channel blocker (NDGA) and the non-specific (NS) calcium channel blockers (FFA and SK&F 9365) abolished PDGF-induced increases in intracellular calcium. This indicates that PDGF causes calcium influx through both non-specific cationic channels and T channels. To study the participation of intracellular calcium stores in this process, we used thapsigargin, caffeine and ryanodine, all of which cause depletion of intracellular calcium stores. The PDGF effect was abolished using both thapsigargin and caffeine but not ryanodine. Collectively, these data indicate that in these human glioma cells PDGF-BB induces release of intracellular calcium from caffeine- and thapsigargin-sensitive calcium stores which in turn lead to further calcium influx through both NS and T channels.

Topics: Becaplermin; Brain Neoplasms; Caffeine; Calcium; Calcium Channel Blockers; Calcium Channels; Calcium Signaling; Calcium-Transporting ATPases; Elapid Venoms; Endoplasmic Reticulum; Enzyme Inhibitors; Flufenamic Acid; Glioma; Humans; Imidazoles; Ion Transport; Masoprocol; Neoplasm Proteins; Nicardipine; Nimodipine; Nitrendipine; omega-Agatoxin IVA; omega-Conotoxin GVIA; omega-Conotoxins; Peptides; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-sis; Ryanodine; Thapsigargin; Tumor Cells, Cultured; Verapamil

C6 glioma - Ca2+ depletion - proliferation arrest morphology change - CDK inhibitor In this study, we investigated the role of the intracellular calcium store in modulating the cellular proliferation and the expression of cell cycle regulatory proteins in cultured C6 glioma cells. By means of microspectrofluorimetry and Ca(2+)-sensitive indicator fura-2, we found that the intracellular Ca2+ pump inhibitors, thapsigargin (TG) irreversibly and 2,5-ditert-butyl-hydroquinone (DBHQ) reversibly depleted the Ca(2+)-store accompanied with the induction of G0/G1 arrest, an increase in glial fibrillary acidic protein (GFAP) expression and morphological changes from a round flat shape to a differentiated spindle-shaped cell. The machinery underlying these changes induced by Ca(2+)-store depletion was investigated. The results indicated that Ca(2+)-store depletion caused an increased expression of p21 and p27 proteins (cyclin-dependent kinase inhibitors), with unchanged mutant p53 protein of C6 cells but reduced amounts of the cell cycle regulators: cyclin-dependent kinase 2 (CDK2), cdc2, cyclin C, cyclin D1, cyclin D3 and proliferating cell nuclear antigen (PCNA) in a time-dependent manner. These findings indicate a new function of the endoplasmic reticulum (ER) Ca2+ store in regulating cellular proliferation rate through altering the expression of p21 and p27 proteins. Moreover, cellular differentiation as revealed by spindle-shaped morphology and induced GFAP expression were also modulated by the ER Ca2+ store. The implication of this finding is that the abnormal growth of cancer cells such as C6 glioma cells may be derived from a signalling of the ER which can be manipulated by depleting the Ca2+ store.

Topics: Blotting, Western; Calcium-Transporting ATPases; CDC2 Protein Kinase; CDC2-CDC28 Kinases; Cell Cycle; Cell Differentiation; Cell Division; Cyclin D1; Cyclin D3; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; Cytosol; Glial Fibrillary Acidic Protein; Glioma; Humans; Hydroquinones; Microfilament Proteins; Muscle Proteins; Protein Serine-Threonine Kinases; Thapsigargin; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Many agonists evoke events in the cell nucleus through the control of Ca2+ signals. Recent studies using isolated nuclei have indicated that the nuclear envelope is a store for nuclear Ca2+. However, the release of Ca2+ directly from the nuclear envelope in living cells has never been reported. In the present study, we have investigated the changes of Ca2+ signals at the cyto-nucleoplasmic interface of rat C6 glioma cells using confocal microscopy. Digital imaging indicates that fluo-3, a Ca2+-sensitive fluorescent probe, was concentrated in or around the nuclear envelope. Our experiments also revealed that C6 cells at rest produced spontaneous Ca2+ spikes in the absence of chemical stimulation. The amplitude of the repetitive Ca2+ spikes was higher at the nuclear envelope than in the whole cell or cytosol. After image subtraction, circular rims of Ca2+ release and uptake were seen at the outer boundary of the nucleus. When the cells were treated with thapsigargin (2 muM), a specific Ca2+-ATPase inhibitor, a long-lasting Ca2+ release was observed at the nuclear envelope. Moreover, most of the released Ca2+ was directed inwardly to the nucleoplasm with little outward diffusion. Our results thus indicate: (1) that the nuclear envelope is a Ca2+ store that possesses the ability to discharge and sequestrate Ca2+; and (2) the Ca2+-releasing channels are present in the inner nuclear membrane.

Topics: Adenosine Triphosphate; Aniline Compounds; Animals; Calcium; Calcium-Transporting ATPases; Endoplasmic Reticulum; Enzyme Inhibitors; Fluorescent Dyes; Glioma; Microscopy, Confocal; Nuclear Envelope; Rats; Signal Transduction; Thapsigargin; Tumor Cells, Cultured; Xanthenes

Antibody to galactocerebroside (GalC) evokes a Ca2+ response in cultured glioma U-87 MG cells. The rise in intracellular calcium [Ca2+]i occurs largely due to the influx of Ca2+ through a plasma membrane channel, though the release of Ca2+ from intracellular stores also contributes. We characterized the channel activated by anti-GalC. The channel activity was transient and the inactivation appeared to be Ca2+ dependent. The channel was impermeant to monovalent ions Na+ and K+ and also to Mn2+. Ni2+ and Co2+ neither permeate through the channel nor inhibit the Ca2+ influx. In contrast Cd2+ the most potent inorganic blocker of Ca2+ channels permeated through this channel. The Ca2+ influx was inhibited by verapamil with IC50 of 65 +/- 8 microM. The Ca2+ influx as well as the intracellular release were markedly inhibited by neomycin sulfate and phorbol dibutyrate, suggesting that the Ca2+ influx may be mediated by IP3 (1). Depletion of intracellular Ca2+ stores by thapsigargin was followed by Ca2+ influx. This represents the capacitative Ca2+ entry pathway and is distinct from the channel activated by anti -GalC.

Topics: Calcium; Calcium Channel Blockers; Calcium Channels; Cations, Divalent; Galactosylceramides; Glioma; Humans; Immunologic Techniques; Ion Channel Gating; Manganese; Thapsigargin; Tumor Cells, Cultured; Verapamil

The ability of adenylyl cyclases to be regulated by physiological transitions in Ca2+ provides a key point for integration of cytosolic Ca2+ concentration ([Ca2+]i) and cAMP signaling. Ca2+-sensitive adenylyl cyclases, whether endogenously or heterologously expressed, require Ca2+ entry for their regulation, rather than Ca2+ release from intracellular stores (Chiono, M., Mahey, R., Tate, G., and Cooper, D. M. F. (1995) J. Biol. Chem. 270, 1149-1155; Fagan, K., Mahey, R., and Cooper, D. M. F. (1996) J. Biol. Chem. 271, 12438-12444). The present study compared the regulation by capacitative Ca2+ entry versus ionophore-mediated Ca2+ entry of an endogenously expressed Ca2+-inhibitable adenylyl cyclase in C6-2B cells. Even in the face of a dramatic [Ca2+]i rise generated by ionophore, Ca2+ entry via capacitative Ca2+ entry channels was solely responsible for the regulation of the adenylyl cyclase. Selective efficacy of BAPTA over equal concentrations of EGTA in blunting the regulation of the cyclase by capacitative Ca2+ entry defined the intimacy between the adenylyl cyclase and the capacitative Ca2+ entry sites. This association could not be impaired by disruption of the cytoskeleton by a variety of strategies. These results not only establish an intimate spatial relationship between an endogenously expressed Ca2+-inhibitable adenylyl cyclase with capacitative Ca2+ entry sites but also provide a physiological role for capacitative Ca2+ entry other than store refilling.

Topics: 4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Biological Transport; Brain; Calcium; Cell Membrane; Colforsin; Cyclic AMP; Egtazic Acid; Glioma; Ionomycin; Ionophores; Kinetics; Rats; Thapsigargin; Tumor Cells, Cultured

The effects of genistein and erbstatin analogue, inhibitors of tyrosine kinase, on Ca2+ mobilization evoked by thapsigargin (TG) were examined in rat glioma C6 cells. Genistein and erbstatin analogue inhibited the Ca2+ release from intracellular pools as well as Ca2+ entry from extracellular medium evoked by TG in a dose-dependent manner. However, they did not affect a Ca2+ entry due to leakage of Ca2+ from extracellular medium into cells. The present results suggest that tyrosine kinase inhibitors inhibit capacitative Ca2+ entry due to the inhibition of both Ca2+ entry itself and Ca2+ release in rat glioma C6 cells.

Topics: Animals; Calcium; Enzyme Inhibitors; Genistein; Glioma; Hydroquinones; Protein-Tyrosine Kinases; Rats; Thapsigargin; Tumor Cells, Cultured

Changes in capacitative Ca2+ entry were studied in neuroblastoma x glioma NG108-15 cells with fura-2 fluorescence measurements in the following three culture conditions. The application of thapsigargin (250 nM) with a Ca2+-free solution depleted intracellular Ca2+ stores and the capacitative Ca2+ entry was induced by the addition of extracellular Ca2+ in the cells cultured in the medium for proliferation. The capacitative Ca2+ entry decreased in the cells cultured in the medium for neuronal differentiation. When these cells resumed proliferation after changing the culture media to the initial medium for proliferation, the capacitative Ca2+ entry increased again and exceeded the level in the initial proliferation state. These results suggested that the capacitative Ca2+ entry occurred more intensely at the proliferation state than at the neuronally differentiated state.

Topics: Animals; Calcium; Cell Differentiation; Cell Division; Culture Media; Glioma; Hybrid Cells; Neuroblastoma; Neurons; Thapsigargin

The influence of cooling on the intracellular concentration of Ca2+ ([Ca2+]i) was tested in cell lines expressing chemical receptors. First, when ATP was externally added to rat basophilic leukemia (RBL-2H3) cells, cooling from 37 degrees C to 27 degrees C induced a transient rapid increase in [Ca2+]i. In the absence of extracellular Ca2+, the [Ca2+]i response was induced whereas an inhibitor of microsomal Ca2+ ATPase, thapsigargin, largely abolished the [Ca2+]i response, suggesting that the internal Ca2+ store liberate the Ca2+. A purinergic receptor antagonist, suramin, completely inhibited the [Ca2+]i response to the cooling. Secondly, when serotonin (5-HT) was added to rat glioma C6BU-1 cells, the cooling induced a transient increase in [Ca2+]. This [Ca2+]i response was induced in the absence of external Ca2+, suggesting that the internal Ca2+ stores liberate the Ca2+. These results raise the possibility that some G protein-coupled receptors are sensitive to cooling in the presence of agonist for the receptor.

Topics: Adenosine Triphosphate; Aniline Compounds; Animals; Calcium; Calcium-Transporting ATPases; Cold Temperature; Fluorescent Dyes; Glioma; GTP-Binding Proteins; Kinetics; Leukemia, Basophilic, Acute; Microsomes; Rats; Receptors, Cell Surface; Serotonin; Signal Transduction; Suramin; Thapsigargin; Time Factors; Tumor Cells, Cultured; Xanthenes

Forced expression of gap junction proteins, connexins, enables gap junction-deficient cell lines to propagate intercellular calcium waves. Here, we show that ATP secretion from the poorly coupled cell lines, C6 glioma, HeLa, and U373 glioblastoma, is potentiated 5- to 15-fold by connexin expression. ATP release required purinergic receptor-activated intracellular Ca2+ mobilization and was inhibited by Cl- channel blockers. Calcium wave propagation also was reduced by purinergic receptor antagonists and by Cl- channel blockers but insensitive to gap junction inhibitors. These observations suggest that cell-to-cell signaling associated with connexin expression results from enhanced ATP release and not, as previously believed, from an increase in intercellular coupling.

Topics: Adenosine Triphosphatases; Animals; Astrocytes; Calcium; Cells, Cultured; Cerebral Cortex; Connexin 43; Connexins; Gap Junction beta-1 Protein; Glioblastoma; Glioma; HeLa Cells; Humans; Microscopy, Fluorescence; Rats; Signal Transduction; Thapsigargin; Transfection; Tumor Cells, Cultured

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

Topics: Animals; Bradykinin; Calcium; Dose-Response Relationship, Drug; Enzyme Inhibitors; Glioma; Hybrid Cells; Marine Toxins; Mice; Neuroblastoma; Okadaic Acid; Oxazoles; Phosphoric Monoester Hydrolases; Phosphorylation; Signal Transduction; Thapsigargin; Type C Phospholipases

The effects of tetrandrine (TET), a Ca2+ antagonist of Chinese herbal origin, and hernandezine (HER), a structural analogue of TET, on Ca2+ mobilization were studied in rat glioma C6 cells. TET and HER alone did not affect the resting cytoplasmic Ca2+ concentration ([Ca2+]i). TET and HER inhibited the peak and sustained elevation of [Ca2+]i induced by bombesin and thapsigargin (TG), a microsomal Ca2+ ATPase inhibitor, in a dose-dependent manner. The doses of TET or HER needed to abolish the sustained and peak increase in [Ca2+]i induced by bombesin and TG were 30 microM and 300 microM, respectively. TET and HER did not increase inositol 1,4,5-trisphosphate (IP3) accumulation by themselves but inhibited IP3 accumulation elevated by bombesin. In permeabilized C6 cells, the addition of IP3 and TG released Ca2+ from intracellular stores. Pretreatment with TET or HER abolished Ca2+ release from intracellular stores induced by bombesin and TG. In the absence of extracellular Ca2+, the addition of 3 mM Ca2+ to extracellular medium slightly increased [Ca2+]i, which indicated Ca2+ entry due to leakage of Ca2+ at the plasma membrane but not Ca2+ influx through Ca2+ channels. TET and HER did not affect this leakage entry of Ca2+. The present results suggest that TET and HER inhibit Ca2+ release from intracellular stores as well as Ca2+ entry from extracellular medium evoked by bombesin and TG. In addition, TET and HER inhibit IP3 accumulation induced by bombesin in rat glioma C6 cells.

Topics: Alkaloids; Animals; Antineoplastic Agents, Phytogenic; Benzylisoquinolines; Bombesin; Calcium; Calcium Channel Blockers; Cell Division; Cell Membrane; Cell Membrane Permeability; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Enzyme Inhibitors; Glioma; Inositol 1,4,5-Trisphosphate; Rats; Thapsigargin; Tumor Cells, Cultured

In glioma C6 cells, extracellular ATP generates inositol 1,4,5-trisphosphate (InsP3), indicating the presence of purinergic receptors coupled to phosphoinositide turnover. To identify the effect of ATP (acting via InsP3) and thapsigargin (acting without InsP3 production as a specific inhibitor of the endoplasmic reticulum Ca(2+)-ATPase) on intracellular Ca2+ pools we used video imaging of Fura-2 loaded into single, intact glioma C6 cells. It has been shown that ATP and thapsigargin initiate Ca2+ response consistent with the capacitative model of Ca2+ influx. When the cells were stimulated by increasing concentrations of ATP (1, 10, 50 and 100 microM) the graded, quantal Ca2+ response was observed. In the absence of extracellular Ca2+ thapsigargin and ionomycin-releasable Ca2+ pools are overlapping, demonstrating that Ca2+ stores are located mainly in the endoplasmic reticulum. After maximal Ca2+ mobilization by ATP, thapsigargin causes further increase in cytosolic Ca2+ concentration, whereas emptying of thapsigargin-sensitive intracellular stores prevents any further Ca2+ release by ATP. Thus, the thapsigargin-sensitive intracellular pool of Ca2+ in glioma C6 cells seems to be larger than that sensitive to InsP3. Two hypothesis to explain this result are proposed. One postulates a presence of two different Ca2+ pools, sensitive and insensitive to InsP3 and both discharged by thapsigargin, and the other, the same intracellular pool of Ca2+ completely emptying by thapsigargin and only partially by InsP3. These results may contribute to understanding the mechanism of Ca2+ signalling mediated by ATP, the most potent intracellular Ca2+ mobilizing agonist in all types of glial cells.

Topics: Adenosine Triphosphate; Calcium; Drug Evaluation, Preclinical; Endoplasmic Reticulum; Enzyme Inhibitors; Glioma; Humans; Inositol 1,4,5-Trisphosphate; Signal Transduction; Thapsigargin; Tumor Cells, Cultured

It has been found that sphingosine and sphingosylphosphorylcholine (amphiphilic cations) have a stimulatory, and cholesterol 3-sulfate (an amphiphilic anion), an inhibitory, effect on [14C]serine incorporation into phosphatidylserine in glioma C6 and rat liver microsomes. In glioma intact cells sphingosine stimulates phosphatidylserine synthesis in a process independent of protein kinase C, but suppressed by thapsigargin. We suggest that the stimulation of the enzyme occurs by the interaction of amphiphilic cations with the membrane cosubstrate phospholipids, leading to a charge redistribution on their phosphate groups, and hence facilitating the enzyme action. A new hypothesis concerning the mechanism of the serine base exchange reaction is discussed.

Topics: Animals; Cholesterol Esters; Glioma; Kinetics; Microsomes; Microsomes, Liver; Nitrogenous Group Transferases; Phosphatidylserines; Phosphorylcholine; Protein Kinase C; Rats; Sphingosine; Tetradecanoylphorbol Acetate; Thapsigargin; Tumor Cells, Cultured

The temporal and spatial changes of intracellular free calcium ([Ca2+]i) within the cytosol and nucleis of C6 glioma cells have been investigated with laser confocal scanning microscopy to evaluate the current view that Ca2+ ions pass freely through nuclear pores by diffusion. Our results indicate that localized cytosolic Ca2+ release, which appeared as puffs, spread with an apparent diffusion rate of 0.35 +/- 0.07 microns/sec (n = 44). This release was followed by an immediate Ca2+ uptake at the resting stage. Following the treatment with thapsigargin, an inhibitor of microsomal Ca(2+)-ATPase, release of nuclear Ca2+ from certain nuclear hot zones and nuclear envelope was obtained. Most of the nuclear Ca2+ released were confined to the nuclear boundary, but a slow migration of Ca2+ towards the cytosol was observed. The apparent diffusion rate of this Ca2+ release is 0.015 microns/sec. By contrast, the inward spread into nucleus occurred with a diffusion rate of 0.04 microns/sec. From these diffusion rates and other experimental evidence, we conclude that the movement of Ca2+ at the nucleocytosolic interface is more than a simple diffusion process and the interface is a barrier to Ca2+ movement.

Topics: Animals; Calcium; Calcium-Transporting ATPases; Cell Compartmentation; Diffusion; Enzyme Inhibitors; Glioma; Microscopy, Confocal; Microscopy, Fluorescence; Nuclear Envelope; Rats; Terpenes; Thapsigargin; Tumor Cells, Cultured

Long-term superfusion with bradykinin causes oscillations of cytosolic Ca2+ activity ([Ca2+]i) in Fura-2 loaded rat glioma cells. The [Ca2+]i rise is associated with synchronous plasma membrane hyperpolarization oscillating with a frequency of 0.8-1.8 per min. The initial large transient [Ca2+]i rise, induced immediately with bradykinin admission results from InsP3-mediated Ca2+ release, whereas the subsequent oscillations depend mainly on Ca2+ influx, as demonstrated: (i) by blockade of [Ca2+]i oscillations by reduction of [Ca2+]ex' or addition of Ca(2+)-channel blockers; and (ii) evidence from Mn2+ quench experiments. Suppression of [Ca2+]i oscillations with high K+ depolarization and with block of Ca(2+)-dependent K+ channels proves that membrane hyperpolarization is required for Ca2+ influx during the oscillation. Ca2+ release from intracellular stores by inhibitors of endoplasmic reticulum Ca(2+)-ATPase attenuates or blocks the [Ca2+]i oscillations. This suggests that bradykinin-induced Ca2+ influx is controlled by the filling state of the stores. The [Ca2+]i oscillations are suppressed by hypertonic medium and enhanced by hypotonic medium. Cell swelling enhances Ca2+ influx. We propose the following model for generation of the oscillations in the glial cell line: InsP3-induced Ca2+ release from internal stores periodically evokes Ca2+ influx through Ca(2+)-permeable cation channels. Hyperpolarization of the plasma membrane due to the activation of Ca(2+)-dependent K+ channels enhances the Ca2+ influx. The concomitant K+ efflux could lead to cell shrinkage which suppresses Ca2+ influx. Cell volume and membrane potential probably serve as feedback regulators during the [Ca2+]i oscillations.

Topics: Animals; Bradykinin; Calcium; Cell Size; Enzyme Inhibitors; Extracellular Matrix; Glioma; Membrane Potentials; Osmosis; Rats; Terpenes; Thapsigargin; Tumor Cells, Cultured

The effects of 2,5-di-tert-butylhydroquinone (DBHQ) and thimerosal on phosphatidylserine synthesis by the base exchange reaction and on calcium mobilization in intact glioma C6 cells were compared with that of thapsigargin, a selective inhibitor of the endoplasmic reticulum Ca(2+)-ATPase. It has been found that all these agents inhibit phosphatidylserine synthesis by 70%, but their effectiveness are different. The data show that this inhibition is caused by Ca2+ depletion of the endoplasmic reticulum, indicating that phosphatidylserine synthesis requires high concentration of Ca2+ within this structure. On this basis and on literature data, a new model for the localization of the serine base exchange enzyme in the endoplasmic reticulum membrane is proposed.

Topics: Brain Neoplasms; Calcium; Calcium-Transporting ATPases; Endoplasmic Reticulum; Enzyme Inhibitors; Glioma; Hydroquinones; Ionomycin; Phosphatidylserines; Terpenes; Thapsigargin; Thimerosal; Tumor Cells, Cultured

Transcription mechanisms regulating nerve growth factor (NGF) gene expression in the CNS are yet to be thoroughly understood. We have used C6-2B rat glioma cells to characterize the signal transduction pathways that contribute to transcriptional and posttranscriptional regulation of NGF mRNA. Because the NGF promoter contains an AP-1 consensus sequence, we have investigated whether increases in AP-1 binding activity correlate with enhanced NGF mRNA expression. Gel mobility shift assays using an oligonucleotide homologous to the AP-1 responsive element of the rat NGF gene (AP-1NGF) revealed that 12-O-tetradecanoyl phorbol-13-acetate (TPA) and, to a lesser extent, isoproterenol (ISO) and thapsigargin, a microsomal Ca(2+)-ATPase inhibitor, stimulated binding to AP-1NGF within 2 h. All of these stimuli increased NGF mRNA levels within 3 h. Cycloheximide pretreatment blocked the TPA and ISO-mediated binding to AP-1NGF suggesting that de novo synthesis of c-Fos/c-Jun may be required for the transcriptional regulation of NGF gene. Nuclear run-on assays and NGF mRNA decay studies revealed that TPA increases NGF transcription whereas ISO affects both transcription and mRNA stabilization. We propose that (i) different signal transduction mechanisms regulate the expression of the NGF gene in cells derived from the CNS, and (ii) both mRNA transcription and stability account for the cAMP-mediated increase in NGF mRNA levels.

Topics: Animals; Base Sequence; Cell Line; Cell Nucleus; Enzyme Inhibitors; Fibroblast Growth Factor 2; Glioma; Isoproterenol; Molecular Sequence Data; Nerve Growth Factors; Oligodeoxyribonucleotides; Rats; RNA Processing, Post-Transcriptional; RNA, Messenger; Signal Transduction; Terpenes; Tetradecanoylphorbol Acetate; Thapsigargin; Transcription Factor AP-1; Transcription, Genetic; Tumor Cells, Cultured

The effect of sphingosine on intracellular calcium signalling in glioma C6 cells was studied with Fura-2 video imaging technique. Sphingosine had a direct effect on changes in cytosolic Ca2+ concentration only when applied at high concentration of 100 microM, causing the cytosolic Ca2+ level to rise. However, at a much lower concentration of 15 microM sphingosine diminished calcium responses triggered by thapsigargin (a specific inhibitor of calcium pump in the endoplasmic reticulum) and ionomycin (calcium ionophore). Since responses to thapsigargin and ionomycin were blocked in Ca(2+)-free medium, we postulate that sphingosine is acting on the intracellular calcium stores. Additionally, sphingosine (at 15 microM and 100 microM) markedly decreases thapsigargin-induced sustained elevation in cytosolic Ca2+ concentration, indicating its inhibitory effect on thapsigargin-evoked Ca2+ influx. Sphingosine is a known inhibitor of protein kinase C and the involvement of this enzyme is postulated in the modulatory effects of sphingosine on intracellular calcium dynamics.

Topics: Animals; Calcium; Calcium-Transporting ATPases; Cell Line; Cytosol; Enzyme Inhibitors; Fluorescent Dyes; Fura-2; Glioma; Ionomycin; Kinetics; Signal Transduction; Sphingosine; Terpenes; Thapsigargin; Tumor Cells, Cultured; Video Recording

Glioma C6 cells treated with 12-0-tetradecanoyl-phorbol-13-acetate, TPA (10 nM and 100 nM) manifested slow increase in intracellular calcium concentration ([Ca2+]i), dependent upon both Ca2+ release from intracellular stores and Ca2+ entry, and ranging from 50 to 500 nM in different cells. The effect of TPA was abolished by the down-regulation procedure and by protein kinase C inhibitors, such as staurosporine (100 nM), suramin (100 microM), and sphingosine (100 microM), pointing to a role of protein kinase C (PKC) in this process. On the other hand, thapsigargin (100 nM), a selective inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, produced a rapid increase in [Ca2+]i (up to 800 nM). This increase consisted of a transient initial phase followed by sustained elevation in [Ca2+]i, typical of Ca2+ release from intracellular stores and of Ca2+ entry, respectively. However, when the cells were exposed to TPA (100 nM) prior to thapsigargin (100 nM), then thapsigargin produced only a transient rise in [Ca2+]i. We suggest that TPA, a PKC activator, affects thapsigargin-induced Ca2+ entry, probably by PKC-mediated changes in cytoskeleton structures.

Topics: Animals; Biological Transport; Calcium; Calcium-Transporting ATPases; Cytoskeleton; Endoplasmic Reticulum; Glioma; Mice; Neoplasm Proteins; Nerve Tissue Proteins; Protein Kinase C; Signal Transduction; Terpenes; Tetradecanoylphorbol Acetate; Thapsigargin; Tumor Cells, Cultured

To determine whether NG108-15 cells contain a functional Na+/Ca2+ exchanger, we isotonically replaced extracellular Na+ with N-methyl-D-glucamine (NMG) and measured the effect on cytosolic Ca2+ concentration ([Ca2+]i) using the fluorescent Ca2+ indicator fura 2. Replacement with NMG alone had no effect on basal [Ca2+]i or the rise in [Ca2+]i evoked by 80 mM K+ or 10 microM bradykinin, but caused a larger [Ca2+]i increase when thapsigargin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) were added to the cells; this enhanced [Ca2+]i increase could be reversed by adding Na+ back to the bathing buffer. The elevation in [Ca2+]i induced by thapsigargin and FCCP was inversely proportional to extracellular Na+ concentration. Furthermore, the exchanger operated in the reverse mode, as measured by either [Ca2+]i change or 45Ca2+ uptake. An 810 bp cDNA fragment of the exchanger was amplified by PCR; it differed by a single amino acid residue from the corresponding segment of the rat brain Na+/Ca2+ exchanger. These data suggest that a functioning Na+/Ca2+ exchanger exists in NG108-15 cells.

Topics: Base Sequence; Calcium; Calcium-Transporting ATPases; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Carrier Proteins; DNA Primers; DNA, Complementary; Glioma; Hybrid Cells; Molecular Sequence Data; Neuroblastoma; Sodium; Sodium-Calcium Exchanger; Terpenes; Thapsigargin; Tumor Cells, Cultured

Elevation of cytosolic free Ca2+ inhibits the type VI adenylyl cyclase that predominates in C6-2B cells. However, it is not known whether there is any selective requirement for Ca2+ entry or release for inhibition of cAMP accumulation to occur. In the present study, the effectiveness of intracellular Ca2+ release evoked by three independent methods (thapsigargin, ionomycin, and UTP) was compared with the capacitative Ca2+ entry that was triggered by these treatments. In each situation, only Ca2+ entry could inhibit cAMP accumulation (La3+ ions blocked the effect); Ca2+ release, which was substantial in some cases, was without effect. A moderate inhibition, as was elicited by a modest degree of Ca2+ entry, could be rendered substantial in the absence of phosphodiesterase inhibitors. Such conditions more closely mimic the physiological situation of normal cells. These results are particularly significant, in demonstrating not only that Ca2+ entry mediates the inhibitory effects of Ca2+ on cAMP accumulation, but also that diffuse elevations in [Ca2+]i are ineffective in modulating cAMP synthesis. This property suggests that, as with certain Ca(2+)-sensitive ion channels, Ca(2+)-sensitive adenylyl cyclases may be functionally colocalized with Ca2+ entry channels.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Calcium; Cyclic AMP; Glioma; Terpenes; Thapsigargin; Tumor Cells, Cultured; Uridine Triphosphate

Phospholipid metabolism was studied in N1E-115 neuroblastoma and C6 glioma cells exposed to thapsigargin, a selective inhibitor of endoplasmic reticulum Ca(2+)-ATPase that raises the cytosolic free Ca2+ concentration [Ca2+]i. Thapsigargin caused only a transient increase of [Ca2+]i (< 1 min) in N1E-115 cells similar in magnitude and duration to agonist-induced calcium release mediated by inositol trisphosphate. Sustained elevation of [Ca2+]i due to influx of extracellular calcium, as occurs in most other cell lines including C6 cells, did not occur in N1E-115 cells. Increased uptake of inorganic phosphate (Pi) associated calcium influx was observed in C6 but not in N1E-115 cells. Thapsigargin affected phospholipid synthesis in both cell lines, most likely by inhibiting phosphatidic acid phosphohydrolase as indicated by diversion of [3H]oleic acid incorporation from triacylglycerol to phospholipid synthesis and stimulation of [32P]Pi incorporation into anionic phospholipids at the expense of phosphatidylcholine synthesis. The response to increased phosphatidate/phosphatidyl-CMP availability was cell specific. Thapsigargin (> 100 nM) selectively stimulated phosphatidylglycerol synthesis 20-30-fold in N1E-115 neuroblastoma cells while phosphatidylinositol synthesis was increased < 2-fold. In contrast, phosphatidylglycerol was not affected in C6 glioma cells and phosphatidylinositol synthesis was stimulated 8-fold by thapsigargin (> 1 microM). Agonist-stimulated calcium release did not increase phosphatidylglycerol synthesis in N1E-115 cells. Thapsigargin-stimulated phosphatidylglycerol synthesis and agonist-stimulated phosphatidylinositol synthesis could occur at the same time. Similar results were obtained with TMB-8, an inhibitor of intracellular Ca2+ release that decreases diacylglycerol utilization by blocking choline uptake and phosphatidylcholine synthesis without affecting resting [Ca2+]i. Thus [Ca2+]i does not directly mediate the effects of thapsigargin, TMB-8 or agonist stimulation on anionic phospholipid metabolism. These additional effects may limit the use of thapsigargin to assess Ca(2+)-dependence of phospholipid metabolism associated with Ca(2+)-mediated signal transduction.

Topics: Animals; Calcium; Gallic Acid; Glioma; Mice; Neuroblastoma; Oleic Acid; Oleic Acids; Phosphates; Phosphatidylglycerols; Phosphatidylinositols; Rats; Terpenes; Thapsigargin; Tumor Cells, Cultured

Various first messengers linked to phospholipase C, including acetylcholine and interleukin 1, regulate the production both of the secreted form of the amyloid protein precursor (APP) and of amyloid beta-protein. We have now identified intracellular signals which are responsible for mediating these effects. We show that activation of phospholipase C may affect APP processing by either of two pathways, one involving an increase in protein kinase C and the other an increase in cytoplasmic calcium levels. The effects of calcium on APP processing appear to be independent of protein kinase C activation. The observed effects of calcium on APP processing may be of therapeutic utility.

Topics: Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Calcium; Calcium-Transporting ATPases; Carbachol; Cell Line; CHO Cells; Cricetinae; Electrophoresis, Polyacrylamide Gel; Glioma; Humans; Immunoblotting; Interleukin-1; Models, Biological; Molecular Weight; Protein Kinase C; Protein Processing, Post-Translational; Receptors, Muscarinic; Terpenes; Thapsigargin; Transfection; Tumor Cells, Cultured

Intact and permeabilized glioma C6 cells were incubated with [14C]serine in media containing low (100 nM) or high (2 mM) [Ca2+] and serine incorporation into phosphatidylserine was examined. In all cases thapsigargin, a blocker of the endoplasmic reticulum Ca(2+)-ATPase, diminished this process, whereas the action of the ionophore A23187 was dependent on the external calcium concentration and time of incubation. In permeabilized cells incubated at 100 nM Ca2+, serine incorporation into phosphatidylserine was diminished when the endoplasmic reticulum Ca2+ levels were lowered by the ionophore. In intact cells incubated at 2 mM CaCl2, addition of A23187 had no effect for the first 30 min and later decreased [14C]serine incorporation. This result seems to be not connected with the degradation of already formed phosphatidylserine, or with an enhanced metabolic conversion of this phospholipid, but with the decrease of its synthesis. The mechanism of this last process appears to be involved in the ionophore-induced perturbation of cellular Ca2+ homeostasis. Our results indicate that phosphatidylserine synthesis is a Ca(2+)-regulated process.

Topics: Animals; Calcimycin; Calcium; Calcium-Transporting ATPases; Cell Membrane Permeability; Glioma; Phosphatidylserines; Serine; Terpenes; Thapsigargin; Tumor Cells, Cultured

ATP-induced changes in the intracellular Ca2+ concentration ([Ca2+]i) in neuroblastoma x glioma hybrid NG108-15 cells were studied. Using the fluorescent Ca2+ indicator fura-2, we have shown that the [Ca2+]i increased in response to ATP. ATP at 3 mM caused the greatest increased in [Ca2+]i, whereas at higher concentrations of ATP the response became smaller. Two nonhydrolyzable ATP analogues, adenosine 5'-thiotriphosphate and 5'-adenylyl-beta, gamma-imidodiphosphate, could not trigger significant [Ca2+]i change, but they could block the ATP effect. Other adenine nucleotides, including ADP, AMP, alpha beta-methylene-ATP, beta, gamma-methylene-ATP, and 2-methylthio-ATP, as well as UTP and adenosine, all had no effect on [Ca2+]i at 3 mM. In the absence of extracellular Ca2+, the effect of ATP was inhibited totally, but could be restored by the addition of Ca2+ to the cells. Upon removal of Mg2+, the maximum increase in [Ca2+]i induced by ATP was enhanced by about 42%. Ca(2+)-channel blockers partially inhibited the ATP-induced [Ca2+]i rise. The ATP-induced [Ca2+]i rise was not affected by thapsigargin pretreatment, though such pretreatment blocked bradykinin-induced [Ca2+]i rise completely. No heterologous desensitization of [Ca2+]i rise was observed between ATP and bradykinin. The magnitude of the [Ca2+]i rise induced by ATP increased between 1.5 and 3.1 times when external Na+ was replaced with Tris, N-methyl-D-glucamine, choline, or Li+. The addition of EGTA or verapamil to cells after their maximum response to ATP immediately lowered the [Ca2+]i to the basal level in Na(+)-containing or Na(+)-free Tris solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine Triphosphate; Adenylyl Imidodiphosphate; Animals; Bradykinin; Calcium; Calcium Channel Blockers; Calcium-Transporting ATPases; Dose-Response Relationship, Drug; Fura-2; Glioma; Hybrid Cells; Kinetics; Neuroblastoma; Nifedipine; omega-Conotoxins; Peptides; Terpenes; Thapsigargin; Verapamil

Endothelin-1 (ET) and ATP mobilize Ca2+ in rat C6 glioma cells by stimulating phosphoinositide turnover. Both agents also inhibit adenylyl cyclase (AC) activity in C6 glioma cells. The goal of this study was to characterize the molecular mechanisms responsible for the inhibition of AC activity. The administration of either ET, ATP, A23187, or thapsigargin to cells simultaneously with isoproterenol for 5 min inhibited isoproterenol-stimulated cAMP synthesis by a maximum of 60%, 91%, 65%, and 68%, respectively. Pretreatment of cells with pertussis toxin (PTX) did not alter the inhibitory effects of A23187 or thapsigargin, whereas the inhibitory effects of ET or ATP were completely eliminated. Removal of extracellular Ca2+ and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'- tetraacetic acid acetoxymethyl ester treatment failed to affect the inhibition caused by ET or ATP, whereas the inhibition caused by A23187 or thapsigargin was completely eliminated in Ca(2+)-free medium and was attenuated by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester treatment. The inhibition by both receptor agonists in the earlier phase (30 sec) of the AC reaction was, however, reduced by using either Ca(2+)-free medium or PTX pretreatment. The administration of 3-isobutyl-1-methylxanthine or Ro 20-1724 suggested that the inhibitory effects of A23187 and thapsigargin were partially due to Ca(2+)-dependent stimulation of PDE activity. Short term treatment with phorbol-12-myristate-13-acetate (PMA) had no effect on isoproterenol-stimulated AC activity. However, the inhibition of cAMP induced by ET or ATP, but not by A23187 or thapsigargin, was diminished by PMA, suggesting that the receptor signal via Gi was blocked by PMA treatment. The antagonistic effect of PMA was blocked by staurosporine. All four agents still inhibited AC activity in cells that had been treated with PMA for 24 hr to deplete protein kinase C. ET produced an additional decrease in AC activity in cells that had been treated with a maximally effective concentration of A23187 or thapsigargin. The ET- or ATP-induced decrease in cAMP levels showed homologous desensitization. These results demonstrate that ETZ receptors and ATP receptors in C6 glioma cells inhibit AC activity primarily by interaction with a PTX-sensitive G(i) and partially by elevation of [Ca(2+)]. Protein kinase C activation is not responsible for agonist-induced inhibition of AC but appears to uncouple the G(i)/AC system activa

Topics: Adenosine Triphosphate; Adenylate Cyclase Toxin; Adenylyl Cyclase Inhibitors; Animals; Calcimycin; Calcium; Drug Interactions; Endothelins; Glioma; GTP-Binding Proteins; Isoproterenol; Pertussis Toxin; Phosphodiesterase Inhibitors; Protein Kinase C; Rats; Terpenes; Thapsigargin; Tumor Cells, Cultured; Virulence Factors, Bordetella

In an NG 108-15 neuroblastoma x glioma hybrid cell suspension, extracellular ATP (via P2-purinergic receptors) and bradykinin stimulated Ins(1,4,5)P3 formation, which was accompanied by an increase in the cytosolic Ca2+ concentration ([Ca2+]i). Leucine enkephalin (EK) also slightly increased [Ca2+]i in the absence, but not in the presence, of apyrase, which hydrolyses extracellular ATP and ADP to AMP. When the cells were stimulated by P2-agonists or bradykinin prior to the application of EK, EK induces a remarkable rise in [Ca2+]i. This P2-agonist- or bradykinin-assisted EK action was also observed in single cells on a coverslip. A decrease in the extracellular Ca2+ concentration only slightly lowered the EK-induced rise in [Ca2+]i, but treatment of the cells with thapsigargin, an agent which depletes Ca2+ in the Ins(1,4,5)P3-sensitive pool, almost completely abolished EK action. The observed permissive stimulation by EK of Ins(1,4,5)P3 formation induced by a P2-agonist or bradykinin may be a primary event for the EK-induced [Ca2+]i rise. These actions of EK were antagonized by naloxone and completely reversed by prior treatment of the cells with pertussis toxin, whereas the toxin hardly affected the actions of P2-agonists and bradykinin themselves. Thus EK can induce phospholipase C activation and subsequent Ca2+ mobilization, provided that the cells have been previously or are simultaneously stimulated by endogenous adenine nucleotides or by externally applied P2-agonists or bradykinin. In this cross-talk mechanism between opioid receptors and these Ca(2+)-mobilizing agonist receptors, pertussis toxin-sensitive G-proteins play a permissive role.

Topics: Adenosine Triphosphate; Bradykinin; Calcium; Enkephalin, Leucine; Enkephalins; Enzyme Activation; Glioma; GTP-Binding Proteins; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Naloxone; Neuroblastoma; Pertussis Toxin; Receptors, Bradykinin; Receptors, Neurotransmitter; Receptors, Opioid; Receptors, Purinergic; Terpenes; Thapsigargin; Tumor Cells, Cultured; Type C Phospholipases; Virulence Factors, Bordetella

In C6-2B rat glioma cells, agonist-stimulated cAMP accumulation is potently inhibited after the stimulation of endogenous bradykinin receptors or stably transfected substance K receptors, coupled to phosphatidylinositol hydrolysis. In the present report, pharmacological tools were used to selectively stimulate either protein kinase C or Ca2+, the two final effectors activated upon phosphatidylinositol hydrolysis, and their role in the inhibition of the C6-2B cell cAMP signaling pathway was investigated. Activation of protein kinase C by an acute treatment with phorbol 12-myristate 13-acetate or L-alpha-1-oleoyl-2-acetyl-sn-3-glycerol did not reduce, but rather enhanced, the cAMP accumulation elicited by forskolin, a direct activator of adenylyl cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. This effect was antagonized by the protein kinase inhibitor H-7 and mimicked by the protein phosphatase inhibitor okadaic acid. Thapsigargin, a selective microsomal Ca(2+)-ATPase inhibitor, evoked a sustained increase in the intracellular free Ca2+ concentration, with an EC50 of 24.8 +/- 4.3 nM, and inhibited the cAMP accumulation induced by the beta-adrenergic receptor agonist isoproterenol with comparable potency (IC50 = 19.3 +/- 0.2 nM), strongly suggesting a causal relationship between the two phenomena. The inhibition by thapsigargin of isoproterenol- or forskolin-stimulated cAMP accumulation was not affected by pertussis toxin or down-regulation or inhibition of protein kinase C. Dantrolene, a blocker of Ca2+ release from intracellular stores, antagonized 1) the Ca2+ transient in response to thapsigargin and substance K and 2) the inhibitory effect of these compounds on isoproterenol- or forskolin-induced cAMP accumulation. Moreover, sequestration of intracellular Ca2+ with the cell-permeable Ca2+ chelator ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid acetoxymethyl ester abolished the cAMP inhibition mediated by thapsigargin. Finally, isoproterenol- or forskolin-stimulated adenylyl cyclase activity in digitonin-permeabilized cells was not affected by either thapsigargin or substance K. These data provide compelling evidence that increases in intracellular free Ca2+ concentration without activation of protein kinase C suffice and are responsible for the inhibition of cAMP accumulation in C6-2B cells.

Topics: Animals; Calcium; Colforsin; Cyclic AMP; Enzyme Activation; Glioma; Hydrolysis; Isoproterenol; Phosphatidylinositols; Protein Kinase C; Rats; Receptors, Adrenergic, beta; Signal Transduction; Terpenes; Tetradecanoylphorbol Acetate; Thapsigargin; Tumor Cells, Cultured

In C6-2B cells, agonist-stimulated cyclic AMP accumulation is inhibited when the cytosolic Ca2+ concentration is increased. We now demonstrate that in C6-2B cells: (i) the early kinetics of the cyclic AMP inhibition by substance K (t1/2 = 35 s) and thapsigargin (t1/2 = 1.6 min) closely mimic the kinetics of the cytosolic Ca2+ increase evoked by either agent (t1/2 = 25 s and 1.5 min respectively); (ii) the Ca2+ rise and cyclic AMP inhibition by substance K or thapsigargin are similarly affected in EGTA-containing medium; (iii) PCR detects type-III and type-VI adenylate cyclase cDNAs, and RNAase protection assays show that the mRNA for type-VI adenylate cyclase, an isoform inhibitable by submicromolar Ca2+ concentrations, is the predominant species, strongly suggesting that type-VI adenylate cyclase is probably the target molecule for Ca(2+)-mediated inhibition of cyclic AMP accumulation.

Topics: Adenylyl Cyclases; Animals; Brain Neoplasms; Calcium; Cyclic AMP; DNA; Glioma; Kinetics; Neurokinin A; Rats; Terpenes; Thapsigargin; Tumor Cells, Cultured

Bradykinin-induced increases in the intracellular free Ca2+ concentration ([Ca2+]i) were recorded in single NG108-15 cells with indo-1-based dual-emission microfluorimetry (50% effective concentration, 16 nM). A 1-min exposure to 30 nM bradykinin completely depleted the inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ store; refilling the store required extracellular Ca2+ (half time, 2 min). Refilling the IP3-sensitive store was completely blocked by 1 microM La3+ and 10 microM nitrendipine, but not 10 microM verapamil, 10 microM flunarizine, 1 microM nitrendipine, or 0.1 microM La3+. Thapsigargin irreversibly depleted the Ca2+ store and prevented its refilling (half-maximal inhibitory concentration, 3 nM). Influx of Ca2+ across the plasma membrane did not increase after depletion of the IP3-sensitive store by exposure to bradykinin, although maintained presence of the agonist produced significant Ca2+ influx. Similarly, Mn2+ and Ba2+ influx, as measured by indo-1 quenching and spectral shifts, did not increase following depletion of IP3-sensitive store. In contrast to depletion of the IP3-sensitive Ca2+ store by bradykinin, thapsigargin (10 nM) treatment produced Ca2+ and Ba2+ influx. We conclude that after Ca2+ mobilization, the IP3-sensitive Ca2+ store in NG108-15 cells is refilled with cytoplasmic Ca2+ via a thapsigargin-sensitive Ca(2+)-Mg(2+)-ATPase. Cytoplasmic Ca2+ is replenished by a persistent leak of Ca2+ across the plasma membrane. This leak is not modulated by the status of the intracellular Ca2+ store. In NG108-15 cells, agonist and thapsigargin-evoked Ca2+ entry are mediated by activation of plasmalemmal Ca2+ channels independent of the status of the IP3-sensitive intracellular Ca2+ store.

Topics: Animals; Bradykinin; Calcium; Calcium-Transporting ATPases; Dose-Response Relationship, Drug; Flunarizine; Glioma; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Mice; Neuroblastoma; Nitrendipine; Terpenes; Thapsigargin; Tumor Cells, Cultured; Verapamil

Phosphatidylserine synthesis was studied in glioma C6 cells with [14C]serine and in the presence or absence of agents which increase the level of [Ca2+]i. It was found that glutamate and acetylcholine inhibited this synthesis by up to 40%, whereas thapsigargin and the ionophore A23187 inhibited by up to 70%. The inhibitory effect of thapsigargin and the A23187 was observed in Ca(2+)-free medium. The data show that the inhibition of this synthesis is caused by the Ca(2+)-depletion from endoplasmic reticulum, suggesting that the synthesis of phosphatidylserine occurs on the luminal side of these structures and can be regulated by transmembrane signaling systems.

Topics: Acetylcholine; Animals; Calcimycin; Calcium; Calcium Chloride; Calcium-Transporting ATPases; Dose-Response Relationship, Drug; Edetic Acid; Fura-2; Glioma; Glutamates; Glutamic Acid; Kinetics; Phosphatidylserines; Terpenes; Thapsigargin; Tumor Cells, Cultured

Continuous superfusion of rat glioma cells with medium containing bradykinin (from 0.2 nM) induced a transient hyperpolarization followed by regular hyperpolarizing oscillations of the membrane potential. Similar repetitive hyperpolarizing oscillations were caused by extracellularly applied bradykinin or muscarine or by intracellularly injected GTP-gamma-S. The frequency of the oscillations was 1 per minute at bradykinin concentrations ranging from 0.2 nM to 2 microM, but the amplitude and duration increased with rising peptide concentration. The muscarine-induced oscillations were blocked by atropine. In the presence of extracellular Ca2+, the substances thapsigargin, 2,5-di(tert-butyl)-1,4-benzohydroquinone (tBuBHQ), and ionomycin reversibly suppressed the bradykinin-induced oscillations. Thapsigargin and tBuBHA, which are known to block the Ca2+ ATPase of endoplasmic reticulum, caused a transient rise in cytosolic Ca2+ activity, monitored with Fura-2, in suspensions of rat glioma cells or of mouse neuroblastoma-rat glioma hybrid cells. After a transient Ca2+ rise caused by thapsigargin, tBuBHQ, or ionomycin, the Ca2+ response to bradykinin which is known to be due to release of Ca2+ from internal stores was suppressed. This indicates that thapsigargin and tBuBHQ deplete internal Ca2+ stores as already seen previously for ionomycin. Thus, the inhibition of the membrane potential oscillations by thapsigargin, tBuBHQ, and ionomycin indicates that the oscillations are associated with activation of InsP3-sensitive Ca2+ stores. In some cells composite oscillation patterns which consisted of two independent oscillations with different amplitudes that overlapped additively were seen. We discuss that this pattern and the concentration dependency of the oscillations could be due to "quantal" Ca2+ release from stores with different inositol 1,4,5-triphosphate sensitivities. Subsidence of the oscillations after omission of extracellular Ca2+ seems to be due to a lack of replenishment of the intracellular stores with Ca2+, which comes from the extracellular compartment.

Topics: Animals; Benzoquinones; Bradykinin; Calcium; Cytosol; Glioma; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Membrane Potentials; Mice; Muscarine; Neuroblastoma; Rats; Terpenes; Thapsigargin; Tumor Cells, Cultured

In NG108-15 cells, bradykinin (BK) and thapsigargin (TG) caused transient increases in a cytosolic free Ca2+ concentration ([Ca2+]i), after which [Ca2+]i elevated by TG only declined to a higher, sustained level than an unstimulated level. In PC12 cells, carbachol (CCh) evoked a transient increase in [Ca2+]i followed by a sustained rise of [Ca2+]i, whereas [Ca2+]i elevated by TG almost maintained its higher level. In the absence of extracellular Ca2+, the sustained elevation of [Ca2+]i induced by each drug we used was abolished. In addition, the rise in [Ca2+]i stimulated by TG was less affected after CCh or BK, whereas CCh or BK caused no increase in [Ca2+]i after TG. TG neither increased cellular inositol phosphates nor modified the inositol phosphates format on stimulated by CCh or BK. We conclude that TG may release Ca2+ from both IP3-sensitive and -insensitive intracellular pools and that some kinds of signalling to link the intracellular Ca2+ pools and Ca2+ entry seem to exist in neuronal cells.

Topics: Animals; Biological Transport; Bradykinin; Calcium; Calcium Channel Blockers; Cell Line; Cell Membrane; Fluorescent Dyes; Fura-2; Glioma; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Kinetics; Neuroblastoma; Nimodipine; omega-Conotoxins; PC12 Cells; Peptides, Cyclic; Terpenes; Thapsigargin