sq-23377 and Glioma

Temozolomide (TMZ), an oral alkylator of the imidazotetrazine family, is used to treat glioma. Whether this drug has any ionic effects in glioma cells remains largely unclear.. With the aid of patch-clamp technology, we investigated the effects of TMZ on the ionic currents in U373 glioma cells. The mRNA expression of KCNN4 (KCa3.1) in U373 glioma cells and TMZ's effect on K+ currents in these KCNN4 siRNA-transfected U373 cells were investigated.. In whole-cell recordings, TMZ decreased the amplitude of voltage-dependent K+ currents (IK) in U373 cells. TMZ-induced IK inhibition was reversed by ionomycin or 1-ethyl-2-benzimidazolinone (1-EBIO). In cell-attached configuration, TMZ concentration-dependently reduced the activity of intermediate-conductance Ca2+-activated K+ (IKCa) channels with an IC50 value of 9.2 µM. Chlorzoxazone or 1-EBIO counteracted the TMZ-induced inhibition of IKCa channels. Although TMZ was unable to modify single-channel conductance, its inhibition of IKCa channels was weakly voltage-dependent and accompanied by a significant prolongation in the slow component of mean closed time. However, neitherlarge-conductance Ca2+-activated (BKCa) nor inwardly rectifying K+ (Kir) channels were affected by TMZ. In current-clamp mode, TMZ depolarized the cell membrane and 1-EBIO reversed TMZ-induced depolarization. TMZ had no effect on IK in KCNN4 siRNA-transfected U373 cells.. In addition to the DNA damage it does, its inhibitory effect on IKCa channels accompanied by membrane depolarization could be an important mechanism underlying TMZ-induced antineoplastic actions.

Topics: Alkylating Agents; Benzimidazoles; Cell Line, Tumor; Dacarbazine; DNA Damage; Glioma; Humans; Intermediate-Conductance Calcium-Activated Potassium Channels; Ionomycin; Large-Conductance Calcium-Activated Potassium Channels; Membrane Potentials; Patch-Clamp Techniques; Potassium Channels, Inwardly Rectifying; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering; Temozolomide

The inhalation anesthetic isoflurane has been shown to induce mitochondrial dysfunction and caspase activation, which may lead to learning and memory impairment. Ginsenoside Rg1 is reported to be neuroprotective. We therefore set out to determine whether ginsenoside Rg1 can attenuate isoflurane-induced caspase activation via inhibiting mitochondrial dysfunction.. We investigated the effects of ginsenoside Rg1 at concentrations of 12.5, 25, and 50 μmol/L and pretreatment times of 12 h and 24 h on isoflurane-induced caspase-3 activation in H4 naïve and stably transfected H4 human neuroglioma cells that express full-length human amyloid precursor protein (APP) (H4-APP cells). For mitochondrial dysfunction, we assessed mitochondrial permeability transition pore (mPTP) and adenosine-5'-triphosphate (ATP) levels. We employed Western blot analysis, chemiluminescence, and flowcytometry.. Here we show that pretreatment with 50 µmol/L ginsenoside Rg1 for 12 h attenuated isoflurane-induced caspase-3 activation and mitochondrial dysfunction in H4-APP cells, while pretreatment with 25 and 50 µmol/L ginsenoside Rg1 for 24 h attenuated isoflurane-induced caspase-3 activation and mitochondrial dysfunction in both H4 naïve and H4-APP cells.. These data suggest that ginsenoside Rg1 may ameliorate isoflurane-induced caspase-3 activation by inhibiting mitochondrial dysfunction. Pending further studies, these findings might recommend the use of ginsenoside Rg1 in preventing and treating isoflurane-induced neurotoxicity.

Topics: Amyloid beta-Protein Precursor; Caspase 3; Cell Line, Tumor; Gene Expression Regulation, Enzymologic; Ginsenosides; Glioma; Humans; Ionomycin; Isoflurane; Mitochondria

Malignant gliomas (MG), including grades III and IV astrocytomas, are the most common adult brain tumors. These tumors are highly aggressive with a median survival of less than 2 years. Nuclear factor I (NFI) is a family of transcription factors that regulates the expression of glial genes in the developing brain. We have previously shown that regulation of the brain fatty acid-binding protein (B-FABP; FABP7) and glial fibrillary acidic protein (GFAP) genes in MG cells is dependent on the phosphorylation state of NFI, with hypophosphorylation of NFI correlating with GFAP and B-FABP expression. Importantly, NFI phosphorylation is dependent on phosphatase activity that is enriched in GFAP/B-FABP+ve cells. Using chromatin immunoprecipitation, we show that NFI occupies the GFAP and B-FABP promoters in NFI-hypophosphorylated GFAP/B-FABP+ve MG cells. NFI occupancy, NFI-dependent transcriptional activity, and NFI phosphorylation are all modulated by the serine/threonine phosphatase calcineurin. Importantly, a cleaved form of calcineurin, associated with increased phosphatase activity, is specifically expressed in NFI-hypophosphorylated GFAP/B-FABP+ve MG cells. Calcineurin in GFAP/B-FABP+ve MG cells localizes to the nucleus. In contrast, calcineurin is primarily found in the cytoplasm of GFAP/B-FABP-ve cells, suggesting a dual mechanism for calcineurin activation in MG. Finally, our results demonstrate that calcineurin expression is up-regulated in areas of high infiltration/migration in grade IV astrocytoma tumor tissue. Our data suggest a critical role for calcineurin in NFI transcriptional regulation and in the determination of MG infiltrative properties.

Topics: Adult; Astrocytoma; Calcineurin; Calcium-Binding Proteins; Cell Line, Tumor; Chromatin Immunoprecipitation; Cyclosporine; Glioma; Humans; Immunohistochemistry; Immunoprecipitation; Ionomycin; NFI Transcription Factors; Phosphorylation; Promoter Regions, Genetic; Protein Binding; Protein Transport

Malignant glioma cells maintain an elevated intracellular pH (pH(i)) within hypoxic-ischemic tumor microenvironments through persistent activation of sodium-proton transport (McLean et al., 2000). Amiloride has been reported to selectively kill human malignant glioma cell lines but not primary astrocytes (Hegde et al., 2004). While amiloride reduces pH(i) of malignant gliomas by inhibiting isoform 1 of sodium-proton exchange (NHE1), direct acidification was shown to be cytostatic rather than cytotoxic. At cytotoxic concentrations, amiloride has multiple drug targets including inhibition of NHE1 and sodium-calcium exchange. Amiloride's glioma cytotoxicity can be explained, at least in part, by dual inhibition of NHE1 and of Na(+)-dependent calcium efflux by isoform 1.1 of the sodium-calcium exchanger (NCX1.1), which increases [Ca(2+)](i) and initiates glioma cell demise. As a result of persistent NHE1 activity, cytosolic free levels of sodium ([Na(+)](i)) in U87 and C6 glioma cells are elevated 3-fold, as compared with normal astrocytes. Basal cytosolic free calcium levels ([Ca(2+)](i)) also are increased 5-fold. 2', 4'-dichlorobenzamil (DCB) inhibits the sodium-dependent calcium transporter (NCX1.1) much more potently than NHE1. DCB was employed in a concentration-dependent fashion in glioma cells to selectively inhibit the forward mode of NCX1.1 at ≤1μM, while dually inhibiting both NHE1 and NCX1.1 at ≥20μM. DCB (1μM) was not cytotoxic to glioma cells, while DCB (20μM) further increased basal elevated levels of [Ca(2+)](i) in glioma cells that was followed by cell demise. Cariporide and SEA0400 are more selective inhibitors of NHE1 and NCX1.1 than amiloride or DCB, respectively. Individually, Cariporide and SEA0400 are not cytotoxic, but in combination induced glioma cell death. Like amiloride, the combination of Cariporide and SEA0400 produced glioma cell death in the absence of demonstrable caspase activation.

Topics: Amiloride; Aniline Compounds; Animals; Astrocytes; Brain Neoplasms; Calcium; Cell Death; Cell Line, Tumor; Cytosol; Glioma; Guanidines; Humans; Hypoxia-Ischemia, Brain; Ionomycin; Ionophores; Personal Space; Phenyl Ethers; Protons; Rats; Rats, Sprague-Dawley; Sodium; Sodium-Calcium Exchanger; Sodium-Hydrogen Exchanger 1; Sodium-Hydrogen Exchangers; Sulfones; Tumor Microenvironment

In mammals, the principal circadian oscillator resides in the hypothalamic suprachiasmatic nucleus. However, the basic components and the ability to generate a circadian rhythm are also characteristic of most peripheral tissues and some cell lines. In our present study, we show that the rat C6 glioma cell line displays circadian oscillations of reporter luciferase bioluminescence driven by the mouse Per2 promoter and of clock-related gene transcripts. Per2::luc expressing C6 cells display circadian rhythm in their bioluminescence levels for more than seven days. In addition, clock and clock-controlled genes show dynamic circadian oscillation in C6 cells after exposure to dexamethasone. It is also significant that Per1 is not induced in C6 cells by a calcium ionophore, which is in stark contrast to Rat-1 cells. The C6 glioma cell line has therefore the potential to be a useful tool in future investigations of the underlying molecular machinery of the circadian clock.

Topics: Animals; Cell Cycle Proteins; Cell Line, Tumor; Chlorocebus aethiops; Circadian Rhythm; Colforsin; COS Cells; Dexamethasone; Glioma; Horses; Humans; Ionomycin; Luminescence; Mice; Nuclear Proteins; Period Circadian Proteins; Promoter Regions, Genetic; Rats; Tetradecanoylphorbol Acetate; Transcription Factors; Up-Regulation

To investigate the potential role of cytokines in promoting Fas ligand (FasL)-expressing colon cancer cells.. Immunohistochemical SABC method was used to observe the expression of Fas receptor and ligand in SW620 colon cancer cell line and Jurkat T cells in order to provide the morphological evidence for the functions of Fas receptor and ligand. To examine the cytotoxicity of effector cells, CytoTox96(r) non-radioactive cytotoxicity assay was adopted to measure the lactate dehydrogenase-releasing value after SW620 cells were co-cultured with Jurkat T lymphocytes.. The FasL of colon cancer SW620 cells was positive. The positive substances were distributed in the cell membrane and cytoplasm. The Fas receptor of colon cancer SW620 cells was negative. The Fas receptor and ligand of Jurkat T lymphocytes turned out to be positive. The positive substances were distributed in the cell membrane. After phytohemagglutinin (PHA)-stimulated Jurkat T lymp-hocytes were co-cultured with phorbol 12-myristate 13-acetate (PMA)-plus-ionomycin-stimulated (for 48 h) SW620 cells or tumor necrosis factor-alpha (TNF-alpha)-stimulated (for 48 h) SW620 cells or unstimulated SW620 cells for 4 h, the cytotoxicity of SW620 cells to PHA-stimulated Jurkat cells at effector-to-target ratios of 10:1, 5:1, 2.5:1, and 1.25:1 was 74.6%, 40.8%, 32.4%, and 10.9% (F = 8.19, P<0.05); or 54.9%, 35.3%, 22.0%, and 10.3% (F = 11.12, P<0.05); or 14.9%, 10.5%, 6.9%, and 5.8% (F = 3.45, P<0.05). After PHA-stimulated Jurkat T lymphocytes were co-cultured with unstimulated SW620 cells for 8 h, the cytotoxicity of SW620 cells to PHA-stimulated Jurkat cells at effector-to-target ratios of 5:1, 2.5:1, and 1.25:1 from the experiment was 83.9%, 74.1%, and 28.5% (F = 137.04, P<0.05) respectively. Non-radioactive cytotoxicity assay showed that the apoptotic rate of Jurkat cells remarkably increased with the increase of planting concentration of SW620 cells and co-culture time after the SW620 cells were co-cultured with the Jurkat T lymphocytes. The cytotoxicity was significantly enhanced by PMA+ionomycin or TNF-alpha.. The FasL expressed in human colon cancer cells may be regulated by endogenous factors in the microenvironment of the host and facilitate the escape of tumor cells from the host immune system.

Topics: Cell Communication; Cell Line, Tumor; Coculture Techniques; Colonic Neoplasms; Cytokines; Cytotoxicity, Immunologic; Fas Ligand Protein; fas Receptor; Glioma; Humans; Immunohistochemistry; Ionomycin; Ionophores; Jurkat Cells; Membrane Glycoproteins; Phytohemagglutinins; T-Lymphocytes; T-Lymphocytes, Regulatory; Tetradecanoylphorbol Acetate

We examined the mechanism of 17beta-estradiol (estrogen)-mediated inhibition of apoptosis in C6 (rat glioma) cells following exposure to hydrogen peroxide (H(2)O(2)). Cells were preincubated with 4 microM estrogen for 2 h and then exposed to 100 microM H(2)O(2) for 24 h. Exposure to H(2)O(2) caused significant increases in intracellular calcium (Ca(2+)), as determined by fura-2, which was attenuated by preincubation with estrogen. H(2)O(2) and ionomycin caused cell death in a dose-dependent manner, as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Preincubation with estrogen restored viability in cells exposed to H(2)O(2) but not in cells exposed to ionomycin. Western blot analysis showed an increase in Bax/Bcl-2 ratio, calpain activity, and caspase-3 activity following treatment with H(2)O(2), and estrogen pretreatment decreased levels of all three. Cell morphology, as evaluated by Wright staining, indicated apoptosis in cells treated with H(2)O(2), and pretreatment with estrogen reduced apoptosis. Results from MTT and Wright staining were further supported by the terminal deoxyribonucleotidyl transferase (TdT)-mediated dUTP Nick End Labeling (TUNEL) assay. These results indicate a role for estrogen in preventing apoptosis in C6 glial cells exposed to H(2)O(2). Our results suggest that estrogen may have a protective role in minimizing glial cell apoptosis in neurological diseases such as demyelinating disease or central nervous system trauma.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Calcium; Calpain; Carrier Proteins; Caspase 3; Caspases; Cell Line; Cell Survival; Dose-Response Relationship, Drug; Estrogens; Fura-2; Glioma; Hydrogen Peroxide; In Situ Nick-End Labeling; Ionomycin; Ionophores; Oxidative Stress; Peptide Fragments; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Receptors, Estrogen; Spectrin; Tetrazolium Salts; Thiazoles; Time Factors

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

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

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 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

Changes in intracellular calcium concentration are important in mediating a wide variety of physiological responses. Recently there has been renewed interest in the use of aequorin, a protein from jellyfish that emits light when calcium is bound, to measure calcium levels in cells. We have loaded populations of cells from the human glioma line, U373MG, with aequorin. Lysis of aequorin-loaded but not control cells with detergent resulted in a luminescence signal that was dependent on extracellular calcium. Aequorin-loaded cells responded to substance P, histamine, or the calcium ionophore, ionomycin, with an increase in luminescence. Signals in response to detergent, ionomycin, or substance P could be detected up to 48 h after cells were loaded with aequorin. Other neurokinin-1 agonists but not agonists at neurokinin-2 or neurokinin-3 receptors produced luminescence signals. Neurokinin-1 antagonists inhibited the substance P-induced signal. The aequorin-loading procedure worked well with U373MG cells but not with AR42J, CHO, IMR-90, or WI-38 cells.

Topics: Aequorin; Calcium; Glioma; Histamine; Humans; Ionomycin; Ionophores; Luminescent Measurements; Receptors, Neurokinin-1; Reproducibility of Results; Substance P; 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

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

1. The pharmacological characteristics of a putative Ca2+ activated K+ channel (IKCa channel) in rat glioma C6 cells were studied in the presence of the Ca2+ ionophore, ionomycin and various K+ channel blockers, 86Rb+ being used as a radioisotopic tracer for K+. 2. The resting 86Rb+ influx into C6 cells was 318 +/- 20 pmol s-1. The threshold for ionomycin activation of 86Rb+ influx was approx. 100 nM. At ionomycin concentrations above the activation threshold, the initial rate of 86Rb+ influx was proportional to ionophore concentration. Ionomycin-activated 86Rb+ flux was saturable (EC50 = 0.62 +/- 0.03 microM) and was not inhibited by ouabain. 3. Intracellular Ca2+ increased within 30 s from a basal level of 42 +/- 2 nM to 233 +/- 17 nM, after addition of 2 microM ionomycin. During this period, intracellular pH fell from 7.03 +/- 0.04 to 6.87 +/- 0.03 and the cell hyperpolarized from -34 +/- 10 mV to -76 +/- 2 mV. 4. Single channel conductance measurements on inside-out patches in physiological K+ solutions identified a 14 +/- 3 pS CA(2+)-activated K+ current between -25 mV and +50 mV. In symmetrical (100 mM) K+, the single channel conductance was 26 pS. 5. Externally applied quinine (IC50 = 0.12 +/- 0.34 mM) and tetraethylammonium chloride (IC50 = 10 +/- 1.9 mM) inhibited 86Rb+ influx into C6 cells in a concentration-dependent manner. Charybdotoxin (IC50 = 0.5 +/- 0.02 nM) and iberiotoxin (IC50 = 800 +/- 150 nM), as well as the crude venoms from the scorpions Leiurus quinquestriatus and Mesobuthus tamulus, also inhibited 86Rb+ influx. In contrast, apamin and toxin I had no inhibitory effects on 86Rb+ flux. A screen of fractions from cation exchange h.p.l.c. of Mesob. tamulus venom revealed the presence of at least four charybdotoxin-like peptides. One of these was iberiotoxin; the other three are novel toxins. 6. The ionomycin-activated 86Rb+ influx into rat C6 glioma cells has proved to be a valuable pharmacological assay for the screening of toxins and crude venoms which modify intermediate conductance, Ca2+ activated K+ channel activity.

Topics: Animals; Brain Neoplasms; Calcium; Charybdotoxin; Chromatography, High Pressure Liquid; Enzyme Inhibitors; Glioma; Hydrogen-Ion Concentration; Ionomycin; Ionophores; Membrane Potentials; Ouabain; Potassium Channels; Rats; Rubidium; Rubidium Radioisotopes; Scorpion Venoms; Tumor Cells, Cultured

Ionomycin stimulated membrane-associated protein kinase Cs (PKCs) activity in C6 rat glioma cells as much as the potent PKCs stimulator 12-O-tetradecanoyl phorbol 13-acetate (TPA). However, while TPA, as expected, powerfully stimulated the phosphorylation of the PKCs' 85-kDa myristoylated alanine-rich protein kinase C substrate (MARCKS) protein, ionomycin unexpectedly did not. Instead, ionomycin reduced the basal MARCKS phosphorylation. Pretreating the glioma cells with ionomycin prevented TPA-stimulated PKCs from phosphorylating the MARCKS protein. The stimulation of membrane PKCs activity and the prevention of MARCKS phosphorylation by ionomycin required external Ca2+ because they were both abolished by adding 5 mM EGTA to the culture medium. Recently (Chakravarthy, B. R., Isaacs, R. J., Morley, P., Durkin, J. P., and Whitfield, J. F. (1995) J. Biol. Chem. 270, 1362-1368), we proposed that Ca2+ x calmodulin complexes block MARCKS phosphorylation by the activated PKCs in keratinocytes stimulated by raising the external Ca2+ concentration. In the present experiments calmodulin prevented MARCKS phosphorylation by TPA-stimulated PKCs in glioma cell lysates, and this blockade was lifted by a calmodulin antagonist, the calmodulin-binding domain peptide. But, physiologically more significant, pretreating intact glioma cells with a cell-permeable calmodulin antagonist, calmidazolium, prevented ionomycin from blocking MARCKS phosphorylation by PKCs in unstimulated and TPA-stimulated cells. The effect of ionomycin on MARCKS phosphorylation was not due to the stimulation of Ca2+ x calmodulin-dependent phosphoprotein phosphatase, calcineurin, because cyclosporin A, a potent inhibitor of this phosphatase, did not stop ionomycin from preventing MARCKS phosphorylation. The ability of ionomycin to prevent TPA-stimulated PKCs from phosphorylating MARCKS depended on whether ionomycin was added before, with, or after TPA. Maximum blockade occurred when ionomycin was added before TPA but was less effective when added with or after TPA. These results indicate that Ca2+ x calmodulin can profoundly affect PKCs' signaling at the substrate level.

Topics: Amino Acid Sequence; Animals; Calcium; Calmodulin; Cattle; Glioma; Intracellular Signaling Peptides and Proteins; Ionomycin; Keratinocytes; Membrane Proteins; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Myristoylated Alanine-Rich C Kinase Substrate; Phosphorylation; Protein Kinase C; Proteins; Rats; Tetradecanoylphorbol Acetate; 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

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

The effects of Ca2+ on the translocation of conventional and new protein kinase C isozymes in intact cells were studied by using C6 glioma cells as a model system. Two conditions which monitor intracellular Ca2+ were performed: one is extracellular Ca(2+)-depletion by treating the cells with physiological saline solution (PSS) without Ca2+ but containing 0.5 mM EGTA, the other is treating the cells with 1 microM ionomycin to induce Ca(2+)-influx. In addition, the TPA and endothelin-1 induced translocations of conventional and new PKC isozymes under these two conditions were also comparatively studied. When the intact cells were treated with Ca(2+)-free, EGTA containing PSS, the membrane-bound conventional PKC alpha (cPKC alpha) was greatly reduced and cytosolic cPKC alpha was slightly increased. However, neither membrane bound nor cytosolic new PKC delta (nPKC delta) was affected by extracellular Ca(2+)-depletion. On the other hand, when the cells were treated with 1 microM ionomycin, the translocation of cPKC alpha itself was observed while nPKC delta was not affected. In extracellular Ca(2+)-depletion, the translocation of cPKC alpha induced by 100 nM TPA still occurred although the extent of translocation was smaller than that induced by TPA under normal Ca2+ conditions; however, that induced by 30 nM ET-1 was blocked. After the cells were treated with 1 microM ionomycin, the translocation of cPKC alpha induced by 30 nM TPA was further increased compared to 1 microM ionomycin or 30 nM TPA alone, while that induced by ET-1 was only slightly further increased. All these results suggested that in intact cells, the activation of cPKC alpha was operated by both the intracellular Ca2+ level and diacylglycerol and that of nPKC delta was operated by diacylglycerol alone as predicted by their properties from purified enzyme or cDNA. In addition, the translocation of cPKC alpha induced by the natural activator ET-1 seemed to be more dependent on Ca2+ than TPA in intact cells.

Topics: Biological Transport; Calcium; Endothelins; Enzyme Activation; Glioma; Ionomycin; Isoenzymes; Protein Kinase C; Tetradecanoylphorbol Acetate; 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

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

Interactions between endothelin-1 (ET)-induced phosphoinositide (PI) hydrolysis and agents that increase Ca2+ influx (i.e. A23187 and ionomycin) or induce depolarization (i.e. KCl) were investigated using C6 glioma. A23187 dose-dependently potentiated ET (30 nM)- and ATP (100 microM)-induced [3H]inositol phosphate (IP) accumulation. This potentiation was associated with an increase in the maximal stimulation elicited by both ET and ATP but their EC50 values were unchanged. This effect of A23187 occurred at concentrations that did not affect basal PI turnover; i.e. 10 nM-3 microM. Ionomycin within the range of 1 nM-1 microM also significantly enhanced ET-induced PI breakdown and this effect was associated with an increase of [Ca2+]i. KCl in a concentration-dependent manner (14.7-54.7 mM) markedly inhibited PI breakdown elicited by ET and ATP, but had much less inhibition on basal activity and no effect on A23187- and ionomycin-induced responses. In parallel, KCl added before or after ET, sharply attenuated the increase of ET-induced [Ca2+]i but did not affect basal level or ionomycin-induced [Ca2+]i response. Neither the potentiation by A23187 nor the inhibition by KCl of ET-induced PI turnover was observed in cultured cerebellar astrocytes. Our results suggest that the cell type-specific regulation by Ca2+ ionophores and KCl on ET-induced PI metabolism is closely related to perturbation of [Ca2+]i.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Calcimycin; Calcium; Dose-Response Relationship, Drug; Endothelins; Glioma; Inositol; Inositol Phosphates; Ionomycin; Kinetics; Phosphatidylinositols; Potassium Chloride; Time Factors; Tumor Cells, Cultured

Patients harboring a malignant brain tumor have been described as being highly immunosuppressed, as evidenced by reduced numbers of T cells and the decreased ability of their lymphocytes to produce interleukin-2 (IL-2). In order to determine whether an intrinsic abnormality exists in the T lymphocytes of glioma patients and to evaluate what role corticosteroids may play in glioma-associated immunosuppression, in vitro T cell proliferative function in the presence of recombinant IL-2 (rIL-2) was examined in age-matched groups of normal control subjects, steroid-free patients with glial tumors, steroid-dependent patients with glial tumors, and steroid-dependent patients with nonglial cerebral tumors. The results demonstrated that, when enriched T cell populations of all brain-tumor patients were stimulated with rIL-2 and phytohemagglutinin (PHA), there were no statistically significant differences between any groups. In contrast, when T cell populations were stimulated with mitogenic combinations of phorbol ester, calcium ionophore, and rIL-2, those from steroid-dependent patients with glial tumors had a significantly lower response than those from normal control subjects, suggesting that a population of T cells capable of responding to phorbol ester/ionomycin and not PHA stimulation is inhibited by corticosteroid therapy in glioma patients. In addition, T cells of four brain-tumor patient/age-matched control subject pairs were stimulated with either phorbol ester/ionomycin or PHA for 24 hours; three of the four patients expressed low-affinity IL-2 receptor levels as high or higher than their respective control subjects, suggesting that IL-2 receptor expression in these patients may be quantitatively normal once the T cell number is corrected. Taken together, these results show that the decreased PHA responsiveness that has been previously reported in lymphocytes of glioma patients is not due to a cellular abnormality within the potentially responsive cells, but rather reflects the reduced proportion of T cells within their peripheral blood which, as a consequence, reduces the level of IL-2 production attained upon activation.

Topics: Adrenal Cortex Hormones; Adult; Aged; Brain Neoplasms; Female; Glioma; Humans; Immune Tolerance; Interleukin-2; Ionomycin; Lymphocyte Activation; Male; Middle Aged; Phorbol 12,13-Dibutyrate; Phytohemagglutinins; Receptors, Interleukin-2; Recombinant Proteins; T-Lymphocytes; Tumor Cells, Cultured

Treatment of rat glioma C6 cells with a beta-adrenergic agonist leads to a rise in cAMP level and a subsequent change in cell morphology from an epithelial to an astrocyte type of appearance. This morphological change is reverted by the addition of thrombin. In 10-15 min the cells acquire their normal epithelial morphology. The reversion by thrombin is inhibited by hirudin, but not by antithrombin III (an inhibitor of the proteolytic action of thrombin). Using the intracellular Ca2(+)-indicator fura-2, we observed that the addition of thrombin to the glioma cells generated a Ca2(+)-signal which was inhibited by pretreatment of the cells with hirudin or with 1 mM neomycin. These results suggest that thrombin uses the phospholipid-inositol pathway to counteract the morphological response, which was induced by activation of the cAMP pathway.

Topics: Animals; Antithrombin III; Astrocytes; Blood; Calcium; Culture Media; Epithelial Cells; Glioma; Hirudins; Ionomycin; Isoproterenol; Microscopy, Electron, Scanning; Neomycin; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Signal Transduction; Tetradecanoylphorbol Acetate; Thrombin; Tumor Cells, Cultured

Ca2+-activated K+ channels in rat glioma C6 cells were investigated using monolayers of these cells in petri dishes. The ion flux through the channels was studied with 86Rb+ after addition of a Ca2+-ionophore to the incubation medium. Both the influx and efflux of 86Rb+ through these Ca2+-activated K+ channels were inhibited by the general anesthetic halothane (at clinical concentrations). Other volatile anesthetics such as isoflurane, enflurane and methoxyflurane also inhibited the Ca2+-activated K+ channels at clinical concentrations. Inhibition of these channels by general anesthetics could have profound effects on signal transmission in the brain.

Topics: Anesthetics; Animals; Calcimycin; Calcium; Cell Line; Ethers; Glioma; Halothane; Ionomycin; Potassium Channels; Rats; Rubidium Radioisotopes; Tumor Cells, Cultured

A study was made of the 86Rb+ influx and efflux through Ca2+-activated K+ channels of intact rat glioma C6 cells after addition of a Ca2+ ionophore to the incubation medium. Half-maximal activation of the channels was obtained at a cytoplasmic Ca2+ concentration of approximately 400 nM. The 86Rb+ ion flux through the Ca2+-activated K+ channels was insensitive to apamin, but was inhibited by low concentrations of charybdotoxin (IC50 = 1.6 nM). This is the first evidence for the presence of charybdotoxin-sensitive Ca2+-activated K+ channels in glial cells.

Topics: Animals; Calcimycin; Calcium; Charybdotoxin; Ethers; Glioma; Ionomycin; Potassium Channels; Rubidium; Rubidium Radioisotopes; Scorpion Venoms; Tumor Cells, Cultured

It has been shown recently that astroglial cells of the mammalian CNS possess receptors for neurotransmitters. In order to analyze what sequences of cellular events occur upon activation of these glial receptors, we utilized a 5-HT receptor in a rat clonal cell of glial origin as a model system. When the C6BU-1 glioma cells were exposed to 5-HT, the cytosolic Ca2+ concentration ([Ca2+]i) was elevated and the cellular content of cGMP was increased in a dose-dependent manner. 5-HT receptor antagonists and a Ca2+ entry blocker suppressed the increases in both [Ca2+]i and cGMP. The magnitude of the cGMP increment depended on the environmental Ca2+ concentration and was totally blocked by Ca2+ depletion. Application of a Ca2+ ionophore increased [Ca2+]i and cGMP. There was a tendency for extremely high [Ca2+]i to suppress the cGMP increment. On the contrary, membrane-permeable cyclic nucleotide analogs failed to increase [Ca2+]i. These results suggest that the following sequence of events occurs in 5-HT-induced C6BU-1 cells: activation of 5-HT receptors, Ca2+ influx, a rise in [Ca2+]i, activation of guanylate cyclase, and, finally, activation of cyclic nucleotide phosphodiesterase.

Topics: Animals; Astrocytes; Brain Neoplasms; Calcium; Calcium Channel Blockers; Clone Cells; Cyclic GMP; Cytosol; Dose-Response Relationship, Drug; Ethers; Glioma; Ionomycin; Nucleotides, Cyclic; Rats; Serotonin; Serotonin Antagonists; Time Factors