calcimycin and Astrocytoma

The calcium binding protein, calbindin-D28k is normally present in neurons. Recently we reported that brain injury and tumor necrosis factors (TNFs) induce calbindin-D28k in astrocytes. TNF-treated calbindin expressing astrocytes were resistant to acidosis and calcium ionophore toxicity, suggesting that calbindin may have a cytoprotective role in astrocytes in the injured brain (M.P. Mattson, B. Cheng, S.A. Baldwin, V.L. Smith-Swintosky, J. Keller, J. Geddes, Scheff, J.W., Christakos, S., Brain injury and tumor necrosis factors induce calbindin-D28k in astrocytes: evidence for a cytoprotective response, J. Neurosci. Res., 42 (1995) 257). In order to obtain direct evidence for a role of calbindin, using the eukaryotic expression vector pREP4, rat calbindin-D28k was stably expressed in C6 rat astocytoma glial cells. Cytotoxicity in response to calcium ionophore or amyloid beta-peptide (which accumulates in the brain in Alzheimer's disease and has been reported to be neurotoxic) was measured by MTT reduction in vector transfected cells and in calbindin transfected clones. Stably expressed calbindin resulted in increased cell survival in the presence of calcium ionophore (1-10 microM) or amyloid beta-peptide (10-100 microM). In addition, the calcium ionophore or amyloid beta-peptide mediated rise in intracellular calcium in vector transfected cells was significantly attenuated in calbindin expressing cells. Apoptotic cell death was detected by the Hoechst method in vector transfected C6 glial cells treated with calcium ionophore or beta-amyloid (34-36% apoptotic cells/culture). The number of apoptotic nuclei was significantly attenuated in similarly treated calbindin-D28k transfected clones (10-13% apoptotic cells/culture; p<0.01). Our results support the involvement of calcium fluxes in apoptosis and suggest that calbindin-D28k, by buffering calcium, can suppress death in apoptosis susceptible cells in the central nervous system.

Topics: Amyloid beta-Peptides; Animals; Apoptosis; Astrocytes; Astrocytoma; Calbindin 1; Calbindins; Calcimycin; Calcium; Cell Division; Gene Expression; Ionophores; Mitochondria; Nerve Tissue Proteins; Phenotype; Plasmids; Rats; S100 Calcium Binding Protein G; Transfection; Tumor Cells, Cultured

Since maitotoxin, a potent marine toxin, is known to cause not only Ca2+ influx but also phosphoinositide hydrolysis, we investigated the Ca2+ dependency of maitotoxin-induced phosphoinositide hydrolysis in 1321N1 human astrocytoma cells. Maitotoxin elicited inositol 1,4,5-trisphosphate accumulation in a time-dependent manner. In [3H]inositol-labeled cells, maitotoxin stimulated phosphoinositide hydrolysis in an extracellular Ca2+-dependent manner. Maitotoxin also caused an intracellular Ca2+ elevation, which was abolished by an intracellular Ca2+ chelater BAPTA-AM. Interestingly, maitotoxin still caused phosphoinositide hydrolysis in the BAPTA-AM-treated cells. These results indicate that maitotoxin-induced phosphoinositide hydrolysis is dependent on extracellular but not intracellular Ca2+ in 1321N1 human astrocytoma cells.

Topics: Astrocytoma; Brain Neoplasms; Calcimycin; Calcium; Chelating Agents; Egtazic Acid; Extracellular Space; Humans; Hydrolysis; Inositol 1,4,5-Trisphosphate; Ionophores; Marine Toxins; Oxocins; Phosphatidylinositols; Tumor Cells, Cultured

In this study, we have identified novel splice variants of the Na+ channel alpha subunit mRNA from cultured rat astrocytes and neuroblastoma cells. These splice variants are characterized by premature truncation or deletion of a segment in the third domain of the Na+ channel alpha subunit. The expression of three splice variants was upregulated by exposure to 1 mM dibutyryl cAMP in spinal cord astrocytes but not in cerebral astrocytes and in B50 and B104 neuroblastoma cells. The calcium ionophore 1 microM A23187, did not influence the expression of splice variants in either astrocytes or neuroblastoma cells. These findings suggest that spinal cord astrocytes may maintain a unique regulatory pathway that participates in the control of Na+ channel mRNA expression.

Topics: Alternative Splicing; Amino Acid Sequence; Animals; Animals, Newborn; Astrocytes; Astrocytoma; Brain; Bucladesine; Calcimycin; Cells, Cultured; Genetic Variation; Macromolecular Substances; Models, Molecular; Molecular Sequence Data; Polymerase Chain Reaction; Protein Conformation; Rats; Rats, Sprague-Dawley; Sodium Channels; Spinal Cord; Up-Regulation

A major source of inflammatory cytokines in the measles virus (MV)-infected brain are astrocytes, which produce a variety of soluble mediators including interferons-alpha/beta (IFN-alpha/beta), interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6). Using the MV-strain Edmonston (ED) and the recombinant MV-strain MGV in which the MV-envelope proteins H and F have been replaced by the vesicular stomatitis virus (VSV) envelope protein G, we investigated IL-6 induction in human U-251 astrocytoma cells in the presence and absence of a MV-specific receptor (CD46) interaction. The CD46-MV interaction did not inhibit the induction of cytokines. Similar multiplicities of infection of MGV induced generally lower levels of IL-6 than MV-ED. UV-inactivated replication-incompetent MV-ED induced low levels of IL-6. In contrast, MGV did not induce IL-6 after inactivation with UV light, indicating that the MV-ED-receptor interaction or the uptake of viral particles by membrane fusion induced IL-6, whereas interaction with the VSV-G receptor and uptake of viral particles by endocytosis did not induce IL-6. Crosslink of the MV-receptor CD46 with antibodies and treatment of cells with purified viral glycoproteins led to the induction of small but significant amounts of IL-6. Our data suggest that triggering of CD46 and associated protein kinases can lead to the induction of low levels of IL-6, whereas the replication of the negative strand RNA virus constitutes the major stimulus leading to the synthesis of high levels of IL-6 in astrocytes.

Topics: Antigens, CD; Astrocytes; Astrocytoma; Calcimycin; Carrier Proteins; Gene Expression Regulation; Hemagglutinins, Viral; Humans; Interleukin-6; Intracellular Signaling Peptides and Proteins; Measles virus; Membrane Cofactor Protein; Membrane Glycoproteins; Poly I-C; Receptor Aggregation; Receptors, Virus; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Ultraviolet Rays; Vesicular stomatitis Indiana virus; Viral Envelope Proteins; Viral Fusion Proteins; Virus Replication

The cholinergic agonist carbachol induced the release of arachidonic acid in the 1321N1 astrocytoma cell line, and this was blocked by atropine, suggesting the involvement of muscarinic receptors. To assess the mechanisms of signalling involved in the response to carbachol, a set of compounds characterized by eliciting responses through different mechanisms was tested. A combination of 4beta-phorbol 12beta-myristate 13alpha-acetate and thapsigargin, an inhibitor of endomembrane Ca2+-ATPase that induces a prolonged elevation of cytosolic Ca2+ concentration, induced an optimal response, suggesting at first glance that both protein kinase C (PKC) and Ca2+ mobilization were involved in the response. This was consistent with the observation that carbachol elicited Ca2+ mobilization and PKC-dependent phosphorylation of cytosolic phospholipase A2 (cPLA2; phosphatide sn-2-acylhydrolase, EC 3.1.1.4) as measured by a decrease in electrophoretic mobility. Nevertheless, the release of arachidonate induced by carbachol was unaltered in media containing decreased concentrations of Ca2+ or in the presence of neomycin, a potent inhibitor of phospholipase C which blocks phosphoinositide turnover and Ca2+ mobilization. Guanosine 5'-[gamma-thio]triphosphate added to the cell-free homogenate induced both [3H]arachidonate release and cPLA2 translocation to the cell membrane fraction in the absence of Ca2+, thus suggesting the existence of an alternative mechanism of cPLA2 translocation dependent on G-proteins and independent of Ca2+ mobilization. From the combination of experiments utilizing biochemical and immunological tools the involvement of cPLA2 was ascertained. In summary, these data indicate the existence in the astrocytoma cell line 1321N1 of a pathway involving the cPLA2 which couples the release of arachidonate to the occupancy of receptors for a neurotransmitter, requires PKC activity and G-proteins and might operate in the absence of Ca2+ mobilization.

Topics: Arachidonic Acid; Astrocytoma; Brain Neoplasms; Calcimycin; Calcium; Carbachol; Carcinogens; Cytosol; Enzyme Activation; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Ionophores; Phospholipases A; Phospholipases A2; Protein Kinase C; Receptors, Muscarinic; Tetradecanoylphorbol Acetate; Thapsigargin; Tumor Cells, Cultured

1. ATP, bradykinin (BK), and A-23187 activated the generation of prostaglandin (PG) E2 and thromboxane (TX) B2 in rabbit astrocytes, but not in human astrocytoma cells (1321N1). 2. In human astrocytoma cells, ATP, BK, and A-23187 could not release [3H]arachidonic acid (AA) from [3H]AA-labeled cells and exogenous AA was not converted to TXB2 and PGE2, suggesting the lack of phospholipase (PL) A2 and cyclooxygenase activities in 1321N1 human astrocytoma cells, although they express TXA2 receptors. 3. In rabbit astrocytes, ATP and BK, but not A-23187, showed increased accumulation of inositol phosphates, indicating that an increase in intracellular Ca2+ concentration alone would not be enough to activate PLC. Furthermore, indomethacin, a cyclooxygenase inhibitor, partially attenuated ATP-induced phosphoinositide hydrolysis, indicating that cyclooxygenase product(s) would secondarily activate PLC in response to ATP.

Topics: Adenosine Triphosphate; Animals; Arachidonic Acid; Astrocytes; Astrocytoma; Bradykinin; Calcimycin; Cells, Cultured; Dose-Response Relationship, Drug; Female; Humans; Male; Phosphatidylinositols; Rabbits; Tumor Cells, Cultured

Substance P (SP) and lipopolysaccharide (LPS) stimulated interleukin-6 (IL-6) gene expression, as well as IL-6 protein secretion in the human astrocytoma cell line U373 MG. Staurosporine, an inhibitor of protein kinase C (PKC), entirely blocked SP- but not LPS-induced IL-6 release. In addition, the down regulation of PKC inhibited the SP response and only marginally altered LPS activation. Differently from SP, LPS-induced IL-6 release was markedly reduced by W7, a calmodulin antagonist. Moreover, SP interacted in a synergistic manner with LPS. Thus, neural (SP) and bacterial (LPS) mediators stimulate U373 MG IL-6 release via distinct, though not antagonistic, activation pathways.

Topics: Alkaloids; Astrocytoma; Bucladesine; Calcimycin; Cholera Toxin; Humans; Indomethacin; Interleukin-6; Lipopolysaccharides; Receptors, Neurokinin-1; Staurosporine; Substance P; Sulfonamides; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Bradykinin causes a concentration-dependent, transient rise in intracellular Ca2+ and a sustained inhibition of forskolin-, dopamine- and 5'-N-ethyl-carboxamidoadenosine (NECA)-stimulated cAMP accumulation in D384 astrocytoma cells. Chelation of intracellular calcium abolished bradykinin's inhibitory effect on cAMP accumulation. Chelating extracellular Ca2+ did not block the initial, but eliminated the sustained inhibition of cAMP accumulation. Increasing Ca2+ influx by calcium ionophore A23187 caused a concentration-dependent inhibition of stimulated cAMP accumulation. A hydroquinone derivative 2,5-di(tert-butyl)-1,4-benzohydroquinone (tBuBHQ), which inhibits microsomal Ca2+ sequestration, did not mimic the effect of bradykinin, although it increased [Ca2+]i even more than A23187 did. The inhibitory effect of bradykinin was not mediated by Ca2+/CaM-dependent stimulation of phosphodiesterase (PDE). Forskolin-stimulated adenylyl cyclase activity was inhibited by Ca2+ (10(-7) to 10(-3) M), both in ethyleneglycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) washed and native D384 plasma membranes. This effect was not altered by calmodulin (CaM) or CaM-antagonists. Bradykinin treatment, which attenuates cAMP accumulation in intact cells, did not do so in plasma membranes. These findings suggest that bradykinin-induced inhibition of cAMP formation in D384 cells requires mobilization of [Ca2+]i and subsequent entry of Ca2+ which directly interacts with a component of the adenylyl cyclase system.

Topics: 1-Methyl-3-isobutylxanthine; Adenosine; Adenosine-5'-(N-ethylcarboxamide); Adenylyl Cyclase Inhibitors; Astrocytoma; Bradykinin; Brain Neoplasms; Calcimycin; Calcium; Calmodulin; Colforsin; Cyclic AMP; Dopamine; Egtazic Acid; Humans; Hydroquinones; Inositol 1,4,5-Trisphosphate; Neoplasm Proteins; Pyrrolidinones; Rolipram; Signal Transduction; Tumor Cells, Cultured

Activation of muscarinic receptors in human astrocytoma (1321N1) cells stimulates phosphoinositide metabolism and calcium mobilization. The muscarinic effect on phosphoinositide turnover is evidenced by increased formation of [3H]inositol 1-phosphate (Ins1P) and by increased [3H]inositol incorporation into PtdIns. The muscarinic effect on calcium mobilization is seen as a large increase in undirectional 45Ca2+ efflux from cells equilibrated with 45Ca2+ and a small increase in unidirectional 45Ca2+ influx. A series of muscarinic agonists was used to explore the relationship between phosphoinositide metabolism and unidirectional 45Ca2+ efflux. The maximal increases in [3H]Ins1P formation produced by carbachol and acetylcholine are similar and are much larger than those caused by oxotremorine and pilocarpine. The effects of these agonists on 45Ca2+ efflux are similar: carbachol and acetylcholine cause equivalent maximal increases in the rate of 45Ca2+ efflux whereas oxotremorine and pilocarpine cause submaximal 45Ca2+ efflux responses. The Kact values of carbachol and acetylcholine for stimulation of [3H]Ins1P formation are 40 microM and 1.5 microM, respectively. These values are only 2- to 3-fold higher than the respective Kact values for stimulating 45Ca2+ efflux. The finding that each of the muscarinic agonists tested has nearly identical efficacy and similar potency for stimulating [3H]Ins1P formation and 45Ca2+ efflux supports the idea that hormonal stimulation of phosphoinositide hydrolysis leads to calcium mobilization.

Topics: Astrocytoma; Atropine; Biological Transport, Active; Calcimycin; Calcium; Carbachol; Cell Line; Humans; Kinetics; Phosphatidylinositols; Receptors, Muscarinic; Tubocurarine

Topics: Animals; Astrocytoma; Calcimycin; Cells, Cultured; Cyclic AMP; Isoproterenol; Neoplasm Proteins; Phosphodiesterase Inhibitors; Phosphorylases; Rats; Receptors, Adrenergic; Receptors, Adrenergic, beta