ryanodine and Astrocytoma

The relationship between nitric oxide (NO) and intracellular Ca2+ in hypoxic-ischemic brain damage is not known in detail. Here we used rat striatal slices perfused under low-oxygen and Ca2+-free conditions and cultured human astrocytoma cells incubated under similar conditions as models to study the dynamics of intracellular NO and Ca2+ in hypoxia-induced tissue damage. Exposure of rat striatal slices for 70 min to low oxygen tension elicited a delayed and sustained increase in the release of 45Ca2+. This was potentiated by the NO donors sodium nitroprusside (SNP) and spermine-NO and inhibited by N-omega-nitro-L-arginine methyl ester (L-NAME) or by the NO scavenger 2-phenyl-4,4,5,5 tetramethylimidazoline-1-oxyl-3-oxide (PTIO). A membrane-permeant form of heparin in combination with either ruthenium red (RR) or ryanodine (RY) also inhibited 45Ca2+ release. In human astrocytoma U-373 MG cells, hypoxia increased intracellular Ca2+ concentration ([Ca2+]i) by 67.2 +/- 13.1% compared to normoxic controls and this effect was inhibited by L-NAME, PTIO or heparin plus RR. In striatal tissue, hypoxia increased NO production and LDH release and both effects were antagonized by L-NAME. Although heparin plus RR or RY antagonized hypoxia-induced increase in LDH release they failed to counteract increased NO production. These data therefore indicate that NO contributes to hypoxic damage through increased intracellular Ca2+ mobilization from endoplasmic reticulum and suggest that the NO-Ca2+ signalling might be a potential therapeutic target in hypoxia-induced neuronal degeneration.

Topics: Animals; Anticoagulants; Astrocytoma; Calcium; Cell Line, Tumor; Corpus Striatum; Cyclic N-Oxides; Dose-Response Relationship, Drug; Drug Combinations; Drug Interactions; Enzyme Inhibitors; Free Radical Scavengers; Fura-2; Heparin; Humans; Hydro-Lyases; Hypoxia; Imidazoles; In Vitro Techniques; Intracellular Space; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Perfusion; Rats; Rats, Sprague-Dawley; Ruthenium; Ryanodine

Numerous studies show that intracellular calcium controls the migration rate of different mobile cell types. We studied migrating astrocytoma cells from two human cell lines, U-87MG and A172, in order to clarify the mechanisms by which calcium potentially influences cell migration. Using the wound-healing model to assay migration, we showed that four distinct components of migration could be distinguished: (i) a Ca(2+)/serum-dependent process; (ii) a Ca(2+)-dependent/serum-independent process; (iii) a Ca(2+)/serum-independent process; (iv) a Ca(2+)-independent/serum-dependent process. In U-87MG cells which lack a Ca(2+)-dependent/serum-independent component, we found that intracellular Ca(2+) oscillations are involved in Ca(2+)-dependent migration. Removing extracellular Ca(2+) greatly decreased the frequency of migration-associated Ca(2+) oscillations. Furthermore, non-selective inhibition of Ca(2+) channels by heavy metals such as Cd(2+) or La(3+) almost completely abolished changes in intracellular Ca(2+) observed during migration, indicating an essential role for Ca(2+) channels in the generation of these Ca(2+) oscillations. However, specific blockers of voltage-gated Ca(2+) channels, including nitrendipine, omega-conotoxin GVIA, omega-conotoxin MVIIC or low concentrations of Ni(2+) were without effect on Ca(2+) oscillations. We examined the role of internal Ca(2+) stores, showing that thapsigargin-sensitive Ca(2+) stores and InsP(3) receptors are involved in Ca(2+) oscillations, unlike ryanodine-sensitive Ca(2+) stores. Detailed analysis of the spatio-temporal aspect of the Ca(2+) oscillations revealed the existence of Ca(2+) waves initiated at the leading cell edge which propagate throughout the cell. Previously, we have shown that the frequency of Ca(2+) oscillations was reduced in the presence of inhibitory antibodies directed against beta3 integrin subunits. A simple model of a Ca(2+) oscillator is proposed, which may explain how the generation of Ca(2+) oscillations is linked to cell migration.

Topics: Astrocytoma; Calcium; Calcium Channel Blockers; Cell Movement; Culture Media; Humans; Neoplastic Cells, Circulating; Ryanodine; Thapsigargin; Tumor Cells, Cultured

In human U373 MG astrocytoma cells, histamine and substance P stimulated similar peak increases in intracellular free calcium concentrations ([Ca2+]i), as measured by single cell imaging of Fura-2 fluorescence. Best-fit EC50 values for the peak Ca2+ response were 1.86 microM for histamine and 0.93 nM for substance P. The histamine Ca2+ response was manifest as either a series of repetitive spikes, or, at higher concentrations, a peak followed by a lower plateau level of Ca2+. In contrast, the substance P response became more transient at higher agonist concentrations. Substance P (10 nM) stimulated a biphasic increase in levels of inositol (1,4,5) trisphosphate (Ins(1,4,5)P3) with a peak of 97 +/- 5 pmoles/mg protein at 10 s. In contrast, the Ins(1,4,5)P3 response to 100 microM histamine was only marginally above basal levels of around 12 pmoles/mg protein. However, concentrations of histamine and substance P giving similar Ins(1,4,5)P3 responses produce similar peak increases in [Ca2+]i. HPLC analysis indicated that histamine stimulated the production of [3H]-Ins(1,4,5)P3 and its metabolites, although the magnitude of response was smaller than that observed with substance P. The initial Ca2+ responses to histamine and substance P did not require the presence of extracellular Ca2+. The Ca2+ response to histamine was unaffected by treatment with ryanodine, and was shifted to areas of lower agonist concentration by thimerosal. These results demonstrate that extremely small increases in Ins(1,4,5)P3 can stimulate large increases in [Ca2+]i in U373 MG cells, and suggest a marked redundancy for Ins(1,4,5)P3 production in the Ca2+ signalling pathway.

Topics: Astrocytoma; Calcium Signaling; Chromatography, High Pressure Liquid; Histamine; Humans; Inositol 1,4,5-Trisphosphate; Ryanodine; Substance P; Temperature; Thimerosal; Tumor Cells, Cultured