sq-23377 and Brain-Neoplasms

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

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

The 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

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

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

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

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

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