sq-23377 and 3--4--dichlorobenzamil

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

Changes in the concentration of cytosolic free calcium ([Ca++]i) play fundamental roles in the initiation and regulation of many neuronal processes. Altered regulation of [Ca++]i has been implicated in the action of some anesthetics. We investigated the effects of nitrous oxide (N2O) on Ca++ mobilization and membrane potential in the human neuroblastoma cell line SK-N-SH. [Ca++]i was monitored by fluorescence spectrophotometry of cells loaded with fura-2 or fluo-3. N2O reversibly suppressed carbachol-stimulated increases in [Ca++]i. N2O also inhibited increases in [Ca++]i induced by calcium ionophore or depolarization suggesting a mechanism involving enhanced efflux or sequestration of cytosolic Ca++. The inhibitory effect of N2O was attenuated when the transmembrane Na+ gradient was altered either by suspending cells in nominally Na(+)-free buffer or by pretreating cells with ouabain. The inhibitory effect of N2O was also attenuated by the Na+/Ca++ exchange inhibitor 3,4-dichlorobenzamil. The effects of N2O on membrane potential were measured fluorimetrically using bis(1,3-dibutylthiobarbituric acid)-trimethine oxonol. In the presence of N2O, resting membrane potential was hyperpolarized, a condition that would favor Ca++ efflux mediated by the electrogenic Na+/Ca++ exchanger. Taken together, these findings indicate that N2O suppresses carbachol-stimulated increases in [Ca++]i by enhancing Na+/Ca++ exchange activity. Enhancement of neuronal Na+/Ca++ exchange may contribute to the anesthetic action of N2O.

Topics: Amiloride; Aniline Compounds; Calcium; Carrier Proteins; Cell Line; Fluorescent Dyes; Fura-2; Humans; Ionomycin; Kinetics; Membrane Potentials; Neuroblastoma; Nitrous Oxide; Ouabain; Sodium; Sodium-Calcium Exchanger; Spectrometry, Fluorescence; Tumor Cells, Cultured; Xanthenes

We have previously demonstrated that activation of the Na(+)-Ca2+ exchanger in the reverse mode causes Ca2+ influx in astrocytes. In addition, we showed that the exchange activity was stimulated by nitric oxide (NO)/cyclic GMP and inhibited by ascorbic acid. The present study demonstrates that the Na(+)-Ca2+ exchanger is involved in agonist-induced Ca2+ signaling in cultured rat astrocytes. The astrocytic intracellular Ca2+ concentration ([Ca2+]i) was increased by L-glutamate, noradrenaline (NA), and ATP, and the increases were all attenuated by the NO generator sodium nitroprusside (SNP). SNP also reduced the ionomycin-induced increase in [Ca2+]i. The NA-induced Ca2+ signal was also attenuated by S-nitroso-L-cysteine and 8-bromo cyclic GMP, whereas it was enhanced by 3,4-dichlorobenzamil, an inhibitor of the Na(+)-Ca2+ exchanger. Treatment of astrocytes with antisense, but not sense, deoxynucleotides to the sequence encoding the Na(+)-Ca2+ exchanger enhanced the ionomycin-induced increase in [Ca2+]i and blocked the effects of SNP and 8-bromo cyclic GMP in reducing the NA-induced Ca2+ signal. Furthermore, the ionomycin-induced Ca2+ signal was enhanced by removal of extracellular Na+ and pretreatment with ascorbic acid. These findings indicate that the Na(+)-Ca2+ exchanger is a target for NO modulation of elevated [Ca2+]i and that the exchanger plays a role in Ca2+ efflux when [Ca2+]i is raised above basal levels in astrocytes.

Topics: Adenosine Triphosphate; Amiloride; Animals; Animals, Newborn; Astrocytes; Base Sequence; Bucladesine; Calcium; Carrier Proteins; Cells, Cultured; Cerebral Cortex; Cyclic GMP; Cysteine; Glutamic Acid; Ionomycin; Kinetics; Nitric Oxide; Nitroprusside; Norepinephrine; Oligonucleotides, Antisense; Phosphatidylinositols; Rats; Rats, Sprague-Dawley; S-Nitrosothiols; Signal Transduction; Sodium-Calcium Exchanger

This study characterized the cytosolic free Ca2+ concentration ([Ca2+]i) in NaCN-treated human A-431 cells. The resting [Ca2+]i was 85 +/- 8 nM (n = 141) in untreated cells at 37 degrees C, determined with the fura-2 fluorescence probe. When cells were treated with NaCN, [Ca2+]i increased in a time- and NaCN concentration-dependent manner. When cells were exposed to 10 mM NaCN for 10 min, [Ca2+]i increased 278 +/- 28% (n = 5) but returned to normal within 45 min after treatment. The [Ca2+]i increase depended on the presence of external Ca2+. La3+ and Cd2+, but not verapamil or nifedipine, inhibited the NaCN-induced [Ca2+]i increase. The NaCN-induced [Ca2+]i increase also depended on external Na+ (K1/2 = 85 mM). The intracellular Na+ concentration, measured with the fluorescence probe SBFI, increased 267 +/- 16% after NaCN treatment. The NaCN-induced [Ca2+]i increase was modulated by treatment with ouabain or veratridine and was completely blocked by tetrodotoxin, amiloride (K1/2 = 5.4 microM), and dichlorobenzamil (K1/2 = 0.28 microM). These results suggest NaCN activates the Na+/Ca2+ exchange system. TMB-8 and ryanodine both partially blocked the increase in [Ca2+]i in the presence of external Ca2+, indicating that Ca2+ release from intracellular pools also occurred after the initial Ca2+ influx. NaCN decreased inositol trisphosphates production. U-73122, bradykinin, or monensin did not prevent the NaCN-induced increase in [Ca2+]i. However, the magnitude of the [Ca2+]i increase caused by NaCN was abolished in ionomycin-treated the [Ca2+]i increase caused by NaCN was abolished in ionomycin-treated cells, indicating that intracellular Ca2+ release induced by NaCN is derived from an ionomycin-sensitive Ca2+ pool. The results suggest that NaCN initially increased Na+ influx, which activated the reverse mode of a Na+/Ca2+ exchanger, leading to an increase in Ca2+ influx. The Ca2+ influx induced a Ca2+ mobilization from only an ionomycin-sensitive intracellular Ca2+ pool containing ryanodine receptors.

Topics: Amiloride; Bradykinin; Calcium; Calcium Channels; Carcinoma, Squamous Cell; Carrier Proteins; Cytosol; Extracellular Space; Gallic Acid; Humans; Inositol Phosphates; Ion Channels; Ionomycin; Monensin; Muscle Proteins; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sodium; Sodium Cyanide; Sodium-Calcium Exchanger; Tumor Cells, Cultured