tetrodotoxin and Astrocytoma

To better understand physiological changes that accompany the neoplastic transition of astrocytes to become astrocytoma cells, we studied biopsies of low-grade, pilocytic astrocytomas. This group of tumors is most prevalent in children and the tumor cells maintain most antigenic features typical of astrocytes. Astrocytoma cells were studied with the use of whole cell patch-clamp recordings in acute biopsy slices from 4-mo- to 14-yr-old pediatric patients. Recordings from 53 cells in six cases of low-grade astrocytomas were compared to either noncancerous peritumoral astrocytes or astrocytes obtained from other surgeries. Astrocytoma cells almost exclusively displayed slowly activating, sustained, tetraethylammonium (TEA)-sensitive outward potassium currents (delayed rectifying potassium currents; IDR) and transient, tetrodotoxin (TTX)-sensitive sodium currents (INa). By contrast, comparison glial cells from peritumoral regions or other surgeries showed IDR and INa, but in addition these cells also expressed transient "A"-type K+ currents and inwardly rectifying K+ currents (IIR), both of which were absent in astrocytoma cells. IIR constituted the predominant conductance in comparison astrocytes and was responsible for a high-resting K+ conductance in these cells. Voltage-activated Na+ currents were observed in 37 of 53 astrocytoma cells. Na+ current densities in astrocytoma cells, on average, were three- to fivefold larger than in comparison astrocytes. Astrocytoma cells expressing INa could be induced to generate slow action potential-like responses (spikes) by current injections. The threshold for generating such spikes was -34 mV (from a holding potential of -70 mV). The spike amplitude and time width were 52.5 mV and 12 ms, respectively. No spikes could be elicited in comparison astrocytes, although some of them expressed Na+ currents of similar size. Comparison of astrocytes to astrocytoma cells suggests that the apparent lack of IIR, which leads to high-input resistance (>500 MOmega), allows glioma cells to be sufficiently depolarized to generate Na+ spikes, whereas the high resting K+ conductance in astrocytes prevents their depolarization and thus generation of spikes. Consistent with this notion, Na+ spikes could be induced in spinal cord astrocytes in culture when IIR was experimentally blocked by 10 microM Ba2+, suggesting that the absence of IIR in astrocytoma cells is primarily responsible for the unusual spiking behavior seen in these glial

Topics: Action Potentials; Astrocytes; Astrocytoma; Brain Neoplasms; Carcinoma; Cell Differentiation; Child; Choroid Plexus Neoplasms; Delayed Rectifier Potassium Channels; Humans; Ion Transport; Neoplasm Proteins; Neoplastic Stem Cells; Nerve Tissue Proteins; Patch-Clamp Techniques; Potassium; Potassium Channel Blockers; Potassium Channels; Potassium Channels, Inwardly Rectifying; Potassium Channels, Voltage-Gated; Sodium Channel Blockers; Sodium Channels; Spinal Cord; Tetraethylammonium; Tetrodotoxin

Mouse hepatitis virus (MHV), a murine coronavirus, utilizes murine carcinoembryonic antigens as receptors. The events that follow virus-receptor binding and eventually lead to virus entry are poorly understood. We studied the possible effects of MHV infection on intracellular calcium in a mouse astrocytoma cell line. Using the calcium-sensitive dye fluo-3 and confocal laser scanning microscopy, we found that MHV strain JHM induced an immediate (within 20 s) and transient (lasting no longer than 2 min) calcium increase in about 5% of the infected cells. The calcium increase was blocked by antibodies against the viral spike protein, suggesting that it was specifically triggered by the interaction of the viral spikes with cells. It was also inhibited by L-type calcium channel blockers and was not detected in calcium-free medium, suggesting that the calcium increase was caused by calcium influx from the extracellular medium. Studies of the kinetics of viral replication by immunofluorescence staining of the viral nucleocapsid protein revealed that at 3 h postinfection there was roughly the same percentage of cells (5%) that produced the viral protein as the percentage of cells that had responded with a calcium signal. This finding and the virus dilution studies together suggest that calcium responders may represent cells that had been infected with multiple viruses and undergone rapid viral replication. Furthermore, calcium channel blockers, including verapamil and cadmium chloride, and the calcium chelator EGTA inhibited virus infection. Therefore, the transient intracellular calcium increase reported here may be an early signaling event associated with virus infection.

Topics: Aniline Compounds; Animals; Astrocytoma; Brain Neoplasms; Cadmium Chloride; Calcium; Calcium Channel Blockers; Calcium Channels; Calcium Channels, L-Type; Fluorescent Dyes; Kinetics; Mice; Microscopy, Confocal; Murine hepatitis virus; Nucleocapsid Proteins; Tetrodotoxin; Time Factors; Tumor Cells, Cultured; Xanthenes

Astrocytes in vitro and in situ have been shown to express voltage-activated ion channels previously thought to be restricted to excitable cells, including voltage-activated Na+, Ca2+, and K+ channels. However, unlike neurons, astrocytes do not generate action potentials, and the functional role of voltage-activated channels in astrocytes has been an enigma. In order to study the function of Na+ channels in glial cells, we carried out ion flux measurements, patch-clamp recordings, and ratiometric imaging of [Na+]i during blockade of Na+ channels on rat spinal cord astrocytes cultured for 7-10 d. Acute blockade of astrocyte Na+ channels by TTX had multiple effects: (1) TTX reduced, in a dose-dependent manner, Na+/K(+)-ATPase activity measured as unidirectional influx of 86Rb+; (2) TTX depolarized astrocyte membrane potential at a rate of approximately 1 mV/min; (3) TTX (100 microM) reduced [Na+]i; and (4) prolonged exposure to micromolar TTX induced astrocyte death. All these effects of TTX could be mimicked by ouabain or strophanthidin, specific blockers of the Na+/K(+)-ATPase. The effects of TTX and ouabain (or strophanthidin) were not additive. These results suggest that TTX-blockable Na+ channels in glial cells serve functions that do not require their participation in action potential electrogenesis; in particular, we propose that glial Na+ channels constitute a "return" pathway for Na+/K(+)-ATPase function, which permits Na+ ions to enter the cells to maintain [Na+]i at concentrations necessary for activity of the Na+/K(+)-ATPase. Since astrocyte Na+/K(+)-ATPase is believed to participate in [K+]o homeostasis in the CNS, the coupling of Na+ flux through voltage-activated Na+ channels to ATPase activity may provide a feedback loop that participates in the regulation of K+ ion levels in the extracellular space.

Topics: Animals; Animals, Newborn; Astrocytes; Astrocytoma; Cell Line; Cells, Cultured; Electrophysiology; Ganglia, Spinal; Glioma; Membrane Potentials; Models, Biological; Ouabain; Rats; Rats, Sprague-Dawley; Rubidium; Sodium; Sodium Channels; Sodium-Potassium-Exchanging ATPase; Strophanthidin; Tetrodotoxin; Time Factors; Tumor Cells, Cultured