tetrodotoxin and Retinoblastoma

1. Whole-cell and perforated-patch tight-seal recording techniques were used to characterize the voltage-dependent membrane conductances of the Y-79 cells, a human retinoblastoma line composed of pluripotential retinal precursor cells. 2. Membrane resistance and capacitance were measured under current clamp, yielding approximate average values of 1.8 G omega and 26 pF, respectively. The cells are electrically excitable, and depolarization above -20 mV triggers slow action potentials. 3. Step depolarization of the membrane under voltage clamp elicits a high-threshold transient inward current, followed by a sustained, larger outward current. The outward current is carried by potassium ions, as determined by its susceptibility to blockage by K-channel antagonists [tetraethylammonium (TEA), Cs, and 4-aminopyridine (4-AP)] and insensitivity to reduction of external chloride concentration. 4. The isolated inward current displayed some unusual properties: its amplitude is directly related to extracellular calcium concentration, and replacement of calcium by magnesium completely abolishes it. However, none of the calcium channel antagonists tested (cadmium, nickel, nifedipine, and amiloride) exerted a substantial blockage. In addition, removal of external sodium or superfusion with tetrodotoxin significantly reduce the size of this current. 5. A single voltage-dependent conductance appears to underlie the inward current, because a variety of manipulations, such as changes in the holding potential, in the extracellular concentration of calcium or sodium, or superfusion with tetrodotoxin, failed to reveal the presence of kinetically distinct components. 6. The results suggest that a single voltage-dependent conductance mechanism underlies the depolarization-activated inward current in Y-79 cells. This channel appears to be primarily permeable to calcium, but with a significant contribution by sodium ions. Its functioning appears to be modulated by extracellular calcium.

Topics: Calcium Channel Blockers; Calcium Channels; Cell Membrane; Electrophysiology; Eye Neoplasms; Humans; Kinetics; Microelectrodes; Retinoblastoma; Sodium Channels; Tetrodotoxin; Tumor Cells, Cultured

1. Neuronlike differentiation of Y-79 retinoblastoma was chemically induced in vitro, by plating the cells onto a poly-D-lysine and laminin substrate. The changes in voltage-dependent conductances after 48-72 h were examined with the whole-cell tight-seal and the perforated-patch recording techniques. 2. Although outward currents carried by potassium ions appeared qualitatively similar before and after differentiation, the depolarization-activated transient inward current displayed a pronounced acceleration of its activation and inactivation kinetics. 3. After differentiation, both the threshold of activation and the steady-state inactivation curve of the inward current are displaced in the negative direction by approximately 10-20 mV as compared with untreated cells. The current attains its peak amplitude in approximately 1 ms at maximum activating voltages, and decays within 3 ms. In contrast, in undifferentiated cells these values are on the order of 6 and 60 ms, respectively. The time to recover from inactivation is also shortened 20-fold in differentiated cells. 4. Unlike the mixed conductance of undifferentiated cells, which requires extracellular calcium, the inward current of the neuronlike differentiated cells is insensitive to manipulations of external calcium. Instead, it can be completely abolished in a reversible way by sodium removal or by micromolar concentrations of tetrodotoxin (TTX) in the bathing solution. As such, it resembles in all salient respects the voltage-dependent sodium conductance of nerve cells. 5. The fast sodium current expressed after neuronal differentiation is not the result of a progressive enhancement of an existing conductance, because no such component is discernible in undifferentiated cells. Moreover, recordings performed in cells at early stages of differentiation also failed to reveal the coexistence of the immature and the differentiated inward currents. 6. A possible account of the present observations is that the native inward current of undifferentiated Y-79 cells may correspond to a precursor form of the mature channel, and the observed developmental changes induced by chemical differentiation could be a consequence of progressive modification of the original channels, rather than expression of a separate class of proteins.

Topics: Butyrates; Calcium Channels; Cell Differentiation; Cell Membrane; Electrophysiology; Eye Neoplasms; Humans; Kinetics; Neurons; Retinoblastoma; Sodium Channels; Tetrodotoxin; Tumor Cells, Cultured

Cell lines of medulloblastoma, retinoblastoma, and neuroblastoma, three childhood tumors derived from neuroectoderm, have been compared with respect to their neuronal properties. Neuroblastoma, a neural crest derivative, has been shown to express specific neuronal enzymes and the action potential sodium ionophore. Cell lines of medulloblastoma and retinoblastoma also express neuronal specific enzymes and therefore are considered to be of neuroblastic origin.

Topics: Acetylcholinesterase; Cell Line; Choline O-Acetyltransferase; Humans; Lithium; Medulloblastoma; Neuroblastoma; Neurons; Retinoblastoma; Tetrodotoxin; Tyrosine 3-Monooxygenase; Veratridine