Tetrodotoxin-sensitive sodium channels in a continuously cultured cell line derived from the adult rat cerebellum

Abstract

A method to establish continuously cultured cell lines from adult cerebellar cortex is reported. Clones (prepared by this procedure) were isolated from cerebellar established cultures at the 25th passage and after 15 months in vitro. One clone (UCHCC1) was mainrained in culture and studied while the others were frozen. The cerebellar cell line UCHCC1 retained a neuronal-like morphology; the addition of dimethylsulfoxide (DMSO) to the culture medium elicited a reproducible morphological 'differentiation' event, characterized mainly by process extension. In 'differentiated' cells, veratridine significantly increased the uptake of 22Na. Such enhanced uptake was blocked by tetrodotoxin (TFX) with a half-maximal inhibitory concentration of 0.9 nM. Binding of an [3H]ethylenediamine derivative of TTX ([3H]en-TTX) to the microsomal fraction prepared from same DMSO-treated cells, showed a single class of receptors with a maximal binding (Bmax) of 173 fmol/mg protein and a K d of 1.1 nM. Thyroid UCHT1 cells and 'undifferentiated' (cultured without DMSO) cerebellar cells, did not show significant effects of veratridine on 22Na-uptake, or [3H]en-TFX binding. The 'differentiated' nerve-like properties, induced by appropriate environmental manipulation, demonstrate the usefulness of cerebeUar UCHCC1 cells as a model system for the developing central neuron. On the other hand, the novel transforming procedure opens new possibilities for obtaining permanent cell lines from other regions of the adult CNS.