THE EFFECTS OF HALOTHANE ON CULTURED MOUSE NEUROBLASTOMA CELLS I. Inhibition of Morphological Differentiation

Mouse neuroblastoma cells (clone NB2a) were cultured in the presence of 0.3-2.1% halothane in the gas phase for up to 72 h. Halothane inhibited neurite extension dose dependently and virtually abolished microspike formation even at the lowest concentration tested. These effects were completely reversible. Electron microscopy demonstrated that microfilaments measuring 40-80 A in diameter are the only fibrous organelles visible within microspikes. When the cells were exposed to halothane, no microfilamentous complexes could be identified in any ceils and the subcortical regions of neurites often appeared devoid of individual microfilaments. Microtubules were still present in neurites after exposure to halothane concentrations at which microfilaments disappeared. However, at concentrations above 1.0%, microtubules gradually appeared to decrease in number. Short-term experiments showed that existing neurites and microspikes rapidly retracted when suddenly exposed to culture medium equilibrated with 1.0% halothane and quickly reformed when the halothane was removed. The inhibition of neuroblastoma cell differentiation by halothane appears to be mediated by disruption of 40-80 A diameter microfilaments. Halothane, a commonly used volatile anesthetic, has been shown to disrupt reversibly the mitotic apparatus (24), to cause retraction of protozoan axonemes (1), and to inhibit cell motility (24, 34). Enlarged microtubular structures measuring about 420 A in diameter have been observed in protozoan axonemes (1) and isolated crayfish axons (13) after halothane treatment. These "macrotubular" structures are thought to represent neopolymerized forms of microtubular protein since they are altered by vinblastine and are colchicine labile (13). Collectively, these observations suggest that halothane can reversibly interfere with fundamental mechanisms responsible for intracellular motility and cellular translocation and can interact with at least some species of microtubule protein (tubulin) to produce alterations of microtubular structure. However, from a morphological standpoint, halothane-induced alterations in peripheral nerve preparations appear highly variable. For example, in isolated rabbit vagus nerves, halothane caused an increase in the number of typical microtubules (12); whereas in isolated crayfish axons, it resulted in a change from typical to macrotubular morphology (13). However, Fink and Kennedy (10) have reported that clinical concentrations of halothane have little effect on the rate of fast axonal transport or on microtubule structure or number in rabbit vagus nerves. In an attempt to reconcile these observations THE JOURNAL OF CELL BIOLOGY • VOLUME 63, 1974 pages 531-540 531 on A ril 0, 2017 D ow nladed fom Published November 1, 1974