Neurosecretory PC12 cells differentiated in vitro by prolonged (at least 2 weeks) treatment with nerve growth factor were exposed to α-latrotoxin and studied by morphological and biochemical techniques. Cell monolayers or suspensions responded to the toxin with a prompt and massive release of neurotransmitter. The dose dependency (Km ~ 5 × 10-10 M) and the maximum release effect (~60% of the stored [3H]dopamine released within 8 min) were not appreciably different from the values found in non-differentiated PC12 cells. Moreover, the concentration dependency of the release was found to correspond closely to that of the [125I]α-latrotoxin binding to its specific sites (the α-latrotoxin receptors). The number of these receptors was over two-fold higher in differentiated than in undifferentiated cells. Since, however, differentiation implies a large increase in cell size and surface area, the receptor density (number/unit area) remained virtually unchanged. By radioautography the α-latrotoxin receptors were found to remain diffusely distributed at the entire surface of differentiated cells even when these were allowed to form synapses with myotubes. This situation is at variance with that demonstrated recently at the frog neuromuscular junction, where α-latrotoxin receptors are exclusively localized at the nerve terminal plasmalemma. Scanning and transmission electron microscopy revealed that the enlargements of neuntes where dense granules are preferentially accumulated-the varicosities and terminals-underwent swelling and extensive disorganization within a few minutes after the application of α-latrotoxin, whereas the cell bodies and the tracts of neurites occupied primarily by micro tubules were less severely affected. The greater sensitivity of varicosities and terminals with respect to the other parts of the differentiated cells, rather than the consequence of a specific addressing of the toxin to these structures, might be due to their vulnerability by toxin-induced events, such as the uncontrolled activation of ion fluxes.
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