A novel calmodulin antagonist, CGS 9343B, modulates calcium-dependent changes in neurite outgrowth and growth cone movements

The neurotransmitter 5-HT alters growth cone motility and neurite elongation in neuron B19, isolated from the buccal ganglion of Helisoma trivolvis (Haydon et al., 1984). The effects of 5-HT are mediated by increases in intracellular calcium levels within the growth cones (Cohan et al., 1987). 5-HT causes a receptor-mediated depolarization of the membrane, which results in the opening of voltage-sensitive calcium channels. The resulting calcium influx decreases both the elongation rate and the total outgrowth of neurites. However, the mechanism(s) mediating these calcium-dependent changes is unclear. As many of the intracellular effects of calcium in eukaryotic cells are mediated by the calcium-binding protein calmodulin, we tested the involvement of such an interaction in the regulation of neurite outgrowth. In these experiments, a new, potent calmodulin antagonist with increased selectivity, CGS 9343B (CGS; Norman et al., 1987), was used to inhibit calmodulin activity during the application of 5-HT to neuron B19. The addition of 100 μM 5-HT to the culture medium resulted in a significant decrease in the rate of neurite elongation and total neurite outgrowth. Administration of CGS to the culture medium at a concentration (1.8 μM) equivalent to its IC₅₀ for calmodulin inhibition completely blocked the inhibitory effects of 100 μM 5-HT, on both neurite elongation and total neurite outgrowth. CGS alone caused a slight decrease in elongation rate but had no significant effect on total out-growth. CGS did not block 5-HT-induced electrical activity, indicating that it was not acting as a 5-HT receptor antagonist. Furthermore, measurements of calcium currents obtained from whole-cell patch-clamp recordings demonstrated that CGS did not block membrane calcium channels at concentrations that suppress the effects of 5-HT on neurite outgrowth. These observations suggest that calmodulin mediates some of the effects of increased calcium on neurite elongation and growth cone movements.