Veratridine delays apoptotic neuronal death induced by NGF deprivation through a Na+-dependent mechanism in cultured rat sympathetic neurons

Abstract Superior-cervical ganglion (SCG) cells dissociated from newborn rats depend on nerve growth factor (NGF) for survival. Membrane depolarization with elevated K+ is known to prevent neuronal death following NGF deprivation and/or to promote survival via a Ca2+-dependent mechanism. Here we have exploited the possibility of whether or not a Na+-dependent pathway for neuronal survival is present in these cells. Veratridine (ec50=40 nM), a voltage-dependent Na+ channel activator, significantly delayed the onset of apoptotic cell death in NGF-deprived SCG neurons that had been cultured for 7 days in the presence of NGF. This effect was blocked completely by Na+ channel blockers including tetrodotoxin (TTX, 1 μM), benzamil (25 μM) and flunarizine (1 μM), but was not attenuated by nimodipine (1 μM), an L-type Ca 2+ channel blocker. The saving effect of veratridine on cultured neurons was observed even in low Ca 2+ media (0–1.0 mM), but was completely abolished in a low Na + medium (38 mM). Sodium-binding benzofuran isophthalate was employed as a fluorescent probe for monitoring the level of cytoplasmic free Na + , which revealed a sustained increase in its level (12.9 mM, 307% of that of control) in response to veratridine (0.75 μ M). The TTX or flunarizine completely blocked veratridine-induced Na + influx in these cultured neurons. Moreover, no appreciable increase in intracellular Ca 2+ was detected under these conditions. Though Na + channels were effectual in SCG neurons which were freshly isolated from newborn rats, the Na + -dependent saving effect of veratridine was not observed in these young neurons. These lines of evidence suggest that the death-suppressing effect of veratridine on cultured SCG neurons depends on the Na + influx via voltage-dependent Na + channels, and suggests the presence of Na + -dependent regulatory mechanism(s) in neuronal survival.