Chronic Depolarization Prevents Programmed Death of Sympathetic Neurons in vitro but Does Mot Support Growth: Requirement for Ca2+ Influx but Not Trk Activation

Abstract

Continuous exposure of many types of neurons in cell culture to elevated concentrations of K+ greatly enhances their survival. This effect has been reported to be mediated by a sustained rise of cytoplasmic free Ca2+ concentration caused by influx of Ca2+ through voltage-gated channels activated by K+-induced chronic depolarization. In this report we investigate the effects of elevated K+ on the programmed death that embryonic rat sympathetic neurons undergo in culture when deprived of NGF. Elevated K+ in the culture medium did not significantly prevent death of NGF-deprived cells until after the third day following plating of embryonic day 21 neurons. On the fifth day after plating, incrementally increasing K+ concentrations in the culture medium from 5 to 100 mM caused chronic depolarization of neurons and had a biphasic effect on survival of NGF-deprived cells. Enhanced survival was steeply related to membrane potential, increasing from no enhanced survival in cells held at potentials between -51 and -34 mV to 90-100% of control survival at about -21 mV. At potentials positive to -21 mV, survival decreased. Associated with the chronic depolarization was a sustained rise of steady-state free Ca2+ concentration that showed a biphasic relationship to membrane potential roughly similar to that exhibited by survival. Steady-state Ca2+ concentration increased with increasingly lower membrane potentials to a peak at about -23 mV (to ≈240 nM from ≈40 HM at about -51 mV) and then decreased at more positive potentials. The elevation of intracellular Ca2+ was largely blocked by dihydropyridine and phenylalkylamine Ca2+ channel antagonists and was potentiated by a dihydropyridine Ca2+ channel agonist. Neither the rise of Ca2+, or survival was affected by the Ca2+ channel antagonist, ω-conotoxin. Therefore, the Ca2+ elevation was probably caused by Ca2+ influx through L-type, but not N-type, channels. Antagonists of L channels blocked both survival and the sustained increase of steady-state free Ca2+ at similar concentrations, suggesting that the relevant factor determining survival of depolarized cells was Ca2+ influx rather than some other effect of depolarization. Surprisingly, however, there was no clear correlation between the sustained rise of Ca2+ and survival. Some membrane potentials that induced similar increases of Ca2+ concentration produced widely different levels of survival. While chronic depolarization promoted survival of neurons in the absence of NGF, cells supported in this manner showed little growth as measured by neurite extension, total cellular protein, and mean somal diameter. Compounds commonly used as calmodulin antagonists blocked survival of depolarized cells at concentrations that did not affect survival of cells maintained in NGF. However, these antagonists appeared to block survival by inhibiting Ca2+ influx rather than through an effect on calmodulin. Exposure to NGF, but not depolarization without NGF, caused activation of the tyrosine kinase activity of Trk, suggesting that depolarization does not promote survival by activating Trk. Both NGF and depolarization caused tyrosine phosphorylation of a protein with a molecular weight of about 44 KDa that may be an extracellular signal-regulated protein kinase (ERK). These data show that increased Ca2+ influx induced by chronic depolarization can substitute for trophic factors in promoting survival of sympathetic neurons that would otherwise undergo programmed death. The data also demonstrate that the relationship between intracellular Ca2+ concentration and survival in depolarized neurons is not as straightforward as previously supposed. Additionally, these results suggest that Ca2+ may promote neuronal survival by activating tyrosine kinases downstream from receptor tyrosine kinases and that the signal transduction pathways for growth and survival are separate.