Evidence that nerve growth factor dependence of sympathetic neurons for survival in vitro may be determined by levels of cytoplasmic free Ca2+.
Developing sympathetic neurons established in the presence of nerve growth factor (NGF) die in vitro after acute withdrawal of NGF. This in vitro model mimics the physiological situation in which neurons die during development or after axotomy when trophic support becomes insufficient. We have previously shown that depolarizing agents including high K+ and cholinergic agonists prevent neuronal death induced by acute deprivation of NGF in vitro. Based on this finding, a Ca2+ set-point hypothesis was proposed for the degree of neuronal dependence on tropic factor in vitro. Here we have examined the validity of this hypothesis by measuring the level of cytoplasmic free Ca2+ ([Ca2+]i) with fura-2 as a probe for monitoring Ca2+. (i) There was a good correlation between cell survival in the absence of NGF and [Ca2+]i levels of young sympathetic neurons (1 week in vitro) chronically exposed to various concentrations of extracellular K+, which shows that 50% survival occurred at approximately 184 nM [Ca2+]i and complete survival, independent of trophic support, occurred at approximately 240 nM [Ca2+]i. (ii) The basal level of [Ca2+]i of sympathetic neurons was relatively low (93.0 +/- 10.5 nM) at days 6-8, then increased with incubation time, and finally reached a plateau level of 241 +/- 7 nM at around week 3, when the neurons became independent of NGF for survival. (iii) Sympathetic neurons maintained in the presence of high or low concentrations of Ca2+ displayed altered trophic dependence. Thus, these findings are consistent with this Ca2+ set-point hypothesis for the degree of NGF dependence of sympathetic neurons for survival in vitro.
Koike
T
T
Department of Natural Science, Saga Medical School, Japan.
Tanaka
S
S
D000818:Animals; D002118:Calcium / Q000502:physiology*; D002470:Cell Survival; D002478:Cells, Cultured; D066298:In Vitro Techniques; D009414:Nerve Growth Factors / Q000502:physiology*; D011188:Potassium / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D013564:Sympathetic Nervous System / Q000166:cytology*
D009414:Nerve Growth Factors; D011188:Potassium; D002118:Calcium