CHRONIC DEPOLARIZATION PREVENTS PROGRAMMED DEATH OF SYMPATHETIC NEURONS IN-VITRO BUT DOGS NOT SUPPORT GROWTH - REQUIREMENT FOR CA2+ INFLUX BUT NOT TRK ACTIVATION

Continuous exposure of many types of neurons in cell culture to elevat ed concentrations of K+ greatly enhances their survival. This effect h as been reported to be mediated by a sustained rise of cytoplasmic fre e Ca2+ concentration caused by influx of Ca2+ through voltage-gated ch annels activated by Kf-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 platin g of embryonic day 21 neurons. On the fifth day after plating, increme ntally increasing K+ concentrations in the culture medium from 5 to 10 0 mM caused chronic depolarization of neurons and had a biphasic effec t on survival of NGF-deprived cells. En- hanced survival was steeply r elated 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 d ecreased. Associated with the chronic depolarization was a sustained r ise of steady-state free Ca2+ concentration that showed a biphasic rel ationship to membrane potential roughly similar to that exhibited by s urvival. Steady-state Ca2+ concentration increased with increasingly l ower membrane potentials to a peak at about -23 mV (to approximate to 240 nM from approximate to 40 nM at about -51 mV) and then decreased a t more positive potentials. The elevation of intracellular Ca2+ was la rgely blocked by dihydropyridine and phenylalkylamine Ca2+ channel ant agonists and was potentiated by a dihydropyridine Ca2+ channel agonist . Neither the rise of Ca2+, or survival was affected by the Ca2+ chann el antagonist, omega-conotoxin. Therefore, the Ca2+ elevation was prob ably caused by Ca2+ influx through L-type, but not N-type, channels. A ntagonists of L channels blocked both survival and the sustained incre ase of steady-state free Ca2+ at similar concentrations, suggesting th at the relevant factor determining survival of depolarized cells was C a2+ influx rather than some other effect of depolarization. Surprising ly, however, there was no clear correlation between the sustained rise of Ca2+ and survival. Some membrane potentials that induced similar i ncreases of Ca2+ concentration produced widely different levels of sur vival. While chronic depolarization promoted survival of neurons in th e absence of NGF, cells supported in this manner showed little growth as measured by neurite extension, total cellular protein, and mean som al diameter. Compounds commonly used as calmodulin antagonists blocked survival of depolarized cells at concentrations that did not affect s urvival of cells maintained in NGF. However, these antagonists appeare d to block survival by inhibiting Ca2+ influx rather than through an e ffect on calmodulin. Exposure to NGF, but not depolarization without N GF, caused activation of the tyrosine kinase activity of Trk, suggesti ng that depolarization does not promote survival by activating Trk. Bo th 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 troph ic factors in promoting survival of sympathetic neurons that would oth erwise undergo programmed death. The data also demonstrate that the re lationship between intracellular Ca2+ concentration and survival in de polarized neurons is not as straightforward as previously supposed. Ad ditionally, these results suggest that Ca2+ may promote neuronal survi val by activating tyrosine kinases downstream from receptor tyrosine k inases and that the signal transduction pathways for growth and surviv al are separate. [Key words: programmed cell death, NGF, neuronal calc ium, apoptosis, tyrosine kinases, Trk]