Modifications of intracellular Ca2+ signalling during nerve growth factor-induced neuronal differentiation of rat adrenal chromaffin cells

Postnatal sympathetic neurons (SNs) and chromaffin cells (CCs) derive from neural crest precursors. CCs can differentiate in vitro into SN-like cells after nerve growth factor (NGF) exposure. This study examines changes of in tracellular Ca2+ homeostasis and dynamics of CCs under conditions that prom ote a neuronal phenotype. Spontaneous Ca2+ fluctuations, a frequent observa tion in early cultures of CCs, diminished after > 10 days in vitro in contr ol cells and ceased in NGF-treated ones. At the same time, Ca2+ rises resul ting from entry upon membrane depolarization, gradually increased both thei r size and peak d[Ca2+](i)/dt, resembling those recorded in SNs. Concomitan tly, caffeine-induced Ca2+ rises, resulting from Ca2+ release from intracel lular stores, increased their size and their peak d[Ca2+](i)/dt by > 1000%, and developed transient and sustained release components, similar to those of SNs. The transient component, linked to regenerative Ca2+ release, appe ared after > 10 days of NGF treatment, suggesting a delayed steep enhanceme nt of Ca2+-induced Ca2+ release (CICR). Immunostaining showed that proteins coded by the three known isoforms of ryanodine receptors (RyRs) are presen t in CCs, but that only RyR(2) increased significantly after NGF treatment. Since the transient release component increased more steeply than RyR(2) i mmunostaining, we suggest that the development of robust CICR requires both an increased expression of RyRs and more efficient functional coupling amo ng them. NGF-induced transdifferentiation of chromaffin cells involves the enhancement of both voltage-gated Ca2+ influx and Ca2+ release from intrace llular stores. These modifications are likely to complement the extensive m orphological and functional reorganization required for the replacement of the endocrine phenotype with the neuronal one.