Ca²⁺ oscillations induced by testosterone enhance neurite outgrowth

Testosterone has short- and long-term roles in regulating neuronal function. Here, we show rapid intracellular androgen receptor-independent effects of testosterone on intracellular Ca²⁺ in neuroblastoma cells. We identified testosterone-induced Ca²⁺ signals that began primarily at the neurite tip, followed by propagation towards the nucleus, which was then repeated to create an oscillatory pattern. The initial transient depended upon production of inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃], but subsequent transients required both extracellular Ca²⁺ influx and Ca²⁺ release from intracellular stores. Inhibition of pertussis toxin-sensitive G-protein receptors or the use of siRNA for the Ins(1,4,5)P₃ receptor type 1 blocked the Ca²⁺ response, whereas inhibition or knock-down of the intracellular androgen receptor was without effect. Cytosolic and nuclear Ca²⁺ were buffered with parvalbumin engineered to be targeted to the cytosol or nucleus. Cytoplasmic parvalbumin blocked Ca²⁺ signaling in both compartments; nuclear parvalbumin blocked only nuclear signals. Expression of a mutant parvalbumin did not modify the testosterone-induced Ca²⁺ signal. Neurite outgrowth in neuroblastoma cells was enhanced by the addition of testosterone. This effect was inhibited when cytosolic Ca²⁺ was buffered and was attenuated when parvalbumin was targeted to the nucleus. Our results are consistent with a fast effect of testosterone, involving Ins(1,4,5)P₃-mediated Ca²⁺ oscillations and support the notion that there is synergism in the pathways used for neuronal cell differentiation involving rapid non-genomic effects and the classical genomic actions of androgens.