P19 embryonal carcinoma cells can generate in vitro progenitors of the three main cell lineages found in the CNS. The signaling pathways underlying the acquisition of differentiated phenotypes by these cells are poorly understood.
We have characterized the participation of purinergic ionotropic (P2X) and metabotropic (P2Y) receptors at transcription and protein levels as well as Ca²⁺ transients in P19 cells which were stimulated with retinoic acid (RA) to differentiate into neurons. In undifferentiated cells the ATP-induced Ca²⁺-response was dependent on Ca²⁺-influx, possibly acting of the P2X₄ subtype, and on Ca²⁺ from inside storage buffers acting probably due to P2Y₂ and P2Y₁ subtype activation, since a,b-methylene ATP and dibenzoyl ATP or the blocker PPADS as specific agonists for P2X_1,3,7 receptors failed to produce signals. Eight days following treatment with RA the ATP-induced Ca²⁺-response was increased and partially sensitive to PPADS. A component of the ATP-induced Ca²⁺-increase was due to mobilization of intracellular Ca²⁺-stores and another to capacitative Ca²⁺-entry. UTP caused an increase in intracellular free Ca²⁺-concentration [Ca²⁺]_i. Thus, during neuronal differentiation ATP acts partially via a P2Y receptor, possibly by P2Y₂ and P2Y₆ subtypes.
As information is often carried in the frequency of action potentials in the mature nervous system, the effect of different Ca²⁺-spike frequencies on P2X receptor and nicotinic acetylcholine receptor (nAChRs) expression was investigated. Here we tested the hypothesis that Ca²⁺-signaling controls differentiation of neural precursors to neuronal phenotypes expressing purinergic receptors and nAChRs. Depolarization or neuronal activity generating Ca²⁺-influx stimulated expression levels of P2X_1,4,6 and P2Y_1,2,4,6 receptors and nAChRs (a_3,4,5,6,7 and b_2,4). Suppressing elevation of Ca²⁺-levels in progenitor neurons also enhanced expression of  P2X₁ and a₃ phenotypes. Expression of neuronal markers NF200 and NF160 was upregulated in progenitor neurons by increasing frequencies of Ca²⁺-spikes, and NF expression was reduced by inhibiting the same Ca²⁺- spikes. Low-frequency global and local Ca²⁺-transients occur more than one time during early stages of differentiation. Thus, spontaneous Ca²⁺-signals are an intrinsic property of differentiating neurosphere-derived precursors. Their frequency specifies the acquisition of a neuronal phenotype.
Supported by FAPESP, CNPq and CAPES.