P19 embryonal carcinoma cells as in vitro model for studying purinergic receptor expression and modulation of N-methyl- d-aspartate–glutamate and acetylcholine receptors during neuronal differentiation

Abstract

The in vitro differentiation of P19 murine embryonal carcinoma cells to neurons resembles developmental stages which are encountered during neuronal development. Three days following induction to neuronal differentiation by retinoic acid, most cells of the P19 population lost expression of the stage specific embryonic antigen (SSEA-1) and expressed the neural progenitor cell specific antigen nestin. Beginning from day 4 of differentiation nestin expression was down-regulated, and expression of neuron-specific enolase as marker of differentiated neurons increased. The molecular mechanisms underlying neuronal differentiation are poorly understood. We have characterized the participation of purinergic ionotropic (P2X) and metabotropic (P2Y) receptors at mRNA transcription and protein levels as well as ATP-induced Ca 2+ transients during neuronal differentiation of P19 cells. Gene and protein expression of P2X 2, P2X 6, P2Y 2, and P2Y 6 receptors increased during the course of differentiation, whereas P2X 3, P2X 4, P2Y 1 and P2Y 4 receptor expression was high in embryonic P19 cells and then decreased following induction of P19 cells to differentiation. P2X 1 receptor protein expression was only detected on days 2 and 4 of differentiation. Although P2X 5 and P2X 7 mRNA transcription was present, no protein expression for this receptor subunit could be detected throughout the differentiation process. In undifferentiated cells, mainly ionotropic P2X receptors contributed to the ATP-induced Ca 2+-response. In neuronal-differentiated P19 cells, the ATP-induced Ca 2+-response was increased and the metabotropic component predominated. Purinergic receptor function is implicated to participate in neuronal maturation, as cholinergic and glutamate– N-methyl- d-aspartate (NMDA) induced calcium responses were affected when cells were differentiated in the presence of purinergic receptor antagonists pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS), suramin or reactive blue-2. Our data suggest that inhibition of P2Y 1 and possibly P2X 2 receptors led to a loss of NMDA receptor activity whereas blockade of possibly P2X 2 and P2Y 2 purinergic receptors during neuronal differentiation of P19 mouse led to inhibition of cholinergic receptor responses.