Regulation of intracellular Ca2+ in response to muscarinic and glutamate receptor agonists during the differentiation of NTERA2 human embryonal carcinoma cells into neurons.

Single cell microfluorimetry was used to study intracellular calcium ion signals (Ca(2+)) evoked by acetylcholine (ACh), glutamate receptor agonists and by KCI-induced membrane depolarization, during neuronal differentiation of the human embryonal carcinoma (EC) cell line, NTERA2. In undifferentiated NTERA2 EC cells, Ca(2+) was elevated in response to ACh, but not to the glutamate receptor agonists NMDA, kainate or AMPA. The ACh-induced rise in Ca(2+) was dependent upon both Ca(2+) influx and Ca(2+) mobilization from cytoplasmic calcium stores. Three other human EC cell lines responded similarly to ACh but not to glutamate or KCI-induced depolarization. In neurons derived from NTERA2 cells by retinoic acid induction, Ca(2+) signals were evoked by ACh, NMDA, kainate and by an elevation of the extracellular KCI concentration. As in undifferentiated EC cells, the ACh-mediated increases in Ca(2+) were governed by both Ca(2+) influx and Ca(2+) mobilization. In contrast, the effects of NMDA, kainate and KCI did not involve intracellular Ca(2+) mobilization. The appearance of glutamate and KCI responsiveness was not detected in non-neuronal differentiated derivatives of NTERA2 cells. Using a number of pharmacologically defined muscarinic receptor antagonists we found that NTERA2 EC cells express M(1), M(3), M(4) and possibly M(5) receptor subtypes linked to changes in Ca(2+), whilst only M(3) and M(5) are present in NTERA2-derived neurons. The results were supported by PCR analysis of the muscarinic mRNA species expressed in the cells. The data demonstrate that differentiation of NTERA2 EC cells into neurons involves the induction of functional glutamate receptors coupled to rises in Ca(2+), and changes in the expression of muscarinic ACh receptor subtypes.