Abstract

Single cell microfluorimetry was used to study intracellular calcium ion signals (Ca2+i) evoked by acetylcholine (ACh), glutamate receptor agonists and by KCl-induced membrane depolarization, during neuronal differentiation of the human embryonal carcinoma (EC) cell line, NTERA2. In undifferentiated NTERA2 EC cells, Ca2+i was elevated in response to ACh, but not to the glutamate receptor agonists NMDA, kainate or AMPA. The ACh-induced rise in Ca2+i was dependent upon both Ca2+ influx and Ca2+ mobilization from cytoplasmic calcium stores. Three other human EC cell lines responded similarly to ACh but not to glutamate for KCl-induced depolarization. In neurons derived from NTERA2 cells by retinoic acid induction, Ca2+i signals were evoked by ACh, NMDA, kainate and by an elevation of the extracellular KCl concentration. As in ur (differentiated EC cells, the ACh-mediated increases in Ca2+i were governed by both Ca2+ influx and Ca2+ mobilization. In contrast, the effects of NMDA, kainate and KCl did not involve intracellular Ca2+ mobilization. The appearance of glutamate and KCl 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 M1, M3, M4 and possibly M5 receptor subtypes linked to changes in Ca2+i, whilst only M3 and M5 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 Ca2+i, find changes in the expression of muscarinic ACh receptor subtypes. © European Neurosci ence Association.