REGULATION OF INTRACELLULAR CA2-RECEPTOR AGONISTS DURING THE DIFFERENTIATION OF NTERA2 HUMAN EMBRYONAL CARCINOMA-CELLS INTO NEURONS( IN RESPONSE TO MUSCARINIC AND GLUTAMATE)

Single cell microfluorimetry was used to study intracellular calcium i on signals ([Ca2+](i)) evoked by acetylcholine (ACh), glutamate recept or agonists and by KCl-induced membrane depolarization, during neurona l differentiation of the human embryonal carcinoma (EC) cell line, NTE RA2. In undifferentiated NTERA2 EC cells, [Ca2+](i) was elevated in re sponse to ACh, but not to the glutamate receptor agonists NMDA, kainat e or AMPA, The ACh-induced rise in [Ca2+](i) was dependent upon both C a2+ influx and Ca2+ mobilization from cytoplasmic calcium stores. Thre e other human EC cell lines responded similarly to ACh but not to glut amate or KCI-induced depolarization. In neurons derived from NTERA2 ce lls by retinoic acid induction, [Ca2+](i) signals were evoked by ACh, NMDA, kainate and by an elevation of the extracellular KCI concentrati on. As in undifferentiated EC cells, the ACh-mediated increases in [Ca 2+](i) were governed by both Ca2+ influx and Ca2+ mobilization. in con trast, the effects of NMDA, kainate and KCI did not involve intracellu lar Ca2+ mobilization. The appearance of glutamate and KCl responsiven ess was not detected in non-neuronal differentiated derivatives of NTE RA2 cells. Using a number of pharmacologically defined muscarinic rece ptor antagonists we found that NTERA2 EC cells express M(1), M(3), M(4 ) and possibly M(5) receptor subtypes linked to changes in [Ca2+](i), whilst only M(3) and M(5) are present in NTERA2-derived neurons. The r esults were supported by PCR analysis of the muscarinic mRNA species e xpressed in the cells, The data demonstrate that differentiation of NT ERA2 EC cells into neurons involves the induction of functional glutam ate receptors coupled to rises in [Ca2+](i), and changes in the expres sion of muscarinic ACh receptor subtypes.