Muscarinic receptor-linked increases in intracellular free Ca2+ as measured with quin-2 and Ca2+ release from monolayers of cells have been measured in the human neuroblastoma cell line SH-SY5Y. Induction of differentiation with the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) leads to a decrease in the sensitivity of the cells to low concentrations of agonists with respect to the induced increase in cytosolic free Ca2+ and stimulation of Ca2+ efflux. N o decrease in agonist binding affinity was observed when the displacement of a labelled antagonist, 3H-NMS, by a non-labelled agonist was studied.
The human neuroblastoma cell line SH-SY5Y (Biedler et al., 1973) undergoes an apparent differentiation towards mature ganglion cells when cultured in the presence of the phorbol ester, TPA (Piihlman et al., 1981). Morphological and biochemical changes induced by TPA include extension of long processes, the occurrence of neurosecretory granules in the cytoplasm, a highly elevated noradrenalin content (Piihlman et al., 19811, a n increased resting plasma membrane potential, and the appearance of voltage-dependent Ca2+ channels (Akerman et al., 1984). We have recently shown that muscarinic agonists increase 4 5 ~a 2 + influx . only into the TPAtreated cells, while no response is observed with cells cultured in the absence of TPA (Scott et al., 1985, 1986). Muscarinic receptor-linked Ca2+ release, however, is observed in cells cultured both in the presence and absence of TPA. Preliminary observations have indicated that receptor-linked Ca2+ release from cells grown in the absence of TPA appears to be at least one order of magnitude more sensitive to the agonist (Scott et al., 1986). The aim of this study was to further examine the differences in the agonist-induced Ca2+ mobilization between TPA-treated and non-treated cells and to investigate whether these differences would be linked to a n altered receptor affinity.
MATERIALS AND METHODS
Reagents
Acetylcholine, methacholine, atropine, muscarine, TPA, and quin-2IAM were purchased from Sigma Chemical Co. (St Louis, MO). 3H-NMS (74 Ci/mmol) and 45CaC12 (10-40 Ci/mmol) were from Amersham (UK).