Dependence of cytosolic calcium in differentiating rat pheochromocytoma cells on calcium channels and intracellular stores.

摘要

1. The rat clonal pheochromocytoma cell line (PC12) was used to study changes in the free intracellular Ca2+ concentration [( Ca2+]i) that are related to the distribution of L-type (dihydropyridine-sensitive) and N-type (omega-conotoxin-sensitive) calcium channels during nerve growth factor (NGF)-induced outgrowth of neurites. Changes in [Ca2+]i during K+ depolarization were recorded by means of Fura-2 single-cell microfluorimetry. 2. The basal [Ca2+]i of cells at rest was not altered by long-term treatment with NGF, neither in the cell bodies nor in the growth cones. K+ depolarization of the cells caused a rise in [Ca2+]i. 3. The dihydropyridine (DHP) nifedipine alone, or together with omega-conotoxin (omega-CgTX), were similarly effective in inhibiting the K(+)-induced increase in [Ca2+]i in untreated and NGF-treated cell bodies, arguing for a preferential distribution of L-type Ca2+ channels in this cell area. By contrast, after 6-7 days exposure to NGF the K(+)-induced initial transient rise of [Ca2+]i in growth cones was very sensitive to omega-CgTX, whereas nifedipine affected only the sustained rise. 4. PC12 cells also contain caffeine- and inositol trisphosphate (IP3)-sensitive intracellular Ca2+ stores. Addition of 30 mM-caffeine caused a fast transient rise in [Ca2+]i. The extent of filling of the caffeine-sensitive pool affected basal [Ca2+]i. These Ca2+ storage sites were empty under normal culture conditions. However, a single K+ depolarization caused filling of the stores, followed by spontaneous depletion (50% in about 5 min) after wash-out of high [K+]o. When the caffeine-sensitive stores were empty, the rise in [Ca2+]i was attenuated during submaximal depolarization. Caffeine-sensitive Ca2+ stores were also present in some growth cones, though with much smaller capacities than in cell bodies. 5. Mobilization of Ca2+ from the IP3-sensitive store, by bradykinin exposure, was found to be independent of the caffeine-sensitive pool. There was no apparent 'cross-talk' between both Ca2+ pools. 6. We conclude that changes in [Ca2+]i in cell bodies depend on both membrane Ca2+ channels and intracellular Ca2+ stores. During NGF-induced differentiation there is a predominance of N-type Ca2+ channels in growth cones, while Ca2+ stores are of minor importance in these structures.