Measurement of intracellular Ca2+ in BC3H-1 muscle cells with Fura-2: relationship to acetylcholine receptor synthesis

Synthesis of acetylcholine receptors (AChR) can be affected by calcium, but the role played by this cation is controversial. The effect of changes in extracellular calcium, Ca2+]o, on AChR synthesis was examined in a cultured mouse muscle cell line, BC3H-1. Reduction of Ca2+]o for long periods (approximately 22 h) leads to a decrease in total surface AChR levels, a finding that is consistent with inhibition of AChR synthesis. A half-maximal reduction in surface AChR levels is observed when Ca2+]o is decreased from 1.8 to approximately 5o microM. Under these conditions, however, total protein synthesis is also largely inhibited, suggesting that the effect of Ca2+]o on AChR synthesis may be relatively non-specific. Increasing Ca2+]i by adding the Ca2+ ionophore, A23187 (in the presence of 1.8 mM Ca2+]o) also gives similar and significant reductions of both AChR and protein synthesis. Since the time course of changes in intracellular calcium ( Ca2+]i) produced by these manoeuvres is unknown, we examined the effects of briefer (1-6 h) reductions in Ca2+]o and achieved a more specific reduction in AChR synthesis. A direct measurement of the changes in Ca2+]i resulting from changes in Ca2+]o was made using the fluorescent indicator Fura-2 and video fluorescence microscopy. Our results show that in BC3H-1 muscle cells the resting intracellular calcium decreases reversibly over 20 min when Ca2+]o is decreased. We suggest that a reduction of Ca2+]i produced by the lower Ca2+]o underlies the reduction in AChR synthesis observed in these experiments.