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

Abstract

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, [Ca 2+] o, on AChR synthesis was examined in a cultured mouse muscle cell line, BC3H-1. Reduction of [Ca 2+] o for long periods (∼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 [Ca 2+] o is decreased from 1.8 to ∼ 50 μ M. Under these conditions, however, total protein synthesis is also largely inhibited, suggesting that the effect of [Ca 2+] o on AChR synthesis may be relatively non-specific. Increasing [Ca 2+] i by adding the Ca 2+ ionophore, A23187 (in the presence of 1.8 mM [Ca 2+] o) also gives similar and significant reductions of both AChR and protein synthesis. Since the time course of changes in intracellular calcium ([Ca 2+] i) produced by these manoeuvres is unknown, we examined the effects of briefer (1–6 h) reductions in [Ca 2+] o and achieved a more specific reduction in AChR synthesis. A direct measurement of the changes in [Ca 2+] i resulting from changes in [Ca 2+] 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 [Ca 2+] o is decreased. We suggest that a reduction of [Ca 2+] i produced by the lower [Ca 2+] o underlies the reduction in AChR synthesis observed in these experiments.