Fig. 7. Repetitive short-lasting cytosolic Ca²⁺ spikes evoked by a low ACh concentration are associated with no or very small changes in the Ca²⁺ concentration inside the ER. (A) (a) Transmitted light picture of the cell investigated, with the two regions of interest identified by the coloured circles. The fluorescence intensity images before (b) and after (c) the establishment of the patch–clamp whole-cell recording configuration as well as after the apical bleaching (d) are shown. (e) The Ca²⁺-sensitive fluorescence intensity from Mag-fluo 4 inside the ER before and during stimulation with 50 nM ACh in both the basal and apical regions (upper part) as well as the Ca²⁺-dependent whole-cell current (lower part). (B) 100 nM ACh generated more substantial Ca²⁺-dependent current oscillations close to the maximal activation of the current with 10 µM ACh. In this particular cell, we observed small decreases in the Ca²⁺ concentration of the ER, recorded using the ratiometric dye Mag-fura 2 (351 and 364 nm excitation) and calibrated as described previously (Park et al., 1999). (C) Measurement of the ER Ca²⁺ concentration (Mag-fluo 4) and the Ca²⁺-dependent current oscillations in a cell exposed to an external Ca²⁺-free solution. In Ca²⁺-free solutions, low doses of ACh (25–100 nM) failed to evoke Ca²⁺ oscillations and 200 nM ACh had to be used to generate repetitive cytosolic Ca²⁺ spikes. Individual short-lasting Ca²⁺-dependent current spikes were not associated with any measurable drop in the Ca²⁺ concentration in the ER store, but after ∼2 min of repetitive spiking the ER Ca²⁺ concentration began to decrease. As the ER was losing Ca²⁺, the spikes became smaller and finally disappeared. After the Ca²⁺ oscillations had ceased, there was a further decrease in the ER Ca²⁺ concentration during a period of non-oscillatory elevation in the cytosolic Ca²⁺ concentration. Finally, a supramaximal ACh (10 µM) application caused a marked further Ca²⁺ release from the ER.