The amount of acetylcholine mobilisable Ca2+ in single smooth muscle cells measured with the exogenous cytoplasmic Ca2+ buffer, Indo-1

Single smooth muscle cells from guinea pig urinary bladder were voltage clamped with patch electrodes containing 1 mM Indo-1. As Indo-1 entered the cell, Delta[Ca2+](i) in response to Ca2+ influx with I-Ca (1 s steps to -10 mV) was progressively decreased. Delta F-410 was used as a measure of the Ca2+ amount bound to Indo-1. Within less than 2 min after establishment of the whole-cell configuration, the fraction of Ca2+ entering the cell with I-Ca which binds to Indo-1 became constant, suggesting that Indo-1 completely overrides the endogenous Ca2+ buffers. Under these conditions, Delta F-410 was satisfactorily fitted with the time integral of I-Ca during 1 s long steps. Acetylcholine (ACh, 50 mu M) was rapidly applied to Indo-1 loaded cells to induce IP3-induced Ca2+ release (IICR), which peaked within about 1 s. From Delta F-410 in response to I-Ca and ACh and from the time integral of I-Ca the amount of Ca2+ released during IICR was estimated to be 680 attomole (680 x 10(-18) mole), corresponding to 230 mu M for 3 pi of accessible cytoplasmic volume.