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 ICa (1 s steps to -10 mV) was progressively decreased. delta F410 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 ICa which binds to Indo-1 became constant, suggesting that Indo-1 completely overrides the endogenous Ca2+ buffers. Under these conditions, delta F410 was satisfactorily fitted with the time integral of ICa during 1 s long steps. Acetylcholine (ACh, 50 microM) was rapidly applied to Indo-1 loaded cells to induce IP3-induced Ca2+ release (IICR), which peaked within about 1 s. From delta F410 in response to ICa and ACh and from the time integral of ICa the amount of Ca2+ released during IICR was estimated to be 680 attomole (680 x 10(-18) mole), corresponding to 230 microM for 3 pl of accessible cytoplasmic volume.