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Use of Indo-1FF for measurements of rapid micromolar cytoplasmic free Ca2+ increments in a single smooth muscle cell.

A low-affinity fluorescent Ca2+ indicator Indo-1FF was used to measure cytoplasmic Ca2+ increments in single smooth muscle cells isolated from the urinary bladder of the guinea-pig. The in vitro Kd of Indo-1FF for Ca2+ measured at the microscope stage was 21 microM. Calibration parameters measured in the cell differed substantially from respective in vitro values suggesting that the properties of the cytoplasmic dye had been altered. Addition of proteins (aldolase or albumin) increased the in vitro F405/F495 ratio close to the range observed intracellularly. Emission spectra of Ca(2+)-free Indo-1FF demonstrated a blue-shift of 29 nm with 10 mg/ml aldolase and 60 nm with 10 mg/ml albumin. The Kd value of Indo-1FF for Ca2+ in vitro was not changed by addition of aldolase (up to 20 mg/ml) and was approximately doubled in the presence of 20 mg/ml albumin. Intracellular calibration either by skinning the cells with beta-escine, 'opening' the cell or by intracellular perfusion of 100 microM free Ca2+ (40 mM DPTA-Ca2+ buffer) suggest that the affinity of intracellular Indo-1FF for Ca2+ is not markedly changed. The Indo-1FF concentration of 20 microM in the patch-pipette was found to be a reasonable compromise between acceptable signal-to-noise ratio and increased cytoplasmic Ca2+ buffering. This is because neither the amplitude nor the time-course of depolarization-induced micromolar Ca2+ increments were significantly changed during cell loading with this concentration of the dye. In contrast to Indo-1 loaded cells where rapid changes of [Ca2+]i were buffered, in Indo-1FF loaded cells ICa evoked rapid (rate of rise 150 microM/s) and large (4-6 microM in 35-60 ms) increments of free Ca2+. This results suggest that [Ca2+]i increments in smooth muscle cells are fast and large.