Figure 2.

Measurement of [InsP₃]_cyt. a, Cells were microinjected with CG-1 and NPE-InsP₃, and CG-1 fluorescence was measured on the confocal microscope. Fluorescence intensity values were converted to relative [Ca²⁺] and plotted against time. The cell was subjected, in turn, to increasing doses of InsP₃ released by UV light flashes of 20–80 ms, 500 nM BK application to the medium, and treatment with 10 μM ionomycin. A typical experiment, in which the neurite signal is followed, is shown here. The gray line connecting the tracing after addition of ionomycin indicates a period during which the cell was refocused. b, For two cells where neurite (blue) and soma (red) are monitored, relative peak [Ca²⁺] for each uncaging event has been plotted against [InsP₃]_uncaged (solid circles). The data has been fit (solid curve) with the Hill equation (R = 0.99). A dashed horizontal line was drawn indicating the [Ca²⁺]_cyt response produced by BK stimulation in each region. A corresponding pair of lines were dropped from the Hill equation curve, indicating the [InsP₃] necessary to produce the BK-induced calcium peak. c, Mean values (± SEM) of estimated InsP₃ production by BK in soma (black) and neurite (gray) are compared in a bar graph (P < 0.001). d, [InsP₃]_cyt was measured in a N1E-115 neuroblastoma cell population during a BK-induced calcium wave using competitive radioligand binding.