Depolarization, intracellular calcium and exocytosis in single vertebrate nerve endings.

We have investigated the temporal relationship between depolarization, elevation of Ca2+]i and exocytosis in single vertebrate neuroendocrine nerve terminals. The change of Ca2+]i and vasopressin release were measured with a time resolution of less than 1 s in response to K(+)-induced depolarization. Exocytosis was also monitored in the whole-terminal patch-clamp configuration by time resolved capacitance measurements while Ca2+]i was simultaneously followed by fura-2 fluorescence measurements. In intact as well as patch-clamped nerve terminals sustained depolarization leads to a sustained rise of Ca2+]i. The rate of vasopressin release from intact nerve terminals rises in parallel with Ca2+]i but then declines rapidly towards basal (t1/2 approximately 15 s) despite the maintained high Ca2+]i indicating that only a limited number of exocytotic vesicles can be released. We demonstrate that in nerve terminals exocytosis can be followed during step depolarization by capacitance measurements. The capacitance increase starts instantaneously whereas Ca2+]i rises with a half time of several hundred milliseconds. An instantaneous steep capacitance increase is followed by a slow increase with a slope of 25-50 fF/s indicating the sequential fusion of predocked and cytoplasmic vesicles. During depolarization the capacitance slope declines to zero with a similar time course as the vasopressin release indicating a decrease in exocytotic activity. Depolarization per se in the absence of a sufficient rise of Ca2+]i does not induce exocytosis but elevation of Ca2+]i in the absence of depolarization is as effective as in its presence. The experiments suggest that a rapid rise of Ca2+]i in a narrow region beneath the plasma membrane induces a burst of exocytotic activity preceding the elevation of bulk Ca2+]i in the whole nerve terminal.