Figure 1.

Kinetics of endocytosis are altered with degree of stimulation of calf chromaffin cells. (A) Patch-clamped calf chromaffin cells were stimulated with depolarizing pulse trains of 10 × 50 msec (with 0.5 sec between each stimulus; a1) or 29 × 75 msec (with 4 sec between each stimulus; a2). Before each depolarization, a prepulse was administered to recruit facilitation L-type Ca²⁺ channels in this preparation (9). Typical Ca²⁺ currents recorded from the first depolarizations in a1 and a2 are shown below the protocols (numbers indicate cumulative Ca²⁺ entry for each protocol; note this is 5.5-fold larger for the sustained versus the transient paradigm). (B) Continuous C_m recordings after formation of whole-cell configuration from a single calf chromaffin cell, sequentially stimulated with the two protocols illustrated in A. (b1) With transient stimulation, exocytosis (rising phase of C_m trace) was immediately followed by RE (falling phase of C_m trace). (b2) After a 12-min recovery period, during which baseline C_m was attained and remained stable, a second round of secretion was elicited with the sustained protocol. Under these conditions, endocytosis was slow, and C_m returned to baseline in ≈9 min (see Table 1 for average values). (Inset) SE was evoked during the first round of stimulation, 3 min after establishing whole-cell configuration. Results from a typical experiment are shown [mean values (n = 12) were peak current (676 ± 44 pA), current integral (767 ± 36 pC), total C_m increase (1,750 ± 125 fF), rate of endocytosis (134 ± 20 fF/min) and endocytosis duration (10 ± 0.6 min), respectively]. (C) Conductance traces accompanying the records in B show that this parameter does not change in parallel and thus does not significantly contaminate the C_m records. In total, >90% of the cells tested expressed both forms of endocytosis.