THE RELATION OF EXOCYTOSIS AND RAPID ENDOCYTOSIS TO CALCIUM-ENTRY EVOKED BY SHORT REPETITIVE DEPOLARIZING PULSES IN RAT MELANOTROPIC CELLS

Melanotropic cells release predocked, large, dense-cored vesicles cont aining oc-melanocyte stimulating hormone in response to calcium entry through voltage-gated calcium channels. Our first objective was to stu dy the relationship between exocytosis, rapid endocytosis, and calcium entry evoked by short step depolarizations in the order of duration o f single action potentials (APs). Exocytosis and rapid endocytosis wer e monitored by capacitance measurements. We show that short step depol arizations (40 msec) evoke the fast release of only similar to 3% of t he predocked release-ready vesicle pool. Second, we asked what the dis tance is between voltage-gated calcium channels and predocked vesicles in these cells by modulating the intracellular buffer capacity, Exocy tosis and rapid endocytosis were differ entially affected by low conce ntrations of the calcium chelator EGTA, EGTA slightly attenuated exocy tosis at 100 mu M relative to 50 mu M, but exocytosis was strongly dep ressed at 400 mu M, showing that calcium ions have to travel a large d istance to stimulate exocytosis. Nevertheless, the efficacy of calcium ions to stimulate exocytosis was constant for pulse durations between 2 and 40 msec, indicating that in melanotropes, exocytosis is related linearly to the amount and duration of calcium entry during a single AP, Rapid endocytosis was already strongly depressed at 100 mu M EGTA, which shows that the process of endocytosis itself is calcium depende nt in melanotropic cells. Furthermore, rapid endocytosis proceeded wit h a time constant of similar to 116 msec at 33 degrees C, which is thr ee times faster than at room temperature. There was a strong correlati on between the amplitude of endocytosis and the amplitude of exocytosi s immediately preceding endocytosis. Both this correlation and the fas t time constant of endocytosis suggest that the exocytotic vesicle is retrieved rapidly.