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The Journal of Neuroscience, January 1, 1998, 18(1):81-92
The Relation of Exocytosis and Rapid Endocytosis to Calcium Entry Evoked by Short Repetitive Depolarizing Pulses in Rat Melanotropic Cells
Huibert D. Mansvelder and Karel S. Kits Research Institute Neurosciences Vrije Universiteit, Faculty of Biology, Membrane Physiology Section, 1081 HV, Amsterdam, The Netherlands
Melanotropic cells release predocked, large, dense-cored vesicles containing
-melanocyte stimulating hormone in response to calcium entry through voltage-gated calcium channels. Our first objective was to study the relationship between exocytosis, rapid endocytosis, and calcium entry evoked by short step depolarizations in the order of duration of single action potentials (APs). Exocytosis and rapid endocytosis were monitored by capacitance measurements. We show that short step depolarizations (40 msec) evoke the fast release of only ~3% of the predocked release-ready vesicle pool. Second, we asked what the distance is between voltage-gated calcium channels and predocked vesicles in these cells by modulating the intracellular buffer capacity. Exocytosis and rapid endocytosis were differentially affected by low concentrations of the calcium chelator EGTA. EGTA slightly attenuated exocytosis at 100 µM relative to 50 µM, but exocytosis was strongly depressed at 400 µM, showing that calcium ions have to travel a large distance 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 µM EGTA, which shows that the process of endocytosis itself is calcium dependent in melanotropic cells. Furthermore, rapid endocytosis proceeded with a time constant of ~116 msec at 33°C, which is three times faster than at room temperature. There was a strong correlation between the amplitude of endocytosis and the amplitude of exocytosis immediately preceding endocytosis. Both this correlation and the fast time constant of endocytosis suggest that the exocytotic vesicle is retrieved rapidly.
Key words: exocytosis; endocytosis; calcium entry; channels; EGTA; BAPTA; pituitary;
Copyright © 1998 Society for Neuroscience 0270-6474/98/18181-12$05.00/0
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