Rapid endocytosis (RE) occurs immediately after an exocytotic burst in adrenal chromaffin cells. Capacitance measurements of endocytosis reveal that recovery of membrane is a biphasic process that is complete within 20 sec. The ultimate extent of membrane retrieval is precisely controlled and capacitance invariably returns to its prestimulation value. The mechanism of RE specifically requires intracellular The physiological and molecular basis of secretion has been studied in some detail (1), but the nature of the endocytotic processes that are coupled to exocytosis is poorly understood. Endocytosis serves the dual function of maintaining cell surface area constant and retrieving vesicular components for recycling. In neurons, synaptic vesicle recycling is rapid and it is thought that endocytosed vesicles are recovered virtually intact and quickly returned to the releasable pool (2). This is a critical feature of neuronal communication as, without recycling, neural firing would rapidly deplete the pool of transmitter-containing vesicles ready for release. Whether a similar phenomenon occurs in other types of secretory cell is unknown, but as these cells may also need to secrete repetitively under various conditions, it seems likely that rapid recycling must be present (3).Recently, studies on several preparations have suggested that rapid endocytotic mechanisms can be assessed by measurement of cell capacitance. By this criterion, adrenal chromaffin cells (4), pituitary melanotrophs (5), and nerve terminals (6) rapidly recapture membrane after secretion. Little is known about the kinetics and regulation of this type of endocytosis, termed rapid endocytosis (RE) to distinguish it from other better-studied phenomena such as receptormediated endocytosis (mediated by clathrin-coated vesicles) or various kinds of pinocytosis (7). We have conducted a detailed analysis of RE in bovine adrenal chromaffin cells subjected to physiological stimulation. We show that it is a kinetically complex and highly regulated process that reproducibly follows each round of secretion. The final amount of membrane recovered seems to be precisely controlled, as most cells return to their prestimulation capacitance value. RE requires intracellular Ca2+ and GTP hydrolysis and we provide direct evidence that the guanine nucleotide binding protein (G protein) dynamin is intimately involved in the process. In contrast, our results suggest that RE does not involve a clathrin-coated vesicle-based mechanism. MATERIALS AND METHODSPreparation of Cells and Patch-Clamp. Chromaffin cells were isolated from calf adrenal medullae, cultured, and patchclamped as described (8). Capacitance was measured by a computer program using a phase-tracking technique (9). Secretion was elicited by a train of 10 depolarizations, from a holding potential of -90 mV to + 10 mV, each lasting 50 msec; 500 msec separated each depolarization. Each depolarization was preceded by a 50-msec prepulse to +120 mV to recruit facilitation Ca2+ channels (8). All experiments...

To determine whether the different types of Ca2+ channels present in the same secretory cell contribute equally to secretion, we used chromaffin cells to analyse the coupling between three distinct types of Ca2+ channel and exocytosis. These are omega-conotoxin-GVIA-sensitive N-type channels, omega-agatoxin-IVA-sensitive P-type Ca2+ channels and dihydropyridine-sensitive facilitation Ca2+ channels, which are normally quiescent but are activated by depolarizing pre-pulses, repetitive depolarizations to physiological potentials, or agents that raise cyclic AMP. We have simultaneously monitored changes in capacitance as an assay of catecholamine secretion, and Ca2+ currents. Although all three types of Ca2+ channel trigger secretion individually, facilitation channels produce much greater secretion for a given size of Ca2+ current, indicating that they are coupled more efficiently to exocytosis. These results indicate that facilitation Ca2+ channels may be physically nearer vesicle release sites. They also show that low efficiency P- and N-type channels could trigger mild release and that high-efficiency facilitation channels may underlie the massive catecholamine release that occurs during the 'fight or flight' response.

To what extent the quantal hypothesis of transmitter release applies to dense-core vesicle (DCV) secretion is unknown. We determined the characteristics of individual secretory events in calf chromaffin cells using catecholamine amperometry combined with different patterns of stimulation. Raising the frequency of action potential trains from 0.25-10 Hz in 2 mM [Ca(2+)]o or [Ca(2+)]o from 0.25-7 mM at 7 Hz elevated the amount released per event (quantal size). With increased stimulation, quantal size rose continuously, not abruptly, suggesting that release efficiency from a single population of DCVs rather than recruitment of different-sized vesicles contributed to the effect. These results suggest that catecholamine secretion does not conform to the quantal model. Inhibition of rapid endocytosis damped secretion in successive episodes, implying an essential role for this process in the recycling of vesicles needed for continuous secretion.

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