By subjecting isolated adrenal medullary cells to intense electric fields of brief duration it is possible to gain access to the cell interior without impairing the ability of the cell to undergo exocytosis. After a single exposure to ~ field of 2kV/cm, ~=200 gsec, adrenal medullary cells behave as if their plasma membrane contains two pores of effective radius 2nm. At 37 ~ these 'equivalent pores' remain patent for up to lhr. The formation and stability of these 'pores' is not affected by the Ca content of the bathing solution. The 'pores' permit externally applied catecholamine and Ca-EGTA to equilibrate rapidly with the cell water.Cells rendered 'leaky' in K glutamate medium containing 5mM Mg-ATP and EGTA to give an ionized Ca close to 10-SM release less than i % of their total catecholamine. These same cells can release up to 30 % of their catecholamine when exposed to 10 s M Ca. This Ca-dependent release is unaffected by Cachannel blockers such as D600. Catecholamine release in response to a calcium challenge only seems to occur during the first few minutes whilst the Ca concentration is changing, and the extent of release depends onthe final Ca concentration achieved. Half-maximal release occurs at about 1 gM Ca, and this value is independent of the EGTA concentration used to buffer the ionized Ca. The relation between ionized Ca and catecholamine release is best fitted by a requirement for 2 Ca ions.Calcium-evoked release of catecholamine is associated with the release of dopamine-fi-hydroxylase (D/~H) but not lactate dehydrogenase. The ratio DflH/catecholamine released is the same as that in stimulated intact cells and perfused glands. The time course of appearance in the external medium of D/~H and catecholamine is identical. Transmission electron microscopy of 'leaky' cells exposed to 10 aM Ca reveals no marked differences from unstimulated intact cells. The cytoplasm of 'leaky' cells exposed to 10-5M Ca contains large membrane-bounded vacuoles. When secretion is caused to take place in the presence of horseradish peroxidase, this marker is found within the vacuoles.Ca-dependent release of both catecholamine and D/~H requires Mg-ATP. Cells equilibrated with Ca in the absence of Mg-ATP can be triggered to undergo exocytosis by the addition of Mg-ATP. In the absence of Mg, ATP alone is ineffective. Of a variety of other nucleotides tested, none is as effective as ATP. Mg-ATP affects the extent of exocytosis and not its apparent affinity for Ca.Replacement of glutamate as the major anion by chloride results in a marked reduction in Ca-dependent release of both catecholamine and D/~H. Chloride causes a small increase in Caindependent release of catecholamine, a large reduction in the extent of exocytosis, and a decrease in the apparent affinity of exocytosis for Ca. Of a variety of anions examined, their order of effectiveness at supporting Ca-dependent exocytosis is glutamate -> acetate -> C1-> Br > SCN -.Exocytosis is not obviously affected by replacing K by Na or sucrose or by altering the pH over the ra...

Annexin 2 is a calcium-dependent phospholipid-binding protein that has been implicated in a number of membrane-related events, including regulated exocytosis. In chromaffin cells, we previously reported that catecholamine secretion requires the translocation and formation of the annexin 2 tetramer near the exocytotic sites. Here, to obtain direct evidence for a role of annexin 2 in exocytosis, we modified its expression level in chromaffin cells by using the Semliki Forest virus expression system. Using a real-time assay for individual cells, we found that the reduction of cytosolic annexin 2, and the consequent decrease of annexin 2 tetramer at the cell periphery, strongly inhibited exocytosis, most likely at an early stage before membrane fusion. Secretion also was severely impaired in cells expressing a chimera that sequestered annexin 2 into cytosolic aggregates. Moreover, we demonstrate that secretagogue-evoked stimulation triggers the formation of lipid rafts in the plasma membrane, essential for exocytosis, and which can be attributed to the annexin 2 tetramer. We propose that annexin 2 acts as a calcium-dependent promoter of lipid microdomains required for structural and spatial organization of the exocytotic machinery.

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Exposure of 'leaky' bovine adrenal medullary cells to the phorbol ester TPA causes a shift in the calciumactivation curve to lower calcium concentrations without altering the levels of secretion at the extremes of the activation curve. These results are consistent with' a role for protein kinase C in exocytosis. Calcium

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