Calcium‐dependent in vitro interaction between bovine adrenal medullary cell membranes and chromaffin granules as a model for exocytosis

Konings, F; Potter, W.P. De

doi:10.1016/0014-5793(81)81043-5

Cited by 30 publications

(4 citation statements)

References 20 publications

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“…There have been a number of reports that Ca-dependent exocytosis can take place in cellfree systems (Davis & Lazarus, 1976;Baker, 1977;Gratzl, Dahl, Russell & Thorn, 1977;Konings & de Potter, 1981), where the reported affinity for Ca is in the same range that we find in 'leaky' cells. Unfortunately, the cell-free systems described to date are rather capricious and have not yet been shown to meet all the strict criteria for exocytosis.…”

Section: )supporting

confidence: 70%

Calcium-dependence of catecholamine release from bovine adrenal medullary cells after exposure to intense electric fields

1982

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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...

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Section: )supporting

confidence: 70%

Calcium-dependence of catecholamine release from bovine adrenal medullary cells after exposure to intense electric fields

1982

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“…However, disadvantages exist with both these approaches. For secretion, for example, where thecellular response involves participation of the plasma membrane, reconstitution experiments have not, with a few notable exceptions (Vacquier, 1975;Davis & Lazarus, 1976;Gratzl et al 1977;Konings & de Potter, 1981), been successful. Even when such reconstitutions have succeeded in other systems (e.g.…”

Section: Introductionmentioning

confidence: 99%

Gaining access to the cytosol: the technique and some applications of electropermeabilization

Knight¹,

Scrutton²

1986

Biochemical Journal

271

100

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“…Several in vitro systems for regulated exocytosis have been described (Davis and Lazarus, !976;Milutinovic et al, 1977;Konings and De Potter, 1981;Whitaker and Baker, 1983), of which the most impressive is the sea urchin'egg system of Jackson and co-workers (Haggerty and Jackson, 1983;Crabb and Jackson;Jackson et al, 1985). Unfortu-nately, even this last system is probably too cumbersome to be applicable to a detailed study of the molecular mechanisms underlying the exocytotic fusion event.…”

mentioning

confidence: 99%

A specific interaction in vitro between pancreatic zymogen granules and plasma membranes: stimulation by G-protein activators but not by Ca2+.

Nadin

Rogers

Tomlinson

et al. 1989

The Journal of Cell Biology

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Abstract. The molecular details of the final step in the process of regulated exoc'ytosis, the fusion of the membrane of the.secretory granule with the plasma membrane, are at present obscure. As a first step in an investigation of this membrane fusion event, we have developed a cell-free assay for the interaction between pancreatic zymogen granules and plasma membranes. We show here that plasma membranes are able to trigger the release of the granule contents, and that this effect is specific to pancreatic membranes, involves membrane fusion, requires membrane proteins, and is stimulated by activators of G-proteins but not by Ca 2+. The assay is simple, reliable, and rapid, and should permit the identification of proteins that are involved in the exocytotic fusion event.ultIr~G the process of regulated exocytosis, cell activation generates intracellular signals that trigger fusion between the membranes of secretory vesicles and the plasma membrane, leading to the release of the vesicle con-

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Calcium‐dependent in vitro interaction between bovine adrenal medullary cell membranes and chromaffin granules as a model for exocytosis

Cited by 30 publications

References 20 publications

Calcium-dependence of catecholamine release from bovine adrenal medullary cells after exposure to intense electric fields

Calcium-dependence of catecholamine release from bovine adrenal medullary cells after exposure to intense electric fields

Gaining access to the cytosol: the technique and some applications of electropermeabilization

A specific interaction in vitro between pancreatic zymogen granules and plasma membranes: stimulation by G-protein activators but not by Ca2+.

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