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

Abstract

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...

[Ca2+]iElevations Detected by BK Channels during Ca2+Influx and Muscarine-Mediated Release of Ca2+from Intracellular Stores in Rat Chromaffin Cells

“…magnitude of the [Ca 2ϩ ] i elevations produced by mAChR activation is less than that produced by influx, the elevation produced by mAChR activation persists much longer and seems to affect a significantly greater portion of the chromaffin cell membrane. Based on estimates of the Ca 2ϩ dependence of exocytosis from bovine chromaffin cells (Knight and Baker, 1982;Augustine and Neher, 1992b) indicating that secretion can be initiated at [Ca 2ϩ ] i lower than 1 M, our results are consistent with previous reports implicating mAChRs in eliciting secretion in rat chromaffin cells (Wakade and Wakade, 1983;Malhotra et al, 1988). This contrasts with bovine chromaffin cells, in which release of Ca 2ϩ from intracellular stores by mAChR activation produces relatively weak elevations in [Ca 2ϩ ] i O'Sullivan et al, 1989) and is ineffective in producing secretion (Fisher et al, 1981;Kim and Westhead, 1989).…”

Further Characterization of Dopamine Release by Permeabilized PC 12 Cells

“…Yet Mg2+ in high amounts inhibited Ca2+-induced release in chromaffin cells permeabilized by high-voltage discharge (Knight and Baker, 1982). The experiments in this study were initially undertaken to look for inhibitory effects of Mg2+ in a-toxin-permeabilized PC 12 cells.…”

Reconstitution of constitutive secretion using semi-intact cells: regulation by GTP but not calcium.

“…The mechanism of regulated secretion has been studied extensively using permeabilized cell systems and patch-clamp techniques (Knight and Baker, 1982;Dunn and Holz, 1983;Vallar et al, 1987;Howell et al, 1987; for reviews see Gomperts, 1990;Almers, 1990;Burgoyne, 1990). These studies have identified calcium and GTP as two key components in this process; calcium and the nonhydrolyzable GTP analogue guanosine 5'-O-(3-thiotriphosphate) (GTPTS) 1 often act synergistically to trigger secretion from storage granules.…”