Fusion of neurotransmitter vesicles with target membrane is calcium independent in a cell-free system.

In adrenal chromaffin cells, stimulation of Ca2+ influx leads to the secretion of neurotransmitters. The intracellular Ca2+ target involved in the fusion of secretory vesicles with the plasma membrane (PM) is still not known. We have reconstituted this fusion in vitro by using chromaffin granules (CGs) and target membrane vesicles and a Ca2(+)-dependent phospholipase A2 (PLA2). Vesicle fusion is measured by a fluorescence dequenching assay with octadecyl rhodamine B used as the marker. CGs fuse with PM vesicles only in the presence of active PLA2. The kinetics of this fusion process depend on the amount of target PM added. Once fusion competence of PM vesicles is achieved by exposure to PLA2 (primed PM vesicles), it is conserved after removal of the PLA2 even in Ca2(+)-free buffer. The kinetics of fusion between these primed PM vesicles and CGs depend on the amount of PM and on the temperature. Further incubation of the PLA2-treated PM vesicles at 30 degrees C in the absence of calcium results in an enhanced fusion competence. During this incubation, the amount of free arachidonic acid liberated by PLA2 decreases, suggesting that during a second process arachidonic acid may be processed to the terminal fusogen. The final steps of secretion can thus be subdivided into a Ca2(+)-dependent and -independent process: first, a Ca2(+)-dependent activation of PLA2 liberating fatty acids from phospholipids and second, a Ca2(+)-independent processing to the terminal fusogen and subsequent Ca2(+)-independent fusion of the CGs with the PM.

Inhibition of exocytosis by intracellularly applied antibodies against a chromaffin granule-binding protein.

Exocytotic secretion requires the interaction and fusion of secretory vesicles with the plasma membrane. This process could be mediated by specific recognition molecules acting as intracellular, membrane-bound receptors and ligands. One possible component of such a recognition site on the plasma membrane is a protein of relative molecular mass (Mr) 51,000 (51K) that has been isolated from bovine adrenal chromaffin cells. This protein binds strongly to chromaffin granules, the secretory vesicles of these cells. To determine the function of this membrane-anchored chromaffin granule-binding protein in exocytosis, we tested the effect of intracellularly injected antibodies on secretion. Here we show, by two independent techniques in two different cell types, that antibodies against this protein inhibit exocytosis. In rat pheochromocytoma cell cultures, monospecific antibodies, applied by erythrocyte ghost fusion, impair the release of 3H-noradrenaline. The same antibodies, introduced into individual chromaffin cells through a patch pipette, block exocytosis, as revealed by the measurement of membrane capacitance. These results demonstrate the functional involvement in exocytosis of a plasma membrane protein with high affinity for secretory vesicles.

Digitonin-permeabilized cells are exocytosis competent.

Release of norepinephrine from PC12 cells can be stimulated by free Ca2+ in micromolar concentrations after permeabilization with 10 micrograms/ml of digitonin. This release is time and temperature dependent, half-maximal at 0.3 microM Ca2+, and, after washing out of endogenous ATP, half-maximal at about 0.5 mM MgATP when exogenously added. Similar results were obtained with bovine adrenal chromaffin cells using the same protocol. Support for the idea that the mechanism of release from both permeabilized cell types is still exocytosis is demonstrated at the electron microscopic level by immunolabeling chromaffin granule membrane antigens that were introduced into the plasma membrane following stimulation. Electron micrographs furthermore demonstrate that chromaffin granules retain typical dense cores after permeabilization, indicating that leakiness of catecholamines from the granules was not a major factor. Pores, formed by digitonin in the plasma membranes, were utilized to introduce antibodies into such exocytosis-competent cells. Anti-actin and anti-chromaffin granule membrane antibodies show a staining pattern similar to conventionally fixed and stained preparations. Our results demonstrate that pores formed by digitonin do not impair the process of exocytosis although they are big enough to allow macromolecules to pass in both directions. The digitonin-permeabilized cell is therefore an ideal in vitro system with which to study the fusion process between chromaffin granules and the plasma membrane.

Docking of chromaffin granules--a necessary step in exocytosis?

Putative docking of secretory vesicles comprising recognition of and attachment to future fusion sites in the plasma membrane has been investigated in chromaffin cells of the bovine adrenal medulla and in rat phaeochromocytoma (PC 12) cells. Upon permeabilization with digitonin, secretion can be stimulated in both cell types by increasing the free Ca2+-concentration to microM levels. Secretory activity can be elicited up to 1 hr after starting permeabilization and despite the loss of soluble cytoplasmic components indicating a stable attachment of granules to the plasma membrane awaiting the trigger for fusion. Docked granules can be observed in the electron microscope in permeabilized PC 12 cells which contain a large proportion of their granules aligned underneath the plasma membrane. The population of putatively docked granules in chromaffin cells cannot be as readily discerned due to the dispersal of granules throughout the cytoplasm. Further experiments comparing PC 12 and chromaffin cells suggest that active docking but not transport of granules can still be performed by permeabilized cells in the presence of Ca2+: a short (2 min) pulse of Ca2+ in PC 12 cells leads to the secretion of almost all releasable hormone over a 15 min observation period whereas, in chromaffin cells, with only a small proportion of granules docked, withdrawal of Ca2+ leads to an immediate halt in secretion. Transport of chromaffin granules from the Golgi to the plasma membrane docking sites seems to depend on a mechanism sensitive to permeabilization. This is shown by the difference in the amount of hormone released from the two permeabilized cell types, reflecting the contrast in the proportion of granules docked to the plasma membrane in PC 12 or chromaffin cells. Neither docking nor the docked state are influenced by cytochalasin B or colchicine. The permeabilized cell system is a valuable technique for the in vitro study of interaction between secretory vesicles and their target membrane.