Characterization of Hormone and Protein Release from a-Toxin-permeabilized Chromaffin Cells in Primary Culture*

Addition of Staphylococcus aureus a-toxin to adult bovine chromaffin cells maintained in primary culture causes permeabilization of cell membrane as shown by the release of intracellular “Rb”. The a-toxin does not provoke a spontaneous release of either catecholamines or chromogranin A, a protein marker of the secretory granule, showing the integrity of the secretory vesicle membrane. However the addition of micromolar free Ca2+ concentration induced the co-release of noradrenaline and chromogranin A. In a-toxin-treated cells, the released chromogranin A could not be sedimented and lactate dehydrogenase was still associated within cells, which provides direct evidence that secretory product is liberated by exocytosis. By contrast, permeabilization of cells with digitonin caused a Ca2+-dependent but also a Ca2+-independent release of secretory product, a dramatic loss of lactate dehydrogenase, as well as release of secretory product in a sedimentable form. Ca2+-dependent exocytosis from a-toxin-permeabilized cells required Mg2+-ATP and did not occur in the presence of other nucleotides. Thus a-toxin is a convenient tool to permeabilize chromaffin cells, and has the advantage of keeping intracellular structures, specifically the exocytotic machinery, intact.