Introduction of Macromolecules into Bovine Adrenal Medullary Chromaffin Cells and Rat Pheochromocytoma Cells (PC12) by Permeabilization with Streptolysin O: Inhibitory Effect of Tetanus Toxin on Catecholamine Secretion

Conditions are described for controlled plasma membrane
permeabilization of rat pheochromocytoma cells (PC12) and cultured
bovine adrenal chromaffin cells by Streptolysin O (SLO). The
transmembrane pores created by SLO invoke rapid efflux of
intracellular 86Rb+ and ATP, and also permit passive diffusion of
proteins, including immunoglobulins, into the cells.
SLO-permeabilized PC12 cells release [3H]dopamine in response to
micromolar concentrations of free Ca2+. Permeabilized adrenal
chromaffin cells present a similar exocytotic response to Ca2+ in
the presence of Mg2+/ ATP. Permeabilized PC12 cells accumulate
antibodies against synaptophysin and calmodulin, but neither
antibody reduces the Ca2+-dependent secretory response. Reduced
tetanus toxin, although ineffective when applied to intact
chromaffin cells, inhibits Ca2+-induced exocytosis by both types of
permeabilized cells studied. Omission of dithiothreitol, toxin
inactivation by boiling, or preincubation with neutralizing
antibodies abolishes the inhibitory effect. The data indicate that
plasma membrane permeabilization by Streptolysin O is a useful tool
to probe and define cellular components that are involved in the
final steps of exocytosis.