Assay for Exocytosis in Permeabilized Cells

Permeabilized secretory cells are widely used to study the final events during secretion by exocytosis. Convenient cellular models are bovine adrenal chromaffin cells in short term culture and the rat pheochromocytoma cell line PC 12. Both cell types take up labeled catecholamines and store them in secretory vesicles, from which they can be released upon stimulation. The released catecholamines can be detected in the supernatant. After permeabilization of the plasma membrane, release of catecholamines can be triggered by micromolar concentrations of Ca.  

A combination of chelators for divalent cations is suitable to buffer the free Ca concentration from 0.1 -100 (iM under experimental conditions. Added Mg and ATP as well as the pH of the medium must be considered, because they alter the equilibrium between Ca and the chelators present. The free Ca and Mg concentrations are calculated by a computer program and controlled by Ca and Mg specific electrodes (Fohr et al., 1993). Each Ca buffer is prepared separately from stock solutions, with a final check of pH, pCa, or pMg. If no Ca electrode is available, the calculated total amount of Ca (as CaCy and Mg (as Mg(CH3COO)2) must be added before the pH adjustment. Buffers can be stored at -20 °C but should be thawed only once, mainly because of decomposition of ATP. 

The experimental procedures for both PC 12 and adrenal chromaffin cells, using alpha-toxin or SLO to permeabilize the plasma membrane, are given below. 

The secretory response decreases with time after permeabilization. The amount of the decrease differs between different cells and preparations. In SLO-permeabilized PC 12 cells, exocytosis can be restored using cytosolic fractions. The free Ca concentration necessary to elicit exocytosis varies between 1 and 10 [xM depending on the cell preparations, but is always 3-5 fold higher in alpha-toxin-permeabilized cells. Since the permeability to Ca is the same in cells treated with alpha-toxin, SLO or digitonin (Fohr etal., 1989, 1991) buffering by intracellular proteins may be the reason tor this variability. 

Application of Alpha-Toxin and Streptolysin O as Tools in Cell Biological Research 263 

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