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Digitonin-permeabilized cells are exocytosis competent
T Schäfer1, U O Karli, E K Gratwohl
1Department of Biochemistry, Biocenter of the University, Basel, Switzerland.
Journal of Neurochemistry
|December 1, 1987
Summary
Stimulating norepinephrine release from digitonin-permeabilized PC12 cells and chromaffin cells with calcium ions demonstrates exocytosis. This method provides an ideal in vitro system for studying granule-plasma membrane fusion.
Area of Science:
- Cell biology
- Neuroscience
- Biochemistry
Background:
- Norepinephrine release is crucial for neuronal communication.
- Understanding the exocytosis mechanism requires suitable experimental models.
Purpose of the Study:
- To investigate norepinephrine release from permeabilized cells.
- To validate digitonin-permeabilized cells as a model for studying exocytosis.
Main Methods:
- PC12 cells and bovine adrenal chromaffin cells were permeabilized with digitonin.
- Calcium ion (Ca2+) and magnesium-adenosine triphosphate (MgATP) concentrations were manipulated.
- Electron microscopy and immunolabeling were used to visualize cellular structures and processes.
Main Results:
- Free Ca2+ stimulated norepinephrine release in a time-, temperature-, and concentration-dependent manner.
- Exogenous MgATP was required for release after endogenous ATP was washed out.
- Electron microscopy confirmed exocytosis and intact chromaffin granules, with digitonin pores allowing macromolecule passage without impairing fusion.
Conclusions:
- Digitonin permeabilization creates an exocytosis-competent cell model.
- This in vitro system is suitable for detailed study of the granule-plasma membrane fusion process in exocytosis.