Membrane Capacitance Measurement: Restoration of Calcium-Dependent Exocytosis Blocked by Botulinum a Neurotoxin in Bovine Chromaffin Cells
Botulinum A neurotoxin (BoNtx A) is a potent inhibitor of calcium-dependent exocytosis in bovine chromaffin cells1. The intracellular target or mechanism of action are not yet known. Exocytosis is characterized by the fusion of vesicles with the plasma membrane followed by the release of their contents into the extracellular space. The signal transducing pathway connecting receptor stimulation and vesicle fusion have not been elucidated in full detail. There is evidence, however, that the intracellular Ca++ concentration plays a key role2. Moreover, exocytosis can be stimulated in staphylococcal α-toxin permeabilised chromaffin cells by elevating the intracellular calcium concentration1,3. Though the intracellular increase of the Ca++ concentration occurred at a late stage within the signal transducing pathway, BoNtx A-treated chromaffin cells failed to secret noradrenaline. The block of exocytosis was resistant even to an enforced stimulation with 100μM Ca++. In contrast to these findings, however, exocytosis in BoNtx A-treated cerebrocortical synaptosomes was restored by intracellular Ca++ in a concentration dependent manner, if there was Ca++ diffusion into the cells through Ca++-ionophore-induced channels4. Similar results were obtained in poisoned motor end-plates. In this preparation the increase in the intracellular Ca++ concentration was achieved by potassium channel blockers5,6. To gain more information about the role of Ca++ in the action of BoNtx A we have taken a different approach. Chromaffin cells were electroporated in BoNtx A- containing culture medium so that the toxin was able to use the short-lived7 membrane openings for diffusion into the cytosol8,9. Exocytosis was determined by measuring changes in membrane capacitance during perfusion of the cytosol with Ca++10,11 The resulting inhibition of hormone release could be overcome by elevated Ca++ concentrations. However, to be effective the Ca++ concentration had to be increased at an early stage of intoxication.
KeywordsChromaffin Cell Concentration Dependent Manner Tetanus Toxin Bovine Chromaffin Cell Bovine Adrenal Chromaffin Cell
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