Rapid Vesicle Replenishment after the Immediately Releasable Pool Exocytosis is tightly linked to Fast Endocytosis, and depends on Basal Calcium and Cortical Actin in Chromaffin Cells

AbstractThe maintenance of the secretory response requires a continuous replenishment of releasable vesicles. It was proposed that the immediately releasable pool (IRP) is important in chromaffin cell secretion during action potentials applied at basal physiological frequencies, because of the proximity of IRP vesicles to voltage dependent Ca2+ channels. However, previous reports showed that IRP replenishment after depletion is too slow to manage such a situation. In this work we used patch ‐clamp measurements of membrane capacitance, confocal imaging of F‐actin distribution and cytosolic Ca2+ measurements with Fura ‐2 to re‐analyze this problem in primary cultures of mouse chromaffin cells. We provide evidence that IRP replenishment has one slow (time constant between 5‐10 s) and one rapid component (time constant between 0.5‐1.5 s) linked to a dynamin‐dependent fast endocytosis. Both, the fast endoc ytosis and the rapid replenishment component were eliminated when 500 nM Ca2+ was added to the internal solution during patch ‐clamp experiments, but they became dominant and accelerated when the cytosolic Ca2+ buffer capacity was increased. In addition, both rapid replenishment and fast endocytosis were retarded when cortical F ‐actin cytoskeleton was disrupted with cytochalasin D. Finally, in permeabilized chromaffin cells stained with rhodamine‐phalloidin, the cortical F‐actin density was reduced when the Ca2+ concentration was increased in a range of 10...
Source: Journal of Neurochemistry - Category: Neuroscience Authors: Tags: ORIGINAL ARTICLE Source Type: research