Low Frequency Stimulation of Mouse Adrenal Slices Reveals a Clathrin‐Independent, Protein Kinase C‐Mediated Endocytic Mechanism

Abstract

Evidence suggests that chromaffin cells employ separate mechanisms for evoked endocytosis and granule recycling when stimulated at basal (approximately 0.5 Hz) and stress-activated (approximately 15 Hz) rates. Previous studies have focused mainly on elucidating the cellular mechanisms responsible for membrane recycling under conditions similar to the stress-activated state and indicate a clathrin/dephosphin-mediated retrieval via coated pits. However, the mechanism for membrane internalisation at basal stimulus intensity remains largely unexplored. We electrically stimulated chromaffin cells in adrenal tissue slices at the sympathetic basal firing rate and measured cell capacitance in the perforated voltage clamp configuration. A new method for the separation of non-secretory from secretory cell capacitance signals is presented. Simultaneous catecholamine release was measured electrochemically to isolate the exocytic from endocytic components of the capacitance responses. Using this approach we demonstrate that firing patterns that mimic basal sympathetic input results in rapid and graded membrane retrieval. We show that block of the calcium-mediated protein phosphatase 2B, a common step in clathrin-mediated processes, did not alter endocytosis elicited at basal firing levels. We further blocked clathrin-mediated retrieval with a clathrin/dephosphin-disrupting peptide (PP-19) and found endocytosis to be blocked at 15 Hz stimulation but complete and indistinguishable from control cells at 0.5 Hz stimulation. Lastly, pharmacological treatments show that conventional isoforms of protein kinase C (cPKC) are required for the 0.5 Hz-evoked retrieval mechanism. From these data we conclude that unlike endocytosis evoked under stress conditions, basal firing activity results in a clathrin-independent rapid membrane retrieval mediated through conventional isoforms of PKC.