The pH-Sensitive Dye Acridine Orange as a Tool to MonitorExocytosis/Endocytosis in Synaptosomes

Abstract : We introduce the use of the pH-sensitive dye acridine orange (AO) to monitor exo/endocytosis of acidic neurotransmitter-containing vesicles in synaptosomes. AO is accumulated exclusively in acidic v-ATPase-dependent bafilomycin (Baf)-sensitive compartments. A fraction of the accumulated AO is rapidly released (fluorescence increase) upon depolarization with KCl in the presence of Ca²⁺. The release (completed in 5-6 s) is followed by reuptake to values below the predepolarization baseline. The reuptake, but not the release, is inhibited by Baf added 5 s prior to KCl. In a similar protocol, Baf does not affect the initial fast phase of glutamate release measured enzymatically, but it abolishes the subsequent slow phase. Thus, the fast AO release corresponds to the rapid phase of glutamate release and the slow phase depends on vesicle cycling. AO reuptake depends in part on the progressive accumulation of acid-loaded vesicles during cycling. Stopping exocytosis at selected times after KCl by Ca²⁺ removal with EGTA evidences endocytosis : Its T_1/2 was 12 ± 0.6 s. The K_A⁺, channel inhibitors 4-aminopyridine (100 μM) and α-dendrotoxin (10-100 nM) are known to induce glutamate release by inducing the firing of Na⁺ channels ; their action is potentiated by the activation of protein kinase C. Also these agents promote a Ca²⁺-dependent AO release, which is prevented by the Na⁺ channel inhibitor tetrodotoxin and potentiated by 4β-phorbol 12-myristate 13-acetate (PMA). With α-dendrotoxin, endocytosis was monitored by stopping exocytosis at selected times with EGTA or alternatively with Cd²⁺ or tetrodotoxin. The T_1/2 of endocytosis, which was unaffected by PMA, was 12 ± 0.4 s with EGTA and Cd²⁺ and 9.5 ± 0.5 s with tetrodotoxin. Protein kinase C activation appeared to facilitate vesicle turnover.