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The pH‐Sensitive Dye Acridine Orange as a Tool to MonitorExocytosis/Endocytosis in Synaptosomes
Author(s) -
Zoccarato Franco,
Cavallini Lucia,
Alexandre Adolfo
Publication year - 1999
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1999.0720625.x
Subject(s) - exocytosis , bafilomycin , endocytosis , chemistry , acridine orange , egta , biophysics , synaptic vesicle , vesicle , channel blocker , synaptosome , biochemistry , calcium , biology , membrane , receptor , apoptosis , organic chemistry , autophagy
: We introduce the use of the pH‐sensitive dye acridineorange (AO) to monitor exo/endocytosis of acidic neurotransmitter‐containingvesicles in synaptosomes. AO is accumulated exclusively in acidicv‐ATPase‐dependent bafilomycin (Baf)‐sensitive compartments. A fraction of theaccumulated AO is rapidly released (fluorescence increase) upon depolarizationwith KCl in the presence of Ca 2+ . The release (completed in 5‐6 s)is followed by reuptake to values below the predepolarization baseline. Thereuptake, but not the release, is inhibited by Baf added 5 s prior to KCl. Ina similar protocol, Baf does not affect the initial fast phase of glutamaterelease measured enzymatically, but it abolishes the subsequent slow phase.Thus, the fast AO release corresponds to the rapid phase of glutamate releaseand the slow phase depends on vesicle cycling. AO reuptake depends in part onthe progressive accumulation of acid‐loaded vesicles during cycling. Stoppingexocytosis at selected times after KCl by Ca 2+ removal with EGTAevidences endocytosis : Its T 1/2 was 12 ± 0.6 s.The K A + , channel inhibitors 4‐aminopyridine (100μ M ) and α‐dendrotoxin (10‐100 n M ) are known toinduce glutamate release by inducing the firing of Na + channels ;their action is potentiated by the activation of protein kinase C. Also theseagents promote a Ca 2+ ‐dependent AO release, which is prevented bythe Na + channel inhibitor tetrodotoxin and potentiated by4β‐phorbol 12‐myristate 13‐acetate (PMA). With α‐dendrotoxin,endocytosis was monitored by stopping exocytosis at selected times with EGTAor alternatively with Cd 2+ or tetrodotoxin. The T 1/2 of endocytosis, which was unaffected by PMA, was 12± 0.4 s with EGTA and Cd 2+ and 9.5 ± 0.5 s with tetrodotoxin. Protein kinase C activation appeared to facilitate vesicle turnover.