PMID- 9930734
OWN - NLM
STAT- MEDLINE
DCOM- 19990211
LR  - 20190630
IS  - 0022-3042 (Print)
IS  - 0022-3042 (Linking)
VI  - 72
IP  - 2
DP  - 1999 Feb
TI  - The pH-sensitive dye acridine orange as a tool to monitor exocytosis/endocytosis 
      in synaptosomes.
PG  - 625-33
AB  - 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 microM) and alpha-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 4beta-phorbol 12-myristate 13-acetate 
      (PMA). With alpha-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.
FAU - Zoccarato, F
AU  - Zoccarato F
AD  - Department of Biological Chemistry, University of Padova, Italy.
FAU - Cavallini, L
AU  - Cavallini L
FAU - Alexandre, A
AU  - Alexandre A
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - England
TA  - J Neurochem
JT  - Journal of neurochemistry
JID - 2985190R
RN  - 0 (Acids)
RN  - 0 (Anti-Bacterial Agents)
RN  - 0 (Chelating Agents)
RN  - 0 (Fluorescent Dyes)
RN  - 0 (Macrolides)
RN  - 0 (Neurotransmitter Agents)
RN  - 00BH33GNGH (Cadmium)
RN  - 3KX376GY7L (Glutamic Acid)
RN  - 4368-28-9 (Tetrodotoxin)
RN  - 526U7A2651 (Egtazic Acid)
RN  - 660YQ98I10 (Potassium Chloride)
RN  - 88899-55-2 (bafilomycin A1)
RN  - BH3B64OKL9 (4-Aminopyridine)
RN  - F30N4O6XVV (Acridine Orange)
RN  - SY7Q814VUP (Calcium)
SB  - IM
MH  - 4-Aminopyridine/pharmacology
MH  - Acids/metabolism
MH  - Acridine Orange/*pharmacokinetics
MH  - Animals
MH  - Anti-Bacterial Agents/pharmacology
MH  - Biological Transport/drug effects/physiology
MH  - Cadmium/pharmacology
MH  - Calcium/metabolism
MH  - Cerebral Cortex/cytology
MH  - Chelating Agents/pharmacology
MH  - Dimerization
MH  - Egtazic Acid/pharmacology
MH  - Endocytosis/*physiology
MH  - Exocytosis/*physiology
MH  - Fluorescent Dyes/*pharmacokinetics
MH  - Glutamic Acid/metabolism
MH  - *Hydrogen-Ion Concentration
MH  - *Macrolides
MH  - Neurons/cytology/physiology
MH  - Neurotransmitter Agents/metabolism
MH  - Potassium Chloride/pharmacology
MH  - Rats
MH  - Synaptic Vesicles/chemistry/metabolism
MH  - Synaptosomes/chemistry/drug effects/*metabolism
MH  - Tetrodotoxin/pharmacology
EDAT- 1999/02/04 00:00
MHDA- 1999/02/04 00:01
CRDT- 1999/02/04 00:00
PHST- 1999/02/04 00:00 [pubmed]
PHST- 1999/02/04 00:01 [medline]
PHST- 1999/02/04 00:00 [entrez]
AID - 10.1046/j.1471-4159.1999.0720625.x [doi]
PST - ppublish
SO  - J Neurochem. 1999 Feb;72(2):625-33. doi: 10.1046/j.1471-4159.1999.0720625.x.