Inhibition of vesicular glutamate storage and exocytotic release by Rose Bengal

Abstract

It had been thought that quantal size in synaptic transmission is invariable. Evidence has been emerging, however, that quantal size can be varied under certain conditions. We present evidence that alteration in vesicular [(3)H]L-glutamate (Glu) content within the synaptosome (a pinched-off nerve ending preparation) leads to a change in the amount of exocytotically released [(3)H]Glu. We found that Rose Bengal, a polyhalogenated fluorescein derivative, is a quite potent membrane-permeant inhibitor (K(i) = 19 nM) of glutamate uptake into isolated synaptic vesicles. This vesicular Glu uptake inhibition was achieved largely without affecting H(+)-pump ATPase. We show that various degrees of reduction elicited by Rose Bengal in [(3)H]Glu in synaptic vesicles inside the synaptosome result in a corresponding decrease in the amount of [(3)H]Glu released in a depolarization- (induced by 4-aminopyridine) and Ca(2+)-dependent manner. In contrast, fluorescein, the halogen-free analog of Rose Bengal, which is devoid of inhibitory activity on vesicular [(3)H]Glu uptake, failed to change the amount of exocytotically released [(3)H]Glu. These observations suggest that glutamate synaptic transmission could be altered by pharmacological intervention of glutamate uptake into synaptic vesicles in the nerve terminal, a new mode of synaptic manipulation for glutamate transmission.

Glycolysis and Glutamate Accumulation into Synaptic Vesicles

“…Glu Content in Synaptic Vesicles within Synaptosomes-[ 3 H]Glu content in synaptic vesicles within the synaptosome was assayed as described previously (42,43) with minor modifications. Synaptosomes (100 g of protein) were suspended in 0.1 ml of oxygenated (95% O 2 , 5% CO 2 ) Krebs-Ringer buffer containing 150 mM NaCl, 2.4 mM KCl, 1. filters to determine the total amount of [ 3 H]Glu taken up by the synaptosomes, and the rest were immediately frozen on dry ice.…”

“…Glu Release from Synaptosomes-Glutamate release from synaptosomes was assayed using the superfusion technique described previously (43), with minor modifications. Synaptosomes (200 g of protein) were suspended in 1.5 ml of oxygenated (95% O 2 /5% CO 2 ) artificial cerebrospinal fluid (ACSF) containing 124 mM NaCl, 5 mM KCl, 2 mM MgSO 4 , 1.25 mM NaH 2 PO 4 , 22 mM NaHCO 3 , and 10 mM D-glucose and preincubated with or without 300 M iodoacetate or 2 M oligomycin at 37°C for 10 min.…”

“…Generation of the membrane potential across the synaptic vesicle membrane was monitored by ATP-induced fluorescence quenching of the membrane potential-sensitive dye oxonol V (Molecular Probes, Inc., Eugene, OR) using a Fluorolog III fluorospectrophotometer (Horiba Jobin Yvon Co., Ltd., Tokyo, Japan) as described previously (32,43). Vesicular ATPase activity was assayed by determining free inorganic phosphate liberated upon incubation of synaptic vesicles (30 g of protein) with ATP, according to the method of Lanzetta et al (45), with minor modifications as described previously (43).…”

“…Vesicular ATPase activity was assayed by determining free inorganic phosphate liberated upon incubation of synaptic vesicles (30 g of protein) with ATP, according to the method of Lanzetta et al (45), with minor modifications as described previously (43).…”

“…In contrast to Na ϩ -dependent plasma membrane Glu uptake, vesicular Glu uptake is insensitive to aspartate (28, 31, 34, 48, 50) but potently inhibited by the membranepermeant polyhalogenated fluorescein Rose Bengal (43). t-ACPD also inhibits vesicular Glu uptake by serving as a substrate for the vesicular Glu transporter (51).…”