Two modes of exocytosis from synaptosomes are differentially regulated by protein phosphatase types 2A and 2B

Abstract

The inhibitors okadaic acid (OA), fostriecin (FOS) and cyclosporin A (CsA), were used to investigate the roles of protein phosphatases in regulating exocytosis in rat brain synaptosomes by measuring glutamate release and the release of the styryl dye FM 2-10. Depolarization was induced by 30 mM KCl, or 0.3 mM or 1 mM 4-aminopyridine (4AP). OA and FOS produced a similar partial inhibition of KCl-and 0.3 mM 4AP-evoked exocytosis in both assays, but had little effect upon exocytosis evoked by 1 mM 4AP. In contrast, CsA had no effect upon KCl-and 0.3 mM 4AP-evoked exocytosis, but significantly enhanced glutamate release but not FM 2-10 dye release evoked by 1 mM 4AP. None of the phosphatase inhibitors changed calcium signals from FURA-2-loaded synaptosomes either before or after depolarization. Pretreatment with 100 nM phorbol 12-myristate 13-acetate abolished the inhibitory effect of OA on exocytosis induced by 0.3 mM 4AP. Taken together, these results show that exocytosis from synaptosomes has a phosphatase-sensitive and phosphatase-insensitive component, and that there are two modes of phosphatase-sensitive exocytosis that can be elicited by different depolarization conditions. Moreover, these two modes are differentially sensitive to phosphatase 2A and 2B.

Impact of Actin Filament Stabilization on Adult Hippocampal and Olfactory Bulb Neurogenesis

“…For example, voltagedependent Ca 2ϩ and NMDA channel activities decrease on actin depolymerization (Johnson and Byerly 1993;Rosenmund and Westbrook 1993;Akopian et al, 2006). Conversely, primary hippocampal neurons cultured from mice lacking gelsolin exhibit enhanced Ca 2ϩ influx after exposure to glutamate (Furukawa et al, 1997 influx and neurotransmitter release because they retain the machinery for the uptake, storage, and exocytosis of neurotransmitters (Fink et al, 2002a;Baldwin et al, 2003). Ca 2ϩ release from intrasyn- ] i increase (Mulkey and Zucker, 1991).…”

Pyridoxine Inhibits Depolarization-Evoked Glutamate Release in Nerve Terminals from Rat Cerebral Cortex: a Possible Neuroprotective Mechanism?

“…When the styryl dye FM1-43 reversibly partitions into the outer leaflet of exposed plasma membrane, its fluorescence increases. By endocytosis, FM1-43 dye can be taken up into the synaptic vesicles, and during subsequent exocytosis, is lost to the extracellular medium, accompanied by a decrease in fluorescence (Baldwin et al, 2003). In Fig.…”

“…Synaptic vesicle fusion with the plasma membrane was measured by use of release of the fluorescent dye FM1-43, as described previously (Baldwin et al, 2003). In brief, synaptosomes (0.5 mg/ml) were incubated in HBM with 1.2 mM CaCl 2 for 2 min at 37°C in a stirred test tube.…”

High throughput analysis of endogenous glutamate release using a fluorescence plate reader

“…Synaptosomes were stored on ice and diluted to 250 g/mL protein in a final volume of 2 mL using Krebs-like solution as described (Baldwin et al, 2003). The glutamate release assay was started by the addition of 1 mM NADP + ±1.2 mM calcium chloride as required.…”

“…For each well of a 96 well plate 25 L synaptosome solution was added to 175 L calcium-free Krebs-like solution prepared as described (Baldwin et al, 2003). After 60 s, 1 mM NADP + was added ±1.2 mM calcium chloride as required.…”