Permeabilized bovine adrenal chromaffin cells have been used to characterize the MgATP requirement of processes preceding exocytosis. Incubation of primary cultures with the membrane‐permeable phenylarsine oxide (PAO) at 20 microM inhibited the phosphorylation of phosphatidylinositol (PtdIns) and completely blocked secretion. This block could be reversed by addition of 2,3‐dimercaptopropanol to the permeabilized cells. Simultaneous addition of [gamma32P]ATP and 2,3‐dimercaptopropanol permitted a comparison between recovery of secretion and phosphorylation of intracellular components. Recovery of secretion closely correlated with phosphorylation of PtdIns and PtdIns4P. Subcellular fractionation of permeabilized cells after recovery of secretion revealed that the majority of newly phosphorylated PtdIns4P was localized on the chromaffin granules. In accordance with these results, PtdIns 4‐kinase activity was found in protein extracts of permeabilized cells as well as associated with purified chromaffin granules, sensitive in both cases to PAO. Additionally, PtdIns 4‐kinase activity in these two assays was inhibited by quercetin. In permeabilized cells, quercetin decreased the levels of labeled PtdIns4P and Ptdlns(4,5)P2 and inhibited secretion. Our data suggest that a chromaffin granule‐associated PtdIns 4‐kinase acts in the priming of exocytosis.

Amphetamineanalogs have emerged as popular recreational drugs of abuse. The number of reports of these substances producing severe acute toxicity and death is increasing. In 'Ecstasy' -associated deaths, focal necrosis in the liver and individual myocytic necrosis has been reported. Furthermore, serotonergic and dopaminergic neuronal cell damage has been observed in experimental amphetamine intoxication in laboratory animals. Here we demonstrate that subchronic exposure to D-amphetamine, methamphetamine, methylenedioxyamphetamine, and methylenedioxymethamphetamine ('Ecstasy') results in significant neurotoxicity in rat neocortical neurons in vitro. This neuronal cell death is accompanied by endonucleosomal DNA cleavage and differential expression of anti- and proapoptotic bcl-xL/S splice variants. In addition, we observed pronounced induction of cell stress-associated transcription factor c-jun and translation initiation inhibitor p97 after amphetamine treatment. These data support that the neurotoxic effects of different amphetamines are extended to rat neocortical neurons and that apoptotic pathways are involved in amphetamine-induced neurotoxicity.

The membrane protein syntaxin participates in several protein-protein interactions that have been implicated in neurotransmitter release. To probe the physiological importance of these interactions, we microinjected into the squid giant presynaptic terminal botulinum toxin C1, which cleaves syntaxin, and the H3 domain of syntaxin, which mediates binding to other proteins. Both reagents inhibited synaptic transmission yet did not affect the number or distribution of synaptic vesicles at the presynaptic active zone. Recombinant H3 domain inhibited the interactions between syntaxin and SNAP-25 that underlie the formation of stable SNARE complexes in vitro. These data support the notion that syntaxin-mediated SNARE complexes are necessary for docked synaptic vesicles to fuse.Synaptic transmission is based on the rapid exocytosis of neurotransmitters from synaptic vesicles (1). Exocytosis occurs at active zones, where vesicles are docked for Ca 2ϩ -triggered fusion with the presynaptic plasma membrane (2). The exocytotic event is coupled to an endocytotic cascade that recycles fused synaptic vesicles for additional rounds of regulated fusion (3). Several proteins of the synaptic vesicle, plasma membrane, and presynaptic cytosol have been implicated in neurotransmitter release (4, 5). Syntaxin-1A, a neuronal homologue of a family of proteins involved in membrane fusion (6-8), plays a key role in exocytosis. First, this plasma membrane protein is a substrate of botulinum C1 (Bot-C1), a toxin that inhibits transmitter release by the proteolytic activity of its disulfide-linked light chain (9). Second, Drosophila lacking syntaxin suffer embryonic lethality and a loss of synaptic transmission (10, 11).In vitro, syntaxin 1A participates in a number of interactions that are believed to be essential for neurotransmitter release. Syntaxin-binding partners include Ca 2ϩ channels, complexin, and synaptotagmin, a putative Ca 2ϩ receptor for transmitter release, as well as mammalian homologues of the Unc13 and secϪ1 protein families (4,5,12). Syntaxin also binds SNAP-25 (synaptosomal associated protein of 25 kDa), a peripheral plasma membrane protein, and synaptobrevin, a major integral membrane protein of synaptic vesicles (13,14). Together these proteins form a stable ternary complex of 7S (15) that resists dissociation by SDS (14). This 7S complex has been proposed to mediate the docking and fusion of synaptic vesicles at the presynaptic plasma membrane, because it binds the fusion catalyzing proteins SNAP (soluble NSF attachment protein) and NSF (for N-ethylmaleimide-sensitive fusion protein) (15, 16). The resulting higher-order SNARE (SNAP receptor) complex of 20S dissociates upon hydrolysis of ATP by NSF, and this dissociation is thought to promote membrane fusion (15-17).To investigate how syntaxin's interactions with presynaptic proteins contribute to the trafficking events that underlie neurotransmitter release, we have perturbed syntaxin function in vivo by microinjecting Bot-C1 and recombinant fragments of synt...