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The Journal of Neuroscience, December 15, 2002, 22(24):10680-10689
Effects of 2-(4-Morpholinyl)-8-Phenyl-4H-1-Benzopyran-4-One on Synaptic Vesicle Cycling at the Frog Neuromuscular Junction
Silvio O. Rizzoli and William J. Betz Department of Physiology and Biophysics, University of Colorado Medical School, Denver, Colorado 80262
Inositol phospholipids are thought to play an important regulatory role in synaptic membrane traffic. We investigated the effects of perturbing 3-phosphoinositide metabolism on neurotransmission at the frog neuromuscular junction. We used the reversible phosphoinositide-3 kinase (PI3K) inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one [LY294002 (LY)] and we examined its effects by intracellular recording, fluorescence imaging with styryl dyes (FM 1-43 and FM 2-10), calcium imaging, and electron microscopy. LY treatment reversibly inhibited vesicle cycling; electron micrographs indicated a dramatic reduction in the number of vesicles, balanced by the appearance of numerous cisternas. LY wash-off reverted the phenotype; terminals were refilled with vesicles, and they resumed normal FM 1-43 uptake and release. Surprisingly, LY treatment also enhanced the frequency of spontaneous release up to 100-fold in a calcium-independent manner. LY evoked similar effects in normal frog Ringer's solution, Ca-free Ringer's solution, and BAPTA AM-pretreated preparations; imaging of nerve terminals loaded with the calcium-sensitive fluorescent dye fluo-3 showed no significant change in fluorescence intensity during LY treatment. FM 1-43 imaging data suggested that LY evoked the cycling of 70-90% of all vesicles. The LY-induced effect on spontaneous release was reproduced by the casein kinase 2 inhibitor 5,6-dichlorobenzimidazole riboside but not, however, by the PI3K inhibitor wortmannin. Because LY has been shown recently to potently inhibit casein kinase 2 as well as PI3K, we hypothesize that casein kinase 2 inhibition is responsible for the enhancement of spontaneous release, whereas PI3K inhibition induces the block of vesicle cycling.
Key words: neuromuscular junction; phosphoinositide 3- kinase; casein kinase 2; exocytosis; endocytosis; LY294002
Copyright © 2002 Society for Neuroscience 0270-6474/02/222410680-10$05.00/0
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