Effects of 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one on Synaptic Vesicle Cycling at the Frog Neuromuscular Junction

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.