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.
Rizzoli
Silvio O
SO
Department of Physiology and Biophysics, University of Colorado Medical School, Denver, Colorado 80262, USA.
Betz
William J
WJ
D000200:Action Potentials / Q000187:drug effects; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D047390:Casein Kinase II; D002867:Chromones / Q000494:pharmacology*; D046508:Culture Techniques; D004004:Dichlororibofuranosylbenzimidazole / Q000494:pharmacology; D004330:Drosophila / Q000502:physiology; D004558:Electric Stimulation; D004705:Endocytosis; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005089:Exocytosis; D005456:Fluorescent Dyes / Q000378:metabolism; D007700:Kinetics; D009025:Morpholines / Q000494:pharmacology*; D009045:Motor Endplate / Q000187:drug effects / Q000502:physiology; D009469:Neuromuscular Junction / Q000378:metabolism* / Q000502:physiology / Q000648:ultrastructure; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D011726:Pyridinium Compounds / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D011894:Rana pipiens; D013570:Synaptic Membranes / Q000378:metabolism; D013572:Synaptic Vesicles / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*
D002867:Chromones; D004791:Enzyme Inhibitors; C073804:FM1 43; D005456:Fluorescent Dyes; D009025:Morpholines; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D004004:Dichlororibofuranosylbenzimidazole; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D002118:Calcium