Regulation of neurotransmitter release

Abstract

Regulated exocytosis is known to be ATP dependent. We examined [ 3 H]-noradrenaline exocytosis from rat brain cortical synaptosomes perforated with streptolysin-0 and compared release in the presence of 5 mM ATP with that of ITP, GTP, CTP or UTP. ITP, GTP and UTP were able to support release to varying degrees, and they were also shown to maintain neurotransmitter efflux to levels comparable to efflux in the presence of ATP. CTP was ineffective in supporting release or maintaining low. The current study investigates the effectiveness of different nucleotides to decrease Ca 2+ -independent release from synaptosomes as well as their ability to support vesicle release. Overall, our data support a role for protein kinase C (PKC) in promoting neurotransmitter release. UTP may up regulate PKC, thereby stimulating exocytosis. We show that ATP is not exclusive in supporting Ca 2+ -dependent exocytosis. ITP, GTP and UTP are shown to support Ca 2+ -dependent exocytosis. Calmodulin (CaM) is a ubiquitous protein that is found in all eukaryotic cells. However, its function in neurotransmitter release is unclear. Stimulating release from perforated synaptosomes with Ca 2+ and Ba 2+ allowed investigation of the role CaM plays in neurotransmitter release. By stimulating release with Ba 2+ one can look at release that is CaM-independent because Ba 2+ does not activate CaM. Release was stimulated with either Ca 2+ or Ba 2+ . In the presence of CaM and Ca 2+ /calmodulin dependent protein kinase II (CaMKII) inhibitors release was not abolished, indicating that release is not CaM-dependent. The CaM inhibitors W-7, W-5, and trifluoperizine (TFP) decreased Ca 2+ -dependent release. The CaMKII KN62 did not significantly decrease Ca 2+ - or Ba 2+ -dependent release. Ca 2+ dependent release decreased slightly in the presence of KN93. Exogenous CaM and ?-CaMKII enhanced release. Our results indicate that CaM is not essential for release, but does enhance the sensitivity of the Ca 2+ trigger.