Inositol derivatives modulate spontaneous transmitter release at the frog neuromuscular junction

Abstract One of the consequences of G-protein-coupled receptor activation is stimulation of phosphoinositol metabolism, leading to the generation of IP 3 and its metabolites 1,3,4,5-tetrakisphosphate (IP 4 ) and inositol 1,2,3,4,5,6-hexakisphosphate (IP 6 ). Previous reports indicate that high inositol polyphosphates (IP 4 and IP 6 ) are involved in clathrin-coated vesicular recycling. In this study, we examined the effects of IP 4 and IP 6 on spontaneous transmitter release in the form of miniature endplate potentials (MEPP) and on enhanced vesicular recycling by high K + at frog motor nerve endings. In resting conditions, IP 4 and IP 6 delivered intracellularly via liposomes, caused concentration-dependent increases in MEPP frequency and amplitude. Pretreatment with the protein kinase A (PKA) inhibitor H-89 or KT 5720 reduced the IP 4 -mediated MEPP frequency increase by 60% and abolished the IP 6 -mediated MEPP frequency increases as well as the enhancement in MEPP amplitude. Pretreatment with antibodies against phosphatidylinositol 3-kinase (PI 3-K), enzyme also associated with clathrin-coated vesicular recycling, did not alter the IP 4 and IP 6 -mediated MEPP frequency increases, but reduced the MEPP amplitude increase by 50%. In our previous reports, IP 3 , but not other second messengers releasing Ca 2+ from internal Ca 2+ stores, is able to enhance the MEPP amplitude. In order to dissociate the effect of Ca 2+ release vs. metabolism to IP 4 and IP 6 , we evaluated the effects of 3-deoxy-3-fluoro-inositol 1,4,5-trisphosphate (3F-IP 3 ), which is not converted to IP 4 or IP 6 . 3F-IP 3 produced an increase then decrease in MEPP frequency and a decrease in MEPP amplitude. In elevated vesicle recycling induced by high K + -Ringer solution, IP 4 and IP 6 have similar effects, except decreasing MEPP frequency at a higher concentration (10 −4 M). We conclude that (1) high inositol polyphosphates may represent a link between IP 3 and cAMP pathways; (2) the IP 3 -induced increase of MEPP amplitude is likely to be due to its high inositol metabolites; (3) PI 3-K is not involved in the IP 4 and IP 6 -mediated MEPP frequency increases, but may be involved in MEPP size.