The release of chemical transmitter from nerve terminals is critically dependent on a transient increase in intracellular Ca2+.6,25 The increase in Ca2+ may be due to influx of Ca2+ from the extracellular fluid15 or release of Ca2+ from intracellular stores such as mitochondria.1,8,18 Whether Ca2+ utilized in transmitter release is liberated from organelles other than mitochondria is uncertain. Smooth endoplasmic reticulum is known to release Ca2+, e.g., on activation by inositol trisphosphate or cyclic adenosine diphosphate-ribose,2 so the possibility exists that Ca2+ from this source may be involved in the events leading to exocytosis. We examined this hypothesis by testing whether inositol trisphosphate and cyclic adenosine diphosphate-ribose modified transmitter release. We used liposomes to deliver these agents into the cytoplasmic compartment and binomial analysis to determine their effects on the quantal components of transmitter release. Administration of inositol trisphosphate (10-4M) caused a rapid, 25% increase in the number of quanta released. This was due to an increase in the number of functional release sites, as the other quantal parameters were unaffected. The effect was reversed with 40min of wash. Virtually identical results were obtained with cyclic adenosine diphosphate-ribose (10-4M). Inositol trisphosphate caused a 10% increase in quantal size, whereas cyclic adenosine diphosphate-ribose had no effect. The results suggest that quantal transmitter release can be increased by Ca2+ released from smooth endoplasmic reticulum upon stimulation by inositol trisphosphate or cyclic adenosine diphosphate-ribose. This may involve priming of synaptic vesicles at the release sites or mobilization of vesicles to the active zone. Inositol trisphosphate may have an additional action to increase the content of transmitter within the vesicles. These findings raise the possibility of a role of endogenous inositol phosphate and smooth endoplasmic reticulum in the regulation of cytoplasmic Ca2+ and transmitter release.

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 (IP4) and inositol 1,2,3,4,5,6-hexakisphosphate (IP6). Previous reports indicate that high inositol polyphosphates (IP4 and IP6) are involved in clathrin-coated vesicular recycling. In this study, we examined the effects of IP4 and IP6 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, IP4 and IP6 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 IP4-mediated MEPP frequency increase by 60% and abolished the IP6-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 IP4 and IP6-mediated MEPP frequency increases, but reduced the MEPP amplitude increase by 50%. In our previous reports, IP3, but not other second messengers releasing Ca2+ from internal Ca2+ stores, is able to enhance the MEPP amplitude. In order to dissociate the effect of Ca2+ release vs. metabolism to IP4 and IP 6, we evaluated the effects of 3-deoxy-3-fluoro-inositol 1,4,5-trisphosphate (3F-IP3), which is not converted to IP 4 or IP6. 3F-IP3 produced an increase then decrease in MEPP frequency and a decrease in MEPP amplitude. In elevated vesicle recycling induced by high K+-Ringer solution, IP4 and IP6 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 IP3 and cAMP pathways; (2) the IP3-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 IP6-mediated MEPP frequency increases, but may be involved in MEPP size.

1. The role of calmodulin (CaM) in transmitter release was investigated using liposomes to deliver CaM and monoclonal antibodies against CaM (antiCaM) directly into the frog motor nerve terminal. 2. Miniature endplate potentials (MEPPs) were recorded in a high K+ solution, and effects on transmitter release were monitored using estimates of the quantal release parameters m (number of quanta released), n (number of functional transmitter release sites), p (mean probability of release), and vars p (spatial variance in p). 3. Administration of CaM, but not heat-inactivated CaM, encapsulated in liposomes (1000 units ml-1) produced an increase in m (25%) that was due to an increase in n. MEPP amplitude was not altered by CaM. 4. Administration of antiCaM, but not heat-inactivated antiCaM, in liposomes (50 μl ml-1) produced a progressive decrease in m (40%) that was associated with decreases in n and p. MEPP amplitude was decreased (15%) after a 25 min lag time, suggesting a separation in time between the decreases in quantal release and quantal size. 5. Bath application of the membrane-permeable CaM antagonist W7 (28 μM) produced a gradual decrease in m (25%) that was associated with a decrease in n. W7 also produced a decrease in MEPP amplitude that paralleled the decrease in m. The decreases in MEPP size and m produced by W7 were both reversed by addition of CaM. 6. Our results suggest that CaM increases transmitter release by mobilizing synaptic vesicles at the frog motor nerve terminal.