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 effect of urotensin II (U-II) on spontaneous transmitter release was examined in the frog to see if the biological activity of this vasoactive peptide extended to neural tissues. 2. In normal Ringer solution, frog and human U-II (fU-II and hU-II, respectively) caused concentration-dependent, reversible increases in miniature endplate potential (MEPP) frequency, with hU-II about 22 times more potent than fU-II. hU-II caused a dose-dependent increase in MEPP amplitude, whereas fU-II caused an increase, followed by a decrease with higher concentrations. 3. Increasing extracellular Ca 2+ three-fold had no effect on the MEPP frequency increase to 25 μM hU-II. Pretreatment with thapsigargin to deplete endoplasmic reticulum Ca 2+ caused a 61% reduction in the MEPP frequency increase to 25 μM hU-II. 4. Pretreatment with the phospholipase C inhibitor U-73122 caused a 93% reduction in the MEPP frequency increase to 25 μM hU-II and a 15% reduction in the increase in MEPP amplitude. Pretreating with antibodies against the inositol 1,4,5-trisphosphate (IP 3) type 1 receptor using liposomal techniques reduced the MEPP frequency increase by 83% but had no effect on MEPP amplitude. 5. Pretreating with protein kinase C inhibitors (bisindolylmaleimide I and III) had no effect on the response to 25 μM hU-II, but pretreating with protein kinase A inhibitors (H-89 and KT5720) reduced the MEPP frequency increase by 88% and completely abolished the increase in MEPP amplitude. 6. Our results show that hU-II is a potent stimulator of spontaneous transmitter release in the frog and that the effect is mediated by IP 3 and cyclic AMP/protein kinase A.