Ca2+ signals mediated by Ins(1,4,5)P-3-gated channels in rat ureteric myocytes

Localized Ca2+-release signals (puffs) and propagated Ca2+ waves were chara cterized in rat ureteric myocytes by confocal microscopy. Ca2+ puffs were e voked by photorelease of low concentrations of Ins(1,4,5)P-3 from a caged p recursor and by low concentrations of acetylcholine; they were also observe d spontaneously in Ca2+-overloaded myocytes. Ca2+ puffs showed some variabi lity in amplitude, time course and spatial spread, suggesting that Ins(1,4, 5)P-3-gated channels exist in clusters containing variable numbers of chann els and that within these clusters a variable number of channels can be rec ruited. Immunodetection of Ins(1,4,5)P-3 receptors revealed the existence o f several spots of fluorescence in the confocal cell sections, supporting t he existence of clusters of Ins(1,4,5)P-3 receptors. Strong Ins(1,4,5)P-3 p hotorelease and high concentrations of acetylcholine induced Ca2+ waves tha t originated from an initiation site and propagated in the whole cell by sp atial recruitment of neighbouring Ca2+-release sites. Both Ca2+ puffs and C a2+ waves were blocked selectively by intracellular applications of heparin and an anti-Ins(1,4,5)P-3-receptor antibody, but were unaffected by ryanod ine and intracellular application of an anti-ryanodine receptor antibody, m RNAs encoding for the three subtypes of Ins(1,4,5)P-3 receptor and subtype 3 of ryanodine receptor were detected in these myocytes, and the maximal bi nding capacity of [H-3]Ins(1,4,5)P-3 was 10- to 12-fold higher than that of [H-3]-ryanodine. These results suggest that Ins(1,4,5)P-3-gated channels m ediate a continuum of Ca2+ signalling in smooth-muscle cells expressing a h igh level of Ins(1,4,5)P-3 receptors and no subtypes 1 and 2 of ryanodine r eceptors.