Ca2+ signals mediated by Ins(1,4,5)P3-gated channels in rat ureteric myocytes

Localized Ca2+-release signals (puffs) and propagated Ca2+ waves were characterized in rat ureteric myocytes by confocal microscopy. Ca2+ puffs were evoked by photorelease of low concentrations of Ins(1,4,5)P-3 from a caged precursor and by low concentrations of acetylcholine; they were also observed spontaneously in Ca2+-overloaded myocytes. Ca2+ puffs showed some variability in amplitude, time course and spatial spread, suggesting that Ins(1,4,5)P-3-gated channels exist in clusters containing variable numbers of channels and that within these clusters a variable number of channels can be recruited. Immunodetection of Ins(1,4,5)P-3 receptors revealed the existence of several spots of fluorescence in the confocal cell sections, supporting the existence of clusters of Ins(1,4,5)P-3 receptors. Strong Ins(1,4,5)P-3 photorelease and high concentrations of acetylcholine induced Ca2+ waves that originated from an initiation site and propagated in the whole cell by spatial recruitment of neighbouring Ca2+-release sites. Both Ca2+ puffs and Ca2+ waves were blocked selectively by intracellular applications of heparin and an anti-Ins(1,4,5)P-3-receptor antibody, but were unaffected by ryanodine and intracellular application of an anti-ryanodine receptor antibody, mRNAs 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 binding 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 mediate a continuum of Ca2+ signalling in smooth-muscle cells expressing a high level of Ins(1,4,5)P-3 receptors and no subtypes 1 and 2 of ryanodine receptors.