Abstract
Localized Ca(2+)-release signals (puffs) and propagated Ca(2+) waves were characterized in rat ureteric myocytes by confocal microscopy. Ca(2+) 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 Ca(2+)-overloaded myocytes. Ca(2+) 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 Ca(2+) waves that originated from an initiation site and propagated in the whole cell by spatial recruitment of neighbouring Ca(2+)-release sites. Both Ca(2+) puffs and Ca(2+) 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 [(3)H]Ins(1,4,5)P(3) was 10- to 12-fold higher than that of [(3)H]ryanodine. These results suggest that Ins(1,4,5)P(3)-gated channels mediate a continuum of Ca(2+) 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.