Crosstalk between ryanodine receptors and IP3 receptors as a factor shaping spontaneous Ca2+-release events in rabbit portal vein myocytes

In smooth muscle cells freshly isolated from rabbit portal vein, there was only one site discharging the majority of spontaneous Ca2+-release events; the activity of this single site was studied using laser scanning confocal imaging after loading the cells with the fluorescent Ca2+ indicator fluo-4 acetoxymethyl ester. Localised spontaneous Ca2+-release events visualised by line-scan imaging revealed two predominant spatiotemporal. patterns: (i) small-amplitude, fast events similar to Ca2+ sparks in cardiomyocytes and (ii) larger and slower events. The sum of two Gaussian profiles was well fitted to the amplitude histogram (peak frequencies at 1.8 and 3.2 F/F-o) and spatial spread (full width at half-maximal amplitude) histogram (peak frequencies at 2 and 3.8 mum) for the 230 localised Ca2+-release events analysed. The existence of two populations of Ca2+-release events was also supported by the histograms of the rise times and half-decay times, which revealed modes at 38 and 65 ms, respectively. Shifting the scan line along the z-axis during imaging from a single discharge site suggested that the appearance of two populations of Ca2+-release events is not due to out-of-focus imaging. Both small and large events persisted upon 3-5 min exposure to 1-5 mum nicardipine, but were abolished after 10-15 min exposure to 50-100 mum ryanodine, 0.1 mum thapsigargin or 10 mum cyclopiazonic acid. Only small-amplitude, fast events persisted in the presence of inhibitors of inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release, 10 mum xestospongin C or 30 mum 2-aminoethoxy-diphenylborate (2-APB), or in the presence of 2.5 mum U-73122 (a phospholipase C (PLC) inhibitor). Coupling between neighbouring Ca2+-release domains giving rise to spontaneous [Ca2+](i) waves was abolished in the presence of 2-APB. Examination of the saltatory propagation of the waves suggested that the critical factor that determines propagation between domains is a time-dependent change in the sensitivity of ryanodine receptors and/or IP3 receptors to Ca2+, which can give rise to 'loose coupling' between release sites. These results suggest that activation Of IP3 receptors (due to the tonic activity of PLC and ongoing production Of IN recruits neighbouring domains of ryanodine receptors, leading to larger Ca2+ releases and saltatory propagation of [Ca2+](i) waves in portal vein myocytes.