Variability in spontaneous subcellular calcium release in guinea‐pig ileum smooth muscle cells

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Abstract
1. Spontaneous, localized transient increases in [Ca2+]i ('Ca2+ sparks') were observed in about 40 % of fluo-3-loaded myocytes examined using laser scanning confocal microscopy. Ca2+ sparks persisted after application of Cd2+ (200 microM), but were abolished by ryanodine (30 microM) or thapsigargin (0.1 microM), suggesting that they arise from the spontaneous activation of ryanodine receptors (RyR) in the sarcoplasmic reticulum (SR). 2. Ca2+ sparks occurred much more frequently at certain sites (or 'frequent discharge sites', FDSs) within any confocal plane of the cell and line-scan imaging revealed a wide variation in their spatial size, amplitude and time course. Some spontaneous local transients were very similar to 'Ca2+ sparks' observed in heart, i.e. lasting approximately 200 ms with a peak fluorescence ratio of 1.75 +/- 0.23 (mean +/- s.d., n = 33). Other events were faster and smaller, lasting only approximately 40 ms with a peak normalized fluorescence of 1.36 +/- 0.09 (mean +/- s.d., n = 28). 3. Spontaneous Ca2+ waves with a wide range of propagation velocities (between 30 and 260 micron s-1) were also observed. In about 60 % of records (n = 33), Ca2+ sparks could be detected at the sites of wave initiation. Waves of elevated [Ca2+]i propagated with non-constant velocity and in some cases terminated. These observations could be explained by heterogeneity in the distribution of subcellular release sites as well as variability in the contribution of each release site to the wave. 4. Spontaneous [Ca2+]i transients in single dispersed visceral smooth muscle cells have a wide spectrum of behaviour that is likely to be the result of spatio-temporal recruitment of smaller local events, probably via a calcium-induced calcium release (CICR) mechanism. The spatial non-uniformity of SR and RyR distribution within the cell may account for the existence of 'frequent discharge sites' firing the majority of the smooth muscle Ca2+ sparks and the wide variation in the Ca2+ wave propagation velocities observed.