Confocal imaging of calcium release events in single smooth muscle cells

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Abstract
Localized [Ca2+]i transients (‘sparks’) first directly detected in cardiac myocytes were considered to represent ‘elementary’ Ca2+‐release events playing a key role during excitation–contraction coupling ( Cheng et al. 1993 ). In this study we employed confocal [Ca2+]i imaging to characterize subcellular calcium signalling in fluo‐3 loaded visceral and vascular smooth muscle cells. In some experiments membrane potential of the myocyte was controlled using whole‐cell patch clamp technique and changes in membrane current were recorded simultaneously with [Ca2+]i imaging. Some local [Ca2+]i transients were very similar to ‘Ca2+ sparks’ observed in heart, i.e. lasting ≈200 ms with a peak fluorescence ratio of 1.75 ± 0.23 (mean ± SD, n = 33). Ca2+ sparks were found to occur in certain preferred locations in the cell, termed frequent discharge sites. Other events were faster and smaller, lasting only ≈40 ms with a peak normalized fluorescence of 1.36 ± 0.09 (mean ± SD, n = 28). A high correlation between spontaneous transient outward currents and spark occurrence was observed. Proliferating waves of elevated [Ca2+]i initiated during membrane depolarization seem to arise from spatio‐temporal recruitment of local Ca2+‐release events. The spatial non‐uniformity of sarcoplasmic reticulum and ryanodine receptor distribution within the cell may account for the existence of ‘frequent discharge sites’ and the wide variation in the Ca2+ wave propagation velocities observed.