Confocal imaging of calcium release events in single smooth muscle cells

Abstract

Localized [Ca2+]i transients ('sparks') first directly detected in cardiac myocytes were considered to represent 'elementary' Ca(2+)-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 approximately 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 approximately 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 Ca(2+)-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.