Simultaneous measurements of changes in sarcoplasmic reticulum and cytosolic [Ca2+] in rat uterine smooth muscle cells

1. The role of the sarcoplasmic reticulum (SR) was investigated in spontaneous and agonist-induced uterine Ca2+ transients, by combining low- (mag-fluo-4) and high-affinity (fura-2) indicators to measure intraluminal SR ([Ca2+](L)) and cytosolic ([Ca2+](i)) calcium concentration, simultaneously, in single smooth muscle cells from pregnant rat uterus. 2. Carbachol or ATP, in the absence of extracellular Ca2+, decreased [Ca2+](L) and increased [Ca2+](i). Although some replenishment (around 50%) occurred in its absence, extracellular Ca2+ was required for full replenishment of the SR Ca2+. 3. In 4/15 cells, ATP evoked oscillations of [Ca2+](i). These were accompanied by successive release and re-uptake of SR Ca2+. Inhibition of the SR Ca2+-ATPase with thapsigargin abolished the oscillations and luminal changes. 4. Spontaneous [Ca2+](i) transients produced no detectable changes in [Ca2+](L). The larger [Ca2+](i) transients evoked by high-K+ depolarisation increased [Ca2+](L). Spontaneous activity was inhibited when [Ca2+](L) was increased. 5. These data show that it is possible to simultaneously measure SR and cytosolic [Ca2+], and to investigate their response to agonist application and spontaneous activity.