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