Simultaneous measurements of changes in sarcoplasmic reticulum and cytosolic [Ca ²⁺] in rat uterine smooth muscle cells

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.