Modulation of agonist-induced Ca²⁺ release by SR Ca²⁺ load: Direct SR and cytosolic Ca²⁺ measurements in rat uterine myocytes

Release of Ca²⁺ from sarcoplasmic reticulum (SR) is one of the most important mechanisms of smooth muscle stimulation by a variety of physiologically active substances. Agonist-induced Ca²⁺ release is considered to be dependent on the Ca²⁺ content of the SR, although the mechanism underlying this dependence is unclear. In the present study, the effect of SR Ca²⁺ load on the amplitude of [Ca²⁺]_i transients elicited by application of the purinergic agonist ATP was examined in uterine smooth muscle cells isolated from pregnant rats. Measurement of intraluminal Ca²⁺ level ([Ca²⁺]_L) using a low affinity Ca indicator, mag-fluo-4, revealed that incubation of cells in a high-Ca²⁺ (10 mM) extracellular solution leads to a substantial increase in [Ca²⁺]_L (SR overload). However, despite increased SR Ca²⁺ content this did not potentiate ATP-induced [Ca²⁺]_i transients. Repetitive applications of ATP in the absence of extracellular Ca²⁺, as well as prolonged incubation in Ca²⁺-free solution without agonist, depleted the [Ca²⁺]_L (SR overload). In contrast to overload, partial depletion of the SR substantially reduced the amplitude of Ca²⁺ release. ATP-induced [Ca²⁺]_i transients were completely abolished when SR Ca²⁺ content was decreased below 80% of its normal value indicating a steep dependence of the IP₃-mediated Ca²⁺ release on the Ca²⁺ load of the store. Our results suggest that in uterine smooth muscle cells decrease in the SR Ca²⁺ load below its normal resting level substantially reduces the IP₃-mediated Ca²⁺ release, while Ca²⁺ overload of the SR has no impact on such release.