Modulation of agonist-induced Ca 2+ release by SR Ca 2+ load: direct SR and cytosolic Ca 2+ measurements in rat uterine myocytes

Abstract

Release of Ca 2+ from sarcoplasmic reticulum (SR) is one of the most important mechanisms of smooth muscle stimulation by a variety of physiologically active substances. Agonist-induced Ca 2+ release is considered to be dependent on the Ca 2+ content of the SR, although the mechanism underlying this dependence is unclear. In the present study, the effect of SR Ca 2+ load on the amplitude of [Ca 2+] i transients elicited by application of the purinergic agonist ATP was examined in uterine smooth muscle cells isolated from pregnant rats. Measurement of intraluminal Ca 2+ level ([Ca 2+] L) using a low affinity Ca indicator, mag-fluo-4, revealed that incubation of cells in a high-Ca 2+ (10 mM) extracellular solution leads to a substantial increase in [Ca 2+] L (SR overload). However, despite increased SR Ca 2+ content this did not potentiate ATP-induced [Ca 2+] i transients. Repetitive applications of ATP in the absence of extracellular Ca 2+, as well as prolonged incubation in Ca 2+–free solution without agonist, depleted the [Ca 2+] L (SR overload). In contrast to overload, partial depletion of the SR substantially reduced the amplitude of Ca 2+ release. ATP-induced [Ca 2+] i transients were completely abolished when SR Ca 2+ content was decreased below 80% of its normal value indicating a steep dependence of the IP 3-mediated Ca 2+ release on the Ca 2+ load of the store. Our results suggest that in uterine smooth muscle cells decrease in the SR Ca 2+ load below its normal resting level substantially reduces the IP 3-mediated Ca 2+ release, while Ca 2+ overload of the SR has no impact on such release.