EFFECTS OF RYANODINE ON ACETYLCHOLINE-INDUCED CA2+ MOBILIZATION IN SINGLE SMOOTH-MUSCLE CELLS OF THE PORCINE CORONARY-ARTERY

To study the essential features of acetylcholine (ACh)- and caffeine-sensitive cellular Ca2+ storage sites in single vascular smooth muscle cells of the porcine coronary artery, the effects of ryanodine on both ACh- and caffeine-induced Ca2+ mobilization were investigated by measuring intracellular Ca2+ concentration ([Ca2+]i) using Fura 2 in Ca2+-containing or Ca2+-free solution. The resting [Ca2+]i of the cells was 122 nM in normal physiological solution and no spontaneous activity was observed. In a solution containing 2.6 mM Ca2+, 10-mu-M ACh or 128 mM K+ produced a phasic, followed by a tonic, increase in [Ca2+]i but 20 mM caffeine produced only a phasic increase. In Ca2+-free solution containing 0.5 mM ethylenebis(oxonitrilo)tetraacetate (EGTA), the resting [Ca2+]i rapidly decreased to 102 nM within 5 min, and 10-mu-M ACh or 20 mM caffeine (but not 128 mM K+) transiently increased [Ca2+]i. Ryanodine (50-mu-M) greatly inhibited the phasic increase in [Ca2+]i induced by 10-mu-M ACh or 5 mM caffeine and increased the time to peak and to the half decay after the peak in the presence or absence of extracellular Ca2+. By contrast, ryanodine (50-mu-M) enhanced the tonic increase in [Ca2+]i induced by 128 mM K+ and also by 10-mu-M ACh in Ca2+-containing solution. In Ca2+-free solution containing 0.5 mM EGTA, ACh (10-mu-M) failed to increase [Ca2+]i following application of 20 mM caffeine. The level of [Ca2+]i induced by 20 mM caffeine was greatly reduced, but not abolished, following application of 10-mu-M ACh in Ca2+-free solution. These results suggest that both ACh and caffeine release Ca2+ from the ryanodine-sensitive sarcoplasmic reticulum (SR) in smooth muscle cells of the porcine coronary artery. The finding that ryanodine significantly increased the resting [Ca2+]i and inhibited the rate of decline of [Ca2+]i following wasthout of high K+ or ACh in Ca2+-containing solution suggests that SR may negatively regulate the resting [Ca2+]i in smooth muscle cells of the porcine coronary artery.