Effects of ryanodine on acetylcholine-induced Ca²⁺ mobilization in single smooth muscle cells of the porcine coronary artery

Abstracts

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 μM 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 μM ACh or 20 mM caffeine (but not 128 mM K⁺) transiently increased [Ca²⁺]_i. Ryanodine (50 μM) greatly inhibited the phasic increase in [Ca²⁺]_i induced by 10 μM 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 μM) enhanced the tonic increase in [Ca²⁺]_i induced by 128 mM K⁺ and also by 10 μM ACh in Ca²⁺-containing solution. In Ca²⁺-free solution containing 0.5 mM EGTA, ACh (10 μM) 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 μM 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.