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 Ca(2+)-containing or Ca(2+)-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 microM 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 Ca(2+)-free solution containing 0.5 mM ethylenebis(oxonitrilo)tetraacetate (EGTA), the resting [Ca2+]i rapidly decreased to 102 nM within 5 min, and 10 microM ACh or 20 mM caffeine (but not 128 mM K+) transiently increased [Ca2+]i. Ryanodine (50 microM) greatly inhibited the phasic increase in [Ca2+]i induced by 10 microM 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 microM) enhanced the tonic increase in [Ca2+]i induced by 128 mM K+ and also by 10 microM ACh in Ca(2+)-containing solution. In Ca(2+)-free solution containing 0.5 mM EGTA, ACh (10 microM) 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 microM ACh in Ca(2+)-free solution.(ABSTRACT TRUNCATED AT 250 WORDS)