Effects of cyclopiazonic acid and ryanodine on cytosolic calcium and contraction in vascular smooth muscle

1 In smooth muscle, both Ca2+ release from the sarcoplasmic reticulum (SR) and Ca2+ influx across the plasma membrane are responsible for the increase in the cytosolic Ca2+ level ([Ca2+](i)). To understand further the role of SR on smooth muscle contraction, the effects of an inhibitor of the SR Ca2+ pump, cyclopiazonic acid (CPA 10 mu M), an inhibitor of the Ca2+-induced Ca2+ release, ryanodine, (10 mu M), and an activator of the Ca2+-induced Ca2+ release, caffeine (20 mM), on [Ca2+](i) and contractile force were examined in the ferret portal vein loaded with a photoprotein, aequorin. 2 CPA induced a small increase in the aequorin signal reaching a maximum at 7 min. Several minutes after the increase in the aequorin signal, muscle tension increased reaching a maximum at 21.5 min. In contrast, ryanodine changed neither the aequorin signal nor contraction. In the presence of ryanodine, caffeine induced a sustained increase in the aequorin signal and transient contraction. After washing ryanodine and caffeine, the aequorin signal and muscle tone returned to their respective control levels. After treatment with ryanodine and caffeine, the second addition of caffeine was almost ineffective whereas CPA still increased the aequorin signal and muscle tension. 3 In the presence of external Ca2+, noradrenaline (NA, 10 mu M) induced a transient increase followed by a sustained increase in the aequorin signal and sustained contraction. In contrast, KCl (70 mM) induced sustained increases in the aequorin signal and sustained contraction. In Ca2+-free solution, NA induced a small transient increase in the aequorin signal and a small transient contraction. These changes were inhibited in the presence of CPA or on pretreatment of the muscle with ryanodine and caffeine. These results suggest that CPA or ryanodine and caffeine depleted Ca2+ in SR. High K+ was ineffective in the absence of external Ca2+. 4 In the presence of external Ca2+ and CPA, NA and high K+ induced larger aequorin signals than in the absence of CPA, whereas the magnitude and shape of the contractions did not change. In contrast, pretreatment with ryanodine and caffeine did not have such an effect. In the muscle pretreated with ryanodine and caffeine, CPA changed the responses to high K+ and NA in a similar manner to that in the muscle without the pretreatment with ryanodine and caffeine. 5 Dissociation of contraction from [Ca2+](i) as measured with aequorin suggests that NA and high K+ increase Ca2+ in two compartments: a compartment containing contractile elements (contractile compartment) and another compartment unrelated to contractile elements (non-contractile compartment). Because CPA augmented the stimulant-induced increase in aequorin signal without changing contraction, the non-contractile compartment may be located near the SR and the CPA-sensitive SR Ca2+ pump may regulate the Ca2+ level in this compartment. However, because CPA changed neither the magnitude nor shape of the contractions in the presence of external Ca2+, the SR Ca2+ pump may have little effect on regulation of Ca2+ level in the contractile compartment. Furthermore, the release of Ca2+ from SR seems to have little effect on the increase in the contractile Ca2+ because ryanodine and caffeine changed neither the aequorin signals nor contractions induced by NA and high K+ in the presence of external Ca2+ in the ferret portal vein.